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Referee #1

Evidence, reproducibility and clarity

In this study, Makino et al. investigate how tension within the neck linker regulates the coordinated stepping of kinesin-1, a dimeric motor protein with two motor domains (or "heads"). Using high-resolution structural analyses, the authors identify a bulge near the neck linker's base in the nucleotide-free head that restricts forward extension, increasing steric hindrance when extended forward. This hindrance, they propose, prevents the tethered head from prematurely binding to the microtubule while the leading, microtubule-bound head awaits ATP. Molecular dynamic simulations and single-molecule fluorescence assays support this steric hindrance-based model, suggesting a mechanism that thermodynamically suppresses off-pathway transitions, thereby guiding kinesin-1's processive movement along microtubules. I recommend acceptance of the manuscript, subject to the following revisions:

1. Abstract: The current abstract is challenging to follow. For instance, the phrase "The detached head preferentially binds to the forward tubulin-binding site after ATP binding, but the mechanism preventing premature binding to the microtubule while awaiting ATP remains unknown" could imply that the tethered head binds ATP, which is misleading. A clearer statement would be: "The detached head preferentially binds to the forward tubulin-binding site after ATP binding to the leading, microtubule-bound head, but the mechanism preventing premature binding to the microtubule while its partner awaits ATP remains unknown."
2. Terminology: In the introduction, consider rephrasing to "...its two motor domains ("heads")."
3. Lines 71-72: The sentences "This mechanism explains how the tethered head preferentially binds to the forward-binding site 'after ATP binding.' However, it does not clarify how the tethered head is prevented from rebinding to the rear-tubulin binding site 'before ATP binding'" could be rephrased for clarity. A suggested revision is: "This mechanism explains how the tethered head preferentially binds to the forward-binding site after ATP binding to the microtubule-bound, leading head. However, it does not clarify how the tethered head is prevented from rebinding to the rear-tubulin binding site before ATP binds to the leading head."
4. Line 98: Consider revising "could release both ADP" to "could release both ADPs" or "could release both ADP molecules."
5. Lines 103-104: The statement "Therefore, these results suggest the tension posed to the neck linker plays a critical role in suppressing microtubule-binding of the tethered head" should be clarified. Since tension only develops in the two-heads-bound state, using "steric hindrance" instead of "tension" may improve precision.
6. Lines 374-375: Replace "...before ATP-binding triggers the forward stepping..." with "...before ATP binding to the leading head triggers the forward stepping..."
7. Tense Consistency: Ensure consistent use of present or past tense throughout the manuscript for clarity.

Significance

The conclusions are supported by the data provided, offering valuable insights into the coordination of kinesin's motor domains during movement. These findings help address a knowledge gap in kinesin stepping mechanics, making this work relevant to researchers studying cytoskeletal motor proteins.

When I think of antisemitic behavior, Nazi Germany is always the first thing to pop into my head. I never knew that the Russians similarly treated Jews, although not as extremely. Exerting leniency on your policy against emigration to drive a specific group of people out of your country is another form of hate. Given the conditions in Russia, I can understand why emigration to the West seemed so appealing. Anything would be better than their current treatment under antisemitic laws in Russia.

This is in reference to the status of Arjuna. It represents his lineage and authority. According to the story of Jada Bharata, he was a powerful, high standing king, but one with high paternal instinct. The story states, Bharata, "...rose from his position of meditation and rescu(ed) the fawn from the water, took it to his cottage, made a fire, and with care and attention fondled the little thing back to life." This shows his compassion of youth, whereas Arjuna is considered a harder, more stubborn head of authority.

Source: The Story Of Jada Bharata. The story of Jada Bharata. (n.d.). https://www.ramakrishnavivekananda.info/vivekananda/volume_4/lectures_and_discourses/the_story_of_jada_bharata.htm

How training loss is compute.

nm563 I also think that since 1 in 20 doesn't display the percentage many will not calculate it off the top of their head and will simply fall into this exact line of thinking stated.

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Reply to the reviewers

We thank the reviewers for the detailed evaluations and thoughtful comments, which have improved the clarity and readability of this manuscript. We have responded to all reviewer comments and incorporated their suggested changes into the text and figures. The line numbers in our response correspond to those in the revised manuscript. We have also included new experimental results suggested by reviewer 2, which further strengthen our main conclusion.

Reviewer #1

1. Introduction, page 3: The statement "Single dimeric kinesin moves processively along microtubules in a hand-over-hand manner by alternately moving the two heads in an 8-nm step toward the plus-end of the microtubule" is inaccurate. The kinesin heads take ~16 nm steps, while the center of mass advances in ~8 nm increments. Please adjust the wording accordingly.
2. Introduction, page 5: In the sentence "These results are consistent with the closed and open conformations of the nucleotide-binding pocket in the rear and front heads of microtubule-bound kinesin dimers observed in cryo-electron microscopy (cryo-EM) studies," I recommend changing the order to align with the previous sentence. The correct order would be "These results are consistent with the open and closed conformations of the nucleotide-binding pocket in the front and rear heads." Response: We thank the reviewer for pointing out our misunderstandings. We have corrected these sentences accordingly (lines 45-47 and lines 111-112).

Reviewer #2

MAJOR CONCERNS Limitations of this study: The authors need to discuss the limitations of their work. 1) They used a cys-lite kinesins mutant and introduced new surface-exposed cysteines. These mutants have lower kcat values than WT. 2) They used fluorescently labeled ATP molecules, which are hydrolyzed 10 times slower than unlabeled nucleotides. 3) They still observe crosslinking under reducing conditions and partial (but almost complete) crosslinking under oxidized conditions. 4)They assumed that cysteine crosslinked orientation mimics the orientation of the neck-linker in the front and rear conditions. The authors clearly pointed to these issues in the Results section. While these assumptions are also supported by several control experiments, the authors need to acknowledge some of these limitations in the Discussion as well.

__Response: __We have now reiterated some of the key caveats in the Discussion, and newly described in the Results section those points not mentioned in the original manuscript that do not affect the conclusion. We also added a summary of the limitations and caveats into the first paragraph of the Discussion section (lines 425-431).

1) We added a sentence in the Results section to describe that the ATP-binding kinetics of the Cys-light mutant remained consistent with previous studies as follows: “First, we demonstrated that k+1 and k-1 of the wild-type head without Cys-modification were unchanged after oxidization (Table 1) and were comparable to those previously reported (Cross, 2004)” (lines 163-166). The reduced kcat values of cysteine pair-added mutants before crosslinking were primarily due to reduced microtubule association rate (data not included in this manuscript). We have added a sentence in the Results section describing the kcat results as follows: “The reduced ATPase activity primarily results from a decreased microtubule association rate (data to be presented elsewhere) with little change in ATP binding or microtubule dissociation rates (Table 1).” (lines 144-146).

2) Fluorescently-labeled ATP was used to determine the ATP off-rates of the E236A mutant monomer and E236A rear head of the E236A/WT heterodimer. Two caveats in these measurements could lead to underestimating the ATP off-rate: 1) The off rate of Alexa-ATP from the head may be reduced compared to unmodified ATP, as Alexa-ATP driven motility showed a 10-fold reduce velocity. 2) The ATP off-rate of the E236A mutant may differ from that of the rear head in the wild-type dimer, since the E236A mutant likely stabilizes the neck linker-docked state more strongly than in the rear head of the wild-type dimer. These points are crucial for evaluating the results of ATP off-rate and the affinity for ATP, so we have added sentences in the Discussion section as follows: “We note, however, that this Kd of ATP may somewhat underestimate the true value in wild-type kinesin for two reasons: first, the E236A mutation likely stabilizes the neck linker-docked, closed state more than in the rear head of the wild-type dimer (Rice et al., 1999), and second, the Alexa-ATP used to measure the ATP off-rate of E236A head showed ~10-fold smaller velocity compared to unmodified ATP, partly due to a slower ATP off-rate (Figure 2____-____figure supplement 3).” (lines 449-454).

3) Under reducing condition, the rear head crosslink contained 30% crosslinked species, while under oxidized condition, the front head crosslink contained 11% un-crosslinked species (Figure 1____-____figure supplement 1). These heterogeneities likely affect the rate constants of k-1 for rear head crosslink and k2 for front head crosslink, as crosslinked and un-crosslinked species showed significantly different rate constants. However, we did not use the rear head crosslink result to determine k-1, since ATP hydrolysis likely occurred before reversible ATP dissociation. Instead, we used E236A monomer to estimate the k-1 of the rear head. In addition, the result for k2 of the front head crosslink was further validated using the E236A/WT heterodimer, which will be described in the next section.

4) This is an important point, and therefore, we conducted experiments using the E236A/WT heterodimer (including new experimental results of ATP binding kinetics of the front head) and obtained consistent results. To address this point, we have revised the following sentences in the Discussion: “In the front head, backward orientation of the neck linker has little effect on ATP binding and dissociation rates, both when measured for a monomer crosslink (Figure 2A, B) and for the front head of a E236A-WT heterodimer (Figure 4B, C, F).” (lines 432-433); “However, we found that the ATP-induced detachment rates from microtubule (k2) were similarly reduced for both the front head crosslink (7.0 s-1; Figure 3A) and the front WT head of the E236A/WT heterodimer (6.3 s-1; Figures 6D), suggesting that a step subsequent to ATP binding is gated in the front head.” (lines 437-441).

Line 238, the authors wrote that "forward constraint on the neck linker in the rear head does not significantly accelerate the detachment from the microtubule." Can the authors comment on why the read-head-like construct has a low affinity for microtubules even in the absence of ATP (Line 220)? I believe that the low affinity of the head in this conformation is more striking (and potentially more important) than the changes they observe in detachment rates. The authors should also consider that they might not be able to reliably measure the changes in the dissociation rate in single molecule assays of this construct (especially if the release rate of the rear head in the oxidized condition increases a lot higher than that of WT). The kymographs show infrequent and brief events, which raises doubts about how reliably they can measure the release rates under those imaging conditions. Higher motor concentrations and faster imaging rates may address this concern.

Response: The low microtubule affinity of the rear-head-like crosslink stems from an extremely slow ADP release rate upon microtubule binding, not from a fast microtubule-detachment rate. Using stopped-flow measurements of microtubule-binding kinetics (microtubule-stimulated mant-ADP release and microtubule association rates), we found that the rear-head-crosslink resulted in a 2,000-fold decrease in the microtubule-stimulated ADP-release rate. This finding also explains the reduced ATPase of the rear-head-crosslink (Figure 1E). Since this low microtubule-affinity state occurs in the ADP-bound state rather than the ATP-bound state, we hypothesized that the neck-linker docked ADP-bound state cannot effectively bind to microtubules, requiring neck-linker undocking for microtubule binding (Mattson-Hoss et al., Proc. Natl. Acad. Sci., 111, 7000-7005 (2014)). While we acknowledge that understanding slow microtubule binding in the neck linker docked state is important for elucidating the mechanism and regulation of microtubule-binding of the head, this paper focuses specifically on the mechanism and regulation of “microtubule-detachment”. We plan to present these microtubule-binding kinetics data in a separate manuscript currently in preparation.

To explain the low microtubule affinity of the rear-head-crosslink, we added this explanation to the text; “because this constraint on the neck linker dramatically reduces the microtubule-activated ADP release rate (data to be presented elsewhere), creating a weak microtubule binding state” (lines 226-228).

Although the rear head crosslinking construct under oxidative condition showed fewer fluorescent spots per kymographs (images) due to its low microtubule binding rate, we collected more than one hundred spots by recording additional microscope movies (N=140; Figure 3-figure supplement 2B), ensuring sufficient data for statistical analysis.

Figure 2: How do the rates shown in Figure 2A-B compare to the previous kinetics studies in the field? The authors compare the dissociation rate of WT measured in rapid mixing experiments to that of E236A in smFRET assays. It is not clear whether these comparisons can be made reliably using different assays. Can the authors perform rapid mixing of E236A or try to determine the rate for the WT from smFRET trajectories?

Response: The results of ATP on/off rates are comparable to the previous stopped flow measurements of ATP binding to monomeric kinesin-1 on microtubule, which are 2-5 µM-1s-1 and ~150 s-1, respectively (summarized in the review by Cross (2004)). We added a sentence as follows: “First, we demonstrated that k+1 and k-1 of the wild-type head without Cys-modification were unchanged after oxidization (Table 1) and were comparable to those previously reported (Cross, 2004).” (lines 163-166).

As the reviewer pointed out, the rapid mixing and smFRET data cannot be directly compared due to the differences in temporal resolution and fluorescent probe used. In Figure 2E (2F in the revised version), we measured ATP dissociation rate for both WT and E236A using smFRET. Due to the lower temporal resolution, we could not accurately determine ATP binding rate using smFRET. Therefore, to compare the ATP binding rate between WT and E236A heads, we now have added stopped-flow measurements of mant-ATP binding to the E236A monomer, as shown in Fig. 2C and Figure 2-supplement 2, and described in the text (lines 182-185).

Line 396: One of the most significant conclusions of this work is that the backward orientation of the neck linker has little effect on ATP binding to the front head. This is only supported by the results shown in Fig. 2A-B. Can the authors perform/analyze smFRET assays on the E236A/WT heterodimer to directly show whether the ATP binding rate to the WT head is affected or not affected by the orientation of the neck linker of the WT head?

Response: We agree with the reviewer that our finding about ATP binding to the front head is potentially significant in the kinesin field, as it has been widely believed that ATP-binding is suppressed in the front head. In our original manuscript, this conclusion was supported only by the measurement of ATP on-rate of the front-head-crosslink, which may differ from the front head of a dimer in which the backward orientation of the neck linker is maintained by the backward strain. Although the reviewer suggested performing smFRET experiments using E236A/WT heterodimer, smFRET have relatively low temporal resolution (50-100 fps) and cannot accurately measure the frequency of ATP binding, so we used this technique only to determine ATP off rates. In this revised manuscript, we now have added stopped-flow experiments to separately measure the ATP binding to the front and rear heads of the E236A/WT heterodimer. By labeling the rear E236A head with a fluorophore to quench the mant-ATP signal bound to the rear head, we successfully measured mant-ATP binding rate to the front head. We found that the ATP-binding rate to the front head was comparable to that of an unconstrained monomer head, providing direct evidence for our conclusion. The revised version includes Fig. 4 A-C (with Figure 4-supplement 2; Figs. 4 and 5 are swapped in order) showing the kinetics of ATP binding to the front and rear heads of the E236A/WT heterodimer, with corresponding text in the result section (lines 315-324).

MINOR CONCERNS Lines 31 and 32: I recommend replacing "ATP affinity" with "ATP binding rate" or "the dissociation of ATP" to be more specific. This is because they do not directly measure the affinity (Kd), but instead measure the on or off rates. Line 41: Replace "cellar" with "cellular". Line 83: The authors should cite Andreasson et al. here.

Response: We have corrected these sentences accordingly (lines 31, 40, 85).

Lines 83-86: It seems this sentence belongs to the next paragraph. It also needs a citation(s).

Response: This statement lacks experimental evidence and may confuse readers, so we have removed it for clarity.

Line 151: It would be helpful to add a conclusion sentence at the end of this paragraph to explain what these results mean to the reader.

Response: A conclusion sentence of this paragraph has been added: “These results demonstrate that neck linker constraints in both forward and rearward orientations inhibit specific steps in the mechanochemical cycle of the head (lines 151-153)”.

Lines 175-180: I recommend combining and shortening these sentences, as follows, to avoid confusing the reader: "To detect the ATP dissociation event of the rear head, we employed a mutant kinesin with a point mutation of E236A in the switch II loop, which almost abolishes ATPase hydrolysis and traps in the microtubule-bound, neck-linker docked state,"

Response: We have corrected these sentences accordingly (line 179-181).

Line 314: "which was rarely observed ...". This is out of place and confusing as is. I recommend moving this sentence after the sentence that ends in Line 295.

Response: This sentence explains how the dark-field microscopy data was analyzed to determine whether the labeled head was in the leading or trailing position before detaching from the microtubule, but the explanation needs clarification. We removed the phrase “which was rarely observed for E236A-WT heterodimer” and simplified this sentence as follows: “Moreover, these observations allow us to distinguish whether the gold-labeled WT head was in the leading or trailing position just before microtubule detachment; the backward displacement of the detached head indicates that the labeled WT head occupied the leading position prior to detachment (Figure 5____-____figure supplement ____1).” (lines 347-351).

Line 300: Can the authors comment on why E236A/WT has a substantially lower ATPase rate than WT homodimer? Is it possible to determine which step in the catalytic cycle is inhibited?

Response: We demonstrated that the k2 (microtubule-detachment rate) of the front head matched the ATP turnover rate of the E236A/WT heterodimer (Figure 6 B and E), suggesting that the inhibited step occurs after ATP binding in the front head. In contrast, the rear E236A head showed virtually no ATP hydrolysis activity, since in high-speed dark field microscopy, we observed forward step caused by rear E236A head detachment from microtubule only rarely, approximately once every few seconds (Figure 5-figure supplement 1). We added a sentence in the text as follows: “As described later, the reduced ATPase rate results from suppressed microtubule detachment of the front WT head, while the rear E236A head is virtually unable to detach from microtubules” (lines 311-313).

Line 323: Is the unbound dwell time unchanged?

Response: The unbound dwell time exhibited a weak ATP-dependence, which we described only in Figure 5-supplement 2 (Figure 4-supplement 2 in the old version). We observed three distinct phases in the unbound dwell time based on mobility differences, with ATP dependence appearing only in the third phase. This finding suggests that ATP binding to the microtubule-bound E236A head is sometimes necessary for the detached WT head to rebind to the forward-tubulin binding site, indicating that the microtubule-bound E236A head occasionally releases ATP during the one-head-bound state (without the forward neck linker strain). To describe the ATP-dependence of the unbound dwell time, we added a sentence in the main text as follows: “In contrast, the dwell time of the unbound state of the gold-labeled WT head showed weak ATP dependence (Figure 5____-____figure supplement 2), indicating that the rear E236A head occasionally releases ATP when the front head detaches from the microtubule and the neck linker of E236A head becomes unconstrainted. This finding further supports the idea that forward neck linker strain plays a crucial role in reducing the reversible ATP release rate.” (lines 372-377).

Line 331: I recommend replacing "ATP-induced detachment" with "nucleotide-induced detachment" for clarity.

Response: We have revised the phrase accordingly (line 371).

Line 344: I recommend replacing "affinity" with "forward strain prevents the release of the nucleotide" or similar to avoid confusion. Forward strain reduces the off-rate of the bound nucleotide, rather than allowing ATP to bind more efficiently to the rear head.

Response: We agree to the reviewer’s comment and have corrected this sentence accordingly (line 338).

Lines 376-385: G7-12 constructs are introduced in Figure 6, but the results in this paragraph are shown in Figure 5. They should be moved to Figure 6 to avoid confusion.

Response: To improve the readability, we have reorganized Figures 4-6, such that all the figure panels related to the neck linker extended mutants are shown in Figure 6; Figure 5D has been moved to Figure 6F.

Line 421: delete "not" before "does not".

Response: We have corrected this typo.

Lines 433-441: Unless I am mistaken, more recent work in the kinesin field showed that backward trajectories of kinesin 1 reported by Carter and Cross are due to slips from the microtubule rather than backward processive runs of the motor.

Response: The slip motion demonstrated by Sudhakar et al. (2021) differs from the backstep motion reported by Carter and Cross (and many other laboratories). Slip motion occurs after kinesin detaches from the microtubule and continues until the bead returns to the trap center. In contrast, backstep motion occurs during processive movement when the trap force either exceeds or approaches the stall force. The kinetics of these motions also differ significantly: slip steps occur with a dwell time of 71 µs and are independent of ATP concentration, while backsteps take ~0.3 s (at 1 mM ATP) and depend on ATP concentration. These differences indicate that slip motion is phenomenologically distinct from backsteps occurring under supra-stall or near-stall force.

Line 474: Replace "suppresses" with "suppressed".

Response: We have corrected this typo.

Figure 4E: I would plot these results with increasing ATP concentration on the x-axis.

Response: We formatted Figure 4E to match Figure 4b from Isojima et al. (Nature Chem. Biol. 2015), to emphasize the difference in ATP dependence of the front and rear head.

Figure 4B: The authors should explain how they distinguish between bound and unbound states in the main text or figure legends. For example, it is not clear how the authors score when the motor rebinds to the microtubule in the first unbinding event shown in Figure 4B (displacement plot).

Response: The method was described in the Materials and Methods section, but we have now described how to distinguish between bound and unbound states in the main text as follows: “Unlike the unbound trailing head of wild-type dimer that showed continuous mobility (Isojima et al., 2016), the unbound WT head of E236A-WT heterodimer exhibited a low-fluctuation state in the middle (Figure 5B, s.d. trace). This low-fluctuation unbound state was distinguishable from the typical microtubule-bound state, having a shorter dwell time of ~5 ms compared to the bound state and positioning backward, closer to the E236A head, relative to the bound state (Figure 5____-____figure supplement 2).” (lines 351-356).

__Reviewer #3____

__

Minor Issues:

• Line 22, Abstract - The phrase "move in a hand-over-hand manner" could be clearer if phrased as "move in a hand-over-hand fashion" to improve readability.

Response: We changed the word “manner” to “process” (line 23).

• Abstract - Neck linker conformation in the leading head: The sentence "We demonstrate that the neck linker conformation in the leading kinesin head increases microtubule affinity without altering ATP affinity" would benefit from defining this conformation as "backward" for clarity.

• Abstract - Neck linker conformation in the trailing head: The sentence "The neck linker conformation in the trailing kinesin head increases ATP affinity by several thousand-fold compared to the leading head, with minimal impact on microtubule affinity" should also clarify that this conformation is "forward."

Response: We have corrected these sentences accordingly (line 30, 32).

• Abstract - Conformation-specific effects: The authors mention conformation-specific effects in the neck linker structure but do not define the neck linker's conformation or the motor domain's (MD) conformation. Clarifying these conformational changes would improve the explanation of how they promote ATP hydrolysis and dissociation of the trailing head before the leading head detaches from the microtubule, thereby providing a kinetic basis for kinesin's coordinated walking mechanism.

Response: We have revised the last sentence of the abstract accordingly by specifying the neck linker’s conformation as follows: ”In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.” (lines 34-37).

• Line 306 - Use of ATP in the E236A-WT heterodimer: In discussing the "ATP-induced detachment rate of the WT head in the E236A-WT heterodimer," the authors should consider justifying their choice of ATP over ADP for inducing microtubule (MT) dissociation. Since ATP typically promotes tighter MT binding and ATP turnover is reduced in forward-positioned WT heads, it may be unclear to some readers why ATP was chosen.

Response: We measured the ATP-induced detachment rate k2 of the front head of the E236A-WT heterodimer to validate our findings from the front-head-crosslinked monomer experiments, which demonstrated reduced k2 after oxidation. To clarify this point, we have now included ATP binding kinetics measurements for both front and rear heads of the E236A-WT heterodimer, as suggested by reviewer 2. These additional data demonstrate consistency between the results from the crosslinked monomer and E236A-WT heterodimer experiments.

• Discussion - Backward-oriented neck linker in the front head: The discussion mentions that the backward-oriented neck linker in the front head reduces its ATP-induced detachment rate, suggesting that a step after ATP binding (e.g., isomerization, ATP hydrolysis, or phosphate release) is gated in the front head. However, the authors do not clarify that the backward neck linker orientation would imply the nucleotide pocket should be open or at least not fully closed, thus inhibiting ATP turnover. This is important because, as demonstrated in other studies, full closure of the nucleotide pocket is linked to neck linker docking. This point should be addressed earlier in the discussion.

Response: We have addressed this point by revising this sentence as follows: “These results are consistent with an inability of the front head to fully close its nucleotide pocket to promote ATP hydrolysis and Pi release (Benoit et al., 2023), as will be discussed later.” (lines 441-443)

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Referee #3

Evidence, reproducibility and clarity

Summary:

Kinesin-1 is a dimeric motor protein that transports cargo along microtubules via an ATP-powered, hand-over-hand stepping mechanism. Its processive movement is driven by coordinated ATPase cycles in the two motor domains (heads), which ensures sequential, alternating microtubule binding and detachment for unidirectional motility. The goal of this study by Niitani et al. was to investigate how the neck linker, a region extending from the motor domain's C-terminus that undergoes large conformational changes, differentially regulates microtubule affinity and nucleotide turnover in each of the two heads. The authors employed a combination of pre-steady-state and single-molecule analyses to separately measure the ATP-binding and microtubule-detachment kinetics of the front and rear heads.

To isolate the kinetics of each head, the authors used disulfide crosslinking to trap the front and rear head states of a monomeric kinesin as well as a heterodimeric kinesin in which one chain was locked in the rear head conformation. These experiments revealed that the backward-facing neck linker of the front head reduces microtubule detachment, while the forward-facing neck linker of the rear head enhances ATP affinity. This finding is consistent with cryo-EM structures of two-head-bound kinesins, which show that the front head has an open nucleotide pocket with the neck linker pulled backward, while the rear head has a closed nucleotide pocket with the neck linker docked and extended toward the front head, regardless of the nucleotide bound.

The authors used pre-steady-state kinetics and single-molecule assays to explore how the neck linker conformation influences kinesin's motility cycle. ATP binding rates to the kinesin head on microtubules were measured through stopped-flow experiments with mant-ATP and nucleotide-free kinesin-microtubule complexes. These results showed that crosslinking the rear head reduced the ATP dissociation rate, while crosslinking the front head had no significant effect on ATP binding kinetics. The dissociation of ATP from the rear head was further investigated by trapping it in a pre-ATP hydrolysis state using a kinesin mutant (E236A) that significantly slows ATP hydrolysis and stabilizes the neck-linker docked state.

The authors also investigated the impact of neck-linker orientation (forward vs. backward) on kinesin detachment from microtubules by measuring turbidity changes after rapidly mixing nucleotide-free, crosslinked kinesin-microtubule complexes with ATP using a stopped-flow apparatus. Their results demonstrated that the forward neck linker conformation in the rear head promotes microtubule detachment, while the backward-oriented neck linker in the front head reduces the detachment rate. This suggests that the neck linker conformation mediates gating of microtubule affinity and nucleotide binding. Additionally, they show that partial docking of the neck linker onto the head is sufficient to partially open the gating mechanism.

To further investigate the role of neck linker tension in front head gating, the authors created an E236A-WT heterodimer. In this dimer, the E236A mutant functions as a long-lived rear head, trapping it in a neck-linker docked state, while the WT head is positioned at the front in the presence of ATP. The analysis of microtubule detachment kinetics and ATPase activity in this heterodimer revealed that although the front WT head can hydrolyze ATP, its catalytic activity is suppressed by the E236A mutant in the rear head.

Overall, this is a biochemically rigorous study that supports recent structural findings, particularly by highlighting the existence and role of the asymmetry of the neck linkers in two-head-bound kinesin dimers and the distinct conformations of their motor domains.

Minor Issues:

• Line 22, Abstract - The phrase "move in a hand-over-hand manner" could be clearer if phrased as "move in a hand-over-hand fashion" to improve readability.
• Abstract - Neck linker conformation in the leading head: The sentence "We demonstrate that the neck linker conformation in the leading kinesin head increases microtubule affinity without altering ATP affinity" would benefit from defining this conformation as "backward" for clarity.
• Abstract - Neck linker conformation in the trailing head: The sentence "The neck linker conformation in the trailing kinesin head increases ATP affinity by several thousand-fold compared to the leading head, with minimal impact on microtubule affinity" should also clarify that this conformation is "forward."
• Abstract - Conformation-specific effects: The authors mention conformation-specific effects in the neck linker structure but do not define the neck linker's conformation or the motor domain's (MD) conformation. Clarifying these conformational changes would improve the explanation of how they promote ATP hydrolysis and dissociation of the trailing head before the leading head detaches from the microtubule, thereby providing a kinetic basis for kinesin's coordinated walking mechanism.
• Line 306 - Use of ATP in the E236A-WT heterodimer: In discussing the "ATP-induced detachment rate of the WT head in the E236A-WT heterodimer," the authors should consider justifying their choice of ATP over ADP for inducing microtubule (MT) dissociation. Since ATP typically promotes tighter MT binding and ATP turnover is reduced in forward-positioned WT heads, it may be unclear to some readers why ATP was chosen.
• Discussion - Backward-oriented neck linker in the front head: The discussion mentions that the backward-oriented neck linker in the front head reduces its ATP-induced detachment rate, suggesting that a step after ATP binding (e.g., isomerization, ATP hydrolysis, or phosphate release) is gated in the front head. However, the authors do not clarify that the backward neck linker orientation would imply the nucleotide pocket should be open or at least not fully closed, thus inhibiting ATP turnover. This is important because, as demonstrated in other studies, full closure of the nucleotide pocket is linked to neck linker docking. This point should be addressed earlier in the discussion.

Significance

As indicated in the notes to the authors, I'm supportive of the work reported and view the a biochemically rigorous approach has been used to understand how the neck linker element has such a significant influence on the chemomechanical cycle of each motor domain.

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Referee #2

Evidence, reproducibility and clarity

This manuscript investigates the role of the neck linker in coordinating the stepping cycles of the two heads of a kinesin-1 motor. Previous studies in the field showed that kinesin walks by alternating stepping of its heads, referred to as hand-over-hand. In this stepping mechanism, the front head of a kinesin dimer must remain bound until the rear head dissociates from the microtubule, moves forward, and rebinds to the tubulin on the plus-end side of the front head. There is a large body of work done to address this question. These studies all point to the central role of the 14 amino acid extension, a neck-linker, which connects the two heads to a common stalk, in coordination of kinesin motility. In a two-head-bound state, the motor domains (heads) are oriented parallel to the microtubule, but the neck linkers are orienting toward each other, thereby, breaking the symmetry in a homodimeric motor. In addition, the neck linkers are quite short, almost stretching to their near contour length to accommodate the microtubule binding of both heads. Previous studies pointed out that either the opposing orientation or the intramolecular tension of the neck linkers coordinate the stepping cycle.

However, we still do not know which step(s) in the chemo-mechanical cycle is controlled by the neck-linker to keep the two heads out of phase. The front head gating model postulates that ATP binding to the front head is gated until the rear head detaches from the microtubule. The rear head gating model proposes that the neck linker accelerates the detachment of the rear head from the microtubule. In this study, the authors use pre-steady state kinetics and smFRET to address this question. They measured ATP binding and microtubule detachment kinetics of kinesin's catalytic domain with neck linker constraints 1) imposed by disulfide crosslinking of the neck linker in monomeric kinesin in backward (rear head-like) and forward (front head-like) orientations, and 2) using the E236A-WT heterodimer to create a two-head microtubule-bound state with the mutant and WT heads occupying the rear and front positions respectively. They found that neck-linker conformation of the rear head reduces the ATP dissociation rate but has little effect on microtubule affinity. In comparison, the neck-linker conformation of the front head does not change ATP binding to the front head, but it reduces ATP-induced detachment of the front head, suggesting that a step after ATP binding (i.e. ATP hydrolysis or Pi release) is gated in the front head.

Major Concerns

Limitations of this study: The authors need to discuss the limitations of their work. 1) They used a cys-lite kinesins mutant and introduced new surface-exposed cysteines. These mutants have lower kcat values than WT. 2) They used fluorescently labeled ATP molecules, which are hydrolyzed 10 times slower than unlabeled nucleotides. 3) They still observe crosslinking under reducing conditions and partial (but almost complete) crosslinking under oxidized conditions. 4)They assumed that cysteine crosslinked orientation mimics the orientation of the neck-linker in the front and rear conditions. The authors clearly pointed to these issues in the Results section. While these assumptions are also supported by several control experiments, the authors need to acknowledge some of these limitations in the Discussion as well.

Line 238, the authors wrote that "forward constraint on the neck linker in the rear head does not significantly accelerate the detachment from the microtubule." Can the authors comment on why the read-head-like construct has a low affinity for microtubules even in the absence of ATP (Line 220)? I believe that the low affinity of the head in this conformation is more striking (and potentially more important) than the changes they observe in detachment rates. The authors should also consider that they might not be able to reliably measure the changes in the dissociation rate in single molecule assays of this construct (especially if the release rate of the rear head in the oxidized condition increases a lot higher than that of WT). The kymographs show infrequent and brief events, which raises doubts about how reliably they can measure the release rates under those imaging conditions. Higher motor concentrations and faster imaging rates may address this concern.

Figure 2: How do the rates shown in Figure 2A-B compare to the previous kinetics studies in the field? The authors compare the dissociation rate of WT measured in rapid mixing experiments to that of E236A in smFRET assays. It is not clear whether these comparisons can be made reliably using different assays. Can the authors perform rapid mixing of E236A or try to determine the rate for the WT from smFRET trajectories?

Line 396: One of the most significant conclusions of this work is that the backward orientation of the neck linker has little effect on ATP binding to the front head. This is only supported by the results shown in Fig. 2A-B. Can the authors perform/analyze smFRET assays on the E236A/WT heterodimer to directly show whether the ATP binding rate to the WT head is affected or not affected by the orientation of the neck linker of the WT head?

Minor Concerns

Lines 31 and 32: I recommend replacing "ATP affinity" with "ATP binding rate" or "the dissociation of ATP" to be more specific. This is because they do not directly measure the affinity (Kd), but instead measure the on or off rates.

Line 41: Replace "cellar" with "cellular".

Line 83: The authors should cite Andreasson et al. here.

Lines 83-86: It seems this sentence belongs to the next paragraph. It also needs a citation(s).

Line 151: It would be helpful to add a conclusion sentence at the end of this paragraph to explain what these results mean to the reader.

Lines 175-180: I recommend combining and shortening these sentences, as follows, to avoid confusing the reader: "To detect the ATP dissociation event of the rear head, we employed a mutant kinesin with a point mutation of E236A in the switch II loop, which almost abolishes ATPase hydrolysis and traps in the microtubule-bound, neck-linker docked state,"

Line 314: "which was rarely observed ...". This is out of place and confusing as is. I recommend moving this sentence after the sentence that ends in Line 295.

Line 300: Can the authors comment on why E236A/WT has a substantially lower ATPase rate than WT homodimer? Is it possible to determine which step in the catalytic cycle is inhibited?

Line 323: Is the unbound dwell time unchanged?

Line 331: I recommend replacing "ATP-induced detachment" with "nucleotide-induced detachment" for clarity.

Line 344: I recommend replacing "affinity" with "forward strain prevents the release of the nucleotide" or similar to avoid confusion. Forward strain reduces the off-rate of the bound nucleotide, rather than allowing ATP to bind more efficiently to the rear head.

Lines 376-385: G7-12 constructs are introduced in Figure 6, but the results in this paragraph are shown in Figure 5. They should be moved to Figure 6 to avoid confusion.

Line 421: delete "not" before "does not".

Lines 433-441: Unless I am mistaken, more recent work in the kinesin field showed that backward trajectories of kinesin 1 reported by Carter and Cross are due to slips from the microtubule rather than backward processive runs of the motor.

Line 474: Replace "suppresses" with "suppressed".

Figure 4E: I would plot these results with increasing ATP concentration on the x-axis.

Figure 4B: The authors should explain how they distinguish between bound and unbound states in the main text or figure legends. For example, it is not clear how the authors score when the motor rebinds to the microtubule in the first unbinding event shown in Figure 4B (displacement plot).

Significance

I believe that this work will make an important contribution to the large body of literature focused on the mechanism of kinesin, which serves as an excellent model system to understand the kinetics and mechanics of a molecular motor. The mechanism proposed by the authors modifies the front-head gating model and is in agreement with recent structural work done on a kinesin dimer bound to a microtubule. Overall, the work is well performed, and the conclusions are well supported by the experimental data. I have several major and minor concerns to improve the clarity of this work and strengthen its conclusions.

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Referee #1

Evidence, reproducibility and clarity

In this study, the authors investigate the molecular mechanism behind kinesin-1's coordinated movement along microtubules, with a focus on how ATP binding, hydrolysis, and microtubule attachment/detachment are regulated in the leading and trailing heads. Using pre-steady state kinetics and single-molecule assays, they show that the neck linker's conformation modulates nucleotide affinity and detachment rates in each head differently, establishing an asynchronous chemo-mechanical cycle that prevents simultaneous detachment. Supported by cryo-EM structural data, their findings suggest that strain-induced conformational changes in the nucleotide-binding pockets are crucial for kinesin's hand-over-hand movement, presenting a detailed kinetic model of its stepping mechanism. The manuscript is well-crafted, technically rigorous, and should be of significant interest to cell biology and cytoskeletal motor researchers. I recommend acceptance with the following minor changes:

1. Introduction, page 3: The statement "Single dimeric kinesin moves processively along microtubules in a hand-over-hand manner by alternately moving the two heads in an 8-nm step toward the plus-end of the microtubule" is inaccurate. The kinesin heads take ~16 nm steps, while the center of mass advances in ~8 nm increments. Please adjust the wording accordingly.
2. Introduction, page 5: In the sentence "These results are consistent with the closed and open conformations of the nucleotide-binding pocket in the rear and front heads of microtubule-bound kinesin dimers observed in cryo-electron microscopy (cryo-EM) studies," I recommend changing the order to align with the previous sentence. The correct order would be "These results are consistent with the open and closed conformations of the nucleotide-binding pocket in the front and rear heads."

Significance

All conclusions are well-supported by the provided data. The findings address a critical gap in our understanding of how kinesin's two motor domains coordinate their movements, offering insights into the molecular basis of its stepping mechanism. This work should be of significant interest to the cytoskeletal research community.

This passage stood out to me because, it makes me want to keep reading what will happen next in the reading. I believe the author added this passage to attract the readers to keep reading and attract their attention. "If a baby wants to die, it will die". Means that if the baby is not taken care of it might not survive. Back in the 1900's mothers clearly did not take care about their children as much as they should. The theme of this story is how mothers that were in extreme poverty had children that they could not take care of and would end up dying, since there was not any other choice. The theme from this story from only from the pages assigned is that poverty, inequality, can change the way people view life.

I think that the ability to create an image is something so imporant when it comes to writing. One of my favorite authors masterfully can create an image in my head and i love listening to him because of it.

I am really bad at not using this kind of passive voice speech. This is mostly because it sound correct in my head rather than the simpler phrase. Although I am no learning when you use it, it just sounds like you're fumbling over your own words.

This is the conclusion

A ton of my interpretation is definitely based off personal and professional experience. But then again it is also based off of the mood that I am in. How much of a threshold do I have for my brain functionality that day? Interpretation and what is being relayed sometime is just to dang hard. Especially if the point isn't being made. Sometimes I'm ok get on with it please? Then I lose interest and stop talking sure I nod my head but if I am at that point, I am already done trying to figure it out.

I feel as if when it comes to the charm box and what's inside that's supposed to heal or help the individual almost take form of God.

I have seen this before on my phone, when it looks for my look-alike, it shows me someone with the same head shape and maybe same facial feature placement, but not really someone who looks like me.

Reviewer #3 (Public review):

This study explores sensory prediction errors in sensory cortex. It focuses on the question of how these signals are shaped by non-hierarchical interactions, specifically multimodal signals arising from same level cortical areas. The authors used 2-photon imaging of mouse auditory cortex in head-fixed mice that were presented with sounds and/or visual stimuli while moving on a ball. First, responses to pure tones, visual stimuli and movement onset were characterized. The authors then made the running speed of the mouse predictive of sound intensity and/or visual flow (closed loop). Mismatches were created through the interruption of sound and/or visual flow for 1 second, disrupting the expected sensory signal. As a control, sensory stimuli recorded during the close loop phase were presented again decoupled from the movement (open loop). The authors suggest that auditory responses to the unpredicted interruption of the sound, which affected neither running speed nor pupil size, reflect mismatch responses. That these mismatch responses were enhanced when the visual flow was congruently interrupted, indicates cross-modal influence of prediction error signals.

This study's strengths are the relevance of the question and the design of the experiment. The authors are experts in the techniques used. Responses to the interruption of the sound are similar in quality, if not quantity, in the predictive and the control situation, yet the contribution of sound offset sensitivity to the observed mismatch responses is not discussed.

I like to call it the invisible pond. Where thoughts just come up. It's interesting, like shower thoughts or letting the subconscious do its thing as you do your work. I like it. It really does come back to having good mood and taking care of your well-being.

Multi-head Latent Attention

Compress the key-value store of tokens, which decreases memory usage during inferencing.

"Whispering" technique of propaganda

there are some similarities between this part and the start of Christian creation. It would be interesting to see side by side in comparison to the Holy Bible

Here, Montesquieu critiques Luis XVI's insecurity, showing how absolutism prioritizes personal power over military success.

This type of situation that hits my 'tism/adhd ticks with a vengeance. I truly struggle with these daily interactions every time i come into contact with them. As someone with a passion for psychology i do fully understand the reason for phatic communion and why humans in general need these human to human transactions, again the true "lone Wolf" comes into play. Why would someone not shorten the wasted time in having a falsified conversation about how you are doing, when neither party has an interest in the response and usually using these interactions as a segway into talking about themselves. I usually throw a head nod, it saves so much time and the same interest insome one is conveyed. I suppose i come across this issue because i am someone that asks you a question because i actually care about the answer you are giving and have no interest in talking about myself in the process. Im working on this with myself but its the hardest thing in the world to be apart of for myself.

this statement pairs well with humans being such pack animals. It is very rare to find a person who is truly a "Lone Wolf". I don't know what else to add to this other then that was the thought that popped into my head. communication absolutely is a shaper or world and reality, of a singular person or a whole wide scale.

I totally agree with this statement here. For me personally I tend to forget things constantly if I don't explicitly write them down. This has forced me to have to carry around journals just containing to do list so that I don't forget what I have to do. I also find it interesting that the author explicitly mentions that it allows him to have those ideas critiqued. No matter how hard you try the first iteration will always need some reworking. This is something that I often forget and need to remind myself of when doing creative work. It is a process, and one that requires a lot of reworking to get right.

I find this an extremely valuable idea to use. We have a multitool in our pockets at all times - I catch ideas in my own head at many various times of day. Sometimes, I'm too preoccupied to catch the whisper and write it down. I need to "externalize" as written here.

I wanted to comment specifically on externalization and just my thoughts on this statement. I believe that externalization, in terms of just getting your ideas out, is extremely important and valuable. I enjoy working on music production, and many times, there are great ideas that you come across that maybe you forget to get in the daw and never make it out of your head. Many times, these ideas are extremely valuable to see where you can improve or what you've done better. I also wanted to express that externalization can also be important in separating yourself from the problem or design. Sometimes, not only thinking of how you would solve it can open another solution that could be crucial to you finding the best solution since perception oftentimes does not beat perception.

I read about a guy (https://www.boredpanda.com/not-everyone-having-internal-monologue/) that did an informal study about people not having an inner monologue. I was a bit shocked. I thought everyone had that voice that they argued with inside their head, sometimes quietly, sometimes aloud while working or driving. It's probably a bit lonely without having that pesky voice blabbing in the background all day.

I am constantly using inner voice dialogue. It is a 24/7 thing. Seems like it never ends. Constanly going over things in my head before they are spoken or written. In the catagory of constant overthinking. Always thinking of the reactions to my actions and so on.

Perceivability Issue – Low Contrast

The text on website lacks sufficient contrast against the white or light-coloured background. This makes it harder for users with visual impairments or low contrast sensitivity to read and navigate the website effectively. Improving the contrast between the text and background would align with WCAG standards and ensure that all users can perceive the content clearly.

the collective memory of empire w/ Britain at the head stopped UK form successfully merging into EU

Toni Morrison on the "serious function of racism"

Authors’ Response (31 October 2024)

GENERAL ASSESSMENT

Pannexin (Panx) hemichannels are a family of heptameric membrane proteins that form pores in the plasma membrane through which ions and relatively large organic molecules can permeate. ATP release through Panx channels during the process of apoptosis is one established biological role of these proteins in the immune system, but they are widely expressed in many cells throughout the body, including the nervous system, and likely play many interesting and important roles that are yet to be defined. Although several structures have now been solved of different Panx subtypes from different species, their biophysical mechanisms remain poorly understood, including what physiological signals control their activation. Electrophysiological measurements of ionic currents flowing in response to Panx channel activation have shown that some subtypes can be activated by strong membrane depolarization or caspase cleavage of the C-terminus. Here, Henze and colleagues set out to identify endogenous activators of Panx channels, focusing on the Panx1 and Panx2 subtypes, by fractionating mouse liver extracts and screening for activation of Panx channels expressed in mammalian cells using whole-cell patch clamp recordings. The authors present a comprehensive examination with robust methodologies and supporting data that demonstrate that lysophospholipids (LPCs) directly Panx-1 and 2 channels. These methodologies include channel mutagenesis, electrophysiology, ATP release and fluorescence assays, molecular modelling, and cryogenic electron microscopy (cryo-EM). Mouse liver extracts were initially used to identify LPC activators, but the authors go on to individually evaluate many different types of LPCs to determine those that are more specific for Panx channel activation. Importantly, the enzymes that endogenously regulate the production of these LPCs were also assessed along with other by-products that were shown not to promote pannexin channel activation. In addition, the authors used synovial fluid from canine patients, which is enriched in LPCs, to highlight the importance of the findings in pathology. Overall, we think this is likely to be a landmark study because it provides strong evidence that LPCs can function as activators of Panx1 and Panx2 channels, linking two established mediators of inflammatory responses and opening an entirely new area for exploring the biological roles of Panx channels. Although the mechanism of LPC activation of Panx channels remains unresolved, this study provides an excellent foundation for future studies and importantly provides clinical relevance.

We thank the reviewers for their time and effort in reviewing our manuscript. Based on their valuable comments and suggestions, we have made substantial revisions. The updated manuscript now includes two new experiments supporting that lysophospholipid-triggered channel activation promotes the release of signaling molecules critical for immune response and demonstrates that this novel class of agonist activates the inflammasome in human macrophages through endogenously expressed Panx1. To better highlight the significance of our findings, we have excluded the cryo-EM panel from this manuscript. We believe these changes address the main concerns raised by the reviewers and enhance the overall clarity and impact of our findings. Below, we provide a point-by-point response to each of the reviewers’ comments.

RECOMMENDATIONS

Essential revisions:

1. The authors present a tremendous amount of data using different approaches, cells and assays along with a written presentation that is quite abbreviated, which may make comprehension challenging for some readers. We would encourage the authors to expand the written presentation to more fully describe the experiments that were done and how the data were analysed so that the 2 key conclusions can be more fully appreciated by readers. A lot of data is also presented in supplemental figures that could be brought into the main figures and more thoroughly presented and discussed.

We appreciate and agree with the reviewers’ observation. Our initial manuscript may have been challenging to follow due to our use of both wild-type and GS-tagged versions of Panx1 from human and frog origins, combined with different fluorescence techniques across cell types. In this revision, we used only human wild-type Panx1 expressed in HEK293S GnTI^- cells, except for activity-guided fractionation experiments, where we used GS-tagged Panx1 expressed in HEK293 cells (Fig. 1). For functional reconstitution studies, we employed YO-PRO-1 uptake assays, as optimizing the Venus-based assay was challenging. We have clarified these exceptions in the main text. We think these adjustments simplify the narrative and ensure an appropriate balance between main and supplemental figures.

1. It would also be useful to present data on the ion selectivity of Panx channels activated by LPC. How does this compare to data obtained when the channel is activated by depolarization? If the two stimuli activate related open states then the ion selectivity may be quite similar, but perhaps not if the two stimuli activate different open states. The authors earlier work in eLife shows interesting shifts in reversal potentials (Vrev) when substituting external chloride with gluconate but not when substituting external sodium with N-methyl-D-glucamine, and these changed with mutations within the external pore of Panx channels. Related measurements comparing channels activated by LPC with membrane depolarization would be valuable for assessing whether similar or distinct open states are activated by LPC and voltage. It would be ideal to make Vrev measurements using a fixed step depolarization to open the channel and then various steps to more negative voltages to measure tail currents in pinpointing Vrev (a so called instantaneous IV).

We fully agree with the reviewer on the importance of ion selectivity experiments. However, comparing the properties of LPC-activated channels with those activated by membrane depolarization presented technical challenges, as LPC appears to stimulate Panx1 in synergy with voltage. Prolonged LPC exposure destabilizes patches, complicating G-V curve acquisition and kinetic analyses. While such experiments could provide mechanistic insights, we think they are beyond the scope of current study.

1. Data is presented for expression of Panx channels in different cell types (HEK vs HEKS GnTI-) and different constructs (Panx1 vs Panx1-GS vs other engineered constructs). The authors have tried to be clear about what was done in each experiment, but it can be challenging for the reader to keep everything straight. The labelling in Fig 1E helps a lot, and we encourage the authors to use that approach systematically throughout. It would also help to clearly identify the cell type and channel construct whenever showing traces, like those in Fig 1D. Doing this systematically throughout all the figures would also make it clear where a control is missing. For example, if labelling for the type of cell was included in Fig 1D it would be immediately clear that a GnTI- vector alone control for WT Panx1 is missing as the vector control shown is for HEK cells and formally that is only a control for Panx2 and 3. Can the authors explain why PLC activates Panx1 overexpressed in HEK293 GnTl- cells but not in HEK293 cells? Is this purely a function of expression levels? If so, it would be good to provide that supporting information.

As mentioned above, we believe our revised version is more straightforward to digest. We have improved labeling and provided explanations where necessary to clarify the manuscript. While Panx1 expression levels are indeed higher in GnTI^- than in HEK293 cells, we are uncertain whether the absence of detectable currents in HEK293 cells is solely due to expression levels. Some post-translational modifications that inhibit Panx1, such as lysine acetylation, may also impact activity. Future studies are needed to explore these mechanisms further.

1. The mVenus quenching experiments are somewhat confusing in the way data are presented. In Fig 2B the y axis is labelled fluorescence (%) but when the channel is closed at time = 0 the value of fluorescence is 0 rather than 100 %, and as the channel opens when LPC is added the values grow towards 100 instead of towards 0 as iodide permeates and quenches. It would be helpful if these types of data could be presented more intuitively. Also, how was the initial rate calculated that is plotted in Fig 2C? It would be helpful to show how this is done in a figure panel somewhere. Why was the initial rate expressed as a percent maximum, what is the maximum and why are the values so low? Why is the effect of CBX so weak in these quenching experiments with Panx1 compared to other assays? This assay is used in a lot of experiments so anything that could be done to bolster confidence is what it reports on would be valuable to readers. Bringing in as many control experiments that have been done, including any that are already published, would be helpful.

We modified the Y-axis in Figure 2 to “Quench (%)” for clarity. The data reflects fluorescence reduction over time, starting from LPC addition, normalized to the maximal decrease observed after Triton-X100 addition (3 minutes), enabling consistent quenching value comparisons. Although the quenching value appears small, normalization against complete cell solubilization provides reproducible comparisons. We do not fully understand why CBX effects vary in Venus quenching experiments, but we speculate that its steroid-like pentacyclic structure may influence the lysophospholipid agonistic effects. As noted in prior studies (DOI: 10.1085/jgp.201511505; DOI: 10.7554/eLife.54670), CBX likely acts as an allosteric modulator rather than a simple pore blocker, potentially contributing to these variations.

