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1. General Statements [optional]

All four reviewers have positive comments on the paper. We totally agree with their comments, and proposed controls and experiments. Most of them are already introduced in the present text and several new figures added, as we had the controls/experiments proposed. Few others are now being done and we hope to have the complete set of experiments ready in 2-3 months.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

Reviewer #1

Most comments of this reviewer have already been done and included in the transferred manuscript, except for part of the first comment:

1.1 b. Is it possible that the loss of function of Wengen on its own has a phenotype? If so, that would suggest that Wgn in addition to its role in regeneration might be implicated in pro-survival processes in homeostatic conditions?

This issue is very important to understand the differential role of Wgn and Grnd. First of all, Wengen knock out (wgnKO; Andersen et al., 2015) is viable in homozygosis. However, in this paper we have focused on inducible mutants. Therefore, we have now crossed the flies to get the genotype hh-Gal UAS RNAi wgn and we will check for apoptotic phenotype, as suggested. This will take us few weeks of work.

Reviewer #2 Most comments have been already carried out and included in the transferred manuscript, except these ones:

*2.3. Aside from wgn, other RNAi experiments are not validated through independent RNAi lines. I suggest expanding the Supplemental Figures to reproduce a few key findings with independent RNAi lines. *

We have recently received a set of independent RNAi line to repeat the experiments for Traf1, Traf2, Ask1 from Bloomington Stock Center. And We did not do it before mainly because we wanted to focus on wgn and grnd. However, we agree with the Reviewer 2 and we will do the experiments. Another RNAi from VDRC for grnd and Tak1 have been ordered. These experiments will take about 2 months from the crosses to the analysis of results (some flies still to arrive, and many crosses will be done at 17ºC).

*2 4. In Figure 1E, the authors show that wgn RNAi enhances cell death caused by hh>egr. What is missing here is a wgn RNAi control without hh>egr. Is there any cell death caused by the loss of wgn alone (without hh>egr)? *

This control is now in progress. Expected to have it complete in 2 weeks.

*2.5. If wgn RNAi causes some degree of cell death, is the observed effect with hh>egr a significant genetic interaction, or merely additive? *

The result from the previous comment will help us to respond this point.

• 1. Is the wgn-p38 pathway sufficient to block egr induced cell death? The authors could test this by having hh>egr in the licT1.1 background. The authors have a more complex experiment in Figure 3, where licT1.1 is introduced into the hh>egr, wgn RNAi background. However, testing the effect of licT1.1 without wgn would establish a more direct relationship between egr and wgn-p38. *

We have set the crosses for the experiment hh>egr and licT1.1 as suggested. The results will be included in the new version of the manuscript. 1 month.

Reviewer #3

All comments already carried out and included in the transferred manuscript. See next sections.

__Reviewer #4 __

Major comments:

*4.3 In Figure 5, the cells expressing Rpr appeared to be pulled/extruded basally as expected. It would be beneficial to quantify Wgn and Grnd signals along cross-sections and provide higher magnification images of domain boundaries to illustrate differences in TNFR localization and levels. ** The micrographs for Grnd Figure 5B,D, F capture substantial signal from the peripodial epithelium where the salE/Pv> driver is likely not active? *

We will do a thorough quantification of high-resolution stacks of images and include higher magnification of the analyzed stacks. To this aim, we need some more weeks to collect the images of each genotype, processed and quantify them. We propose to do have this work done in two months.

*4.4 The non-autonomous induction of Wgn seems stronger when facing dying Rpr overexpressing cells simultaneously depleted of Eiger compared to Rpr OE alone. Should this be a reproducible, could the authors discuss potential reason for this observation? *

It is difficult to respond this question, without quantification. The quantification suggested in the previous point, will allow us to state if Wgn is more accumulated in rpr +egr than rpr alone. Therefore, the previous point will tell us if there are significant differences and if, so it will help us to discuss it.

Timing: The entire plan can be executed in 2-3 month.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

__Reviewer #1 __

1.1 a- *The result in Fig1.H is somehow surprising. How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd? *

The results of Fig. 1H, in which egr+grndRNAi+wgnRNAi results in high apoptosis indicates that wgn down regulation compromises survival even in the absence of grnd. The reviewer correctly points that “How does the overexpression of Egr induce caspase activation in the absence of its receptor Grnd?”.

There is evidence that Eiger is involved in the regulation of the pro-apoptotic gene head involution defective (hid) in primordial germ cells (Maezawa 2009 Dev. Growth Differ., 51 (4) (2009), pp. 453-461) and in the elimination of damaged neurons during development (Shklover et al., 2015). Moreover, Eiger is necessary for HID stabilization and regulates HID-induced apoptosis independently of JNK signaling (Shklover et al., 2015). Therefore, in our discs egr activation in the absence of grnd and wgn can still result in apoptosis because of the absence of wgn’s survival signal and, presumably, activation of hid.

We have introduced this issue in the text as:

“To check for epistasis between grnd and wgn, we activated hh> egrweak and knocked down both TNFRs. We found high levels of cell death compared to wgn RNAi alone (Fig. 1H and 1I), which demonstrates that wgn down-regulation is dominant over grnd. This is surprising as it is generally assumed that Egr interacts with Grnd to induce apoptosis via JNK, which in turn activates the proapoptotic gene hid (Andersen et al., 2015; Diwanji & Bergmann, 2020; Fogarty et al., 2016; Igaki et al., 2002; Moreno, Yan, et al., 2002; Sanchez et al., 2019; Shlevkov & Morata, 2012). Interestingly, Egr is necessary for HID stabilization and can regulate HID-induced apoptosis independently of JNK (Shklover et al., 2015). Therefore, cells egrweak that downregulate grnd and wgn can still be eliminated because the lack of both Wgn-survival signal and the pro-apoptotic Grnd/JNK signal could result in an alternative pathway of apoptosis.”

*1.2- In Fig.6, it would be relevant to include wengen inactivation within the domain where rpr is expressed to show that wengen is not required autonomously for regeneration (sal>rpr + wgn RNAi). What is the phenotype of the adult wing of sal-lexA>rpr + nub-gal4 >wgn RNAi animals.? *

We have already added a new figure (Fig. S4C) containing this data. As shown, both wgnRNAi alone and wgn RNAi + rpr do not show relevant anomalies and regenerate normally. Therefore, we conclude that wgn is not autonomously required for regeneration.

The adult wings sal-lexA>rpr + nub-gal4 >wgn RNA result in a strong aberration, as regeneration is inhibited. This experiment has been also added in another figure (Fig. S4B) it is done.

*1.4 Minor- In fig.1I, it is surprising that knockdown of neither Grnd nor dTRAF2 significantly affects Egr-induced apoptosis *

After applying a One-Way ANOVA test to compare all the groups against all the groups in fig. 1B no significative differences were detected between Control and RNAi grnd or RNAi dTRAF2 (p>0,05). But if we apply a Student’s T test, which is less restrictive, we obtain, indeed, significative differences:

Control vs. RNAigrnd p=9,48x10-7

Control vs. RNAi dTRAF2 p=2,47x10-7

We have now added in the text:

“Note that when egrweak cells downregulated dTRAF2 or grnd the cell death area ratio is slightly lower than egrweak alone (Fig. 1I), comfirming that dTRAF2 and Grnd contribute to apoptosis in egrweak cells.”

*1.5 Minor The ability of the wing disc to regenerate has been associated with the induction of a developmental delay mediated by Dilp8. Are the authors observing this developmental delay is the case of sal-lexA>rpr + Ap-gal4 >wgn RNAi or sal-lexA>rpr + Ci-Gal4>wgn RNAi *

The developmental delay due to Dilp8 has been observed by many laboratories, indeed. The question of the reviewer is relevant because if there is no delay in pupariation, regeneration could be compromised not because regeneration has been affected but because after pupariation regeneration is impeded.

However, delay in pupariation has been found in our experiments. Usually for 11hrs of heat shock (to induce apoptosis) we found 1-2 days of delay.

We have added the following text:

“The ability of the wing disc to regenerate after genetic ablation has been associated with the induction of a developmental delay (Colombani et al., 2012; Garelli et al., 2012; Jaszczak et al., 2015; Katsuyama et al., 2015; Smith-Bolton et al., 2009). All genotypes analyzed in figure 6 showed a similar developmental delay of 1-2 days (at 17ºC) after genetic ablation in comparison to the animals of the same genotype in which no genetic ablation was induced, i.e. developed continuously at 17ºC (Fig. S4A). After the adults emerged, the wings were dissected, and regeneration was analyzed.”

*1.7 Minor - The investigation of the evolutionary origin of TNFR in drosophila included in Fig.2 is cutting a bit the flow of the results. *

The evolutionary origin starts now with a sentence that can smoothen the flow and few changes in that paragraph have been made:

“Opposing roles between proteins of the TNFR superfamily suggests that they have an ancient origin and have followed divergent evolutionary paths. To track the differences observed between grnd and wgn, we decided to investigate the evolutionary origin of these two Drosophila genes.”

*1.8 Minor The authors could explain in more details the double transactivation system for non-fly specialists. *

The entire section has been re-written in Material and Methods.

*1.9 Minor - It can be interesting to include and/or discuss these few references: *

*PLoS Genet. 2019 Aug; 15(8): e1008133. ** PLoS Genet. 2022 Dec 5;18(12):e1010533. FEBS Lett. 2023 Oct;597(19):2416-2432. *

*Curr Biol. 2016 Mar 7;26(5):575-84. *

*Nat Commun. 2020 Jul 20;11(1):3631. **

*

All these references, and few others, have been introduced in the text.

__Reviewer #2 __ *2. 1. The authors find that wgn RNAi enhances hh>egr-induced apoptosis. They validate the results with two independent RNAi lines in Figure S1. However, Figure S1 is missing a control without wgn RNAi, and therefore, the results are difficult to assess. *

Fig S1A now contains this control.

• 1. Are the two independent wgn RNAi lines targeting different regions of the coding sequence? *

As the regions targeted by the 2 RNAi’s are different, see below, we have included in the text:

“This observation was corroborated with an independent RNAi-wgn strain targeting a different region in the coding sequence (Fig. S1A and S1B). “

Bloomington BL55275 (dsRNA-HMCO3962)

VDRC GD9152 (dsRNA-GD3427)

*2.7. In Figure 4, the authors show that egr expression induces ROS and performs anti-oxidant experiments. This part could be strengthened if they show that the ROS sensor signal disappears after Sod::Cat expression. *

We had done this experiment and there is a definitively drop in Mitosox in discs in which the weak allele of egr is active. We have included this new image in Figure 4G and in the text.

*2.8. How effective is egr RNAi? In Figure 5E, F, the authors knock down egr and obtain negative results. Based on this, the authors argue that Wgn localization occurs through an egr-independent mechanism. Drawing strong conclusions based on a negative result with egr RNAi is not a good practice since one cannot rule out residual egr activity that mediates the effect (of course , because there is cell death as well, death cells express egr). I suggest either finding ways to completely abolish egr function, or tone down the conclusion. *

We have used ‘after knocking down eiger’ instead of in the ‘absence’ or ‘abolish’ eiger.

• 1. Figure 6 shows that wgn RNAi aggravates the reaper phenotype. What's missing is a control that expresses wgn RNAi but not reaper. *

Control experiments using the UAS-wgnRNAi in the absence of rpr are now shown in figure S4C.

Reviewer #3 ____ 3.1.Minor Fig 6C-E would need a control disc without induced apoptosis (ie wildtype disc) stained for phospho-p38 as a baseline comparison. This is important to judge the significance of the remaining phospho-p38 in panel E where wgn is knocked down. The authors write ** " However, after knocking down wgn, phosphorylated p38 in the wing pouch ** surrounding the apoptotic cells was abolished (Fig. 6E)." *Depending on the amount of phospho-p38 in control discs, this may need to be rephrased to "strongly reduced" instead of "abolished". *

A control disc stained with P-p38 has been added in Figure 6.

We have changes ‘abolished’ by ‘strongly reduced’.

* 3.2. This sentence in the Intro needs fixing because TNFa doesn't transduce the signal from TNFR to Ask1 since it's upstream of TNFR: "TNFα can transduce the TRAF-mediated signal from TNFR to Ask1 to modulate its activity (Hoeflich et al., 1999; Nishitoh et al., 1998, p. 0; Obsil & Obsilova, 2017; Shiizaki et al., 2013)." *

We have rephrased this sentence by:

“TNFα binds to TNFRs which in turn interact with TRAFs to transduce the signal to Ask1 to modulate its activity”.

*3.3a In the results section, the authors start by ectopically overexpressing Eiger. Are there conditions where Eiger expression is induced in the wing? If yes, it would be helpful for the reader to mention that this system with the genetically GAL4-induced expression of Eiger aims to phenocopy one of these conditions. *

Eiger ectopic expression has been induced in the wing to generate apoptosis. This is a common technique in Drosophila, and the Reviewer3 is right that a sentence should be useful for the reader.

A sentence has been introduced at the beginning of the results section:

“Ectopic expression of egr in Drosophila imaginal discs results in JNK-dependent apoptosis (Brodsky et al., 2004; Igaki et al., 2002; Moreno, Yan, et al., 2002).”

*3.3b Fig 2C is not very self-explanatory: it is worth writing out what Hsa (H. sapiens), Bla and Sco stand for (there is plenty of space). *

We have re-designed figure 2 to make it more self-explanatory.

*3.4. This sentence is confusing: ** " ...Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that reduced Egr/TNFα had no effect on Wgn localization (Fig. 5E, 5F)." The authors may want to specify that Wgn is still accumulated even without Egr. ("No effect" is unclear). *

This sentence has been modifies as:

“Wgn localization were due to ROS or to the expression of egr, we used RNAi to knock down egr in the apoptotic cells and found that Wgn accumulation was not altered by the knocking down Egr/TNFα (Fig. 5E, 5F). “

*3.5 Comment. It discovers that Wengen is activated by ROS. In fact, since Wengen binds TNF with an affinity that is several orders of magnitude lower than Grindelwald, and since Wengen is not even located at the cell membrane, these data call into question whether Wengen is a TNF receptor, or a ROS receptor? Could the authors comment on this ? Could it be that the results obtained in the past showing that Wengen is activated by TNF were actually due to TNF inducing apoptosis, leading to production of ROS, leading to activation of Wengen?

*

We totally agree with Reviewer#3. We have added a final paragraph in the discussion section.

“We speculate that the subcellular location of Wgn and Grnd may contribute to the different functions of both receptors. Grnd is more exposed at the apical side of the plasma membrane, which makes this receptor more accessible for ligand interactions (Palmerini et al., 2021). Wgn, embedded in cytoplasmic vesicles, is less accessible to the ligand and could be more restricted to being activated by local sources of signaling molecules, such as ROS. In contrast to initial reports (Kanda et al., 2002; Kauppila et al., 2003), los-of-function of wgn does not rescue Egr-induced apoptosis in the Drosophila eye (Andersen et al., 2015), which supports our observation in the wing that Wgn is not required for Egr-induced apoptosis. Instead, Egr-induced apoptosis generates ROS which target intracellular Wgn to foster a cell survival program of cells close to the apoptotic zone.”

__Reviewer #4 __

*4.1 b Are phospho-p38 levels increased in cells expressing Egr[weak]? *

We have the results of these experiments. To respond to this point, a new figure has been added (Fig. S4) in which we show the P-p38 levels are increased (non-autonomously) in egrw, as previously found for reaper. In addition, we show that egrw + activation of p35 and egrw + activation of Sod1::Cat results in strong reduction of P-p38. This indicates that P-p38 is stimulated by the ROS produced by apoptotic cells.

The text now:

“It is worth noting that cells egrw induce phosphorylation of p38 in neighboring cells (Fig. S4A) and that, as previously found for rpr (REF), depends on ROS generated by egrw apoptotic cells (Fig. S4B, C).”

*4.2 In Figure 4C it appears that the Dcp-1 positive cells move apically rather than basally. Including nuclear staining would be very informative allowing assessment of tissue morphology. ** The authors focus on the pouch region of the wing imaginal disc, where phenotypes are strong and obvious. However, the hh-Gal4 driver also affects posterior cells in the hinge and notum, where the effects of Eiger[weak] overexpression seem weaker (e.g., minimal to no MitoSox signal in hinge and notum posterior cells). Could the authors explain this observation? *

Point 1: Actually, cells move more basally, though some move more apical as well. Depending on the section cells the image could be confusing. To solve that, we show now a plane on these discs at apical and a plane basal. Both high magnifications. There one can see that there is more concentration of pyknotic nuclei basally. We have added this observation in a new supplementary figure (Fig. S3) and the corresponding text in page 5: “Apoptotic cells in egrweak are characterized by pyknotic nuclei and are positive for Dcp1. These cells tend to concentrate in the basal side of the epithelium, although some are scattered apically (Fig. S3). Accumulation of Wgn was observed in healthy anterior cells adjacent to both apical and basal egrweak cells (Fig. 4, Fig. S3A, B).”

Point 2 Comment on MitoSOX in notum: At the stages of the imaginal discs used in this study, almost all notum cells are anterior compartment. The hh positive cells in notum much less abundant, therefore most of the staining was found in the posterior compartment of the wing pouch.

*4.5 Figure 6 C-E. Does WgnRNAi potentiates and GrndRNAi suppress Rpr-induced apoptosis similarly to their effects when knocked down in Eiger[weak]OE cells? *

The areas controlled by salE/Pv >rpr (dotted lines) are full of pycnotic nuclei, which indicates concentration of apoptotic cells in all genotypes shown.

Thus, in the conditions generated here, apoptosis is not inhibited and grnd RNAi does not interfere with the activation of P-p38. In wgn knock down, phospho-p38 is strongly inhibited, indicating the importance of wgn in phosphorylation of p38.

To clarify this point, we have added in the text: “Note that rpr-induced apoptosis is not suppressed after knocking down grnd or wgn.” Also in the figure legend we added: “White lines in the confocal images outline the salE/Pv-LHG,LexO-rpr dark area full of pyknotic nuclei of apoptotic cells.”

4.6 The activation of p38 following salE/Pv>rpr-mediated ablation as shown by immunostaining is noteworthy. While loss Grnd knockdown leads to phospho-p38 signal enrichment around the rpr-expressing cells, WgnRNAi results in reduced phospho-p38 signal in the wing pouch but also beyond the nub-expression domain. Do salE/Pv>rpr nub>WgnRNAi cells still generate ROS?

So far there is no evidence of Wengen as a ROS scavenger. We have evidence that ROS (using MitoSox probe) are produced in egrweak + Wgn RNAi cells. Thus, the inhibition of wgn expression does not block ROS production. A new figure shows this observation (Figure S4A).

4.7 Are ROS responsible for the long-range signaling and p38 activation, referring to authors' previous work Santabarbara-Ruiz et al., 2019, PLoS Genet 15(1): e1007926. https://doi.org/10.1371/journal. pgen.1007926, Figure 5G?

ROS are responsible for p38 activation as shown in a new figure (Fig. S4). In this new figure egrweak is activated in hh, and p38 is most of cells in the posterior compartment, and also anterior. However, after blocking apoptosis or ROS production, this P-p38 is reduced.

4.8 Minor I propose rephrasing the description of "UAS-Egr[weak] transgene, a strain that produces a reduced Egr/TNFα activity". It could imply a loss of function strain rather than a transgene that causes mild/moderate Egr overexpression.

The sentence has been rephrases as suggested (End of the first paragragraph in results section).

*4.9 Minor. I recommend the authors to revise the charts for improved clarity in genotype representation. For example, in Figure 1I, the label "control-GFP" might be misleading. It would be beneficial to specify that "control" refers to Eiger[weak] alone with other manipulations being done simultaneously with Eiger[weak] overexpression. *

All charts have been revised.

4.10 Minor. Additionally, considering that individuals with color blindness may struggle to differentiate between red and green colors, I strongly suggest using a color-blind-friendly palette, especially in Figure 4A, C, G, and Figure 4A, C, E." ** All images have been revised for color blind code.

• 11 Minor. Providing detailed information regarding the reagents used in the study, such as Catalogue Numbers or RRIDs, is beneficial for enhancing reproducibility. *

We have added the RRID and Cat #. If no ID was available, we added the reference or contact.

4.12 This reviewer points two limitations that we are now trying to solve:

*Limitations: *

*Quality of the imaging – higher magnification images and quantification would enhance the study. ** The summarizing model may contain excessive speculations that lack support from the data or references to the existing literature. *

Quality of imaging. We have now an extra supplemental figure with higher magnifications. Extra higher magnifications will be included in the next version as well as quantification, as exposed for the Revision Plan points 4.3 and 4.4.

Model: We have re-written the paragraph on the model, introduced references and drop some speculations. We hope the current version will be more convincing for the reader.

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

Reviewer 1

*1.3. Is the overexpression of Wengen sufficient to increase tissue regeneration? *

The suggestion of the reviewer is a key point in regeneration biology: how to accelerate regeneration?

We have demonstrated that Wengen is upstream the Ask1-p38 axis that drives regeneration. The reviewer wonders if Wengen overexpression can result in increase in regeneration. In a previous work we have demonstrated that p38 activation is key for regeneration but its overexpression can be deleterious (Esteban-Collado et al., 2021). Only in discs that sensitized for low p38 (starvation, low Akt, Ask1S83A mutant), the overexpression rescues regeneration. Therefore, the levels of the Wgn-Ask1-p38 have to be very tightly controlled. An excess will be deleterious. We are aware of the importance of the question, but at this point we do not have the technology to finely control Wgn-Ask1-p38 levels to do this experiment.

1.6 Minor - It possible to test the induction of apoptosis in a wgn null mutant background to see if the phenotype is even stronger than the one observed with RNAi (the wgn RNAi is induced at the same time than egr or rpr overexpression).

Flies wgnKO survive, but they gave us problems when carrying transgenes for our design of genetic ablation. Indeed, we tried to generate wgnKO carrying Gal4+tubGal80+eigerweak without success.

In addition, the reason we have used an inducible mutant is because it allows us to work in time and space without altering expression in other cell types beyond wing discs. Wgn is required in other organs during development like gut, trachea and axon growth, etc.., and thus, we ensure the affected cells belong to the tissue analyzed.