1. Could provide more information to help rationalize how Yo-Pro-1, which has a charge of +2, can permeate what are thought to be anion favouring Panx channels? We appreciate that the biophysical properties of Panx channel remain mysterious, but it would help to hear how a bit more about the authors thinking. It might also help to cite other papers that have measured Yo-Pro-1 uptake through Panx channels. Was the Strep-tagged construct of Panx1 expressed in GnTI- cells and shown to be functional using electrophysiology?

Our recent study suggest that the electrostatic landscape along the permeation pathway may influence its ion selectivity (DOI: 10.1101/2024.06.13.598903). However, we have not yet fully elucidated how Panx1 permeates both anions and cations. Based on our findings, ion selectivity may vary with activation stimulus intensity and duration. Cation permeation through Panx1 is often demonstrated with YO-PRO-1, which measures uptake over minutes, unlike electrophysiological measurements conducted over milliseconds to seconds. We referenced two representative studies employing YO-PRO-1 to assess Panx1 activity. Whole-cell current measurements from a similar construct with an intracellular loop insertion indicate that our STREP-tagged construct likely retains functional capacity.

1. In Fig 5 panel C, data is presented as the ratio of LPC induced current at -60 mV to that measured at +110 mV in the absence of LPC. What is the rationale for analysing the data this way? It would be helpful to also plot the two values separately for all of the constructs presented so the reader can see whether any of the mutants disproportionately alter LPC induced current relative to depolarization activated current. Also, for all currents shown in the figures, the authors should include a dashed coloured line at zero current, both for the LPC activated currents and the voltage steps.

We used the ratio of LPC-induced current to the current measured at +110 mV to determine whether any of the mutants disproportionately affect LPC-induced current relative to depolarization-activated current. Since the mutants that did not respond to LPC also exhibited smaller voltage-stimulated currents than those that did respond, we reasoned that using this ratio would better capture the information the reviewer is suggesting to gauge. Showing the zero current level may be helpful if the goal was to compare basal currents, which in our experience vary significantly from patch to patch. However, since we are comparing LPC- and voltage-induced currents within the same patch, we believe that including basal current measurements would not add useful information to our study.

Given that new experiments included to further highlight the significance of the discovery of Panx1 agonists, we opted to separate structure-based mechanistic studies from this manuscript and removed this experiment along with the docking and cryo-EM studies.

1. The fragmented NTD density shown in Fig S8 panel A may resemble either lipid density or the average density of both NTD and lipid. For example, Class7 and Class8 in Fig.S8 panel D displayed split densities, which may resemble a phosphate head group and two tails of lipid. A protomer mask may not be the ideal approach to separate different classes of NTD because as shown in Fig S8 panel D, most high-resolution features are located on TM1-4, suggesting that the classification was focused on TM1-4. A more suitable approach would involve using a smaller mask including NTD, TM1, and the neighbouring TM2 region to separate different NTD classes.

We agree with the reviewer and attempted 3D classification using multiple smaller masks including the suggested region. However, the maps remained poorly defined, and we were unable to confidently assign the NTD.

1. The authors don’t discuss whether the LPC-bound structures display changes in the external part of the pore, which is the anion-selective filter and the narrower part of the pore. If there are no conformational changes there, then the present structures cannot explain permeability to large molecules like ATP. In this context, a plot for the pore dimension will be helpful to see differences along the pore between their different structures. It would also be clearer if the authors overlaid maps of protomers to illustrate differences at the NTD and the "selectivity filter."

Both maps show that the narrowest constriction, formed by W74, has a diameter of approximately 9 Å. Previous steered molecular dynamics simulations suggest that ATP can permeate through such a constriction, implying an ion selection mechanism distinct from a simple steric barrier.

1. The time between the addition of LPC to the nanodisc-reconstituted protein and grid preparation is not mentioned. Dynamic diffusion of LPC could result in equal probabilities for the bound and unbound forms. This raises the possibility of finding the Primed state in the LPC-bound state as well. Additionally, can the authors rationalize how LPC might reach the pore region when the channel is in the closed state before the application of LPC?

We appreciate the reviewer’s insight. We incubated LPC and nanodisc-reconstituted protein for 30 minutes, speculating that LPC approaches the pore similarly to other lipids in prior structures. In separate studies, we are optimizing conditions to capture more defined conformations.

1. In the cryo-EM map of the “resting” state (EMDB-21150), a part of the density was interpreted as NTD flipped to the intracellular side. This density, however, is poorly defined, and not connected to the S1 helix, raising concerns about whether this density corresponds to the NTD as seen in the “resting” state structure (PDB-ID: 6VD7). In addition, some residues in the C-terminus (after K333 in frog PANX1) are missing from the atomic model. Some of these residues are predicted by AlphaFold2 to form a short alpha helix and are shown to form a short alpha helix in some published PANX1 structures. Interestingly, in both the AF2 model and 6WBF, this short alpha helix is located approximately in the weak density that the authors suggest represents the “flipped” NTD. We encourage the authors to be cautious in interpreting this part as the “flipped” NTD without further validation or justification.

We agree that the density corresponding the extended NTD into the cytoplasm is relatively weak. In our recent study, we compared two Panx1 structures with or without the mentioned C-terminal helix and found evidence suggesting the likelihood of NTD extension (DOI: 10.1101/2024.06.13.598903). Nevertheless, to prevent potential confusion, we have removed the cryo-EM panel from this manuscript.

1. Since the authors did not observe densities of bound PLC in the cryo-EM map, it is important to acknowledge in the text the inherent limitations of using docking and mutagenesis methods to locate where PLC binds.

Thank you for the suggestion. We have removed this section to avoid potential confusion.

Optional suggestions:

1. The authors used MeOH to extract mouse liver for reversed-phase chromatography. Was the study designed to focus on hydrophobic compounds that likely bind to the TMD? Panx1 has both ECD and ICD with substantial sizes that could interact with water soluble compounds? Also, the use of whole-cell recordings to screen fractions would not likely identify polar compounds that interact with the cytoplasmic part of the TMD? It would be useful for the authors to comment on these aspects of their screen and provide their rationale for fractionating liver rather than other tissues.

We have added a rationale in line 90, stating: “The soluble fractions were excluded from this study, as the most polar fraction induced strong channel activities in the absence of exogenously expressed pannexins.” Additionally, we have included a figure to support this rationale (Fig. S1A).

1. The authors show that LPCs reversibly increase inward currents at a holding voltage of -60 mV (not always specified in legends) in cells expressing Panx1 and 2, and then show families of currents activated by depolarizing voltage steps in the absence of LPC without asking what happens when you depolarize the membrane after LPC activation? If LPCs can be applied for long enough without disrupting recordings, it would be valuable to obtain both I-V relations and G-V relations before and after LPC activation of Panx channels. Does LPC disproportionately increase current at some voltages compared to others? Is the outward rectification reduced by LPC? Does Vrev remain unchanged (see point above)? Its hard to predict what would be observed, but almost any outcome from these experiments would suggest additional experiments to explore the extent to which the open states activated by LPC and depolarization are similar or distinct.

Unfortunately, in our hands, the prolonged application of lysolipids at concentrations necessary to achieve significant currents tends to destabilize the patch. This makes it challenging to obtain G-V curves or perform the previously mentioned kinetic analyses. We believe this destabilization may be due to lysolipids’ surfactant-like qualities, which can disrupt the giga seal. Additionally, prolonged exposure seems to cause channel desensitization, which could be another confounding factor.

1. From the results presented, the authors cannot rule out that mutagenesis-induced insensitivity of Panx channels to LPCs results from allosteric perturbations in the channels rather than direct binding/gating by LPCs. In Fig 5 panel A-C, the authors introduced double mutants on TM1 and TM2 to interfere with LPC binding, however, the double mutants may also disrupt the interaction network formed within NTD, TM1, and TM2. This disruption could potentially rearrange the conformation of NTD, favouring the resting closed state. Three double Asn mutants, which abolished LPC induced current, also exhibited lower currents through voltage activation in Fig 5S, raising the possibility the mutant channels fail to activate in response to LPC due to an increased energy barrier. One way to gain further insight would be to mutate residues in NTD that interact with those substituted by the three double Asn mutants and to measuring currents from both voltage activation and LPC activation. Such results might help to elucidate whether the three double Asn mutants interfere with LPC binding. It would also be important to show that the voltage-activated currents in Fig. S5 are sensitive to CBX?

Thank you for the comment, with which we agree. Our initial intention was to use the mutagenesis studies to experimentally support the docking study. Due to uncertainties associated with the presented cryo-EM maps, we have decided to remove this study from the current manuscript. We will consider the proposed experiments in a future study.

1. Could the authors elaborate on how LPC opens Panx1 by altering the conformation of the NTDs in an uncoordinated manner, going from “primed” state to the “active” state. In the “primed” state, the NTDs seem to be ordered by forming interactions with the TMD, thus resulting in the largest (possible?) pore size around the NTDs. In contrast, in the “active” state, the authors suggest that the NTDs are fragmented as a result of uncoordinated rearrangement, which conceivably will lead to a reduction in pore size around NTDs (isn’t it?). It is therefore not intuitive to understand why a conformation with a smaller pore size represents an “active” state.

We believe the uncoordinated arrangement of NTDs is dynamic, allowing for potential variations in pore size during the activated conformation. Alternatively, NTD movement may be coupled with conformational changes in TM1 and the extracellular domain, which in turn could alter the electrostatic properties of the permeation pathway. We believe a functional study exploring this mechanism would be more appropriately presented as a separate study.

1. Can the authors provide a positive control for these negative results presented in Fig S1B and C?

The positive results are presented in Fig. 1D and E.

1. Raw images in Fig S6 and Fig S7 should contain units of measurement.

Thank you for pointing this out.

1. It may be beneficial to show the superposition between primed state and activated state in both protomer and overall structure. In addition, superposition between primed state and PDB 7F8J.

We attempted to superimpose the cryo-EM maps; however, visually highlighting the differences in figure format proved challenging. Higher-resolution maps would allow for model building, which would more effectively convey these distinctions.

1. Including particles number in each class in Fig S8 panel C and D would help in evaluating the quality of classification.

Noted.

1. A table for cryo-EM statistics should be included.

Thanks, noted.

1. n values are often provided as a range within legends but it would be better to provide individual values for each dataset. In many figures you can see most of the data points, which is great, but it would be easy to add n values to the plots themselves, perhaps in parentheses above the data points.

While we agree that transparency is essential, adding n-values to each graph would make some figures less clear and potentially harder to interpret in this case. We believe that the dot plots, n-value range, and statistical analysis provide adequate support for our claims.

1. The way caspase activation of Panx channels is presented in the introduction could be viewed as dismissive or inflammatory for those who have studied that mechanism. We think the caspase activation literature is quite convincing and there is no need to be dismissive when pointing out that there are good reasons to believe that other mechanisms of activation likely exist. We encourage you to revise the introduction accordingly.

Thank you for this comment. Although we intended to support the caspase activation mechanism in our introduction, we understand that the reviewer’s interpretation indicates a need for clarification. We hope the revised introduction removes any perception of dismissiveness.

1. Why is the patient data in Fig 4F normalized differently than everything else? Once the above issues with mVenus quenching data are clarified, it would be good to be systematic and use the same approach here.

For Fig. 4F, we used a distinct normalization method to account for substantial day-to-day variation in experiments involving body fluids. Notably, we did not apply this normalization to other experimental panels due to their considerably lower day-to-day variation.

1. What was the rational for using the structure from ref 35 in the docking task?

The docking task utilized the human orthologue with a flipped-up NTD. We believe that this flipped-up conformation is likely the active form that responds to lysolipids. As our functional experiments primarily use the human orthologue for biological relevance, this structure choice is consistent. Our docking data shows that LPC does not dock at this site when using a construct with the downward-flipped NTD.

1. Perhaps better to refer to double Asn ‘substitutions’ rather than as ‘mutations’ because that makes one think they are Asn in the wt protein.

Done.

1. From Fig S1, we gather that Panx2 is much larger than Panx1 and 3. If that is the case, its worth noting that to readers somewhere.

We have added the molecular weight of each subtype in the figure legend.

1. Please provide holding voltages and zero current levels in all figures presenting currents.

We provided holding voltages. However, the zero current levels vary among the examples presented, making direct comparisons difficult. Since we are comparing currents with and without LPC, we believe that indicating zero current levels is unnecessary for this study.

1. While the authors successfully establish lysophospholipid-gating of Panx1 and Panx2, Panx3 appears unaffected. It may be advisable to be more specific in the title of the article.

We are uncertain whether Panx3 is unaffected by lysophospholipids, as we have not observed activation of this subtype under any tested conditions.

(This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

Consolidated Peer Review Report (20 December 2023)

GENERAL ASSESSMENT

Pannexin (Panx) hemichannels are a family of heptameric membrane proteins that form pores in the plasma membrane through which ions and relatively large organic molecules can permeate. ATP release through Panx channels during the process of apoptosis is one established biological role of these proteins in the immune system, but they are widely expressed in many cells throughout the body, including the nervous system, and likely play many interesting and important roles that are yet to be defined. Although several structures have now been solved of different Panx subtypes from different species, their biophysical mechanisms remain poorly understood, including what physiological signals control their activation. Electrophysiological measurements of ionic currents flowing in response to Panx channel activation have shown that some subtypes can be activated by strong membrane depolarization or caspase cleavage of the C-terminus. Here, Henze and colleagues set out to identify endogenous activators of Panx channels, focusing on the Panx1 and Panx2 subtypes, by fractionating mouse liver extracts and screening for activation of Panx channels expressed in mammalian cells using whole-cell patch clamp recordings. The authors present a comprehensive examination with robust methodologies and supporting data that demonstrate that lysophospholipids (LPCs) directly Panx-1 and 2 channels. These methodologies include channel mutagenesis, electrophysiology, ATP release and fluorescence assays, molecular modelling, and cryogenic electron microscopy (cryo-EM). Mouse liver extracts were initially used to identify LPC activators, but the authors go on to individually evaluate many different types of LPCs to determine those that are more specific for Panx channel activation. Importantly, the enzymes that endogenously regulate the production of these LPCs were also assessed along with other by-products that were shown not to promote pannexin channel activation. In addition, the authors used synovial fluid from canine patients, which is enriched in LPCs, to highlight the importance of the findings in pathology. Overall, we think this is likely to be a landmark study because it provides strong evidence that LPCs can function as activators of Panx1 and Panx2 channels, linking two established mediators of inflammatory responses and opening an entirely new area for exploring the biological roles of Panx channels. Although the mechanism of LPC activation of Panx channels remains unresolved, this study provides an excellent foundation for future studies and importantly provides clinical relevance.

RECOMMENDATIONS

Essential revisions:

1. The authors present a tremendous amount of data using different approaches, cells and assays along with a written presentation that is quite abbreviated, which may make comprehension challenging for some readers. We would encourage the authors to expand the written presentation to more fully describe the experiments that were done and how the data were analysed so that the key conclusions can be more fully appreciated by readers. A lot of data is also presented in supplemental figures that could be brought into the main figures and more thoroughly presented and discussed.
2. It would also be useful to present data on the ion selectivity of Panx channels activated by LPC. How does this compare to data obtained when the channel is activated by depolarization? If the two stimuli activate related open states then the ion selectivity may be quite similar, but perhaps not if the two stimuli activate different open states. The authors earlier work in eLife shows interesting shifts in reversal potentials (Vrev) when substituting external chloride with gluconate but not when substituting external sodium with N-methyl-D-glucamine, and these changed with mutations within the external pore of Panx channels. Related measurements comparing channels activated by LPC with membrane depolarization would be valuable for assessing whether similar or distinct open states are activated by LPC and voltage. It would be ideal to make Vrev measurements using a fixed step depolarization to open the channel and then various steps to more negative voltages to measure tail currents in pinpointing Vrev (a so called instantaneous IV).
3. Data is presented for expression of Panx channels in different cell types (HEK vs HEKS GnTI-) and different constructs (Panx1 vs Panx1-GS vs other engineered constructs). The authors have tried to be clear about what was done in each experiment, but it can be challenging for the reader to keep everything straight. The labelling in Fig 1E helps a lot, and we encourage the authors to use that approach systematically throughout. It would also help to clearly identify the cell type and channel construct whenever showing traces, like those in Fig 1D. Doing this systematically throughout all the figures would also make it clear where a control is missing. For example, if labelling for the type of cell was included in Fig 1D it would be immediately clear that a GnTI- vector alone control for WT Panx1 is missing as the vector control shown is for HEK cells and formally that is only a control for Panx2 and 3. Can the authors explain why PLC activates Panx1 overexpressed in HEK293 GnTl- cells but not in HEK293 cells? Is this purely a function of expression levels? If so, it would be good to provide that supporting information.
4. The mVenus quenching experiments are somewhat confusing in the way data are presented. In Fig 2B the y axis is labelled fluorescence (%) but when the channel is closed at time = 0 the value of fluorescence is 0 rather than 100 %, and as the channel opens when LPC is added the values grow towards 100 instead of towards 0 as iodide permeates and quenches. It would be helpful if these types of data could be presented more intuitively. Also, how was the initial rate calculated that is plotted in Fig 2C? It would be helpful to show how this is done in a figure panel somewhere. Why was the initial rate expressed as a percent maximum, what is the maximum and why are the values so low? Why is the effect of CBX so weak in these quenching experiments with Panx1 compared to other assays? This assay is used in a lot of experiments so anything that could be done to bolster confidence is what it reports on would be valuable to readers. Bringing in as many control experiments that have been done, including any that are already published, would be helpful.
5. Could provide more information to help rationalize how Yo-Pro-1, which has a charge of +2, can permeate what are thought to be anion favouring Panx channels? We appreciate that the biophysical properties of Panx channel remain mysterious, but it would help to hear how a bit more about the authors thinking. It might also help to cite other papers that have measured Yo-Pro-1 uptake through Panx channels. Was the Strep-tagged construct of Panx1 expressed in GnTI- cells and shown to be functional using electrophysiology?
6. In Fig 5 panel C, data is presented as the ratio of LPC induced current at -60 mV to that measured at +110 mV in the absence of LPC. What is the rationale for analysing the data this way? It would be helpful to also plot the two values separately for all of the constructs presented so the reader can see whether any of the mutants disproportionately alter LPC induced current relative to depolarization activated current. Also, for all currents shown in the figures, the authors should include a dashed coloured line at zero current, both for the LPC activated currents and the voltage steps.
7. The fragmented NTD density shown in Fig S8 panel A may resemble either lipid density or the average density of both NTD and lipid. For example, Class7 and Class8 in Fig.S8 panel D displayed split densities, which may resemble a phosphate head group and two tails of lipid. A protomer mask may not be the ideal approach to separate different classes of NTD because as shown in Fig S8 panel D, most high-resolution features are located on TM1-4, suggesting that the classification was focused on TM1-4. A more suitable approach would involve using a smaller mask including NTD, TM1, and the neighbouring TM2 region to separate different NTD classes.
8. The authors don’t discuss whether the LPC-bound structures display changes in the external part of the pore, which is the anion-selective filter and the narrower part of the pore. If there are no conformational changes there, then the present structures cannot explain permeability to large molecules like ATP. In this context, a plot for the pore dimension will be helpful to see differences along the pore between their different structures. It would also be clearer if the authors overlaid maps of protomers to illustrate differences at the NTD and the "selectivity filter."
9. The time between the addition of LPC to the nanodisc-reconstituted protein and grid preparation is not mentioned. Dynamic diffusion of LPC could result in equal probabilities for the bound and unbound forms. This raises the possibility of finding the Primed state in the LPC-bound state as well. Additionally, can the authors rationalize how LPC might reach the pore region when the channel is in the closed state before the application of LPC?
10. In the cryo-EM map of the “resting” state (EMDB-21150), a part of the density was interpreted as NTD flipped to the intracellular side. This density, however, is poorly defined, and not connected to the S1 helix, raising concerns about whether this density corresponds to the NTD as seen in the “resting” state structure (PDB-ID: 6VD7). In addition, some residues in the C-terminus (after K333 in frog PANX1) are missing from the atomic model. Some of these residues are predicted by AlphaFold2 to form a short alpha helix and are shown to form a short alpha helix in some published PANX1 structures. Interestingly, in both the AF2 model and 6WBF, this short alpha helix is located approximately in the weak density that the authors suggest represents the “flipped” NTD. We encourage the authors to be cautious in interpreting this part as the “flipped” NTD without further validation or justification.
11. Since the authors did not observe densities of bound PLC in the cryo-EM map, it is important to acknowledge in the text the inherent limitations of using docking and mutagenesis methods to locate where PLC binds.

Optional suggestions:

1. The authors used MeOH to extract mouse liver for reversed-phase chromatography. Was the study designed to focus on hydrophobic compounds that likely bind to the TMD? Panx1 has both ECD and ICD with substantial sizes that could interact with water soluble compounds? Also, the use of whole-cell recordings to screen fractions would not likely identify polar compounds that interact with the cytoplasmic part of the TMD? It would be useful for the authors to comment on these aspects of their screen and provide their rationale for fractionating liver rather than other tissues.
2. The authors show that LPCs reversibly increase inward currents at a holding voltage of -60 mV (not always specified in legends) in cells expressing Panx1 and 2, and then show families of currents activated by depolarizing voltage steps in the absence of LPC without asking what happens when you depolarize the membrane after LPC activation? If LPCs can be applied for long enough without disrupting recordings, it would be valuable to obtain both I-V relations and G-V relations before and after LPC activation of Panx channels. Does LPC disproportionately increase current at some voltages compared to others? Is the outward rectification reduced by LPC? Does Vrev remain unchanged (see point above)? Its hard to predict what would be observed, but almost any outcome from these experiments would suggest additional experiments to explore the extent to which the open states activated by LPC and depolarization are similar or distinct.
3. From the results presented, the authors cannot rule out that mutagenesis-induced insensitivity of Panx channels to LPCs results from allosteric perturbations in the channels rather than direct binding/gating by LPCs. In Fig 5 panel A-C, the authors introduced double mutants on TM1 and TM2 to interfere with LPC binding, however, the double mutants may also disrupt the interaction network formed within NTD, TM1, and TM2. This disruption could potentially rearrange the conformation of NTD, favouring the resting closed state. Three double Asn mutants, which abolished LPC induced current, also exhibited lower currents through voltage activation in Fig 5S, raising the possibility the mutant channels fail to activate in response to LPC due to an increased energy barrier. One way to gain further insight would be to mutate residues in NTD that interact with those substituted by the three double Asn mutants and to measuring currents from both voltage activation and LPC activation. Such results might help to elucidate whether the three double Asn mutants interfere with LPC binding. It would also be important to show that the voltage-activated currents in Fig. S5 are sensitive to CBX?
4. Could the authors elaborate on how LPC opens Panx1 by altering the conformation of the NTDs in an uncoordinated manner, going from “primed” state to the “active” state. In the “primed” state, the NTDs seem to be ordered by forming interactions with the TMD, thus resulting in the largest (possible?) pore size around the NTDs. In contrast, in the “active” state, the authors suggest that the NTDs are fragmented as a result of uncoordinated rearrangement, which conceivably will lead to a reduction in pore size around NTDs (isn’t it?). It is therefore not intuitive to understand why a conformation with a smaller pore size represents an “active” state.
5. Can the authors provide a positive control for these negative results presented in Fig S1B and C?
6. Raw images in Fig S6 and Fig S7 should contain units of measurement.
7. It may be beneficial to show the superposition between primed state and activated state in both protomer and overall structure. In addition, superposition between primed state and PDB 7F8J.
8. Including particles number in each class in Fig S8 panel C and D would help in evaluating the quality of classification.
9. A table for cryo-EM statistics should be included.
10. n values are often provided as a range within legends but it would be better to provide individual values for each dataset. In many figures you can see most of the data points, which is great, but it would be easy to add n values to the plots themselves, perhaps in parentheses above the data points.
11. The way caspase activation of Panx channels is presented in the introduction could be viewed as dismissive or inflammatory for those who have studied that mechanism. We think the caspase activation literature is quite convincing and there is no need to be dismissive when pointing out that there are good reasons to believe that other mechanisms of activation likely exist. We encourage you to revise the introduction accordingly.
12. Why is the patient data in Fig 4F normalized differently than everything else? Once the above issues with mVenus quenching data are clarified, it would be good to be systematic and use the same approach here.
13. What was the rational for using the structure from ref 35 in the docking task?
14. Perhaps better to refer to double Asn ‘substitutions’ rather than as ‘mutations’ because that makes one think they are Asn in the wt protein.
15. From Fig S1, we gather that Panx2 is much larger than Panx1 and 3. If that is the case, its worth noting that to readers somewhere.
16. Please provide holding voltages and zero current levels in all figures presenting currents.
17. While the authors successfully establish lysophospholipid-gating of Panx1 and Panx2, Panx3 appears unaffected. It may be advisable to be more specific in the title of the article.

REVIEWING TEAM

Reviewed by:

Jorge Contreras, Professor, University of California, Davis, USA: electrophysiology and ion channel mechanisms

Wei Lü, Associate Professor, Department of Structural Biology, Van Andel Institute, USA: ion channel mechanisms, X-ray crystallography and cryo-EM

Xiaofeng Tan, Research Fellow, NINDS, NIH, USA: structural biology (X-ray crystallography and cryo-electron microscopy) and ion channel mechanisms

Kenton J. Swartz, Senior Investigator, NINDS, NIH, USA: ion channel structure and mechanisms, chemical biology and biophysics, electrophysiology and fluorescence spectroscopy

Curated by:

Kenton J. Swartz, Senior Investigator, NINDS, NIH, USA

(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Comments concerning minor and presentational issues have been omitted for brevity.)

The costumes look cool and I like how he integrated three forms of art into one Is he still doing this to this day? Since it's been 12 years since this interview Holy shit I almost applied to the art institute of chicago The bright and vibrant colors are nice and I wonder if he chooses colors on purpose I relate to what he said about his ideas and how it can feel like theyre just bursting and filling his head.

Your life looks amazing on paper.

You're smart, creative, healthy, travel to amazing places and have a loving family and friends.

But inside, you...

Have I hit the nail on the head?

Disconcerting childhood.

Woodson's use of the word "zapped" apropos of the speaker's conceptions signifies that their disappearance was instantaneous. Given that these notions regarded his mother's demise, toward whom he presumably was highly affectionate, and he was intending to write poetry, necessitating profound and often introspective thought, one may conjecture that these ideas had great psychological magnitude; thus, the fact that such significant thoughts were erased so suddenly and completely by an argument that did not concern him—a menial extraneous phenomenon—illuminates that diversions can effortlessly alter one's intentions or how they are executed.

These 3 lines that convey that the author has so much things like swarming his brain that he is at one time that he has no idea what to do anymore.

Sometimes when confilict is happening around you, it gets hard to focus.

People have this pre notion of what they think feminism is or what it should look like instead of actually researching it or even trying to wrap they head around what else it can be or why it even came about and it all due to history or men being on top and women being on the bottom.

When he author describe what their sister is doing as a radical feminist I think that's how most feminist are seen as a stereotype and that's why many people shy away from them because of their interpretation of how they make look "crazy" or rebellious.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging. To further support the authors' claims, several improvements are necessary, particularly in the methods of quantification and the points that should be demonstrated quantitatively. It is crucial to investigate the correlation between aging and the proteins eIF2β and eIF2α.

Thank you so much for your review and comments. We included analyses of protein levels of eIF2α, eIF2β, and eIF2γ at 7 days and 21 days (Figure 4D). The manuscript was revised as below;

Lines 242-245 ‘As for the other subunits of eIF2 complex, proteome analysis did not detect a significant difference in the protein levels of eIF2α and eIF2γ between milton knockdown and control flies at 7 and 21 days (Figure 4D).’

Reviewer #2 (Public Review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of Milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria.

The manuscript has several weaknesses. The reader should take extra care while reading this manuscript and when acknowledging the findings and the model in this manuscript.

The defect in autophagy by the depletion of axonal mitochondria is one of the main claims in the paper. The authors should work more on describing their results of LC3-II/LC3-I ratio, as there are multiple ways to interpret the LC3 blotting for the autophagy assessment. Lysosomal defects result in the accumulation of LC3-II thus the LC3-II/LC3-I ratio gets higher. On the other hand, the defect in the early steps of autophagosome formation could result in a lower LC3-II/LC3-I ratio. From the results of the actual blotting, the LC3-I abundance is the source of the major difference for all conditions (Milton RNAi and eIF2β overexpression and depletion). In the text, the authors simply state the observation of their LC3 blotting. The manuscript lacks an explanation of how to evaluate the LC3-II/LC3-I ratio. Also, the manuscript lacks an elaboration on what the results of the LC3 blotting indicate about the state of autophagy by the depletion of axonal mitochondria.

Thank you for pointing it out, and we apologize for an insufficient description of the result. We included quantitation of the levels of LC3-I and LC3-II in Figure 2A, 2D, 3D, 6B and 7B. As the reviewer pointed out, changes in the LC3-II/LC3-I ratio do not necessarily indicate autophagy defects. However, since p62 accumulation (Figure 2B, 2E, 3E, 6C, 7C in the original manuscript), these results collectively suggest that autophagy is lowered. We revised the manuscript to include this discussion as below:

Lines 174-186 ‘During autophagy progression, LC3 is conjugated with phosphatidylethanolamine to form LC3-II, which localizes to isolation membranes and autophagosomes. LC3-I accumulation occurs when autophagosome formation is impaired, and LC3-II accumulation is associated with lysosomal defects(31,32). p62 is an autophagy substrate, and its accumulation suggests autophagic defects(31,32). We found that milton knockdown increased LC3-I, and the LC3-II/LC3-I ratio was lower in milton knockdown flies than in control flies at 14-day-old (Figure 2A). We also analyzed p62 levels in head lysates sequentially extracted using detergents with different stringencies (1% Triton X-100 and 2% SDS). Western blotting revealed that p62 levels were increased in the brains of 14-day-old of milton knockdown flies (Figure 2B). The increase in the p62 level was significant in the Triton X-100-soluble fraction but not in the SDS-soluble fraction (Figure 2B), suggesting that depletion of axonal mitochondria impairs the degradation of less-aggregated proteins.’

Line 189-190 : ‘At 30 day-old, LC3-I was still higher, and the LC3-II/LC3-I ratio was lower, in milton knockdown compared to the control (Figure 2D).’

Line 199-201: ‘However, in contrast with milton knockdown, Pfk knockdown did not affect the levels of LC3-I, LC3-II or the LC3-II/LC3-I ratio (Figure 3D).’

Line 275-281: ‘Neuronal overexpression of eIF2β increased LC3-II, while the LC3-II/LC3-I ratio was not significantly different (Figure 6A and B). Overexpression of eIF2β significantly increased the p62 level in the Triton X-100-soluble fraction (Figure 6C, 4-fold vs. control, p < 0.005 (1% Triton X-100)) but not in the SDS-soluble fraction (Figure 6C, 2-fold vs. control, p = 0.062 (2% SDS)), as observed in brains of milton knockdown flies (Figure 2B). These data suggest that neuronal overexpression of eIF2β accumulates autophagic substrates.’

Line 307-315: ‘Neuronal knockdown of milton causes accumulation of autophagic substrate p62 in the Triton X-100-soluble fraction (Figure 2B), and we tested if lowering eIF2β ameliorates it. We found that eIF2β heterozygosity caused a mild increase in LC3-I levels and decreases in LC3-II levels, resulting in a significantly lower LC3-II/LC3-I ratio in milton knockdown flies (Figure 7B). eIF2β heterozygosity decreased the p62 level in the Triton X-100-soluble fraction in the brains of milton knockdown flies (Figure 7C). The p62 level in the SDS-soluble fraction, which is not sensitive to milton knockdown (Figure 2B), was not affected (Figure 7C). These results suggest that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.’

Another main point of the paper is the up-regulation of eIF2β by depleting the axonal mitochondria leads to the proteostasis crisis. This claim is formed by the findings from the proteome analyses. The authors should have presented their proteomic data with much thorough presentation and explanation. As in the experiment scheme shown in Figure 4A, the author did two proteome analyses: one from the 7-day-old sample and the other from the 21-day-old sample. The manuscript only shows a plot of the result from the 7-day-old sample, but that of the result from the 21-day-old sample. For the 21-day-old sample, the authors only provided data in the supplemental table, in which the abundance ratio of eIF2β from the 21-day-old sample is 0.753, meaning eIF2β is depleted in the 21-day-old sample. The authors should have explained the impact of the eIF2β depletion in the 21-day-old sample, so the reader could fully understand the authors' interpretation of the role of eIF2β on proteostasis.

Thank you for pointing it out. We included plots of the results of 21-day-old proteome as a part of the main figure (Figure 4C). As the reviewer pointed out, eIF2β protein levels are reduced at the 21-day-old. Since a reduction in the eIF2_β_ ameliorated milton knockdown-induced locomotor defects in aged flies (Figure 7D), the reduction in eIF2β observed in the 21-day-old milton knockdown flies is not likely to negatively contribute to milton knockdown-induced defects. We included this discussion in the manuscript as below:

Lines 337-341:‘eIF2β protein levels are reduced at the 21-day-old; however, since a reduction in the eIF2β ameliorated milton knockdown-induced locomotor defects in aged flies (Figure 7), the reduction in eIF2β observed in the 21-day-old is not likely to negatively contribute to milton knockdown-induced defects.’

The manuscript consists of several weaknesses in its data and explanation regarding translation.

(1) The authors are likely misunderstanding the effect of phosphorylation of eIF2α on translation. The P-eIF2α is inhibitory for translation initiation. However, the authors seem to be mistaken that the down-regulation of P-eIF2α inhibits translation.

We are sorry for our insufficient explanation in the previous version. As the reviewer pointed out, it is well known that the phosphorylated form of eIF2α inhibits translation initiation. Neuronal knockdown of milton caused a reduction in p-eIF2α (Figure 4J and K), and it also lowered translation (Figure 5); the relationship between these two events is currently unclear. We do not think that a reduction in the p-eIF2α suppressed translation; rather, we propose that the unbalance of expression levels of the components of eIF2 complexes negatively affects translation. We revised discussion sections to describe our interpretation more in detail as below:

Line 368-378: ‘eIF2β is a component of eIF2, which meditates translational regulation and ISR initiation. When ISR is activated, phosphorylated eIF2α suppresses global translation and induces translation of ATF4, which mediates transcription of autophagy-related genes(39,40). Since ISR can positively regulate autophagy, we suspected that suppression of ISR underlies a reduction in autophagic protein degradation. We found neuronal knockdown of milton reduced phosphorylated eIF2α, suggesting that ISR is reduced (Figure 4). However, we also found that global translation was reduced (Figure 5). It may be possible that increased levels of eIF2β disrupt the eIF2 complex or alter its functions. The stoichiometric mismatch caused by an imbalance of eIF2 components may inhibit ISR induction. Supporting this model, we found that eIF2β upregulation reduced the levels of p-eIF2α (Figure 6).’

We have revised the graphical abstract and removed the eIF2 complex since its role in the loss of proteostasis caused by milton knockdown has not been elucidated yet.

(2) The result of polysome profiling in Figure 4H is implausible. By 10%-25% sucrose density gradient, polysomes are not expected to be observed. The authors should have used a gradient with much denser sucrose, such as 10-50%.

Thank you for pointing it out. It was a mistake of 10-50%, and we apologize for the oversight. It was corrected (Figure 5).

(3) Also on the polysome profiling, as in the method section, the authors seemed to fractionate ultra-centrifuged samples from top to bottom and then measured A260 by a plate reader. In that case, the authors should have provided a line plot with individual data points, not the smoothly connected ones in the manuscript.

Thank you for pointing it out. We revised the graph (Figure 5).

(4) For both the results from polysome profiling and puromycin incorporation (Figure 4H and I), the difference between control siRNA and Milton siRNA are subtle, if not nonexistent. This might arise from the lack of spatial resolution in their experiment as the authors used head lysate for these data but the ratio of Phospho-eIF2α/eIF2α only changes in the axons, based on their results in Figure 4E-G. The authors could have attempted to capture the spatial resolution for the axonal translation to see the difference between control siRNA and Milton siRNA.

Thank you for your comment. We agree that it would be an interesting experiment, but it will take a considerable amount of time to analyze axonal translation with spatial resolution. We will try to include such analyses in the future. For this manuscript, we revised the discussion section to include the reviewer's suggestion as below;

Lines 351-353: ‘Further analyses to dissect the effects of milton knockdown on proteostasis and translation in the cell body and axon by experiments with spatial resolution would be needed.’

Recommendations for the authors:

From the Reviewing Editor:

As the Reviewing Editor, I have read your manuscript and the associated peer reviews. I have concerns about publishing this work in its current form. I think that your manuscript cannot claim to have found a novel function of eIF2beta because of technical uncertainties and conceptual problems that should be addressed.

Thank you so much for your review and comments. We addressed all the concerns raised by the reviewers. Point-by-point responses are listed below.

First, your manuscript is based partly on what appears to be a mistaken understanding of the mechanistic basis of the ISR. Specifically, eIF2 is a heterotrimeric complex of alpha, beta, and gamma subunits. When eIF2a is phosphorylated, the heterotrimer adopts a new conformation. This conformation directly binds and inhibits eIF2B, the decameric GEF that exchanges the GDP bound to the gamma subunit of the eIF2 complex for GTP. Unless I misunderstood your paper, you seem to propose that decreasing levels of phospho-eIF2a will inhibit translation, but this is backward from what we know about the ISR.

Thank you for your insightful comment, and we are sorry for the confusion. We did not mean to propose that decreasing levels of phospho-eIF2_a_ inhibits translation. We apologize for our insufficient explanation, which might have caused a misunderstanding (Lines 312-318 in the original version). We agree with the reviewer that ‘mismatch due to elevated eIF2-beta could change the behavior of the ISR’. We revised the text in the result section as follows:

Lines 259-264 (in the Result section) ‘Phosphorylation of eIF2α induces conformational changes in the eIF2 complex and inhibits global translation(36). To analyze the effects of milton knockdown on translation, we performed polysome gradient centrifugation to examine the level of ribosome binding to mRNA. Since p-eIF2α was downregulated, we hypothesized that milton knockdown would enhance translation. However, unexpectedly, we found that milton knockdown significantly reduced the level of mRNAs associated with polysomes (Figure 5A and B).’

Lines 368-378 (in the Discussion section): ‘eIF2β is a component of eIF2, which meditates translational regulation and ISR initiation. When ISR is activated, phosphorylated eIF2α suppresses global translation and induces translation of ATF4, which mediates transcription of autophagy-related genes(39,40). Since ISR can positively regulate autophagy, we suspected that suppression of ISR underlies a reduction in autophagic protein degradation. We found neuronal knockdown of milton reduced phosphorylated eIF2α, suggesting that ISR is reduced (Figure 4). However, we also found that global translation was reduced (Figure 5). It may be possible that increased levels of eIF2β disrupt the eIF2 complex or alter its functions. The stoichiometric mismatch caused by an imbalance of eIF2 components may inhibit ISR induction. Supporting this model, we found that eIF2β upregulation reduced the levels of p-eIF2α (Figure 6).’

It may be possible that a stoichiometric mismatch due to elevated eIF2-beta could change the behavior of the ISR, but your paper doesn't adequately address the expression levels of all three eIF2 subunits: alpha, beta, and gamma. The proteomic data shown in Fig 4B is unconvincing on its own because the changes in the beta subunit are subtle. The Western blot in Figure 4C suggests that the KD changes the mass or mobility of the beta subunit, and most importantly, there are no Western blots measuring the levels of eIF2a, eIF2a-phospho, or eIF2-gamma.

We appreciate the reviewer’s comment and agree that the stoichiometric mismatch due to elevated eIF2β may interfere with ISR. We found overexpression of eIF2β lowered p-eIF2 alpha (Figure S2 in V1), which supports this model. We included this data in the main figure in the revised manuscript (Figure 6D) and revised the text as below:

Lines 279-281: ‘Since milton knockdown reduced the p-eIF2α level (Figure 4K), we asked whether an increase in eIF2β affects p-eIF2α. Neuronal overexpression of eIF2β did not affect the eIF2α level but significantly decreased the p-eIF2α level (Figure 6D, E).’

Expression data of eIF2α and eIF2γ from proteomic analyses has been extracted from proteome analyses and included as a table (Figure 4D). Western blots of phospho-eIF2a (Figure S1 in V1) in the main figure (Figure 4G). The result section was revised as below;

Lines 242-245: ‘As for the other subunits of eIF2 complex, proteome analysis did not detect a significant difference in the protein levels of eIF2α and eIF2γ between milton knockdown and control flies at 7 and 21 days (Figure 4D).’

Reviewer #1 (Recommendations For The Authors):

L125-128: In this section, while the efficiency of Milton knockdown is referenced from a previous publication, it is necessary to also mention that the Miro knockdown has been similarly reported in the literature. Additionally, the Methods section lacks details on the Miro RNAi line used, and Table 2 does not include the genotype for Miro RNAi. This information should be included for clarity and completeness.

Thank you for pointing it out. Knockdown efficiency with this strain has been reported (Iijima-Ando et al., PLoS Genet, 2012). We revised the text to include citation and knockdown efficiency as follows:

Lines 139-147: ‘There was no significant increase in ubiquitinated proteins in milton knockdown flies at 1-day old, suggesting that the accumulation of ubiquitinated proteins caused by milton knockdown is age-dependent (Figure S1). We also analyzed the effect of the neuronal knockdown of Miro, a partner of milton, on the accumulation of ubiquitin-positive proteins. Since severe knockdown of Miro in neurons causes lethality, we used UAS-Miro RNAi strain with low knockdown efficiency, whose expression driven by elav-GAL4 caused 30% reduction of Miro mRNA in head extract(24). Although there was a tendency for increased ubiquitin-positive puncta in Miro knockdown brains, the difference was not significant (Figure 1B, p>0.05 between control RNAi and Miro RNAi). These data suggest that the depletion of axonal mitochondria induced by milton knockdown leads to the accumulation of ubiquitinated proteins before neurodegeneration occurs.’

L132-L136: The current phrasing in this section suggests an increase in ubiquitinated proteins for both Milton and Miro knockdowns. However, since there is no significant difference noted for Miro, it is incorrect to state an increase in ubiquitin-positive puncta. Furthermore, combining the results of Milton knockdown to claim an increase in ubiquitinated proteins prior to neurodegeneration is misleading. At the very least, the expression here needs to be moderated to accurately reflect the findings.

Thank you for pointing it out. We revised the text as above.

L137-L141: Results in Figure 1 indicate that Milton knockdown leads to an increase in ubiquitinated proteins at 14 days, while Miro knockdown shows no difference from the control at either 14 or 30 days. Conversely, both the control and Miro exhibit an increase in ubiquitinated proteins with aging, but this trend does not seem to apply to Milton knockdown. This observation suggests that Milton KD may not affect the changes in protein quality control associated with aging. It implies that Milton's function might be more related to protein homeostasis in younger cells, or that changes due to aging might overshadow the effects of Milton knockdown. These interpretations should be included in the Results or Discussion sections for a more comprehensive analysis.

Thank you for your insightful comment. We revised the text to include those points as follows:

Lines 152-153: ‘These results suggest that depletion of axonal mitochondria may have more impact on proteostasis in young neurons than in old neurons.’

Lines 355-362: ‘The depletion of axonal mitochondria and accumulation of abnormal proteins are both characteristics of aged brains(37,38). Our results suggest that the loss of axonal mitochondria is an event upstream of proteostasis collapse during aging. Neuronal knockdown of milton had more impact on proteostasis in young neurons than the old neurons (Figure 1). Proteome analyses also showed that age-related pathways, such as immune responses, are enhanced in young flies with milton knockdown (Table 2). The reduction in axonal transport of mitochondria may be one of the triggering events of age-related changes and accelerates the onset of aging in the brain.’

L143 : Please remove the erroneously included quotation mark.

Thank you for pointing it out. We corrected it.

L145-L147:

- While it is understood that Milton knockdown results in a reduction of mitochondria in axons, as reported previously and seemingly indicated in Figure 1E, this paper repeatedly refers to axonal depletion of mitochondria. Therefore, it would be beneficial to quantitatively assess the number of mitochondria in the axonal terminals located in the lamina via electron microscopy. Such quantification would robustly reinforce the argument that mitochondrial absence in axons is a consequence of Milton knockdown.

Thank you for pointing it out. We included quantitation of the number of mitochondria in the synaptic terminals (Figure 1E).

The text and figure legend was revised accordingly:

Lines 156-157: ‘As previously reported(24), the number of mitochondria in presynaptic terminals decreased in milton knockdown (Figure 1E).’

- The knockdown of Milton is known to reduce mitochondrial transport from an early stage, but what about swelling? By observing swelling at 1 day and 14 days, it may be possible to confirm the onset of swelling and discuss its correlation with the accumulation of ubiquitinated proteins.

Quantitation of axonal swelling has also been included (Figure 1F).

We appreciate reviewer’s comments on the correlation between the accumulation of ubiquitinated proteins and axonal swelling. Axonal swelling was not observed at 3-days-old (Iijima-Ando et al., PLoS Genetics, 2012), indicating that axonal swelling is an age-dependent event. Dense materials are found in swollen axons more often than in normal axons, suggesting a positive correlation between disruption of proteostasis and axonal damage. It would be interesting to analyze the time course of events further; however, we feel it is beyond the scope of this manuscript. We revised the text as below to include this discussion:

Lines 157-159: ‘The swelling of presynaptic terminals, characterized by the enlargement and roundness, was not reported at 3-day-old(24) but observed at this age with about 4% of total presynaptic terminals (Figure 1F, asterisks).’

Lines 162-167: ‘Dense materials are rarely found in age-matched control neurons, indicating that milton knockdown induces abnormal protein accumulation in the presynaptic terminals (Figure 1G and H). In milton knockdown neurons, dense materials are found in swollen presynaptic terminals more often than in presynaptic terminals without swelling, suggesting a positive correlation between the disruption of proteostasis and axonal damage (Figure 1G).’

Lines 362-365: ‘Disruption of proteostasis is expected to contribute neurodegeneration(38), and it would be interesting to analyze the sequence of protein accumulation and axonal degeneration in milton knockdown ((24,29) and Figure 1) in detail with higher time resolution.’

L147-L151: Though Figures 1F and 1G provide qualitative representations, it is advisable to quantitatively assess whether dense materials significantly accumulate. Such quantitative analysis would be required to verify the accumulation of dense materials in the context of the study.

Thank you for pointing it out. We included quantitation of the number of neurons with dense material (Figure 1G). We revised the manuscript as follows:

Line 161-163: ‘Dense materials are rarely found in age-matched control neurons, indicating that milton knockdown induces abnormal protein accumulation in the presynaptic terminals (Figure 1G and H).’

Regarding Figure 1B, C:

- Even though the count of puncta in the whole brain appears to be fewer than 400, the magnification of the optic lobe suggests a substantial presence of puncta. Please clarify in the Methods section what constitutes a puncta and whether the quantification in the whole brain is based on a 2D or 3D analysis. Detail the methodology used for quantification.

Thank you for your comment. We revised the method section to include more details as below:

Lines 434-437: ‘Quantitative analysis was performed using ImageJ (National Institutes of Health) with maximum projection images derived from Z-stack images acquired with same settings. Puncta was identified with mean intensity and area using ImageJ.’

- What about 1-day-old specimens? Does Milton knockdown already show an increase in ubiquitinated protein accumulation at this early stage? Investigating whether ubiquitin-protein accumulation is involved in aging promotion or is already prevalent during developmental stages is a necessary experiment.

Thank you for your comment. We carried out immunostaining with an anti-ubiquitin antibody in the brains at 1-day-old. No significant difference was detected between the control and milton knockdown. This result has been included as Figure S1 in the revised manuscript. The result section was revised as below:

Line 136-139 ‘There was no significant increase in ubiquitinated proteins in milton knockdown flies at 1-day old, suggesting that the accumulation of ubiquitinated proteins caused by milton knockdown is age-dependent (Figure S1).’

For Figure 1E: In the Electron Microscopy section of the Methods, define how swollen axons were identified and describe the quantification methodology used.

Thank you for your comment. Swollen axons are, unlike normal axons, round in shape and enlarged. We revised the text as below;

Lines 157-160: ‘The swelling of presynaptic terminals, characterized by the enlargement and roundness, was not reported at 3-day-old(24) but observed at this age with about 4% of total presynaptic terminals (Figure 1F, asterisks).’

Lines 683-684, Figure 1 legend: ‘Swollen presynaptic terminals (asterisks in (F)), characterized by the enlargement and higher circularity, were found more frequently in milton knockdown neurons.’

L218-L219: Throughout the text, the expression 'eIF2β is "upregulated" in response to Milton knockdown' is frequently used. However, considering the presented results, it might be more accurate to interpret that under the condition of Milton knockdown, eIF2β is not undergoing degradation but rather remains stable.

Thank you for pointing it out. We replaced ‘upregulated’ with ‘increased’ throughout the text.

L234-L235: On what basis is the conclusion drawn that there is a reduction? Given that three experiments have been conducted, it would be possible and more convincing to quantify the results to determine if there is a significant decrease.

Thank you for pointing it out. We quantified the AUC of polysome fraction and carried out statistical analysis. There is a significant decrease in polysome in milton knockdown, and this result has been included in Figure 5B. We revised the figure and the legend accordingly.

L236: 5H-> 4H

Thank you for pointing it out, and we are sorry for the confusion. We corrected it.

L238-L239: Since there is no significant difference observed, it may not be accurate to interpret a reduction in puromycin incorporation.

Thank you for pointing it out. As described above, quantification of polysome fractions showed that milton knockdown significantly reduce polysome (Figure 5B). We revised the manuscript as below;

Lines 263-264: ‘However, unexpectedly, we found that milton knockdown significantly reduced the level of mRNAs associated with polysomes (Figure 5A and B).’

Figure 5D and Figure 6D: Climbing assays have been conducted, but I believe experiments should also be performed to examine whether overexpression or heterozygous mutants of eIF2β induce or suppress degeneration.

Thank you for pointing it out. We analyzed the eyes with eIF2_β_ overexpression for neurodegeneration. Although there was a tendency of elevated neurodegeneration in the retina with eIF2_β_ overexpression, the difference between control and eIF2_β_ overexpression did not reach statistical significance (Figure S2). This result has been included as Figure S2 in the revised manuscript, and the following sentences have been included in the text:

Lines 288-293: ‘We asked if eIF2β overexpression causes neurodegeneration, as depletion of axonal mitochondria in the photoreceptor neurons causes axon degeneration in an age-dependent manner(24). eIF2β overexpression in photoreceptor neurons tends to increase neurodegeneration in aged flies, while it was not statistically significant (p>0.05, Figure S2).’

L271-L272: The results in Figure 6B are surprising. I anticipated a greater increase compared to the Milton knockdown alone. While p62 appears to be reduced, it is not clear why these results lead to the conclusion that lowering eIF2β rescues autophagic impairment. Please add a discussion section to address this point.