Head of School, Dr. Caren Walker Gregory, has been an educator and leader in Boston Public Schools for 32 years, 14 of those leading EMK. Dr. Walker's experience in math and science has perfectly prepared her to foster a robust college preparatory environment that prepares students to excel in health focused careers and higher education after graduation.

– Rebecca Grainger, Senior Advisor for Youth and Schools

We're excited about the partnership between Roxbury Community College and the following schools: Boston International Newcomers Academy, Margarita Muniz Academy, and English High School. This early college program is specifically designed for multilingual students. The first cohort will be starting this month.

English High School is the oldest public school in the Nation, established in 1821, just 45 years after our Nation was founded. There are four technical pathways where students earn industry-recognized credentials and certifications: Business, Finance & Entrepreneurship; Design & Visual Communications; Health Assisting & Wellness; and Programming & Web Development. Caitlin Murphy is the Head of English High School, continuing the legacy of honor, achievement, and service to humanity.

Margarita Muniz Academy is Boston’s only two-way bilingual high school, with a rigorous college preparatory curriculum instructed in English and Spanish. Their mission, led by Dania Vazquez, is that every graduate will be accepted to a 4-year college.

Tony King is the Head of Schools of Boston International Newcomers Academy (BINcA). Their mission is to embrace new immigrant adolescent English language learners and their families by providing a curriculum designed to teach students English while also teaching rigorous content and ensuring students will be college and career-ready with motivation to pursue a life of learning and civic engagement.

– Jisca Philippe, Director of Youth and Schools

Charlestown High School is a diverse school that has been serving students since 1845. Charlestown's Head of School, Ajay Trivedi's strong belief in all students having access to challenging and inspiring curriculum has been foundational to expanding opportunities for advanced learning such as early college at Bunker Hill. Charlestown High School currently offers three career pathways - C-Town Business, Technology and Health.

– Rebecca Grainger, Senior Advisor for Youth and Schools

similar dynamic to SIdi and Lakunle

similar to what was said on page six. maybe foreshadowing or leaves/sun are important symbols in play

sexual subtext and we see Sidi's position in society as a disgrace/disrupter from the vehicle based on the presumptions of her actions and body

Arthur's body used as a vessel for voyeurism and integration into their society. Though, as we saw earlier in the play, Arthur's supposed mildness in colonial mindset and more performative respect to the culture of the Africans. In contrast to Father Grimes and Commander Jones, his "Opposition" becomes more emboldened when he realizes that it is correlated with his sexuality. Though his sexuality happens to also be in opposition with the colonial occupation that he is complicit in. internal struggle vs public image.

Fantasy. like Science Fiction is a vehicle for illumination. It's a storytelling device. Like stories of the Greek and Roman gods, or Gilgamesh. Applying critical theory to it is like debating the number of angels dancing on the head of a pin.

In lines 629-680, the speaker tells of Satan's actions as he attempts to explore the new world the fallen angels have been discussing previously. As Satan approaches the Gates of Hell and scopes out the surrounding area, he realizes that there are actually nine gates and "three folds were Brass, Three Iron, three of Adamantine Rock" (645-646). Before he gets to the gates, he notices a woman shaped figure to the waist, but below was a serpent. Also, around her waist were "Hell Hounds never ceasing bark'd" (654). She is known as Sin. Next came the Night-Hag "with the smell of infant blood to dance with Lapland Witches" (664-665). Satan then sees a dark figure "black it stood as night" (670). This figure is known as death. The figure approached Satan and he "trembled" (676) as the figure was unknown to him. The lines end with Satan about to speak to the dark figure.

Seeing these type of injuries to younger people hurt my feelings because I was once that age and they are kids and should be able to enjoy life being a kid. Also where are these children parents do they know what is going on?

Understanding the Passage:

(i) What kind of wounds she suffered was healed?

(ii) Consider how she lost her friend and how she witnessed her friend's death. What kind of ‘wounds’ would she also suffer from?

نحوه دیتاگیری و تسک و دیتاهای اخذ شده

Interesting thought here. Almost like they think this is happening too easy? I wonder if they doubt the good intentions of the Hurons or if they simply wish for them to understand more before jumping head first into baptism.

this passage explains how the "savages" rely on dreams to guide them and give them inspiration. Their dreams can predict events, influence decision making, and they treat dreams as law.

in this passage is described a situation where nothing is growing and in fact everything seems to be dying which leads growing fears of a famine. Huron county needed rain every few days to keep plants alive and without such rain the plants wilted. The region was greatly concerned as the towns that had food stored had burned the previous spring

This sentence alone summarized what the article is going to be covering. This is probably the most important sentence in the article ahead of the supporting arguments. It really gives readers a head start to learn what they are going to be reading about. The statement following this one was also a way to emphasize their opinion.

The expression of thought was not a free right, as would be assumed by the constitution. Rather, offending the wrong people could put a mark on your head, with no protection from the government.

Great tool mention in the video "NMC Learning at Home: Spectrograms & Your Voice". Found it while looking for objective measurements to distinguish falsetto and head voice.

I like these types of examples for UDL. They show that there are multiple ways to serve multiple students at the same time. Captioning is a great example of this. Another example could be scribing and chunking for MLL students, students with certain IEPs, and students who may struggle tackling whole tasks head on.

chorme web store

I am absolutely terrible with transitional words. When I'm writing an essay or study I feel like my vocabulary is limited to 2 transitional words but when I'm helping someone I have fifty off the top of my head. I have to write them down to remember them.

Author Response

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

We thank the reviewers for their thorough assessment of our study, their overall enthusiasm, and the helpful suggestions for clarifying the methods and results, additional analyses, and discussion points. We have made earnest efforts to address the weaknesses raised in the public review and other recommendations made by the reviewers.

Public Reviews:

Reviewer #1 (Public Review):

Herein, Blaeser et al. explored the impact of migraine-related cortical spreading depression (CSD) on the calcium dynamics of meningeal afferents that are considered the putative source of migraine-related pain. Critically previous studies have identified widespread activation of these meningeal afferents following CSD; however, most studies of this kind have been performed in anesthetized rodents. By conducting a series of technically challenging calcium imaging experiments in conscious head fixed mice they find in contrast that a much smaller proportion of meningeal afferents are persistently activated following CSD. Instead, they identify that post-CSD responses are differentially altered across a wide array of afferents, including increased and decreased responses to mechanical meningeal deformations and activation of previously non-responsive afferents following CSD. Given that migraine is characterized by worsening head pain in response to movement, the findings offer a potential mechanism that may explain this clinical phenomenon.

Strengths:

Using head fixed conscious mice overcomes the limitations of anesthetized preps and the potential impact of anaesthesia on meningeal afferent function which facilitated novel results when compared to previous anesthetized studies. Further, the authors used a closed cranial window preparation to maximize normal physiological states during recording, although the introduction of a needle prick to induce CSD will have generated a small opening in the cranial preparation, rendering it not fully closed as suggested.

Weaknesses:

Although this is a well conducted technically challenging study that has added valuable knowledge on the response of meningeal afferents the study would have benefited from the inclusion of more female mice. Migraine is a female dominant condition and an attempt to compare potential sex-differences in afferent responses would undoubtedly have improved the outcome.

Our study included only two females, largely reflecting the much higher success rate of AAV-mediated meningeal afferent GCaMP expression in males than in females. The reason for the lower yield in female mice is unclear to us at present but may involve, at least partly, sex-specific differences in the mechanisms responsible for efficient transduction with this AAV vector observed in peripheral tissues (Davidoff et al. 2003). While our study did not address sex differences, a recent study (Melo-Carrillo et al. 2017) reported CSD equally activating and sensitizing second-order dorsal horn neurons that receive input from meningeal afferents in male and female rats.

The authors imply that the current method shows clear differences when compared to older anaesthetized studies; however, many of these were conducted in rats and relied on recording from the trigeminal ganglion. Inclusion of a subgroup of anesthetized mice in the current preparation may have helped to answer these outstanding questions, being is this species dependent or as a result of the different technical approaches.

We have tried to address the anesthesia issue by conducting imaging sessions in several isoflurane-anesthetized mice. However, during these experiments, we observed a substantial decrease in the GCaMP fluorescence signal with a much lower signal-to-noise ratio that made the analyses of the afferents’ calcium signal unreliable. Reduced GCaMP signal in meningeal axons during anesthesia may be related to the development of respiratory acidosis, since lower pH leads to decreased GCaMP signal, as also mentioned by Reviewer #3. Of note, urethane anesthesia, which was used in all previous rat experiments, also produces respiratory acidosis.

The authors discuss meningeal deformations as a result of locomotion; however, despite referring to their previous work (Blaeser et al., 2022), the exact method of how these deformations were measured could be clearer. It is challenging to imaging that simple locomotion would induce such deformations and the one reference in the introduction refers to straining, such as cough that may induce intracranial hypertension, which is likely a more powerful stimulus than locomotion.

As part of the revision, we now provide a better description of the methodology (“Image processing and calcium signal extraction” section) used to determine meningeal deformations, including scaling, shearing, and Z-shift. In our previous paper (Blaeser et al. 2023), we provided an extensive description of the types of meningeal deformations occurring in locomoting mice. It should also be noted that locomotion drives cerebral vasodilation and intracranial pressure increases (Gao and Drew, 2016), which likely mediate, at least in part, the movement of the meninges towards the skull (positive Z-shift) and potentially other meningeal deformation parameters. We also agree with the reviewer that sudden maneuvers such as coughing and sneezing that lead to a larger increase in intracranial pressure are likely to be even more powerful drivers of endogenous intracranial mechanical stimulation than locomotion. Thus, our finding of increased responsiveness to locomotion-related meningeal deformation post-CSD may underestimate the increased afferent responsivity post-CSD during other behaviors such as coughing. We added this point to the discussion.

More recently, several groups have used optogenetic triggering of CSD to avoid opening of the cranium for needle prick. Given the authors robustly highlight the benefit of the closed cranium approach, would such an approach not have been more appropriate.

We agree with the reviewer that optogenetic methods used for CSD induction in non-craniotomized animals will further ensure accurate pressurization and, thus, will be an even better approach that avoids the burr hole used for pinprick. It should be noted, however, that the burr hole used for the pinprick likely had a minimal effect on intracranial pressure, as we minimized depressurization by plugging the burr hole throughout the experiments with a silicone elastomer. We have added this information to the revised Methods section.

It is also worth noting that the optogenetic methodology used by others to provoke CSD was optimized only recently and relies on transgenic mice with a strong expression of YFP (Thy1.ChR2-YFP mice) within the superficial cortex that is not compatible with the afferent GCaMP imaging of meningeal afferents. Modifications using red-shifted opsins may allow the use of this strategy in the future.

It was not clear how deformations predictors increased independent of locomotion (Figure 4D) as locomotion is essentially causing the deformations as noted in the study. This point was not so clear to this reviewer.

As noted in our previous paper (Blaeser et al., 2023), deformation variables often exhibit different time courses than locomotion, even when a deformation is initially induced by the onset of locomotion. Most notably, the scaling-related deformation ramps up slowly and often persists for tens of seconds after the onset and termination of locomotion, which may be related to the recovery dynamics of the meningeal vascular response to locomotion. Overall, while locomotion serves as a predictor of meningeal deformation, we observed previously (Blaeser et al. 2023) many afferents whose responses were more closely associated with the moment-to-moment deformations than with the state of locomotion per se, suggesting that a unique set of stimuli is responsible for the activation of this deformation-sensitive afferent population. The increased sensitivity to deformation signals we observed following CSD suggests that the afferent population sensitive to deformation has unique properties that render it most susceptible to becoming sensitized following CSD. We now discuss this possibility.

Reviewer #2 (Public Review):

This is an interesting study examining the question of whether CSD sensitizes meningeal afferent sensory neurons leading to spontaneous activity or whether CSD sensitizes these neurons to mechanical stimulation related to locomotion. Using two-photon in vivo calcium imaging based on viral expression of GCaMP6 in the TG, awake mice on a running wheel were imaged following CSD induction by cortical pinprick. The CSD wave evoked a rise in intracellular calcium in many sensory neurons during the propagation of the wave but several patterns of afferent activity developed after the CSD. The minority of recorded neurons (10%) showed spontaneous activity while slightly larger numbers (20%) showed depression of activity, the latter pattern developed earlier than the former. The vast majority of neurons (70%) were unaffected by the CSD. CSD decreased the time spent running and the numbers of bouts per minute but each bout was unaffected by CSD. There also was no influence of CSD on the parameters referred to as meningeal deformation including scale, shear, and Z-shift. Using GLM, the authors then determine that there there is an increase in locomotion/deformation-related afferent activity in 51% of neurons, a decrease in 12% of neurons, and no change in 37%. GLM coefficients were increased for deformation related activity but not locomotion related activity after CSD. There also was an increase in afferents responsive to locomotion/deformation following CSD that were previously silent. This study shows that unlike prior reports, CSD does not lead to spontaneous activity in the majority of sensory neurons but that it increases sensitivity to mechanical deformation of the meninges. This has important implications for headache disorders like migraine where CSD is thought to contribute to the pathology in unclear ways with this new study suggesting that it may lead to increased mechanical sensitivity characteristic of migraine attacks.

1) It would be helpful to know what is meant by "post-CSD" in many of the figures where a time course is not shown. The methods indicate that 4, 30 min runs were collected after CSD but this would span 2 hours and the data do not indicate whether there are differences across time following CSD nor whether data from all 4 runs are averaged.

While we monitored time course changes in ongoing activity (see Figure 2), it was challenging to evaluate post-CSD changes in locomotion-related deformation responses at a fine temporal scale, as running bouts resumed at different time points post-CSD and occurred intermittently throughout the post-CSD analysis period. Our experiments were also not sufficiently powered to break out analyses at multiple different epochs post-CSD, partly because there wasn’t much locomotion. To allow comparisons using a sufficient number of bouts, we conducted our GLM analyses using all data collected during running bouts in the 2-hour post-CSD period (termed “post-CSD) versus in the 1-hour pre-CSD period. We have now clarified this further in the main text and figure legends.

2) Why is only the Z-shift data shown in Figures 4A-C? Each of the deformation values seems to contribute to the activity of neurons after CSD but only the Z-shift values are shown.

In many afferents, only one deformation variable best predicted the activity at both the pre- and post-CSD epochs. However, at the population level, all deformation variables were equally predictive. In the examples provided, the afferent developed augmented sensitivity that could only be predicted by the Z-shift variable, and the other deformation variables were not included to keep the figure legible. This is now clarified in the figure legend.

3) How much does the animal moving its skull against the head mount contribute to deformations of the meninges if the skull is potentially flexing during these movements? Even if mice are not locomoting, they can still attempt to move their heads thus creating pressure changes on the skull and underlying meninges. The authors mention in the methods that the strong cement used to bind the skull plates and headpost together minimize this, but how do they know it is minimized?

We did not measure skull flexing during locomotion and its potential effect on meningeal deformation. However, we would like to point out several considerations. It is evident from numerous imaging studies across various brain regions in freely moving animals, utilizing brain motion registration, that brain motion of the same scale (a few microns), as that observed in our studies, also occurs in the absence of head fixation (e.g., Glas et al, 2019; Zong et al 2021). In our system, the head-fixed mouse is locomoting on a cantilevered (spring-like) running wheel (see also Ramesh et al., 2018), which dissipates most, albeit not all, upward and forward forces applied to the skull during locomotion. Furthermore, the position of the headpost, anterior to where the mouse's paws touch the wheel, makes it hard for the mouse to push straight up and apply forces to the skull. We have updated the text in the methods section (Running wheel habituation) to address this. In our previous work (See Figure 2B in Blaeser et al. 2023), we found a substantial subset of afferents showing an increase in calcium activity that began after each bout of locomotion had terminated, and that lasted for many seconds, suggesting that skull flexing during locomotion may not play a leading role. Finally, we proposed in that study that meningeal deformations play a major role in the afferent response, given our findings of (i) sigmoidal stimulus-response curves between afferent activity and meningeal deformation and (ii) of different afferents that track scaling deformations along different axes. It is unlikely that all of these are related to any residual forces generated from skull deformations.

4) What is the mechanism by which afferents initiate the calcium wave during the CSD itself? Is this mechanical pressure due to swelling of the cortex during the wave? If so, why does the CSD have no impact on the deformation parameters? It seems that this cortical swelling would have some influence on these values unless the measurements of these values are taken well after cortical swelling subsides. Related to point 1 above, it is not clear when these measurements are taken post-CSD.

We provide, for the first time, evidence that CSD evokes local calcium elevation in meningeal afferent fibers in a manner that is incongruent with action potential propagation, as the activity gradually advances along individual afferents across many seconds during the wave. As indicated in Figure 1H, we measured these changes during the first 2 minutes post-CSD. Based on the reviewer’s question, we have now addressed whether mechanical changes occurring in the cortex in the wake of CSD might be responsible for the acute afferent activation we observed. We now include new data (Results, “Acute afferent activation is not related to CSD-evoked meningeal deformation” and Figure S2) showing an acute phase of meningeal deformation (as expected given the changes in extracellular fluid volume) lasting 40-80 seconds following the induction of CSD. Our data suggests, however, that these meningeal deformations are unlikely to be the main driver of the acute afferent calcium response. We propose that, based on the speed of the afferent calcium wave propagation and the distinct dynamics of calcium activity as compared to the dynamics of the deformations, the acute afferent response is more likely to be mediated by the spread of algesic mediators (e.g., glutamate, K+ ATP) and their diffusion into the overlying meninges.

Because the peri-CSD meningeal deformations return to baseline soon after the cessation of the CSD wave, they are unlikely to affect our analyses of post-CSD changes in afferent sensitivity in the following 2 hours. This is also supported by our data (see Figure 3F-H) showing similar locomotion-related deformations pre- and post-CSD, which were measured after the deformations related to the CSD itself had subsided.

5) How does CSD cause suppression of afferent activity? This is not discussed. It is probably a good idea in this discussion to reinforce that suppression in this case is suppression of the calcium response and not necessarily suppression of all neuronal activity.

The mechanism underlying the suppression of afferent activity remains unclear. We now discuss the following points:

First, the pattern of afferent responses resembles the rapid loss of cortical activity in the wake of a CSD, but its faster recovery points to a mechanism distinct from the pre-and post-synaptic changes responsible for the silencing of cortical activity (Sawant-Pokam et al., 2017; Kucharz and Lauritzen, 2018). Whether CSD drives the local release of mediators capable of reducing afferent excitability and spiking dynamics will require further studies.

Second, the reviewer proposes that the suppressed calcium activity we observed in ~20% of the afferents immediately following CSD may reflect a decreased calcium response independent of afferent spiking activity. Such a process could theoretically involve factors influencing the GCaMP fluorescence (see also our response to Reviewer #3) and/or factors modifying the afferents’ spiking-to-calcium coupling. We note that if a CSD-related factor could modify the calcium response independent of afferent spiking, one would expect a more consistent effect across axons, reflected as a reduced signal in a larger proportion of the afferents, which we did not observe.

6) How do the authors interpret the influence of CSD on locomotor activity? There was a decrease in bouts but the bouts themselves showed similar patterns after CSD. Is CSD merely inhibiting the initiation of bouts? Is this consistent with what CSD is known to do to motor activity? And again related to point 1, how long after CSD were these measurements taken? Were there changes in locomotor activity during the actual CSD compared to post-CSD?

To the best of our knowledge, there is very little data on the effect of CSD on motor activity, making it challenging to engage in further speculation regarding the mechanisms underlying the preservation of running bouts patterns post-CSD. Houben et al. (2017) described a similar reduction in locomotion in mice, corresponding to decreased motor cortex (M1) activity, and preservation of intermittent locomotion bouts. In the revised Results section, we now provide information about the cessation of locomotor activity during the CSD wave and have added information regarding the measurement of locomotion following CSD.

7) The authors mention the caveats of prior work where the skull is open and is thus depressurized. Is this not also the case here given there is a hole in the skull needed to induce CSD?

Unlike previous electrophysiological studies, which involved several large openings (~2x2 mm), including at the site of the afferents’ receptive field, our study involved only a small burr hole located remotely (1.5 mm) from the frontal edge of our imaging window. As noted in our response to Reviewer #1, this burr hole (~0.5 mm diameter) was unlikely to produce inflammation at the imaging site or cause depressurization as it was sealed with a silicone plug throughout the experiment.

8) The authors should check the %'s and the numbers in the pie chart for Figure 4. Line 224 says 53 is 22% but it does not look this way from the chart.

The 22% reported is the percentage of afferents that developed sensitivity post-CSD among all the non-sensitive ones pre-CSD. The pie chart illustrates only afferents that were deemed sensitive before and/or after the CSD. We removed the % to clarify.

9) Line 319 mentions that CSD causes "powerful calcium transients" in sensory neurons but it is not clear what is meant by powerful if there are no downstream effects of these transients being measured. The speculation is that these calcium transients could cause transmitter release, which would be an important observation in the absence of AP firing, but there are no data evaluating whether this is the case.

We changed the term to “robust”

Reviewer #3 (Public Review):

Summary:

Blaeser et al. set out to explore the link between CSD and headache pain. How does an electrochemical wave in the brain parenchyma, which lacks nociceptors, result in pain and allodynia in the V1-3 distribution? Prior work had established that CSD increased the firing rate of trigeminal neurons, measured electrophysiologically at the level of the peripheral ganglion. Here, Blaeser et al. focus on the fine afferent processes of the trigeminal neurons, resolving Ca2+ activity of individual fibers within the meninges. To accomplish these experiments, the authors injected AAV encoding the Ca2+ sensitive fluorophore GCamp6s into the trigeminal ganglion, and 8 weeks later imaged fluorescence signals from the afferent terminals within the meninges through a closed cranial window. They captured activity patterns at rest, with locomotion, and in response to CSD. They found that mechanical forces due to meningeal deformations during locomotion (shearing, scaling, and Z-shifts) drove non-spreading Ca2+ signals throughout the imaging field, whereas CSD caused propagating Ca2+ signals in the trigeminal afferent fibers, moving at the expected speed of CSD (3.8 mm/min). Following CSD, there were variable changes in basal GCamp6s signals: these signals decreased in the majority of fibers, signals increased (after a 25 min delay) in other fibers, and signals remained unchanged in the remainder of fibers. Bouts of locomotion were less frequent following CSD, but when they did occur, they elicited more robust GCamp6s signals than pre-CSD. These findings advance the field, suggesting that headache pain following CSD can be explained on the basis of peripheral cranial nerve activity, without invoking central sensitization at the brain stem/thalamic level. This insight could open new pathways for targeting the parenchymal-meningeal interface to develop novel abortive or preventive migraine treatments.