Thank you for pointing it out. We apologize for the unclear description of the result. Milton knockdown flies show p62 accumulation (Figure 2), and deleting one copy of eIF2beta in milton knockdown background reduced p62 accumulation (Figure 7C). We revised the text as below:

Lines 307-315: ‘Neuronal knockdown of milton causes accumulation of autophagic substrate p62 in the Triton X-100-soluble fraction (Figure 2B), and we tested if lowering eIF2β ameliorates it. We found that eIF2β heterozygosity caused a mild increase in LC3-I levels and decreases in LC3-II levels, resulting in a significantly lower LC3-II/LC3-I ratio in milton knockdown flies (Figure 7B). eIF2β heterozygosity decreased the p62 level in the Triton X-100-soluble fraction in the brains of milton knockdown flies (Figure 7C). The p62 level in the SDS-soluble fraction, which is not sensitive to milton knockdown (Figure 2B), was not affected (Figure 7C). These results suggest that suppression of eIF2β ameliorates the impairment of autophagy caused by milton knockdown.’

L369: Please specify the source of the anti-ubiquitin antibody used.

Thank you for pointing it out. We included the antibody information in the method section.

Figure 7: While the relationship between Milton knockdown and the eIF2β and eIF2α proteins has been elucidated through the authors' efforts, I would like to see an investigation into whether eIF2β is upregulated and eIF2α phosphorylation is reduced in simply aged Drosophila. This would help us understand the correlation between aging and eIF2 protein dynamics.

Thank you for your comment. We agree that it is an important question, and we are working on it. However, we feel that it is beyond the scope of the current manuscript.

L645-L646: If the mushroom body is identified using mito-GFP, then include mito-GFP in the genotype listed in Supplementary Table 2.

We are sorry for the oversight. We corrected it in Supplementary Table 2.

Additionally, while it is presumed that the mito-GFP signal decreases in axons with Milton RNAi, how was the lobe tips area accurately selected for analysis? Please include these details along with a comprehensive description of the quantification methodology in the Methods section.

Thank you for your comment. Although the mito-GFP signal in the axon is weak in the milton knockdown neurons, it is sufficient to distinguish the mushroom body structure from the background. We revised the method section to include this information in the method section:

Line 437-438: ‘For eIF2α and p-eIF2α immunostaining, the mushroom body was detected by mitoGFP expression.’

Reviewer #2 (Public review):

Summary:

In this manuscript, Mackie et al. investigate gustatory behavior and the neural basis of gustation in the predatory nematode Pristionchus pacificus. First, they show that the behavioral preferences of P. pacificus for gustatory cues differ from those reported for C. elegans. Next, they investigate the molecular mechanisms of salt sensing in P. pacificus. They show that although the C. elegans transcription factor gene che-1 is expressed specifically in the ASE neurons, the P. pacificus che-1 gene is expressed in the Ppa-ASE and Ppa-AFD neurons. Moreover, che-1 plays a less critical role in salt chemotaxis in P. pacificus than C. elegans. Chemogenetic silencing of Ppa-ASE and Ppa-AFD neurons results in more severe chemotaxis defects. The authors then use calcium imaging to show that both Ppa-ASE and Ppa-AFD neurons respond to salt stimuli. Calcium imaging experiments also reveal that the left and right Ppa-ASE neurons respond differently to salts, despite the fact that P. pacificus lacks lsy-6, a microRNA that is important for ASE left/right asymmetry in C. elegans. Finally, the authors show that the receptor guanylate cyclase gene Ppa-gcy-23.3 is expressed in the right Ppa-ASE neuron (Ppa-ASER) but not the left Ppa-ASE neuron (Ppa-ASEL) and is required for some of the gustatory responses of Ppa-ASER, further confirming that the Ppa-ASE neurons are asymmetric and suggesting that Ppa-GCY-23.3 is a gustatory receptor. Overall, this work provides insight into the evolution of gustation across nematode species. It illustrates how sensory neuron response properties and molecular mechanisms of cell fate determination can evolve to mediate species-specific behaviors. However, the paper would be greatly strengthened by a direct comparison of calcium responses to gustatory cues in C. elegans and P. pacificus, since the comparison currently relies entirely on published data for C. elegans, where the imaging parameters likely differ. In addition, the conclusions regarding Ppa-AFD neuron function would benefit from additional confirmation of AFD neuron identity. Finally, how prior salt exposure influences gustatory behavior and neural activity in P. pacificus is not discussed.

Strengths:

(1) This study provides exciting new insights into how gustatory behaviors and mechanisms differ in nematode species with different lifestyles and ecological niches. The results from salt chemotaxis experiments suggest that P. pacificus shows distinct gustatory preferences from C. elegans. Calcium imaging from Ppa-ASE neurons suggests that the response properties of the ASE neurons differ between the two species. In addition, an analysis of the expression and function of the transcription factor Ppa-che-1 reveals that mechanisms of ASE cell fate determination differ in C. elegans and P. pacificus, although the ASE neurons play a critical role in salt sensing in both species. Thus, the authors identify several differences in gustatory system development and function across nematode species.

(2) This is the first calcium imaging study of P. pacificus, and it offers some of the first insights into the evolution of gustatory neuron function across nematode species.

(3) This study addresses the mechanisms that lead to left/right asymmetry in nematodes. It reveals that the ASER and ASEL neurons differ in their response properties, but this asymmetry is achieved by molecular mechanisms that are at least partly distinct from those that operate in C. elegans. Notably, ASEL/R asymmetry in P. pacificus is achieved despite the lack of a P. pacificus lsy-6 homolog.

Weaknesses:

(1) The authors observe only weak attraction of C. elegans to NaCl. These results raise the question of whether the weak attraction observed is the result of the prior salt environment experienced by the worms. More generally, this study does not address how prior exposure to gustatory cues shapes gustatory responses in P. pacificus. Is salt sensing in P. pacificus subject to the same type of experience-dependent modulation as salt sensing in C. elegans?

(2) A key finding of this paper is that the Ppa-CHE-1 transcription factor is expressed in the Ppa-AFD neurons as well as the Ppa-ASE neurons, despite the fact that Ce-CHE-1 is expressed specifically in Ce-ASE. However, additional verification of Ppa-AFD neuron identity is required. Based on the image shown in the manuscript, it is difficult to unequivocally identify the second pair of CHE-1-positive head neurons as the Ppa-AFD neurons. Ppa-AFD neuron identity could be verified by confocal imaging of the CHE-1-positive neurons, co-expression of Ppa-che-1p::GFP with a likely AFD reporter, thermotaxis assays with Ppa-che-1 mutants, and/or calcium imaging from the putative Ppa-AFD neurons.

(3) Loss of Ppa-che-1 causes a less severe phenotype than loss of Ce-che-1. However, the loss of Ppa-che-1::RFP expression in ASE but not AFD raises the question of whether there might be additional start sites in the Ppa-che-1 gene downstream of the mutation sites. It would be helpful to know whether there are multiple isoforms of Ppa-che-1, and if so, whether the exon with the introduced frameshift is present in all isoforms and results in complete loss of Ppa-CHE-1 protein.

(4) The authors show that silencing Ppa-ASE has a dramatic effect on salt chemotaxis behavior. However, these data lack control with histamine-treated wild-type animals, with the result that the phenotype of Ppa-ASE-silenced animals could result from exposure to histamine dihydrochloride. This is an especially important control in the context of salt sensing, where histamine dihydrochloride could alter behavioral responses to other salts.

(5) The calcium imaging data in the paper suggest that the Ppa-ASE and Ce-ASE neurons respond differently to salt solutions. However, to make this point, a direct comparison of calcium responses in C. elegans and P. pacificus using the same calcium indicator is required. By relying on previously published C. elegans data, it is difficult to know how differences in growth conditions or imaging conditions affect ASE responses. In addition, the paper would be strengthened by additional quantitative analysis of the calcium imaging data. For example, the paper states that 25 mM NH4Cl evokes a greater response in ASEL than 250 mM NH4Cl, but a quantitative comparison of the maximum responses to the two stimuli is not shown.

(6) It would be helpful to examine, or at least discuss, the other P. pacificus paralogs of Ce-gcy-22. Are they expressed in Ppa-ASER? How similar are the different paralogs? Additional discussion of the Ppa-gcy-22 gene expansion in P. pacificus would be especially helpful with respect to understanding the relatively minor phenotype of the Ppa-gcy-22.3 mutants.

(7) The calcium imaging data from Ppa-ASE is quite variable. It would be helpful to discuss this variability. It would also be helpful to clarify how the ASEL and ASER neurons are being conclusively identified during calcium imaging.

(8) More information about how the animals were treated prior to calcium imaging would be helpful. In particular, were they exposed to salt solutions prior to imaging? In addition, the animals are in an M9 buffer during imaging - does this affect calcium responses in Ppa-ASE and Ppa-AFD? More information about salt exposure, and how this affects neuron responses, would be very helpful.

(9) In Figure 6, the authors say that Ppa-gcy-22.3::GFP expression is absent in the Ppa-che-1(ot5012) mutant. However, based on the figure, it looks like there is some expression remaining. Is there a residual expression of Ppa-gcy-22.3::GFP in ASE or possibly ectopic expression in AFD? Does Ppa-che-1 regulate rGC expression in AFD? It would be helpful to address the role of Ppa-che-1 in AFD neuron differentiation.

Author response:

We very much appreciate the reviewers’ and editor’s overall positive responses to our manuscript "Evolution of lateralized gustation in nematodes".

Reviewer #1:

The mechanism of lsy-6-independent establishment of ASEL/R asymmetry in P. pacificus remains uncharacterized. 

We thank the reviewer for recognizing the novel contributions of our work in revealing the existence of alternative pathways for establishing neuronal lateral asymmetry despite the absence of the lsy-6 miRNA in a divergent nematode species. We are certainly encouraged now to search for genetic factors that abolish asymmetric expression of gcy-22.3.

Reviewer #2:

(1) The authors observe only weak attraction of C. elegans to NaCl. These results raise the question of whether the weak attraction observed is the result of the prior salt environment experienced by the worms. More generally, this study does not address how prior exposure to gustatory cues shapes gustatory responses in P. pacificus. Is salt sensing in P. pacificus subject to the same type of experience-dependent modulation as salt sensing in C. elegans? 

Proposed revision: For our live imaging experiments, we had not considered if starved P. pacificus animals in the presence of salt may exhibit responses different from a well-fed state. However, we will venture to address the effect of experience-dependent modulation in P. pacificus chemotaxis behavior using NH4Cl.

(2) A key finding of this paper is that the Ppa-CHE-1 transcription factor is expressed in the Ppa-AFD neurons as well as the Ppa-ASE neurons, despite the fact that Ce-CHE-1 is expressed specifically in Ce-ASE. However, additional verification of Ppa-AFD neuron identity is required. Based on the image shown in the manuscript, it is difficult to unequivocally identify the second pair of CHE-1-positive head neurons as the Ppa-AFD neurons. Ppa-AFD neuron identity could be verified by confocal imaging of the CHE-1-positive neurons, co-expression of Ppa-che-1p::GFP with a likely AFD reporter, thermotaxis assays with Ppa-che-1 mutants, and/or calcium imaging from the putative Ppa-AFD neurons. 

We are happy to provide additional evidence to confirm Ppa-AFD neuron identity since the expression of Ppa-CHE-1 in non-ASE amphid neurons is one of the major differences between the two nematode specie

Proposed revision: We will provide results showing the Ppa-ttx-1::gfp reporter expression in finger-like neuronal endings and Ppa-_TTX-1::ALFA co-localization with _Ppa-che-1::gfp in the putative AFD neurons and discuss the possible role of Ppa-CHE-1 in AFD differentiation. We attempted to obtain AFD markers using several reporter strains. However, Ppa-gcy-8.1p::gfp(csuEx101) (PPA24212) showed no expression while Ppa-gcy-8.2p::gfp(csuEx100) (PPA41407) showed only expression in pharyngeal cells.

(4) The authors show that silencing Ppa-ASE has a dramatic effect on salt chemotaxis behavior. However, these data lack control with histamine-treated wild-type animals, with the result that the phenotype of Ppa-ASE-silenced animals could result from exposure to histamine dihydrochloride. This is an especially important control in the context of salt sensing, where histamine dihydrochloride could alter behavioral responses to other salts. 

Proposed revision: Thank you for noticing this oversight. The control for histamine-treated wild-type worms in the Ppa-ASE silencing experiments was inadvertently left out in the original submission. Because the HisCl transgene is on a randomly segregating transgene array, we have scored worms with and without the transgene expressing the co-injection marker (Ppa-egl-20p::rfp expressed in the tail) to show that the presence of the transgene is necessary for the knockdown of NH4Br attraction.

We will also address most of the other more minor suggestions and clarifications sought by the reviewers.

I believe this point to be very valid when brainstorming. Scenarios are very essential to design processes because they can simulate what perspectives could be. It can also help you with the bumps in the road before you start anything. I relate to this statement a lot because I often create scenarios in my head because I want to include as many as I can when taking any action.

We are accepting what impact technology is having on us. Is this the future we want to have? Do we want to go into work and find no one to talk to or listen to except for the voice in our head? This is not the future I want for myself or others...

The writer tells the reader understanding who the audience is, & the reader's needs and level of understanding will help the writer to specifically communicate what needs to be said. *not going over the reader's head.

First thing that popped in my head was an instruction manual because those are usually clear and as if someone if helping you complete a task.

1. What parts impacted you the most. Reading this article I found that the most crazy part was that it was caused by medical staff and not a patient that was not correctly discussed and screened on what not to do. The patient was injured unknowingly by the oxygen tank by medical staff that should be trained in what to do and what not to do around MRI operations.

2. Review of what safety measures were overlooked and or ignored. The nurse did not take proper safety measures and/ or was not trained in the knowledge needed to know what they can and can not do around an MRI. With this in motion the nurse brought in a oxygen tank that was not suitable for the magnet and resulted in the injury and death of a patient.

3. An assessment of your current safety practices and a pledge of how you intend to view safety in the future.

Within reading this article and many others. I will take every step and measure to enforce every safety measure given. I do not want the possible injury or death of a person be at my fault due to lack of safety. I will make sure to keep my coworkers and my patients safe by keeping these articles in mind and remembering how being forgetful can harm someone.

I found this to be an interesting idea. During my time in this master's degree, I have begun seeing the positive side of AI in learning. Before it all, I was one of those people that said "AI is bad and students are never going to learn because they will probably just cheat with AI." Yet learning to use it to help inside of lessons has shown me how it can be a positive source for students to use. Yet this section telling us not to necessarily include it or exclude it in teaching, but to simply to design instruction for our students to learn makes me reset my head a bit. I am reading this as "add AI if it helps to enhance the learning experience. Do not add it just because you can. Do not force it." It makes me think more when developing lessons that I will only want to use it if it comes to my mind when figuring out what to do.

This section makes me reflect on the common misconception that tools like GenAI or even Google can replace human knowledge. As an educator, I see how tempting it might be for students (and even teachers) to rely heavily on these tools, but this dependency can create significant gaps in critical thinking and problem-solving. The quote about interpreting information resonates with me because technology can provide data, but understanding and applying it require skills and context that only humans bring.

Personally, I agree with the statement that solving complex problems requires more than just finding information online. It reminds me of situations in my professional role where I’ve had to assess the validity of data or consider the nuances of a problem—something no search engine or AI can do without my input and expertise. GenAI can be a powerful assistant, but the “knowledge in our head” is what allows us to navigate ambiguity and discern quality.

I wonder if relying too much on tools like ChatGPT might weaken students’ ability to critically evaluate information or even know where to start when they don’t have a foundation of knowledge. While GenAI can support learning, I see a real danger if we let it replace the essential process of building and applying our understanding. What do you think—is there a way to balance using these tools without diminishing the development of core skills?

I want to show people a clear narative yet sometimes my abstrations in my head makes my art sometimes hard to read to otgers. There is also the idea about chosin the forbidden fruit and not going back to where you once livied. I will not go back to how i did think, this art is my forbidden fruit

Ophelia bursts in to talk to her father and describes what has just happened. Hamlet came to her and in a frantic state and started acting mad right before her eyes. The strange description of Hamlet seems to be in consequent to his experiences with the ghost of Old Hamlets soul.

I don't tend to spend a lot of time trying to label assessments as "formative" vs "summative", but I also have the luxury of not having to spend much time thinking about giving letter grades. From a teaching and learning standpoint, I always just have the words of my mentor going through my head is that for learning to happen there generally needs to be "practice with feedback" (presumably followed by more practice and feedback until the task is mastered). This presumes that a teacher is providing a task that is aligned with the desired learning objective and then is giving the student an opportunity to demonstrate their performance and receives useful feedback about that performance so that the student's next attempt at the performance can be better. This is where I think mastery grading excels.

Example showing that the audience will be many different people from many different backgrounds, therefore meaning that the technical writting should be readable and understandable by everyone.

eLife Assessment

Bowler et al. present a software/hardware system for behavioral control of navigation-based virtual reality experiments, particularly suited for pairing with 2-photon imaging but applicable to a variety of techniques. This system represents a valuable contribution to the field of behavioral and systems neuroscience, as it provides a standardized, easy to implement, and flexible system that could be adopted across multiple laboratories. The authors provide compelling evidence of the functionality of their system by reporting benchmark tests and demonstrating hippocampal activity patterns consistent with standards in the field. This work will be of interest to systems neuroscientists looking to integrate flexible head-fixed behavioral control with neural data acquisition.

Reviewer #1 (Public review):

Summary:

Bowler et al. present a thoroughly tested system for modularized behavioral control of navigation-based experiments, particularly suited for pairing with 2-photon imaging but applicable to a variety of techniques. This system, which they name behaviorMate, represents an important methodological contribution to the field of behavioral and systems neuroscience. As the authors note, behavioral control paradigms vary widely across laboratories in terms of hardware and software utilized and often require specialized technical knowledge to make changes to these systems. Having a standardized, easy to implement, and flexible system that can be used by many groups is therefore highly desirable.

Strengths:

The present manuscript provides compelling evidence of the functionality and applicability of behaviorMate. The authors report benchmark tests for high-fidelity, real-time update speed between the animal's movement and the behavioral control, on both the treadmill-based and virtual reality (VR) setups. The VR system relies on Unity, a common game development engine, but implements all scene generation and customizability in the authors' behaviorMate and VRMate software, which circumvents the need for users to program task logic in C# in Unity. Further, the authors nicely demonstrate and quantify reliable hippocampal place cell coding in both setups, using synchronized 2-photon imaging. This place cell characterization also provides a concrete comparison between the place cell properties observed in treadmill-based navigation vs. visual VR in a single study, which itself is a valuable contribution to the field.

Weaknesses: None noted.

Documentation for installing and operating behaviorMate is available via the authors' lab website and Github, linked in the manuscript.

The authors have addressed all of my requests for clarification from the previous round of review. This work will be of great interest to systems neuroscientists looking to integrate flexible head-fixed behavioral control with neural data acquisition.

Reviewer #2 (Public review):

The authors present behaviorMate, an open-source behavior control system including a central GUI and compatible treadmill and display components. Notably, the system utilize the "Intranet of things" scheme and the components communicate through local network, making the system modular, which in turn allows user to configure the setup to suit their experimental needs. Overall, behaviorMate is a useful resource for researchers performing head-fixed VR imaging studies involving 1D navigation tasks, as the commercial alternatives are often expensive and inflexible to modify.

One major utility of behaviorMate is an open-source alternative to commercial behavior apparatus for head-fixed imaging studies involving 1D navigation tasks. The documentation, BOM, CAD files, circuit design, source and compiled software, along with the manuscript, create an invaluable resource for neuroscience researcher looking to set up a budget-friendly VR and head-fixed imaging rig. Some features of behaviorMate, including the computer vision-based calibration of treadmill, and the decentralized, Android-based display devices, are very innovative approaches and can be quite useful in practical settings.

behaviorMate can also be used as a set of generic schema and communication protocols that allows the users to incorporate recording and stimulation devices during a head-fixed imaging experiment. Due to the "Intranet of things" approach taken in the design, any hardware that supports UDP communication can in theory be incorporated into the system. In terms of current capability, behaviorMate supports experimental contingencies based on animal position and time and synchronization with external recording devices using a TTL start signal. Further customization involving more complicated experimental contingencies, more accurate recording synchronization (for example with ephys recording devices), incorporation of novel behavior and high-speed neural recording hardware beyond GPIO signaling would require modification of the Java source and custom hardware implementation. Modification to the Java source of behaviorMate can be performed with basic familiarity with object-oriented programming using the Java programming language, and a JavaFX-based plugin system is under development to make such customizations more approachable for users.

In summary, the manuscript presents a well-developed and useful open-source behavior control system for head-fixed VR imaging experiments with innovative features.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public Review):

(1) As VRMate (a component of behaviorMate) is written using Unity, what is the main advantage of using behaviorMate/VRMate compared to using Unity alone paired with Arduinos (e.g. Campbell et al. 2018), or compared to using an existing toolbox to interface with Unity (e.g. Alsbury-Nealy et al. 2022, DOI: 10.3758/s13428-021-01664-9)? For instance, one disadvantage of using Unity alone is that it requires programming in C# to code the task logic. It was not entirely clear whether VRMate circumvents this disadvantage somehow -- does it allow customization of task logic and scenery in the GUI? Does VRMate add other features and/or usability compared to Unity alone? It would be helpful if the authors could expand on this topic briefly.

We have updated the manuscript (lines 412-422) to clarify the benefits of separating the VR system as an isolated program and a UI that can be run independently. We argue that “…the recommended behaviorMate architecture has several important advantages. Firstly, by rendering each viewing angle of a scene on a dedicated device, performance is improved by splitting the computational costs across several inexpensive devices rather than requiring specialized or expensive graphics cards in order to run…, the overall system becomes more modular and easier to debug [and] implementing task logic in Unity would require understanding Object-Oriented Programming and C# … which is not always accessible to researchers that are typically more familiar with scripting in Python and Matlab.”

VRMate receives detailed configuration info from behaviorMate at runtime as to which VR objects to display and receives position updates during experiments. Any other necessary information about triggering rewards or presenting non-VR cues is still handled by the UI so no editing of Unity is necessary. Scene configuration information is in the same JSON format as the settings files for behaviorMate, additionally there are Unity Editor scripts which are provided in the VRmate repository which permit customizing scenes through a “drag and drop” interface and then writing the scene configuration files programmatically. Users interested in these features should see our github page to find example scene.vr files and download the VRMate repository (including the editor scripts).  We provided 4 vr contexts, as well as a settings file that uses one of them which can be found on the behaviorMate github page (https://github.com/losonczylab/behaviorMate) in the “vr_contexts” and “example_settigs_files” directories. These examples are provided to assist VRMate users in getting set up and could provide a more detailed example of how VRMate and behaviorMate interact.

(2) The section on "context lists", lines 163-186, seemed to describe an important component of the system, but this section was challenging to follow and readers may find the terminology confusing. Perhaps this section could benefit from an accompanying figure or flow chart, if these terms are important to understand.

We maintain the use of the term context and context list in order to maintain a degree of parity with the java code. However, we have updated lines 173-175 to define the term context for the behaviorMate system: “... a context is grouping of one or more stimuli that get activated concurrently. For many experiments it is desirable to have multiple contexts that are triggered at various locations and times in order to construct distinct or novel environments.”

a. Relatedly, "context" is used to refer to both when the animal enters a particular state in the task like a reward zone ("reward context", line 447) and also to describe a set of characteristics of an environment (Figure 3G), akin to how "context" is often used in the navigation literature. To avoid confusion, one possibility would be to use "environment" instead of "context" in Figure 3G, and/or consider using a word like "state" instead of "context" when referring to the activation of different stimuli.

Thank you for the suggestion. We have updated Figure 3G to say “Environment” in order to avoid confusion.

(3) Given the authors' goal of providing a system that is easily synchronizable with neural data acquisition, especially with 2-photon imaging, I wonder if they could expand on the following features:

a. The authors mention that behaviorMate can send a TTL to trigger scanning on the 2P scope (line 202), which is a very useful feature. Can it also easily generate a TTL for each frame of the VR display and/or each sample of the animal's movement? Such TTLs can be critical for synchronizing the imaging with behavior and accounting for variability in the VR frame rate or sampling rate.

Different experimental demands require varying levels of precision in this kind of synchronization signals. For this reason, we have opted against a “one-size fits all” for synchronization with physiology data in behaviorMate. Importantly this keeps the individual rig costs low which can be useful when constructing setups specifically for use when training animals. behaviorMate will log TTL pulses sent to GPIO pins setup as sensors, and can be configured to generate TTL pulses at regular intervals. Additionally all UDP packets received by the UI are time stamped and logged. We also include the output of the arduino millis() function in all UDP packets which can be used for further investigation of clock drift between system components. Importantly, since the system is event driven there cannot be accumulating drift across running experiments between the behaviorMate UI and networked components such as the VR system.

For these reasons, we have not needed to implement a VR frame synchronization TTL for any of our experiments, however, one could extend VRMate to send "sync" packets back to behaviorMate to log when each frame was displayed precisely or TTL pulses (if using the same ODROID hardware we recommend in the standard setup for rendering scenes). This would be useful if it is important to account for slight changes in the frame rate at which the scenes are displayed. However, splitting rendering of large scenes between several devices results in fast update times and our testing and benchmarks indicate that display updates are smooth and continuous enough to appear coupled to movement updates from the behavioral apparatus and sufficient for engaging navigational circuits in the brain.

b. Is there a limit to the number of I/O ports on the system? This might be worth explicitly mentioning.

We have updated lines 219-220 in the manuscript to provide this information: Sensors and actuators can be connected to the controller using one of the 13 digital or 5 analog input/output connectors.

c. In the VR version, if each display is run by a separate Android computer, is there any risk of clock drift between displays? Or is this circumvented by centralized control of the rendering onset via the "real-time computer"?

This risk is mitigated by the real-time computer/UI sending position updates to the VR displays. The maximum amount scenes can be out of sync is limited because they will all recalibrate on every position update – which occurs multiple times per second as the animal is moving. Moreover, because position updates are constantly being sent by behaviorMate to VRMate and VRMate is immediately updating the scene according to this position, the most the scene can become out of sync with the mouse's position is proportional to the maximum latency multiplied by the running speed of the mouse. For experiments focusing on eliciting an experience of navigation, such a degree of asynchrony is almost always negligible. For other experimental demands it could be possible to incorporate more precise frame timing information but this was not necessary for our use case and likely for most other use cases. Additionally, refer to the response to comment 3a.

Reviewer #2 (Public review):

(1) The central controlling logic is coupled with GUI and an event loop, without a documented plugin system. It's not clear whether arbitrary code can be executed together with the GUI, hence it's not clear how much the functionality of the GUI can be easily extended without substantial change to the source code of the GUI. For example, if the user wants to perform custom real-time analysis on the behavior data (potentially for closed-loop stimulation), it's not clear how to easily incorporate the analysis into the main GUI/control program.

Without any edits to the existing source code behaviorMate is highly customizable through the settings files, which allow users to combine the existing contexts and decorators in arbitrary combinations. Therefore, users have been able to perform a wide variety of 1D navigation tasks, well beyond our anticipated use cases by generating novel settings files. The typical method for providing closed-loop stimulation would be to set up a context which is triggered by animal behavior using decorators (e.g. based on position, lap number and time) and then trigger the stimulation with a TTL pulse. Rarely, if users require a behavioral condition not currently implemented or composable out of existing decorators, it would require generating custom code in Java to extend the UI. Performing such edits requires only knowledge of basic object-oriented programming in Java and generating a single subclass of either the BasicContextList or ContextListDecorator classes. In addition, the JavaFX (under development) version of behaviorMate incorporates a plugin which doesn't require recompiling the code in order to make these changes. However, since the JavaFX software is currently under development, documentation does not yet exist. All software is open-sourced and available on github.com for users interested in generating plugins or altering the source code.

We have added the additional caveat to the manuscript in order to clarify this point (Line 197-202): “However, if the available set of decorators is not enough to implement the required task logic, some modifications to the source code may be necessary. These modifications, in most cases, would be very simple and only a basic understanding of object-oriented programming is required. A case where this might be needed would be performing novel customized real-time analysis on behavior data and activating a stimulus based on the result”

(2) The JSON messaging protocol lacks API documentation. It's not clear what the exact syntax is, supported key/value pairs, and expected response/behavior of the JSON messages. Hence, it's not clear how to develop new hardware that can communicate with the behaviorMate system.

The most common approach for adding novel hardware is to use TTL pulses (or accept an emitted TTL pulse to read sensor states). This type of hardware addition  is possible through the existing GPIO without the need to interact with the software or JSON API. Users looking to take advantage of the ability to set up and configure novel behavioral paradigms without the need to write any software would be limited to adding hardware which could be triggered with and report to the UI with a TTL pulse (however fairly complex actions could be triggered this way).

For users looking to develop more customized hardware solutions that interact closely with the UI or GPIO board, additional documentation on the JSON messaging protocol has been added to the behaviormate-utils repository (https://github.com/losonczylab/behaviormate_utils). Additionally, we have added a link to this repository in the Supplemental Materials section (line 971) and referenced this in the manuscript (line 217) to make it easier for readers to find this information.

Furthermore, developers looking to add completely novel components to the UI  can implement the interface described by Context.java in order to exchange custom messages with hardware. (described  in the JavaDoc: https://www.losonczylab.org/behaviorMate-1.0.0/)  These messages would be defined within the custom context and interact with the custom hardware (meaning the interested developer would make a novel addition to the messaging API). Additionally, it should be noted that without editing any software, any UDP packets sent to behaviorMate from an IP address specified in the settings will get time stamped and logged in the stored behavioral data file meaning that are a large variety of hardware implementation solutions using both standard UDP messaging and through TTL pulses that can work with behaviorMate with minimal effort. Finally, see response to R2.1 for a discussion of the JavaFX version of the behaviorMatee UI including plugin support.

(3) It seems the existing control hardware and the JSON messaging only support GPIO/TTL types of input/output, which limits the applicability of the system to more complicated sensor/controller hardware. The authors mentioned that hardware like Arduino natively supports serial protocols like I2C or SPI, but it's not clear how they are handled and translated to JSON messages.

We provide an implementation for an I2C-based capacitance lick detector which interested developers may wish to copy if support for novel I2C or SPI. Users with less development experience wishing to expand the hardware capabilities of  behaviorMatecould also develop adapters which can be triggered  on a TTL input/output. Additionally, more information about the JSON API and how messages are transmitted to the PC by the arduino is described in point (2) and the expanded online documentation.

a. Additionally, because it's unclear how easy to incorporate arbitrary hardware with behaviorMate, the "Intranet of things" approach seems to lose attraction. Since currently, the manuscript focuses mainly on a specific set of hardware designed for a specific type of experiment, it's not clear what are the advantages of implementing communication over a local network as opposed to the typical connections using USB.

As opposed to serial communication protocols as typical with USB, networking protocols seamlessly function based on asynchronous message passing. Messages may be routed internally (e.g. to a PCs localhost address, i.e. 0.0.0..0) or to a variety of external hardware (e.g. using IP addresses such as those in the range 192.168.1.2 - 192.168.1.254). Furthermore, network-based communication allows modules, such as VR, to be added easily. behavoirMate systems can be easily expanded using low-cost Ethernet switches and consume only a single network adapter on the PC (e.g. not limited by the number of physical USB ports). Furthermore, UDP message passing is implemented in almost all modern programming languages in a platform independent manner (meaning that the same software can run on OSX, Windows, and Linux). Lastly, as we have pointed out (Line 117) a variety of tools exist for inspecting network packets and debugging; meaning that it is possible to run behaviorMate with simulated hardware for testing and debugging.

The IOT nature of behaviorMate means there is no requirement for novel hardware to be implemented  using an arduino,  since any system capable of  UDP communication can  be configured. For example, VRMate is usually run on Odroid C4s, however one could easily create a system using Raspberry Pis or even additional PCs. behaviorMate is agnostic to the format of the UDP messages, but packaging any data in the JSON format for consistency would be encouraged. If a new hardware is a sensor that has input requiring it to be time stamped and logged then all that is needed is to add the IP address and port information to the ‘controllers’ list in a behaviorMate settings file. If more complex interactions are needed with novel hardware than a custom implementation of ContextList.java may be required (see response to R2.2). However, the provided UdpComms.java class could be used to easily send/receive messages from custom Context.java subclasses.

Solutions for highly customized hardware do require basic familiarity with object-oriented programming using the Java programming language. However, in our experience most behavioral experiments do not require these kinds of modifications. The majority of 1D navigation tasks, which behaviorMate is currently best suited to control, require touch/motion sensors, LEDs, speakers, or solenoid valves,  which are easily controlled by the existing GPIO implementation. It is unlikely that custom subclasses would even be needed.

Reviewer #3 (Public review):

(1) While using UDP for data transmission can enhance speed, it is thought that it lacks reliability. Are there error-checking mechanisms in place to ensure reliable communication, given its criticality alongside speed?

The provided GPIO/behavior controller implementation sends acknowledgement packets in response to all incoming messages as well as start and stop messages for contexts and “valves”. In this way the UI can update to reflect both requested state changes as well as when they actually happen (although there is rarely a perceptible gap between these two states unless something is unplugged or not functioning). See Line 85 in the revised manuscript “acknowledgement packets are used to ensure reliable message delivery to and from connected hardware”.

(2) Considering this year's price policy changes in Unity, could this impact the system's operations?

VRMate is not affected by the recent changes in pricing structure of the Unity project.

The existing compiled VRMate software does not need to be regenerated to update VR scenes, or implement new task logic (since this is handled by the behaviorMate GUI). Therefore, the VRMate program is robust to any future pricing changes or other restructuring of the Unity program and does not rely on continued support of Unity. Additionally, while the solution presented in VRMate has many benefits, a developer could easily adapt any open-source VR Maze project to receive the UDP-based position updates from behaviorMate or develop their own novel VR solutions.

(3) Also, does the Arduino offer sufficient precision for ephys recording, particularly with a 10ms check?

Electrophysiology recording hardware typically has additional I/O channels which can provide assistance with tracking behavior/synchronization at a high resolution. While behaviorMate could still be used to trigger reward valves, either the ephys hardware or some additional high-speed DAQ would be recommended to maintain accurately with high-speed physiology data. behaviorMate could still be set up as normal to provide closed and open-loop task control at behaviorally relevant timescales alongside a DAQ circuit recording events at a consistent temporal resolution. While this would increase the relative cost of the individual recording setup, identical rigs for training animals could still be configured without the DAQ circuit avoiding unnecessary cost and complexity.

(4) Could you clarify the purpose of the Sync Pulse? In line 291, it suggests additional cues (potentially represented by the Sync Pulse) are needed to align the treadmill screens, which appear to be directed towards the Real-Time computer. Given that event alignment occurs in the GPIO, the connection of the Sync Pulse to the Real-Time Controller in Figure 1 seems confusing.

A number of methods exist for synchronizing recording devices like microscopes or electrophysiology recordings with behaviorMate’s time-stamped logs of actuators and sensors. For example, the GPIO circuit can be configured to send sync triggers, or receive timing signals as input. Alternatively a dedicated circuit could record frame start signals and relay them to the PC to be logged independently of the GPIO (enabling a high-resolution post-hoc alignment of the time stamps). The optimal method to use varies based on the needs of the experiment. Our setups have a dedicated BNC output and specification in the settings file that sends a TTL pulse at the start of an experiment in order to trigger 2p imaging setups (see line 224, specifically that this is a detail of “our” 2p imaging setup). We provide this information as it might be useful suggesting how to have both behavior and physiology data start recording at the same time. We do not intend this to be the only solution for alignment. Figure 1 indicates an “optional” circuit for capturing a high speed sync pulse and providing time stamps back to the real time PC. This is another option that might be useful for certain setups (or especially for establishing benchmarks between behavior and physiology recordings). In our setup event alignment does not exclusively occur on the GPIO.

a. Additionally, why is there a separate circuit for the treadmill that connects to the UI computer instead of the GPIO? It might be beneficial to elaborate on the rationale behind this decision in line 260.

Event alignment does not occur on the GPIO, separating concerns between position tracking and more general input/output features which improves performance and simplifies debugging.  In this sense we maintain a single event loop on the Arduino, avoiding the need to either run multithreaded operations or rely extensively on interrupts which can cause unpredictable code execution (e.g. when multiple interrupts occur at the same time). Our position tracking circuit is therefore coupled to a separate,low-cost arduino mini which has the singular responsibility of position-tracking.

b. Moreover, should scenarios involving pupil and body camera recordings connect to the Analog input in the PCB or the real-time computer for optimal data handling and processing?

Pupil and body camera recordings would be independent data streams which can be recorded separately from behaviorMate. Aligning these forms of full motion video could require frame triggers which could be configured on the GPIO board using single TTL like outputs or by configuring a valve to be “pulsed” which is a provided type customization.

We also note that a more advanced developer could easily leverage camera signals to provide closed loop control by writing an independent module that sends UDP packets to behavoirMate. For example a separate computer vision based position tracking module could be written in any preferred language and use UDP messaging to send body tracking updates to the UI without editing any of the behaviorMate source code (and even used for updating 1D location).

(5) Given that all references, as far as I can see, come from the same lab, are there other labs capable of implementing this system at a similar optimal level?

To date two additional labs have published using behaviorMate, the Soltez and Henn labs (see revised lines 341-342). Since behaviorMate has only recently been published and made available open source, only external collaborators of the Losonczy lab have had access to the software and design files needed to do this. These collaborators did, however, set up their own behavioral setups in separate locations with minimal direct support from the authors–similar to what would be available to anyone seeking to set a behaviorMate system would find online on our github page or by posting to the message board.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(4) To provide additional context for the significance of this work, additional citations would be helpful to demonstrate a ubiquitous need for a system like behaviorMate. This was most needed in the paragraph from lines 46-65, specifically for each sentence after line 55, where the authors discuss existing variants on head-fixed behavioral paradigms. For instance, for the clause "but olfactory and auditory stimuli have also been utilized at regular virtual distance intervals to enrich the experience with more salient cues", suggested citations include Radvansky & Dombeck 2018 (DOI: 10.1038/s41467-018-03262-4), Fischler-Ruiz et al. 2021 (DOI: 10.1016/j.neuron.2021.09.055).

We thank the reviewer for the suggested missing citations and have updated the manuscript accordingly (see line 58).

(5) In addition, it would also be helpful to clarify behaviorMate's implementation in other laboratories. On line 304 the authors mention "other labs" but the following list of citations is almost exclusively from the Losonczy lab. Perhaps the citations just need to be split across the sentence for clarity? E.g. "has been validated by our experimental paradigms" (citation set 1) "and successfully implemented in other labs as well" (citation set 2).

We have split the citation set as suggested (see lines 338-342).

Minor Comments:

(6) In the paragraph starting line 153 and in Fig. 2, please clarify what is meant by "trial" vs. "experiment". In many navigational tasks, "trial" refers to an individual lap in the environment, but here "trial" seems to refer to the whole behavioral session (i.e. synonymous with "experiment"?).

In our software implementation we had originally used “trial” to refer to an imaging session rather than experiment (and have made updates to start moving to the more conventional lexicon). To avoid confusion we have remove this use of “trial” throughout the manuscript and replaced with “experiment” whenever possible

(7) This is very minor, but in Figure 3 and 4, I don't believe the gavage needle is actually shown in the image. This is likely to avoid clutter but might be confusing to some readers, so it may be helpful to have a small inset diagram showing how the needle would be mounted.

We assessed the image both with and without the gavage needle and found the version in the original (without) to be easier to read and less cluttered and therefore maintained that version in the manuscript.

(8) In Figure 5 legend, please list n for mice and cells.

We have updated the Figure 5 legend to indicate that for panels C-G, n=6 mice (all mice were recorded in both VR and TM systems), 3253 cells in VR classified as significantly tuned place cells VR, and 6101 tuned cells in TM,

(9) Line 414: It is not necessary to tilt the entire animal and running wheel as long as the head-bar clamp and objective can rotate to align the imaging window with the objective's plane of focus. Perhaps the authors can just clarify the availability of this option if users have a microscope with a rotatable objective/scan head.

We have added the suggested caveat to the manuscript in order to clarify when the goniometers might be useful (see lines 281-288).

(10) Figure S1 and S2 could be referenced explicitly in the main text with their related main figures.

We have added explicit references to figures S1 and S2 in the relevant sections (see lines 443, 460  and 570)

(11) On line 532-533, is there a citation for "proximal visual cues and tactile cues (which are speculated to be more salient than visual cues)"?

We have added citations to both Knierim & Rao 2003 and Renaudineau et al. 2007 which discuss the differential impact of proximal vs distal cues during navigation as well as Sofroniew et al. 2014 which describe how mice navigate more naturally in a tactile VR setup as opposed to purely visual ones.

(12) There is a typo at the end of the Figure 2 legend, where it should say "Arduino Mini."

This typo has been fixed.

Reviewer #2 (Recommendations For The Authors):

(4) As mentioned in the public review: what is the major advantage of taking the IoT approaches as opposed to USB connections to the host computer, especially when behaviorMate relies on a central master computer regardless? The authors mentioned the readability of the JSON messages, making the system easier to debug. However, the flip side of that is the efficiency of data transmission. Although the bandwidth/latency is usually more than enough for transmitting data and commands for behavior devices, the efficiency may become a problem when neural recording devices (imaging or electrophysiology) need to be included in the system.

behaviorMate is not intended to do everything, and is limited to mainly controlling behavior and providing some synchronizing TTL style triggers. In this way the system can easily and inexpensively be replicated across multiple recording setups; particularly this is useful for constructing additional animal training setups. The system is very much sufficient for capturing behavioral inputs at relevant timescales (see the benchmarks in Figures 3 and 4 as well as the position correlated neural activity in Figures 5 and 6 for demonstration of this). Additional hardware might be needed to align the behaviorMate output with neural data for example a high-speed DAQ or input channels on electrophysiology recording setups could be utilized (if provided). As all recording setups are different the ideal solution would depend on details which are hard to anticipate. We do not mean to convey that the full neural data would be transmitted to the behaviorMate system (especially using the JSON/UDP communications that behaviorMate relies on).

(5) The author mentioned labView. A popular open-source alternative is bonsai (https://github.com/bonsai-rx/bonsai). Both include a graphical-based programming interface that allows the users to easily reconfigure the hardware system, which behaviorMate seems to lack. Additionally, autopilot (https://github.com/auto-pi-lot/autopilot) is a very relevant project that utilizes a local network for multiple behavior devices but focuses more on P2P communication and rigorously defines the API/schema/communication protocols for devices to be compatible. I think it's important to include a discussion on how behaviorMate compares to previous works like these, especially what new features behaviorMate introduces.

We believe that behaviorMate provides a more opinionated and complete solution than the projects mentioned. A wide variety of 1D navigational paradigms can be constructed in behaviorMate without the need to write any novel software. For example, bonsai is a “visual programming language” and would require experimenters to construct a custom implementation of each of their experiments. We have opted to use Java for the UI with distributed computations across modules in various languages. Given the IOT methodology it would be possible to use any number of programming languages or APIs; a large number of design decisions were made  when building the project and we have opted to not include this level of detail in the manuscript in order to maintain readability. We strongly believe in using non-proprietary and open source projects, when possible, which is why the comparison with LabView based solutions was included in the introduction. Also, we have added a reference to the autopilot reference to the section of the introduction where this is discussed.

(6) One of the reasons labView/bonsai are popular is they are inherently parallel and can simultaneously respond to events from different hardware sources. While the JSON events in behaviorMate are asynchronous in nature, the handling of those events seems to happen only in a main event loop coupled with GUI, which is sequential by nature. Is there any multi-threading/multi-processing capability of behaviorMate? If so it's an important feature to highlight. If not I think it's important to discuss the potential limitation of the current implementation.

IOT solutions are inherently concurrent since the computation is distributed. Additional parallelism could be added by further distributing concerns between additional independent modules running on independent hardware. The UI has an eventloop which aggregates inputs and then updates contexts based on the current state of those inputs sequentially. This sort of a “snapshot” of the current state is necessary to reason about when the start certain contexts based on their settings and applied decorators. While the behaviorMate UI uses multithreading libraries in Java to be more performant in certain cases, the degree to which this represents true vs “virtual” concurrency would depend on the individual PC architecture it is run on and how the operating system allocates resources. For this reason, we have argued in the manuscript that behaviorMate is sufficient for controlling experiments at behaviorally relevant timescales, and have presented both benchmarks and discussed different synchronization approaches and permit users to determine if this is sufficient for their needs.

(7) The context list is an interesting and innovative approach to abstract behavior contingencies into a data structure, but it's not currently discussed in depth. I think it's worth highlighting how the context list can be used to cover a wide range of common behavior experimental contingencies with detailed examples (line 185 might be a good example to give). It's also important to discuss the limitation, as currently the context lists seem to only support contingencies based purely on space and time, without support for more complicated behavior metrics (e.g. deliver reward only after X% correct).

To access more complex behavior metrics during runtime, custom context list decorators would need to be implemented. While this is less common in the sort of 1D navigational behaviors the project was originally designed to control, adding novel decorators is a simple process that only requires basic object oriented programming knowledge. As discussed we are also implementing a plugin-architecture in the JavaFX update to streamline these types of additions.

Minor Comments:

(8) In line 202, the author suggests that a single TTL pulse is sent to mark the start of a recording session, and this is used to synchronize behavior data with imaging data later. In other words, there are no synchronization signals for every single sample/frame. This approach either assumes the behavior recording and imaging are running on the same clock or assumes evenly distributed recording samples over the whole recording period. Is this the case? If so, please include a discussion on limitations and alternative approaches supported by behaviorMate. If not, please clarify how exactly synchronization is done with one TTL pulse.

While the TTL pulse triggers the start of neural data in our setups, various options exist for controlling for the described clock drift across experiments and the appropriate one depends on the type of recordings made, frame rate duration of recording etc. Therefore behaviorMate leaves open many options for synchronization at different time scales (e.g. the adding a frame-sync circuit as shown in Figure 1 or sending TTL pulses to the same DAQ recording electrophysiology data).  Expanded consideration of different synchronization methods has been included in the manuscript (see lines 224-238).

(9) Is the computer vision-based calibration included as part of the GUI functionality? Please clarify. If it is part of the GUI, it's worth highlighting as a very useful feature.

The computer vision-based benchmarking is not included in the GUI. It is in the form of a script made specifically for this paper. However for treadmill-based experiments behaviorMate has other calibration tools built into it (see line 301-303).

(10) I went through the source code of the Arduino firmware, and it seems most "open X for Y duration" functions are implemented using the delay function. If this is indeed the case, it's generally a bad idea since delay completely pauses the execution and any events happening during the delay period may be missed. As an alternative, please consider approaches comparing timestamps or using interrupts.

We have avoided the use of interrupts on the GPIO due to the potential for unpredictable code execution. There is a delay which is only just executed if the duration is 10 ms or less as we cannot guarantee precision of the arduino eventloop cycling faster than this. Durations longer than 10 ms would be time stamped and non-blocking. We have adjusted this MAX_WAIT to be specified as a macro so it can be more easily adjusted (or set to 0).

(11) Figure 3 B, C, D, and Figure 4 D, E suffer from noticeable low resolution.

We have converted Figure 3B, C, D and 4C, D, E to vector graphics in order to improve the resolution.

(12) Figure 4C is missing, which is an important figure.

This figure appeared when we rendered and submitted the manuscript. We apologize if the figure was generated such that it did not load properly in all pdf viewers. The panel appears correctly in the online eLife version of the manuscript. Additionally, we have checked the revision in Preview on Mac OS as well as Adobe Acrobat and the built-in viewer in Chrome and all figure panels appear in each so we hope this issue has been resolved.

(13) There are thin white grid lines on all heatmaps. I don't think they are necessary.

The grid lines have been removed from the heatmaps  as suggested.

(14) Line 562 "sometimes devices directly communicate with each other for performance reasons", I didn't find any elaboration on the P2P communication in the main text. This is potentially worth highlighting as it's one of the advantages of taking the IoT approaches.

In our implementation it was not necessary to rely on P2P communication beyond what is indicated in Figure 1. The direct communication referred to in line 562 is meant only to refer to the examples expanded on in the rest of the paragraph i.e. the behavior controller may signal the microscope directly using a TTL signal without looping back to the UI. As necessary users could implement UDP message passing between devices, but this is outside the scope of what we present in the manuscript.

(15) Line 147 "Notably, due to the systems modular architecture, different UIs could be implemented in any programming language and swapped in without impacting the rest of the system.", this claim feels unsupported without a detailed discussion of how new code can be incorporated in the GUI (plugin system).

This comment refers to the idea of implementing “different UIs”. This would entail users desiring to take advantage of the JSON messaging API and the proposed electronics while fully implementing their own interface. In order to facilitate this option we have improved documentation of the messaging API posted in the README file accompanying the arduino source code. We have added reference to the supplemental materials where readers can find a link to the JSON API implementation to clarify this point.

Additionally, while a plugin system is available in the JavaFX version of behaviorMate, this project is currently under development and will update the online documentation as this project matures, but is unrelated to the intended claim about completely swapping out the UI.

Reviewer #3 (Recommendations For The Authors):

(6) Figure 1 - the terminology for each item is slightly different in the text and the figure. I think making the exact match can make it easier for the reader.

- Real-time computer (figure) vs real-time controller (ln88).

The manuscript was adjusted to match figure terminology.

- The position controller (ln565) - position tracking (Figure).

We have updated Figure 1 to highlight that the position controller does the position tracking.

- Maybe add a Behavior Controller next to the GPIO box in Figure 1.

We updated Figure 1 to highlight that the Behavior Controller performs the GPIO responsibility such that "Behavior Controller" and "GPIO circuit" may be used interchangeably.

- Position tracking (fig) and position controller (subtitle - ln209).

We updated Figure 1 to highlight that the position controller does the position tracking.

- Sync Pulse is not explained in the text.

The caption for Figure 1 has been updated to better explain the Sync pulse and additional systems boxes

(7) For Figure 3B/C: What is the number of data points? It would be nice to see the real population, possibly using a swarm plot instead of box plots. How likely are these outliers to occur?

In order to better characterize the distributions presented in our benchmarking data we have added mean and standard deviation information the plots 3 and 4. For Figure 3B: 0.0025 +/- 0.1128, Figure 3C: 12.9749 +/- 7.6581, Figure 4C: 66.0500 +/- 15.6994, Figure 4E: 4.1258 +/- 3.2558.

what is the parameters of the attention layer and the transformer? Seems like no mentioning

Parents after a certain age become a burden to their children which gives added pressure to already struggling young couples and families.

Believe that everything is false, nothing you have known is like what it is. You have no senses, body, shape, movement, and location. Those are just simple things in your head. What is certain for sure? Perhaps nothing, that may be the certain truth.

Unhouseled means not having Eucharist (a Christian service) administered (OED). He also said he died when his sins were the greatest, meaning that Claudius' assassination is partly to blame for Hamlet being in hell.