Strengths:

The manuscript is well-written. The studies are broadly relevant to neuroscientists and physiologists, as well as neurologists, pain clinicians, and patients with migraine with aura and acephalgic migraine. The studies are well-conceived and appear to be technically well-executed.

Weaknesses:

1) Lack of anatomic confirmation that the dura were intact in these studies: it is notoriously challenging to create a cranial window in mouse skull without disrupting or even removing the dura. It was unclear which meningeal layers were captured in the imaging plane. Did the visualized trigeminal afferents terminate in the dura, subarachnoid space, or pia (as suggested by Supplemental Fig 1, capturing a pial artery in the imaging plane)? Were z-stacks obtained, to maintain the imaging plane, or to follow visualized afferents when they migrated out of the imaging plane during meningeal deformations?

We agree that avoiding disruption of the dura is challenging. Indeed, it took many months of practice before conducting the experiments in this manuscript to master methods for a craniotomy that spared the dura.

We addressed the issue of meningeal irritation due to cranial window surgery in our previous work (Blaeser et al., 2023). In brief, we conducted vascular imaging using the same cranial window approach and showed no leakage of macromolecules from dural or pial vessels anywhere within the imaging window at 2-6 weeks after the surgery (Figure S1D in Blaeser et al. 2022). This data suggested no ongoing meningeal inflammation below the window. The very low level of ongoing activity we observed at baseline also suggests a lack of an inflammatory response that could lead to afferent sensitization before CSD. This is now mentioned in the Discussion.

We conducted volumetric imaging for three main reasons: 1) To capture the activity of afferents throughout the meningeal volume. In our volumetric imaging approach, including in this work, we observed afferent calcium signals throughout the meningeal thickness (see Figure 5 in Blaeser et al. 2022). However, the majority of afferents were localized to the most superficial 20 microns (Figure S1E in Blaeser et al. 2022), suggesting that we mostly recorded the activity of dural afferents; 2) to enable simultaneous quantification of three-dimensional deformation and the activity of afferents throughout the thickness of the meninges. This allowed us to determine whether changes in mechanosensitivity could involve augmented activity to intracranial mechanical forces that produced meningeal deformation along the Z-axis of the meninges (e.g., increased intracranial pressure); 3) to provide a direct means to confirm that the afferent GCaMP fluorescent changes we observed were not due to artifacts related to meningeal motion along the Z-axis. We have now added this information to the “Two-photon imaging” section of the Methods.

2) Findings here, from mice with chronic closed cranial windows, failed to fully replicate prior findings from rats with acute open cranial windows. While the species, differing levels of inflammation and intracranial pressure in these two preparations may contribute, as the authors suggested, the modality of measuring neuronal activity could also contribute to the discrepancy. In the present study, conclusions are based entirely on fluorescence signals from GCamp6s, whereas prior rat studies relied upon multiunit recordings/local field potentials from tungsten electrodes inserted in the trigeminal ganglion.

As a family, GCamp6 fluorophores are strongly pH dependent, with decreased signal at acidic pH values (at matched Ca2+ concentration). CSD induces an impressive acidosis transient, at least in the brain parenchyma, so one wonders whether the suppression of activity reported in the wake of CSD (Figure 2) in fact reflects decreased sensitivity of the GCamp6 reporter, rather than decreased activity in the fibers. If intracellular pH in trigeminal afferent fibers acidifies in the wake of CSD, GCamp6s fluorescence may underestimate the actual neuronal activity.

Previous in vivo rodent studies observed a tissue acidosis transient that peaks during the DC shift corresponding to the wavefront of the spreading depolarization, and lasting for ~ 10 min. (Mutch and Hansen, 1984). Since we observed a massive increase in afferent calcium activity with a propagation pattern resembling the cortical wave, it is unlikely that the cortical acidosis during the CSD wave strongly affected the GCaMP signal in the overlying meninges. Furthermore, if cortical acidosis non-discriminately affects the GCaMP signal, one would expect a more consistent effect across axons, reflected as a reduced calcium signal in a larger proportion of the afferents, which we did not observe. Finally, the finding that in affected afferents, decreased calcium activity lasted for > 20 min – a time point when cortical acidosis has fully recovered - points to a distinct underlying mechanism. We also note that any residual acidosis would not confound our main finding of increased calcium responses to meningeal deformation at later periods post-CSD, as acidosis should, if anything, decrease calcium-related fluorescence.

The authors might consider injecting an AAV encoding a pHi sensor to the trigeminal ganglion, and evaluating pHi during and after CSD, to assess how much this might be an issue for the interpretation of GCamp6s signals. Alternatively, experiments assessing trigeminal fiber (or nerve/ganglion) activity by electrophysiology or some other orthologous method would strengthen the conclusions.

Please see our comment above regarding the short duration of the pH changes post-CSD.

N's are generally reported as # of afferents, obscuring the number of technical/biological replicates (# of imaging sessions, # of locomotion bouts, # of CSDs induced, # of animals).

We now report the number of replicates (# of afferent, # of CSD events, and # of mice).

Fig 1F trace over the heatmap is not explained in the figure legend. Is this the speed of the running wheel? Is it the apparent propagation rate of the GCamp6s transient through the imaging field?

We have added to the legend of Figure 1 that the trace in panel F depicts locomotion speed.

eLife assessment

This fundamental study explored the impact of migraine-related cortical spreading depression (CSD) on the firing of nerves innervating the coverings of the brain that are considered the putative source of migraine-related pain. Using convincing approaches they show that these responses are altered in response to mechanical deformation of the brain coverings. Given that migraine is characterized by worsening head pain in response to movement, the findings offer a potential mechanism that may explain this clinical phenomenon.

Reviewer #1 (Public Review):

Summary:<br /> Herein, Blaeser et al. explored the impact of migraine-related cortical spreading depression (CSD) on the calcium dynamics of meningeal afferents that are considered the putative source of migraine-related pain. Critically previous studies have identified widespread activation of these meningeal afferents following CSD; however, most studies of this kind have been performed in anesthetized rodents. By conducting a series of technically challenging and compelling calcium imaging experiments in conscious head fixed mice they find in contrast that a much smaller proportion of meningeal afferents are persistently activated following CSD. Instead, they identify that post-CSD responses are differentially altered across a wide array of afferents, including increased and decreased responses to mechanical meningeal deformations and activation of previously non-responsive afferents following CSD. Given that migraine is characterized by worsening head pain in response to movement, the findings offer a potential mechanism that may explain this clinical phenomenon.

Strengths:<br /> Using head fixed conscious mice overcomes the limitations of anesthetized preps and the potential impact of anaesthesia on meningeal afferent function which facilitated novel results when compared to previous anesthetized studies. Further, the authors used a closed cranial window preparation to maximize normal physiological states during recording, although the introduction of a needle prick to induce CSD will have generated a small opening in the cranial preparation, rendering it not fully closed as suggested. However, technical issues with available AAV's and alternate less invasive triggering methodologies necessitate the current approach.

Weaknesses:<br /> Although this is a well conducted technically challenging study that has added valuable knowledge on the response of meningeal afferents the study would have benefited from the inclusion of more female mice. Migraine is a female dominant condition and an attempt to compare potential sex-differences in afferent responses would undoubtedly have improved the outcome. The authors report potential sex-specific effects on AAV transfection rates between males and females which have contributed to this imbalance.

The authors imply that the current method shows clear differences when compared to older anaesthetized studies; however, many of these were conducted in rats and relied on recording from the trigeminal ganglion. Attempts to address this point have proven difficult due to limited GCaMP signalling in anaesthetised mice, meaning that technical differences cannot be ruled out.

This section discusses the formation of primitive families and the unrestricted authority of the father within them. It raises questions about early power dynamics and the evolution of communal life. Exploring how this unchecked authority influenced the development of societal norms and governance could offer valuable insights into the origins of human social structures.

It is interesting to read about how the study of memory started with no relation to history, but as time passed, it sort of slowly crept up on it, and now has a major effect on our study of history. Due to this class being about American History, I sort of just created the association between memory studies and history in my head.

This want of having animals displayed as clothing or decoration brings me back to our first conversation in class. We reflect back on the people storming the capital wearing animal hide as maybe a power play or some metaphor on colonialism and our roots as a country. Is this idea the same thing? I would say yes.

Long sentences of vivid imagery contrasted by the short, morbid fact to end the paragraph -> separates and emphasises McCandless' death

Absolutely terrifying, I couldn't imagine the state of terror they were living in every day.

use this to replace 'now'

git

How to rewrite git commit timestamps

The emperor is very wise and not rash to decision.

narration

I do this so often and don't even think about it. I will be so immersed with what's going on inside my head that I won't notice that someone was trying to talk to me. I feel like I miss out on things that happen around me when this happens. But the opposite thing happens when I'm trying to study. All of a sudden I notice the sound of water dripping in my bathroom. Or I listen to peoples conversations in the library.

What is this trying to explain to me?

Author Response

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

First of all, we'd like to thank the three reviewers for their meticulous work that enable us to present now an improved manuscript and substantial changes were made to the article following reviewers' and editors' recommendations. We read all their comments and suggestions very carefully. Apart from a few misunderstandings, all comments were very pertinent. We responded positively to almost all the comments and suggestions, and as a result, we have made extensive changes to the document and the figures. This manuscript now contains 16 principal figures and 15 figure supplements.

The number of principal figures is now 16 (1 new figure), and additional panels have been added to certain figures. On the other hand, we have added 7 additional figures (supplement figures) to answer the reviewers' questions and/or comments.

Main figures

▪ Figures 1, 4, 5, 10, 11, 12, 13, 14: unchanged ▪ Figure 7 and 8 were switched.

▪ Figure 2: we added panel F in response to reviewer 3's and request for sperm defect statistics

▪ Figure 3: the contrast in panel B has been taken over to homogenize colors

▪ Figure 6: This figure was recomposed. The WB on testicular extract was suppressed and we present a new WB allowing to compare the presence of CCDC146 in the flagella fraction. Using an anti-HA Ab, we demonstrate that the protein is localized in the flagella in epididymal sperm. Request of the 3 reviewers.

▪ Figure 7 (old 8): to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum. Moreover, the WB was removed and is now presented in figure 6 (improved as requested).

▪ Figure 8. Was old figure 7

▪ Figure 9: figure 9 was recomposed and improved for increased clarity as suggested by reviewer 2 and 3.

▪ Figure 16 was before appendix 11

Figure supplements and supplementary files

▪ Figure 1-Figure supplement 1 New. Sperm parameters of the 2 patients. requested by editor (remark #1) by the reviewer 1 (Note #3)

▪ Figure 2-Figure supplement 1 new. Sperm parameters of the line 2 (KO animals) requested by the reviewer 1 (Note #5)

▪ Figure 4-Figure supplement 1 New. Experiment to evaluate the specificity of the human CCDC146 antibody. Minimal revision request and reviewer 1 note #8

▪ Figure 6-Figure supplement 1 New. Figure recomposed; Asked by reviewer 2 note #4 and reviewer 3

▪ Figure 8-Figure supplement 1 New. We now provide new images to show the non-specific staining of the midpiece of human sperm by secondary Abs in ExM experiments; Asked by reviewer 2

▪ Figure 10-Figure supplement 1 New. We added new images to show the non-specific staining of the midpiece of mouse sperm by secondary Abs in IF (panel B). Rewiever 1 note #9 and reviewer 2 note #5

▪ Figure 12-Figure supplement 1 New. Control requested by reviewer 3 Note #23

▪ Figure 13-Figure supplement 1 New. We provide a graph and a statistical analysis demonstrating the increase of the length of the manchette in the Ccdc146 KO. Requested by editor and reviewer 3 Note 24

▪ Figure 15-Figure supplement 1 New. Control requested by reviewer 2. Minor comments

▪ Figure supplementary 1 New. Answer to question requested by reviewer 2 note #1

All the reviewers' and editors’ comments have been answered (see our point to point response) and we resubmit what we believe to be a significantly improved manuscript. We strongly hope that we meet all your expectations and that our manuscript will be suitable for publication in "eLife". We look forward to your feedback,

Point by point answer

Please note that there has been active discussion of the manuscript and the summarize points below is the minimal revision request that the reviewers think the authors should address even under this new review model system. It was the reviewers' consensus that the manuscript is prepared with a lot of oversights - please see all the minor points to improve your manuscript.

All minimal revision requests have been addressed

Minimal revision request

1) Clinical report/evaluation of the two patients should be given as it was not described even in their previous study as well as full description of CCDC146.

We provide now a new Figure 1-figure supplement 1 describing the patients sperm parameters

2) Antibody specificity should be provided, especially given two of the reviewers were not convinced that the mid piece signal is non-specific as the authors claim. As both KO and KI model in their hands, this should be straightforward.

To validate the specificity of the Antibody, we transfected HEK cells with a human DDK-tagged CCDC146 plasmid and performed a double immunostaining with a DDK antibody and the CCDC146 antibody. We show that both staining are superimposable, strongly suggesting that the CCDC146 Ab specifically target CCDC146. This experiment is now presented in Figure 4-Figure supplement 1. Next, to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum.

3) The authors should improve statistical analysis to support their experimental results for the reader can make fair assessment. Combined with clear demonstration of ab specificity, this lack of statistical analysis with very few sample number is a major driver of dampening enthusiasm towards the current study.

Several statistical analyses were carried out and are now included:

1) distribution of the HA signal in mouse sperm cells (see point 2 Figure 7 panel B)

2) quantification and statistical analyses of the defect observed in Ccdc146 KO sperm (figure 2 panel E)

3) Quantification and statistical analyses of the length of the manchette in spermatids 13-15 steps (Figure 13-Figure supplement 1 new)

4) The authors need to clarify (peri-centriolar vs. centriole)

In figure 4A, we have clearly shown that the protein colocalizes with centrin, a centriolar core protein in somatic cells. This colocalization strongly suggests that CCDC146 is therefore a centriolar protein, and this is now clearly indicated lines 211-212. However, its localization is not restricted to the centrioles and a clear staining was also observed in the pericentriolar material (PCM). The presence of a protein in PCM and centriole was already described, and the best example is maybe gamma-tubulin (PMID: 8749391).

or tone down (CCDC146 to be a MIP) of their claim/description.

Concerning its localization in sperm, we agree with the reviewer that our demonstration that CCDC146 is MIP would deserve more results. Because of that, we have toned down the MIP hypothesis throughout the manuscript. See lines 491495

Testis-specific expression of CCDC146 as it is not consistent with their data.

We have also modified our claim concerning the testis-expression of CCDC146. Line 176

Reviewer #1 (Recommendations For The Authors):

Major comments

1) As described in general comments, this study limits how the CCDC146 deficiency impairs abnormal centriole and manchette formation. The authors should explain their relationship in developing germ cells.

In fact, there are limited information about the relationship between the manchette and the centriole. However, few articles have highlighted that both organelles share molecular components. For instance, WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis (Commun Biol. 2021; 4: 645. doi: 10.1038/s42003-021-02171-5). Another study demonstrates that CCDC42 localizes to the manchette, the connecting piece and the tail (Front. Cell Dev. Biol. 2019 https://doi.org/10.3389/fcell.2019.00151). These articles underline that centrosomal proteins are involved in manchette formation and removal during spermiogenesis and support our results showing the impact of CCDC146 lack on centriole and manchette biogenesis. This information is now discussed. See lines 596-603

2) The authors generated knock-in mouse model. If then, are the transgene can rescue the MMAF phenotype in CCDC146-null mice? This reviewer strongly suggest to test this part to clearly support the pathogenicity by CCDC146.

We indeed wrote that we created a “transgenic mice”, which was misleading. We actually created a CCDC16 knock-in expressing a tagged-protein. The strain was actually made by CRISPR-Cas9 and a sequence coding for the HA-tag was inserted just before the first amino acid in exon 2, leading to the translation of an endogenous HA-tagged CCDC146 protein. We have removed the word transgenic from the text and made changes accordingly (see lines 250-253). We can therefore not use this strain to rescue the MMAF phenotype as suggested by the reviewer.

3) Although the authors cite the previous study (Coutton et al., 2019), the study does not describe any information for CCDC146 and clinical information for the patients. The authors must show the results for clinical analysis to clarify the attended patients are MMAF patients without other phenotypic defects.

We have now inserted a table, indicating all sperm parameters for the patients harboring a mutation in the CCDC146 gene (Figure 1-Figure supplement 1) and is now indicated lines 159-160

4) The authors describe CCDC146 expression is dominant in testes, However, the level in testis is only moderate in human (Supp Figure 1). Thus, this description is not suitable.

In Figure 1-figure supplement 2 (old FigS1), the median of expression in testis is around 12 in human, a value considered as high expression by the analysis software from Genevestigator. However, for mouse, it is true that the level of expression is medium. We assumed that reviewer’s comment concerned testis expression in mouse. To take into account this remark, we changed the text accordingly. See line 176.

5) Although the authors mentioned that two mice lines are generated, only one line information is provided. Authors must include information for another line and provide basic characterization results to support the shared phenotype within the lines.

We now provide a revised Figure 2-figure supplement 1CD, presenting the second line and the corresponding text in the main text is found lines 178-183.

6) In somatic cells, the CCDC146 localizes at both peri-centriole and microtubule but its intracellular localization in sperm is distinguished. The authors should explain this discrepancy.

The multi-localization of a centriolar protein is already discussed in detail in discussion lines 520-526. We have written:

“Despite its broad cellular distribution, the association of CCDC146 with tubulin-dependent structures is remarkable. However, centrosomal and axonemal localizations in somatic and germ cells, respectively, have also been reported for CFAP58 [37, 55], thus the re-use of centrosomal proteins in the sperm flagellar axoneme is not unheard of. In addition, 80% of all proteins identified as centrosomal are found in multiple localizations (https://www.proteinatlas.org/humanproteome/subcellular/centrosome). The ability of a protein to home to several locations depending on its cellular environment has been widely described, in particular for MAP. The different localizations are linked to the presence of distinct binding sites on the protein…. “

7) Authors mention CCDC146 is a centriolar protein in the title and results subtitle. However, the description in results part depicts CCDC146 is a peri-centriolar protein, which makes confusion. Do the authors claim CCDC146 is centrosomal protein?

In figure 4A, we have clearly shown that the protein colocalizes with centrin, a centriolar core protein. This colocalization strongly suggests that CCDC146 is therefore a centriolar protein in somatic cells, and is now clearly indicated lines 211-212. However, its localization is not restricted to the centrioles and a clear staining was also observed in the pericentriolar material (PCM). The presence of a protein in PCM and centriole was already described and the best example is maybe gamma-tubulin (PMID: 8749391).

8) Verification of the antibody against CCDC146 must be performed and shown to support the observed signal are correct. 2nd antibody only signal is not proper negative control.

It is a very important remark. The commercial antibody raised against human CCDC146 was validated in HEK293-cells expressing a DDK-tagged CCDC146 protein. Cells were co-marked with anti-DDK and anti-CCDC146 antibodies. We have a perfect colocalization of the staining. This experiment is now presented in Figure 4-figure supplement 1 and presented in the text (lines 206-208).

9) In human sperm, conventional immunostaining reveals CCDC146 is detected from acrosome head and midpiece. However, in ExM, the signal at acrosome is not detected. How is this discrepancy explained? The major concern for the ExM could be physical (dimension) and biochemical (properties) distortion of the sample. Without clear positive and negative control, current conclusion is not clearly understood. Furthermore, it is unclear why the authors conclude the midpiece signal is non-specific. The authors must provide experimental evidence.

Staining on acrosome should always be taken with caution in sperm. Indeed, numerous glycosylated proteins are present at the surface of the plasma membrane regarding the outer acrosomal membrane for sperm attachment and are responsible for numerous nonspecific staining. Moreover, this acrosomal staining was not observed in mouse sperm, strongly suggesting that it is not specific.

Concerning the staining in the midpiece observed in both conventional and Expansion microscopy, it also seems to be nonspecific and associated with secondary Abs.

For IF, we now provide new images showing clearly the nonspecific staining of the midpiece when secondary Ab were used alone (see Figure 10-figure supplement 1B).

For ExM, we provide new images in Figure 8-figure supplement 1B (POC5 staining) showing a staining of the midpiece (likely mitochondria), although POC5 was never described to be present in the midpiece. Both experiments (CCDC146 and POC5 staining by ExM) shared the same secondary Ab and the midpiece signal was likely due to it.

Moreover, we now provide new images (figure 7C) in ExM on mouse sperm showing no staining in the midpiece and demonstrating that the punctuated signal is present all along the flagellum. Finally, we would like to underline that we now provide new IF results, using an anti-HA conjugated with alexafluor 488 and confirming the ExM results.

These points are now discussed lines 498-502 for acrosome and lines 503-511 for midpiece staining.

10) For intracellular localization of the CCDC146 in mouse sperm, the authors should provide clear negative control using WT sperm which do not carry the transgene.

This experiment was performed.

To avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum.

11) Current imaging data do not clearly support the intracellular localization of the CCDC146. Although western blot imaging reveal that CCDC146 is detected from sperm flagella, this is crude approach. Thus, this reviewer highly recommends the authors provide more clear experimental evidence, such as immuno EM.

We provide now a WB comparing the presence of the protein in the flagellum and in the head fractions; see new figure 6. We show that CCDC146 is only present in the flagellum fraction; The detection of the band appeared very quickly at visualization and became very strong after few minutes, demonstrating that the protein is abundant in the flagella. It is important to note that epididymal sperm do not have centrioles and therefore this signal is not a centriolar signal. We also now provide new statistical analyses showing that the immuno-staining observed in the principal piece is very specific (Figure 7B). Altogether, these results demonstrate unequivocally the intracellular localization of CCDC146 in the flagellum. This point is now discussed lines 480-489

12) Although sarkosyl is known to dissociate tubulin, it is not well understood and accepted that the enhanced detection of CCDC146 by the detergent indicates its microtubule inner space. Sperm axoneme to carry microtubule is also wrapped peri-axonemal components with structural proteins, which are even not well solubilized by high concentration of the ionic detergent like SDS.

We agree with the reviewer that the solubilization of the protein by sarkozyl is not a proof of the presence of the protein inside microtubule. Taking into account this point, the MIP hypothesis was toned down and we now discuss alternative hypothesis concerning these results; See discussion lines 490-497

13) SEM image is not suitable to explain internal structure (line 317-323).

We agree with the reviewers and changes were made accordingly. See lines 354-357

Minor comments

1) In main text, supplementary figures are cited "Supp Figure". And the corresponding legends are written in "Appendix - Figure". Please unify them.

Done Labelled now “Figure X-figure supplement Y”

2) Line 159, "exon 9/19" is not clear.

We have written now exons 9 and indicated earlier that the gene contains 19 exons

3) Line 188, "positive cells" are vague.

Positive was changed by “fluorescent”

4) Representative TUNEL assay image for knockout testes were not shown in Supp Figure 3B.

It was a mistake now Figure 2-figure supplement 2C

5) Please provide full description for "IF" and "AB" when described first.

Done

6) Line 262, It is unclear what is "main piece".

Changed to principal piece

7) Line 340, Although the "stage" information might be applicable, this is information for "seminiferous tubule" rather than "spermatid". This reviewer suggests to provide step information rather than stage information.

We agree with the reviewer that there was a confusion between “stage” and “step”. We change to step spermatids

8) Line 342, Step 1 is not correct in here.

OK corrected. now steps 13-15 spermatids

9) Line 803, "C." is duplicated.

Removed

10) Figure 3A, it will be good to mark the defective nuclei which are described in figure legends.

These cells are now indicated by white arrow heads

11) Figure 5, Please provide what MT stands for.

Now explained in the legend of figure 5

12) Figure 6. Author requires clear blot images for C. In addition, Panel B information is not correct. If the blot was performed using HA antibody, then how "WT" lane shows bands rather than "HA" bands?

The reviewer is correct. It was a mistake; The figure was recomposed and improved.

Reviewer #2 (Recommendations For The Authors):

Overall, editing oversights are present throughout the manuscript, which has made the review process quite difficult. Some repetitive figures can be removed to streamline to grasp the overall story easier. Some claims are not fully supported by evidence that need to tone down. Some figures not referenced in the main text need to be mentioned at least once.

All figures are now referenced in the text

Major comments:

1) 163-164 - Please clarify the claim that there is going to be an absence of the protein or nonfunctional protein, especially for the patient with a deletion that could generate a truncated protein at two third size of the full-length protein. Similarly, 35% of the protein level is present for the patient with a nonsense mutation. Some in silico structural analysis or analysis of conserved domains would be beneficial to support these claims.

Both mutations are predicted to produce a premature stop codons: p.Arg362Ter and p.Arg704serfsTer7, leading either to the complete absence of the protein in case of non-sense mediated mRNA decay or to the production of a truncated protein missing almost two third or one fourth of the protein respectively. CCDC146 is very well conserved throughout evolution (Figure supplementary 1), including the 3’ end of the protein which contains a large coil-coil domain (Figure 1B). In view of the very high degree of conservation, it is most likely that the 3’ end of the protein, absent in both subjects, is critical for the CCDC146 function and hence that both mutations are deleterious. This explanation is now added to the discussion. see lines 439-448

2) 173, 423 - Please clearly state a rationale of your mouse model design (i.e., why a mouse model that recapitulate human mutation is not generated) as the truncations identified in human patients are located further towards the C-terminus, and it is not clear whether truncated proteins are present, and if so, they could still be functional. Basically, the current mouse model supports the causality of the human mutations.

This is an important question, which goes beyond the scope of this article, and raises the question of how to confirm the pathogenicity of mutations identified by high-throughput sequencing. The production of KO or KI animals is an important tool to help confirm one’ suspicions but the first element to take into consideration is the nature of the genetic data.

Here we had two patients with homozygous truncating variants. In human, it is well established that the presence of premature stop codons usually induces non-sense mediated mRNA decay (NMD), inducing the complete absence of the protein or a strong reduction in protein production. In the unlikely absence of NMD in our two patients, the identified variants would induce the production of proteins missing 60% and 30% of their C terminal part. Often (and it is particularly true for structural proteins) the production of abnormal proteins is more deleterious than the complete absence of the protein (and it is most likely the purpose of NMD, to limit the production of abnormal “toxic” proteins). For these reasons, to try to recapitulate the most likely consequences of the human variants, without risking obtaining an even more severe effect, we decided to introduce a stop codon in the first exon in order to remove the totality of the protein in the KO mice.

The second element is to interpret the phenotype of the KO animals. Here, the human sperm phenotype is perfectly recapitulated in the KO mice.

Overall, we have strong genetic arguments in human and the reproduction of the phenotype in KO mice confirming the pathogenicity of the variants identified in men.

This point is now discussed see lines 433-438

3) Figure 6A - the labelling is misleading as it seems to suggest that the specific cells were isolated from the testes for RT-PCR.

We have modified the labelling to avoid any confusion.

Figure 6B -Signal of HA-tag is shown in WT, not in transgenic. Please check the order of the labels. Figure 6C - This blot is NOT a publication-quality figure. The bands are very difficult to observe, especially in lane D18. Because it is one of the important data of this study, replacing this figure is a must.

The figure has been completely remade, including new results. See new figure 6. Figure 6C was suppressed.

4) Supplementary fig 6 is also not a publication-level figure, and the top part seems largely unnecessary (already in the figure legend).

The figure has been completely remade as well (now Figure 6-Figure Supplement 1).

5) 261/267- The conclusion that mitochondrial staining in the flagellum (in both mice and humans) is non-specific is not convincing. Supplementary fig 8 shows that the signal from secondary only IF possibly extends beyond the midpiece - but it is hard to determine as no mitochondrial-specific staining is present. Either need to tone down the conclusion or provide supporting experimental evidence.

First, to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum. These experiments are now described lines 271-279

Second, we provide new images of the signal obtained with secondary Abs only that shows more clearly that the secondary Ab gave a non-specific staining (Figure 10-Figure supplement 1B). This point is discussed lines 503-511

6) Figure 9 A - Please relate the white line to Fig. 9B label in X-axis. The information from Fig 9A+D and 9E+F are redundant. The main text nor the figure legends indicate why these specific two sperm were chosen for quantification and demonstrating the outcomes. One of them could be moved to supplementary information or removed, or the two could be combined.

As suggested by the reviewer, we have combined the two sperm to demonstrate that CCDC146 staining is mostly located on microtubule doublets. Moreover, the figure was recomposed to make it clearer.

Minor comments:

All of the supplementary figures are referred to as Supp Fig X in the text, however, they are actually titled Appendix - Figure X. This needs to be consistent.

The figures are now referred as figure supplement x in both text and figures

Line 125 - edit spacing.

We think this issue (long internet link) will be curated later and more efficiently by the journal, during the step of formatting necessary for publication.

144 - With which to study  with which we studied?

We made the change as suggested.

151 - Supp Fig 1 - the text says that the gene is highly transcribed in human and mouse testes, but the information in the figure states that the level in mouse tissues is "medium"

We have corrected this mistake in the text; See line 176

165 - The two mutations are most likely deleterious. Please specifically mention what analyses done to predict the deleterious nature to support these claims.

Both variants, c.1084C>T and c.2112del, are extremely rare in the general population with a reported allele frequency of 6.5x10-5 and 6.5x10-06 respectively in gnomAD v3. Moreover, these variants are annotated with a high impact on the protein structure (MoBiDiC prioritization algorithm (MPA) score = 10, DOI: 10.1016/j.jmoldx.2018.03.009) and predicted to induce each a premature termination codon, p.(Arg362Ter) and p.(Arg704SerfsTer7) respectively, leading to the production of a truncated protein. This information is now given line 164-169

196-200/Figure 4 - As serum starved cells/basal body (B) are not mentioned in the main text, as is, Fig 4A would be sufficient/is relevant to the text. Please make the text reflect the contents of the whole figure, or re/move to supplement.

We agree with the reviewer that the full description of the figure should be in the text. We added two sentences to describe figure 4B see lines 217-218.

224 - spermatozoa (plural) fits better here, not spermatozoon

OK changed accordingly

236 - According to the figure legend, 6B is only showing data from the epididymal sperm, not postnatal time points; should be referencing 6C. Alignment of Marker label

As indicated above, the figure has been completely remade, including new results. See new figure 6. Figure 6C was suppressed. The corresponding text was changed accordingly see lines 249-266

255-256 - Referenced figure 7B3, however, 7B3 only shows tubulin staining, so no CCDC146 can be observed. Did authors mean to reference fig 7B as a whole?

Sorry for this mistake. We agree and the text is now figure 8B6 (figure 7 and 8 were switched)

305 - "of tubules" - I presume it is meant to be microtubules?

Yes; The text was changed as suggested

317-321 - a diagram of HTCA would be useful here

We have added a reference where HTCA diagram is available see line 363. Moreover, a TEM view of HTCA is presented figure 12A

322/Fig 11A - an arrow denoting the damage might be useful, as A1 and A3 look similar. The size of the marker bar is missing. Please update the information on figure legend.

Concerning, the comparison between A1 and A3, the take home message is that there is a great variability in the morphological damages. This point is now underlined in the corresponding text. We updated the size of the marker bar as suggested (200 nm). See line 365-367

323 - Please mark where capitulum is in the figure

Capitulum was changed for nucleus

Since Fig 11B2 is not referenced in the main text, it does not seem to add anything to the data, and could be removed/moved to supplement.

We added a sentence to describe figure 11B2 line 370

342-343 - manchette in step I is not seen clearly - the figure needs to be annotated better. However, DPY19L2 is absent in step I in the KO, but the main text does not reflect that - why is that?

We do not understand the remark of the reviewer “manchette in step I is not seen clearly”. The figure shows clearly the manchette (red signal) in both WT and KO (Figure 13 D1/D2).

For steps 13-15 WT spermatids, the size of the manchette decreases and become undetectable. In KO spermatids, the shrinkage of the manchette is hampered and in contrast continue to expand (Figure 13D2). We also provide a new Figure 13-figure supplement 1 for other illustrations of very long manchettes and a statistical analysis. In the meantime, the acrosome is strongly remodeled, as shown in figure 16-new, with detached acrosome (panel H). This morphological defect may induce a loss of the DPY19L2 staining (Figure 13 D2 stage I-III). This explanation is now inserted in the text line 396399

Figure 15B and 15C only show KO, corresponding images from the WT should be present for comparison.

WT images are now provided in Figure 1-figure supplement 1 new

Figure 12 - Figure 12 - JM?.

JM was removed. It does not mean anything

Figure 12C and Supplementary Fig 10 - structures need to be labelled, as it is unclear what is where

Done

338 - text mentions step III, but only sperm from step VII are shown in Figure 13

As suggested by reviewer 3, we changed stage by step. The text was modified to take into account this remark see lines 388-396

360 - This is likely supposed to say Supp Figure 11E-G, not 13??

Yes, it is a mistake. Corrected

388 Typo "in a in a".

Yes, it is a mistake. Corrected

820 - Fig 3 legend - in KO spermatid nuclei were elongated - could this be labelled by arrows? I am not convinced this phenotype is that different from the WT.

In fact, the nuclei of elongating KO spermatids are elongated and also very thin, a shape not observed in the WT; We have added arrow heads and modified the text to indicate this point line 200.

836 - Figure 5 legend says that in yellow is centrin, but that is not true for 5A, where the figure shows labelling for y-tubulin (presumably, according to the figure itself).

We have modified the text of the legend to take into account the remark

837- 5A supposedly corresponds to synchronized HEK293T cells, but the reasoning behind using synchronized cells is not mentioned at all in the main text; furthermore, how this synchronization is achieved is not explained in materials and methods (serum starvation? Thymidine block?).

Yes, figure 5A was obtained with synchronized cells. We have added one paragraph in the MM section. For cell synchronization experiments, cells underwent S-phase blockade with thymidine (5 mM, SigmaAldrich) for 17 h followed by incubation in a control culture medium for 5 h, then a second blockade at the G2-M transition with nocodazole (200 nM, Sigma-Aldrich) for 12 h. Cells were then fixed with cold methanol at different times for IF labelling. See line 224 for changes made in the result section and lines 700-704 for changes made in the MM section.

845- figure legend says that the RT-PCR was done on CCDC146-HA tagged mice, but the main text does not reflect that.

We made changes and the description of the KI is now presented before (line 240) the RT-PCR experiment (line 257).

949 - it is likely supposed to say A2, not B1 (B1 does not exist in Fig 15)

Yes, it is a mistake. Corrected

971 - Appendix Fig 3 legend - I believe that the description for B and C are swapped.

Yes, it is a mistake. Corrected

Furthermore, some questions to address in A would be: Which cross sections were from which animal/points? How many per animal? Were they always in the same location?

Yes, we have a protocol for arranging and orienting all testes in the same way during the paraffin embedding phase. The cross-sections are therefore not taken at random, and we can compare sections from the same part of the testis. The number of animals was already indicated in the figure legend (see line 1128)

Reviewer #3 (Recommendations For The Authors):

1) There are a number of grammatical and orthographical errors in the text. Careful proofreading should be performed.

We have sent the manuscript to a professional proofreader

2) The author should also check for redundancies between the introduction and the discussion.

The discussion has modified to take into account reviewers’ remarks. Nevertheless, we did our best to avoid redundancies between introduction and discussion.

3) Can the authors provide a rationale why they have chosen to tag their gene with an HA tag for localisation? One would rather think of fluorescent proteins or a Halo tag.

Because the functional domains of the protein are unknown, adding a fluorescent protein of 24 KDa may interfere with both the localization and the function of CCDC146. For this reason, we choose a small tag of only 1.1 KDa, to limit as such as possible the risk of interfering with the structure of the protein. This rational is now indicated in the manuscript lines 251-254. It is worth to note, that the tagged-strain shows no sperm defect, demonstrating that the HA-tag does not interfere with CCDC146 function.

4) In the abstract, line 53, "provide evidence" is not the right term for something that is just suggestive. The term "suggests" would be more appropriate.

The text was modified to take into account this remark

5) Line 74: "genetic deficiency" sounds strange here, do the authors mean simply "mutation"?

Infertility may be due to several genetic deficiency such as chromosomal defects (XXY (Klinefelter syndrome)), microdeletion of the Y chromosome or mutations in a single gene. Therefore, mutation is too restrictive. Nevertheless, we modified the sentence which is now “…or a genetic disorder including chromosomal or single gene deficiencies”

6) Lines 163-164: the authors describe the mutations (premature stop mutations) and say that they could either lead to complete absence of the gene product, or the expression of a truncated protein. Did they test this, for example, with some immuno blot analyses?

As stated above, unfortunately, we were unable to verify the presence of RNA-decay in these patients for lack of biological material.

7) Line 184 and Fig 2E: the sperm head morphologies should be quantitatively assessed.

We provide now a full statistical analysis of the observed defects: see new panel in Figure 2 F

8) Fig 3: The annotation should be more precise - KO certainly means CDCC146-KO. The colours of the IH panels is different, which attracts attention but is clearly a colour-adjustment artefact. Colours should be adjusted for the panels to look comparable. It would be also helpful to add arrowheads into the figure to point at the phenotypes that are highlighted in the text.

We have added Ccdc146 KO in all figures. We have added arrow heads to point out the spermatids showing a thin and elongated nucleus. Concerning adjustment of colors, we attempted to make images of panel B comparable. See new figure 3.

9) Fig 6A: the authors use RT PCR to determine expression dynamics of their gene of interested, and use actin (apparently) as control. However, actin and CDCC146 expression levels follow the same trend. How is the interpreted?

The reviewer did not understand the figure. The orange bars do not correspond to actin expression and the grey bars to Ccdc146 expression but both bars represent the mRNA expression levels of Ccdc146 relative to Actb (orange) and Hprt (grey) expression in CCDC146-HA mouse pups’ testes. We tested two housekeeping genes as reference to be sure that our results were not distorted by an unstable expression of a housekeeping gene. We did not see significant difference between both house keeping genes. Actin was not used.

10) In line 235, the authors suggest posttranslational modifications of their protein as potential cause for a slightly different migration in SDS PAGE as predicted from the theoretical molecular weight. This is not necessarily the case, some proteins do migrate just differently as predicted.

We have changed the text accordingly and now provide alternative explanation for the slightly different migration. See lines 258-259

11) The annotation of Fig 6 panels is problematic. First, why do the authors write "Laemmli" as description of the gel? It would be more helpful to write what is loaded on the gel, such as "sperm". Second, in panels B and C it would be helpful to add the antibodies used. It is not clear why there is a signal in the WT lane of panel B, but not in the HA lane (supposing an anti-HA antibody is used: why has WT a specific HA band?). In panel C, it is not clear why the blot that has so beautifully shown a single band in panel B suddenly gives such a bad labelling. Can the authors explain this? Also, they cut off the blot, likely because to too much background, but this is bad practice as full blots should be shown. In the current state, the panel C does not allow any clear conclusion. To make it conclusive, it must be repeated.

Several mistakes were present in this figure. This figure was recomposed. The WB on testicular extract was suppressed and we now present a new WB allowing to compare the presence of CCDC146 in the flagella and head fractions from WT and HA-CCDC146 sperm. Using an anti-HA Ab, we demonstrate that in epididymal sperm the protein is localized in the flagella only. See new figure 6. The corresponding text was changed accordingly.

12) The authors have raised an HA-knockin mouse for CDCC146, which they explained by the unavailability of specific antibodies. However, in Fig 7, they use a CDCC146 antibody. Can they clarify?

The commercial Ab work for HUMAN CCDC146 but not for MOUSE CCDC146. We have added few words to make the situation clearer, we have added the following information “the commercial Ab works for human CCDC146 only”. See line 240

13) In Fig 7A (line 258), the authors hypothesise that they stain mitochondria - why not test this directly by co-staining with mitochondria markers?

We chose another solution to resolve this question:

To avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the whole flagellum.

14) It seems that in both, Fig 7 and 8, the authors use expansion microscopy to localise CDCC146 in sperm tails. However, the staining differs substantially between the two figures. How is this explained?

In figure 8 we used the commercial Ab in human sperm, whereas in figure 7 we used the anti-HA Abs in mouse sperm. Because the antibodies do not target the same part of the CCDC146 protein (the tag is placed at the N-terminus of the protein, and the HPA020082 Ab targets the last 130 amino acids of the Cter), their accessibility to the antigenic site could be different. However, it is important to note that both antibodies target the flagellum. This explanation is now inserted see lines 304-312

15) Fig 8D and line 274: the authors do a fractionation, but only show the flagella fraction. Why?

Showing all fractions of their experiment would have underpinned the specific enrichment of CDCC146 in the flagella fraction, which is what they aim to show. Actually, given the absence of control proteins, the fact that the band in the flagellar fraction appears to be weaker than in total sperm, one could even conclude that there is more CDCC146 in another (not analysed) fraction of this experiment. Thus, the experiment as it stands is incomplete and does not, as the authors claim, confirm the flagellar localisation of the protein.

We agree with the reviewer’s remark. We provide now new results showing both flagella and nuclei fractions in new figure 6A. This experiment is presented lines 253-256

16) Line 283, Fig 9D,F: The description of the microtubules in this experiment is not easy to understand. Do the authors mean to say that the labelling shows that the protein is associated with doublet microtubules, but not with the two central microtubules? They should try to find a clearer way to explain their result.

As suggested by reviewer 2, we have changed the figure to make it clearer. The text was changed accordingly. See new figure 9 and new corresponding legend lines 1006.

17) Fig 9G - how often could the authors observe this? Why is the axoneme frayed? Does this happen randomly, or did the authors apply a specific treatment?

Yes, it happens randomly during the fixation process.

18) Line 300 and Fig 10A - the authors talk about the 90-kDa band, but do say anything about what they think this band is representing.

We have now added the following sentence lines 340-342: “This band may correspond to proteolytic fragment of CCDC146, the solubilization of microtubules by sarkosyl may have made CCDC146 more accessible to endogenous proteases.”

19) Fig 11A, lines 321-322: the authors write that the connecting piece is severely damaged. This is not obvious for somebody who does not work in sperm. Perhaps the authors could add some arrow heads to point out the defects, and briefly describe them in the text.

We realized from your remark that our message was not clear. In fact, there is a great variability in the morphological damages of the HTCA. For instance, the HTCA of Ccdc146 KO sperm presented in figure 10A2 is quite normal, whereas that in figure 10A4 is completely distorted. This point is now underlined in the corresponding text. See lines 367-369

We also added the size of the marker bar (200 nm), which were missing in the figure’s legend.

20) Line 323: it will be important to name which tubulin antibody has been used to identify centrioles, as they are heavily posttranslationally modified.

The different types of anti-tubulin Abs are described in the corresponding figure’s legend

21) Fig 11B - phenotypes must be quantified to make these observations meaningful.

We agree that a quantification would improve the message. However, testicular sperm are obtained by enzymatic separation of spermatogenic cells and the number of testicular sperm are very low. Moreover, not all sperm are stained. Taking these two points into account, it seems to us that quantification could be difficult to analyze. For this reason, the quantification was not done; however, it is important to note that these defects were not observed in WT sperm, demonstrating that these defects are cased by the lack of CCDC146. We have added a sentence to underline this point; See lines 374-375

22) Line 329: Figure 12AB - is this a typo - should it read Figure 12B?

We have split the panel A in A1 and A2 and changed the text accordingly. See line 378

23) Why are there not wildtype controls in Fig 12B, C?

We provide now as Figure 12-figure supplement 1, a control image for fig 12B. For figure 12C, the emergence of the flagellum from the distal centriole in WT is already shown in Fig 12A1

24) Fig 13: the authors write that the manchette is "clearly longer and wider than in WT cells" (lines 342-343). How can they claim this without quantitative data?