The ghost informs Hamlet that he cannot share what the afterlife is like because of a curse that will make Hamlet's head basically explode. He also says that in the daylight he is constantly burning in flames until the sins that he committed when he was alive have burned out of him and during the night time he is able to roam freely

Reviewer #1 (Public Review):

Summary:

This work uses transgenic reporter lines to isolate entpd5a+ cells representing classical osteoblasts in the head and non-classical (osterix-) notochordal sheath cells. The authors also include entpd5a- cells, col2a1a+ cells to represent the closely associated cartilage cells. In a combination of ATAC and RNA-Seq analysis, the genome-wide transcriptomic and chromatin status of each cell population is characterized, validating their methodology and providing fundamental insights into the nature of each cell type, especially the less well-studied notochordal sheath cells. Using these data, the authors then turn to a thorough and convincing analysis of the regulatory regions that control the expression of the entpd5a gene in each cell population. Determination of transcriptional activities in developing zebrafish, again combined with ATAC data and expression data of putative regulators, results in a compelling and detailed picture of the regulatory mechanisms governing the expression of this crucial gene.

Strengths:

The major strength of this paper is the clever combination of RNA-Seq and ATAC analysis, further combined with functional transcriptional analysis of the regulatory elements of one crucial gene. This results in a very compelling story.

Weaknesses:

No major weaknesses were identified, except for all the follow-up experiments that one can think of, but that would be outside of the scope of this paper.

Reviewer #2 (Public Review):

Summary:

Complementary to mammalian models, zebrafish has emerged as a powerful system to study vertebrate development and to serve as a go-to model for many human disorders. All vertebrates share the ancestral capacity to form a skeleton. Teleost fish models have been a key model to understand the foundations of skeletal development and plasticity, pairing with more classical work in amniotes such as the chicken and mouse. However, the genetic foundation of the diversity of skeletal programs in teleosts has been hampered by mapping similarities from amniotes back and not objectively establishing more ancestral states. This is most obvious in systematic, objective analysis of transcriptional regulation and tissue specification in differentiated skeletal tissues. Thus, the molecular events regulating bone-producing cells in teleosts have remained largely elusive. In this study, Petratou et al. leverage spatial experimental delineation of specific skeletal tissues -- that they term 'classical' vs 'non-classical' osteoblasts -- with associated cartilage of the endo/peri-chondrial skeleton and inter-segmental regions of the forming spine during development of the zebrafish, to delineate molecular specification of these cells by current chromatin and transcriptome analysis. The authors further show functional evidence of the utility of these datasets to identify functional enhancer regions delineating entp5 expression in 'classical' or 'non-classical' osteoblast populations. By integration with paired RNA-seq, they delineate broad patterns of transcriptional regulation of these populations as well as specific details of regional regulation via predictive binding sites within ATACseq profiles. Overall the paper was very well written and provides an essential contribution to the field that will provide a foundation to promote modeling of skeletal development and disease in an evolutionary and developmentally informed manner.

Strengths:

Taken together, this study provides a comprehensive resource of ATAC-seq and RNA-seq data that will be very useful for a wide variety of researchers studying skeletal development and bone pathologies. The authors show specificity in the different skeletal lineages and show the utility of the broad datasets for defining regulatory control of gene regulation in these different lineages, providing a foundation for hypothesis testing of not only agents of skeletal change in evolution but also function of genes and variations of unknown significance as it pertains to disease modeling in zebrafish. The paper is excellently written, integrating a complex history and experimental analysis into a useful and coherent whole. The terminology of 'classical' and 'non-classical' will be useful for the community in discussing the biology of skeletal lineages and their regulation.

Weaknesses:

Two items arose that were not critical weaknesses but areas for extending the description of methods and integration into the existing data on the role of non-classical osteoblasts and establishment/canalization of this lineage of skeletal cells.

(1) In reading the text it was unclear how specific the authors' experimental dissection of the head/trunk was in isolating different entp5a osteoblast populations. Obviously, this was successful given the specificity in DEG of results, however, analysis of contaminating cells/lineages in each population would be useful - e.g. using specific marker genes to assess. The text uses terms such as 'specific to' and 'enriched in' without seemingly grounded meaning of the accuracy of these comments. Is it really specific - e.g. not seen in one or other dataset - or is there some experimental variation in this?

(2) Further, it would be valuable to discuss NSC-specific genes such as calymmin (Peskin 2020) which has species and lineage-specific regulation of non-classical osteoblasts likely being a key mechanistic node for ratcheting centra-specific patterning of the spine in teleost fishes. What are dynamics observed in this gene in datasets between the different populations, especially when compared with paralogues - are there obvious cis-regulatory changes that correlate with the co-option of this gene in the early regulation of non-classical osteoblasts? The addition of this analysis/discussion would anchor discussions of the differential between different osteoblasts lineages in the paper.

Reviewer #1 (Public review):

Summary:

This work uses transgenic reporter lines to isolate entpd5a+ cells representing classical osteoblasts in the head and non-classical (osterix-) notochordal sheath cells. The authors also include entpd5a- cells, col2a1a+ cells to represent the closely associated cartilage cells. In a combination of ATAC and RNA-Seq analysis, the genome-wide transcriptomic and chromatin status of each cell population is characterized, validating their methodology and providing fundamental insights into the nature of each cell type, especially the less well-studied notochordal sheath cells. Using these data, the authors then turn to a thorough, and convincing analysis of the regulatory regions that control the expression of the entpd5a gene in each cell population. Determination of transcriptional activities in developing zebrafish, again combined with ATAC data and expression data of putative regulators results in a compelling, and detailed picture of the regulatory mechanisms governing expression of this crucial gene.

Strengths:

The major strength of this paper is the clever combination of RNA-Seq and ATAC analysis, further combined with functional transcriptional analysis of the regulatory elements of one crucial gene. This results in a very compelling story.

Weaknesses:

No major weakness, except for all the follow-up experiments that one can think of, but that would be outside of the scope of this paper.

Comments on revisions:

The description of Supplementary Figure 1 is still confusing: in the results section, it says "We photo converted and directly imaged entpd5a:Kaede positive embryos starting from the 15 somite- stage (s), when we could first detect the fluorophore along the newly-formed notochord progenitor cells (Suppl. Fig. 1E). We repeated photoconversion and imaging at 18, 21 and 24s (Suppl. Fig. 1F-H). ...(Suppl. Fig 1E)"<br /> In the response, the authors say "we could see new Kaede expression under the control of the entpd5a promoter region within 1.5 hours of photoconversion, as shown in Suppl. Figure 1E-H."<br /> In the legend to Suppl. Fig. 1, it says "Using the entpd5a:Kaede photoconversion line we first detect entpd5a expression at the 15 somite-stage (E). Following the same embryo, active expression of the gene continues until prior to 24 hpf (F-H)."<br /> So my questions are: -was there a delay between photoconversion and imaging - was the same delay used for all pictures - was there indeed additional photoconversion for Fig.1 F-H before imaging?<br /> This could be stated in Materials and Methods, and maybe in the legend to Suppl. Fig. 1

All other issues have been addressed.

Reviewer #2 (Public review):

Summary:

Complementary to mammalian models, zebrafish has emerged as a powerful system to study vertebrate development and serve as a go-to model for many human disorders. All vertebrates share the ancestral capacity to form a skeleton. Teleost fish models have been a key model to understand the foundations of skeletal development and plasticity, pairing with more classical work in amniotes such as the chicken and mouse. However, the genetic foundation of the diversity of skeletal programs in teleosts have been hampered by mapping similarities from amniotes back and not objectively establishing more ancestral states. This is most obvious in systematic, objective analysis of transcriptional regulation and tissue specification in differentiated skeletal tissues. Thus, the molecular events regulating bone-producing cells in teleosts have remained largely elusive. In this study, Petratou et al. leverage spatial experimental delineation of specific skeletal tissues -- that they term 'classical' vs 'non-classical' osteoblasts -- with associated cartilage of the endo/peri-chondrial skeleton and inter-segmental regions of the forming spine during development of the zebrafish, to delineate molecular specification of these cells by current chromatin and transcriptome analysis. The authors further show functional evidence of the utility of these datasets to identify functional enhancer regions delineating entp5 expression delineated in 'classical' or 'non-classical' osteoblast populations. By integration with paired RNA-seq, they delineate broad patterns of transcriptional regulation of these populations as well as specific detail of regional regulation via predictive binding sites within ATACseq profiles. Overall the paper was very well written and provides an essential contribution to the field that will provide a foundation to promote modeling of skeletal development and disease in an evolutionary and developmentally informed manner.

Strengths:

Taken together, this study provides a comprehensive resource of ATAC-seq and RNA-seq data that will be very useful for a wide variety of researchers studying skeletal development and bone pathologies. The authors show specificity in the different skeletal lineages and show utility of the broad datasets for defining regulatory control of gene regulation in these different lineages, providing the foundation for hypothesis testing of not only agents of skeletal change in evolution but also function of genes and variations of unknown significance as it pertains to disease modeling in zebrafish. The paper is excellently written, integrating a complex history and experimental analysis into a useful and coherent whole. The terminology of 'classical' and 'non-classical' will be useful for the community in discussing biology of skeletal lineages and their regulation.

Weaknesses:

Two items arose that proposed areas for extending the description to integrate the data into the existing data on role of non-classical osteobasts and establishment/canalization of this lineage of skeletal cells.

(1) It was unclear how specific the authors' experimental dissection of head/trunk was in isolating different entp5a osteoblast populations. Obviously, this was successful given the specificity in DEG of results, however an analysis of contaminating cells/lineages in each population would be useful - e.g. maybe use specific marker genes to assess. The text uses terms such as 'specific to' and 'enriched in' without seemingly grounded meaning of the accuracy of these comments. Is it really specific e.g. not seen in one or other dataset, or is there some experimental variation in this?

(2) Further, it would be valuable to discuss NSC-specific genes such as calymmin (Peskin 2020) which has species and lineage specific regulation of non-classical osteoblasts likely being a key mechanistic node for ratcheting centra-specific patterning of the spine in teleost fishes. What are dynamics observed in this gene in datasets between the different populations, especially when compared with paralogues - is there obvious cis-regulatory changes that correlate with the co-option of this gene in early regulation of non-classical osteoblasts? The addition of this analysis/discussion would anchor discussions of a differential between different osteoblasts lineages in the paper.

Comments on revisions: All issues have been addressed.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work uses transgenic reporter lines to isolate entpd5a+ cells representing classical osteoblasts in the head and non-classical (osterix-) notochordal sheath cells. The authors also include entpd5a- cells, col2a1a+ cells to represent the closely associated cartilage cells. In a combination of ATAC and RNA-Seq analysis, the genome-wide transcriptomic and chromatin status of each cell population is characterized, validating their methodology and providing fundamental insights into the nature of each cell type, especially the less well-studied notochordal sheath cells. Using these data, the authors then turn to a thorough and convincing analysis of the regulatory regions that control the expression of the entpd5a gene in each cell population. Determination of transcriptional activities in developing zebrafish, again combined with ATAC data and expression data of putative regulators, results in a compelling and detailed picture of the regulatory mechanisms governing the expression of this crucial gene.

Strengths:

The major strength of this paper is the clever combination of RNA-Seq and ATAC analysis, further combined with functional transcriptional analysis of the regulatory elements of one crucial gene. This results in a very compelling story.

Weaknesses:

No major weaknesses were identified, except for all the follow-up experiments that one can think of, but that would be outside of the scope of this paper.

Reviewer #2 (Public Review):

Summary:

Complementary to mammalian models, zebrafish has emerged as a powerful system to study vertebrate development and to serve as a go-to model for many human disorders. All vertebrates share the ancestral capacity to form a skeleton. Teleost fish models have been a key model to understand the foundations of skeletal development and plasticity, pairing with more classical work in amniotes such as the chicken and mouse. However, the genetic foundation of the diversity of skeletal programs in teleosts has been hampered by mapping similarities from amniotes back and not objectively establishing more ancestral states. This is most obvious in systematic, objective analysis of transcriptional regulation and tissue specification in differentiated skeletal tissues. Thus, the molecular events regulating bone-producing cells in teleosts have remained largely elusive. In this study, Petratou et al. leverage spatial experimental delineation of specific skeletal tissues -- that they term 'classical' vs 'non-classical' osteoblasts -- with associated cartilage of the endo/peri-chondrial skeleton and inter-segmental regions of the forming spine during development of the zebrafish, to delineate molecular specification of these cells by current chromatin and transcriptome analysis. The authors further show functional evidence of the utility of these datasets to identify functional enhancer regions delineating entp5 expression in 'classical' or 'non-classical' osteoblast populations. By integration with paired RNA-seq, they delineate broad patterns of transcriptional regulation of these populations as well as specific details of regional regulation via predictive binding sites within ATACseq profiles. Overall the paper was very well written and provides an essential contribution to the field that will provide a foundation to promote modeling of skeletal development and disease in an evolutionary and developmentally informed manner.

Strengths:

Taken together, this study provides a comprehensive resource of ATAC-seq and RNA-seq data that will be very useful for a wide variety of researchers studying skeletal development and bone pathologies. The authors show specificity in the different skeletal lineages and show the utility of the broad datasets for defining regulatory control of gene regulation in these different lineages, providing a foundation for hypothesis testing of not only agents of skeletal change in evolution but also function of genes and variations of unknown significance as it pertains to disease modeling in zebrafish. The paper is excellently written, integrating a complex history and experimental analysis into a useful and coherent whole. The terminology of 'classical' and 'non-classical' will be useful for the community in discussing the biology of skeletal lineages and their regulation.

Weaknesses:

Two items arose that were not critical weaknesses but areas for extending the description of methods and integration into the existing data on the role of non-classical osteoblasts and establishment/canalization of this lineage of skeletal cells.

(1) In reading the text it was unclear how specific the authors' experimental dissection of the head/trunk was in isolating different entp5a osteoblast populations. Obviously, this was successful given the specificity in DEG of results, however, analysis of contaminating cells/lineages in each population would be useful - e.g. using specific marker genes to assess. The text uses terms such as 'specific to' and 'enriched in' without seemingly grounded meaning of the accuracy of these comments. Is it really specific - e.g. not seen in one or other dataset - or is there some experimental variation in this?

We thank the reviewer for pointing this out. Given that the separation from head and trunk is done manually, there will be some experimental variability. We have used anatomical hallmarks (cleithrum and swim bladder), and therefore would expect the variability to be small. Regarding classical osteoblasts contaminating trunk tissue, head removal was consistently performed using the aforementioned anatomical hallmarks in a manner that ensures that the cleithrum does not remain in the trunk tissue.  In order to alleviate concerns regarding trunk cell populations contaminating cranial populations, and to further clarify our strategy, we add the following statement to the Materials and Methods section: “The procedure does not allow for a complete separation of notochordal non-classical osteoblasts from cranial classical osteoblasts, as the notochord extends into the cranium. However, the amount of sheath cells in that portion of the notochord is negligible, compared both to the number of classical (cranial) osteoblasts in head samples, and to notochord cells isolated in trunk samples.”

(2) Further, it would be valuable to discuss NSC-specific genes such as calymmin (Peskin 2020) which has species and lineage-specific regulation of non-classical osteoblasts likely being a key mechanistic node for ratcheting centra-specific patterning of the spine in teleost fishes. What are dynamics observed in this gene in datasets between the different populations, especially when compared with paralogues - are there obvious cis-regulatory changes that correlate with the co-option of this gene in the early regulation of non-classical osteoblasts? The addition of this analysis/discussion would anchor discussions of the differential between different osteoblasts lineages in the paper.

This is an interesting concept and idea, that we will consider in a possible revision or, if requiring substantial additional efforts, in a possible new research line. An excellent starting point for further studies using our datasets.

Reviewer #3 (Public Review):

Summary:

This study characterizes classical and nonclassical osteoblasts as both types were analyzed independently (integrated ATAC-seq and RNAseq). It was found that gene expression in classical and nonclassical osteoblasts is not regulated in the same way. In classical osteoblasts, Dlx family factors seem to play an important role, while Hox family factors are involved in the regulation of spinal ossification by nonclassical osteoblasts. In the second part of the study, the authors focus on the promoter structure of entpd5a. Through the identification of enhancers, they reveal complex modes of regulation of the gene. The authors suggest candidate transcription factors that likely act on the identified enhancer elements. All the results taken together provide comprehensive new insights into the process of bone development, and point to spatio-temporally regulated promoter/enhancer interactions taking place at the entpd5a locus.

Strengths:

The authors have succeeded in justifying a sound and consistent buildup of their experiments, and meaningfully integrating the results into the design of each of their follow-up experiments. The data are solid, insightfully presented, and the conclusion valid. This makes this manuscript of great value and interest to those studying (fundamental) skeletal biology.

Weaknesses:

The study is solidly constructed, the manuscript is clearly written and the discussion is meaningful - I see no real weaknesses.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Minor issues that may need to be addressed or detailed:

Supplementary Figures 1I-J, text page 4, line 24: "photoconversion and imaging": this needs some more detailed description: green fluorescent cells should be actively expressing Kaede, but only if there is a delay between photoconversion and imaging. What is the reason that Supplementary Figure 1F shows mainly green fluorescent cells, contrary to 1G-J?

In our experiments, we could see new Kaede expression under the control of the entpd5a promoter region within 1.5 hours of photoconversion, as shown in Suppl. Figure 1E-H, suggesting that this time window was sufficient for protein generation. The reason for Suppl. Fig 1F showing more green fluorescence we believe relates to the high rate of transcriptional activity at that stage, in the entirety of the notochord progenitor cells. In addition, this is an effect which we attribute to the relatively small number of cells producing red fluorescence at that stage, due to photoconversion of only a few Kaede+ cells at the 15 somites stage (Suppl. Fig. 1E). Therefore, the masking effect of the green fluorescence by the red is not as significant as in G and H, where the red fluorescence resulting from photoconversion right after imaging at 18s and 21s, respectively, significantly overlaps with new green fluorescence. This can be seen in the image as the presence of orange fluorescence in G and H, instead of the clear red shown in E, I and J.

In addition to this, we would like to point out that in Suppl. Fig. 1I, J the reason that green fluorescence is only detected in the ventral region of the notochord, is because the promoter of entpd5a only remains active in the ventral-most sheath cells at that stage. This is stated in the results section of the main text, first subsection, paragraph 3. The reason for this very interesting, strictly localised expression pattern remains unclear.

Somewhat intriguing: green fluorescence in Figure 1B, C (osx:GAL4FF) and Supplementary Figure 1C (entpd5a:GAL4FF) in the CNS? Would that be an artefact of the GAL4FF/UAS:GFP system?

We are confident that the fluorescence pointed out by the reviewer is not an artefact of the GAL4FF/UAS system, for a few reasons. Firstly, osx (Sp7) has been shown to be expressed and to function in the nervous system in mice (Park et al, BBRC, 2011; Elbaz et al, Neuron, 2023). Secondly, not only osx, but also entpd5a can be readily detected in a subset of cranial and spinal neurons in early development using the entpd5a:GAL4FF; UAS:GFP transgenic line (Suppl. Fig 1C). Finally, when establishing transgenic lines with the entpd5a(1.1):GFP construct, expression was almost invariably present in diverse elements of the nervous system, but not in bone (data not shown). This led us to hypothesise that the minimal promoter of entpd5a (and possibly also that of osx) is activated by transcription factors active in the nervous system, and this effect is likely controlled by the surrounding enhancers, but also the genome location. It is unclear at present what the endogenous neural expression of the two genes is like, and we did not further investigate this in this study, as the focus was on the skeleton.

Figure 2: What exactly is "Corrected Total Cell Fluorescence"? Is it green + red fluorescence?

We thank the reviewer for pointing out the absence of more information on this. Corrected total cell fluorescence does not correspond to green+ red fluorescence, rather it is calculated as follows for a single channel:

CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings)

More details can be found in the following website: https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html

We have edited the Materials and Methods section under “Imaging and image analysis” to include the aforementioned information.

Page 11, line 34: The authors may have missed the recently published "Raman et al., Biomolecules 2024 Vol. 14; doi:10.3390/biom14020139" describing RNA-Seq in 4 dpf osterix+ osteoblasts.

We thank the reviewer for drawing our attention to the Raman et al publication. The reference has now been added in the manuscript.

Figure 5A and B: use a higher resolution version to make the numbers and gene names more readable. Figures 5C and 6A could also use a larger font for the text and numbers.

High resolution files are now included with the revised manuscript, which should significantly help in making figures more easily readable. Although we agree with the reviewer that larger fonts would improve readability, due to the nature of the graphs (very small spaces in some cases, where the numbers would have to fit) this would not be easy to achieve. However, we believe that this issue will be resolved with the availability of higher resolution files. If readability remains a concern, we would be happy to attempt re-organising the graphs to allow for larger fonts.

Reviewer #2 (Recommendations For The Authors):

I suggest no further experiments, but do suggest that a few points be clarified.

In the Discussion, the text "the less evolved osteoblasts of fish and amphibians..." is not accurate. These cells are not less evolved as they represent an independent lineage to tetrapods that have evolved with different stresses for a similar time. However, as teleost fishes and amphibians share characteristics and all share a common ancestor, these signatures represent a putative ancestral state of skeletal differentiation not seen in amniotes, including humans.

We thank the reviewer for pointing out the unfortunate phrasing. The text has now been modified as follows: “Specifically, the osteoblasts of teleost fish and amphibians, whose characteristics are putatively closer to a more ancestral state of skeletal differentiation compared to amniotes, appear to share gene expression with chondrocytes”.

The title could potentially be shortened to reach a broader audience by removing the initial clause of 'integration of ATAC and RNA seq' as this is a commonly performed analysis - "Chromatin and transcriptomic signature in classical and non-classical zebrafish osteoblasts indicate mechanisms of ancestral skeletal differentiation" is more descriptive of the findings and not focused on the method.

We have discussed this internally, but would prefer to retain the current title. The reason is (1) because we would like to see our methodology and datasets be used as platform for further studies, and the current title, in our opinion, facilitates this. In regards to replacing “mechanisms of entpd5a regulation” with “mechanisms of ancestral skeletal differentiation”, we think this does not give an accurate description of our work, which is primarily focused on elucidating entpd5a promoter dynamics.

All datasets should be made available as soon as possible for use in the field.

The datasets (raw and processed) are available on the GEO database. The corresponding accession numbers can be found in our data availability statement.

Minor comments:

(1) Figure 1A. The labels are missing for grey and light blue structures.

These structures are together making up the “notochord sheath”, which is comprised of the basal lamina (grey), the medial layer of fibrillar collagen (light blue) and the outer layer of loosely arranged matrix (lighter blue). We modified the figure legend to indicate that the three layers all correspond to the notochord sheath.

(2) Figure 2A. The constructs in the lower part of the panel are not discussed in the legend and seem out of place in terms of data type and analysis.

We would argue that indicating which non-coding regions and which ATAC peaks were responsible for driving GFP expression in each construct aids in a better understanding of our results. We thank the reviewer for pointing out the lack of mention of these constructs in the figure legend. This issue has now been resolved.

(3) Be wary of red/green color combinations, especially in the figures where these are juxtaposed with each other.

We apologise for the use of red/green colour. Although it is not possible for this manuscript to change the colour patterns, we will make sure to avoid the use of these colours in conjunction in the future.

(4) The use of fish as a term should be classified as teleost fish, as authors are not addressing non-teleost basal ray-finned fishes or the fact that tetrapods are within bony fishes overall.

This is well spotted, we have now remedied this by editing the manuscript. Where the term “fish” was used, we now state “teleost fish”.

(5) Age information is missing in several Figures (e.g. 1D and 2C).

In some of the figures space constrains did not allow for including the stage on the figure itself. However, we have made sure that in those cases the stage is incorporated in the figure legend.

(6) The resolution of several Figures (e.g. Figure 5 and Supplementary Figure 3) is low.

We address this issue by providing high resolution figures with the revised manuscript.

(7) In the sentence (top page before Discussion) "The same conclusion was reached upon isolation from these three..", it was unclear what 'upon isolation' referred to.

We agree with the reviewer that this phrasing is unclear. To enhance clarity, the manuscript now reads as follows: “The same conclusion was reached upon isolation of the DEGs highlighted by our RNA-seq results, from the three aforementioned groups of genes associated with ATAC peaks for each cell population.”

Reviewer #3 (Public review):

Xiaoyu Wu and colleagues examined the potential role in sleep of a Drosophila ribosomal RNA methyltransferase, mettl5. Based on sleep defects reported in CRISPR generated mutants, the authors performed both RNA-seq and Ribo-seq analyses of head tissue from mutants and compared to control animals collected at the same time point. While these data were subjected to a thorough analysis, it was difficult to understand the relative direction of differential expression between the two genotypes. In any case, a major conclusion was that the mutant showed altered expression of circadian clock genes, and that the altered expression of the period gene in particular accounted for the sleep defect reported in the mettl5 mutant. As noted above, a strength of this work is its relevance to a human developmental disorder as well as the transcriptomic and ribosomal profiling of the mutant. However, there are numerous weaknesses in the manuscript, most of which stem from misinterpretation of the findings, some methodological approaches, and also a lack of method detail provided. The authors seemed to have missed a major phenotype associated with the mettl5 mutant, which is that it caused a significant increase in period length, which was apparent even in a light: dark cycle. Thus the effect of the mutant on clock gene expression more likely contributed to this phenotype than any associated with changes in sleep behavior.

Reviewer #1 (Public review):

Summary:

In this manuscript by Bimbard et al., a new method to perform stable recordings over long periods of time with neuropixels as well as the technical details on how the electrodes can be explanted for a follow up reuse is provided. I think the description of all parts of the method are very clear, and the validation analyses (n of units per day over time, RMS over recording days...) are very convincing. I however missed a stronger emphasis on why this could provide a big impact on the ephys community, by enabling new analyses, new behavior correlation studies or neurophysiological mechanisms across temporal scales that were previously inaccessible with high temporal resolution (i.e. not with imaging).

Strengths:

Open source method. Validation across laboratories. Across species (mice and rats) demonstration of its use and in different behavioral conditions (head-fixed and freely moving). The implant offers a major advance compared to previous methods and that will help the community generate richer datasets.

Weaknesses:

None noted.

Reviewer #2 (Public review):

Summary:

This work by Bimbard et al., introduces a new implant for Neuropixels probes. While Neuropixels probes have critically improved and extended our ability to record the activity of a large number of neurons with high temporal resolution, the use of these expensive devices in chronic experiments has so far been hampered by the difficulty of safely implanting them and, importantly, to explant and reuse them after conclusion of the experiment. The authors present a newly designed two-part implant, consisting of a docking and a payload module, that allows for secure implantation and straightforward recovery of the probes. The implant is lightweight, making it amenable for use in mice and rats, and customizable. The authors provide schematics and files for printing of the implants, which can be easily modified and adapted to custom experiments by researchers with little to no design experience. Importantly, the authors demonstrate the successful use of this implant across multiple use cases, in head-fixed and freely moving experiments, in mice and rats, with different versions of Neuropixels probes and across 8 different labs. Taken together, the presented implants promise to make chronic Neuropixels recordings and long-term studies of neuronal activity significantly easier and attainable for both current and future Neuropixels users.

Strengths:<br /> - The implants have been successfully tested across 8 different laboratories, in mice and rats, in head-fixed and freely moving conditions and have been adapted in multiple ways for a number of distinct experiments.<br /> - Implants are easily customizable and authors provide a straightforward approach for customization across multiple design dimensions even for researchers not experienced in design.<br /> - The authors provide clear and straightforward descriptions of the construction, implantation and explant of the described implants.<br /> - The split of the implant into a docking and payload module makes reuse even in different experiments (using different docking modules) easy.<br /> - The authors demonstrate that implants can be re-used multiple times and still allow for high-quality recordings.<br /> - The authors show that the chronic implantations allow for the tracking of individual neurons across days and weeks (using additional software tracking solutions), which is critical for a large number of experiments requiring the description of neuronal activity, e.g. throughout learning processes.<br /> - The authors show that implanted animals can even perform complex behavioral tasks, with no apparent reduction in their performance.

Author response:

The following is the authors’ response to the original reviews.

Reviewer 1 (Public Review):

Summary:

In this manuscript by Bimbard et al., a new method to perform stable recordings over long periods of time with neuropixels, as well as the technical details on how the electrodes can be explanted for follow-up reuse, is provided. I think the description of all parts of the method is very clear, and the validation analyses (n of units per day over time, RMS over recording days...) are very convincing. I however missed a stronger emphasis on why this could provide a big impact on the ephys community, by enabling new analyses, new behavior correlation studies, or neurophysiological mechanisms across temporal scales.

Strengths:

Open source method. Validation across laboratories. Across species (mice and rats) demonstration of its use and in different behavioral conditions (head-fixed and freely moving).

Weaknesses:

Weak emphasis on what can be enabled with this new method that didn't exist before.

We thank the reviewer for highlighting the limited discussion around scientific impact. Our implant has several advantages which combine to make it much more accessible than previous solutions. This enables a variety of recording configurations that would not have been possible with previous designs, facilitating recordings from a wider range of brain regions, animals, and experimental setups. In short, there are three key advances which we now emphasise in the manuscript:

Adaptability: The CAD files can be readily adapted to a wide range of configurations (implantation depth, angle, position of headstage, etc.). Labs have already modified the design for their needs, and re-shared with the community (Discussion, Para 5).

Weight: Because of the lightweight design, experimenters can i) perform complex and demanding freely moving tasks as we exemplify in the manuscript, and ii) implant female and water restricted mice while respecting animal welfare weight limitations (Flexible design, Para 1).

Cost: At ~$10, our implant is significantly cheaper than published alternatives, which makes it affordable to more labs and means that testing modifications is cost-effective (Discussion, Para 4).

Reviewer 1 (Recommendations For The Authors):

- Differences between mice and rats seem very significant. Although this is probably not surprising, I suggest that the authors comment on this to make it clear to anyone trying to use in different species that are not quantified in the main figures.

The reviewer is correct—there are qualitative differences between mice and rats, particularly with respect to the unit median amplitude. We have added a comment in the discussion to highlight these inter-species variations (Discussion, Para 7)

- Another comment that would be useful to have would be how to tackle the problem of tracking the same neuron across days. Even if currently impossible, it could be useful to provide discussion along those lines as to where future improvements (either in hardware or software) can be made.

We thank the reviewer for highlighting this. Figure. 5 does show data from tracking the same neuron across days (and even months). We have modified the language to make this clear.

Reviewer 2 (Public Review):  

Summary:

This work by Bimbard et al., introduces a new implant for Neuropixels probes. While Neuropixels probes have critically improved and extended our ability to record the activity of a large number of neurons with high temporal resolution, the use of these expensive devices in chronic experiments has so far been hampered by the difficulty of safely implanting them and, importantly, to explant and reuse them after conclusion of the experiment. The authors present a newly designed two-part implant, consisting of a docking and a payload module, that allows for secure implantation and straightforward recovery of the probes. The implant is lightweight, making it amenable for use in mice and rats, and customizable. The authors provide schematics and files for printing of the implants, which can be easily modified and adapted to custom experiments by researchers with little to no design experience. Importantly, the authors demonstrate the successful use of this implant across multiple use cases, in head-fixed and freely moving experiments, in mice and rats, with different versions of Neuropixels probes, and across 8 different labs. Taken together, the presented implants promise to make chronic Neuropixel recordings and long-term studies of neuronal activity significantly easier and attainable for both current and future Neuropixels users.

Strengths:

The implants have been successfully tested across 8 different laboratories, in mice and rats, in headfixed and freely moving conditions, and have been adapted in multiple ways for a number of distinct experiments.

Implants are easily customizable and the authors provide a straightforward approach for customization across multiple design dimensions even for researchers not experienced in design.

The authors provide clear and straightforward descriptions of the construction, implantation, and explant of the described implants.

The split of the implant into a docking and payload module makes reuse even in different experiments (using different docking modules) easy.

The authors demonstrate that implants can be re-used multiple times and still allow for high-quality recordings.

The authors show that the chronic implantations allow for the tracking of individual neurons across days and weeks (using additional software tracking solutions), which is critical for a large number of experiments requiring the description of neuronal activity, e.g. throughout learning processes.

The authors show that implanted animals can even perform complex behavioral tasks, with no apparent reduction in their performance.

Weaknesses:

While implanted animals can still perform complex behavioral tasks, the authors describe that the implants may reduce the animals' mobility, as measured by prolonged reaction times. However, the presented data does not allow us to judge whether this effect is specifically due to the presented implant or whether any implant or just tethering of the animals per se would have the same effects.

The reviewer is correct: some of the differences in mouse reaction time could be due to the tether rather than the implant. As these experiments were also performed in water-restricted female mice with the heavier Neuropixels 1.0 implant, our data represent the maximal impact of the implant, and we have highlighted this point in the revision (Freely behaving animals, Para 2).  

While the authors make certain comparisons to other, previously published approaches for chronic implantation and re-use of Neuropixels probes, it is hard to make conclusive comparisons and judge the advantages of the current implant. For example, while the authors emphasize that the lower weight of their implant allows them to perform recordings in mice (and is surely advantageous), the previously described, heavier implants they mention (Steinmetz et al., 2021; van Daal et al., 2021), have also been used in mice. Whether the weight difference makes a difference in practice therefore remains somewhat unclear.

The reviewer is correct: without a direct comparison, we cannot be certain that our smaller, lighter implant improves behavioural results (although this is supported by the literature, e.g. Newman et al, 2023). However, the reduced weight of our implant is critical for several laboratories represented in this manuscript due to animal welfare requirements. Indeed, in van Daal et al the authors “recommend a [mouse] weight of >25 g for implanting Neuropixels 1.0 probes.” This limit precludes using (the vast majority of) female mice, or water-restricted animals. Conversely, our implant can be routinely used with lighter, water-restricted male and female mice. We emphasised this point in the revision (Discussion, Para 2).

The non-permanent integration of the headstages into the implant, while allowing for the use of the same headstage for multiple animals in parallel, requires repeated connections and does not provide strong protection for the implant. This may especially be an issue for the use in rats, requiring additional protective components as in the presented rat experiments.

We apologise for not clarifying the various headstage holder options in the manuscript and we have now addressed this in the revision (Freely behaving animals, Para 1&2). Our repository has headstage holder designs (in the XtraModifications/Mouse_FreelyMoving folder). This allows leaving the headstage on the implant, and thus minimize the number of connections (albeit increasing the weight for the mouse). Indeed, mice recorded while performing the task described in our manuscript had the head-stage semi-permanently integrated to the implant, and we now highlight this in the revision (Freely behaving animals, Para 1).

Reviewer 2 (Recommendations For The Authors): 

The description of the different versions of the head-stage holders should be more clear, listing also advantages/disadvantages of the different solutions. It would be also useful if the authors could comment on the use of these head-stage holders in rats, since they do not seem to offer much protection.

We thank the reviewer for this point, and we have added notes to the manuscript to clarify the various advantages of the different headstage-holders, and that the headstage can be permanently attached to the implant (Freely behaving animals, Para 1&2). This is the primary advantage of these solutions compared with the minimal implant—at the expense of increasing the implant weight.  

The reviewer’s concerns regarding the lack of protection for implants in rats is well-placed, and we now emphasise that these experiments benefited from the additional protection of an external 3D casing, which is likely critical for use in larger animals (Freely behaving animals, Para 1).

While re-used probes seem to show similar yields across multiple uses (Figure 4C), it seems as if there is a much higher variability of the yield for probes that are used for the first (maybe also second) time. There are probes with much higher than average yields, but it seems none of the re-used probes show such high yields. Is this a real effect? Is this because the high-yield probes happened to have not been used multiple times? Is there an analysis the authors could provide to reduce the concern that yields may generally be lower for re-used probes/that there are no very high yields for re-used probes?

We understand the reviewer’s concern with respect to Figure 4C, however, the re-use of any given probe was determined only by the experimental needs of the project. It is therefore not possible that there is a relationship between probes selected for re-use and unit-yield. We now specify this in the revised legend of Figure 4C. This variability (and the consistency in yield across uses) likely stems from differences between labs, brain region, and implantation protocol.

The authors claim that a 'large fraction' of units could be tracked for the entire duration of the experiment (Figure 5A,B). They mention in the discussion that quantification can be found in a different manuscript (van Beest et al., 2023), but this should also be quantified here in at least some more detail, also for other animals in addition to the one mouse which was recorded for ~100 days. What fraction can be held for different durations? What is the average holding time, etc.?

We agree with the reviewer, and have now added new panels quantifying the probability and reliability of tracking a neuron across days (Figure 5E-F). We also comment on the change in tracking probability across time, and its variability across recordings (Stability, Para 4).

Reviewer 3 (Public Reviews):

Summary:

In this manuscript, Bimbard and colleagues describe a new implant apparatus called "Apollo Implant", which should facilitate recording in freely moving rodents (mice and rats) using Neuropixels probes. The authors collected data from both mice and rats, they used 3 different versions of Neuropixels, multiple labs have already adopted this method, which is impressive. They openly share their CAD designs and surgery protocol to further facilitate the adaptation of their method.

Strengths:

Overall, the "Apollo Implant" is easy to use and adapt, as it has been used in other laboratories successfully and custom modifications are already available. The device is reproducible using common 3D printing services and can be easily modified thanks to its CAD design (the video explaining this is extremely helpful). The weight and price are amazing compared to other systems for rigid silicon probes allowing a wide range of use of the "Apollo Implant".

Weaknesses:

The "Apollo Implant" can only handle Neuropixels probes. It cannot hold other widely used and commercially available silicon probes. Certain angles and distances are not possible in their current form (distance between probes 1.8 to 4mm, implantation depth 2-6.5 mm, or angle of insertion up to 20 degrees).

As we now discuss in the manuscript (Discussion, Para 4), one implant accommodating the diversity of the existing probes is beyond the scope of this project. However, because the design is adaptable, groups should be able to modify the current version of the implant to adapt to their electrodes’ size and format (and can highlight any issues in the Github “Discussions” area).

With Neuropixels, the current range of depths covers practically all trajectories in the mouse brain. In rats, where deeper penetrations may be useful, the experimenter can attach the probe at a lower point in the payload module to expose more of the shank. We now specify this in the Github repository.  

We have now extended the range of inter-probe distances from a maximum of 4 mm to 6.5 mm. Distances beyond this may be better served by 2 implants, and smaller distances could be achieved by attaching two probes on the same side of the docking module. These points are now specified in the revised manuscript (Flexible design, Para 2).

Reviewer 3 (Recommendations For The Authors):

I have only a few questions and suggestions:

Is it possible to create step-by-step instructions for explantation (similar to Figure-1 with CAD schematics)? You mention that payload holder is attached to a micromanipulator, but it is unclear how this is achieved. How was the payload secured with a screw (which screw)? My understanding is that as you turn the screw in the payload holder, it will grab onto the payload module from both sides, but the screw is not in contact with the payload module, correct? I found the screw type on your GitHub, but it would be great if you could add a bill of materials in a table format, so readers don't have to jump between GitHub and article.

We have now added a bill of materials to the revised manuscript (Implant design and materials, Para 2), although up-to-date links are still provided on the Github repository due to changing availability.

What happens if you do a dual probe implant and cannot avoid blood vessels in one or both of the craniotomies due to the pre-defined geometry? Is this a frequent issue? How can you overcome this during the surgery?

Blood vessels can be difficult to avoid in some cases, but we are typically able to rotate/reposition the probes to solve this issue. In some cases, with 4-shank probes, the blood vessel can be positioned between individual probe shanks. We now detail this in the revised manuscript (Assembly and implantation, Para 3).

I assume if the head is not aligned (line-332) the probe can break during recovery. Have you experienced this during explanation?

As we now specify in the manuscript (Explantation, Para 2), we are careful when explanting the probe to avoid this issue, and due to the flexibility of the shanks, it does not appear to be a major concern.

Why did you remove the UV glue (line 435)? How can you level the skull? I assume you have covered bregma and lambda in the first surgery which can create an uneven surface to measure even after you remove the UV glue.

We thank the reviewer for highlighting this omission from the methods. We now explain (Implantation, Carandini-Harris laboratory) that the UV-glue is completely removed during the second surgery, and the skull is cleaned and scored. This improves the adhesion of the dental cement, and allows for reliable levelling of the skull.

In line 112 you mentioned that the number of recorded neurons was stable; however, you found a 3% mean decrease in unit count per day (line 120). Stability is great until day 10 (in Figure 4A), but it deteriorates quickly after that. I think it would help readers if you could add the mean{plus minus}SEM of recorded units in the text for days 1-10, days 11-50, and days 51-100 (using the data from Figure 4A).

We have now added Supplementary Figure 4 to show unit count across bins of days, and a corresponding comment in the text (Stability, Para 2).

A full survey of the probe (Figure 4B) means that you recorded neuronal activity across 4-5000 channels (depending on how many channels were in the brain). While it is clear that a full probe survey can reduce the number of animals needed for a study, it is also clear in this figure that by day 25 you can record ~300 neurons on 4000 channels. It would be great to discuss this in the discussion and give a balanced view of the long-term stability of these recordings.

Overall, keeping a large number of units for a long time still remains a challenge. Here, we could record on average 85 neurons per bank during the first 10 days, and then only 45 after 50 days. It is important to note that our quantification averages across all banks recorded, including those in a ventricle or partly outside of the brain. Thus, our results represent a lower estimate of the total neurons recorded. Our new Supplementary Figure 4 helps to highlight the diversity of neuron number recorded per animal. Further improvements in surgical techniques and spike sorting will likely improve stability further and we have now added this comment in the manuscript (Stability, Para 2). For example, we observed excellent stability in a mouse where the craniotomy was stabilized with KwikSil (Supplementary Figure 5).

The RMS value was around 20 uV in some of the recordings, and according to Figure 4G it is around 16 uV on average. Is it safe to accept putative single units with 20 uV amplitudes, when the baseline noise level is this close to the spike peak-to-peak amplitude?

On average, less than 1% of the units selected using all the other metrics except the amplitude had an amplitude below 30 µV, and 2.6% below 50 µV. Increasing the threshold to 30 µV, or even 50 µV, did not affect the results. We have now added this comment in the Methods (Data processing, Para 3).

Can you add the waveform and ISIH of the example unit from day 106 to Figure 5?

We have now added 4 units tracked up to day 106 in Figure 5.  

Could you move Supplementary Figure 3A to Figure 4? The number of units is more valuable information than the RMS noise level. I understand that you don't have such a nice coverage of all the days as in Figure 3 and 4, but you might be able to group for the first 3 days and the last 3 days (and if data is available, the middle 3 days) as a boxplot. The goal would be for the reader to be able to see whether there is any change in the number of single units over time.

We agree with the reviewer, the number of units is more valuable. We had included this information in Figure 4A-F, but we have made edits to the text to make it clearer that this is what is being shown. The data from Figure 3A is already contained within Figure 4, but in 3A the data is separated by individual labs.

Product numbers are missing in multiple places: line-285 (screw), line-288 (screw), line-290 (screw), line-309 (manipulator), line-374 (gold pin and silver wire), line-384 (Mill-Max), line-394 (silver wire), and many more. It would be great if you could add all these details, so people can replicate your protocol.

We thank the reviewer for highlighting this, and we have added details of screw thread-size and length to relevant parts of the manuscript, although any type of screw can be used. Similarly, other components are non-specific (e.g. multiple silver-wire diameters were used across labs), so we have not included specific product numbers for general consumer items (like screws and silver wires) to avoid indicating that a specific part must be purchased.

While it is great to see lab-specific methods, I am not sure in their current form it helps to understand the protocol better. The information is conveyed in different ways (I assume these were written by different people), in different orders, and in different depths (some mention probe implant location relative to bregma and midline, some don't). There are many different glues, epoxies, cement, wires, and pins. I would recommend rewriting these methods sections under a unified template, so it is easier to follow.

We thank the reviewer for this suggestion and we have rewritten this section of the methods accordingly. We now use a template structure to simplify the comparisons between labs: the same template is used for each lab in each section (payload module assembly, implantation, and data acquisition).

Line-307: why is a skull screw optional for grounding? What did you use for ground and reference if not a ground screw?

We now specify in the manuscript that during head-fixed experiments, the animal’s headplate can be used for grounding, and combined with internal referencing provided by the Neuropixels, yielded lownoise recordings (Implantation protocol, Methods).

It's interesting that he sees the church as this objectively good being and idea, and that people have done damage to her, though she is still good, just downtrodden. Rather than the people being the church and their actions being representative, thus painting the church itself as bad.

I think the fact that the words have no other meaning to him except the sequence of their letters may be the key to his win. He has no intellectual baggage to distract him, he just sees the letters he has and fits them in like a puzzle.

I teach Community Nutrition Program Development, and explaining that evaluation is the most important aspect of any program design is always a challenge. It's hard to wrap your head around how meaningless a campaign (or in this case, curriculum) would be without a way to evaluate it.

Reviewer #2 (Public Review):

Kanie et all have carried out a tour-de-force effort to further understand the hierarchy and function of centriole distal appendages in ciliogenesis. They made a thorough effort to understand the localization of all the known distal appendage proteins. To examine the distal appendage hierarchy, they used an automated analysis of centrosomal localization. It is not clear how this was quantified and pictures are not shown. They used CEP170, a marker for subdistal appendages, to define a mask around centrioles. It is not clear how the experiment was analyzed and normalized. The techniques used in this study cannot be compared with those commonly used in the field which normally include storm and other super-resolution techniques (which are less prone to artifacts) and correlated electron microscopy. Thus, it is not possible to make a head-to-head comparison. The lack of rescue experiments further weakens the conclusions of this paper.

oh true! want to do this more as well. sometimes, my brain doesn't even think. if i need a hot drink, my brain defaults to coffee which of course is always a bad idea

Stare into the soul. As for me i will look at this deep dark hole in my head. Wondering why i am like this.

To identify carbohydrates (and lipids in general besides phospholipids), you can count the number of C, H, and O present in the molecule.

The number of carbons and oxygens should be equal, whereas the number of hydrogens should be double this amount.

You can also identify lipids by looking for the polar "head" and nonpolar "tail"!

question does this make lipids have a specialized interaction with the phospholipid bilayer of a cell?

Ophelia says that his actions and words align for good, and he treats her very well, with the love in his heart. Ophelia is clearly head over heels for him, and has continued to defend Hamlet over and over whilst her father expresses his concerns. I feel as if this will end up hurting Ophelia, as she is unable to step away from her feelings and see dangerous possibilities.

Atlas losing his freedom, punished to eternally hold up the heavens, this shows losing something Humanity and myself personally consider extremely important. Poor Atlas.

This highlights a paradox in American education. While the system aims to equalize opportunities, wealthier families can use their resources to give their children a head start, creating inequality despite the ideal of equal chance for all.

This paragraph explores the fundamental paradox at the heart of the American Dream as it relates to education. Although the Dream advocates for equal opportunities, systemic inequalities such as wealth disparities and unequal access to quality schools—create barriers. The paradox lies in how education, intended to equalize opportunities, can also perpetuate inequalities when families prioritize their children’s advantage over collective fairness.

When first reading this section, I first disagreed with how one's success depends on those of their parents or guardians. In my opinion, true success comes from how determined you are to achieving your goals. My parents weren't able to attend college, they had barely reached middle school, but in my eyes I still view them as successful. They had one goal which was to pursue a better future for themselves and their children in an unknown place and they succeeded in that. I view them ass successful for how passionate and determined they were which to me is what I aspire my success to stem from.

This highlights how people are set up for success or failure at birth, in a more light-hearted way. It states that "the success of one generation depends at least partly on the success of their parents or guardians." In other words, one of the main ways you will succeed in life is if you're parents/guardians already are. And if i'm not mistaken, the "only way" to do that is almost like you're pre-destined for it. I find this both inaccurate and accurate. Yes, I do believe that those who have parents that are successful struggle less in more ways than one--not just in their education. Specifically, it is easier to fail when you do not have the right resources and/or support. This is not to say that all of those who are less fortunate fail--it just makes it 10X as difficult.

I highly agree and find this to be true especially being a freshman and examining results of college acceptances of my classmates. There are especially large disparities as regardless of the accessibility of public school, it is unavoidable that some students will have more resources available to them allowing them to have an easier time getting through school compared to other students who have lower socioeconomic status and as a result have less resources available to them. Additionally, I also find it to be especially true how schools in higher income areas have more resources. For instance, I noticed how a high school in a higher income neighborhood had much more clubs and diverse career pathways for students that I would have appreciated, compared to my own school in a lower income area which had few clubs and pathway options I could choose from.

many families do not have the privilege of picking a home and area code based off if the schools are good or bad they are limited to picking a address at which they are able to afford. the statement of how far behind your child is when they get to kindergarden also favors the wealthy because many parents aren't able to send their child to a day care before school starts where they can get a head start along with many families work full time and are not able to teach their children at home before kindergarden.

Early education is critical for setting students up for success, but access to high-quality programs like Head Start is often limited to wealthier families. it shows how early disadvantages accumulate, leaving poor students "behind" before they even start elementary school, how harsh the society is.

I totally agree with this. Many wealthy children have access to resources that low-income students don't have. They often have parents who can help them with school and guide them through college. While the government has made programs to help lower-income children through programs like Head Start, I feel that wealthier children still have far more resources and privileges when it comes to education. So I don't think there is really going to a equal ground when it comes to educations between lower income and higher income students.

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Reply to the reviewers

We thank the reviewers for their general comment and for the critical evaluation of our analyses and results interpretation. Their comments greatly helped us to improve the manuscript.

• *

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: An analysis of an Arabidopsis VSP13 presumed lipid transport is provided. The analysis pretty much follows similar studies done on yeast and human homologs. Key findings are the identification of multiple products from the locus due to differential splicing, analysis of lipid binding and transport properties, subcellular location, tissue specific promoter activity, mutant analysis suggesting a role in lipid remodeling following phosphate deprivation, but no physiological or growth defects of the mutants. Major points: The paper is generally written and documented, the experiments are well conducted and follow established protocols. The following major points should be considered:

1. There are complementary lipid binding assays that should be considered such as liposome binding assays, or lipid/western dot blots. All of these might give slightly different results and may inform a consensus. Of course, non-membrane lipids such as TAG cannot be tested in a liposome assay.

Concerning lipid transfer proteins (LTPs), it is important to differentiate the lipid binding capacity related to the transport specificity (which lipids are transported by a LTP?) from the lipid binding capacity linked to the targeting of a LTP to a specific membrane (a LTP can bind a specific lipid via a domain distinct from the lipid transfer domain to be targeted in cells, but will not transport this lipid). Both aspects are of high interest to be determined. Our goal here was to focus on the identification of the lipids bound to AtVPS13M1 and to be likely transported, which is why we used a truncation (1-335) corresponding to the N-term part of the hydrophobic tunnel. Liposome binding assays and lipid dot blots are necessary to answer the question of the membrane binding capacity of the protein. We think that this aspect is out of the scope of the current article as it will require to express and purify other AtVPS13M1 domains that are known to bind lipids such as the two PH domains and the C2. This will be the scope of future investigations in our lab.

Similarly, lipid transfer based only on fluorophore-labeled lipids may be misleading because the fluorophore could affect binding. It is mentioned that the protein in this assay is tethered by 3xHis to the liposomes. Un less I ma missing something, I do not understand how that should work. This needs to be better explained.