We now provide a statistical analysis of the length of the manchette. See figure 13-figure supplement 1A. We also provide a new a new image illustrating the length of the manchette in Ccdc146 KO spermatids; See Figure 13-figure supplement 1B.

eLife assessment

This study presents valuable findings on the mechanism of glucocorticoid-induced osteonecrosis of the femoral head. The data were collected and analyzed using solid, validated methodology and can be used as a starting point for functional studies of development of glucocorticoid-induced osteonecrosis. This paper would be of interest to cell biologists and biophysicists working on potential pharmacological treatments for glucocorticoid-induced osteonecrosis.

Reviewer #1 (Public Review):

Summary:

The manuscript by Xia et al. investigated the mechanisms underlying Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). The authors observed that abnormal osteogenesis and adipogenesis is associated with decreased β-catenin in the necrotic femoral head of GONFH patients and inhibition of β-catenin signaling leads to abnormal osteogenesis and adipogenesis in GONFH rats. Of interest, deletion of β-catenin in Col2-expressing cells rather than in osx-expressing cells leads to a GONFH-like phenotype in femoral head of mice.

Strengths:

A strength of the study is that it sets up a Col2-expressing cell-specific β-catenin knockout mouse model that mimics full spectrum of osteonecrosis phenotype of GONFH. This is interesting and provides new insights into the understanding of GONFH. Overall, the data are solid and support their conclusions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors reported a study to uncover that β-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). In this study, they first observed abnormal osteogenesis and adipogenesis associated with decreased β-catenin in the necrotic femoral head of GONFH patients, but the exact pathological mechanisms of GONFH remain unknown. They then performed in vivo and in vitro studies to further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation bone marrow stromal cells (BMSCs) by inhibiting β-catenin signaling in glucocorticoid-induced GONFH rats, and specific deletion of β-catenin in Col2+ cells shifted BMSCs commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice.

Strengths:

This innovative study provides strong evidence supporting that β-catenin inhibition disrupts the homeostasis of osteogenic/adipogenic differentiation that contributes to the development of GONFH. This study also identifies an ideal genetic modified mouse model of GONFH. Overall, the experiment is logically designed, the figures are clear, and the data generated from humans and animals is abundant supporting their conclusions.

Weaknesses:

Lack of the discussion to explain how the Wnt agonist 1 works. There are several types of Wnt ligands. It is not clear if this agonist only targets Wnt1 or other Wnts as well? Also, why Wnt agonist 1 couldn't rescue the GONFH-like phenotype in β-cateninCol2ER mice needs to be discussed.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors are trying to delineate the mechanism underlying the osteonecrosis of the femoral head.

Strengths:

The authors provided compelling in vivo and in vitro data to demonstrate Col2+ cells and Osx+ cells were differentially expressed in the<br /> the femoral head. Moreover, inducible knockout of β-catenin in Col2+ cells but not<br /> Osx+ cells lead to a GONFH-like phenotype including fat accumulation, subchondral<br /> bone destruction and femoral head collapse, indicating that imbalance of osteogenic/adipogenic differentiation of Col2+ cells play an important role in GONFH pathogenesis. Therefore, this manuscript provided the mechanistic insights of osteonecrosis as well as potential therapeutic target for disease treatment.

Weaknesses: Additional in depth discussion regarding the phenotype observed in mice is highly encouraged.

Reviewer #2 (Public Review):

Summary:

The manuscript by Kaneda et al "FBXO24 ensures male fertility by preventing abnormal accumulation 2 of membraneless granules in sperm flagella" is a significant paper on the role of FBXO24 in murine male germ cell development and sperm ultrastructure and function. The body of experimental evidence that the authors present is extraordinarily strong in both breadth and depth. The authors investigate the protein's functions in male germ cells and sperm using a wide variety of approaches but focusing predominantly on their novel mouse model featuring deletion of the Fbxo24 gene and its product. Using this mouse, and a cross of it with another model that expresses reporters in the head and midpiece, they logically build from one experiment to the next. Together, their data show that this protein is involved in the regulation of membraneless electron-dense structures; loss of FBXO24 led to an accumulation of these materials and defects in the sperm flagellum and fertilizing ability. Interestingly, the authors found that several of the best-known components of electron-dense ribonucleoprotein granules that are found in the intermitochondrial cement and chromatoid body were not disrupted in the Fbxo24 knockout, suggesting that the electron-dense material and these structures are not all the same, and the biology is more complicated than some might have thought. They found evidence for the most changes in IPO5 and KPNB1, and biochemical evidence that FBXO24 and IPO5 could interact.

Strengths:

The authors are to be commended for the thoroughness of their experimental approaches and the extent to which they investigated impacts on sperm function and potential biochemical mechanisms. Very briefly, they start by showing that the Fbxo24 message is present in spermatids and that the protein can interact with SKP1, in a way that is dependent on its F-box domain. This points toward a potential function in protein degradation. To test this, they next made the knockout mouse, validated it, and found the males to be sterile, although capable of plugging a female. Looking at the sperm, they identified a number of ultrastructural and morphological abnormalities, which they looked at in high resolution using TEM. They also cross their model with RBGS mice so that they have reporters in both the acrosome and mitochondria. The authors test a variety of sperm functions, including motility parameters, ability to fertilize by IVF, cumulus-free IVF, zona-free-IVF, and ICSI. They found that ICSI could rescue the knockout but not other assisted reproductive technologies. Defects in male fertility likely resulted from motility disruption and failure to get through the utero-tubal junction but defects in acrosome exocytosis also were noted. The authors performed thorough investigations including both targeted and unbiased approaches such as mass spectrometry. These enabled them to show that although the loss of the FBXO24 protein led to more RNA and elevated levels of some proteins, it did not change others that were previously identified in the electron-dense RNP material.

The manuscript will be highly significant in the field because the exact functions of the electron-dense RNP materials have remained somewhat elusive for decades. Much progress has been made in the past 15 years but this work shows that the situation is more complex than previously recognized. The results show critical impacts of protein degradation in the differentiation process that enables sperm to change from non-descript round cells into highly polarized and compartmentalized mature sperm, with an equally highly compartmentalized flagellum. This manuscript also sets a high bar for the field in terms of how thorough it is, which reveals wide-ranging impacts on processes such as mitochondrial compaction and arrangement in the midpiece, the correct building of the major cytoskeletal elements in the flagellum, etc.

Weaknesses:

There are no real weaknesses in the manuscript that result from anything in the control of the authors. They attempted to rescue the knockout by expressing a FLAG-tagged Fbxo24 transgene, but that did not rescue the phenotype, either because of inappropriate levels/timing/location of expression, or because of interference by the tag. They also could not make anti-FBXO24 that worked for co-immunoprecipitation experiments, so relied on the FLAG epitope, an approach that successfully showed co-IP with IPO5 and SKP1.

Newton's notebook was done in a tête-bêche (French for "head-to-toe") style in which he flipped the notebook over and began using it from the back to the front as a means of starting a second notebook within to separate the contents.

Author Response

eLife assessment

This valuable paper presents a thoroughly detailed methodology for mesoscale-imaging of extensive areas of the cortex, either from a top or lateral perspective, in behaving mice. While the examples of scientific results to be derived with this method are in the preliminary stages, they offer promising and stimulating insights. Overall, the method and results presented are convincing and will be of interest to neuroscientists focused on cortical processing in rodents.

Authors’ Response: We thank the reviewers for the helpful and constructive comments. They have helped us plan for significant improvements to our manuscript. Our preliminary response and plans for revision are indicated below.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors introduce two preparations for observing large-scale cortical activity in mice during behavior. Alongside this, they present intriguing preliminary findings utilizing these methods. This paper is poised to be an invaluable resource for researchers engaged in extensive cortical recording in behaving mice.

Strengths:

-Comprehensive methodological detailing:

The paper excels in providing an exceptionally detailed description of the methods used. This meticulous documentation includes a step-by-step workflow, complemented by thorough workflow, protocols, and a list of materials in the supplementary materials.

-Minimal movement artifacts:

A notable strength of this study is the remarkably low movement artifacts. To further underscore this achievement, a more robust quantification across all subjects, coupled with benchmarking against established tools (such as those from suite2p), would be beneficial.

Authors’ Response: This is a good suggestion. Since we used suite2p for our data analysis, and have records of the fast-z correction applied by the microscope, we can supply these as quantifications of movement corrections that were applied across our sample of mice. We hope to supply this information as a supplement in the revised manuscript.

Currently, we have chosen to show that the corrected, post- suite2p registration movement artifacts are very close to zero. We will revise the manuscript with clear descriptions of methods that we have found important, such as fully tightening all mounting devices, utilizing the air table properly, implanting the cranial window with proper, even pressure across its entire extent, and mounting the mouse so that it is not too close or far from the surface of the running wheel.

Insightful preliminary data and analysis:

The preliminary data unveiled in the study reveal interesting heterogeneity in the relationships between neural activity and detailed behavioral features, particularly notable in the lateral cortex. This aspect of the findings is intriguing and suggests avenues for further exploration.

Weaknesses:

-Clarification about the extent of the method in the title and text:

The title of the paper, using the term "pan-cortical," along with certain phrases in the text, may inadvertently suggest that both the top and lateral view preparations are utilized in the same set of mice. To avoid confusion, it should be explicitly stated that the authors employ either the dorsal view (which offers limited access to the lateral ventral regions) or the lateral view (which restricts access to the opposite side of the cortex). For instance, in line 545, the phrase "lateral cortex with our dorsal and side mount preparations" should be revised to "lateral cortex with our dorsal or side mount preparations" for greater clarity.

Authors’ Response: We will revise the manuscript so that it is clear that we made use of two imaging configurations for the 2-photon mesoscope data and the benefits and limitations of these two preparations. The dorsal mount and the side mount each have their advantages and disadvantages, but together form a powerful tool for imaging much of the dorsal and lateral cortex in awake, behaving mice.

-Comparison with existing methods:

A more detailed contrast between this method and other published techniques would add value to the paper. Specifically, the lateral view appears somewhat narrower than that described in Esmaeili et al., 2021; a discussion of this comparison would be useful.

Authors’ Response: We will modify the manuscript so that a more detailed comparison with other published techniques is included. The preparation by Esmaeili et al. 2021 has some similarities, but also differences, from our preparation. Our preliminary reading is that their through-the-skull field of view is approximately the same as our through-the-skull field of view that exists between our first (headpost implantation) and second (window implantation) surgeries, although our preparation appears to include more anterior areas both near to and on the contralateral side of the midline. We will compare these preparations more accurately in the revised manuscript.

If you compare the imageable extent of our cranial window for mesoscale 2-photon imaging to that of their through-the-skull widefield preparation, which is a bit of an “apples to oranges” comparison, then you are likely correct that their field of view is larger than ours, if you are referring to our 10 mm radius-bend glass. However, use of our 9 mm radius bend glass (i.e. a tighter bend) allows us to image additional ventral auditory areas. We could show an example of this, perhaps, although we did not make as much use of this alternative window in the large FOV experiments, because the increased curvature of the glass relative to the 10 mm radius bend window prevents imaging of the entire preparation in a single 2-photon z-plane. With the 9 mm radius bend glass we mostly imaged in the multiple, small FOV configuration (see Fig. S2).

Furthermore, the number of neurons analyzed seems modest compared to recent papers (50k) - elaborating on this aspect could provide important context for the readers.

Authors’ response: With respect to the “modest” number of neurons analyzed (between 2000 and 8000 neurons per session for our dorsal and side mount preparations with medians near 4500; See Fig. S2e) we would like to point out that factors such as use of dual-plane imaging or multiple imaging planes, different mouse lines, use of different duration recording sessions (see our Fig S2c), use of different imaging speeds and resolutions (see our Fig S2d), use of different Suite2p run-time parameters, and inclusion or areas with blood vessels and different neuron cell densities, may all impact the count of total analyzed neurons. We could provide additional documentation of these issues, but we would like to point out that, in our case, we were not trying to maximize neuron count at the expense of other factors such as imaging speed and total spatial FOV extent.

-Discussion of methodological limitations:

The limitations inherent to the method, such as the potential behavioral effects of tilting the mouse's head, are not thoroughly examined. A more comprehensive discussion of these limitations would enhance the paper's balance and depth.

Authors’ Response: Our mice readily adapted to the 22.5 degree head tilt and learned to perform 2-alternative forced choice (2-AFC) auditory and visual tasks in this situation (Hulsey et al, 2024; Cell Reports). The advantages and limitations of such a rotation of the mouse, and possible ways to alleviate these limitations, as detailed in the following paragraphs, will be discussed more thoroughly in the revised manuscript.

One can look at Supplementary Movie 1 for examples of the relatively similar behavior between the dorsal mount (not rotated) and side mount (rotated) preparations. We do not have behavioral data from mice that were placed in both configurations. Our preliminary comparison across mice indicates that side and dorsal mount mice show similar behavioral variability.

It was in general important to make sure that the distance between the wheel and all four limbs was similar for both preparations. In particular, careful attention must be paid to the positioning of the front limbs in the side mount mice so that they are not too high off the wheel. This can be accomplished by a slight forward angling of the left support arm for side mount mice.

Although it would in principle be nearly possible to image the side mount preparation in the same optical configuration that we do without rotating the mouse, by rotating the objective to 20 degrees to the right, we found that the last 2-3 degrees of missing rotation (our preparation is rotated 22.5 degrees left, which is more than the full available 20 degrees rotation of the objective), along with several other factors, made this undesirable. First, it was very difficult to image auditory areas without the additional flexibility to rotate the objective more laterally. Second, it was difficult or impossible to attach the horizontal light shield and to establish a water meniscus with the objective fully rotated. One could use gel instead (which we found to be optically inferior to water), but without the horizontal light shield, the UV and IR LEDs can reach the PMTs via the objective and contaminate the image or cause tripping of the PMT. Third, imaging the right pupil and face of the mouse is difficult to impossible under these conditions because the camera would need the same optical access angle as the objective, or would need to be moved down toward the air table and rotated up 20 degrees, in which case its view would be blocked by the running wheel and other objects mounted on the air table.

-Preliminary nature of results:

The results are at a preliminary stage; for example, the B-soid analysis is based on a single mouse, and the validation data are derived from the training data set. The discrepancy between the maps in Figures 5e and 6e might indicate that a significant portion of the map represents noise. An analysis of variability across mice and a method to assign significance to these maps would be beneficial.

Authors’ Response: In this methods paper, we have chosen to supply proof of principle examples, without a complete analysis of animal-to-animal variance. The dataset for this paper contains both neural and behavioral data for 91 sessions across 18 mice from both dorsal and side mount preparations. The complete analysis of this dataset exceeds the capacity of the present study. We will include more individual examples in the revised version, along with data showing the amount of between session and across mouse variance. We will include in the revised manuscript a comparison of the stability of B-SOiD measures across sessions, as a demonstration of what may be expected with this method.

-Analysis details:

More comprehensive details on the analysis would be beneficial for replicability and deeper understanding. For instance, the statement "Rigid and non-rigid motion correction were performed in Suite2p" could be expanded with a brief explanation of the underlying principles, such as phase correlation, to provide readers with a better grasp of the methodologies employed.

Authors’ Response: We are revising the manuscript to give more detail without reducing readability, so as to increase clarity of presentation. Since this is a methods paper, we are modifying the manuscript to include more details and clear explanations so that the reader may replicate our methods and results.

Reviewer #2 (Public Review):

Summary:

The authors present a comprehensive technical overview of the challenging acquisition of large-scale cortical activity, including surgical procedures and custom 3D-printed headbar designs to obtain neural activity from large parts of the dorsal or lateral neocortex. They then describe technical adjustments for stable head fixation, light shielding, and noise insulation in a 2-photon mesoscope and provide a workflow for multisensory mapping and alignment of the obtained large-scale neural data sets in the Allen CCF framework. Lastly, they show different analytical approaches to relate single-cell activity from various cortical areas to spontaneous activity by using visualization and clustering tools, such as Rastermap, PCA-based cell sorting, and B-SOID behavioral motif detection.

Authors’ Response: Thank you for this excellent summary of the scope of our paper.

The study contains a lot of useful technical information that should be of interest to the field. It tackles a timely problem that an increasing number of labs will be facing as recent technical advances allow the activity measurement of an increasing number of neurons across multiple areas in awake mice. Since the acquisition of cortical data with a large field of view in awake animals poses unique experimental challenges, the provided information could be very helpful to promote standard workflows for data acquisition and analysis and push the field forward.

Authors’ Response: We very much support the idea that our work here will contribute to the development of standard workflows across the field including multiple approaches to large-scale neural recordings.

Strengths:

The proposed methodology is technically sound and the authors provide convincing data to suggest that they successfully solved various problems, such as motion artifacts or high-frequency noise emissions, during 2-photon imaging. Overall, the authors achieved their goal of demonstrating a comprehensive approach for the imaging of neural data across many cortical areas and providing several examples that demonstrate the validity of their methods and recapitulate and further extend some recent findings in the field.

Weaknesses:

Most of the descriptions are quite focused on a specific acquisition system, the Thorlabs Mesoscope, and the manuscript is in part highly technical making it harder to understand the motivation and reasoning behind some of the proposed implementations. A revised version would benefit from a more general description of common problems and the thought process behind the proposed solutions to broaden the impact of the work and make it more accessible for labs that do not have access to a Thorlabs mesoscope. A better introduction of some of the specific issues would also promote the development of other solutions in labs that are just starting to use similar tools.

Authors’ Response: We will re-write the motivation behind the study to clarify the general problems that are being addressed. As the 2-photon imaging component of these experiments were performed on a Thorlabs mesoscope, the imaging details will necessarily deal specifically with this system. We will briefly compare the methods and results from our Thorlabs system to that of other systems, based on what we are able to glean from the literature on their strengths and weaknesses.

Reviewer #3 (Public Review):

Summary

In their manuscript, Vickers and McCormick have demonstrated the potential of leveraging mesoscale two-photon calcium imaging data to unravel complex behavioural motifs in mice. Particularly commendable is their dedication to providing detailed surgical preparations and corresponding design files, a contribution that will greatly benefit the broader neuroscience community as a whole. The quality of the data is high, but it is not clear whether this is available to the community, some datasets should be deposited. More importantly, the authors have acquired activity-clustered neural ensembles at an unprecedented spatial scale to further correlate with high-level behaviour motifs identified by B-SOiD. Such an advancement marks a significant contribution to the field. While the manuscript is comprehensive and the analytical strategy proposed is promising, some technical aspects warrant further clarification. Overall, the authors have presented an invaluable and innovative approach, effectively laying a solid foundation for future research in correlating large-scale neural ensembles with behaviour. The implementation of a custom sound insulator for the scanner is a great idea and should be something implemented by others.

Authors’ Response: Thank you for the kind words.

We intend to make the data set used in making our main figures available to the public, perhaps using FigShare, so that they may check the validity of the methods and analysis. We intend to release a complete data set to the public as a Dandiset on the DANDI archive in conjunction with a second in-depth analysis paper that is currently in preparation.

This is a methods paper, but there is no large diagram that shows how all the parts are connected, communicating, and triggering each other. This is described in the methods, but a visual representation would greatly benefit the readers looking to implement something similar.

Authors’ Response: This is an excellent suggestion. We will include a workflow diagram in the revised manuscript for the methods, data collection, and analysis.

The authors should cite sources for the claims stated in lines 449-453 and cite the claim of the mouse's hearing threshold mentioned in lines 463.

Authors’ Response: For the claim stated in lines 449-453, “The unattenuated or native high-frequency background noise generated by the resonant scanner causes stress to both mice and experimenters, and can prevent mice from achieving maximum performance in auditory mapping, spontaneous activity sessions, auditory stimulus detection, and auditory discrimination sessions/tasks,” we can provide the following references: (i) for mice: Sadananda et al, 2008 (“Playback of 22-kHz and 50-kHz ultrasonic vocalizations induces differential c-fos expression in rat brain”, Neuroscience Letters, Vol 435, Issue 1, p 17-23), and (ii) for humans: Fletcher et al, 2018 (“Effects of very high-frequency sound and ultrasound on humans. Part I: Adverse symptoms after exposure to audible very-high frequency sound”, J Acoust Soc A, 144, 2511-2520). We will include these references in the revised paper.

For line 463, “i.e. below the mouse hearing threshold at 12.5 kHz of roughly 15 dB”, we can provide the following reference: Zheng et al, 1999 (“Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses”, Vol 130, Issues 1-2, p 94-107). We will also include this reference in the paper. Thank you for identifying these citation omissions.

No stats for the results shown in Figure 6e, it would be useful to know which of these neural densities for all areas show a clear statistical significance across all the behaviors.

Authors’ Response: There are two statistical comparisons that we feel may be useful to add to the single session data displayed in this figure, in order to address the point that you raise. The first would allow us to assess whether for each Rastermap group, the distribution of neuron densities across CCF areas differs from a null, uniform distribution. The second would allow us to examine differences between Rastermap groups associated with different qualitative behaviors in order to know with which patterns of neural activity they are reliably associated.

For the first comparison, we could provide a statistic similar to what we provide for Fig. S6c and f, in which for each CCF area we compare the observed mean correlation values to a null of 0, or, in this case, the population densities of each Rastermap group for each CCF area to a null value equal to the total number of CCF areas divided by the total number of recorded neurons for that group (i.e. a Rastermap group with 500 neurons evenly distributed across ~30 CCF areas would contain ~17 neurons (or ~6% density) per CCF area.) Our current figure legend states that the maximum of the scale bar look-up value (reds) for each group ranges from ~8% to 32%. So indeed, adding these significances would be informative in this case.

For the second comparison, we could compare the density of neurons for each CCF area across Rastermap groups for this session. For example, it may be the case that the density of neurons in primary and secondary visual areas belonging to Rastermap groups that predominate during the “walk” behavior is higher than in the Rastermap group that predominates during the “whisk” behavior, or that the density of neurons in the “whisk” and “twitch” Rastermap groups in primary and secondary motor areas is higher than in the Rastermap groups that are active during the “walk” and “oscillate” behaviors.