We truly agree with Reviewer 1 that the presence of a fluorophore could affect lipid binding to the protein. In this assay, lipids are labeled on their polar head and it is therefore difficult to conclude about the specificity of our protein in term of transport. This assay is used as a qualitative assay to show that AtVPS13M1(1-335) is able to transfer lipids in vitro, and in the manuscript, we did not make any conclusion about its transport specificity based on this assay, but rather used the binding assay to assess the binding, and likely transport, specificity of AtVPS13M1. FRET-based assay is a well-accepted assay in the lipid transfer community to easily probe lipid transport in vitro and has been used in the past to assess transfer capacity of different proteins, including for VPS13 proteins (for examples, see (Kumar et al., 2018; Hanna et al., 2022; Valverde et al., 2019)).

To be able to transfer lipids from one liposome to another, both liposomes have to be in close proximity. Therefore, we attached our protein on donor acceptors, to favor the transport of the fluorescent lipids from the donor to the acceptor liposomes. Then, we progressively increased acceptor liposomes concentration to favor liposome proximity and the chance to have lipid transfer. We added a scheme on Figure 3B of the revised version of the manuscript to clarify the principle of the assay. In addition, we provided further control experiments suggested by Reviewers 2 and 3 showing that the fluorescence signal intensity depend on AtVPS13M1(1-335) protein concentration and that no fluorescence increase is measured with a control protein (Tom20.3) (see Figure 3C-D of the revised manuscript).

The in vivo lipid binding assay could be obscured by the fact that the protein was produced in insect cells and lipid binding occurs during the producing. What is the evidence that added plants calli lipids can replace lipids already present during isolation.

Actually we don’t really know whether the insect cells lipids initially bound to AtVPS13M1(1-335) are replaced by calli lipids or whether they bound to still available lipid binding sites on the protein. But we have two main lines of evidence showing that our purified protein can bind plant lipids even in the presence of insect cells lipids: 1) our protein can bind SQDG and MGDG, two plants specific lipids, and 2) as explained p.8 (lines 243-254), lipids coming from both organisms have a specific acyl-chain composition, with insect cells fatty acids mainly composed of C16 and C18 with 0 or 1 unsaturation whereas plant lipids can have up to 3 unsaturations. By analyzing and presenting on the histograms lipid species from insect cells, calli and those bound to AtVPS13M1(1-335), we were able to conclude that for all the lipid classes besides PS, a wide range of lipid species deriving from both organisms was bound to our protein. The data about the lipid species bound to AtVPS13M1(1-335) are presented in Figure 2E and S2.

The effects on lipid composition of the mutants are not very drastic from what I can tell. Furthermore, how does this fit with the lipid composition of mitochondria where the protein appears to be mostly located?

It is true that lipid composition variations in the mutants are not drastic but still statistically significant. As a general point in the field of lipid transfer, it is not very common to have major changes in total lipidome on single mutants of lipid transfer proteins because of a high redundancy of lipid transport pathway in cells. This is particularly true for VPS13 proteins, as exemplified by multiple studies. Major lipid phenotypes can be revealed in specific conditions, such as phosphate starvation in our case, or when looking at specific organelles or specific tissues and/or developmental stages. This is explained and illustrated by examples in the discussion part p. 16 (line 526-532). In addition, as suggested by Reviewer 3, we performed further lipid analysis on calli and also on rosettes under Pi starvation and found a similar trend (Figure 4 and S4 of the revised version of the manuscript). Thus, we believe that, even if not drastic, these variations during Pi starvation are a real phenotype of our mutants.

As we found that our protein is located at the mitochondrial surface, we agree that Reviewer 1’s suggestion to perform lipidomic analyses on isolated mitochondria will be of high interest but this will be the scope of future studies that we will performed in our lab. First, we would like to identify all the organelles at which AtVPS13M1 is localized before performing subfractionations of these different organelles from the same pool of cell cultures grown in presence or absence of phosphate.

For the localization of the fusion protein, has it been tested whether the furoin is functional? This should be tested (e.g. by reversion of lipid composition).

As we did not observe major developmental phenotypes in our mutants, complementation should be indeed tested by performing lipidomic analyses in calli or plants grown in presence or absence of Pi, which is a time-consuming and expensive experiment. Because we used the fusions mainly for tissue expression study and subcellular localization and not for functional analyses, we believe that this is not an essential control to be performed for this work.

It is speculated that different splice forms are located to different compartments. Can that be tested and used to explain the observed subcellular location patterns?

Indeed some splice forms can modify the sequence of domains putatively involved in protein localization. This could be tested by producing synthetic constructs with one specific exon organization, which is challenging according to the size of AtVPS13M1 cDNA (around 12kb). In addition, our long-read sequencing experiment and PCR analyses revealed the existence of six transcripts, a major one representing around 92% and the five others representing less than 2.5% (Figure 1D). Among the five less abundant transcripts, four produce proteins with a premature stop codon and are likely to arise from splicing defects as explained in the discussion part p. 15 (lines 488-496). One produces a full-length protein with an additional loop in the VAB domain but because of the low abundance of this alternative transcript (1.4%), we believe it does not contribute significantly to the major localization we observed in plants and did not attend to analyze its localization.

GUS fusion data only probe promoter activity but not all levels of gene expression. That caveat should be discussed.

We are aware of this drawback and that is the reason why we fused the GUS enzyme directly to our protein expressed under its native locus (i.e. with endogenous promoter and exons/introns) as depicted in Figure 5A. Therefore, our construction allows to assess directly AtVPS13M1 protein level in plant tissues.

Minor points: 1. Extraplastidic DGDG and export from chloroplasts following phosphate derivation was first reported in PMID: 10973486.

We added this reference in the text.

Check throughout the correct usage of gene expression as genes are expressed and proteins produced.

Many thanks for this remark, we modified the text accordingly

In general, the paper is too long. Redundancies between introduction, results and discussion should be removed to streamline.

We reduced the text to avoid redundancy.

I suggest to redraw the excel graphs to increase line thickness and enlarge font size to increase presentation and readability.

We tried as much as we can to enlarge graphs and font size increasing readability.

Reviewer #1 (Significance (Required)):

Significance: Interorganellar lipid trafficking is an important topic and especially under studied in plants. Identifying components involved represents significant progress in the field. Similarly, lipid remodeling following phosphate derivation is an important phenomenon and the current advances our understanding.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: The manuscript "AtVPS13M1 is involved in lipid remodelling in low phosphate and is located at the mitochondria surface in plants" by Leterme et al. identifies the protein VPS13M1 as a lipid transporter in Arabidopsis thaliana with important functions during phosphate starvation. The researchers were able to localise this protein to mitochondria via GFP-targeting in Arabidopsis. Although VPS13 proteins are well described in yeast and mammals, highlighting their importance in many vital cellular processes, there is very little information on them in plants. This manuscript provides new insights into plant VPS13 proteins and contributes to a better understanding of these proteins and their role in abiotic stress responses, such as phosphate starvation.

Major points: - Please describe and define the domains of the VPS13M1 protein in detail, providing also a figure for that. Figure 1 is mainly describing possible splice variants, whereas the characteristics of the protein are missing.

We have added information on AtVPS13M1 domain organization in the introduction (p.4, lines 103-109) and referred to Figure 1A that described protein domain organization. We did not added too much details as plant VPS13 protein domains organization was extensively described in two previous studies cited several times in the manuscript (Leterme et al., 2023; Levine, 2022).

• Please compare the expression level of VPS13M1 in the presence and in the absence of phosphate.

Many thanks for this suggestion. We performed qRT-PCR analyses of AtVPS13M1 from mRNA extracted from calli grown six days in presence and absence of phosphate. The results obtained did not reveal variations in mRNA level. The results were added in Figure S1A of the revised version of the manuscript and discussed in p.5 (lines 154-156).

• Page 9, second paragraph: Here, the lipid transport capability of AtVPS13M1 is described. Varying concentrations of this recombinant protein should be used in this test. Further, it is not highlighted, that a truncated version of VSP13M1 is able to transport lipids. This is surprising, since this truncated version is less than 10% of the total protein (only aa 1-335).

We agree with reviewer 2 that increasing protein concentration is an important control to perform. We included an experiment with an increasing quantity of protein (2X and 4X) in the revised version of the manuscript and showed that the signal intensity increased faster when protein concentration is higher (Figure 3D of the revised manuscript). As requested by Reviewer 3, we also included a negative control with Tom20.3 to show that the signal increase after the addition of AtVPS13M1(1-335) is specific to this protein (Figure 3C of the revised manuscript).

The transport ability of the N-terminal part of VPS13 was demonstrated in yeast and mammals VPS13D (Kumar et al., 2018; Wang et al., 2021). We highlighted this p. 7 (lines 213-218) of the revised version of the manuscript. This is explained by the inherent structure of VPS13 proteins that are composed of several repeats of the same domain type called RBG (for repeating β-groove), each forming a β-sheet with a hydrophobic surface. The higher the number of RBG repeats, the longer the hydrophobic tunnel is. The (1-335) N-terminal region corresponds to two RBG unit repeats forming a “small” tunnel able to bind and transfer lipids. The number of RBG repeats has influence on the quantity of lipids bound per protein in vitro, the longest the protein is, the highest the number of lipid molecules bound is (Kumar et al., 2018), but the effect on protein length on in vitro lipid transfer capacity has not been investigated yet to the best of our knowledge.

• Also, for phenotype analysis, T-DNA insertion mutants are used that still contain VPS13M1 transcripts. Although protein fragments where not detected by proteomic analysis, this might be due to low sensitivity of the proteomic assay. Further the lipid transport domain of VPS13M1 (aa 1-335) might not be affected by the T-DNA insertions at all. Here more detailed analysis needs to be done to prove that indeed loss-of protein function occurs in the mutants.

We do not have other methods than proteomic to test whether our mutants are KO or not. We tried unsuccessfully to produce antibodies. Mass spectrometry is the most sensitive method but the absence of detection indeed does not mean the absence of the protein. From proteomic data, we can conclude that at least, our mutants present a decrease in AtVPS13M1 protein level, thus we called them “knock down” in the revised version of the manuscript and added the following sentence p. 9 (lines 297-300): “As the absence of detection of a protein by mass spectrometry-based proteomics does not allow us to strictly claim the absence of this protein in the sample, we concluded that AtVPS13M1 expression in both atvps13m1-1 and atvps13m1-4 was below the detection limit and consider them as knock down (KD) for AtVPS13M1.”

• Localisation in mitochondria: As the Yepet signal is very weak, a control image of not transfected plant tissue needs to be included. Otherwise, it might be hard to distinguish the Yepet signal from background signal. The localisation data presented in Figure 5 does not allow the conclusion that VPS13M1 is localized at the surface of mitochondria as stated in the title. It only indicates (provided respective controls see above) that VPS13M1 is in mitochondria. Please provide more detailed analysis such as targeting to tobacco protoplasts, immunoblots or in vitro protein import assays. Also test +Pi vs. -Pi to see if VPS13M1 localisation is altered in dependence of Pi.

Indeed our Yepet signal is not very strong but on the experiments we performed on Col0 non-transformed plants, we did not very often see fluorescence background in the leaves’ vascular tissue, that is why we focused our study on this tissue. We sometimes observed some background signals in some cells that are clearly different from AtVPS13M1-3xYepet signals and never co-localized with mitochondria. Examples of these aspecific signals are presented in Figure S6E of the revised version of the manuscript.

We agree with reviewer 2 that our confocal images suggested, but not demonstrated, a localization at the surface of mitochondria. To confirm the localization, we generated calli cell cultures from AtVPS13M1-3xYepet lines and performed subcellular fractionations and western blot analyses confirming that AtVPS13M1 was indeed enriched in mitochondria and also in microsomal fractions (Figure 6G of the revised version). Then we performed mild proteolytic digestion of the isolated mitochondria with thermolysin and show that AtVPS13M1 was degraded, as the outer membrane protein Tom20.3, but not the inner membrane protein AtMic60, showing that AtVPS13M1 is indeed at the surface of mitochondria (Figure 5H of the revised manuscript). We believe that this experiment, in addition to the confocal images showing a signal around mitochondria, convincingly demonstrates that AtVPS13M1 is located at the surface of mitochondria.

The localization of AtVPS13M1 under Pi starvation is a very important question that we tried to investigate without success. Indeed, we intended to perform confocal imaging on seedlings grown in liquid media to easily perform Pi starvation as described for the analysis of AtVPS13M1 tissue expression with β-glucuronidase constructs. However, the level of fluorescence background was very high in seedlings and no clear differences between non-transformed and AtVPS13M1-3xYepet lines were observed, even in root tips where the protein is supposed to be the most highly expressed according to β-glucuronidase assays. Example of images obtained are presented in Figure R1. We concluded that the level of expression of our construct was too low in seedlings. The constructions of lines with a higher AtVPS13M1 expression level, by changing the promotor, to better analyze AtVPS13M1 in different tissues or in response to Pi starvation will be the scope of future work in our laboratory in order to investigate AtVPS13M1 localization under low Pi.

Phenotype analysis needs to be done under Pi stress and not under cold stress! Further, root architecture and root growth should also be done under Pi depletion. Here the title is also misleading, it is not at all clear why the authors switch from phosphate starvation to cold stress.

In the revised version of the manuscript, we analyzed the seedlings root growth of two mutants (atvps13m1-3 and m1-4) under low Pi and did not notice significant differences (Figure 7E, S7D of the revised version). We analyzed growth under cold stress because this stress also promotes remodeling of lipids, but we agree that it goes beyond the scope of this article that is focused on Pi starvation and we removed this part from the revised manuscript.

Minor points: Page 3, line 1: what does the abbreviation VPS stand for?

The definition of VPS (Vacuolar Protein Sorting) was added.

Page 3, line 1: change "amino acids residues" to "amino acid residues"

This was done.

Page 3, line 8 - 12: please rewrite this sentence. You write, that because of their distribution VPS13 proteins do exhibit many important physiological roles. The opposite is true: They are widely distributed in the cell because of their involvement in many physiological processes.

We changed the sentence to “ VPS13 proteins localize to a wide variety of membranes and membrane contact sites (MCSs) in yeast and human (Dziurdzik and Conibear, 2021). This broad distribution on different organelles and MCSs is important to sustain their important roles in numerous cellular and organellar processes such as meiosis and sporulation, maintenance of actin skeleton and cell morphology, mitochondrial function, regulation of cellular phosphatidylinositol phosphates level and biogenesis of autophagosome and acrosome (Dziurdzik and Conibear, 2021; Hanna et al., 2023; Leonzino et al., 2021).”

Page 6, line6: change "cDNA obtained from A. thaliana" to "cDNA generated from A. thaliana.

This was done.

Page 6, line 10: change" 7.6kb" to "7.6 kb"

This was done.

Page 7: address this question: can the isoforms form functional VPS13 proteins? This might help to postulate whether these isoforms are a result of defective splicing events.

We addressed this aspect in the discussion p.15 at lines 486-502.

Figure 2 B: Change "AtVPS13M1"to "AtVPS13M1(1-335)"

This was done.

Figure 2, legend: -put a blank before µM in each case.

This was done.

-Change 0,125µM to 0.125 µM

This was done.

-what does "in absence (A-0µM)" mean?

This means that the Acceptor liposomes are at 0 µM. To clarify, we changed it to “Acceptor 0 µM” in the revised version of the manuscript (Figure 3C).

-Which statistical analysis was employed?

We performed a non-parametric Mann-Whitney test in the revised version of the manuscript. This was indicated in the legend.

-Further, rewrite the sentence "Mass spectrometry (MS) analysis of lipids bound to AtVPS13M1(1-335) or Tom20 (negative control) after incubation with calli total lipids. Results are expresses in nmol of lipids per nmol of proteins (C) or in mol% (D)". -"C" and "D" are not directly comparable, as in "C" no Tom20 was used and in "C" no insect cells were used.

-Further, in "D" the experimental setup is not clear. AtVPS13(1-335) is supposed to be purified protein after incubation with calli lipids (figure 2, A). Further, in the same figure, lipid composition of "insect cells" and "calli-Pi" are compared àwhy? Please clarify this.

C and D are two different representations of the same results providing different types of information. In C., the results are expressed in nmol of lipids / nmol of proteins to assess 1) that the level of lipids found in AtVPS13M1(1-335) purifications is significantly higher than what we can expect from the background (assessed using Tom20) and 2) what are the classes of lipids that associate or not to AtVPS13M1(1-335). In D. the lipid distribution in mol% is presented for AtVPS13M1(1-335) as well as for total extracts from calli and insect cells to be able to compare if one lipid class is particularly enriched or not in AtVPS13M1(1-335) purifications compared to the initial extracts with which the protein was incubated. As an example, it allows to deduce that the absence of DGDG detected in the AtVPS13M1(1-335) purifications is not linked to a low level of DGDG in the calli extract, because it represented around 15 mol%, but likely to a weak affinity of the protein for this lipid. We did not represent the Tom20 lipid distribution on this graph because it represents background of lipid binding to the purification column and might suggest that Tom20 binds lipids. We changed the legend in this way and hope that it is clearer now: “C-D. Mass spectrometry (MS) analysis of lipids bound to AtVPS13M1(1-335) or Tom20 (negative control) after incubation with calli total lipids and repurification. Results are expresses in nmol of lipids per nmol of proteins in order to analyze the absolute quantity of the different lipid classes bound to AtVPS13M1(1-335) compared to Tom20 negative control (C), and in mol% to assess the global distribution of lipid classes in AtVPS13M1(1-335) purifications compared to the total lipid extract of insect cells and calli (D).”

Figure 3: -t-test requires a normal distribution of the data. This is not possible for an n=3. Please use an adequate analysis.

We performed more replicates and used non-parametric Mann-Whitney analyses in the revised version of the manuscript.

-Please clarify the meaning of the letters on the top of the bars in the legend.

This corresponded to the significance of t-tests performed in the first version of the manuscript that were reported in Table S3. As in the new version we performed Mann-Whitney tests, we highlighted the significance by stars and in the figure legends.

Please, make it clear that two figures belong to C.

This was clarified in the legend.

-Reorganise the order of figure 3 (AàBàCàD)

Because of the configuration of the different histograms presented in the figure, we were not able to change the order but we believed that the graphs can be easily red this way.

Page 10, 3. Paragraph: since the finding, that no peptides were found in the VSP13M1 ko lines, although transcription was not altered, is surprising, please include the proteomic data in the supplement

Proteomic data were deposited on PRIDE with the identifier PXD052019. They will remain not publicly accessible until the acceptance of the manuscript.

Page 11, line 17: The in vitro experiments showed a low affinity of VSP13M1 towards galactolipids. It is further claimed that this is consistent with the finding of the AtVSP13M1 Ko line in vivo, that in absence of PI, no change in DGDG content could be observed. However, the "absence" of VSP13M1 in vivo might still result in a bigger VSP13M1 protein, than the truncated form (1-335) used for the in vitro experiments

It is true that our in vitro experiments were performed only with a portion of AtVPS13M1 and that the length of the protein could influence protein binding specificity. We removed this assessment from the manuscript.

Page 13, lane 8: you should reconsider the use of a triple Yepet tag: If two or more identical fluorescent molecules are in close proximity, their fluorescence emission is quenched, which results in a weak signal (as the one that you obtained). See: Zhuang et al. 2000 (PNAS) Fluorescence quenching: A tool for single-molecule protein-folding study

Many thanks to point this paper. We use a triple Yepet because AtVPS13M1 has a very low level of expression and because this strategy was used successfully to visualize proteins for which the signal was below the detection level with a single GFP (Zhou et al., 2011). The quenching of the 3xYepet might also depend on the conformation they adopt on the targeting protein.

Page 13, line 14: change 1µm to 1 µm

This was done.

Page 13, line 29: please reduce the sentence to the first part: if A does not colocalize with B, it is not necessary to mention that B does not colocalise with A.

The sentence was modified accordingly.

Page 14, 2. Paragraph: it is not conclusive that phenotype analysis is suddenly conducted with plants under cold stress, since everything was about Pi-starvation and the role of VSP13M1. Lipid remodelling under Pi stress completely differs from the lipid remodelling under cold stress.

We eliminated this part in the revised version of the manuscript.

Page 14, line 20: change figure to Figure

This was done.

Page 07, line 17: change artifact to artefact

This was done.

Reviewer #2 (Significance (Required)):

General assessment: The paper is well written and technically sound. However, some points could be identified, that definitely need a revision. Overall, we got the impression that so far, the data gathered are still quite preliminary and need some more detailed investigations prior to publication (see major points).

Advance: The study definitely fills a gap of knowledge since not much is known on the function of plant VPS13 proteins so far.

Audience: The study is of very high interest to the plant lipid community but as well of general interest for Plant Molecular Biology and intracellular transport.

Our expertise: Plant membrane transport and lipid homeostasis.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The manuscript by Leterme et al. (2024) describes the characterization of VPS13M1 from Arabidopsis. VPS13 proteins have been analyzed in yeast and animals, where they establish lipid transfer connections between organelles, but not much is known about VPS13 proteins in plants. First, different splicing forms were characterized, and the form A was identified as the most relevant one with 92% of the transcripts. The protein (just N-terminal 335 amino acids out of ca. 3000 amino acids) was expressed in insect cells and purified. Next, the protein was used for lipid binding assays with NBD-labeled lipids followed by analysis in polyacrylamide gel electrophoresis. VPS13M1 bound to PC, PE, PS and PA. Then, the protein from insect cells was incubated with Arabidopsis callus lipids, and lipids bound to VPS13M1 analyzed by LC-MS/MS. Lipid transfer between liposomes was measured by the change in fluorescence in donor liposomes derived from two labeled lipids after addition of the protein caused by lipid transfer and dilution to acceptor liposomes. T-DNA insertion mutants were isolated and the lipids measured in callus derived from these mutants. Protein localization in different plant organs was recorded with a GUS fusion construct transferred into transgenic plants. The protein was localized to mitochondria using a VPS13M1-Yepet fusion construct transferred into mutant plants. The mutant plants show no visible difference to wild type, even when the plants were grown under stress conditions like low temperature. The main message of the title is that VPS13M1 localizes to the mitochondria which is well documented, and it is involved in lipid remodeling under low phosphate conditions.

The lipid transfer assay shown in Figure 2F lacks a negative control. This would be the experiment with donor and acceptor liposomes in the presence of another protein like Tom20.

Many thanks for this suggestion. In the revised version of the manuscript, we performed a fluorescent lipid transport assay with Tom20.3 in the presence of 25 µM of donor liposomes and 1.5 mM of acceptor liposomes, the condition for which we observed a maximum of transport for AtVPS13M1(1-335). As expected, no fluorescence increase was observed. The results are presented in the Figure 3C of the revised manuscript.

The lipid data (Fig. 3 and Fig. S4) do not sufficiently support the second claim, i.e. that the protein is involved in lipid remodeling under low P. Data in Fig. 3C are derived from only 3 replicates and in Fig. S4 from only 2 replicas with considerable error bars. Having only 2 replicates is definitely not sufficient. Fig. 3C shows a suppression in the decrease in PE and PC at 4 d of P deprivation (significant for two mutants for PE, for only one for PC). Fig. S4A shows suppression of the decrease in PC at 6 d after P deprivation (significant for both mutants), but no significant effect on PE. Fig. 4SB shows no significant change in PE or PC at -P after 8 d of P deprivation. The data are not consistent. There are also problems with the statistics in Fig. 3 and Fig. S4. The authors used T-test, but place letters a, b, c on top of the bars. Usually, asterisks should be used to indicate significant differences. Data indicate medians and ranges, not mean and SD. In Fig. S4, how can you indicate median and range if you have only 2 replicates? Why did the authors use callus for lipid measurements? Why not use leaves and root tissues? What does adjusted nmol mean? What does the dashed line at 1.05 on the y axis mean? Taken together, I suggest to repeat lipid measurements with leaves and roots from plantets grown under +P and -P conditions in tissue culture with 5 replcates. Significant differences can be analyzed on the level of absolute (nmol per mg FW/DW) or relative (%) amounts.

Here are our answers to concerns about the design of our lipidomics experiments:

We used calli for lipid measurement because it is very easy to control growth conditions and to performed phosphate starvation from this cell cultures. The second reason is that it is a non-photosynthetic tissue with a high level of phospholipids and a low level of galactoglycerolipids and it is easier to monitor the modification of the balance phospholipids/galactoglycerolipids in this system. The lipid analysis on calli at 4 days of growth in presence or absence of Pi were performed on 3 biological replicates but on two different mutants (atvps13m-1 and m1-3) and we drew our conclusions based on variations that were significant for both mutants. In the revised version of the manuscript, we performed further lipidomic analyses on calli from Col0 and another mutant (atvps13m1-2) after 6 days of growth in presence or absence of Pi (Figure 4E, S4A-C, n=4-5) and added new data on a photosynthetic tissue (rosettes) from Col0 and atvps13m1-3 mutant. For rosettes analysis, seeds were germinated 4 days in plates with 1 mM Pi and then transferred on plates with 1 mM or 5 µM of Pi. Rosettes were harvested and lipids analyzed after 6 days (Figure 4F-G, S4D, n=4-5). All the data were represented with medians and ranges because we believe that median is less sensitive to extreme values than mean and might better represent what is occurring. Ranges highlight the minimal and maximal value of the data analyzed and we believe it is a representative view of the variability we obtained between biological samples.

Lipid measurement are done by mass spectrometry. As it was already reported, mass spectrometry quantification is not trivial as the intensity of the response depends on the nature of the molecule (for a review, see (Jouhet et al., 2024)). To counteract this ionisation problem, we developed a method with an external standard that we called Quantified Control (QC) corresponding to an A. thaliana callus lipid extract for which the precised lipid composition was determined by TLC and GC-FID. All our MS signals were “adjusted” to the signal of this QC as described in (Jouhet et al., 2017). Therefore our lipid measurement are in adjusted nmol. In material and method we modified the sentence accordingly p22 lines 720-723: “Lipid amounts (pmol) were adjusted for response differences between internal standards and endogenous lipids and by comparison with a quality control (QC).” This allows to represent all the lipid classes on a same graph and to have an estimation of the lipid classes distribution. To assess the significance of our results, we used in the revised version of the manuscript non-parametric Mann-Whitney tests and added stars representing the p-value on charts. This was indicated in the figure legends.

Here are our answers to concerns about the interpretation of our lipidomics experiments:

To summarize, in the revised version of the manuscript, lipid analyses were performed in calli from 3 different mutants (two at day 4, one at day 6) and in the rosettes from one of these mutants. All the results are presented in Figure 4 and S4. In all the experiments, we found that in +Pi, there is no major modifications in the lipid content or composition. In –Pi, we found that the total glycerolipid content is always higher in the mutant compared to the Col0, whatever the tissue or mutant considered (Figure 4A and S4A, D). In calli, this higher increase in lipid content is mainly due to an accumulation of phospholipids and in rosettes, of galactolipids. Because of high variability between our biological replicates, we did not always found significant differences in the absolute amount of lipids in –Pi. However, the analysis of the fold change in lipid content in –Pi vs +Pi always pointed toward a reduced extent of phospholipid degradation. We also added in these graphs the fold change for the total phospholipids and total galactolipids contents in the revised version of the manuscript. We believe that the new analyses we performed strengthen our conclusion about the role of AtVPS13M1 in phospholipid degradation and not on the recycling of precursors backbone to feed galactoglycerolipids synthesis at the chloroplast envelope.

Page 9, line 15: Please use the standard form of abbreviations of lipid molecular species with colon, e.g. PC32:0, not PC32-0

The lipid species nomenclature has been changed accordingly.

Page 11, line 4, (atvps13m1.1 and m1.3: please indicate the existence of mutant alleles with dashes, i.e. (atvps13m1-1 and atvps13m1-3

Names of the mutants have been changed accordingly.

Page 14, line 21: which line is indicated by atvps13m1.2-4? What does -4 indicate here?

This indicates that mutants m1-2 to m1-4 were analyzed.

Page 16, line 25: many abbreviations used here are very specific and not well known to the general audience e.g. ONT, IR, PTC, NMD etc. I think it is OK to mention them here, but still use the full terms, given that they are not used very frequently in the manuscript.

We kept ONT abbreviation because it was cited many times in both the results and discussion part. IR, PTC and NMD were cited only in the discussion and were eliminated.

Page 19, line 11. The authors cite Hsueh et al and Yang et al for LPTD1 playing a role in lipid homeostasis during P deficiency. But Yang et al. described the function of a SEC14 protein in Arabidopsis and rice during P deficiency. Is SEC14 related to LPTD1?

Many thanks for noticing this mistake. We removed the citation Yang et al. in the revised version of the manuscript.

Reference Tangpranomkorn et al. 2022: In the text, it says that this is a preprint, but in the Reference list, this is indicated with "Plant Biology" as Journal. In the internet, I could only find this manuscript in bioRxiv.

This manuscript was accepted in “New Phytologist” in December 2024 and is now cited accordingly in the new version of the manuscript.

Reviewer #3 (Significance (Required)):

The manuscript by Leterme et al describes the characterization of the lipid binding and transport protein VTPS13M1 from Arabidopsis. I think that the liposome assay needs to be done with a negative control. Furthermore, I have major concerns with the lipid data in Fig. 3C and Fig. S4. These lipid data of the current manuscript need to be redone. I do not agree that the lipid data allow the conclusion that "AtVPS13M1 is involved in lipid remodeling in low phosphate" as stated in the title.

References cited in this document:

Dziurdzik, S.K., and E. Conibear. 2021. The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites. Int J Mol Sci. 22:2905. doi:10.3390/ijms22062905.

Hanna, M., A. Guillén-Samander, and P. De Camilli. 2023. RBG Motif Bridge-Like Lipid Transport Proteins: Structure, Functions, and Open Questions. Annu Rev Cell Dev Biol. 39:409–434. doi:10.1146/annurev-cellbio-120420-014634.

Hanna, M.G., P.H. Suen, Y. Wu, K.M. Reinisch, and P. De Camilli. 2022. SHIP164 is a chorein motif lipid transfer protein that controls endosome–Golgi membrane traffic. Journal of Cell Biology. 221:e202111018. doi:10.1083/jcb.202111018.

Jouhet, J., E. Alves, Y. Boutté, S. Darnet, F. Domergue, T. Durand, P. Fischer, L. Fouillen, M. Grube, J. Joubès, U. Kalnenieks, J.M. Kargul, I. Khozin-Goldberg, C. Leblanc, S. Letsiou, J. Lupette, G.V. Markov, I. Medina, T. Melo, P. Mojzeš, S. Momchilova, S. Mongrand, A.S.P. Moreira, B.B. Neves, C. Oger, F. Rey, S. Santaeufemia, H. Schaller, G. Schleyer, Z. Tietel, G. Zammit, C. Ziv, and R. Domingues. 2024. Plant and algal lipidomes: Analysis, composition, and their societal significance. Progress in Lipid Research. 96:101290. doi:10.1016/j.plipres.2024.101290.

Jouhet, J., J. Lupette, O. Clerc, L. Magneschi, M. Bedhomme, S. Collin, S. Roy, E. Maréchal, and F. Rébeillé. 2017. LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation. PLoS ONE. 12:e0182423. doi:10.1371/journal.pone.0182423.

Kumar, N., M. Leonzino, W. Hancock-Cerutti, F.A. Horenkamp, P. Li, J.A. Lees, H. Wheeler, K.M. Reinisch, and P. De Camilli. 2018. VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites. J Cell Biol. 217:3625–3639. doi:10.1083/jcb.201807019.

Leonzino, M., K.M. Reinisch, and P. De Camilli. 2021. Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1866:159003. doi:10.1016/j.bbalip.2021.159003.

Leterme, S., O. Bastien, R.A. Cigliano, A. Amato, and M. Michaud. 2023. Phylogenetic and Structural Analyses of VPS13 Proteins in Archaeplastida Reveal Their Complex Evolutionary History in Viridiplantae. Contact (Thousand Oaks). 6:1–23. doi:10.1177/25152564231211976.

Levine, T.P. 2022. Sequence Analysis and Structural Predictions of Lipid Transfer Bridges in the Repeating Beta Groove (RBG) Superfamily Reveal Past and Present Domain Variations Affecting Form, Function and Interactions of VPS13, ATG2, SHIP164, Hobbit and Tweek. Contact. 5:251525642211343. doi:10.1177/25152564221134328.

Valverde, D.P., S. Yu, V. Boggavarapu, N. Kumar, J.A. Lees, T. Walz, K.M. Reinisch, and T.J. Melia. 2019. ATG2 transports lipids to promote autophagosome biogenesis. J Cell Biol. 218:1787–1798. doi:10.1083/jcb.201811139.

Wang, J., N. Fang, J. Xiong, Y. Du, Y. Cao, and W.-K. Ji. 2021. An ESCRT-dependent step in fatty acid transfer from lipid droplets to mitochondria through VPS13D−TSG101 interactions. Nat Commun. 12:1252. doi:10.1038/s41467-021-21525-5.

Zhou, R., L.M. Benavente, A.N. Stepanova, and J.M. Alonso. 2011. A recombineering-based gene tagging system for Arabidopsis. Plant J. 66:712–723. doi:10.1111/j.1365-313X.2011.04524.x.

star

super interesting

Author response:

The following is the authors’ response to the current reviews.

Reviewer #1 (Public review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with two lipid compositions similar to native viral membranes. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region. The revised manuscript demonstrates that these lipid interactions are robust to alterations in membrane composition and rigidity. However, it does not address the reverse-that phospholipids known experimentally not to associate with these antibodies (if any such lipids exist) also fail to interact in MD simulations.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. These simulations recapitulate lipid binding interactions solved in published crystallographic studies but also lead to the discovery of a novel lipid binding site the authors term the "Loading Site", which could guide future experiments with this antibody.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. These CG simulations , which cannot resolve atomistic interactions, are nonetheless compelling because negative controls (ab 13h11, BSA) that should not associate with membrane indeed sample significantly less membrane.

Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive, creative, and biologically inspired. Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #2 (Public review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The use of multiscale MD simulations allows for a detailed exploration of the system at different time and length scales. The combination of MD simulations and structural bioinformatics provides a comprehensive approach to validate the identified binding sites. Finally, the steered MD simulations offer quantitative insights into the binding strength between the membrane and bnAbs.

While the simulations and analyses provide qualitative insights into the binding interactions, they do not offer a quantitative assessment of energetics. The coarse-grained simulations exhibit artifacts and thus require careful analysis.

This study contributes to a deeper understanding of the molecular mechanisms underlying bnAb recognition of the HIV-1 envelope. The insights gained from this work could inform the design of more potent and broadly neutralizing antibodies.

Recommendations for the authors:

Reviewing Editor:

We recommend the authors remove the figure and section related to bnAb LN01, perform additional analysis (e.g., further expanding on the differences in antibody binding in the presence or absence of antigen), and present this as a separate manuscript in a follow-up study.

We consider the analysis of a bnAb with a transmembrane antigen and of LN01 as essential to the manuscript and novel results.  Study of LN01 provides many insights unique from the other MPER bnAbs in this study.  We agree further characterization of LN01 and bnAbs with transmembrane antigen or full-length Env are intriguing and necessary to complete the full mechanistic understanding of lipid-associated antibodies.  LN01 section in this paper is novel in the field and demonstrates the preliminary evidence motivating further work, which we agree are beyond the scope of this already long detailed study.

Reviewer #1 (Recommendations for the authors):

I appreciate the degree to which the authors responded to my previous points raised in the private review, including edits where I might have missed something in the manuscript or relevant literature. I imagine such a point-by-point response was quite onerous. Thank you also for balancing presentation/clarity with content/rigor considering the large information content of this manuscript; in silico results are inherently hard to present given the delicate balance between rigorous validation and novel information content. I apologize if I repeat points raised and addressed previously and commend the authors on their revised study, which is much improved in clarity; any additional revisions are of course entirely at your discretion.

"...now having more diversity in lipid headgroup chemistries" references the wrong figure-it should be: Figure 2-figure supplement 2A-C. The incorrect figure is also referenced again several sentences down: "...relevant CDR and framework surface loops..."

Thank you for pointing out this error. We have corrected figure references.

"One shared conformational difference observed for these bnAbs the higher cholesterol bilayers was slightly more extensive and broader interaction profiles as well as modestly deeper embedding of the relevant CDR and framework surfaces loops" please rephrase

Thank you for this suggestion.  We rephrased this for improved clarity and flow. 

"These results bolster the feasibility for using all-atom MD as an in silico platform to explore differential phospholipid affinity at these sites (i.e., specificity studies) and influence on antibody preferred conformation as membrane composition and lipid chemistry are systematically varied" Please tone down these speculations-you have demonstrated that simulations are robust to different headgroup chemistries but have not provided evidence for the exclusion of lipids that are known not to associate with these antibodies.

We rephrased this speculation to highlight the potential of this application. We also emphasize future studies that would be required to achieve this application in the following sentence.

“These results motivate use of all-atom MD as an in silico approach for exploring differential phospholipid affinity at these sites…”

Figure 2A: Specify which PDB entry corresponds to the displayed crystal structures in the main figure or caption.

We clarified these PDB entries in the figure caption. 

Check reference formatting in supplemental figures when generating VOR.

I am not sure how relevant this might be to the claims of Figure 2-figure supplement 3, but AlphaFold3-based phospholigand docking might provide an additional orthogonal approach if relevant ligand(s) are available for such analysis (particularly for the newly proposed 10E8 POPC complex).

Thank you for this suggestion.  AI/ML based prediction methods like AF3 and RoseTTAFold All-Atom (RFAA) are interesting new methods that have come since our initial submission.   We’ve decided these experiments are beyond the scope of this already long and detailed study. We have added a sentence suggesting use of these methods in future work.

"We next studied bnAb LN01 to interrogate differences" --> this transition still reads a bit unclear. Why shift gears and change antibodies? Also, while you do go into its interactions both +/- antigen, there's no lead into the simulation initialization with and without antigen to guide the reader into the comparisons you will draw in the figure. Also, the order of information presentation is a bit strange, where the rationale for choosing a single monomeric helix is brought up in the middle of the paragraph instead of at the beginning of the section. In the next paragraph, it goes back to the initialization of the membrane composition again, which feels a bit disorganized-I do appreciate the unique challenge of having to weave through so much quality data! In fact, if you were to conduct simulations of membrane + antigen vs. membrane + LN01 vs. membrane + LN01 + antigen, I am tempted to say that this could be removed from this manuscript and flow better as a paper in and of itself.

We thank the reviewer for the suggestion to improve the writing style.  We feel this section adds a lot of value to the manuscript, so we will keep it in the paper and improved the transition as well as rationale.  

We selected to study the additional antibody LN01 and the monomeric MPER-TM antigen conformation because of the existing structural evidence available without additional creative model building.  This rationale has been updated in the new text.  

We changd the order of information as suggested, moving the rationale for antigen fragment earlier in the paragraph followed by the background of the lipids sites from the crystal that can lead into simulation set-up.  We clarified the simulation initialization was similar for systems with and without antigen in the opening sentence of the paragraph

"previously observed snorkeling and hydration of TM Arg686" --> Is this R696 (numbering could be different based on the particular Env)?

Thank you for noting this typo, we have corrected the numbering.

Potential font color issue with Figure 3-Figure supplement 1 B and part of A text-could be fixed in typesetting.

The discussion reads very well. Is it possible to direct antibody maturation, even in an engineered context, towards membrane affinity without increasing immunogenic polyreactivity? This is mentioned very briefly and cited with ref 36, but I would be interested in the author's thoughts on this topic.

We thank the reviewer for the insightful idea to explore in future work.  Our conclusion alludes to possibly artificially evolving membrane affinity studied by MD, as done in vitro by Nieva and co-workers.  Because the hypothetical nature, we’ve chosen not to elaborate on those ideas from this manuscript.

Reviewer #2 (Recommendations for the authors):

To ensure reproducibility and facilitate further research, the authors should publicly deposit the code for running the MD simulations and analyses (e.g., on GitHub) along with the underlying data used in the study (e.g., on Zenodo.org).

We appreciate the consideration for open-source code and analysis. Representative code and simulation trajectories were uploaded to the following repositories:

https://github.com/cmaillie98/mper_bnAbs.git

https://zenodo.org/records/13830877

—-

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with a lipid composition similar to the native virion. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. Additional contacts and conformational restraints imposed by ectodomain regions of the envelope glycoprotein, however, remain unaddressed-the size of such simulations likely runs into technical limitations including sampling and compute time.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive. However, given the large amount of data presented within the manuscript, the text would benefit from clearer subsections that delineate discrete mechanistic discoveries, particularly for experimentalists interested in antibody discovery and design. One area the paper does not address involves the polyreactivity associated with membrane binding antibodies-MD simulations and/or pulling velocity experiments with model membranes of different compositions, with and without model antigens, would be needed. Finally, given the challenges in initializing these simulations and their limitations, the text regarding their generalized use for discovery, rather than mechanism, could be toned down.

Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #2 (Public Review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The conclusions from the paper are mostly well supported by the simulations, however, they remain very descriptive and the key findings should be better described and validated. In particular:

It has been shown that the lipid composition of HIV membrane is rich in cholesterol [1], which accounts for almost 50% molar ratio. The authors use a very different composition and should therefore provide a reference. It has been shown for 4E10 that the change in lipid composition affects dynamics of the binding. The robustness of the results to changes of the lipid composition should also be reported.

The real advantage of the multiscale approach (coarse grained (CG) simulation followed by a back-mapped all atom simulation) remains unclear. In most cases, the binding mode in the CG simulations seem to be an artifact.

The results reported in this study should be better compared to available experimental data. For example how does the approach angle compare to cryo-EM structure of the bnAbs engaging with the MPER region, e.g. [2-3]? How do these results from this study compare to previous molecular dynamics studies, e.g.[4-5]?

References<br /> (1) Brügger, Britta, et al. "The HIV lipidome: a raft with an unusual composition." Proceedings of the National Academy of Sciences 103.8 (2006): 2641-2646.<br /> (2) Rantalainen, Kimmo, et al. "HIV-1 envelope and MPER antibody structures in lipid assemblies." Cell Reports 31.4 (2020).<br /> (3) Yang, Shuang, et al. "Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack." Nature Communications 13.1 (2022): 6393.<br /> (4) Carravilla, Pablo, et al. "The bilayer collective properties govern the interaction of an HIV-1 antibody with the viral membrane." Biophysical Journal 118.1 (2020): 44-56.<br /> (5) Pinto, Dora, et al. "Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01." Cell host & microbe 26.5 (2019): 623-637.

Considering reviewer suggestions, we slightly reorganized the results section into specific sub-sections with headings and changed the order in which key results were presented to allow the subsequent analysis more accessible for readers.  Supplemental materials were redistributed into eLife format, having each supplemental item grouped to a corresponding main figure. Many slightly detail modifications were made to figures (mostly supplemental items) without changing their character, such as clearer axes labels or revised annotations within panels.

The major additions within the results sections based on the reviews were:

(1) An expanded the comparison between our simulation analyses to previous simulations and to existing cryo-EM structural evidence for MPER antibodies’ membrane orientation the context of full-length antigen, resulting in new supplemental figure panels.

(2) New atomistic simulations of 10E8, PGZL1, and 4E10 evaluating the phospholipid binding predictions in a different lipid composition more closely modeling HIV membranes.

Minor edits to the analyses and interpretations include:

(1) Outlining the geometric components contributing to variance in substates after clustering the atomistic 10E8, 4E10, and PGZL1 simulations.

(2) Better defining the variance and durability of membrane interactions within and across systems in the coarse grain methods section.

(3) Removed interpretations in the original results sections regarding polyreactivity and energetics for MPER bnAbs that were not explicitly supported by data.   

(4) More context of the prevenance of bnAb loop geometries in structural informatics section

(5) Rationale for the choice of the continuous helix MPER-TM conformation in LN01-antigen conformations, and citations to previous gp41 TM simulations.

(6) Removed language on the novelty of the coarse grain and steered pulling simulations as newly developed approaches; tempering the potential discriminating power and applications of those approaches, in light of their limitations.

The discussion was revised to provide more novel context of the results within the field, including discussing direct relevance of the simulation methods for evaluating immune tolerance mechanisms and into antibody engineering.   We have shared custom scripts used for molecular dynamics analysis on github (https://github.com/cmaillie98/mper_bnAbs.git) and uploaded trajectories to a public repository hosted on Zenodo (https://zenodo.org/records/13830877).

Recommendations for the authors:

Below, I provide an extensive list of minor edits associated with the text and figures for the authors to consider. I provide these with the hope of increasing the accessibility of the manuscript to broader audiences but leave changes to the discretion of the authors.

Text/clarity

Figure 1 main text

The main text discussing Figure 1 is disorganized, making the analysis difficult to follow. I would suggest the following: moving the sentence, "4E10 and PG2L1 are structurally homologous" immediately after the paragraph discussing the simulation initiation. Then, add a sentence that directly compares their experimental affinity, neutralization, and polyreactivity of 4E10 and PG2L1 (later, an unintroduced idea pops up, "These patterns may in part explain 4E10's greater polyreactivity"). Next, lead into the discussion of the MD simulation data with something to the effect of: "Given these similarities, we first compared mechanisms of membrane insertion between 4E10 and PG2L1 to bolster confidence in our predictions". Later, the sentence "Across 4E10 and PGZL1 simulations, the bound lipid phosphates"

We thank the reviewer for the suggestion and we have restructured the beginning of the results to implement this style: to first introduce then discuss the comparative PGZL1 & 4E10 results, i.e. Figure 1 plus associated supplements.

In the background and the introduction text leading up to Figure 1, CDR-H3 is discussed at length, however, the first figure focuses almost entirely on how CDR-H1 coordinates a lipid phosphate headgroup. Are there experimental mutations in this loop that do not affect affinity (e.g., to a soluble gp41 peptide), but do affect neutralization (like the WAWA mutation for CDR-H3, discussed later)?

We have altered the Introduction (para 2) and Results (4E10/PGZL1 sub-section) to give more balanced discussion of CDRs H1 & H3.  That includes referencing experimental data addressing the reviewer’s question; a PGZL1 clone H4K3 where mutations to CDRH1 were introduced and shown have minimal impact on affinity to MPER peptide via ELISA and BLI, but those mutant bnAbs had significantly reduced neutralization efficacy (PMC6879610).

The sentence "These phospholipid binding events were highly stable, typically persisting for hundreds of nanoseconds" should be moved down to immediately precede, "[However], in a PGZL1 simulation, we observed a". This would be a good place for a paragraph break following, "Thus, these bnABs constitutively", since this block of text is very long.

Similarly, the sentence and parts of the section, "Likewise, the interactions coordinating the lipid phosphate oxygens at CDR-H1" more appropriately belongs immediately before or after the sentence, "Our simulations uncover the CDR-lipid interactions that are the most feasible".

Thank you for the detailed guidance in reorganizing the Figure 1 results.  We followed the advice to directly compare 4E10 and PGZL1 results separately from 10E8, moving those sections of text appropriately.  New paragraph breaks were added to improve accessibility and flow of concepts throughout the Results.

In the sentence, "our simulations uncover CDR-lipid interactions that are the most feasible and biologically relevant in the context of a full [HIV] lipid bilayer... validation to which of the many possible ions" à have you confidently determined lipid binding and positioning outside of the site validated in figure 1? Which site(s) are these referencing? The next two sentences then introduce two new ideas on the loop backbone stability then lead into lipid exchange, which is a bit jarring.

We have adjusted the language concerning the putative ions/lipids electron density across the many PGZL1 and 4E10 crystal structures, and additionally make the explicit point that we confidently determined the lack of lipid binding outside of the site focused on in Figure 1.

“… both bnAbs showed strong hotspots for a lipid phosphate bound within the CDR-H1 loops, with minimal phospholipid or cholesterol ordering around the proteins elsewhere.  The simulated lipid phosphates bound within CDR-H1 have exceptional overlap with electron densities and atomic details of modelled headgroups from respective lipid-soaked co-crystal structures…”

Figure 2 main text

"We similarly investigated bnAb 10E8" - Please make this a separate subheader, the block text is very long up to this point.

Thank you for the suggestion. We introduced a sub-header to separate work on 10E8 all-atom simulations.

"we observed a POPC complexed with... modelled as headgroup phosphoglycerol anions..." - please cite the references within the text.

Thank you for pointing out this missing reference, we added the appropriate reference.

"One striking and novel observation" - please remove the phrase "striking" throughout, for following best practices in scientific writing (PMC10212555)-this is generally well-done throughout.

We removed “striking” from our text per your suggestion.

"This CDR-L1 site highlights... (>500 fold) across HIV strains" - How much do R29 and Y32 also contribute to antigen binding and the conformation of this loop? These mutants also decreased Kd by approximately 20X, and based on the co-crystal structure with the TM antigen (PDB: 4XCC), seem to play a more direct role in antigen contact. Additionally, these residues should be highlighted on a figure, otherwise it's difficult to understand why they are important for membrane association.

We thank the reviewer for deep engagement to these supporting experimental details.  The R29A+Y32A 10E8 mutant referenced in the text showed only 4-fold Kd increase, a modest change for an SPR binding experiment.  Whereas R29E+Y32E 10E8 mutant resulted in 40x Kd increase, the “20x” the reviewer refers to.  Both 10E8 mutants showed similar drastically reduced breadth and potency of over 2 orders of magnitude on average.

These mutated CDR-L1 residues are not directly involved in antigen contact and adopt the same loop helix conformation when antigen is bound.  A minor impact on antigen binding affinity could be due altering pre-organization of CDR loops upon losing interactions from the Tyr & Arg sidechains - particularly Tyr31 in contact with CDR-H3.

As per the suggestion, clearer annotated figure panel denoting these sidechains has been added to Figure 2-Figure Supplement 1 for 10E8 analysis.

"Structural searches querying... identified between 10^5 and 2*10^6..." - why is this value represented as such a large range? Does this depend on the parameters used for analysis? Please clarify.

Additionally, how prevalent are any random loop conformations compared to the ones you searched? It's otherwise difficult to attribute number of occurrences within the 2 A cutoff to biological significance, as this number is not put in context.

We appreciate the reviewers comment to contextualize the range and relative frequency of the bnAb loop conformations.   RMSD and length of loop are the key parameters, which can be controlled by searching reference loops of similar length.  The main point of the backbone-level searching is simply to imply the bnAb loops are not particularly rare when comparing loops of similar length.   

We did as was suggested and added comparison to random loops of the same length to the main text, including a new Supplementary Table 4.   

“…identified between 105 to 2∙106 geometrically similar sub-segments within natural proteins (<2 Å RMSD)40, reflecting they are relatively prevalent (not rare) in the protein universe, comparing well with frequency of other surface loops of similar length in antibodies (Supplementary Table 3).”

"We next examined the geometries" could start after its own new subheading. Moreover, while there's an emphasis on tilt for neutralization, there is not a figure clearly modelling the proposed Env tilt compared to the relatively planar bilayer. It would be helpful to have an additional panel somewhere that shows the orientation of the antibody (e.g., a representative pose) in the simulations relative to an appropriately curved membrane, Env, the binding conformation of the antibody to Env, and apo Env, given the tilting observed in PMID: 32348769 and theorized in PMC5338832. What additional conformational changes or tilting need to occur between the antibodies and Env to accomplish binding to their respective epitopes?

Thank you for outlining an interesting element to consider in our analysis of a multi-step binding mechanism for MPER antibodies. We added additional figure panels in the supplement to outline the similarities and differences between our simulations and Fabs with the inferred membranes in cryo-EM experiments of full-length HIV Env.  The simulated Fabs’ angles are very similar with only minor tilting to match the cryo-EM antibody-membrane geometries. 

We added Figure 1-figure supplement 1A & Figure 2-figure supplement 2A, and alter to text to reflect this:

“The primary difference is Env-bound Fabs in cryo-EM adopt slightly more shallow approach angles (~15_°_) relative to the bilayer normal.  The simulated bnAbs in isolation prefer orientations slightly more upright, but presenting CDRs at approximately the same depth and orientation.  Thus, these bnAbs appear pre-disposed in their membrane surface conformations, needing only a minor tilt to form the membrane-antibody-antigen neutralization complex.”   

Env tilt dynamics and membrane curvature of natural virions may reconcile some of these differences.  Recent in situ tomography of Full-length Env in pseudo-virions corroborates our approximation of flat bilayers over the short length scales around Env.

The sentence "we next examined the geometries" mentions "potential energy cost, if any, for reorienting...". However, there's no further discussions of geometry or energy cost within this section. Please rephrase, or move this figure to main and increase discussion associated with the various conformational ensembles, their geometry, and their phospholipid association.

As the reviewer highlights, the unbiased simulations and our analysis do not explicitly evaluate energetics.  We removed this phrase, and now only allude to the minimal energy barrier between the similar geometric conformations, relative to the tilting & access requirements for antigen binding mechanism.

“The apparent barrier for re-orientation is likely much less energetically constraining than shielding glycans and accessibility of MPER”

".. describing the spectrum of surface-bound conformations" cites the wrong figure.

Thank you for noticing this error; we correct the figure reference to (Figure 2-figure supplement 4).

Please comment on the significance of how global clustering (Fig. S5A-C) was similar for 4E10 and PGZL1, but different for 10E8 (e.g., blue, orange, and yellow clusters for 4E10 and PHZL1 versus cyan, red, and green clusters for 10E8). As the cyan cluster seems to be much closer in Euclidian space to the 4E10/PGZL1 clusters, it might warrant additional analysis. What do these clusters represent in terms of structure/conformation? How do these clusters differ in membrane insertion as in (A)?

We are grateful you identify analysis in the geometric clustering section that may be of interest to other readers. We have added additional supplementary table (Table 2) to detail the CDR loop membrane insertion and global Fab angles which describe each cluster, to demonstrate their similarities and differences.  We also better describe how global clustering was similar for 4E10 and PGZL1, but different for 10E8 in the relevant results section<br /> The cyan cluster is not close in structure to 4E10/PGZL1 clusters.  We note the original figure panel had an error.  The updated Figure 2-supplement 4B shows the correct Euclidian distance hierarchy with an early split between 4e10/pgzl1 and 10e8 clusters.

Figure 3 main text

The start of this section, "We next studied bnAb LN01...", is a good place for a new subheader.

We have added an additional subheader here: Antigen influence on membrane bound conformations and lipid binding sites for LN01

There should be a sentence in the main text defining the replicate setup and production MD run time. Is the apo and complex based on a published structure? How do you embed the MPER? Is the apo structure docked to membrane like in 4E10? The MD setup could also be better delineated within the methods.

The first two paragraphs in this section have been updated to clarify the relevant simulations configuration and Fab membrane docking prediction details. 

The procedure was the same for predicting an initial membrane insertion, albeit now we use the LN01-TM complex and the calculation will account for the membrane burial of the the TM domain and MPER fragment.  As mentioned, LN01 is predicted as inserted with CDR loops insert similarly with or without the TM-MPER fragment.  The geometry differs from PGZL1/4E10 and 10E8, denoted by the text.

Please comment on the oligomerization state of the antigen used in the MD simulation: how does the simulation differ from a crossed MPER as observed in an MPER antibody-bound Env cryo-EM structure (PMID: 32348769), a three-helix bundle (PMC7210310), or single transmembrane helix (PMC6121722)? How does the model MPER monomer embed in the membrane compared to simulations with a trimeric MPER (PMC6035291, PMID: 33882664)-namely, key arginine residues such as R696?

We thank the reviewer for pointing out critical underlying rationale for modeling this TM-MPER-LN01 complex which we have corrected in the revised draft. The range of potential conformations and display of MPER based on TM domain organization could easily be its own paper – we in fact have a manuscript in preparation on the topic.  

The updated text expands the rationale for choosing the monomeric uninterrupted helix form of the MPER-TM model antigen (para 1 of LN01 section). The alternative conformations we did not to explore are called out, with references provided by the reviewer.

The discussion qualified that the MPER presentation is likely oversimplified here, noting MPER display in the full-length Env trimer will vary in different conformational states or membrane environments. However, the only cryo-EM structures of full-length ENV with TM domains resolved have this continuous helix MPER-TM conformation – seen both within crossing TM dimers or dissociated TM monomers.

Are there additional analyses that can validate the dynamics of the MPER monomer in the membrane and relative to LN01? Such as key contacts you would expect to maintain over the duration of the MD simulation?

We also increased description of this TM domain’s behavior, dynamics (tilt, orientation, Arg696 snorkeling, and complex w LN01) to provide a clearer picture of the simulation results – which aligns with past MD of the gp41 TM domain as a monomer (para 2 of LN01 section).  As well, we noted key LN01-MPER contacts that were maintained.

How does the model MPER modulate membrane properties like lipid density and lipid proximities near LN01?

We checked and didn’t notice differences for the types of lipids (chol, etc) proximal to the MPER-TM or the CDR loops versus the bulk lipid bilayer distributions.  Due to the already long & detailed nature of this manuscript, we elect not to include discussion on this topic.

Supplemental figure 1H-I would be better positioned as a figure 3-associated supplemental figure.

We rearranged to follow the eLife format and have paired supplemental panels with their most relevant main figures.

Figure 3F/H reference a "loading site" but this site is defined much later in the text, which was confusing.

Thank you for pointing out this source of confusion, we rearranged our discussion to reflect the order in which we present data in figures.

What evidence suggests that lipids "quickly exchange from the Loading site into the X-ray site by diffusion"? I do not gather this from Figure S1H/I.

We have rearranged the loading side and x-ray site RMSD maps in Figure 3-Figure supplement 1 to better illustrate how a lipid exchanges between these sites.

Figure 4 main text

The authors assert that in the CG simulations, restraints, "[maintain] Fab tertiary and quaternary structure". However, backbone RMSD does not directly assert this claim-an additional analysis of the key interfacial residues between chains, or geometric analysis between the chains, would better support this claim.

Thank you for pointing this point.  We rephrased to add that the major sidechain contacts between heavy and light chain persist, in addition to backbone RMSD, to describe how these Fabs maintain the fold stably in CG representation. 

In several cases, CG models sample and then dissociate from the membrane. In the text, the authors mention, "course-grained models can distinguishing unfavorable and favorable membrane-bound conformations". Is there a particular orientation that causes/favors membrane association and dissociation? This analysis could look at conformations immediately preceding association and dissociation to give clues as to what orientation(s) favor each state.

Thank you for suggesting this interesting analysis.  Clustering analysis of associated states are presented in Figure 5, Figure 5-Figure Supplement 1, and Figure 6, which show all CDR and framework loop directed insertion.  This feature is currently described in the main text.  

We did not find strong correlation of specific orientations as “pre-dissociation” states or ineffective non-inserting “scanning” events.  We revised the key sentence to reflect the major take away – that non-CDR alternative conformations did not insert and most of those having CDRs inserted in a different manner than all-atom simulations also were prone to dissociate:

“Given that non-CDR directed and alternative CDR-embedded orientations readily dissociate, we conclude that course-grained models can distinguish unfavorable and favorable membrane-bound conformations to an extent that provides utility for characterizing antibody-bilayer interaction mechanisms.”

Figure 6 main text

"For 4E10, trajectories initiated from all three geometries..." only two geometries are shown for each antibody. Please include all three on the plot.

The plots include markers for all three geometries for 4E10, highlighted in stars or with letters on the density plots of angles sampled (Figure 6B,C)

"Aligning a full-length IgG... unlikely that two Fabs simultaneously..." Are there theoretical conformations in which two Fabs could simultaneously associate with membrane? If this was physiological or could be designed rationally, could an antibody benefit further from avidity?

Our modeling suggests the theoretical conformations having two Fabs on the membrane are infeasible.  It’s even less likely multiple Env antigens could be engaged by one IgG.  We have revised the text to express this more clearly.

Figure 7 main text

"An intermediate... showed a modest reduction in affinity..." what affinity does PGZL1 have for this antigen?

The preceding sentence for this information: “Mature PGZL1 has relatively high affinity to the MPER epitope peptide (Kd = 10 nM) and demonstrates great breadth and potency, neutralizing 84% of a 130 strain panel “

Figures

Figure 1

It would be helpful to have an additional panel at the top of this figure further zoomed out showing the orientation of the antibody (e.g., a representative pose) in the simulations relative to an appropriately curved membrane, Env, the binding conformation of the antibody to Env, and apo Env, given the tilting observed in PMID: 32348769 and theorized in PMC5338832. What additional conformational changes or tilting need to occur between the antibodies and Env to accomplish binding to their respective epitopes?

Thank you for the suggestion to include this analysis.  We have added to the text reflecting this information, as well as making new supplemental panels for 4E10 and 10E8 that we compare simulated 4E10 and 10E8 Fab conformations to cryoEM density maps with Fabs bound to full-length HIV Env. Figure 1-figure supplement 1A & Figure 2-figure supplement 2A

In Figure 1, space permitting, it would be helpful to annotate the distances between the phosphates and side chains (similarly, for Figure S1A).

To avoid the overloading the Main figure panels with text, those relevant distances are listed in the methods sections.  Those distances are used to define the “bound” lipid phosphate state.  Generally, we note the interactions are within hydrogen bonding distance.

Annotating "Replicate 1" and "Replicate 2" on the left side of Figure 1C/D would make this figure immediately intuitive.

We have added these labels.

Figure caption 1C: Please clarify the threshold/definition of a contact used to binarize "bound" versus "unbound" (for example, "mean distance cutoff of 2A between the phosphate oxygen and the COM of CDR-H1") [on further reading of the methods section, this criterion is quite involved and might benefit from: a sentence that includes "see methods"]. Additionally, C could use a sentence explaining the bar such as in E, "Phosphate binding is mapped to above each MD trajectory" Please define FR-H3 in the figure caption for E/F.

We have added these details to the figure caption.

Because Figure 1 is aggregated simulation time, it would be helpful to also represent the data as individual replicates or incorporate this information to calculate standard deviations/statistics (e.g., 1 microsecond max using the replicates to compute a standard deviation).

We believe the current quantification & display of data via sharing all trajectories is sufficient to convey the major point for how often each CDR-phosholipid binding site it occupied.  Further tracking and statistics of inter-atomic distances will likely be too tedious & add minimal value. There is some dynamics of the phosphate oxygens between the polar within the CDR site but our “bound” state definitions sufficiently describe the key participating interactions are made.

Figure 2

For A, it would be helpful to annotate the yellow and blue mesh on the figure itself.

We have defined the orange phosphate and blue choline densities.

Also, where are R29 and Y32 relative to this site? In the X-ray panels, Y38 is not shown, and the box delineating the zoom-in is almost imperceptible.

Thank you for this suggestion to include those amino acids which are referenced in the text as critical sites where mutation impacts function. To clarify, Y32 is the pdb numbering for residue Y38 in IMGT numbering. We have added a panel to Figure 2-Figure Supplement 1 having a cartoon graphic of 10E8 loop groove with sidechains & annotating R29 and Y38, staying consistent with out use of IMGT numbering in the manuscript.

Figure 3

It might read clearer to have "LN01+MPER-TM" and "LN01-Apo" in the middle of A/B and C/D, respectively, and a dotted line delineating the left and right side of the figure panels.

We have added these details to the figure for clarity for readers.

It would be helpful to show some critical interactions that are discussed in the text, such as the salt bridge with K31, by labeling these on the figure (e.g., in E-H).

We drafted figure panels with dashed lines to indicate those key interactions.  However, they became almost imperceptible and overloaded with annotations that distracted from the overall details.  For K31, the interaction occurs in LN01 crystal structures readers can refer to.

Why are axes cut off for J?

We corrected this.

Please re-define K/L plots as in Figure 1, and explain abbreviations.

We updated the figure caption to reflect these changes.

Figure 4

The caption for panel A states that the Fab begins in solvent 1-2 nm above the bilayer, but the main text states 0.5-2 nm.

We have reconciled this difference and listed the correct distances: 0.5-2nm.

Please label the y-axis as "Replicate" for relevant figure panels so that they are more immediately interpretable.

This label has been added.

A legend with "membrane-associated" and "non-associated" within the figure would be helpful. Additionally, the average percent membrane associated, with a standard deviation, should be shown (Similar to 1C, albeit with the statistics).

This legend has been added.  We also added the additional statistical metrics requested to strengthen our analysis.

The text references "10, 14, and 12 extended insertion events" for the three antibody-based simulations. How do you define "extended insertion events"? Would breaking this into average insertion time and standard deviation better highlight the association differences between MPER antibodies and controls, in addition to the variability due to difference random initialization?

We thank the reviewer for the insightful suggestion on how to better organize quantitative analysis to support the method. Supplemental Table 3 includes these numbers.

Figure 5

The analysis in Fig. S6C could be included here as a main figure.

The drafted revised figure adding S6C to Figure 5 made for too much information.  Likewise, putting this panel S6C separated it from the parent clustering data of S6B, so we decided to keep these figures separated.  The S6 figure is now Figure 5-figure supplement 1.

Figure 6

Please annotate membrane insertion on E as %.

These are phosphate binding RMSD/occupancy vs time.  The panels are now too small to annotate by %.  The qualitative presentation is sufficient at this stage.  The quantitative % are listed in-line within text when relevant to support assertions made. 

Please use the figure caption to explain why certain clusters (e.g., 10E8 cluster A, artifact, Fig. S6E) are not included in panel E.

We have added this information in the figure caption.

Figure 7

Please show all points on the box and whisker plots (panels E and F), and perform appropriate statistical tests to see if means are significantly different (these are mentioned in the text, but should be annotated on the graph and mentioned within the figure caption).

We have changed these plots to show all data points along with relevant statistical comparisons. The figure captions describe unpaired t-test statistical tests used.

Figure S1

G, H, and I do not belong here-they should be moved to accompany their relevant text section, which associates with Figure 3. It would be helpful to associate this with Figure 3 in the eLife format, "Figure 3-Supplemental Figure 1" or its equivalent.

It's very difficult to distinguish the green and blue circles on panel G.

We darkened the shading and added outline for better visualization

Subfigure I is missing a caption, could be included with H: "(H,I) Additional replicates for LN01+TM (H) and LN01 (I)".

We corrected this as suggested.

Why is H only 3 simulations and not 4? Does it not have a lipid in the x-ray site? Also, the caption states "(top, green)" and "(bottom, cyan)", but the green vs. cyan figures are organized on the left and right. Additional labels within the figure would help make this more intuitive.

If the point of H and I is to illustrate that POPC exchanges between the X-ray and loading sites, this is unclear from the figure. Consider clarifying these figures.

Thank you for describing the confusion in this figure, we have added labels to clarify.

Figure S2 (panels split between revised Figure 4 associated figure supplements)

The LN01 figures should likely follow later so that they can associate with Figure 3, despite being a similar analysis.

We corrected supplements to eLife format so supplements are associated with relevant main figures.

Figure S3 (panels split between revised Figure 1 & 2 associated figure supplements)

As hydrophobicity is discussed as a driving factor for residue insertion, it would be helpful to have a rolling hydrophobicity chart underneath each plot to make this claim obvious.

We prefer the current format, due to the worry of having too much information in these already data-rich panels.  As well, residues are not apolar but are deeply inserted.

Figure S4 (panels split between revised Figure 1 & 2 associated figure supplements)

It would be helpful to label the relevant loops on these figures.

We have labeled loops for clarity.

Do any of these loops have minor contacts with Env in the structure?

The 4E10 and PGZL1 CDRH-1 loop does not directly contact bound MPER peptides bound in crystal structures. 

FRL-3 and CDR-H1 in 10E8 do not contact the MPER peptide antigen component based on x-ray crystal structures.

Do motif contacts with lipid involve minor contacts with additional loops other than those displayed in this figure?

The phosphate-loop interactions in motifs used as query bait here are mediated solely by the backbone and side chain interactions of the loops displayed. We visually inspected most matches and did not see any “consensus” additional peripheral interactions common across each potential instance in the unrelated proteins.  The supplied Supplemental Table 2 contains the information if a reader wanted to conduct a detailed search. 

Why is there such a difference between the loop conformation adopted in the X-ray structure and that in the MD simulation, and why does this lead to the large observed differences in ligand-binding structure matches?

We thank the reviewer for carefully noting our error in labeling of CDR loop and framework region input queries. We revised the labeling to clarify the issue.

The is minimal structural difference between the loops in x-ray and MD.

Figure S5 (Figure 2-Figure supplement 4)

This figure is not colorblind friendly-it would be helpful to change to such a pallet as the data are interesting, but uninterpretable to some.

We have left this figure the same.

"Susbstates" - "Substates"

Corrected, thank you.

Panel B is uninterpretable-please break the axis so that the Euclidian distances can be represented accurately but the histograms can be interpreted.

We have adjusted axis for this plot to better illustrate the cluster thresholds.

The clusters in D-H should be analyzed in greater depth. What is the structural relevance of these clusters other than differences in phospholipid occupancy in (I)? Snapshots of representative poses for each cluster could help clarify these differences.

We have adjusted the text to describe the geometric differences in each of those clusters that result in the different exceptionally lower propensities for forming the key phospholipid interaction.  

The figure caption should make it clear that 3 μS of aggregate simulation time is being used here instead of 4 μS to start with unique tilt initializations. E.g., "unique starting membrane-bound conformations (0 degrees, -15 degrees, 15 degrees initialization relative to the docked pose)". Further, why was the particular 0-degree replicate chosen while the other was thrown out? Or was this information averaged? Why is the full 4 μS then used for D-I?

We thank the reviewer for noting these details.  We didn’t want to bias the differential between 10E8 and 4E10/PGZL1 by including the replicate simulations.  The analysis was mainly intended to achieve more coarse resolution distinction between 10E8 and the similar PGZL1/4E10.  

In the subsequent clustering of individual bnAb simulation groups, the replicate 0 degree simulations had sufficiently different geometric sampling and unique lipid binding behavior that we though it should be used (4 us total) to achieve finer conformational resolution for each bnAb.

Figure S6 (now Figure 5-Figure Supplement 1)

Please label the CDRs in C and provide a color key like in other figures. Also, please label the y-axes. This figure could move to main below 5B with the clusters "A,B,C" labeled on 5B.

We have added the axes labels and color key legend.  We retained a minimal CDR loop labeling scheme for the more throughput interaction profiles here where colored sections in the residue axes denote CDR loop regions.

Figure S7 (Figure 7 Figure Supplement 1)

Panels A and B would likely read better if swapped.

We have swapped these panels for a better flow.

For panel C, please display mean and standard deviation, and compare these values with an appropriate statistical test.

This is already displayed in main figure, we have removed it from supplement.

For E and F, please clarify from which trajectory(s) you are extracting this conformation from. Are these the global mean/representative poses? How do they compare to other geometrically distinct clusters?

The requested information was added to supplemental figure caption.  These are frames from 2 distinct time points selected phosphate bound frames from 0-degree tilt replicates for both 4E10 and 10E8, representing at least 2 distinct macroscopic substates differing in global light chain and heavy chain orientation towards the membrane. 

Table S2 (now Supplementary Table 3)

Please add details for the 13h11 simulation.

Additionally, please add average contact time and their standard deviation to the table, rather than just the aggregated total time. This will highlight the variability associated with the random initializations of each simulation.

We have added the details for 13h11 and the requested analysis (average aggregated time +/- standard deviation and average time per association event +- standard deviation) to supplement our summary statistics for this method.

Reviewer #2 (Recommendations For The Authors):

(1) The structure of the manuscript should be improved. For example, almost half of the introduction (three paragraphs) summarize the results. I found it hard to navigate all the data and specific interactions described in the result section. Furthermore, the claims at the end of several sections seem unsupported. Especially for the generalization of the approach. This should be moved to the discussion section. The discussion is pretty general and does not provide much context to the results presented in this study.

We have significantly reorganized the results section to improve the flow of the manuscript and accessibility for readers, especially the first sections of all-atom simulations. We also removed claims not directly supported by data from our results, and expanded on some of these concepts in the discussion to make some more novel context to the result.

(2) The author should cite more rigorously previous work and refrain from using the term "develop" to describe the simple use of a well established method. E.g. Several studies have investigated membrane protein interactions e.g. [1], membrane protein-bilayer self-assembly [2], steered molecular dynamics [3], etc.

Thank you for identifying relevant work for the simulations that set precedent for our novel application to antibody-membrane interactions.  We have removed language about development of simulation methods from the text and now better reference the precedent simulation methods used here.

(3) Have the authors considered estimating the PMF by combining the steered MD simulation through the application of Jarzynski's equality?

We performed from preliminary PMFs for Fab-membrane binding, but saw it was taking upward of 40 us to reach convergence.  Steered simulations focus on a key lipid may be easier.

Although PMFs are beyond the scope of this work, we added proposals & allusion to their utility as the next steps for more rigorous quantification of fab-membrane interactions.

Minor

(4) The term "integrative modeling" is usually used for computational pipelines which incorporate experimental data. Multiscale modeling would be more appropriate for this study.

We altered descriptions throughout the manuscript to reflect this comment.

(5) Units to report the force in the steered molecular dynamics are incorrect. They should be 98.

We changed axes and results to correctly report this unit.

(6) Labels for axes of several graphs are not missing.

We added labels to all axes of graphs, except for a few where stacked labels can be easily interpreted to save space and reduce complexity in figures.

(7) Figure 3 K & L is this really < 1% of total? The term "total" should also be clarified.

Thank you for pointing this out, we changed the % labels to be correct with axes from 0-100%. We clarified total in the figure caption.

(8) The font size in figures should be uniformized.

This suggestion has been applied

(9) Time needed for steered MD should be reported in CPUh and not hours (page 17).

We removed comments on explicit time measurements for our simulations.

(10) Version of Martini force field is missing in methods section

We used Martini 2.6 and added this to the methods.

References

(1) Prunotto, Alessio, et al. "Molecular bases of the membrane association mechanism potentiating antibiotic resistance by New Delhi metallo-β-lactamase 1." ACS infectious diseases 6.10 (2020): 2719-2731.

(2) Scott, Kathryn A., et al. "Coarse-grained MD simulations of membrane protein-bilayer self-assembly." Structure 16.4 (2008): 621-630.

(3) Izrailev, S., et al. "Computational molecular dynamics: challenges, methods, ideas. Chapter 1. Steered molecular dynamics." (1997).

Reviewer #2 (Public review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The use of multiscale MD simulations allows for a detailed exploration of the system at different time and length scales. The combination of MD simulations and structural bioinformatics provides a comprehensive approach to validate the identified binding sites. Finally, the steered MD simulations offer quantitative insights into the binding strength between the membrane and bnAbs.

While the simulations and analyses provide qualitative insights into the binding interactions, they do not offer a quantitative assessment of energetics. The coarse-grained simulations exhibit artifacts and thus require careful analysis.

This study contributes to a deeper understanding of the molecular mechanisms underlying bnAb recognition of the HIV-1 envelope. The insights gained from this work could inform the design of more potent and broadly neutralizing antibodies.

Reviewer #1 (Public review):

The authors in this paper investigate the nature of the activity in the rodent EPN during a simple freely moving cue-reward association task. Given that primate literature suggest movement coding whereas other primate and rodent studies suggest mainly reward outcome coding in the EPNs, it is important try to tease apart the two views. Through careful analysis of behavior kinematics, position, and the neural activity in the EPNs, the authors reveal an interesting and complex relationship between the EPN and mouse behavior.

Strengths:

(1) The authors use a novel freely moving task to study EPN activity, which displays rich movement trajectories and kinematics. Given that previous studies have mostly looked at reward coding during head fixed behavior, this study adds a valuable dataset to the literature.

(2) The neural analysis is rich and thorough. Both single neuron level and population level (i.e. PCA) analysis are employed to reveal what EPN encodes.

Discussion:<br /> EPN is one of the major output nuclei of the basal ganglia. What information is present within EPN is still unclear, and under investigation. The authors have used electrophysiology to determine the nature of information present within EPN that is likely to be valuable to the field. Future studies should try to address whether this information is specific to certain cell types within EPN or whether there is topography within EPN that reflects the kinematic information present within EPN. This will require more careful dissection of EPN activity based on anatomy. Future experiments should also consider tasks that isolate a single limb (i.e. joystick tasks) in order to better understand the kinematic encoding of forelimb movement. This, combined with recording in forelimb encoding region of EPN, should give us insights into the nature of kinematic control of EPN. Overall, this study will be useful to inspire future investigations in the function of EPN.

Reviewer #2 (Public review):

This paper examined how the activity of neurons in the entopeduncular nucleus (EPN) of mice relates to kinematics, value, and reward. The authors recorded neural activity during an auditory cued two-alternative choice task, allowing them to examine how neuronal firing relates to specific movements like licking or paw movements, as well as how contextual factors like task stage or proximity to a goal influence the coding of kinematic and spatiotemporal features. The data shows that the firing of individual neurons is linked to kinematic features such as lick or step cycles. However, the majority of neurons exhibited activity related to both movement types, suggesting that EPN neuronal activity does not merely reflect muscle-level representations. This contradicts what would be expected from traditional action selection or action specification models of the basal ganglia.

The authors also show that spatiotemporal variables account for more variability compared to kinematic features alone. Using demixed Principal Component Analysis, they reveal that at the population level, the three principal components explaining the most variance were related to specific temporal or spatial features of the task, such as ramping activity as mice approached reward ports, rather than trial outcome or specific actions. Notably, this activity was present in neurons whose firing was also modulated by kinematic features, demonstrating that individual EPN neurons integrate multiple features. A weakness is that what the spatiotemporal activity reflects is not well specified. The authors suggest some may relate to action value due to greater modulation when approaching a reward port, but acknowledge action value is not well parametrized or separated from variables like reward expectation.

A key goal was to determine whether activity related to expected value and reward delivery arose from a distinct population of EPN neurons or was also present in neurons modulated by kinematic and spatiotemporal features. In contrast to previous studies (Hong & Hikosaka 2008 and Stephenson-Jones et al., 2016), the current data reveals that individual neurons can exhibit modulation by both reward and kinematic parameters. Two potential differences may explain this discrepancy: First, the previous studies used head-fixed recordings, where it may have been easier to isolate movement versus reward-related responses. Second, those studies observed prominent phasic responses to the delivery or omission of expected rewards - responses that are present but not common in the current paper. This suggests a possibility that the VGlut2+ EPN neurons that project to the LHb were under/not sampled, antidromic or optogenetic tagging would have been needed to confirm the identity of the populations that were recorded. Alternatively, in the head-fixed recordings, kinematic/spatial coding may have gone undetected due to the forced immobility.

Overall, this paper offers needed insight into how the basal ganglia output encodes behavior. The EPN recordings from freely moving mice clearly demonstrate that individual neurons integrate reward, kinematic, and spatiotemporal features, challenging traditional models. However, the specific relationship between the spatiotemporal activity and factors like action value remains unclear.

Author response:

The following is the authors’ response to the previous reviews.

eLife Assessment

This study presents valuable findings on the potential of short-movie viewing fMRI protocol to explore the functional and topographical organization of the visual system in awake infants and toddlers. Although the data are compelling given the difficulty of studying this population, the evidence presented is incomplete and would be strengthened by additional analyses to support the authors' claims. This study will be of interest to cognitive neuroscientists and developmental psychologists, especially those interested in using fMRI to investigate brain organisation in pediatric and clinical populations with limited fMRI tolerance.

We are grateful for the thorough and thoughtful reviews. We have provided point-bypoint responses to the reviewers’ comments, but first, we summarize the major revisions here. We believe these revisions have substantially improved the clarity of the writing and impact of the results.

Regarding the framing of the paper, we have made the following major changes in response to the reviews:

(1) We have clarified that our goal in this paper was to show that movie data contains topographic, fine-grained details of the infant visual cortex. In the revision, we now state clearly that our results should not be taken as evidence that movies could replace retinotopy and have reworded parts of the manuscript that could mislead the reader in this regard.

(2) We have added extensive details to the (admittedly) complex methods to make them more approachable. An example of this change is that we have reorganized the figure explaining the Shared Response Modelling methods to divide the analytic steps more clearly.

(3) We have clarified the intermediate products contributing to the results by adding 6 supplementary figures that show the gradients for each IC or SRM movie and each infant participant.

In response to the reviews, we have conducted several major analyses to support our findings further:

(1) To verify that our analyses can identify fine-grained organization, we have manually traced and labeled adult data, and then performed the same analyses on them. The results from this additional dataset validate that these analyses can recover fine-grained organization of the visual cortex from movie data.

(2) To further explore how visual maps derived from movies compare to alternative methods, we performed an anatomical alignment control analysis. We show that high-quality maps can be predicted from other participants using anatomical alignment.

(3) To test the contribution of motion to the homotopy analyses, we regressed out the motion effects in these analyses. We found qualitatively similar results to our main analyses, suggesting motion did not play a substantial role.

(4) To test the contribution of data quantity to the homotopy analyses, we correlated the amount of movie data collected from each participant with the homotopy results. We did not find a relationship between data quantity and the homotopy results. 

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Ellis et al. investigated the functional and topographical organization of the visual cortex in infants and toddlers, as evidenced by movie-viewing data. They build directly on prior research that revealed topographic maps in infants who completed a retinotopy task, claiming that even a limited amount of rich, naturalistic movie-viewing data is sufficient to reveal this organization, within and across participants. Generating this evidence required methodological innovations to acquire high-quality fMRI data from awake infants (which have been described by this group, and elsewhere) and analytical creativity. The authors provide evidence for structured functional responses in infant visual cortex at multiple levels of analyses; homotopic brain regions (defined based on a retinotopy task) responded more similarly to one another than to other brain regions in visual cortex during movie-viewing; ICA applied to movie-viewing data revealed components that were identifiable as spatial frequency, and to a lesser degree, meridian maps, and shared response modeling analyses suggested that visual cortex responses were similar across infants/toddlers, as well as across infants/toddlers and adults. These results are suggestive of fairly mature functional response profiles in the visual cortex in infants/toddlers and highlight the potential of movie-viewing data for studying finer-grained aspects of functional brain responses, but further evidence is necessary to support their claims and the study motivation needs refining, in light of prior research.

Strengths:

- This study links the authors' prior evidence for retinotopic organization of visual cortex in human infants (Ellis et al., 2021) and research by others using movie-viewing fMRI experiments with adults to reveal retinotopic organization (Knapen, 2021).

- Awake infant fMRI data are rare, time-consuming, and expensive to collect; they are therefore of high value to the community. The raw and preprocessed fMRI and anatomical data analyzed will be made publicly available.

We are grateful to the reviewer for their clear and thoughtful description of the strengths of the paper, as well as their helpful outlining of areas we could improve.

Weaknesses:

- The Methods are at times difficult to understand and in some cases seem inappropriate for the conclusions drawn. For example, I believe that the movie-defined ICA components were validated using independent data from the retinotopy task, but this was a point of confusion among reviewers. 

We acknowledge the complexity of the methods and wish to clarify them as best as possible for the reviewers and the readers. We have extensively revised the methods and results sections to help avoid potential misunderstandings. For instance, we have revamped the figure and caption describing the SRM pipeline (Figure 5).

To answer the stated confusion directly, the ICA components were derived from the movie data and validated on the (completely independent) retinotopy data. There were no additional tasks. The following text in the paper explains this point:

“To assess the selected component maps, we correlated the gradients (described above) of the task-evoked and component maps. This test uses independent data: the components were defined based on movie data and validated against task-evoked retinotopic maps.” Pg. 11

In either case: more analyses should be done to support the conclusion that the components identified from the movie reproduce retinotopic maps (for example, by comparing the performance of movie-viewing maps to available alternatives (anatomical ROIs, group-defined ROIs). 

Before addressing this suggestion, we want to restate our conclusions: features of the retinotopic organization of infant visual cortex could be predicted from movie data. We did not conclude that movie data could ‘reproduce’ retinotopic maps in the sense that they would be a replacement. We recognize that this was not clear in our original manuscript and have clarified this point throughout, including in this section of the discussion:

“To be clear, we are not suggesting that movies work well enough to replace a retinotopy task when accurate maps are needed. For instance, even though ICA found components that were highly correlated with the spatial frequency map, we also selected some components that turned out to have lower correlations. Without knowing the ground truth from a retinotopy task, there would be no way to weed these out. Additionally, anatomical alignment (i.e., averaging the maps from other participants and anatomically aligning them to a held-out participant) resulted in maps that were highly similar to the ground truth. Indeed, we previously[23] found that adult-defined visual areas were moderately similar to infants. While functional alignment with adults can outperform anatomical alignment methods in similar analyses[27], here we find that functional alignment is inferior to anatomical alignment. Thus, if the goal is to define visual areas in an infant that lacks task-based retinotopy, anatomical alignment of other participants’ retinotopic maps is superior to using movie-based analyses, at least as we tested it.” Pg. 21

As per the reviewer’s suggestion and alluded to in the paragraph above, we have created anatomically aligned visual maps, providing an analogous test to the betweenparticipant analyses like SRM. We find that these maps are highly similar to the ground truth. We describe this result in a new section of the results:

“We performed an anatomical alignment analog of the functional alignment (SRM) approach. This analysis serves as a benchmark for predicting visual maps using taskbased data, rather than movie data, from other participants. For each infant participant, we aggregated all other infant or adult participants as a reference. The retinotopic maps from these reference participants were anatomically aligned to the standard surface template, and then averaged. These averages served as predictions of the maps in the test participant, akin to SRM, and were analyzed equivalently (i.e., correlating the gradients in the predicted map with the gradients in the task-based map). These correlations (Table S4) are significantly higher than for functional alignment (using infants to predict spatial frequency, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.44, CI=[0.32–0.58], p<.001; using infants to predict meridians, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.61, CI=[0.47–0.74], p<.001; using adults to predict spatial frequency, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.31, CI=[0.21–0.42], p<.001; using adults to predict meridians, anatomical alignment > functional alignment: ∆_{Fisher Z} M=0.49, CI=[0.39–0.60], p<.001). This suggests that even if SRM shows that movies can be used to produce retinotopic maps that are significantly similar to a participant, these maps are not as good as those that can be produced by anatomical alignment of the maps from other participants without any movie data.” Pg. 16–17

Also, the ROIs used for the homotopy analyses were defined based on the retinotopic task rather than based on movie-viewing data alone - leaving it unclear whether movie-viewing data alone can be used to recover functionally distinct regions within the visual cortex.

We agree with the reviewer that our approach does not test whether movie-viewing data alone can be used to recover functionally distinct regions. The goal of the homotopy analyses was to identify whether there was functional differentiation of visual areas in the infant brain while they watch movies. This was a novel question that provides positive evidence that these regions are functionally distinct. In subsequent analyses, we show that when these areas are defined anatomically, rather than functionally, they also show differentiated function (e.g., Figure 2). Nonetheless, our intention was not to use the homotopy analyses to define the regions. We have added text to clarify the goal and novelty of this analysis.

“Although these analyses cannot define visual maps, they test whether visual areas have different functional signatures.” Pg. 6

Additionally, even if the goal were to define areas based on homotopy, we believe the power of that analysis would be questionable. We would need to use a large amount of the movie data to define the areas, leaving a low-powered dataset to test whether their function is differentiated by these movie-based areas.

- The authors previously reported on retinotopic organization of the visual cortex in human infants (Ellis et al., 2021) and suggest that the feasibility of using movie-viewing experiments to recover these topographic maps is still in question. They point out that movies may not fully sample the stimulus parameters necessary for revealing topographic maps/areas in the visual cortex, or the time-resolution constraints of fMRI might limit the use of movie stimuli, or the rich, uncontrolled nature of movies might make them inferior to stimuli that are designed for retinotopic mapping, or might lead to variable attention between participants that makes measuring the structure of visual responses across individuals challenging. This motivation doesn't sufficiently highlight the importance or value of testing this question in infants. Further, it's unclear if/how this motivation takes into account prior research using movie-viewing fMRI experiments to reveal retinotopic organization in adults (e.g., Knapen, 2021). Given the evidence for retinotopic organization in infants and evidence for the use of movie-viewing experiments in adults, an alternative framing of the novel contribution of this study is that it tests whether retinotopic organization is measurable using a limited amount of movie-viewing data (i.e., a methodological stress test). The study motivation and discussion could be strengthened by more attention to relevant work with adults and/or more explanation of the importance of testing this question in infants (is the reason to test this question in infants purely methodological - i.e., as a way to negate the need for retinotopic tasks in subsequent research, given the time constraints of scanning human infants?).

We are grateful to the reviewer for giving us the opportunity to clarify the innovations of this research. We believe that this research contributes to our understanding of how infants process dynamic stimuli, demonstrates the viability and utility of movie experiments in infants, and highlights the potential for new movie-based analyses (e.g., SRM). We have now consolidated these motivations in the introduction to more clearly motivate this work:

“The primary goal of the current study is to investigate whether movie-watching data recapitulates the organization of visual cortex. Movies drive strong and naturalistic responses in sensory regions while minimizing task demands[12, 13, 24] and thus are a proxy for typical experience. In adults, movies and resting-state data have been used to characterize the visual cortex in a data-driven fashion[25–27]. Movies have been useful in awake infant fMRI for studying event segmentation[28], functional alignment[29], and brain networks[30]. However, this past work did not address the granularity and specificity of cortical organization that movies evoke. For example, movies evoke similar activity in infants in anatomically aligned visual areas[28], but it remains unclear whether responses to movie content differ between visual areas (e.g., is there more similarity of function within visual areas than between31). Moreover, it is unknown whether structure within visual areas, namely visual maps, contributes substantially to visual evoked activity. Additionally, we wish to test whether methods for functional alignment can be used with infants. Functional alignment finds a mapping between participants using functional activity – rather than anatomy – and in adults can improve signal-to-noise, enhance across participant prediction, and enable unique analyses[27, 32–34].” Pg. 3-4

Furthermore, the introduction culminates in the following statement on what the analyses will tell us about the nature of movie-driven activity in infants:

“These three analyses assess key indicators of the mature visual system: functional specialization between areas, organization within areas, and consistency between individuals.” Pg. 5

Furthermore, in the discussion we revisit these motivations and elaborate on them further:

[Regarding homotopy:] “This suggests that visual areas are functionally differentiated in infancy and that this function is shared across hemispheres[31].” Pg. 19

[Regarding ICA:] “This means that the retinotopic organization of the infant brain accounts for a detectable amount of variance in visual activity, otherwise components resembling these maps would not be discoverable.” Pg. 19–20

[Regarding SRM:] “This is initial evidence that functional alignment may be useful for enhancing signal quality, like it has in adults[27,32,33], or revealing changing function over development[45].” Pg. 21

Additionally, we have expanded our discussion of relevant work that uses similar methods such as the excellent research from Knapen (2021) and others:

“In adults, movies and resting-state data have been used to characterize the visual cortex in a data-driven fashion[25-27].” Pg. 4

“We next explored whether movies can reveal fine-grained organization within visual areas by using independent components analysis (ICA) to propose visual maps in individual infant brains[25,26,35,42,43].” Pg. 9

Reviewer #2 (Public Review):

Summary:

This manuscript shows evidence from a dataset with awake movie-watching in infants, that the infant brain contains areas with distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. However, substantial new analyses would be required to support the novel claim that movie-watching data in infants can be used to identify retinotopic areas or to capture within-area functional organization.

Strengths:

The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. These data position the authors to test their novel claim, that movie-watching data in infants can be used to identify retinotopic areas.

Weaknesses:

To claim that movie-watching data can identify retinotopic regions, the authors should provide evidence for two claims:

- Retinotopic areas defined based only on movie-watching data, predict retinotopic responses in independent retinotopy-task-driven data.

- Defining retinotopic areas based on the infant's own movie-watching response is more accurate than alternative approaches that don't require any movie-watching data, like anatomical parcellations or shared response activation from independent groups of participants.

We thank the reviewer for their comments. Before addressing their suggestions, we wish to clarify that we do not claim that movie data can be used to identify retinotopic areas, but instead that movie data captures components of the within and between visual area organization as defined by retinotopic mapping. We recognize that this was not clear in our original manuscript and have clarified this point throughout, including in this section of the discussion:

“To be clear, we are not suggesting that movies work well enough to replace a retinotopy task when accurate maps are needed. For instance, even though ICA found components that were highly correlated with the spatial frequency map, we also selected some components that turned out to have lower correlations. Without knowing the ground truth from a retinotopy task, there would be no way to weed these out. Additionally, anatomical alignment (i.e., averaging the maps from other participants and anatomically aligning them to a held-out participant) resulted in maps that were highly similar to the ground truth. Indeed, we previously[23] found that adult-defined visual areas were moderately similar to infants. While functional alignment with adults can outperform anatomical alignment methods in similar analyses[27], here we find that functional alignment with infants is inferior to anatomical alignment. Thus, if the goal is to define visual areas in an infant that lacks task-based retinotopy, anatomical alignment of other participants’ retinotopic maps is superior to using movie-based analyses, at least as we tested it.” Pg. 21

In response to the reviewer’s suggestion, we compare the maps identified by SRM to the averaged, anatomically aligned maps from infants. We find that these maps are highly similar to the task-based ground truth and we describe this result in a new section:

“We performed an anatomical alignment analog of the functional alignment (SRM) approach. This analysis serves as a benchmark for predicting visual maps using taskbased data, rather than movie data, from other participants. For each infant participant, we aggregated all other infant or adult participants as a reference. The retinotopic maps from these reference participants were anatomically aligned to the standard surface template, and then averaged. These averages served as predictions of the maps in the test participant, akin to SRM, and were analyzed equivalently (i.e., correlating the gradients in the predicted map with the gradients in the task-based map). These correlations (Table S4) are significantly higher than for functional alignment (using infants to predict spatial frequency, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.44, CI=[0.32–0.58], p<.001; using infants to predict meridians, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.61, CI=[0.47–0.74], p<.001; using adults to predict spatial frequency, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.31, CI=[0.21–0.42], p<.001; using adults to predict meridians, anatomical alignment < functional alignment: ∆_{Fisher Z} M=0.49, CI=[0.39–0.60], p<.001). This suggests that even if SRM shows that movies can be used to produce retinotopic maps that are significantly similar to a participant, these maps are not as good as those that can be produced by anatomical alignment of the maps from other participants without any movie data.” Pg. 16–17

Note that we do not compare the anatomically aligned maps with the ICA maps statistically. This is because these analyses are not comparable: ICA is run withinparticipant whereas anatomical alignment is necessarily between-participant — either infant or adults. Nonetheless, an interested reader can refer to the Table where we report the results of anatomical alignment and see that anatomical alignment outperforms ICA in terms of the correlation between the predicted and task-based maps.

Both of these analyses are possible, using the (valuable!) data that these authors have collected, but these are not the analyses that the authors have done so far. Instead, the authors report the inverse of (1): regions identified by the retinotopy task can be used to predict responses in the movies. The authors report one part of (2), shared responses from other participants can be used to predict individual infants' responses in the movies, but they do not test whether movie data from the same individual infant can be used to make better predictions of the retinotopy task data, than the shared response maps.

So to be clear, to support the claims of this paper, I recommend that the authors use the retinotopic task responses in each individual infant as the independent "Test" data, and compare the accuracy in predicting those responses, based on:

-  The same infant's movie-watching data, analysed with MELODIC, when blind experimenters select components for the SF and meridian boundaries with no access to the ground-truth retinotopy data.

-  Anatomical parcellations in the same infant.

-  Shared response maps from groups of other infants or adults.

-  (If possible, ICA of resting state data, in the same infant, or from independent groups of infants).

Or, possibly, combinations of these techniques.

If the infant's own movie-watching data leads to improved predictions of the infant's retinotopic task-driven response, relative to these existing alternatives that don't require movie-watching data from the same infant, then the authors' main claim will be supported.

These are excellent suggestions for additional analyses to test the suitability for moviebased maps to replace task-based maps. We hope it is now clear that it was never our intention to claim that movie-based data could replace task-based methods. We want to emphasize that the discoveries made in this paper — that movies evoke fine-grained organization in infant visual cortex — do not rely on movie-based maps being better than alternative methods for producing maps, such as the newly added anatomical alignment.

The proposed analysis above solves a critical problem with the analyses presented in the current manuscript: the data used to generate maps is identical to the data used to validate those maps. For the task-evoked maps, the same data are used to draw the lines along gradients and then test for gradient organization. For the component maps, the maps are manually selected to show the clearest gradients among many noisy options, and then the same data are tested for gradient organization. This is a double-dipping error. To fix this problem, the data must be split into independent train and test subsets.

We appreciate the reviewer’s concern; however, we believe it is a result of a miscommunication in our analytic strategy. We have now provided more details on the analyses to clarify how double-dipping was avoided. 

To summarize, a retinotopy task produced visual maps that were used to trace both area boundaries and gradients across the areas. These data were then fixed and unchanged, and we make no claims about the nature of these maps in this paper, other than to treat them as the ground truth to be used as a benchmark in our analyses. The movie data, which are collected independently from the same infant in the session, used the boundaries from the retinotopy task (in the case of homotopy) or were compared with the maps from the retinotopy task (in the case of ICA and SRM). In other words, the statement that “the data used to generate maps is identical to the data used to validate those maps” is incorrect because we generated the maps with a retinotopy task and validated the maps with the movie data. This means no double dipping occurred.

Perhaps a cause of the reviewer’s interpretation is that the gradients used in the analysis are not clearly described. We now provide this additional description:  “Using the same manually traced lines from the retinotopy task, we measured the intensity gradients in each component from the movie-watching data. We can then use the gradients of intensity in the retinotopy task-defined maps as a benchmark for comparison with the ICA-derived maps.” Pg. 10

Regarding the SRM analyses, we take great pains to avoid the possibility of data contamination. To emphasize how independent the SRM analysis is, the prediction of the retinotopic map from the test participant does not use their retinotopy data at all; in fact, the predicted maps could be made before that participant’s retinotopy data were ever collected. To make this prediction for a test participant, we need to learn the inversion of the SRM, but this only uses the movie data of the test participant. Hence, there is no double-dipping in the SRM analyses. We have elaborated on this point in the revision, and we remade the figure and its caption to clarify this point:

We also have updated the description of these results to emphasize how double-dipping was avoided:

“We then mapped the held-out participant's movie data into the learned shared space without changing the shared space (Figure 5c). In other words, the shared response model was learned and frozen before the held-out participant’s data was considered.

This approach has been used and validated in prior SRM studies[45].” Pg. 14

The reviewer suggests that manually choosing components from ICA is double-dipping. Although the reviewer is correct that the manual selection of components in ICA means that the components chosen ought to be good candidates, we are testing whether those choices were good by evaluating those components against the task-based maps that were not used for the ICA. Our statistical analyses evaluate whether the components chosen were better than the components that would have been chosen by random chance. Critically: all decisions about selecting the components happen before the components are compared to the retinotopic maps. Hence there is no double-dipping in the selection of components, as the choice of candidate ICA maps is not informed by the ground-truth retinotopic maps. We now clarify what the goal of this process is in the results:

“Success in this process requires that 1) retinotopic organization accounts for sufficient variance in visual activity to be identified by ICA and 2) experimenters can accurately identify these components.” Pg. 10

The reviewer also alludes to a concern that the researcher selecting the maps was not blind to the ground-truth retinotopic maps from participants and this could have influenced the results. In such a scenario, the researcher could have selected components that have the gradients of activity in the places that the infant has as ground truth. The researcher who made the selection of components (CTE) is one of the researchers who originally traced the areas in the participants approximately a year prior to the identification of ICs. The researcher selecting the components didn’t use the ground-truth retinotopic maps as reference, nor did they pay attention to the participant IDs when sorting the IC components. Indeed, they weren’t trying to find participant specific maps per se, but rather aimed to find good candidate retinotopic maps in general. In the case of the newly added adult analyses, the ICs were selected before the retinotopic mapping was reviewed or traced; hence, no knowledge about the participant-specific ground truth could have influenced the selection of ICs. Even with this process from adults, we find results of comparable strength as we found in infants, as shown below. Nonetheless, there is a possibility that this researcher’s previous experience of tracing the infant maps could have influenced their choice of components at the participant-specific level. If so, it was a small effect since the components the researcher selected were far from the best possible options (i.e., rankings of the selected components averaged in the 64th percentile for spatial frequency maps and the 68th percentile for meridian maps). We believe all reasonable steps were taken to mitigate bias in the selection of ICs.

Reviewer #3 (Public Review):

The manuscript reports data collected in awake toddlers recording BOLD while watching videos. The authors analyse the BOLD time series using two different statistical approaches, both very complex but do not require any a priori determination of the movie features or contents to be associated with regressors. The two main messages are that 1) toddlers have occipital visual areas very similar to adults, given that an SRM model derived from adult BOLD is consistent with the infant brains as well; 2) the retinotopic organization and the spatial frequency selectivity of the occipital maps derived by applying correlation analysis are consistent with the maps obtained by standard and conventional mapping.

Clearly, the data are important, and the author has achieved important and original results. However, the manuscript is totally unclear and very difficult to follow; the figures are not informative; the reader needs to trust the authors because no data to verify the output of the statistical analysis are presented (localization maps with proper statistics) nor so any validation of the statistical analysis provided. Indeed what I think that manuscript means, or better what I understood, may be very far from what the authors want to present, given how obscure the methods and the result presentation are.

In the present form, this reviewer considers that the manuscript needs to be totally rewritten, the results presented each technique with appropriate validation or comparison that the reader can evaluate.

We are grateful to the reviewer for the chance to improve the paper. We have broken their review into three parts: clarification of the methods, validation of the analyses, and enhancing the visualization.

Clarification of the methods

We acknowledge that the methods we employed are complex and uncommon in many fields of neuroimaging. That said, numerous papers have conducted these analyses on adults (Beckman et al., 2005; Butt et al., 2015; Guntupalli et al., 2016; Haak & Beckman, 2018; Knapen, 2021; Lu et al., 2017) and non-human primates (Arcaro & Livingstone, 2017; Moeller et al., 2009). We have redoubled our efforts in the revision to make the methods as clear as possible, expanding on the original text and providing intuitions where possible. These changes have been added throughout and are too vast in number to repeat here, especially without context, but we hope that readers will have an easier time following the analyses now. 