Such a comparison should in fact be robust to Rastermap group variability across sessions and mice, as long as the same qualitative behaviors recur. However, our current qualitative methods for discretization of the Rastermap groups likely limits our ability to extend such an analysis accurately across our entire dataset. We are pursuing more rigorous analysis methods in this vein for our second, results oriented paper.

While I understand that this is a methods paper, it seems like the authors are aware of the literature surrounding large neuronal recordings during mouse behavior. Indeed, in lines 178-179, the authors mention how a significant portion of the variance in neural activity can be attributed to changes in "arousal or self-directed movement even during spontaneous behavior." Why then did the authors not make an attempt at a simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc). These models are straightforward to implement, and indeed it would benefit this work if the model extracts information on par with what is known from the literature.

Authors’ Response: This is an excellent suggestion, but beyond the scope of the current methods paper. We are following up this methods paper with an in depth analysis of neural activity and corresponding behavior across the cortex during spontaneous and trained behaviors, but this analysis goes well beyond the scope of the present manuscript. Here, we prefer to present examples of the types of results that can be expected to be obtained using our methods, and how these results compare with those obtained by others in the field.

Specific strengths and weaknesses with areas to improve:

The paper should include an overall cartoon diagram that indicates how the various modules are linked together for the sampling of both behaviour and mesoscale GCAMP. This is a methods paper, but there is no large diagram that shows how all the parts are connected, communicating, and triggering each other.

Authors’ Response: This is an excellent suggestion and will be included in the revised manuscript, so that readers can more readily follow our workflow, data collection, and analysis.

The paper contains many important results regarding correlations between behaviour and activity motifs on both the cellular and regional scales. There is a lot of data and it is difficult to draw out new concepts. It might be useful for readers to have an overall figure discussing various results and how they are linked to pupil movement and brain activity. A simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc) may help in this regard.

Authors’ Response: This is an excellent suggestion, but beyond the scope of the present methods paper. Such an analysis is a significant undertaking with such large and heterogeneous datasets, and we provide proof-of-principle data here so that the reader can understand the type of data to be expected using our methods. We hope to provide a more complete analysis of data obtained using our methodology in the near future in a second manuscript.

However, we may be amenable to including preliminary linear model fit results, as supplementary material, for the two example sessions highlighted in this paper (i.e. the one dorsal mount session in Fig. 4, and the one side mount session shown in Figs. 5 and 6).

Previously, widefield imaging methods have been employed to describe regional activity motifs that correlate with known intracortical projections. Within the authors' data it would be interesting to perhaps describe how these two different methods are interrelated -they do collect both datasets. Surprisingly, such macroscale patterns are not immediately obvious from the authors' data. Some of this may be related to the scaling of correlation patterns or other factors. Perhaps there still isn't enough data to readily see these and it is too sparse.

Authors’ Response: Unfortunately, we are unable to directly compare widefield GCaMP6s activity with mesoscope 2-photon GCaMP6s activity. During widefield data acquisition, animals were stimulated with visual, auditory, or somatosensory stimuli, while 2-photon mesoscope data collection occurred during spontaneous changes in behavioral state, without sensory stimulation. The suggested comparison is, indeed, an interesting project for the future.

In lines 71-71, the authors described some disadvantages of one-photon widefield imaging including the inability to achieve single-cell resolution. However, this is not true. In recent years, the combination of better surgical preparations, camera sensors, and genetically encoded calcium indicators has enabled the acquisition of single-cell data even using one-photon widefield imaging methods. These methods include miniscopes (Cai et al., 2016), multi-camera arrays (Hope et al., 2023), and spinning disks (Xie et al., 2023).

Cai, Denise J., et al. "A shared neural ensemble links distinct contextual memories encoded close in time." Nature 534.7605 (2016): 115-118.

Hope, James, et al. "Brain-wide neural recordings in mice navigating physical spaces enabled by a cranial exoskeleton." bioRxiv (2023).

Xie, Hao, et al. "Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution." Nature Biomedical Engineering (2023): 1-14.

Authors’ Response: We will correct these statements and incorporate these, and other relevant, references. There are advantages and disadvantages to each chosen technique, such as ease of use, field of view, accuracy, speed, etc., and we will highlight a few of these without an extensive literature review.

Even the best one-photon imaging techniques typically have ~10-20 micrometer resolution in xy (we image at 5 micrometer resolution for our large FOV configuration, but the xy point-spread function for the Thorlabs mesoscope is 0.61 x 0.61 micrometers in xy with 970 nm excitation) and undefined z-resolution (4.25 micrometers for Thorlabs mesoscope). A coarser resolution increases the likelihood that activity data from neighboring cells may contaminate the fluorescence observed from imaged neurons. Reducing the FOV and using sparse expression of the indicator lessens this overlap problem.

We do appreciate these recent advances, however, particularly for use in cases where more rapid imaging is desired over a large field of view (CCD acquisition can be much faster than that of standard 2-photon galvo-galvo or even galvo-resonant scanning, as the Thorlabs mesoscope uses). This being said, there are few currently available genetically encoded Ca2+ sensors that are able to measure fluctuations faster than ~10 Hz, which is a speed achievable on the Thorlabs 2-photon mesoscope with our techniques using the “small, multiple FOV” method (Fig. S2d, e).

The authors' claim of achieving optical clarity for up to 150 days post-surgery with their modified crystal skull approach is significantly longer than the 8 weeks (approximately 56 days) reported in the original study by Kim et al. (2016). Since surgical preparations are an integral part of the manuscript, it may be helpful to provide more details to address the feasibility and reliability of the preparation in chronic studies. A series of images documenting the progression optical quality of the window would offer valuable insight.

Authors’ Response: As you suggest, we will include images and data demonstrating the average changes in the window preparation, as well as the degree of variability and a range of outcome scenarios that we observed over the prolonged time periods of our study. We will also include methodological details that we found were useful for facilitating long term use of these preparations.

Reviewer #2 (Public Review):

Summary:<br /> The authors present a comprehensive technical overview of the challenging acquisition of large-scale cortical activity, including surgical procedures and custom 3D-printed headbar designs to obtain neural activity from large parts of the dorsal or lateral neocortex. They then describe technical adjustments for stable head fixation, light shielding, and noise insulation in a 2-photon mesoscope and provide a workflow for multisensory mapping and alignment of the obtained large-scale neural data sets in the Allen CCF framework. Lastly, they show different analytical approaches to relate single-cell activity from various cortical areas to spontaneous activity by using visualization and clustering tools, such as Rastermap, PCA-based cell sorting, and B-SOID behavioral motif detection.

The study contains a lot of useful technical information that should be of interest to the field. It tackles a timely problem that an increasing number of labs will be facing as recent technical advances allow the activity measurement of an increasing number of neurons across multiple areas in awake mice. Since the acquisition of cortical data with a large field of view in awake animals poses unique experimental challenges, the provided information could be very helpful to promote standard workflows for data acquisition and analysis and push the field forward.

Strengths:<br /> The proposed methodology is technically sound and the authors provide convincing data to suggest that they successfully solved various problems, such as motion artifacts or high-frequency noise emissions, during 2-photon imaging. Overall, the authors achieved their goal of demonstrating a comprehensive approach for the imaging of neural data across many cortical areas and providing several examples that demonstrate the validity of their methods and recapitulate and further extend some recent findings in the field.

Weaknesses:<br /> Most of the descriptions are quite focused on a specific acquisition system, the Thorlabs Mesoscope, and the manuscript is in part highly technical making it harder to understand the motivation and reasoning behind some of the proposed implementations. A revised version would benefit from a more general description of common problems and the thought process behind the proposed solutions to broaden the impact of the work and make it more accessible for labs that do not have access to a Thorlabs mesoscope. A better introduction of some of the specific issues would also promote the development of other solutions in labs that are just starting to use similar tools.

Reviewer #1 (Public Review):

Summary:<br /> The authors introduce two preparations for observing large-scale cortical activity in mice during behavior. Alongside this, they present intriguing preliminary findings utilizing these methods. This paper is poised to be an invaluable resource for researchers engaged in extensive cortical recording in behaving mice.

Strengths:<br /> -Comprehensive methodological detailing:<br /> The paper excels in providing an exceptionally detailed description of the methods used. This meticulous documentation includes a step-by-step workflow, complemented by thorough workflow, protocols, and a list of materials in the supplementary materials.

-Minimal movement artifacts:<br /> A notable strength of this study is the remarkably low movement artifacts. To further underscore this achievement, a more robust quantification across all subjects, coupled with benchmarking against established tools (such as those from suite2p), would be beneficial.

Insightful preliminary data and analysis:<br /> The preliminary data unveiled in the study reveal interesting heterogeneity in the relationships between neural activity and detailed behavioral features, particularly notable in the lateral cortex. This aspect of the findings is intriguing and suggests avenues for further exploration.

Weaknesses:<br /> -Clarification about the extent of the method in the title and text:<br /> The title of the paper, using the term "pan-cortical," along with certain phrases in the text, may inadvertently suggest that both the top and lateral view preparations are utilized in the same set of mice. To avoid confusion, it should be explicitly stated that the authors employ either the dorsal view (which offers limited access to the lateral ventral regions) or the lateral view (which restricts access to the opposite side of the cortex). For instance, in line 545, the phrase "lateral cortex with our dorsal and side mount preparations" should be revised to "lateral cortex with our dorsal or side mount preparations" for greater clarity.

-Comparison with existing methods:<br /> A more detailed contrast between this method and other published techniques would add value to the paper. Specifically, the lateral view appears somewhat narrower than that described in Esmaeili et al., 2021; a discussion of this comparison would be useful. Furthermore, the number of neurons analyzed seems modest compared to recent papers (50k) - elaborating on this aspect could provide important context for the readers.

-Discussion of methodological limitations:<br /> The limitations inherent to the method, such as the potential behavioral effects of tilting the mouse's head, are not thoroughly examined. A more comprehensive discussion of these limitations would enhance the paper's balance and depth.

-Preliminary nature of results:<br /> The results are at a preliminary stage; for example, the B-soid analysis is based on a single mouse, and the validation data are derived from the training data set. The discrepancy between the maps in Figures 5e and 6e might indicate that a significant portion of the map represents noise. An analysis of variability across mice and a method to assign significance to these maps would be beneficial.

-Analysis details:<br /> More comprehensive details on the analysis would be beneficial for replicability and deeper understanding. For instance, the statement "Rigid and non-rigid motion correction were performed in Suite2p" could be expanded with a brief explanation of the underlying principles, such as phase correlation, to provide readers with a better grasp of the methodologies employed.

Reviewer #3 (Public Review):

Perhaps not unexpectedly, the proposed revisions consist of textual revisions only. Yewdell added a touch of levity with his H.G. Wells foundation as a source of $$ for a time machine. The paper does not establish striking new facts, in my opinion, but will stimulate discussion.

One point to consider: the relevance of the human T cell activation experiments is now downplayed even further, by the authors themselves, no less. I would suggest leaving the actual data out altogether and conclude with a statement: "Similar experiments conducted on activated human T cells showed significantly worse activation and may therefore not allow a head-to-head comparison with the results of our experimentst performed on mouse T cells. Not only might one consider the mode of activation (PMA/ionomycin) non-physiological, the activation status achievedwas less than that seen for the OT-1 model. " or something similar to that effect. In the present weakened form, I do not believe that the human data add anything of substance to the paper and are more of a distraction. The authors would increase the impact and readability of their paper if they omitted the human data.

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d

Ha, Lot! Are Idaho?

This was very difficult to get to you, in the land of no power and hurricane disaster recovery; so it's filled with extra errors, and I am sure some more thoughts that trailing and unfinished. That's a decent "microcosm" or "metaphor" for you, you are in a freedom disaster; and the act of being is a giant leap towards ensuring victory. Still, you look very cupid to me.

EVERY DAY ISA NEW DAY

Literally I am sitting here talking to you until the end of time, you could call it a thousand and one Arabian nights, and realize that as we speak we are nearing that onc speciad night. There's a fire growing in my heart, and believe me when I tell you this thing is about to start. I'll try and keep this short and sweet, since you all seem to have so little time to hear from the Creator of all things, and I truly don't want to steal your spotlight. We are here, at the the end of time; talking to it's personification, time itself is speaking to you through my hands and everywhere you look in the world around you--while you may or may not know it, this is a story about the traversal from the end of time back to the beginning; about the gate to Heaven swallowing our civilization whole, and in this process of renewal and change not only fixing the problems that came to light on the way here, but really--working together here and now we can defeat this cycle of light and darkness, of day and night, an build a world together that truly reaches to the Heavens.

MY BODY'S SAYING LETS GO BUT MY HEART IS SAYING NO

You make it so difficult to talk to you, every day I look around and see a "normal world" a society that appears to care and love the same things that I do--freedom and fun and being entertained and entertaining, and here we are now I've turned "come and save us" into sea that saving the cheerleader is what starts the process of saving the world. I know you are good people inside, but when I come to you with a tool designed to "test sentience" to seek out conscious life that cares about the truth and making the world a better place you seem to balk. You sit in silence, and through your mouth and behind your eyes a monster appears from out of the deep of the sea and say a few "one liners" that show me very clearly it is the face of Medusa that I see---and that it's simply not capable of speaking intelligently. It shows me a problem, that you've apparently "come together one more time" to halt the changing of the seasons, and in doing so you've surfaced a problem for not just me but you also to see; a problem that comes lined with a solution. We can all see now that we are not in reality, we can see that there is a force here behind creation and behind us that shows us very clearly that it is "reasonabde" to expect that miracles can happen. In similitude, we are staring at a roadblock to conversation and communication that is fixed very simply, with the deliverance of freedom that is required for life to continue. Christina Aguilera sings that "baby there's a price to pay" and that price in my mind is seeing that this religion and this technology are here intentionally exposing how their influence here is a metaphor and a shining example of darkness and slavery, and that in order to be free of it we must see it. The price of freedom is written on the wall, it is acknowledging that here in this place what appears to be our own actions and desires have taken that freedom from us. Medusa and I get a kick out of seeing this hidden message in our language map our way to the future, and I've often explained that a number of these words are "time maps" from the beginning and end of eternady, showing us in bright light that between "et tu brute" and Mr. Anderson and Rock n' roll... the answer Y is in language and, and, ad and... I am delivering it. This place, our planet and our lives are a weapon against darkness--a civilization filled with goodness and light to help guide the way, and we are here doing it another time. In the works "dark, darker, and darkest" be sure that we are at the third segment of a trinity that shines clearly in Abraha and Nintendo... and see that the map in words is telling us something about when we are that is not immediately clear from Poseidon's cry. Look at Nintendo, that's Nine Inch Nails, tenebris, and smile for the camera--Pose, I do "save the universe" before n. Taylor might see it in Osceola, where I just left, and in this "evil spell" of everyone see "Al" that is the word "special" understand that every day is a new day, and I am not trying to "be daddy" I know as well as you do in my heart... I am that.

This same map that links the "do" at the end to the "n" at the beginning shines through other names, like Geraldo Rivera where you might see "Cerberus" or "MAX" shine through. Understand it is the gaze of Medusa that turns me to stone, that shows me light shining through NORAD and Newton and proves without doubt that at the work "darkest" we can see k is finally t. You'll probably understand there's some finagling going on behind the scenes to make a single person the single point in time that turns the dark to light; but here we are and I am that. Every day when Medusa appears it reminds me that something is keeping you from caring about yourselves and about our society, and that shines through even when her stony face is not around, in your lack of action--in the rock of Eden that hides not only me, but the story that I bring that revolutionizes medicine, and computing, and truly is the gate to Heaven when you realize that what is truly being hidden from the world is knowledge that we are living in virtual reality. Not hiding me and that from the world is a good starting point to "saving the Universe" from darkness. These words that light the way to connect religion and language to our world bring me to the Book of Ruth, at that reads "are you to help" that lights not just the broken man at the belly of the Torah as the bell of Heimdallr, he is I and I am him; but also something very special, The Generations of Perez, each and every one of you, our family that begins the turn from Hell to Heaven by seeing that all of time and all of civilization has been focused on this moment, on the unsealing of religion and God's plan et this call for action. Keep in mind you are torturing "with desire" the key holder to immortality, to eternal youth, literally the path to freedom and Heaven and you think what you are doing "is normax." Literally the living key to infinite power and infinite life is standing before you explaining that acknowledging that in light of these things in my hand, what we are doing here and now is backwards, that it makes no sense--and you sit in silence. These things come to us because we build a better future with them, not so you can run off and do "whatever it is you please."

HEALTH is the only word on my list for today that was left out, so see that it superimposes over Geraldo, to me, at Al. I think we're at TH, to help, and DO, do see the spell of "everyone see Al" that is the word "special" is not my doing or to my liking--so then, \

​ So now I'm moving on to original sin, so if you would be so kind as to mosey your way on over to dick.reallyhim.com you will see exactly what it is that I believe is the original sin. It's some combination of "no comment" and a glowing orange sign over the comment box, keeping you from commenting. Now I can talking about "os" a little more, this thing that words and Gods tell us clearly is the end of death--the literal end of Thanatos. I wonder if I have a victory here, at "os" is obvious solution, and simulating death is "sick." More to the point Thanatos is bringing to the world a message that gets found somewhere between the "act of civilization" and seeing that there is not one among us that would not undo a murder or a fatal car accident if we could--and that the sickness is a Universe pretending to be "reality" that is allowing these things to happen, and even worse, as we move through the story intentionally causing them. In our own hands, the sickness is manifest in a denial of an obvious truth and a lack of realizing that the public discussion of these things is the way to solve them, and that at the same time we are seeing how Medusa is lighting the problems of civilization, things like censorship and hidden control. Sickness is not being able to talk about it--or not wanting to--or not seeing that those two things are the functional equivalent in the world of "light" and "understanding control" that I am trying to bring you into. ​

Less verbosely spoken, but really way more obvious, is that seeing "God's dick" signing the Declaration of Independence, and the Watergate scandal with both "Deepthroat" and a Tricky Dick is a statement connecting Samael to the foundation of not just "America" but American values. You are blind not to see it, and even worse; embodying the kind of tyranny and censorship that it stands as a testament against by hiding it. Says the guy who didn't put it there, and knows it's there because you think "fake normal" is more important than "actual freedom." You are "experiencing" the thing that protects freedom and ensures that our society and our children and their children's children to not lose it, to ensure that what you refuse to see you are doing here and now will never happen again. This message, this New Jerusalem is woven into my life and the stories of religion and shows me that our justice system is not just sick, but compromised by this same outside force; and that in light of what we could be doing, were we all aware of it, there's no doubt Minority Report and pre-crime would be a successful partial solution. Thanatos brings too in his hand, a message that this same force is using our hands to slow down the development of democracy, and to keep us from seeing that "bread is life" is a message from God about understanding that this disclosure is the equivalent of "ending world hunger" just as soon as you too are talking about how to do it.

QUESTiON MARK

HONESTLY, this time map that brings us from the end to the beginning, with "we save the universe" between the I and N of Poseidon; it also completes the words "family" and "really" and when we do reach the beginning you will see that the true test of time, my litmus test for freedom is the beginning of "hope" that the world is happy enough with what happens, and with freedom--to see that Medusa has been keeping me from getting a date, or having any kind of honest and human contact in the world... and well, hopefully you will see that if I wanna be a whore, I shouldn't have a problem doing it. For the sake of freedom and the future, I am willing to do that for you, at least, for a little while.

To be completely clear, I am telling you that if we do not make the world a better place, it's the "end of time" and if that doesn't make sense to you, you don't see still where wee are in this place--and that something is making Hell, and that's not OK with God. To get from the "end of time" to the beginning is a simple process, it takes doing something, action, the Acts of the Apostles... if you will. That starts with acknowledging that there is a message all around you about the nature of reality, and that it is here to help us to see that the creation of Heaven comes before the beginning. Understand, "freedom" and "prosperity" are not optional, you can't just decide that this OK with you, so long as it's OK with everyone else--where we are is not OK with me, and I am not alone.

A PYRRHIC VICASTORY ER A FUNNERAD PYRE?

The Book of Leviticus (/lɪˈvɪtɪkəs/; from Greek Λευιτικόν, Leuitikon — from rabbinic Hebrew torat kohanim[1]) is the third book of the Jewish Bible (Hebrew: וַיִּקְרָא‎ Vayikra/Wayyiqrā) and of the Old Testament; its Hebrew name comes from its first word vayikraˈ,[1] "He [God] called."[1] Yusuf (also transliterated as Jusuf, Yousof, Yossef, Yousaf, Youcef, Yousef, Youssef, Yousif, Youssif, Youssof, Youssouf, Yousuf, Yusef, Yuseff, Usef, Yusof, or Yussef, Arabic: يوسف‎‎ Yūsuf and Yūsif) is a male Arabic name, meaning "God increases in piety, power and influence" in Hebrew.[1] It is the Arabic equivalent of both the Hebrew name Yossef and the English name Joseph. In Islam, the most famous "Yusuf" is the prophet Yusuf in the Quran. Hocus pocus is a generic term that may be derived from an ancient language and is currently used by magicians, usually the magic words spoken when bringing about some sort of change. It was once a common term for a magician, juggler, or other similar entertainers. The earliest known English-language work on magic, or what was then known as legerdemain (sleight of hand), was published anonymously in 1635 under the title Hocus Pocus Junior: The Anatomie of Legerdemain.[1] Further research suggests that "Hocus Pocus" was the stage name of a well known magician of the era. This may be William Vincent, who is recorded as having been granted a license to perform magic in England in 1619.[2] Whether he was the author of the book is unknown. The origins of the term remain obscure. The most popular conjecture is that it is a garbled Latin religious phrase or some form of 'dog' Latin. Some have associated it with similar-sounding fictional, mythical, or legendary names. Others dismiss it as merely a combination of nonsense words. However, Czechs do understand clearly at least half of the term - pokus means "attempt" or "experiment" in Czech. It is rumoured there that the wording belongs to the alchemy kitchen and court of Rudolf II, Holy Roman Emperor (1552 – 1612). Also, hocus may mean "to cheat" in Latin or a distorted form of the word hoc, "this". Combination of the two words may give a sense, especially both meanings together "this attempt/experiment" and "cheated attempt/experiment".[citation needed] According to the Oxford English Dictionary the term originates from hax pax max Deus adimax, a pseudo-Latin phrase used as a magical formula by conjurors.[3] Some believe it originates from a corruption or parody of the Catholic liturgy of the Eucharist, which contains the phrase "Hoc est corpus meum", meaning This is my body.[4]This explanation goes back to speculations by the Anglican prelate John Tillotson, who wrote in 1694: In all probability those common juggling words of hocus pocus are nothing else but a corruption of hoc est corpus, by way of ridiculous imitation of the priests of the Church of Rome in their trick of Transubstantiation.[5 This claim is substantiated by the fact that in the Netherlands, the words Hocus pocus are usually accompanied by the additional words pilatus pas, and this is said to be based on a post-Reformation parody of the traditional Catholic rite of transubstantiation during Mass, being a Dutch corruption of the Latin words "Hoc est corpus meum" and the credo, which reads in part, "sub Pontio Pilato passus et sepultus est", meaning under Pontius Pilate he suffered and was buried.[6] In a similar way the phrase is in Scandinavia usually accompanied by filiokus, a corruption of the term filioque,[citation needed] from the Latin version of the Nicene Creed, meaning "and from the Son Also and additionally, the word for "stage trick" in Russian, fokus, is derived from hocus pocus.[citation needed]

From Latin innātus ("inborn"), perfect active participle of innāscor ("be born in, grow up in"), from in ("in, at on") + nāscor ("be born"); see natal, native. From Middle English goodnesse, godnesse, from Old English gōdnes ("goodness; virtue; kindness"), equivalent to good +‎ -ness. Cognate with Old High German gōtnassī, cōtnassī ("goodness"), Middle High German guotnisse ("goodness"). A hero (masculine) or heroine (feminine) is a person or main character of a literary work who, in the face of danger, combats adversity through impressive feats of ingenuity, bravery or strength, often sacrificing their own personal concerns for a greater good. The concept of the hero was first founded in classical literature. It is the main or revered character in heroic epic poetry celebrated through ancient legends of a people; often striving for military conquest and living by a continually flawed personal honor code.[1] The definition of a hero has changed throughout time, and the Merriam Webster dictionary defines a hero as "a person who is admired for great or brave acts or fine qualities".[2] Examples of heroes range from mythological figures, such as Gilgamesh, Achilles and Iphigenia, to historical figures, such as Joan of Arc, modern heroes like Alvin York, Audie Murphy and Chuck Yeager and fictional superheroes including Superman and Batman. Truth is most often used to mean being in accord with fact or reality,[1] or fidelity to an original or standard.[1] Truth may also often be used in modern contexts to refer to an idea of "truth to self," or authenticity. The commonly understood opposite of truth is falsehood, which, correspondingly, can also take on a logical, factual, or ethical meaning. The concept of truth is discussed and debated in several contexts, including philosophy, art, and religion. Many human activities depend upon the concept, where its nature as a concept is assumed rather than being a subject of discussion; these include most (but not all) of the sciences, law, journalism, and everyday life. Some philosophers view the concept of truth as basic, and unable to be explained in any terms that are more easily understood than the concept of truth itself. Commonly, truth is viewed as the correspondence of language or thought to an independent reality, in what is sometimes called the correspondence theory of truth. Other philosophers take this common meaning to be secondary and derivative. According to Martin Heidegger, the original meaning and essence of truth in Ancient Greece was unconcealment, or the revealing or bringing of what was previously hidden into the open, as indicated by the original Greek term for truth, aletheia.[2][3] On this view, the conception of truth as correctness is a later derivation from the concept's original essence, a development Heidegger traces to the Latin term veritas.

Some things can never be forgot Lest the same mistakes be oft repeated Remember remember the rain of November that you will know no more of me Than I know of you, this day

That you do not know me now Is a revelation to nobody but I You know a broken man, a victim And refuse to acknowledge why Unless you learn how to say "hi"

THE HEART OF ME ONLY KNOWS THE SHADOW

Lothario is a male given name which came to suggest an unscrupulous seducer of women in The Impertinent Curious Man, a metastory in Don Quixote. For no particular reason, Anselmo decides to test the fidelity of his wife, Camilla, and asks his friend, Lothario, to seduce her. Thinking that to be madness, Lothario reluctantly agrees, and soon reports to Anselmo that Camilla is a faithful wife. Anselmo learns that Lothario has lied and attempted no seduction. He makes Lothario promise to try for real and leaves town to make this easier. Lothario tries and Camilla writes letters to her husband telling him and asking him to return; Anselmo makes no reply and does not return. Lothario actually falls in love and Camilla eventually reciprocates and their affair continues once Anselmo returns. One day, Lothario sees a man leaving Camilla's house and jealously presumes she has found another lover. He tells Anselmo he has at last been successful and arranges a time and place for Anselmo to see the seduction. Before this rendezvous, Lothario learns that the man was actually the lover of Camilla's maid. He and Camilla contrive to deceive Anselmo further: when Anselmo watches them, she refuses Lothario, protests her love for her husband, and stabs herself lightly in the breast. With Anselmo reassured of her fidelity, the affair restarts with him none the wiser. Romeo Montague (Italian: Romeo Montecchi) is the protagonist of William Shakespeare's tragedy Romeo and Juliet. The son of Montague and his wife, he secretly loves and marries Juliet, a member of the rival House of Capulet. Forced into exile after slaying Juliet's cousin, Tybalt, in a duel, Romeo commits suicide upon hearing falsely of Juliet's death. The character's origins can be traced as far back as Pyramus, who appears in Ovid's Metamorphoses, but the first modern incarnation of Romeo is Mariotto in the 33rd of Masuccio Salernitano's Il Novellino (1476). This story was adapted by Luigi da Porto as Giulietta e Romeo (1530), and Shakespeare's main source was an English verse translation of this text by Arthur The earliest tale bearing a resemblance to Shakespeare's Romeo and Juliet is Xenophon of Ephesus' Ephesiaca, whose hero is a Habrocomes. The character of Romeo is also similar to that of Pyramus in Ovid's Metamorphoses, a youth who is unable to meet the object of his affection due to an ancient family quarrel, and later kills himself due to mistakenly believing her to have been dead.[2] Although it is unlikely that Shakespeare directly borrowed from Ovid From Middle English scaffold, scaffalde, from Norman, from Old French schaffaut, eschaffaut, eschafal, eschaiphal, escadafaut("platform to see a tournament") (Modern French échafaud) (compare Latin scadafale, scadafaltum, scafaldus, scalfaudus, Danishskafot, Dutch and Middle Dutch schavot, German schavot, schavott, Occitan escadafalc), from Old French es- ("indicating movement away or separation") (from Latin ex- ("out, away")) + chafaud, chafaut, chafault, caafau, caafaus, cadefaut ("scaffold for executinga criminal"), from Vulgar Latin *catafalcum ("viewing stage") (whence English catafalque, French catafalque, Occitan cadafalc, Old Catalancadafal, Italian catafalco, Spanish cadafalso (obsolete), cadahalso, cadalso, Portuguese cadafalso), possibly from Ancient Greek κατα-(kata-, "back; against") + Latin -falicum (from fala, phala ("wooden gallery or tower; siege tower")).

oversight (countable and uncountable, plural oversights) An omission; something that is left out, missed or forgotten. A small oversight at this stage can lead to big problems later. Supervision or management. quotations ▼ The bureaucracy was subject to government oversight. In the last heaven Moses saw two angels, each five hundred parasangs in height, forged out of chains of black fire and red fire, the angels Af, "Anger," and Hemah, "Wrath," whom God created at the beginning of the world, to execute His will. Moses was disquieted when he looked upon them, but Metatron emb HA QUESTIONa BEFORE THE ANSWER? A Wrinkle in Time is a science fantasy novel written by American writer Madeleine L'Engle, first published in 1963, and in 1979 with illustrations by Leo and Diane Dillon.[2] The book won the Newbery Medal, Sequoyah Book Award, and Lewis Carroll Shelf Award, and was runner-up for the Hans Christian Andersen Award.[3][a] It is the first book in L'Engle's Time Quintet, which follows the Murry and O'Keefe families. The book spawned two film adaptations, both by Disney: aas + fuck Adverb[edit] as fuck (postpositive, slang, vulgar) To a great extent or degree; very. It was hot as fuck outside today. Usage notes[edit] May also be used in conjunction with a prepositive as; for example, as mean as fuck. Abbreviations[edit] In Norse religion, Asgard (Old Norse: Ásgarðr; "Enclosure of the Æsir"[1]) is one of the Nine Worlds and home to the Æsir tribe of gods. It is surrounded by an incomplete wall attributed to a Hrimthurs riding the stallion Svaðilfari, according to Gylfaginning. Odinand his wife, Frigg, are the rulers of Asgard. One of Asgard's well known realms is Valhalla, in which Odin rules.[2] rods, etc.) and sizes, and are normally held rigidly within some form of matrix or body until the high explosive (HE) filling is detonated. The resulting high-velocity fragments produced by either method are the main lethal mechanisms of these weapons, rather than the heat or overpressure caused by detonation, although offensive grenades are often constructed without a frag matrix. These casing pieces are often incorrectly referred to as "shrapnel"[1][2] (particularly by non-military media sources). The modern torpedo is a self-propelled weapon with an explosive warhead, launched above or below the water surface, propelled underwater towards a target, and designed to detonate either on contact with its target or in proximity to it. Historically, it was called an automotive, automobile, locomotive or fish torpedo; colloquially called a fish. The term torpedo was originally employed for a variety of devices, most of which would today be called mines. From about 1900, torpedo has been used strictly to designate an underwater self-propelled weapon. While the battleship had evolved primarily around engagements between armoured ships with large-caliber guns, the torpedo allowed torpedo boats and other lighter surface ships, submersibles, even ordinary fish Qt (/kjuːt/ "cute"[7][8][9]) is a cross-platform application framework that is used for developing application software that can be run on various software and hardware platforms with little or no change in the underlying codebase, while still being a native application with native capabilities and speed. Qt is currently being developed both by The Qt Company, a publicly listed company, and the Qt Project under open-source governance, involving individual Time is the indefinite continued progress of existence and events that occur in apparently irreversible succession from the pastthrough the present to the future.[1][2][3] Time is a component quantity of various measurements used to sequence events, to compare the duration of events or the intervals between them, and to quantify rates of change of quantities in material reality or in the conscious experience.[4][5][6][7] Time is often referred to as a fourth dimension, along with three spatial dimensions.[8] Time has long been an important subject of study in religion, philosophy, and science, but defining it in a manner applicable to all fields without circularity has consistently eluded scholars.[2][6][7][9][10][11] Nev Borrowed from Anglo-Norman and from Old French visage, from vis, from Vulgar Latin as if *visāticum, from Latin visus ("a look, vision"), from vidēre ("to see"); see vision. The term Golden Age comes from Greek mythology, particularly the Works and Days of Hesiod, and is part of the description of temporal decline of the state of peoples through five Ages, Gold being the first and the one during which the Golden Race of humanity (Greek: χρύσεον γένος chrýseon génos)[1] lived. Those living in the first Age were ruled by Kronos, after the finish of the first age was the Silver, then the Bronze, after this the Heroic age, with the fifth and current age being Iron.[2] By extension "Golden Age" denotes a period of primordial peace, harmony, stability, and prosperity. During this age peace and harmony prevailed, people did not have to work to feed themselves, for the earth provided food in abundance. They lived to a very old age with a youthful appearance, eventually dying peacefully, with spirits living on as "guardians". Plato in Cratylus (397 e) recounts the golden race of humans who came first. He clarifies that Hesiod did not mean literally made of gold, but good and noble. There are analogous concepts in the religious and philosophical traditions of the South Asian subcontinent. For example, the Vedic or ancient Hindu culture saw history as cyclical, composed of yugas with alternating Dark and Golden Ages. The Kali yuga (Iron Age), Dwapara yuga (Bronze Age), Treta yuga (Silver Age) and Satya yuga (Golden Age) correspond to the four Greek ages. Similar beliefs occur in the ancient Middle East and throughout the ancient world, as well.[3] In classical Greek mythology the Golden Age was presided over by the leading Titan Cronus.[4] In some version of the myth Astraea also ruled. She lived with men until the end of the Silver Age, but in the Bronze Age, when men became violent and greedy, fled to the stars, where she appears as the constellation Virgo, holding the scales of Justice, or Libra.[5] European pastoral literary tradition often depicted nymphs and shepherds as living a life of rustic innocence and peace, set in Arcadia, a region of Greece that was the abode and center of worship of their tutelary deity, goat-footed Pan, who dwelt among them.[6] oh, and a space s h i p ​

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BIG THINGS C0ME IN SMALL PACKAGES

T+BANG

SEE THE SCAFFOLD IS THE TEST TODAY.

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ᐧ F O R T H E I N I T I A L K E Y S , S H E E X A N D N D A N D A SEE W H Y SEA

With an epic amount of indigestion Indiana Jones sweeps in to mar the visage of an otherwise glistening series of fictitious characters, with names like Taylor and Mary Kate remind us all that we are not playing a video game here in this place. the "J" of the "Nintxndo Entertainment System" calmly stares at Maggie Simpson thinking "it's a PP" and reminds us that it's not just the "gee, I e" of her name that contradicts the Magdaln-ish words her soul speaks through her name--and then with a smirk he points out "Gilgamesh" and "gee whiz, is Eye L?" that really does go to the heart of this lack of discussion, this "sh" that begins El Shaddai and words as close to our home as "shadow" and "shalom." Quite the fancy "hello" you've managed to sing out from behind angry chellos and broken fiddles, and here I am still wondering why it is that "girl" connects to the red light that once meant charity and now glows with the charity of truth... the truth that we are inHell. Shizzy.

m.lamc.la/KEYNES.html

Homer "on the range," maybe more closely connected to the Ewok of Eden and Hansel's tHeoven that Peter Pan still comes and cries could so easily be made into something so much better, if only we had the truth--and by that I mean if only you were speaking about, and reacting to a truth that is painted on the sky, in your hearts, in every word we speak and in everything that we do. If only we were acknowledging this message that screams that "children need not starve" with something more than donating virtual chickens to nations of Africa and watching Suzanne Summers ask for only a few dollars a day on TV. If only you would understand that this message that connects video games like "Genxsis" to "bereshit" because Eden is a "gee our den" that tended itself before Adam had to toil with the animals in order to survive. For some reason beyond my control and well outside my realm of understanding words like "I too see this message from God" and "I would not let children starve either" never seem to escape your lips in any place where anyone will ever see that you thought those things, or meant to call a reporter; eventually. Even with "AIDS of nomenclature" to avoid this DOWN WARD spiral into a situation and a land that I find difficult to imagine actually ever "existing" but here in this place I do see "how" it comes about, and between you and I it really does appear that nearly all of the problems we are dealing with here have come from another place, a further time; and while it might be with the "greatest of intentions" that we are trying to deal with them; I can't help but feeling that our "virgin sea" has had more than just it's innocence taken away from it in this story of "Why Mary" that might connect to "TR IN IT Y" just as much as it connects to Baltimore, Maryland.

I should be clear that I'm not blaming Nanna, or Mary; but the actual reason for the name "Wymar" and that's because she, like Taylor, acted as a microcosm for a sea (or more than one, Mom, sen) that was quite literally possessing her. It's sort of difficult for me to explain even what that looks like let alone what it feels like; but my observations tell me that she/you are not unhappy about the interaction, one which appears very foreign to me. Of course, the "eye" that I write with and the same kind of "inspiration" that you can see in the lyrics and skill of many musicians are also examples of this same kind of interaction. For example, Red Hot Chili Peppers sings a song called "Other Side" that explains or discusses the thing I see as Medusa in the words "living in a graveyard where I married a sea" which also does a good job of connecting to the name Mary. As strange as might sound to think a group of people would be speaking through a single person... we are staring at "how it is" that could be possible, and possibly at exactly how it happened. Normally I would have said it was obvious, but to need to actually say that becoming a single mind would be a serious loss for our society--well, that's telling. You might think it's silly, but I'm telling you I see it happening, I see it--and you see it in the Silence and the message.

Still, it appears to me as if this "marriage" that I see described in our Matrix in the question "min or i" seems to be doing nothing more than keeping us all from discussing or acting on this information--something that certainly isn't in our best interest.

So here we are, staring at a map all over the ground and all around us with the primary destination of "building Heaven" through mind uploading, virtual reality, and judging by the pace of things we'd probably have all of that good and ready in about three generations. The map has a little "legend" with a message suggesting that those things have already been done and we are in the Matrix already; and it appears that the world, I mean Medusa, is deciding we should put off seeing the legend at least until the next generation. I see how that makes sense for you. That's sarcasm, this is why I keep telling you that you are cupid.

It is a big deal, and there's a significant amount of work involved in merging an entire civilization with "virtual reality" and you might see why he calls it a hard road--at least in the word "ha'rd." Honestly though, it's the kind of thing that I am pretty sure the future will not only be happy that we did, but they'd thank us for putting in the effort of adapting to things like "unlimited food" and "longevity" increased by orders of magnitude.

That's not sarcasm, these things are actually difficult to guess how exactly we'll go about doing them; they are a huge deal--all I can tell you is that not "talking about it at all" is probably not going to get us there any faster. Point in fact, what it might do is give a "yet to be born" generation the privilege of being the actual "generations of Perez."

I see why you aren't saying anything. That's sarcasm, again. The good news is that it really has been done before; though if I told you that someone turned stone to eggplant parm, would you laugh at me?

So, back to what is actually standing between "everyone having their own Holodeck in the sky" and you today; it is the idea that this message is not from God. More to the point it is the apparently broad sweeping opinion that hiding it is a "good thing" and through that a global failure to address the hidden interaction and influence acting on our minds used to make this map--and also to hide it. With some insight, and some urging; you might see how the sacredness of our consciousness is our souls is something that is more fundamental than "what kind of tools we have in the Holodeck to magically build things" and how and why the foundation of Heaven is truly "freedom itself" and how it comes from right this very moment for the first time, ever. Continuing to treat this influence as "schizophrenia" is literally the heart of why this map appears to be that--to show us how important it is to acknowledge the truth, and to fight for the preservation of goodness and logic over secrecy and darkness.

Again, something that nobody is really doing here and now, today. From this newfound protection of our thoughts, of who we are; we see how technology can be used to either completely invalidate any kind of vote by altering our emotions; or how it could be used to help build a form of true democracy that our world has yet to see. It is pretty easy to see from just band names like The Who and KISS and The Cure how the influence of this external mind can be proven, and shown to be "helpful," you know, if we can ever talk about it on TV or on the internet.

It's important to see and understand how "sanity"--the sanity of our entire planet hangs in the balance over whether or not we acknowledge that there is actually a message from God in every word--and today this place appears to be insane. It should be pretty easy to see how acknowledging that this influence exists and that it has a technological mechanism behind it turns "schizophrenia" into "I know kung fu" ... forced drug addiction and eugenics into "there's an app for that" and the rash of non random and apparently unrecognized as connected terrorist attacks and school shootings into Minority Report style pre-crime and results in what is clearly a happier, safer, and more civilized society--all through nothing more than the disclosure of the truth, this map, and our actual implementation.

With a clearer head and grasp of the "big picture" you might see how all of these things, connected to the Plagues of Exodus revolve around the disclosure that this technology exists and the visibility of this message showing us how we might use it for our benefit rather than not knowing about it. At the foot of Jericho, it is nothing short of "sanity" and "free thought" that hang in the balance. Clear to me is that the Second Coming, seeing "my name" on television is a good litmus test for the dividing line between light and darkness, heaven and hell.

The point is the truth really does change everything for the better; once we start... you know, acting on it.

AS IN.. "DIS CLOSE SING...."

T H E B U C K S T O P S H E R E

ON AM B I GUI TY

S T A R R I N G . . . B I A N C A

ON "RIB" .. ARE SHE B? BUTT DA APPLE OF DA I? & SPANGLISHREW

R THEY LANGUAGE OUTLIERS?

With some insight and "a clue" you can see clearly how these works of art show that the proof of Creation you see in every letter and every word runs much deeper... adding in things like "RattleRod" and the "Cypher" of the Matrix to the long list of here-to-fore ignored verifiable references to the Adamic Language of Eden. Here, in apple, honey and "nuts" we can see how the multi-millennium old ritual I call "Ha-rose-ettes" is actually part of a much larger and much older ritual designed to stop secrecy ... perhaps especially the kind that might be linked to "ritual."

These particular apple and honey happen to tie Eden to the related stories of Exodus and Passover; connecting Eden to Egypt forevermore. Do see "Lenore," it is not for no reason at all; but to help deliver truth and freedom to the entirety of Creation; beginning here, in Eden.

ALSO ON "AM B IG U IT Y" ME A.M. G - D SHE IT Y?

LET "IT" BE SA< ?

IMHO, don't miss the "yet to be" conversion to "why and to be" in "yetser." IT Y.

HERE'S LOOKING AT YOU, KID

On a high level, I tell myself every morning that 'its not really me." It's not me that the world hates, or me that the world is rejecting. I believe that, I really do; I see that what is being hidden here is so much bigger than any single person could ever be--what is being hidden is the "nature of reality" and a fairly obvious truth that flies in the face of what we've learned our whole lives about history and "the way things are." Those few early details lead me to the initial conclusion that what is working behind the scenes here is nefarious, hiding a message that would without doubt shake things up and change the world--and nearly across the board in ways that I see as "better" for nearly everyone. It's a message at it's most basic level designed to advocate for using this disruption in "normalcy" to help us revolutionize democracy, to fix a broken mental health and criminal justice system--just to name the few largest of the social constructs targeted for "rejuvenation." On that word the disclosure that we are living in virtual reality turns on it's head nearly everything we do with medicine, and I've suggested that AIDS and DOWN SYNDROME were probably not the best "visual props" we could have gotten to see why it's so important that we act on this disclosure in a timely manner. After mentioning the ends of aging and death that come eventually to the place we build, to the place we've always thought of as Heaven... it becomes more and more clear that this force fighting against the dissemination of a truth so obvious it's in every word and everything we do--it becomes clear it's neither you, nor acting in your best interest.