Additionally, we updated Figures 3 and 5 in which the main ICA and SRM analyses are described. For instance, in Figure 3’s caption we now add details about how the gradient analyses were performed on the components: 

“We used the same lines that were manually traced on the task-evoked map to assess the change in the component’s response. We found a monotonic trend within area from medial to lateral, just like we see in the ground truth.” Pg. 11

Regarding Figure 5, we reconsidered the best way to explain the SRM analyses and decided it would be helpful to partition the diagram into steps, reflecting the analytic process. These updates have been added to Figure 5, and the caption has been updated accordingly.

We hope that these changes have improved the clarity of the methods. For readers interested in learning more, we encourage them to either read the methods-focused papers that debut the analyses (e.g., Chen et al., 2015), read the papers applying the methods (e.g., Guntupalli et al., 2016), or read the annotated code we publicly release which implements these pipelines and can be used to replicate the findings.

Validation of the analyses

One of the requests the reviewer makes is to validate our analyses. Our initial approach was to lean on papers that have used these methods in adults or primates (e.g., Arcaro, & Livingstone, 2017; Beckman et al., 2005; Butt et al., 2015; Guntupalli et al., 2016; Haak & Beckman, 2018; Knapen, 2021; Moeller et al., 2009) where the underlying organization and neurophysiology is established. However, we have made changes to these methods that differ from their original usage (e.g., we used SRM rather than hyperalignment, we use meridian mapping rather than traveling wave retinotopy, we use movie-watching data rather than rest). Hence, the specifics of our design and pipeline warrant validation. 

To add further validation, we have rerun the main analyses on an adult sample. We collected 8 adult participants who completed the same retinotopy task and a large subset of the movies that infants saw. These participants were run under maximally similar conditions to infants (i.e., scanned using the same parameters and without the top of the head-coil) and were preprocessed using the same pipeline. Given that the relationship between adult visual maps and movie-driven (or resting-state) analyses has been shown in many studies (Beckman et al., 2005; Butt et al., 2015; Guntupalli et al., 2016; Haak & Beckman, 2018; Knapen, 2021; Lu et al., 2017), these adult data serve as a validation of our analysis pipeline. These adult participants were included in the original manuscript; however, they were previously only used to support the SRM analyses (i.e., can adults be used to predict infant visual maps). The adult results are described before any results with infants, as a way to engender confidence. Moreover, we have provided new supplementary figures of the adult results that we hope will be integrated with the article when viewing it online, such that it will be easy to compare infant and adult results, as per the reviewer’s request. 

As per the figures and captions below, the analyses were all successful with the adult participants: 1) Homotopic correlations are higher than correlations between comparable areas in other streams or areas that are more distant within stream. 2) A multidimensional scaling depiction of the data shows that areas in the dorsal and ventral stream are dissimilar. 3) Using independent components analysis on the movie data, we identified components that are highly correlated with the retinotopy task-based spatial frequency and meridian maps. 4) Using shared response modeling on the movie data, we predicted maps that are highly correlated with the retinotopy task-based spatial frequency and meridian maps.

These supplementary analyses are underpowered for between-group comparisons, so we do not statistically compare the results between infants and adults. Nonetheless, the pattern of adult results is comparable overall to the infant results. 

We believe these adult results provide a useful validation that the infant analyses we performed can recover fine-grained organization.

Enhancing the visualization

The reviewer raises an additional concern about the lack of visualization of the results. We recognize that the plots of the summary statistics do not provide information about the intermediate analyses. Indeed, we think the summary statistics can understate the degree of similarity between the components or predicted visual maps and the ground truth. Hence, we have added 6 new supplementary figures showing the intensity gradients for the following analyses: 1. spatial frequency prediction using ICA, 2. meridian prediction using ICA, 3. spatial frequency prediction using infant SRM, 4. meridian prediction using infant SRM, 5. spatial frequency prediction using adult SRM, and 6. meridian prediction using adult SRM.

We hope that these visualizations are helpful. It is possible that the reviewer wishes us to also visually present the raw maps from the ICA and SRM, akin to what we show in Figure 3A and 3B. We believe this is out of scope of this paper: of the 1140 components that were identified by ICA, we selected 36 for spatial frequency and 17 for meridian maps. We also created 20 predicted maps for spatial frequency and 20 predicted meridian maps using SRM. This would result in the depiction of 93 subfigures, requiring at least 15 new full-page supplementary figures to display with adequate resolution. Instead, we encourage the reader to access this content themselves: we have made the code to recreate the analyses publicly available, as well as both the raw and preprocessed data for these analyses, including the data for each of these selected maps.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) As mentioned in the public review, the authors should consider incorporating relevant adult fMRI research into the Introduction and explain the importance of testing this question in infants.

Our public response describes the several citations to relevant adult research we have added, and have provided further motivation for the project.

(2) The authors should conduct additional analyses to support their conclusion that movie data alone can generate accurate retinotopic maps (i.e., by comparing this approach to other available alternatives).

We have clarified in our public response that we did not wish to conclude that movie data alone can generate accurate retinotopic maps, and have made substantial edits to the text to emphasize this. Thus, because this claim is already not supported by our analyses, we do not think it is necessary to test it further.

(3) The authors should re-do the homotopy analyses using movie-defined ROIs (i.e., by splitting the movie-viewing data into independent folds for functional ROI definition and analyses).

As stated above, defining ROIs based on the movie content is not the intended goal of this project. Even if that were the general goal, we do not believe that it would be appropriate to run this specific analysis with the data we collected. Firstly, halving the data for ROI definition (e.g., using half the movie data to identify and trace areas, and then use those areas in the homotopy analysis to run on the other half of data) would qualitatively change the power of the analyses described here. Secondly, we would be unable to define areas beyond hV4/V3AB with confidence, since our retinotopic mapping only affords specification of early visual cortex. Thus we could not conduct the MDS analyses shown in Figure 2.

(4) If the authors agree that a primary contribution of this study and paper is to showcase what is possible to do with a limited amount of movie-viewing data, then they should make it clearer, sooner, how much usable movie data they have from infants. They could also consider conducting additional analyses to determine the minimum amount of fMRI data necessary to reveal the same detailed characteristics of functional responses in the visual cortex.

We agree it would be good to highlight the amount of movie data used. When the infant data is first introduced in the results section, we now state the durations:

“All available movies from each session were included (Table S2), with an average duration of 540.7s (range: 186--1116s).” Pg. 5

Additionally, we have added a homotopy analysis that describes the contribution of data quantity to the results observed. We compare the amount of data collected with the magnitude of same vs. different stream effect (Figure 1B) and within stream distance effect (Figure 1C). We find no effect of movie duration in the sample we tested, as reported below:

“We found no evidence that the variability in movie duration per participant correlated with this difference [of same stream vs. different stream] (r=0.08, p=.700).” Pg. 6-7

“There was no correlation between movie duration and the effect (Same > Adjacent: r=-0.01, p=.965, Adjacent > Distal: r=-0.09, p=.740).” Pg. 7

(5) If any of the methodological approaches are novel, the authors should make this clear. In particular, has the approach of visually inspecting and categorizing components generated from ICA and movie data been done before, in adults/other contexts?

The methods we employed are similar to others, as described in the public review.

However, changes were necessary to apply them to infant samples. For instance, Guntupalli et al. (2016) used hyperalignment to predict the visual maps of adult participants, whereas we use SRM. SRM and hyperalignment have the same goal — find a maximally aligned representation between participants based on brain function — but their implementation is different. The application of functional alignment to infants is novel, as is their use in movie data that is relatively short by comparison to standard adult data. Indeed, this is the most thorough demonstration that SRM — or any functional alignment procedure — can be usefully applied to infant data, awake or sleeping. We have clarified this point in the discussion.

“This is initial evidence that functional alignment may be useful for enhancing signal quality, like it has in adults[27,32,33], or revealing changing function over development[45], which may prove especially useful for infant fMRI[52].” Pg. 21

(6) The authors found that meridian maps were less identifiable from ICA and movie data and suggest that this may be because these maps are more susceptible to noise or gaze variability. If this is the case, you might predict that these maps are more identifiable in adult data. The authors could consider running additional analyses with their adult participants to better understand this result.

As described in the manuscript, we hypothesize that meridian maps are more difficult to identify than spatial frequency maps because meridian maps are a less smooth, more fine-grained map than spatial frequency. Indeed, it has previously been reported (Moeller et al., 2009) that similar procedures can result in meridian maps that are constituted by multiple independent components (e.g., a component sensitive to horizontal orientations, and a separate component sensitive to vertical components). Nonetheless, we have now conducted the ICA procedure on adult participants and again find it is easier to identify spatial frequency components compared to meridian maps, as reported in the public review.

Minor corrections:

(1) Typo: Figure 3 title: "Example retintopic task vs. ICA-based spatial frequency maps.".

Fixed

(2) Given the age range of the participants, consider using "infants and toddlers"? (Not to diminish the results at all; on the contrary, I think it is perhaps even more impressive to obtain awake fMRI data from ~1-2-year-olds). Example: Figure 3 legend: "A) Spatial frequency map of a 17.1-monthold infant.".

We agree with the reviewer that there is disagreement about the age range at which a child starts being considered a toddler. We have changed the terms in places where we refer to a toddler in particular (e.g., the figure caption the reviewer highlights) and added the phrase “infants and toddlers” in places where appropriate. Nonetheless, we have kept “infants” in some places, particularly those where we are comparing the sample to adults. Adding “and toddlers” could imply three samples being compared which would confuse the reader.

(3) Figure 6 legend: The following text should be omitted as there is no bar plot in this figure: "The bar plot is the average across participants. The error bar is the standard error across participants.".

Fixed

(4) Table S1 legend: Missing first single quote: Runs'.

Fixed

Reviewer #2 (Recommendations For The Authors):

I request that this paper cite more of the existing literature on the fMRI of human infants and toddlers using task-driven and resting-state data. For example, early studies by (first authors) Biagi, Dehaene-Lambertz, Cusack, and Fransson, and more recent studies by Chen, Cabral, Truzzi, Deen, and Kosakowski.

We have added several new citations of recent task-based and resting state studies to the second sentence of the main text:

“Despite the recent growth in infant fMRI[1-6], one of the most important obstacles facing this research is that infants are unable to maintain focus for long periods of time and struggle to complete traditional cognitive tasks[7].”

Reviewer #3 (Recommendations For The Authors):

In the following, I report some of my main perplexities, but many more may arise when the material is presented more clearly.

The age of the children varies from 5 months to about 2 years. While the developmental literature suggests that between 1 and 2 years children have a visual system nearly adult-like, below that age some areas may be very immature. I would split the sample and perhaps attempt to validate the adult SRM model with the youngest children (and those can be called infants).

We recognize the substantial age variability in our sample, which is why we report participant-specific data in our figures. While splitting up the data into age bins might reveal age effects, we do not think we can perform adequately powered null hypothesis testing of the age trend. In order to investigate the contribution of age, larger samples will be needed. That said, we can see from the data that we have reported that any effect of age is likely small. To elaborate: Figures 4 and 6 report the participant-specific data points and order the participants by age. There are no clear linear trends in these plots, thus there are no strong age effects.

More broadly, we do not think there is a principled way to divide the participants by age. The reviewer suggests that the visual system is immature before the first year of life and mature afterward; however, such claims are the exact motivation for the type of work we are doing here, and the verdict is still out. Indeed, the conclusion of our earlier work reporting retinotopy in infants (Ellis et al., 2021) suggests that the organization of the early visual cortex in infants as young as 5 months — the youngest infant in our sample — is surprisingly adult-like.

The title cannot refer to infants given the age span.

There is disagreement in the field about the age at which it is appropriate to refer to children as infants. In this paper, and in our prior work, we followed the practice of the most attended infant cognition conference and society, the International Congress of Infant Studies (ICIS), which considers infants as those aged between 0-3 years old, for the purposes of their conference. Indeed, we have never received this concern across dozens of prior reviews for previous papers covering a similar age range. That said, we understand the spirit of the reviewer’s comment and now refer to the sample as “infants and toddlers” and to older individuals in our sample as “toddlers” wherever it is appropriate (the younger individuals would fairly be considered “infants” under any definition).

Figure 1 is clear and an interesting approach. Please also show the average correlation maps on the cortical surface.

While we would like to create a figure as requested, we are unsure how to depict an area-by-area correlation map on the cortical surface. One option would be to generate a seed-based map in which we take an area and depict the correlation of that seed (e.g., vV1) with all other voxels. This approach would result in 8 maps for just the task-defined areas, and 17 maps for anatomically-defined areas. Hence, we believe this is out of scope of this paper, but an interested reader could easily generate these maps from the data we have released.

Figure 2 results are not easily interpretable. Ventral and dorsal V1-V3 areas represent upper or lower VF respectively. Higher dorsal and ventral areas represent both upper and lower VF, so we should predict an equal distance between the two streams. Again, how can we verify that it is not a result of some artifacts?

In adults, visual areas differ in their functional response properties along multiple dimensions, including spatial coding. The dorsal/ventral stream hypothesis is derived from the idea that areas in each stream support different functions, independent of spatial coding. The MDS analysis did not attempt to isolate the specific contribution of spatial representations of each area but instead tested the similarity of function that is evoked in naturalistic viewing. Other covariance-based analyses specifically isolate the contribution of spatial representations (Haak et al., 2013); however, they use a much more constrained analysis than what was implemented here. The fact that we find broad differentiation of dorsal and ventral visual areas in infants is consistent with adults (Haak & Beckman, 2018) and neonate non-human primates (Arcaro & Livingstone, 2017). 

Nonetheless, we recognize that we did not mention the differences in visual field properties across areas and what that means. If visual field properties alone drove the functional response then we would expect to see a clustering of areas based on the visual field they represent (e.g., hV4 and V3AB should have similar representations). Since we did not see that, and instead saw organization by visual stream, the result is interesting and thus warrants reporting. We now mention this difference in visual fields in the manuscript to highlight the surprising nature of the result.

“This separation between streams is striking when considering that it happens despite differences in visual field representations across areas: while dorsal V1 and ventral V1 represent the lower and upper visual field, respectively, V3A/B and hV4 both have full visual field maps. These visual field representations can be detected in adults[41]; however, they are often not the primary driver of function[39]. We see that in infants too: hV4 and V3A/B represent the same visual space yet have distinct functional profiles.” Pg. 8

The reviewer raises a concern that the MDS result may be spurious and caused by noise. Below, we present three reasons why we believe these results are not accounted for by artifacts but instead reflect real functional differentiation in the visual cortex. 

(1) Figure 2 is a visualization of the similarity matrix presented in Figure S1. In Figure S1, we report the significance testing we performed to confirm that the patterns differentiating dorsal and ventral streams — as well as adjacent areas from distal areas — are statistically reliable across participants. If an artifact accounted for the result then it would have to be a kind of systematic noise that is consistent across participants.

(2) One of the main sources of noise (both systematic and non-systematic) with infant fMRI is motion. Homotopy is a within-participant analysis that could be biased by motion. To assess whether motion accounts for the results, we took a conservative approach of regressing out the framewise motion (i.e., how much movement there is between fMRI volumes) from the comparisons of the functional activity in regions. Although the correlations numerically decreased with this procedure, they were qualitatively similar to the analysis that does not regress out motion:

“Additionally, if we control for motion in the correlation between areas --- in case motion transients drive consistent activity across areas --- then the effects described here are negligibly different (Figure S5).” Pg. 7

(3) We recognize that despite these analyses, it would be helpful to see what this pattern looks like in adults where we know more about the visual field properties and the function of dorsal and ventral streams. This has been done previously (e.g., Haak & Beckman, 2018), but we have now run those analyses on adults in our sample, as described in the public review. As with infants, there are reliable differences in the homotopy between streams (Figure S1). The MDS results show that the adult data was more complex than the infant data, since it was best described by 3 dimensions rather than 2. Nonetheless, there is a rotation of the MDS such that the structure of the ventral and dorsal streams is also dissociable. 

Figure 3 also raises several alternative interpretations. The spatial frequency component in B has strong activity ONLY at the extreme border of the VF and this is probably the origin of the strong correlation. I understand that it is only one subject, but this brings the need to show all subjects and to report the correlation. Also, it is important to show the putative average ICA for retinotopy and spatial frequencies across subjects and for adults. All methods should be validated on adults where we have clear data for retinotopy and spatial frequency.

The reviewer notes that the component in Figure 3 shows strong negative response in the periphery. It is often the case, as reported elsewhere (Moeller et al., 2009), that ICA extracts portions of visual maps. To make a full visual map would require combining components into a composite (e.g., a component that has a high response in the periphery and another component that has a high response in the fovea). If we were to claim that this component, or others like it, could replace the need for retinotopic mapping, then we would want to produce these composite maps; however, our conclusion in this project is that the topographic information of retinotopic maps manifest in individual components of ICA. For this purpose, the analysis we perform adequately assesses this topography.

Regarding the request to show the results for all subjects, we address this in the public response and repeat it here briefly: we have added 6 new figures to show results akin to Figure 3C and D. It is impractical to show the equivalent of Figure 3A and B for all participants, yet we do release the data necessary to see to visualize these maps easily.

Finally, the reviewer suggests that we validate the analyses on adult participants. As shown in Figure S3 and reported in the public response, we now run these analyses on adult participants and observe qualitatively similar results to infants.

How much was the variation in the presumed spatial frequency map? Is it consistent with the acuity range? 5-month-old infants should have an acuity of around 10c/deg, depending on the mean luminance of the scene.

The reviewer highlights an important weakness of conducting ICA: we cannot put units on the degree of variation we see in components. We now highlight this weakness in the discussion:

“Another limitation is that ICA does not provide a scale to the variation: although we find a correlation between gradients of spatial frequency in the ground truth and the selected component, we cannot use the component alone to infer the spatial frequency selectivity of any part of cortex. In other words, we cannot infer units of spatial frequency sensitivity from the components alone.” Pg. 20

Figure 5 pipeline is totally obscure. I presumed that I understood, but as it is it is useless. All methods should be clearly described, and the intermediate results should be illustrated in figures and appropriately discussed. Using such blind analyses in infants in principle may not be appropriate and this needs to be verified. Overall all these techniques rely on correlation activities that are all biased by head movement, eye movement, and probably the dummy sucking. All those movements need to be estimated and correlated with the variability of the results. It is a strong assumption that the techniques should work in infants, given the presence of movements.

We recognize that the SRM methods are complex. Given this feedback, we remade Figure 5 with explicit steps for the process and updated the caption (as reported in the public review).

Regarding the validation of these methods, we have added SRM analyses from adults and find comparable results. This means that using these methods on adults with comparable amounts of data as what we collected from infants can predict maps that are highly similar to the real maps. Even so, it is not a given that these methods are valid in infants. We present two considerations in this regard. 

First, as part of the SRM analyses reported in the manuscript, we show that control analyses are significantly worse than the real analyses (indicated by the lines on Figure 6). To clarify the control analysis: we break the mapping (i.e., flip the order of the data so that it is backwards) between the test participant and the training participants used to create the SRM. The fact that this control analysis is significantly worse indicates that SRM is learning meaningful representations that matter for retinotopy. 

Second, we believe that this paper is a validation of SRM for infants. Infant fMRI is a nascent field and SRM has the potential to increase the signal quality in this population. We hope that readers will see these analyses as a proof of concept that SRM can be used in their work with infants. We have stated this contribution in the paper now.

“Additionally, we wish to test whether methods for functional alignment can be used with infants. Functional alignment finds a mapping between participants using functional activity -- rather than anatomy -- and in adults can improve signal-to-noise, enhance across participant prediction, and enable unique analyses[27,32-34].” Pg. 4

“This is initial evidence that functional alignment may be useful for enhancing signal quality, like it has in adults[27,32,33], or revealing changing function over development[45].” Pg. 21

Regarding the reviewer’s concern that motion may bias the results, we wish to emphasize the nature of the analyses being conducted here: we are using data from a group of participants to predict the neural responses in a held-out participant. For motion to explain consistency between participants, the motion would need to be timelocked across participants. Even if motion was time-locked during movie watching, motion will impair the formation of an adequate model that can contain retinotopic information. Thus, motion should only hurt the ability for a shared response to be found that can be used for predicting retinotopic maps. Hence, the results we observed are despite motion and other sources of noise.

What is M??? is it simply the mean value??? If not, how it is estimated?

M is an abbreviation for mean. We have now expanded the abbreviation the first time we use it.

Figure 6 should be integrated with map activity where the individual area correlation should be illustrated. Probably fitting SMR adult works well for early cortical areas, but not for more ventral and associative, and the correlation should be evaluated for the different masks.

With the addition of plots showing the gradients for each participant and each movie (Figures S10–S13) we hope we have addressed this concern. We additionally want to clarify that the regions we tested in the analysis in Figure 6 are only the early visual areas V1, V2, V3, V3A/B, and hV4. The adult validation analyses show that SRM works well for predicting the visual maps in these areas. Nonetheless, it is an interesting question for future research with more extensive retinotopic mapping in infants to see if SRM can predict maps beyond extrastriate cortex.

Occipital masks have never been described or shown.

The occipital mask is from the MNI probabilistic structural atlas (Mazziotta et al., 2001), as reported in the original version and is shared with the public data release. We have added the additional detail that the probabilistic atlas is thresholded at 0% in order to be liberally inclusive. 

“We used the occipital mask from the MNI structural atlas[63] in standard space -- defined liberally to include any voxel with an above zero probability of being labelled as the occipital lobe -- and used the inverted transform to put it into native functional space.” Pg. 27–28

Methods lack the main explanation of the procedures and software description.

We hope that the additions we have made to address this reviewer’s concerns have provided better explanations for our procedures. Additionally, as part of the data and code release, we thoroughly explain all of the software needed to recreate the results we have observed here.

Reviewer #1 (Public review):

Summary:

This study by Howe and colleagues investigates the role of the posterolateral cortical amygdala (plCoA) in mediating innate responses to odors, specifically attraction and aversion. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. They show that specific glutamatergic neurons in the anterior and posterior regions of plCoA are responsible for driving attraction and avoidance, respectively, and that these neurons project to distinct downstream regions, including the medial amygdala and nucleus accumbens, to control these responses.

Strengths:

The major strength of the study is the thoroughness of the experimental approach, which combines advanced techniques in neural manipulation and mapping with high-resolution molecular profiling. The identification of a topographically organized circuit in plCoA and the connection between molecularly defined populations and distinct behaviors is a notable contribution to understanding the neural basis of innate motivational responses. Additionally, the use of functional manipulations adds depth to the findings, offering valuable insights into the functionality of specific neuronal populations.

Weaknesses:

There are some weaknesses in the study's methods and interpretation. The lack of clarity regarding the behavior of the mice during head-fixed imaging experiments raises the possibility that restricted behavior could explain the absence of valence encoding at the population level. Furthermore, while the authors employ chemogenetic inhibition of specific pathways, the rationale for this choice over optogenetic inhibition is not fully addressed, and this could potentially affect the interpretation of the results. Additionally, the choice of the mplCoA for manipulation, rather than the more directly implicated anterior and posterior subregions, is not well-explained, which could undermine the conclusions drawn about the topographic organization of plCoA.

Despite these concerns, the work provides significant insights into the neural circuits underlying innate behaviors and opens new avenues for further research. The findings are particularly relevant for understanding the neural basis of motivational behaviors in response to sensory stimuli, and the methods used could be valuable for researchers studying similar circuits in other brain regions. If the authors address the methodological issues raised, this work could have a substantial impact on the field, contributing to both basic neuroscience and translational research on the neural control of behavior.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work aims to understand the role of thalamus POm in dorsal lateral striatum (DLS) projection in learning a sensorimotor associative task. The authors first confirm that POm forms "en passant" synapses with some of the DLS neuronal subtypes. They then perform a go/no-go associative task that consists of the mouse learning to discriminate between two different textures and to associate one of them with an action. During this task, they either record the activity of the POm to DLS axons using endoscopy or silence their activity. They report that POm axons in the DLS are activated around the sensory stimulus but that the activity is not modulated by the reward. Last, they showed that silencing the POm axons at the level of DLS slows down learning the task.

The authors show convincing evidence of projections from POm to DLS and that POm inputs to DLS code for whisking whatever the outcome of the task is. However, their results do not allow us to conclude if more neurons are recruited during the learning process or if the already activated fibres get activated more strongly. Last, because POm fibres in the DLS are also projecting to S1, silencing the POm fibres in the DLS could have affected inputs in S1 as well and therefore, the slowdown in acquiring the task is not necessarily specific to the POm to DLS pathway.

We thank the reviewer for these constructive comments. The points are addressed below.  

Strengths:

One of the main strengths of the paper is to go from slice electrophysiology to behaviour to get an in-depth characterization of one pathway. The authors did a comprehensive description of the POm projections to the DLS using transgenic mice to unambiguously identify the DLS neuronal population. They also used a carefully designed sensorimotor association task, and they exploited the results in depth.

It is a very nice effort to have measured the activity of the axons in the DLS not only after the mice have learned the task but throughout the learning process. It shows the progressive increase of activity of POm axons in the DLS, which could imply that there is a progressive strengthening of the pathway. The results show convincingly that POm axons in the DLS are not activated by the outcome of the task but by the whisker activity, and that this activity on average increases with learning.

Weaknesses:

One of the main targets of the striatum from thalamic input are the cholinergic neurons that weren't investigated here, is there information that could be provided?

This is true of the parafascicular (Pf) thalamic nucleus, which has been well studied in this context. However, there is much less known about the striatal projections of other thalamic nuclei, including POm, and their inputs to cholinergic neurons. Anatomical tracing evidence from Klug et al. (2018), which mapped brain-wide inputs to striatal cholinergic (ChAT) interneurons, suggests that Pf provides the majority of thalamic innervation of striatal ChAT neurons compared to other thalamic nuclei. Many other thalamic nuclei, including POm, showed very little of no labeling, suggesting weak innervation of ChAT interneurons. However, it is possible that these thalamic nuclei, including POm, do provide functional innervation of ChAT interneurons that is not sufficiently assessed by anatomical tracing. Understanding the innervation patterns of POm-striatal projections beyond the three cell types we have studied here would be an important area of further study.

It is interesting to know that the POm projects to all neuronal types in the DLS, but this information is not used further down the manuscript so the only take-home message of Figure 1 is that the axons that they image or silence in the DLS are indeed connected to DLS neurons and not just passing fibres. In this line, are these axons the same as the ones projecting to S1? If this is the case, why would we expect a different behaviour of the axon activity at the DLS level compared to S1?

Tracing of single POm axons by Ohno et al. (2012) indicated that POm axons form a branched collateral that innervates striatum, while the main axon continues in the rostral-dorsal direction to innervate cortex. We think it is reasonable, based on the morphology, that our optogenetic suppression experiment restricted the suppression of glutamate release to this branch and avoided the other branches of the axon that project to cortex. However, testing this would require monitoring S1 activity during the POm-striatal axon suppression, which we did not do in this study.

It is a very interesting question whether there could be different axon activity behavior in striatum versus S1. There is surprising evidence that POm synaptic terminals are different sizes in S1 and M1 and show different synaptic physiological properties depending on these cortical projection targets (Casas-Torremocha et al., 2022). Based on this, it is possible that POm-striatal synapses show distinct properties compared to cortex; however, this will need to be tested in future work.

The authors used endoscopy to measure the POm axons in the DLS activity, which makes it impossible to know if the progressive increase of POm response is due to an increase of activity from each individual neuron or if new neurons are progressively recruited in the process.

This is a good point. It would be necessary to perform chronic two-photon imaging of POm neurons (or chronic electrophysiological recordings) to determine whether the activity of individual neurons increased versus whether individual neuron activity levels remained similar but new neurons became active with learning. Even under baseline conditions, it is not known in detail what fraction of the population of POm neurons is active during sensory processing or behavior, highlighting how much is still to be discovered in this exciting area of neuroscience.

The picture presented in Figure 4 of the stimulation site is slightly concerning as there are hardly any fibres in neocortical layer 1 while there seems to be quite a lot of them in layer 4, suggesting that the animal here was injected in the VB. This is especially striking as the implantation and projection sites presented in Figures 1 and 2 are very clean and consistent with POm injection.

Although this image was selected to demonstrate the position of the POm injection site and optical fiber implant above striatal axons, the reviewer is correct that there appears to be mixed labeling of axons in L4 and L5a. In some cases, there was expression slightly outside the border of POm (see Fig. 1B, right), which might explain the cortical innervation pattern in this figure. While cortically bound VPM axons pass through the striatum, they do not form synaptic terminals until reaching the cortex (Hunnicutt et al., 2016). If, as may be the case, inhibitory opsins suppress release of neurotransmitter at synaptic terminals more effectively than action potential propagation in axons, it may be likely that optogenetic suppression of POm-striatal terminals is more effective than suppression of action potentials in off-target-labelled VPM axons of passage. Ideally, we could compare effects of suppression of POm-striatal synapses with POm-cortical synapses and VPM-cortical synapses, but this was outside the bandwidth of the present study.

Reviewer #2 (Public Review):

Summary:

Yonk and colleagues show that the posterior medial thalamus (POm), which is interconnected with sensory and motor systems, projects directly to major categories of neurons in the striatum, including direct and indirect pathway MSNs, and PV interneurons. Activity in POm-striatal neurons during a sensory-based learning task indicates a relationship between reward expectation and arousal. Inhibition of these neurons slows reaction to stimuli and overall learning. This circuit is positioned to feed salient event activation to the striatum to set the stage for effective learning and action selection.

Strengths:

The results are well presented and offer interesting insight into an understudied thalamostriatal circuit. In general, this work is important as part of a general need for an increased understanding of thalamostriatal circuits in complex learning and action selection processes, which have generally received less attention than corticostriatal systems.

Weaknesses:

There could be a stronger connection between the connectivity part of the data - showing that POm neurons context D1, D2, and PV neurons in the striatum but with some different properties - and the functional side of the project. One wonders whether the POm neurons projecting to these subtypes or striatal neurons have unique signaling properties related to learning, or if there is a uniform, bulk signal sent to the striatum. This is not a weakness per se, as it's reasonable for these questions to be answered in future papers.

We are very interested to understand the potentially distinct learning-related synaptic and circuit changes that potentially occur at the POm synapses with D1- and D2-SPNs and PV interneurons, and other striatal cell types. We agree that this would be an important topic for further investigation.

All the in vivo activity-related conclusions stem from data from just 5 mice, which is a relatively small sample set. Optogenetic groups are also on the small side.

We appreciate this point and agree that higher N can be important for observing robust effects. A factor of our experiments that helped reduce the number of animals used was the longitudinal design, with repeated measures in the same subjects. This allowed for the internal control of comparing learning effects in the same subject from naïve to expert stages and therefore increased robustness. Even with relatively small group sizes, results were statistically significant, suggesting that the use of more mice was unnecessary, which we considered consistent with best practice in the use of animals in research. We also note that our group sizes were consistent with other studies in the field.  

Reviewer #3 (Public Review):

Yonk and colleagues investigate the role of the thalamostriatal pathway. Specifically, they studied the interaction of the posterior thalamic nucleus (PO) and the dorsolateral striatum in the mouse. First, they characterize connectivity by recording DLS neurons in in-vitro slices and optogenetically activating PO terminals. PO is observed to establish depressing synapses onto D1 and D2 spiny neurons as well as PV neurons. Second, the image PO axons are imaged by fiber photometry in mice trained to discriminate textures. Initially, no trial-locked activity is observed, but as the mice learn PO develops responses timed to the audio cue that marks the start of the trial and precedes touch. PO does appear to encode the tactile stimulus type or outcome. Optogenetic suppression of PO terminals in striatum slow task acquisition. The authors conclude that PO provides a "behaviorally relevant arousal-related signal" and that this signal "primes" striatal circuitry for sensory processing.

A great strength of this paper is its timeliness. Thalamostriatal processing has received almost no attention in the past, and the field has become very interested in the possible functions of PO. Additionally, the experiments exploit multiple cutting-edge techniques.

There seem to be some technical/analytical weaknesses. The in vitro experiments appear to have some contamination of nearby thalamic nuclei by the virus delivering the opsin, which could change the interpretation. Some of the statistical analyses of these data also appear inappropriate. The correlative analysis of Pom activity in vivo, licking, and pupil could be more convincingly done.

The bigger weakness is conceptual - why should striatal circuitry need "priming" by the thalamus in order to process sensory stimuli? Why would such circuitry even be necessary? Why is a sensory signal from the cortex insufficient? Why should the animal more slowly learn the task? How does this fit with existing ideas of striatal plasticity? It is unclear from the experiments that the thalamostriatal pathway exists for priming sensory processing. In fact, the optogenetic suppression of the thalamostriatal pathway seems to speak against that idea.

We thank the reviewer for these constructive comments. The points are addressed below.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

Do POm neurons innervate CINs also? The connection between the PF thalamus and CINs is mentioned in a couple of places - one question is how unique are the input patterns for the POm versus adjacent sensorimotor thalamic regions, including the PF? This isn't a weakness per se but knowing the answer to that question would help in forming a more complete picture of how these different thalamostriatal circuits do or do not contribute uniquely to learning and action selection.

Anatomical tracing evidence from Klug et al. (2018), which mapped brain-wide inputs to striatal cholinergic (ChAT) interneurons, suggests that Pf provides the majority of thalamic innervation of striatal ChAT neurons compared to other thalamic nuclei. Many other thalamic nuclei, including POm, showed very little or no labeling, suggesting weak innervation of ChAT interneurons. However, it is possible that these thalamic nuclei, including POm, do provide functional innervation of ChAT interneurons that is not sufficiently assessed by anatomical tracing.

Another difference between Pf and other thalamic nuclei (likely including POm) comes from anatomical tracing evidence (Smith et al., 2014; PMID: 24523677) which indicates that Pf inputs form the majority of their synapses onto dendritic shafts of SPNs, while other thalamic nuclei form synapses onto dendritic spines. Understanding the innervation patterns of POm-striatal projections beyond the three cell types we have studied here, including ChAT neurons and subcellular localization, would be an important area of further study.

It would be useful to know to what extent these POm-striatum neurons are activated generally during movement, versus this discrimination task specifically.

We agree that distinguishing general movement-related activity from task-specific activity would be very useful. Earlier work (Petty et al., 2021) showed a close relationship between POm neuron activity, spontaneous (task-free) whisker movements, and pupil-indexed arousal in head-restrained mice. Oram et al. (2024; PMID: 39003286) recently recorded VPM and POm in freely moving mice during natural movements, finding that activity of both nuclei correlated with head and whisker movements. These studies indicate that POm is generally coactive with exploratory head and whisker movements.

During task performance, the situation may change with training and attentional effects. For example, Petty and Bruno (2024) (https://elifesciences.org/reviewed-preprints/97188) showed that POm activity correlates more closely with task demands than tactile or visual stimulus modality. Our data indicate that POm axonal signals are increased at trial start during anticipation of tactile stimulus delivery and through the sensory discrimination period, then decrease to baseline levels during licking and water reward collection (Fig. 3). Results of Petty and Bruno (2024) together with ours suggest that POm is particularly active during the context of behaviorally relevant task performance. Thus, we think it is likely that, while pupil dilation indexes general movement and arousal, POm activity is more specific to movement and arousal associated with task engagement and behavioral performance. We have strengthened this point in the Discussion.

Many of the data panels and text for legends/axes are quite small, and the stroke on line art is quite faint - overall figures could be improved from a readability standpoint.

We thank the reviewer for their careful attention to the figures. 

Reviewer #3 (Recommendations For The Authors):

Major

(1) Page 4, the Results regarding PSP and distance from injection site. The r-squared is the wrong thing to look at to test for a relationship. One should look at the p-value on the coefficient corresponding to the slope. The p-value is probably significant given the figures, in which case there may be a relationship contrary to what is stated. All the low r-squared value says is that, if there is a relationship, it does not explain a lot of the PSP variability.

We thank the reviewer for alerting us this oversight. We have included the p value (p = 0.0293) in the figure and legend, and indicated that the relationship is “small but significant”.

(2) Figure 1B suggests that the virus injections extend beyond POm and into other thalamic structures. Do any of the results change if the injections contaminating other nuclei are excluded from the analysis? I am not suggesting the authors change the figures/analyses. I am simply suggesting they double-check.

We selected for injections that were predominantly expressing in POm as determined by post-hoc histological analysis (see Fig. 1, right). As above, we think that axons of passage that do not form striatal synapses are less likely to be suppressed than axons with terminals; however, this would need to be determined in further experiments. Because the preponderance of expression is within POm, we think the results would be similar even with a stricter selection criterion. 

(3) The authors conclude that POm and licking are not correlated (bottom of page 6 pertaining to Figures 3A-F). The danger of these analyses is that they assume that GCaMP8 is a perfect linear reporter of POm spikes. The reliability of GCaMP8 has been quantified in some cell types, but not thalamic neurons, which have relatively higher firing rates.

The reviewer is correct that the relationship between GCaMP8 fluorescence changes and spiking has not been sufficiently characterized in thalamic neurons, and that this would be important to do.

What if the indicator is simply saturated late into the trial (after the average reaction time)? It would look like there is no response and one would conclude no correlation, but there could be a very strong correlation.

While saturation is worthy of concern, the signal dynamics here argue against this possibility. The reason is that the signal increased in the early part of the trial and decreased by the end. If saturation was an issue, this would have been apparent during the initial increase. When the signal decreased in amplitude at the end of the trial, this indicates that the signal is not saturated because it is returning from a point closer to its maximum (and is becoming less saturated).

Also, what happens between trials? Are the correlations the same, stronger, weaker? Ideally, the authors would analyze the data during and between trials.

Between trials the signal did not show further changes in baseline beyond what was displayed at the start and end of behavioral trials. There were no consistent increases or decreases in signals between trials, except perhaps during strong whisking bouts. This is anecdotal because we did not analyze between-trial data. However, it is interesting and important to note that signals increased dramatically in amplitude from naïve, early learning to expert behavioral performance (Fig. 3), highlighting that POm-axonal signals relate to behavioral engagement and performance rather than spontaneous behaviors.  

(4) Axonal activity could also appear more correlated with the pupil than licking because pupil dynamics are slow like the dynamics of calcium indicators. These kernels could artificially inflate the correlation. Ideally, the authors could consider these temporal effects. Perhaps they could deconvolve the temporal profiles of calcium and pupil before correlating? Or equivalently incorporate the profiles into their analysis?

We analyzed the lick probability histograms, which had a temporal profile similar to the calcium signals (Fig. 3D,E), ruling out concerns about effects of temporal effects on correlations. It is also worth noting that we observed changes in correlations between calcium signals and pupil with learning stage (Fig. 3I), even though the temporal profiles (signal dynamics) are not changing. Thus, temporal effects of the signals themselves are not the driver of correlations, but rather the changes in relative timing between calcium signals and pupil, as occur with learning.

(5) The authors conclude that PO provides a "behaviorally relevant arousal-related signal" and that this signal "primes" striatal circuitry for sensory processing. The data here support the first part. It is not clear that the data support the second part, largely because it is vague what "priming" of sensory processing or "a key role in the initial stages of action selection (p.9) even means here. Why would such circuitry even be necessary? Why is a sensory signal from the cortex insufficient? Why should the animal more slowly learn the task? How does this fit with existing ideas of striatal plasticity? Some conceptual proposals from the authors, even if speculative and not offered as a conclusion, would be helpful.

We appreciate these good points and have added further consideration and revision of the concept of priming and potential roles in an extensively revised Discussion section.

(6) The photometry shows that PO turns on about 2 seconds before the texture presentation. PO's activity seems locked to the auditory cue, not the texture (Figure 2). This means that the attempt to suppress the thalamostriatal pathway with JAWS (Figure 4) is rather late, isn't it? Some PO signals surely go through. This seems to contradict the idea of priming above. It would be good if the authors could factor this into their narrative. Perhaps labelling the time of the auditory cue in Figure 4C would also be helpful.

The start of texture presentation (movement of the texture panel toward the mouse) and auditory cue occur at the same time. To clarify this, we added a label “start tone” in Figure 4C and also in Figure 2C.

For optogenetic (JAWS) suppression, we intentionally chose a time window between start tone onset and texture presentation, because our photometry experiments showed that this was when the preponderance of the signal occurred. However, the reviewer is correct that our chosen optogenetic suppression (JAWS) onset occurs shortly after the photometry signal has already started, potentially leaving the early photometry signal un-suppressed. Our motivation for choosing a restricted time window surrounding the texture presentation time was 1) to minimize illumination and potential heating of brain tissue; 2) to target a time window that avoids the auditory cue but covers stimulus presentation. We did not want to extend the duration of the suppression to before the trial started, because this could produce task-non-specific effects, such as distraction or loss of attention before the start of the trial.

Even if some signal were getting through before suppression, we don’t think this contradicts the possibility of ‘priming’, because the process underlying priming would still be disrupted even if not totally suppressed. This would alter the temporal relationship between POm-striatal inputs and further corticostriatal inputs (from S1 and M1 cortex, for example). We have included further consideration of these points and possible relation to the priming concept in the Discussion.

Minor

(1) Page 5, "the sensitivity metric is artificially increased". What do you mean "artificially"? The mice are discriminating better. It is true that either a change in HR or FAR can cause the sensitivity metric to change, but there is nothing artificial or misleading about this.

We removed the word artificial and clarified our definition of behaviorally Expert in this context:

“Mice were considered Expert once they had reached ≥ 0.80 Hit Rate and ≤ 0.30 FA Rate for two consecutive sessions in lieu of a strict sensitivity (d’) threshold; we found this definition more intuitive because d’ is enhanced as Hit Rate and FA Rate approach their extremes (0 or 1)”

(2) Page 7, "Upon segmentation (Figure S4G-J)". Do you mean "segregation by trial outcome"?

Corrected.

(3) Page 9, "POm projections may have discrete target-specific functions, such that POm-striatal inputs may play a distinct role in sensorimotor behavior compared to POm-cortical inputs". Would POm-cortical inputs not also be sensorimotor? The somatosensory cortex contains a lot of corticostriatal cells. It also has various direct and indirect links to the motor cortex as well.

We have clarified the wording here to convey the possibility that POm signals could be received and processed differently by striatal versus cortical circuitry, and have moved this statement to later in the discussion for better elaboration.

(4) The Methods state that male and female mice were used. Why not say how many of each and whether or not there are any sex-specific differences?

We added the following information to the Methods:

The number of male and female mice were as follows, by experiment type: 6 male, 4 female (electrophysiology); 3 male, 2 female (fiber photometry); 4 male, 5 female (optogenetics). Data were not analyzed for sex differences.

Reviewer #1 (Public review):

Summary:

In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modelling to demonstrate that EPNSst+ neural activity represents movement, choice direction and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

Strengths and Weaknesses:

Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modelling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results. To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Fig 1 F-J, Supp 1 F,K etc i.e via inclusion of faint hash lines connecting individual data points across variables. Additionally, Fig 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms. Finally, the authors claimed calcium activity increased following ipsilateral movements. However, figure 3C clearly shows that both SXcontra and SXisi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point? Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high. Vs low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results are striking. However, this relies on co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected. Alternative means of glutamate packaging (other VGluT isoforms, other transporters etc) could also compensate for the partial absence of VGluT2, which should be discussed. The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitations is transparently discussed by the authors.

Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduces generalizability and to some extent the impact of their findings.

Comments on the latest version:

Specifically, they have included more thorough analyses to address several concerns related to interpreting activity patterns of EPSst+ neurons. The authors clearly point out that calcium activity increased during ipsilateral movements, and the increase was statistically larger during the choice phase (Figure 2 supplement 1F-G), indicating that these neurons may represent movement and additional factors (e.g. executive decision-making). Correspondingly, we appreciate the thorough explanation of using a GLM model to determine which behavioural variables contribute to observed physiological signals and adding the example reconstructed signal with direction and reward variables omitted in Figure 3 supplements 1 and 2.

Although no new manipulation experiment is added to the manuscript, the authors respond to common critiques related to testing the behavioural effect after the manipulations in well-trained mice. The discussion related to technical limitations, possible compensatory mechanisms and alternative interpretations is thorough and overall satisfying. Based on the behaviour modeling results, the authors speculate that animals need to integrate more evidence from the past to guide choice in a more uncertain environment (70/30 version), instead of adopting a 'win-stay, lose-shift' strategy in the more deterministic 90/10 version. The authors expand the discussion, but the possibility that EPNSst+ neurons contribute to task performance in well-trained animals under uncertainty is not directly tested. Along with other alternative explanations discussed in the manuscript, we think the paper is valuable literature for future studies to understand the basal ganglia circuits in learning and decision-making.

Reviewer #2 (Public review):

Summary:

This paper aimed to determine the role EP sst+ neurons play in a probabilistic switching task.

Strengths:

- The in vivo recording of the EP sst+ neurons activity in the task is one of the strongest parts of this paper. Previous work had recorded from the EP-LHb population in rodents and primates in head fixed configurations, the recordings of this population in a freely moving context is a valuable addition to these studies and has highlighted more clearly that these neurons respond both at the time of choice and outcome.

- The use of a refined intersectional technique to record specifically the EP sst+ neurons is also an important strength of the paper. This is because previous work has shown that there are two genetically different types of glutamatergic EP neurons that project to the LHb. Previous work had not distinguished between these types in their recordings so the current results showing that the bidirectional value signaling is present in the EP sst+ population is valuable.

Weaknesses:

- One of the main weaknesses of the paper is to do with how the effect of the EP sst+ neurons on the behavior was assessed.

o All the manipulations (blocking synaptic release and blocking glutamatergic transmission) are chronic and more importantly the mice are given weeks of training after the manipulation before the behavioral effect is assessed. This means that as the authors point out in their discussion the mice will have time to adjust to the behavioral manipulation and compensate for the manipulations. The results do show that mice can adapt to these chronic manipulations and that the EP sst+ are not required to perform the task. What is unclear is whether the mice have compensated for the loss of EP sst+ neurons and whether they play a role in the task under normal conditions. Acute manipulations or chronic manipulations without additional training would be needed to assess this.

o Another weakness is that the effect of the manipulations was assessed in the 90/10 contingency version of the task. Under these contingencies, mice integrate past outcomes over fewer trials to determine their choice and animals act closer to a simple win-stay-lose switch strategy. Due to this it is unclear if the EP sst+ neurons would play a role in the task when they must integrate over a larger number of conditions in the less deterministic 70/30 version of the task. Indeed it is not clear that lesioning any other regions involved in evaluation of action outcomes such as VTA dopamine neurons, that encode reward prediction errors, would have any deficit when assessed in this way. Due to this, it's not clear if the mice have adapted to solve the task without evaluating action outcomes at all and are just acting in a more deterministic lose switch manner that would not presumably involve any of the circuitry in evaluating action outcomes.

- The authors conclude that they do not see any evidence for bidirectional prediction errors. It is not possible to conclude this. First, they see a large response in the EP sst+ neurons to the omission of an expected reward. This is what would be expected of a negative reward prediction error. There are much more specific well controlled tests for this that are commonplace in head-fixed and freely moving paradigms that could be tested to probe this. The authors do look at the effect of previous trials on the response and do not see strong consistent results, but this is not a strong formal test of what would be expected of a prediction error, either a positive or negative. The other way they assess this is by looking at the size of the responses in different recording sessions with different reward contingencies. They claim that the size of the reward expectation and prediction error should scale with the different reward probabilities. If all the reward probabilities were present in the same session this should be true as lots of others have shown for RPE. Because however this data was taken from different sessions it is not expected that the responses should scale, this is because reward prediction errors have been shown to adaptively scale to cover the range of values on offer (Tobler et al., Science 2005). A better test of positive prediction error would be to give a larger than expected reward on a subset of trials. Either way there is already evidence that responses reflect a negative prediction error in their data and more specific tests would be needed to formally rule in or out prediction error coding especially as previous recordings have shown it is present in previous primate and rodent recordings.

- There are a lot of variables in the GLM that occur extremely close in time such as the entry and exit of a port. If two variables occur closely in time and are always correlated it will be difficult if not impossible for a regression model to assign weights accurately to each event. This is not a large issue, but it is misleading to have regression kernels for port entry and exits unless the authors can show these are separable due to behavioral jitter or a lack of correlation under specific conditions, which does not seem to be the case.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modeling to demonstrate that EPNSst+ neural activity represents movement, choice direction, and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

Strengths and Weaknesses:

Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modeling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results.

(1) To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Figures 1 F-J, Supplementary Figures 1 F, K, etc i.e via the inclusion of faint hash lines connecting individual data points across variables.

Thank you for the suggestion. The same mouse is now represented in Fig 1 and Fig 1—Figure Supplement 1 as a darkened circle so it can be followed across different panels. Photometry from this mouse was used as sample date in Figure 2b and Figure 2—figure supplement 1a-b.

(2) Additionally, Figure 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms.

We have now added analyses and reconstructed photometry signals from GLMs excluding important predictors in Figure 3—figure supplement 1 and 2. We use the model where both “Direction and reward” were omitted as predictors for the GLM and showed photometry reconstructions aligned to behavioral events used for the full model (Figure 3—figure supplement 1) and partial model (Figure 3—figure supplement 2) to compare model performance.  

(3) Finally, the authors claimed calcium activity increased following ipsilateral movements. However, Figure 3C clearly shows that both SXcontra and SXipsi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point?

We observe that calcium activity increases during ipsilateral choices as the animal moves toward the ipsilateral side port (e.g. CX_ipsi to SE_ipsi; Fig 2C and Fig 3C). The animal also makes other ipsiversive movements not during the “choice” phase of a trial such as when it is returning to the center port following a contralateral choice (e.g. SX_Contra to CE; Fig 2—figure supplement 1F and Fig 3C). We also observe an increase in calcium activity during these ipsiversive movements (e.g. SX_Contra to CE), but they are not as large as those observed during the choice phase (Fig 2—figure supplement 1G). Therefore, during the choice phase of a trial, activity contains signals related to ipsilateral movement and additional factors (e.g. executive decision making).    

(4) Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high vs. low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

Unfortunately, neither licks nor locomotion were recorded during the behavioral sessions when photometry was recorded. In Figure 2—figure supplement 1a we now show individual trials sorted by trial duration (time elapsed between CE and SE) to illustrate the dynamics of the photometry signal on fast vs slow trials within a session.  

(5) There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results is striking. However, this relies on the co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected.

For the Tet-tox experiments in Figure 4 we estimate approximately 70±15% of EP^Sst+ neurons expressed Tet-tox based on our histological counts and published stereological counts in EP (Miyamoto and Fukuda, 2015). It is true that channelrhodopsin expression will not overlap 100% with cells infected by the other virus, indeed our in vitro synaptic physiology shows small residual postsynaptic currents following optogenetic stimulation either from incomplete blockade of synaptic release or neurons that expressed channelrhodopsin but not Tettx (Figure 4—figure supplement 1J-K). The same is shown for CRISPR mediated deletion of Slc17a6 (Fig 5 – Fig supplement 1J-K).  

(6) Alternative means of glutamate packaging (other VGluT isoforms, other transporters, etc) could also compensate for the partial absence of VGluT2, which should be discussed.

While single cell sequencing (Wallace et al, 2017) has shown EP^Sst+ neurons do not express Slc17a7/8 (vGlut1 or vGlut3) it is possible that these genes could be upregulated following CRISPR mediated deletion of Slc17a6, however we do not see evidence of this with our in vitro synaptic physiology (EPSCs are significant suppressed, Figure 5 – Fig supplement 1J-K) and therefore can conclude it is highly unlikely to occur to a significant degree in our experiments. This is now included in the Discussion.

(7) The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well-learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitation is transparently discussed by the authors.

As mentioned above, it is possible that a requirement for EP^Sst+ neurons could be revealed if the experiment was conducted with different parameters (either different reward probabilities, fluctuating reward probabilities within a session, or withholding additional training during viral expression). It is difficult to predict which version of the task, if any, would be most likely to reveal a requirement for EP^Sst+ neurons based on our results. We favor testing for EP^Sst+ function using a new behavioral paradigm that allows us to carefully examine task learning following EP manipulations in an independent study.

(8) Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization, it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

In a previous study we confirmed that EP^Sst+ neurons project exclusively to the LHb using cell-type specific rabies infection and examining all reported downstream regions for axon collaterals (Wallace et al 2017, Suppl. Fig 6F-G). When EP^Sst+ neurons were labeled we did not observe axon collaterals in known targets of EP such as ventro-antero lateral thalamus, red nucleus, parafasicular nucleus of the thalamus, or the pedunculopontine tegmental nucleus, only in the LHb. Additionally, using single cell tracing techniques, others have shown EP neurons that exclusively project to the LHb (Parent et al, 2001).

Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduce generalizability and to some extent the impact of their findings.

Reviewer #2 (Public Review):

Summary:

This paper aimed to determine the role EP sst+ neurons play in a probabilistic switching task.

Strengths:

The in vivo recording of the EP sst+ neuron activity in the task is one of the strongest parts of this paper. Previous work had recorded from the EP-LHb population in rodents and primates in head-fixed configurations, the recordings of this population in a freely moving context is a valuable addition to these studies and has highlighted more clearly that these neurons respond both at the time of choice and outcome.

The use of a refined intersectional technique to record specifically the EP sst+ neurons is also an important strength of the paper. This is because previous work has shown that there are two genetically different types of glutamatergic EP neurons that project to the LHb. Previous work had not distinguished between these types in their recordings so the current results showing that the bidirectional value signaling is present in the EP sst+ population is valuable.

Weaknesses:

(1) One of the main weaknesses of the paper is to do with how the effect of the EP sst+ neurons on the behavior was assessed.

(a) All the manipulations (blocking synaptic release and blocking glutamatergic transmission) are chronic and more importantly the mice are given weeks of training after the manipulation before the behavioral effect is assessed. This means that as the authors point out in their discussion the mice will have time to adjust to the behavioral manipulation and compensate for the manipulations. The results do show that mice can adapt to these chronic manipulations and that the EP sst+ are not required to perform the task. What is unclear is whether the mice have compensated for the loss of EP sst+ neurons and whether they play a role in the task under normal conditions. Acute manipulations or chronic manipulations without additional training would be needed to assess this.

Unfortunately, when mice are given a three week break from behavioral training (the time required to allow for adequate viral expression) behavioral performance on the task (p(highport), p(switch), trial number, trial time, etc.) is significantly degraded. Animals do eventually recover to previous performance levels, but this takes place during a 4-5 day “relearning” period. Here we sought to examine if EP^Sst+ neurons are required for continued task performance and chose to continue to train the animals following viral injection to avoid the “relearning” period that occurs following an extended break from behavioral training which may have made it difficult to interpret changes in behavioral performance due to the viral manipulation vs relearning.  

Acute manipulations were not used because we planned to compare complete synaptic ablation (Tettx) and single neurotransmitter ablation (CRISPR Slc17a6) over similar time courses and we know of no acute manipulation that could achieve single neurotransmitter ablation. 

(b) Another weakness is that the effect of the manipulations was assessed in the 90/10 contingency version of the task. Under these contingencies, mice integrate past outcomes over fewer trials to determine their choice and animals act closer to a simple win-stay-lose switch strategy. Due to this, it is unclear if the EP sst+ neurons would play a role in the task when they must integrate over a larger number of conditions in the less deterministic 70/30 version of the task.

It is possible that a requirement for EP^Sst+ neurons could be revealed if the experiment was conducted with different parameters (either different reward probabilities, fluctuating reward probabilities within a session, or withholding additional training during viral expression). It is difficult to predict which version of the task, if any, would be most likely to reveal a requirement for EP^Sst+ neurons based on our results. We favor testing for EP^Sst+ function using a new behavioral paradigm that allows us to carefully examine task learning following EP manipulations in an independent study.

The authors show an intriguing result that the EP sst+ neurons are excited when mice make an ipsilateral movement in the task either toward or away from the center port. This is referred to as a choice response, but it could be a movement response or related to the predicted value of a specific action. Recordings while mice perform movement outside the task or well-controlled value manipulations within the session would be needed to really refine what these responses are related to.

If activity of EP^Sst+ neurons included a predicted value component, we would expect to see a change in activity during ipsilateral movements when the previous trial was rewarded vs unrewarded. This is examined in Fig 2—figure suppl. 2C, where we compare EP^Sst+ responses during ipsilateral trials when the previous trials were either rewarded (blue) or unrewarded (gray). We show that EP^Sst+ activity prior to side port entry (SE) is identical in these two trial types indicating that EP^Sst+ neurons do not show evidence of predicted value of an action in this context. Therefore, we conclude that increased EP^Sst+ activity during ipsilateral trials is primarily related to ipsilateral movement following CX (we call this the “choice” phase of the trial). We also show that other ipsiversive movements outside of the “choice” phase of a trial (such as the return to center port following a contralateral trial) show a smaller but significant increase in activity (Figure 2—figure supplement 1F-G). Therefore, whereas the activity observed during ipsilateral choice contains signals related to ipsilateral movement and additional factors, our data suggest that predicted value is not one of those factors. We will clarify this point and our definition of “choice” in the narrative.  

(2) The authors conclude that they do not see any evidence for bidirectional prediction errors. It is not possible to conclude this. First, they see a large response in the EP sst+ neurons to the omission of an expected reward. This is what would be expected of a negative reward prediction error. There are much more specific well-controlled tests for this that are commonplace in head-fixed and freely moving paradigms that could be tested to probe this. The authors do look at the effect of previous trials on the response and do not see strong consistent results, but this is not a strong formal test of what would be expected of a prediction error, either a positive or negative. The other way they assess this is by looking at the size of the responses in different recording sessions with different reward contingencies. They claim that the size of the reward expectation and prediction error should scale with the different reward probabilities. If all the reward probabilities were present in the same session this should be true as lots of others have shown for RPE. Because however this data was taken from different sessions it is not expected that the responses should scale, this is because reward prediction errors have been shown to adaptively scale to cover the range of values on offer (Tobler et al., Science 2005). A better test of positive prediction error would be to give a larger-than-expected reward on a subset of trials. Either way, there is already evidence that responses reflect a negative prediction error in their data and more specific tests would be needed to formally rule in or out prediction error coding especially as previous recordings have shown it is present in previous primate and rodent recordings.

We do not conclude that we see no evidence for RPE and the reviewer is correct in stating that a large increase in EP^Sst+ activity following omission of an expected reward would be expected of a negative reward prediction error. However, this observation alone is not strong enough evidence that EP^Sst+ neurons signal RPE. When we looked for additional evidence of RPE within our experiments we did not find consistent demonstrations of its existence in our data. When performing photometry measurements of dopamine release in the striatum, RPE signals are readily observed with a task identical to ours using trial history to as a modifier of reward prediction (Chantranupong, et al 2023). Of course, there could be a weaker more heterogeneous RPE signal in EP^Sst+ neurons that we cannot detect with our methods. As we state in the discussion, RPE signals may be present in a subset of individual neurons (as observed in Stephenson-Jones et al, 2016 and Hong and Hikosaka, 2008) which are below our detection threshold using fiber photometry. Additionally, Hong and Hikosaka, 2008 show that LHb-projecting GPi neurons show both positive and negative reward modulations which may obscure observation of RPE signals with photometry recordings that arise from population activity of genetically defined neurons.   

(3) There are a lot of variables in the GLM that occur extremely close in time such as the entry and exit of a port. If two variables occur closely in time and are always correlated it will be difficult if not impossible for a regression model to assign weights accurately to each event. This is not a large issue, but it is misleading to have regression kernels for port entry and exits unless the authors can show these are separable due to behavioral jitter or a lack of correlation under specific conditions, which does not seem to be the case.

It is true that two variables that are always correlated are redundant in a GLM. For example, center entry (CE) and center exit (CX) occur in quick succession in most trials and are highly correlated (Figure 1C). For this reason, when only one is removed as a predictor from the model but not the other there is a very small change in the MSE of the fit (Figure 3E, -CE or -CX). However, when both are removed model performance decreases further indicating that center-port nose-pokes do contribute to model performance (Figure 3E, -CE/CX). Due to the presence/absence of reward following side port entry there is substantial behavioral jitter (due to water consumption in rewarded trials) that the SE and SX are not always correlated, therefore the model performs worse when either are omitted alone, but even worse still when both SE/SX are omitted together (Figure 3E, -SE/SX). We will update Figure 3 and the narrative to make this more explicit.

Reviewer #3 (Public Review):

Summary:

The authors find that Sst-EPN neurons, which project to the lateral habenula, encode information about response directionality (left vs right) and outcome (rewarded vs unrewarded). Surprisingly, impairment of vesicular signaling in these neurons onto their LHb targets did not impair probabilistic choice behavior.

Strengths:

Strengths of the current work include extremely detailed and thorough analysis of data at all levels, not only of the physiological data but also an uncommonly thorough analysis of behavioral response patterns.

Weaknesses:

Overall, I saw very few weaknesses, with only two issues, both of which should be possible to address without new experiments:

(1) The authors note that the neural response difference between rewarded and unrewarded trials is not an RPE, as it is not affected by reward probability. However, the authors also show the neural difference is partly driven by the rapid motoric withdrawal from the port. Since there is also a response component that remains different apart from this motoric difference (Figure 2, Supplementary Figure 1E), it seems this is what needs to be analyzed with respect to reward probability, to truly determine whether there is no RPE component. Was this done?

We thank the reviewer for this comment, we believe this is particularly important for unrewarded trials as SE and SX occur in rapid succession. In Figure 2—figure supplement 2A-B we now show the photometry signal from Rewarded and Unrewarded ipsilateral trials aligned to SX for different reward probabilities. We quantify the signals for different reward probabilities during a 500ms window immediately prior to SX but find no differences between groups.  

(2) The current study reaches very different conclusions than a 2016 study by Stephenson-Jones and colleagues despite using a similar behavioral task to study the same Sst-EPN-LHb circuit. This is potentially very interesting, and the new findings likely shed important light on how this circuit really works. Hence, I would have liked to hear more of the authors' thoughts about possible explanations of the differences. I acknowledge that a full answer might not be possible, but in-depth elaboration would help the reader put the current findings in the context of the earlier work, and give a better sense of what work still needs to be done in the future to fully understand this circuit.

For example, the authors suggest that the Sst-EPN-LHb circuit might be involved in initial learning, but play less of a role in well-trained animals, thereby explaining the lack of observed behavioral effect. However, it is my understanding that the probabilistic switching task forces animals to continually update learned contingencies, rendering this explanation somewhat less persuasive, at least not without further elaboration (e.g. maybe the authors think it plays a role before the animals learn to switch?).

Also, as I understand it, the 2016 study used manipulations that likely impaired phasic activity patterns, e.g. precisely timed optogenetic activation/inhibition, and/or deletion of GABA/glutamate receptors. In contrast, the current study's manipulations - blockade of vesicle release using tetanus toxin or deletion of VGlut2, would likely have blocked both phasic and tonic activity patterns. Do the authors think this factor, or any others they are aware of, could be relevant?

We have added further discussion of the Stephenson-Jones, et al 2016 study as well as the Lazaridis, et al 2019 study which shows no effect of phasic stimulation of EP when specifically manipulating EP^Sst+ (vGat+/vGlut2+) neurons rather than vGlut2+ neurons as in the Stephenson-Jones study.  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

In some places, there seems to be a mismatch between referenced figures and texts. For example:

(1) The authors described that 'This increase in activity was seen for all three reward probabilities tested (90/10, 80/20, and 70/30) and occurred while the animal was engaged in ipsiversive movements as similar increases were observed following side exit (SX) on contralateral trials as the animal was moving from the contralateral side port back to the center port (Figure 2-Figure Supplement 1c)', but supplement 1c is not about calcium dynamics around the SX event. I presume they mean Figure 2-Figure Supplement 1d.

Yes, this will be corrected in the revised manuscript.

(2) The authors explained that increased EPSst+ neuronal activity following an unrewarded outcome was partially due to the rapid withdrawal of the animal's snout following an unrewarded outcome however, differences in rewarded and unrewarded trials were still distinguishable when signals were aligned to side port exit indicating that these increases in EPSst+ neuronal activity on unrewarded trials were a combination of outcome evaluation (unrewarded) and side port withdrawal occurring in quick succession (SX, Figure 2 - Figure Supplement 1d). I presume that they mean Figure 2 - Figure Supplement 1e.

Yes, this will be corrected in the revised manuscript.

Minor suggestions related to specific figure presentation are below:

Figure 2 and supplement figures:

(1) Figure 2B: the authors may consider presenting outcome-related signals recorded from all trials, including both ipsilateral and contralateral events, and align signals to SE when reward consumption presumably begins, rather than aligning to CE.

We have added sample recordings from ipsilateral and contralateral trials and sorted them by trial duration to allow for clearer presentation of activity following CE and SE (Figure 2—figure supplement 1a-b).

(2) The authors described that 'This increase in activity was seen for all three reward probabilities tested (90/10, 80/20, and 70/30) and occurred while the animal was engaged in ipsiversive movements as similar increases were observed following side exit (SX) on contralateral trials as the animal was moving from the contralateral side port back to the center port (Figure 2-Figure Supplement 1c)', but supplement 1c is not about calcium dynamics around the SX event. I presume they mean Figure 2-Figure Supplement 1d.

Yes, this will be corrected in the revised manuscript.

(3) The authors explained that increased EPSst+ neuronal activity following an unrewarded outcome was partially due to the rapid withdrawal of the animal's snout following an unrewarded outcome however, differences in rewarded and unrewarded trials were still distinguishable when signals were aligned to side port exit indicating that these increases in EPSst+ neuronal activity on unrewarded trials were a combination of outcome evaluation (unrewarded) and side port withdrawal occurring in quick succession (SX, Figure 2 -Figure Supplement 1d). I presume that they mean Figure 2 -Figure Supplement 1e.

Yes, this will be corrected in the revised manuscript.

Figure 3 and supplement figures:

(1) Figure 3C-F: it is hard to compare the amplitude of calcium signals between different behaviour events without a uniform y-axis.

The scale for the y-axis on Figure 3C-D is uniform for all panels. Figure 3E is also uniform for all boxplots. The reviewer may be referring to Figure 2C-F, but the y-axis for all of the photometry data is uniform for all panels and the horizontal line represents zero. The y-axis for the quantification on the right of each panel is scaled to the max/min for each comparison.

(2) Figure 3E is difficult to follow. The authors explained that the 'SE' variable is generated by collapsing the ipsilateral and contralateral port entries, and hence the variable has no choice of direction information. I assumed that the 'SX', 'CE', and 'CX' variables are generated similarly. It is not clear if this is the case for the 'side', 'centre' and 'choice' variables. The authors explained that 'omitting center port entry/exit together or individually also resulted in decreased GLM performance but to a smaller degree than the omission of choice direction (Figure 3e, "-Center")'. My understanding is that they created the Centre variable by collapsing ipsilateral and contralateral centre port entry/exit together. The Centre variable should have no choice of direction information. How is the Center variable generated differently from omitting centre port entry/exit together? I would ask the authors to explain the model and different variables a bit more thoroughly in the text.

We apologize for the confusion. All ten variables used to train the full GLM are listed in Fig. 3C. In Figure 3E variable(s) were omitted to test how they contributed to GLM performance (data labeled “None” is the full model with all variables). Omitted variables are now defined as follows: -Rew = Rew+Unrew removed, -Direction = Ipsi/Contra designation removed and collapsed into CE, CX, SE, SX, -Direction & Rew = Ipsi/Contra info removed from all variables + Rew/Unrew removed, -CE/CX = Ipsi/Contra CE and CX removed, -CE = Ipsi/contra CE removed, -CX = Ipsi/contra CX removed, -SE/SX = Ipsi/Contra SE and SX removed, -SE = Ipsi/contra SE removed, -SX = Ipsi/contra SX removed. This clarification has also been added to the Generalized Linear Model section of Materials and Methods.

Figure 5 and supplement figures:

There are no representative and summary figures show the specificity and efficiency of oChief-tdTomato or Tetx-GFP expression. Body weight changes following virus injection are not well described.

A representative image of Tettx GFP expression are shown in Fig. 4A and percent of infected EP^Sst+ neurons is described in the text (70±15.1% (mean±SD), 1070±230 neurons/animal, n=6 mice). Most oChief-tdTom animals were used for post-hoc electrophysiology experiments and careful quantification of viral expression was not possible. However, Slc17a6 deletion was confirmed in these animals (Fig. 5 – Fig supplement 1J-K) to confirm the manipulation was effective in the experimental group. A representative image of oChief-tdTom expression is shown in Fig. 5A.

We now mention the body weight changes observed following Tettx injection in the narrative.

Reviewer #2 (Recommendations For The Authors):

(1) In the RFLR section you state that "this variable decays...", a variable can't decay only the value of a variable can change. Also, it is not mentioned what variable is being discussed. There are lots of variables in the model so this should be made clear.

We now state, “This variable (β) changes over trials and is updated with new evidence from each new trial’s choice and outcome with an additional bias towards or away from its most recent choice (Figure 1-figure supplement 2A-C).”

(2) I couldn't find in the results section, or the methods section the details for the Tet tx experiments, were mice trained and tested on 90/10 only? Were they trained while the virus was expressing etc? This should be added.

In the methods section we state, ”For experiments where we manipulated synaptic release in EP^Sst+ neurons (Figures 4-5) we trained mice (reward probabilities 90/10, no transparent barrier present) to the following criteria for the 5 days prior to virus injection: 1) p(highport) per session was greater than or equal to 0.80 with a variance less than 0.003, 2) p(switch) per session was less than or equal to 0.15 with a variance less than 0.001, 3) the p(left port) was between 0.45-0.55 with a variance less than 0.005, and 4) the animal performed at least 200 trials in a session. The mean and variance for these measurements was calculated across the five session immediately preceding surgery. The criterion were determined by comparing performance profiles in separate animals and chosen based on when animals first showed stable and plateaued behavioral performance. Following surgery, mice were allowed to recover for 3 days and then continued to train for 3 weeks during viral expression. Data collected during the 5 day pre-surgery period was then compared to data collected for 10 sessions following the 3 weeks allotted for viral expression (i.e. days 22-31 post-surgery).”

Reviewer #3 (Recommendations For The Authors):

(1) The kernel in Figure 3C shows an activation prior to CE on "contra" trials that is not apparent in Figure 2C which shows no activation prior to CE on either contra or ipsi trials. Given that movement directionality prior to CE is dictated by the choice on the PREVIOUS trial, is the "contra" condition in 3C actually based on the previous trial? If so, this should be clarified.

On most “contra” trials the animal is making an ipsiversive movement just prior to CE as it returns to the center from the contralateral side-port (as most trials are no “switch” trials). Therefore, an increase in activity is expected and shown most clearly following SX for contralateral trials in Fig 2 –Fig suppl 1F. A significant increase in activity prior to CE on contra trials compared to ipsi trials can also be seen in Fig 2C, its just not as large a change as the increase observed following CE for ipsi. trials. The comparison between activity observed during the two types of ipsiversive movements is now shown directly in Figure 2—figure supplement 1G.

(2) Paragraph 7 of the discussion uses a phrase "by-in-large", which probably should be "by and large".

Thank you for the correction.

Editor's note:

Should you choose to revise your manuscript, if you have not already done so, please include full statistical reporting including exact p-values wherever possible alongside the summary statistics (test statistic and df) and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05 in the main manuscript.

Readers would also benefit from coding individual data points by sex and noting N/sex.

Sex breakdown has been added to figure legends for each experiment, full statistical reporting is now also include in the figure legends.

Reviewer #2 (Public review):

Summary:

In this work, Gupta & Murphy present several parallel efforts. On one side, they present the hardware and software they use to build a head-fixed mouse experimental setup that they use to track in "real-time" the calcium activity in one or two spots at the surface of the cortex. On the other side, the present another setup that they use to take advantage of the "real-time" version of DeepLabCut with their mice. The hardware and software that they used/develop is described at length, both in the article and in a companion GitHub repository. Next, they present experimental work that they have done with these two setups, training mice to max out a virtual cursor to obtain a reward, by taking advantage of auditory tone feedback that is provided to the mice as they modulate either (1) their local cortical calcium activity, or (2) their limb position.

Strengths:

This work illustrates the fact that thanks to readily available experimental building blocks, body movement and calcium imaging can be carried using readily available components, including imaging the brain using an incredibly cheap consumer electronics RGB camera (RGB Raspberry Pi Camera). It is a useful source of information for researchers that may be interested in building a similar setup, given the highly detailed overview of the system. Finally, it further confirms previous findings regarding the operant conditioning of the calcium dynamics at the surface of the cortex (Clancy et al. 2020) and suggests an alternative based on deeplabcut to the motor tasks that aim to image the brain at the mesoscale during forelimb movements (Quarta et al. 2022).

Weaknesses:

This work covers 3 separate research endeavors: (1) The development of two separate setups, their corresponding software. (2) A study that is highly inspired from the Clancy et al. 2020 paper on the modulation of the local cortical activity measured through a mesoscale calcium imaging setup. (3) A study of the mesoscale dynamics of the cortex during forelimb movements learning. Sadly, the analyses of the physiological data appears uncomplete, and more generally the paper tends to offer overstatements regarding several points:<br /> - In contrast to the introductory statements of the article, closed-loop physiology in rodents is a well-established research topic. Beyond auditory feedback, this includes optogenetic feedback (O'Connor et al. 2013, Abbasi et al. 2018, 2023), electrical feedback in hippocampus (Girardeau et al. 2009), and much more.<br /> - The behavioral setups that are presented are representative of the state of the art in the field of mesoscale imaging/head fixed behavior community, rather than a highly innovative design. In particular, the closed-loop latency that they achieve (>60 ms) may be perceived by the mice. This is in contrast with other available closed-loop setups.<br /> - Through the paper, there are several statements that point out how important it is to carry out this work in a closed-loop setting with an auditory feedback, but sadly there is no "no feedback" control in cortical conditioning experiments, while there is a no-feedback condition in the forelimb movement study, which shows that learning of the task can be achieved in the absence of feedback.<br /> - The analysis of the closed-loop neuronal data behavior lacks controls. Increased performance can be achieved by modulating actively only one of the two ROIs, this is not clearly analyzed (for instance looking at the timing of the calcium signal modulation across the two ROIs. It seems that overall ROIs1 and 2 covariate, in contrast to Clancy et al. 2020. How can this be explained?

Reviewer #3 (Public review):

Summary:

The study demonstrates the effectiveness of a cost-effective closed-loop feedback system for modulating brain activity and behavior in head-fixed mice. Authors have tested real-time closed-loop feedback system in head-fixed mice two types of graded feedback: 1) Closed-loop neurofeedback (CLNF), where feedback is derived from neuronal activity (calcium imaging), and 2) Closed-loop movement feedback (CLMF), where feedback is based on observed body movement. It is a python based opensource system, and authors call it CLoPy. The authors also claim to provide all software, hardware schematics, and protocols to adapt it to various experimental scenarios. This system is capable and can be adapted for a wide use case scenario.

Authors have shown that their system can control both positive (water drop) and negative reinforcement (buzzer-vibrator). This study also shows that using the close loop system mice have shown better performance, learnt arbitrary task and can adapt to change in the rule as well. By integrating real-time feedback based on cortical GCaMP imaging and behavior tracking authors have provided strong evidence that such closed-loop systems can be instrumental in exploring the dynamic interplay between brain activity and behavior.

Strengths:

Simplicity of feedback systems designed. Simplicity of implementation and potential adoption.

Weaknesses:

Long latencies, due to slow Ca2+ dynamics and slow imaging (15 FPS), may limit the application of the system.

Major comments:

(1) Page 5 paragraph 1: "We tested our CLNF system on Raspberry Pi for its compactness, general-purpose input/output (GPIO) programmability, and wide community support, while the CLMF system was tested on an Nvidia Jetson GPU device." Can these programs and hardware be integrated with windows-based system and a microcontroller (Arduino/ Tency). As for the broad adaptability that's what a lot of labs would already have (please comment/discuss)?

(2) Hardware Constraints: The reliance on Raspberry Pi and Nvidia Jetson (is expensive) for real-time processing could introduce latency issues (~63 ms for CLNF and ~67 ms for CLMF). This latency might limit precision for faster or more complex behaviors, which authors should discuss in the discussion section.

(3) Neurofeedback Specificity: The task focuses on mesoscale imaging and ignores finer spatiotemporal details. Sub-second events might be significant in more nuanced behaviors. Can this be discussed in the discussion section?

(4) The activity over 6s is being averaged to determine if the threshold is being crossed before the reward is delivered. This is a rather long duration of time during which the mice may be exhibiting stereotyped behaviors that may result in the changes in DFF that are being observed. It would be interesting for the authors to compare (if data is available) the behavior of the mice in trials where they successfully crossed the threshold for reward delivery and in those trials where the threshold was not breached. How is this different from spontaneous behavior and behaviors exhibited when they are performing the test with CLNF?

Virtual Hermans - Lucas Dul

Lucas Dul does an overview of affordable and available tools for typewriter repair as well as more advanced

Basic Tools

• screwdriver sets
  • Carpenter screwdrivers (come to a point) the point can slip and causecam out screws
  • Hollow ground - provide the most amount of torque and prevent cam-out problems (also called gunsmith He uses the 0623 Chapman set (the number is the date of international typewriter day) The large tip can be problematic
  • long reach screwdriver
  • magnetic screwdriver
  • tempered stainless steel ruler (as a screwdriver, especially
  • microdrivers (usually used for eyeglasses or electronics)
• spring hooks (push/pull)
  • Fixture from an embroidery set with length for getting length
  • grab hooks
• pliers
  • standard needle nose pliers
  • 45-90 degree pliers (he uses more often)
  • wire cutters (for modifying springs in machines and modifying links in machines)
  • parallel draw pieces (with heavy duty cutters)
• Mechanics' wrench set
  • prefer cast ones
  • socket screwdrivers (fixed hex screwdriver) expecially for shift adjustment on the Royal Ps
  • Chapman's has a mini rachet 1/4" socket in it's 0623 set
• Forceps especially a long pair for IBM Selectrics (via Duane Jensen)
• Tweezers
• Blowtorches
  • alcohol torches (for heating and bending metal)
  • soldering, brazing, and heat shrinking
  • small butane torch (cigarette lighter use)
• Oilers with needlepoint applicator (he uses sewing machine oil)
  • One can use the surface tension of the oil to place a dot on the tip of a scewdriver (flat head) and then place the dot within a machine with reasonable precision
• MIG Pliers - have cutouts for taking rubber off of old feedrollers (otherwise these pliers are used for welding); he describes it as the nutcracker of the typewriter world
• strap wrench (especially for removing platen knobs to prevent damage)
• knife (butter knife)
  • razor blade for trimming rubber (otherwise too thin for other applications)
• flashlight (simple is fine)
• marker (Sharpie)
  • marking orientation of removed parts (washes off with alcohol)
• hammer
• retainer clip pliers (especially for IBMs, Brothers, Swintecs) with spare e- and c-clips (some have thumbscrews for minimizing damage to clips) openers are more useful than "closers"
• Bristol wrenches - looks like Allen Keys, but with star cross section for bristol locks in IBM machines
• Hand crank (for IBM Selectrics) thread into the operational cam shaft

Intermediate Tools

• segment bearing rod (good for removing individual typebars)
• drinking straw for ball bearings on royal portables and S-C portables and flat tops staggered 1/2" ball bearings with orbital gear (star-shaped) - snip opposite sides to insert orbital ring and ball bearing for holding and placement in typewriter
• carpenter's pencil for marking

Advanced/Specialty tools

• t-bender for forming metal (exp. thin pieces)
• 9 jaw pliers for bending typebars
• peening pliers (for manipulating and stretching materials)
• wheel benders (he doesn't use often)
• eyelete tool for putting eyelets in typewritter ribbon
• files (small/cheap) widen gaps inside of type guides when necessary or thinning out tight pieces

Very specialized

• Type slug solder jig or solder guide (30:52)
• keyring pliers ($400 and above to purchase)
• multimeter for checking circuit components on electric models. Primarily using Ohm setting to see if current is passing through parts, otherwise they're broken.

Honorable mentions

• center punch for drilling points and new screws
• dental mirror for looking into machines
• spring gauge to set 2lbs for desktop and 1lb for portables

Q&A

air compressors are useful for cleaning

Don't damage screws on older machines.

US used imperial screws until 1940/50s and machines after are all metric.

3 dessert island tools<br /> - screwdrivers, pliers, spring hook

-

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this work, a screening platform is presented for rapid and cost-effective screening of candidate genes involved in Fragile Bone Disorders. The authors validate the approach of using crispants, generating FO mosaic mutants, to evaluate the function of specific target genes in this particular condition. The design of the guide RNAs is convincingly described, while the effectiveness of the method is evaluated to 60% to 92% of the respective target genes being presumably inactivated. Thus, injected F0 larvae can be directly used to investigate the consequences of this inactivation.

Skeletal formation is then evaluated at 7dpf and 14dpf, first using a transgenic reporter line revealing fluorescent osteoblasts, and second using alizarin-red staining of mineralized structures. In general, it appears that the osteoblast-positive areas are more often affected in the crispants compared to the mineralized areas, an observation that appears to correlate with the observed reduced expression of bglap, a marker for mature osteoblasts, and the increased expression of col1a1a in more immature osteoblasts.

Finally, the injected fish (except two lines that revealed high mortality) are also analyzed at 90dpf, using alizarin red staining and micro-CT analysis, revealing an increased incidence of skeletal deformities in the vertebral arches, fractures, as well as vertebral fusions and compressions for all crispants except those for daam2. Finally, the Tissue Mineral Density (TMD) as determined by micro-CT is proposed as an important marker for investigating genes involved in osteoporosis.

Taken together, this manuscript is well presented, the data are clear and well analyzed, and the methods are well described. It makes a compelling case for using the crispant technology to screen the function of candidate genes in a specific condition, as shown here for bone disorders.

Strengths:

Strengths are the clever combination of existing technologies from different fields to build a screening platform. All the required methods are comprehe Zebrafish tanks_13062024nsively described.

We would like to thank the reviewer for highlighting the strengths of our paper.  

Weaknesses:

One may have wished to bring one or two of the crispants to the stage of bona fide mutants, to confirm the results of the screening, however, this is done for some of the tested genes as laid out in the discussion.

We thank the reviewer for their comment. We would like to point out that indeed similar phenotypes have been observed in existing models, as mentioned in the discussion section.

Reviewer #2 (Public review):

Summary:

More and more genes and genetic loci are being linked to bone fragility disorders like osteoporosis and osteogenesis imperfecta through GWAS and clinical sequencing. In this study, the authors seek to develop a pipeline for validating these new candidate genes using crispant screening in zebrafish. Candidates were selected based on GWAS bone density evidence (4 genes) or linkage to OI cases plus some aspect of bone biology (6 genes). NGS was performed on embryos injected with different gRNAs/Cas9 to confirm high mutagenic efficacy and off-target cutting was verified to be low. Bone growth, mineralization, density, and gene expression levels were carefully measured and compared across crispants using a battery of assays at three different stages.

Strengths:

(1) The pipeline would be straightforward to replicate in other labs, and the study could thus make a real contribution towards resolving the major bottleneck of candidate gene validation.

(2) The study is clearly written and extensively quantified.

(3) The discussion attempts to place the phenotypes of different crispant lines into the context of what is already known about each gene's function.

(4) There is added value in seeing the results for the different crispant lines side by side for each assay.

We would like to thank the reviewer for highlighting the strengths of our paper.  

Weaknesses:

(1) The study uses only well-established methods and is strategy-driven rather than question/hypothesis-driven.

We thank the reviewer for this correct remark. The mayor aim of this study was to establish a workflow for rapid in vivo functional screening of candidate genes across a broad range of FBDs. 

(2) Some of the measurements are inadequately normalized and not as specific to bone as suggested:

(a) The measurements of surface area covered by osteoblasts or mineralized bone (Figure 1) should be normalized to body size. The authors note that such measures provide "insight into the formation of new skeletal tissue during early development" and reflect "the quantity of osteoblasts within a given structure and [is] a measure of the formation of bone matrix." I agree in principle, but these measures are also secondarily impacted by the overall growth and health of the larva. The surface area data are normalized to the control but not to the size/length of each fish - the esr1 line in particular appears quite developmentally advanced in some of the images shown, which could easily explain the larger bone areas. The fact that the images in Figure S5 were not all taken at the same magnification further complicates this interpretation.

We thank the reviewer for this detailed and insightful remark. We agree with the reviewer and recognize that the results may be influenced by size differences. However, we do not normalize for size, as variations in growth were considered as part of the phenotypic outcome. This consideration has been addressed in the discussion section.

Line 335-338: ‘Although the measurements of osteoblast-positive and mineralized surface areas may be influenced by size differences among some of the crispants, normalization to size parameters was not conducted, as variations in growth were considered integral to the phenotypic outcome.’

Line 369: ‘Phenotypic variability in these zebrafish larvae can be attributed to several factors, including crispant mosaicism, allele heterogeneity, environmental factors, differences in genomic background and development, and slightly variable imaging positioning.’

(b) Some of the genes evaluated by RT-PCR in Figure 2 are expressed in other tissues in addition to bone (as are the candidate genes themselves); because whole-body samples were used for these assays, there is a nonzero possibility that observed changes may be rooted in other, non-skeletal cell types.

We thank the reviewer for this valuable comment. We acknowledge that the genes assessed by RT-PCR are expressed in other tissues beyond bone. This consideration has been addressed in the discussion section.

Line 362-365: “However, it is important to note that the genes evaluated by RT-PCR are not exclusively expressed in bone tissue. Since whole-body samples were used for expression analysis, there is a possibility that the observed changes in gene expression may be influenced by other non-skeletal cell types”.

(3) Though the assays evaluate bone development and quality at several levels, it is still difficult to synthesize all the results for a given gene into a coherent model of its requirement.

We appreciate the reviewer’s  remark. We acknowledge that the results for the larval stages exhibit variability, making it challenging to synthesize them into a coherent model. However, it is important to emphasize that all adult crispant consistently display a skeletal phenotype. Consequently, the feasibility and reproducibility of this screening method are primarily focusing on the adult stages. This consideration has been addressed in the discussion section of the manuscript.

Line 391-399: ‘In adult crispants, the skeletal phenotype was generally more penetrant. All crispants showed malformed arches, a majority displayed vertebral fractures and fusions and some crispants exhibited distinct quantitative variations in vertebral body measurements. This confirmed the role of the selected genes in skeletal development and homeostasis and their involvement in skeletal disease and established the crispant approach as a valid approach for rapidly providing in vivo gene function data to support candidate gene identification.’

(4) Several additional caveats to crispant analyses are worth noting:

(a) False negatives, i.e. individual fish may not carry many (or any!) mutant alleles. The crispant individuals used for most assays here were not directly genotyped, and no control appears to have been used to confirm successful injection. The authors therefore cannot rule out that some individuals were not, in fact, mutagenized at the loci of interest, potentially due to human error. While this doesn't invalidate the results, it is worth acknowledging the limitation.

We thank the reviewer for this valuable remark. We recognize the fact that working with crispants has certain limitations, including the possibility that some individuals may carry few or no mutant alleles. To address this issue, we use 10 individual crispants during the larval stage and 5 during the adult stage. Although some individuals may lack the mutant alleles, using multiple fish helps reduce the risk of false negatives.

Furthermore, we perform NGS analysis on pools of 10 embryos from the same injection clutch as the fish used in the various assays to assess the indel efficiency. While there remains a possibility of false negatives, the overall indel efficiency, as indicated by our NGS analysis,  is high (>90%), thereby reducing the likelihood of having crispants with very low indel efficiency. We included this in the discussion.

Line 387-390: ‘While there remains a possibility of false negatives, the overall indel efficiency, as indicated by our NGS analysis,  is high (>90%), thereby reducing the likelihood of having crispants with very low indel efficiency.’

(b) Many/most loci identified through GWAS are non-coding and not easily associated with a nearby gene. The authors should discuss whether their coding gene-focused pipeline could be applied in such cases and how that might work.

The authors thank the reviewer for this insightful comment. Our study is focused on strong candidate genes rather than non-coding variants. We recognize that the use of this workflow poses challenges for analyzing non-coding variants, which represents a limitation of the crispant approach. We have addressed this issue in the discussion section of the manuscript.

Line 131: ‘Gene-based’

Line 453: ‘Gene-based’

Line 311-314: ‘It is important to note that this study focused on candidate genes for osteoporosis, not on the role of specific variants identified in GWAS studies. Non-coding variants for instance, which are often identified in GWAS studies,  present significant challenges in terms of functional validation and interpretation.’

Reviewer #3 (Public review):

Summary:

The manuscript "Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility" describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). The authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observed varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes.

A major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested.

We would like to thank the reviewer for highlighting the strengths of our paper.  

A major weakness is a lack of new insights into underlying mechanisms that may contribute to disease phenotypes, nor any clear commonalities across gene sets. This was most evident in the qRT-PCR analysis of select skeletal developmental genes, which all showed varied changes in fold and direction, but with little insight into the implications of the results.

We thank the reviewer for this insightful remark. We want to emphasize that this study focusses on establishing a new screening method for candidate genes involved in FBDs, rather than investigating the underlying mechanisms contributing to disease phenotypes. However, to investigate the underlying mechanisms in these crispants, the creation of bona fide mutants is necessary. We have included this consideration in the discussion.

Furthermore, we acknowledge that the results for the larval stages exhibit variability, which can complicate the interpretation of these findings. This is particularly true for the RT-PCR analysis, where whole-body samples were used, raising the possibility that other tissues may influence the expression results. Therefore, our primary focus is on the adult stages, as all crispants display a skeletal phenotype at this age. We have elaborated on this point in the discussion.

Line 462-463: ‘Moreover, to explore the underlying mechanisms contributing to disease phenotypes, it is essential to establish stable knockout mutants derived from the crispants’.

Line 391-399: ‘In adult crispants, the skeletal phenotype was generally more penetrant. All crispants showed malformed arches, a majority displayed vertebral fractures and fusions and some crispants exhibited distinct quantitative variations in vertebral body measurements. This confirmed the role of the selected genes in skeletal development and homeostasis and their involvement in skeletal disease and established the crispant approach as a valid approach for rapidly providing in vivo gene function data to support candidate gene identification.’

Ultimately, the authors were able to show their approach is capable of connecting candidate genes with perturbation of skeletal phenotypes. It was surprising that all four GWAS candidate genes (which presumably were lower confidence) also produced a result.

We appreciate the reviewer’s comment. We would like to direct attention to the discussion section, where we offer a possible explanation for the observation that all four GWAS candidate genes produce a skeletal phenotype.

Line 460-410: 'The more pronounced and earlier phenotypes in these zebrafish crispants are most likely attributed to the quasi knock-out state of the studied genes, while more common less impactful variants in the same genes result in typical late-onset osteoporosis (Laine et al., 2013) . This phenomenon is also observed in knock-out mouse models for these genes (Melville et al., 2014)(Coughlin et al., 2019).’

These authors have previously demonstrated that crispants recapitulate skeletal phenotypes of stable mutant lines for a single gene, somewhat reducing the novelty of the study.

We thank the reviewer for this comment and appreciate their concern. We have indeed demonstrated that crispants can recapitulate the skeletal phenotypes observed in stable mutant lines for the osteoporosis gene LRP5. However, we would like to highlight that the current study represents the first large-scale screening of candidate genes associated with bone disorders, including genes related to both OI and osteoporosis. We have included this information in both the abstract and the discussion

Line 60-62: ‘We advocate for a novel comprehensive approach that integrates various techniques and evaluates distinct skeletal and molecular profiles across different developmental and adult stages.’

Line 456-457: ‘While this work represents a pioneering effort in establishing a screening platform for skeletal diseases, it offers opportunities for future improvement and refinement.’

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Figure 1a: what does the differential shading of the bone elements represent? Explain in the legend.

The differential shading doesn't represent anything specific. It's simply used to enhance the visual appeal and to help distinguish between the different structures. We removed the shading in the figure.

(2) Supplementary Figures 2-5: should the numbering of these figures be also in order of appearance in the text? I understand that the authors prefer to associate the transgenic and the alizarin red-stained specimens, however, the reading would be easier that way.

We changed this accordingly.

(3) Lines 275-276: "no significant differences in standard length (Figure 4a)": should be Figure 4b.

The suggested changes are incorporated in the manuscript.

Line 276-277: ‘Among the eight crispants that successfully matured into adulthood, none exhibited significant differences in standard length and head size (n=5 fish per crispant) (Figure 4b).’

(4) Line 277 "larger eye diameter": should be Figure 4b.

The suggested changes are incorporated in the manuscript.

Line 378: ‘However, esr1 crispants were observed to have notably larger eye diameters (Figure 4b).’

(5) Line 280: "no obvious abnormalities were detected (Figure 4b,c)": should be Figure 4a, c. Note that the authors may reconsider the a, b, c numbering in Figure 4 by inverting a and b.

The suggested changes are incorporated in the manuscript.

Line 278-281: ‘All these crispants demonstrated various abnormalities in the caudal part of the vertebral column such as fusions, compressions, fractures, or arch malformations, except for daam2 crispants where no obvious abnormalities were detected (Figure 4a,c; Supplementary Figure 6).’

(6) Table 2: This table, which recapitulates all the results presented in the manuscript, is in the end the centerpiece of the work. It is however difficult to read in its present form. Three suggestions:

- Transpose it such that each gene has its own column, and the lines give the results for the different measurements

- Place the measurements that result in "ns" for all crispants at the end (bottom) of the table.

- Maybe bring the measurements at 7dpf, 14dpf, and 90 dpf together.

We agree with the reviewer and have added a new table where we transposed the data. However, we chose not to place the measurements that resulted in 'ns' for all crispants at the end of the table, as we believe it is important to track the evolution of the phenotype over time. Where possible, we have grouped the measurements for 7 dpf and 14 dpf together.

Reviewer #2 (Recommendations for the authors):

(1) It would help to justify why these particular area measurements are appropriate for this set of candidate genes, which were selected based on putative links to bone quality rather than bone development.

The selected methods are among the most commonly used to evaluate bone phenotypes. They are straightforward to reproduce, as well as cost- and time-effective. The strength of this approach lies in its use of simple, reproducible techniques that form the foundation for characterizing bone development.  Although the candidate genes were chosen based on their putative links to bone quality, early skeletal phenotypes can already be observed during bone development.

The mineralized surface area of the total head and specific head structures was selected to evaluate the degree of mineralization in early skeletal development, as mineralization is a direct indicator of bone formation. Additionally, the osteoblast-positive surface areas were measured to provide insight into the formation of new skeletal tissue during early development. Osteoblasts, as active bone-forming cells, are essential for understanding bone growth and the dynamics of skeletal phenotypes.

Examples in the manuscript:

Line 212-214: ‘The osteoblast-positive areas in both the total head and the opercle were then quantified to gain insight into the formation of new skeletal tissue during early development.’

Line 221-223: ‘Subsequently, Alizarin Red S (ARS) staining was conducted on the same 7 and 14 dpf crispant zebrafish larvae in order to evaluate the degree of mineralization in the early skeletal structures.’

(2) Reword: The opercle bone is the earliest forming bone of the opercular series, and appears to be what the authors are referring to as the "operculum" at 7-14 dpf. The operculum is the larger structure (gill cover) in which the opercle is embedded. It would be more accurate to simply refer to the opercle at these stages.

We agree with this comment and changed the text accordingly.

(3) Define BMD and TMD at first usage.

BMD and TMD are now defined in the manuscript.

Line 41-43: ‘Six genes associated with severe recessive forms of Osteogenesis Imperfecta (OI) and four genes associated with bone mineral density (BMD), a key osteoporosis indicator, identified through genome-wide association studies (GWAS) were selected.’

Line 286-288: ‘For each of the vertebral centra, the length, tissue mineral density (TMD), volume, and thickness were determined and tested for statistical differences between groups using a regression-based statistical test (Supplementary Figure 7).’

(4) It would be helpful to note the grouping of candidates into OI vs. BMD GWAS throughout the figures.

We agree with this comment and added this to all figure legends.

‘The first four genes are associated with the pathogenesis of osteoporosis, while the last six are linked to osteogenesis imperfecta’

Reviewer #3 (Recommendations for the authors):

Major points:

(1) For the Results, it would be useful to the Reader to justify the selection of human candidate genes and their associated zebrafish orthologs to model skeletal functions. For example, what are variants identified from human studies, and do they impact functional domains? Are these domains and/or proteins conserved between humans/zebrafish? Is there evidence of skeletal expression in humans/zebrafish?

Supplementary Table 4 lists the selected human candidate genes with reported mutations and/or polymorphisms associated with both skeletal and non-skeletal phenotypes. The table also includes additional findings from studies in mice and zebrafish. An extra column was now added to indicate gene conservation between human and zebrafish. We consulted UniProt (https://www.uniprot.org) and ZFIN (https://zfin.org) to assess the skeletal expression of these genes in human and zebrafish. All genes showed expression in the trabecular bone and/or bone marrow in humans, as well as in bone elements in zebrafish. We added this in the discussion.

Line 309: ‘All selected genes show skeletal expression in both human and zebrafish.’

Supplemental table 4 legend: ‘The conservation between human and zebrafish is reported in the last column.’

As part of this, some version of Supplementary Table 4 might be included as a main display to introduce the targeted genes, ideally separated by rare (recessive OI) vs. common disease (osteoporosis). In the case of common disease and GWAS hits, how did authors narrow in on candidate genes (which often have Mbp-scale associated regions spanning multiple genes)? Further, what is the evidence that the mechanism of action of the GWAS variant is haploinsufficiency modeled by their crispant zebrafish?

We have kept Supplementary Table 4 in the supplementary material but have referred to it earlier in the manuscript’s introduction. Consequently, the table has been renumbered from ‘Supplementary Table 4’  to ‘Supplementary Table 1’.

The selection of genes potentially involved in the pathogenesis of osteoporosis is based on the data from the GWAS catalog, which annotates SNPs using the Ensemble mapping pipeline. The available annotation on their online search interface includes any Ensemble genes to which a SNP maps, or the closest upstream and downstream gene within a 50kb window. Four genes were selected for this screening method based on the criteria outlined in the results section. In this study, we aim to evaluate the general involvement of specific genes in bone metabolism, rather than to model a specific variant.

Line 135-136 and 309-311: ‘An overview of the selected genes with observed mutant phenotypes in human, mice and zebrafish is provided in Supplementary Table 1.’

(2) Using the crispant approach does not impact maternally-deposited RNAs that would dampen early developmental phenotypes. Considering the higher variability in larval phenotypes, perhaps the maternal effect plays a role. The authors might investigate developmental expression profiles of their genes using existing RNA-seq datasets such as from White et al (doi: 10.7554/eLife.30860).

We thank the reviewer for this comment and agree with the possibility that maternally-deposited RNAs might have an impact on early developmental phenotypes. We included this in the discussion.

Line 369-372: ‘Phenotypic variability in these zebrafish larvae can be attributed to several factors, including crispant mosaicism, allele heterogeneity, environmental factors, differences in genomic background and development, maternally-deposited RNAs, and slightly variable imaging positioning.’

(3) While making comparisons within a clutch of mutant vs scrambled control is crucial, it is also important to ensure phenotypes are not specific to a single clutch. Do phenotypes remain consistent across different crosses/clutches?

Yes, phenotypes remain consistent across different crosses and clutches. We included images from a second clutch in the Supplementary material (Supplementary Figure 8) and refereed to it in the discussion.

Line 394-397: ‘Additionally, these skeletal malformations were consistently observed in a second clutch of crispants (Supplementary Figure 8), underscoring the reproducibility of these phenotypic features across independent clutches.’

(4) Understanding that antibodies may not exist for many of the selected genes for zebrafish, authors should verify haploinsufficiency using an RT-qPCR of targeted genes in crispants vs. controls.

We appreciate the reviewer’s suggestion to use RT-qPCR to examine expression levels of the targeted genes in crispants. However, previous experience suggests that relying on RNA expression to verify haploinsufficiency in zebrafish can be challenging. In zebrafish KO mutants, RT-qPCR often still detects gene transcripts, potentially due to incomplete nonsense-mediated decay (NMD) of the mutated mRNA, which may allow residual expression even in the absence of functional protein. As a more definitive approach, we prefer to use antibodies to confirm haploinsufficiency at the protein level. However, as the reviewer noted, generating and applying specific antibodies in zebrafish remains challenging.

(5) Please indicate how parametric vs. non-parametric statistical tests were selected for datasets.

We initially selected the parametric unpaired t-test, assuming the data were normally distributed with similar variances between groups. We verified the assumption of equal variances using the F-test, which was not significant across all assays. However, we did not assess the normality of the data directly, meaning we cannot confirm the normality assumption required for the t-test. Given this, we have opted to use the non-parametric Mann-Whitney U test, which does not require assumptions of normality, to ensure the robustness of our statistical analyses. We changed the Figures, the figure legends and the text accordingly.

(6) In the figures and tables, I recommend adding notation showing the grouping of the first four genes as GWAS osteoporosis, the next three genes as osteoblast differentiation, the next two genes as bone mineralization, and the final gene as collagen transport to orient the reader. One might expect there to be a clustering of phenotypic outcomes based on the selection of genes, and it would be easier to follow this. This would be particularly useful to include in Table 2.

Our primary objective is to assess the feasibility and reproducibility of the crispant screen rather than performing an in-depth pathway analysis or categorizing genes by biological processes. For this purpose, we have organized candidate genes based on their relevance to osteoporosis and Osteogenesis Imperfecta, without subdividing them further. We have clarified this focus in the figure legends, as suggested in an earlier recommendation.

(7) For Figure 1, consider adding a smaller zoomed version of 1a embedded in each sub-figure with each measured element highlighted to improve readability.

We agree with this comment and changed the figure accordingly.

Minor points:

(1) Table 2 could be simplified to improve readability. The headers have redundancies across columns with varied time points and could be merged.

The suggested changes are incorporated in the manuscript (see earlier comment about this).

(2) "BMD" is not defined in the Abstract. This is a personal preference, but there were numerous abbreviations in the text that made it difficult to follow at times.

The suggested changes are incorporated in the manuscript (see earlier comment about this).

He was head of the Carter Center.

• https://en.m.wikipedia.org/wiki/Robert_Pastor