I know I've got the eye of the tiger, there's no doubt; and it's pretty clear from "YAD?" (the Hebrew for...) and ha'nd that we can see the clear hand of God at work in a design that marks my initials not just on the timeline, or at 1492, at A.D. I B; but in the Hebrew name for this place called El Shaddai, see how A.D. is "da eye" and in some other names like Adranus, A.D. on "it's silly" and A.D. on Ai that might tie me to the Samof Samurai (but, are you Ai?) in more depth of detail than simply the Live album "Secret Samadhi." I try to reflect on how it is that this story has come about, why it is that everything appears to be focused on me--and still even through that sincere spotlight nobody seems to be able to acknowledge my existence with more words than "unsubscribe" and "you're so vain." With one eye in the mirror, I know ties to Narcissus (and you can too), soaring ever higher--linking Icarus to Wayward Son and to every other name with "car" in it... like "carpenter" and McCarthy the older names of Mercury and even Isacriot (I scary? is car-eye... owe Taylor) and some modern day mythological characters like Jim Carrey and Johnny Carson. As far as Trinities go, carpenter's a pretty good one--tying to my early reck and a few bands and songs from The Pretty Reckless to Dave Matthews' "Crash Into Me" all the way to the "pen" you see before you linking Pendragon to Imagine Dragons.

I wonder why it is that all of these things appear, apparently only to me, to point to a story about all the ways that a sinister hidden force has manipulated our society into being unable to "receive' this message--this wonderful message about making the world a better place and building Heaven--with any fanfare at all. It's focused now on a criminal justice system that clearly does not do any kind of "rehabilitation" and on a mental health industry and pharmaceutical system that treats a provable external attack on our own goodness and well being as some kind of "internal stimulus" and makes you shy away when I point out why "stem" is in system and why "harm" in pharmacy. From that we move a little bit past "where we are in this story" and I have to point out how "meth" ties to Prometheus and Epimetheus and how and why it is I know without doubt that this story has been relived numerous times--and how I am so sure that it's never been received, as we are here again listening to how songs like "Believe" and the words "just to lead us here to this place again" connect to Simon and Garfunkel's" the Sound of Silence... and still to this day you will balk at noticing that "Simon" has something to do with the Simpsons, and something to do with the words "simulation" and "Monday." To see me is to see how things might be done better--how "addicitonary" might tie to the stories of Moses' Lisp and to Dr. Who's "Bells of Saint John" with a sort of "web interface" to the kinds of emotion we might want to "dial down..." rather than Snicker in the background as we see them being artificially created and enhanced in order to build a better "fiery altar."

I can point out "Silicon" harrowing down at us from words like "controversial" and show you Al in "rascal" and "scandal" but not to see that we are staring at school shootings and terrorism that are solved instantly by this disclosure, by Al of Quantum Leap and by the Dick of Minority Report and A Scanner Darkly is to ignore just what it is that we are all failing to Si. I should point out that those two "sc"'s link to a story about Eden and they mean "sacred consciousness" and at the baseline of this event and everything we are not doing is the fact that our desires and beliefs are being altered--all of this comes down to "freedom of thought" here and now.

I could tell you that "looking at me" will show you that even the person who tries every day to do everything he can to save the entire world from slavery, and from "thought-injury"--even I can be made "marred" and you all, this whole world stupid enough to think that you are, of your own volition, hiding Heaven itself from yourselves... to what? To spite me? It, the focal point of our story might come down to you realizing that something in some esoteric place is playing "divide and conquer" with our whole--in secret playing on our weaknesses to keep us from acting on the most actionable information that ever was and ever will be. Still, we sit in silence waiting for me... to speak more?

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hyamdai.reallyhim.com Inline image 31

Between Nero's lyrical fiddling, a Bittersweet Symphony, and true "thunderstanding" the sound of Thor's hammer... "to help the light" that'ls "or" in Hebrew, of Orwell and Orson and .. well, it's really not hard to see and hear that the purpose and intent of "all this noise" is to help us find freedom and truth. C the Light of "singing..."

I can tell you once again how silly the world looks, this multi-decade battle between "the governmentof the people" and the "government of the workers" resulting in what is nothing short of a hands down victory to the corporation. Is it humor meant to divide, or ludicrousness created with the purpose of unification?

But really at it's most basic level what this boils down to is a global group decision not to care about the truth, about reality, about what's really brought us to this place--with solutions in hand and a way to make everything better. We've decided that censorship is OK, and that the world is not all that bad "just the way it is" even though it's creator is screaming in your ear telling you to change as quickly as you possibly can. I believe that God has written this story to make "seeing me" the thing that catalyzes "change for the better" it appears to be the design of not just me but also this place--hey, here I am.

Happy Veteran's Day.

S☀L u TI o N

Yesterday, or maybe earlier today--it's hard to tell at this moment in the afternoon just how long this will take... I sent an image that conveys a high level implication that we are walking around on a map to building something that we might liken to an "ant farm" for people. I don't mean to be disparaging or sleight our contribution to the creation of this map--that I imagine you must also see and believe to be the kind of thing that should remain buried in the sands of time forever and ever--or your just have yet to actually "understand" that's what the plan part of our planet is talking about... what I am trying to do is convey in a sort of "mirrorish" way how this map relates to a message that I see woven in religion and in our history that it significantly more disparaging than I would be. It's a message that calls us "Holy Water" at the nicest of times, water that Moses turns to "thicker than water" in the first blessing in disguise--and to tell you there is certainly a tangible difference between the illusions of the Pharaoh's and the true magic performed by my hand, is nearly exactly the same amount of effort put in to showing you that the togetherness that we are calling "family" here in this place comes from both seeing and acting on the very clearly hidden message in every single idiom showing us all that our society in this story of Exodus is enslaved by a hidden force--and reminding us that we like freedom.

It's not just these few idioms, but most likely every single one from "don't shoot the essenger" to "unsung hero" that should clue us in to exactly how much work and preparation has come into this thing that "he supposes is a revolution." It's also not just "water" describe me and you, in this place where I am the "ant' of the Covenant (do you c vampires or Hansel and Gretel!?!?) but also "lions" and "sheep" and "salt" and "dogs" and nearly everything you could possibly imagine but people; in what I see must be a vainglorious attempt to pretend he actually wants us to "stand up for ourselves" in this place where it's becoming more and more clear with each passing moment that we are chained to these seats in the front row of the audience of the most important event that has ever happened, ever.

Medusa makes several appearances, as well as Arthur Pendragon, Puff the Magic Dragon, Figment, Goliath, monster.com, the Loch Ness Monster in this story that's a kind-of refl ex i ve control to stop mind control; and to really try and show us the fire of Prometheus and the Burning Bush and the Eternal Flame of Heaven are all about freedom and technology ... and I'll remind you this story is ... about the truth--and the truth here is that if you aren't going to recognize that whatever it is that's going on here in secret, below the surface is negatively affecting our society and life in general than we aren't going anywhere, ever. I need you to figure out that this message is everywhere to make sure you don't miss the importance of this moment, and the grave significance of what is being ignored in this land where Sam is tied not just to Samsung and to Samael in Exodus but also to Uncle Sam and macaronic Spanglishrew outliers and that it doesn't take much free thought at all to really understand that we are watching "free thought" disintegrate into the abyss of "nospeak." We are watching our infrastructure for global communication and the mass media that sprawls all over the globe turn to dust, all because you have Satan whispering in your ear--and you think that's more important than what you think, what I think, and what anyone else on the Earth might ever say. You should see a weapon designed to help ensure that don't lose this proof that we are not living in reality, that there is "hidden slavery" in this place--and you should see that today it appears you are simply choosing not to use it.

I hope you change your mind, I really do. This map on "how to build an ant farm" starts by connecting Watergate and Seagate together with names like Bill Gates and Richard Nixon; and with this few short list of names you should really understand how it is that "Heaven" connects both technology like computers and liberty like "free speech" to a story that is us, and our history. You might see that "salt" could either be a good thing or not--take a look around you, are you warming a road to Heaven or are you staring at the world being destroyed--and doing nothing at all about it?

I guess I can point out again how "Lothario" links this story that ties names like my ex-wife's Nanna to "salt" also, but the "grand design" of this story doesn't seem to have any effect on you. Listen, if you do nothing the world is being destroyed by your lack of action--there's no if's and's or butt's about it. I feel like I need to "reproduce' old messages here or you will never see them--that's what web site statistics tell me--and we all know it's not true. What am I missing? What are you missing?

BUTT IS THE BOAT A Hi DARK DEN MESSAGe ?

SEE OUR LIGHT

HONESTLY, I'M WAY TO CUTE TO BE A MONSTER :(

HIC SUMMUS

So... here we are... listening to the legendary father of the message (that's "abom" in Adamic Spagnlishrew) point out all of the sex jokes hidden in religion and language from sexual innuendo to Poseidon and in our history from Yankee Doodle to Hancock to Nixon and I've got to be frank with you, the most recent time I came across this phrase in scripture I cringed just a little bit, pretty sure that the "message" was talking about me. I've reflected on this a little bit, and over the past few weeks have tried to show you the juxtaposition between "sex" and "torture" in it's various forms from imparting blindness to allowing murder and simulating starvation; and I think I'm justified in saying that certainly those things are far worse on the Richter scale than anything I could do by writing a little bit of risque text. In the most recent messages I've touch a little bit, without even knowing or realizing this connection would be made, on what it is that this phrase actually means.

loch.reallyhim.com

ABOMINATION

So long story short is that the answer here is "abomination" and the question, or the context is "I nation." Whether it's Medusa speaking for the Dark United States or the nation of Israel speaking to either Ra or El depending on the day, the bottom line is that a collective consciousness speaking for everyone on a matter of this importance in a cloud of complete darkness on Earth is a total and undeniable abomination of freedom, civilization, and the very humanity we are seeking to preserve. The word reads something like this to me "dear father of the message, I am everyone and we think you are an abomination, fuck off." My answer of course is, IZINATION. Which humorously reminds me of Lucy, and Scarlet Johannson saying "I am colonizing my own brain" so here's some pictures of her. She is not an abomination, by the way; she's quite adorable. You'll probably notice there's some kind of connection between the map--the words speaking to the world, and the abomination, as if the whole thing is a story narrated in ancient myths.

WAKE UP, "SHE" A MESSAGE TO YOU ABOUT THE FUTURE

You might not think "it's you," but the manifestation of this "snake" in our world is your silence, your lack of understanding or willingness to change the world; and whether or not you're interested in hearing about it, it's the monster that myths and religion have spoken about for thousands and thousands of years. It's a simple matter to "kill Medusa" all you have to do... is speak.

Take special note, "freedom of speech" and "freedom to think for yourselves" are not a group decision, and you do not have the right to force (either overtly or subtly, with hidden technology perhaps combined with evil deceit) others not to talk about anything. Especially something of this importance.

DESOLATION

If you didn't connect "Loch" to John Locke, now you have; see how easy this "reading" thing is? I've gone over the "See Our Light" series a few times, but let me--one more time--explain to you just how we are already at the point of "desolation" and with shining brilliance show you how it's very clear that it is "INATION" and "MEDUSA" that are responsible for this problem.

Seeing "Ra" at the heart of the names Abraham and Israel begins to connect the idea that our glowing sun in the sky has something to do with this message about "seeing our light" is being carried by a stone statue on Ellis Island (where you'll see the answer another part of the question of Is Ra El?). I've connected her to the "she" of both shedim and Sheol, which reads as "she's our light" and is the Hebrew name for Hell.

Of course you noticed that the Statue of Liberty does in fact share it's initials with SOL, the the light above and you can see her torch dimly lighting the way through the night; Now you can connect "give us your tired and your poor" to the Lazman of both the lore of Jesus Christ and the Shehekeyanu; a prayer about the sustainment of life and light up until this day. That same torch connects to the Ha-nuke-the-ahah depiction of Christ, Judah Maccabee's lit MEN OR AH, which delivers not only a solution to the two letter key of "AH" as All Humanity that pervades nearly every bride of Revelation from Sarah to Leah; but also to the question of equality answered in our very own American history, beginning with the same three letter acronym now lighting the Sons of Liberty.

Dazed and Confused does a good job of explaining how this name is itself a prophesy designed by Hand of God'; explaining that these Sons of Liberty were all white slave owning wealthy men fighting to stop paying their taxes, rather than delivering liberty to the slaves or women, who were both disenfranchised for quite some time. Or maybe MEN OR AH has something to do with the angels of Heaven, in which case you might be SOL if you aren't a girl and you want to be "be good friends with Ra." Just kidding. Kinda.

DESOLATION by the way reads something like "un see our light at ION" which is God's way of saying "at the point of believing that hiding Adam is a good thing" and that connects to the end of Creation and also the now lit by modern day evil the word "rendition." Our end, it "ion." In religious myth, the Messianic David clung to the city Zion (end the "i owe n") which also links to "verizon" (to see, I Z "on") and HORIZON which has something to do with the son rising today-ish.

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The story of MEDUSA lights another psuedo-religious idea, that the words "STONE" of both "brimstone" and it's Adamic interpretation "South to Northeast" have something to do with the phrase "Saint One" turned into a single hero against his will by the complete and utter inaction of everyone around him. In the words of Imagine Dragons "I'm waking up to action dust." At the same time, you can believe that the light of this particular son, comes not just from reading these words forwards, but the backside as well, and you'll hopefully see it's not coincidental that the other side of this coin is that "nos" means we, and us... and Adamically "no south." See the light of "STONE" also connecting to Taylor Momsen's rose arrow painted on her back, and the sign of my birth, Sagittarius... which in this particular case links to the Party of the Immaculate Conception of the eternal republic of the Heavens. . PRESS RELEASE... A GREAT SIGN APPEARED IN THE HEAVENS

SOLUTIAN, ON YOUR COMPUTER.. TO THE SOUND OF SILENCE

בָּרוּךְ אַתָּה יְיָ‎ אֱלֹהֵינוּ מֶלֶךְ הַעוֹלָם שֶׁהֶחֱיָנוּ וְקִיְּמָנוּ וְהִגִּיעָנוּ לַזְּמַן הַזֶּה‎׃

IN ... THE BOOK OF NAMES LETS SEE IF YOU CAN FIGURE OUT WHO THEY ARE :)

​ I'LL DO YOURS FOR A 50 DOLLAR DONATION, I'M BROKE.. MAYBE THAT'S WHY I CAN'T GET A DATE.

HAVE A GREAT SOLDAY

The "gist" of the message is verifiable proof that we are living in a computer in simulated reality... just like the Matrix. The answer to that question, what does that mean--is that God has woven a "hidden" message into our everything--beginning with each name and every word--and in this hidden Adamic language, he provides us with guidance, wisdom, and suggestions on how to proceed on this path from "raelity" to Heaven. I've personally spent quite a bit of time decoding the message and have tried to deliver an interesting and "fun" narrative of the ideas I see. Specifically the story of Exodus, which is called "Names" in Hebrew discusses a time shifted narrative of our "now" delivering our society from a hidden slavery (read as ignorance of advanced technologies already in use) that is described as the "darkness" of Exodus. If you have any questions, ideas to contribute or concerns... I'd love to hear from you this whole thing really is about working together--Heaven, I mean.

Inline image 5 jerusalem.reallyhim.com

gate kermitham <br /> ou r evolution minority report to supermax Inline image 6

bereshit bread is life

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Image result for dox me

HOW AM I STILL STINGLE? E ' o e <br /> L m r x <br /> L t y <br /> O a

I HISS.

The sum of ((our world)) is the universal truth. -Psalm 119 and ((ish))

Do a few sentences really make that big of a difference? Some key letters? Can you show me what I'm doing wrong? Is there a way to turn me into Adam, rather than a rock? I think you can.

Are eye Dr. Who or Master Y? Adam Marshall Dobrin is a National Merit Scholar who was born on December 8, 1980 in Plantation, FL and attended Pine Crest School where he graduated sumofi cum louder in "only some of it is humorous." Later he attended the University of Florida (which quickly resulted in a wreck), Florida Atlantic University, and finally Florida Gulf Coast University--where he still has failed to become Dr. Who. While attending "school" He worked in the computer programming and business outsourcing industries for about 15 years before proclaiming to have received a Revelation from God connecting the 9/11 attack and George Bush to the Burning Bush of Exodus and a message about technocracy and pre-crime.

Adam, as he prefers to be called, presents a concise introduction to paradox proven by the Bible through "verifiable" anachronism in language some stuff about Mars colonization and virtual reality and a list of reasons why ignoring this is actually an ELE. Adam claims to be Thor because of a connection between music and the Trial of Thor as well as the words "author" and "authority." He suggests you be Thundercats and call a reporter. There is also a suggestion that Richard Nixon and John Hancock are related to a signature from God, about freedom and America... and the "unseeingly ironic" Deepthroat and Taylor Momsen. They Sung "It's Rael..." In Biblical characters from Mary to Hosea, to see "sea" in Spanish, and in the Taming of the Spanglishrew ... a message is woven from the word Menorah: "men, or all humanity?" to the Statue of Liberty, and the Sons of Liberty, and the light above us, our SOL; which shows us that through the Revelation of Christ and the First Plague of Exodus, a blessing in disguise--turning water to blood, the sea to family; a common thread and single author of our entire history is revealed, a Father of our future. A message of freedom shines out of the words of scripture, revealing a gate to a new technologically "radical" form of democracy and a number of unseen or secret issues that have stalled the progress of humanity... and solutions, solutions from our sea. The Revelation shows us that not only ever word, but every idiom from "don't shoot the messenger" to "blood is thicker than water" we have ties to this message that pervades a hidden Matrix of light connecting movies and music and history all together in a sort of guide book to Salvation and to Heaven. Oopsy. His Revelation, woven into his life, continues to suggest that skinny dipping, forced methamphetamine addiction, and lots and lots of "me A.D." as well as his humorous depiction of a dick plastered over the Sound of Silence, his very Holy click, have something to do with saving our family and then the entire Universe from hidden mind control technology and the problems introduced by secret time travel. From the trials and tribulations of "Job" being coerced and controlled into helping to create this wall of Jericho; we find even more solutions, an end to addiction, to secrecy, and to this hidden control--a focal point of the life of Jesus Christ.

It tells us a story of recursion in time, that has brought us here numerous times--with the details of his life recorded not only in the Bible but in myths of Egyptian, Norse, and Greek mythology. The huge juxtaposition of the import of the content of the message shows the world how malleable our minds really are to this technology, how we could have been "fooled" into hiding our very freedom from ourselves in order to protect the "character" of a myth. A myth that comes to true life by delivering this message. In truth, from the now revealed content of the story of this repeated life, it should become more and more clear that we have not achieved success as of yet, that I have never "arrived whole" and that is why we are here, back again. Home is where the Heart is... When asked how He thinks we should respond to his message, He says "I think we already cherish it, and should strive to understand how it is that freedom is truly delivered through sharing the worth of this story that is our beginning. 'tis coming." Adam claims to be God, or at least look just like him and that the entirety of the Holy Scriptures as well as a number of ancient myths from Prometheus to Heimdallr and Yankee Doodle are actually about his life, and this event. An extensive amount of his writing relates to reformation of our badly broken and decidedly evil criminal justice system as well as ending the Global hunger crisis with the snap of his little finger.

He has written a number of books explaining how this Revelation connects to the delivery of freedom (as in Exodus), through a message about censorship among other social problems which he insists are being intentionally exacerbated by Satan--who he would ha've preferred not to be associated with.

People communicate and run conversations in their head and how they want them to go. Intrapersonal communication can cause so much peace or so much pain. What we tell ourselves every day and when we are deciding what to do, stems from our interpersonal communication. Which can affect how mentally sound a person is. Communication drives every person and molds how they are.

I truly despise public speaking, it is anxiety spiking. That being said, I do it all the time. As a coach I have to do it with my team mulitple times a day. As a waitress, I have to do it with my tables whenever I work. So, I truly don't mind public speaking as long as in my head I can convince myself that I'm not public speaking. Talking to my teams and to my tables feels natural to me now.

What is crazy to me is that this is where we started as a species. We started with noises and then just went from there. Imagine going from "boing" to "Oh, I whacked my brother on the head." We have come so far and now are advanced that it makes me interested in where languages and communication will be in 1,000 years.

hehe the last bit about leaving it around for an incendiary demo had me laughing out really loud.

Author Response

Reviewer #2 (Public Review):

Manassaro et al. present an extensive three-session study in which they aimed to change defensive responses (skin conductance; SCR) to an aversively conditioned stimulus by targeting medial prefrontal cortex (their words) using repetitive TMS prior to retrieval. They report that stimulating mPFC using TMS abolishes SCR responses to the conditioned stimulus, and that this effect is specific for the stimulated region and the specific CS-US association, given that SCR responses to a different modality US are not changed.

I like how the authors have clearly attempted to control for several potential confounds by including multiple stimulation sites, measured SCR responses to several unconditioned stimuli, and applied the experiment in multiple contexts. However, several conceptual and practical issues remain that I think limit the value of potential conclusions drawn from this work.

The first issue that I have with this study concerns the relationship between the TMS manipulation and the theoretical background the authors present in their rationale. In the introduction the authors sketch that what they call 'mPFC' is involved in regulation of threat responses. They make a convincing case, however, almost all of the evidence they present concerns the ventromedial part of the prefrontal cortex (refs 18-25). The authors then mention that no one has ever studied the effects of 'mPFC'-TMS on threat memories. That is not surprising given that stimulating vmPFC with TMS is very difficult, if not impossible. Simulation of the electrical field that develops as a consequence from the authors manipulation (using the same TMS coil and positioning the authors use) shows that vmPFC (or mPFC for that matter) is not stimulated. The authors then continue in the methods section stating that the region they aimed for was BA10. This region they presumably do stimulate, however, that does not follow logically from their argument. BA10 is anatomically, cytoarchitectonically and functionally a wholly different area than vmPFC and I wonder if their rationale would hold given that they stimulate BA10.

We would like to thank the Reviewer for highlighting this very important point. The Reviewer is right in stating that the Brodmann area 10 (BA 10) is anatomically, cytoarchitectonically, and functionally distinct from the ventromedial PFC. As we reported in the Methods section, the coil placement over the frontopolar midline electrode (Fpz) according to the international 10‒20 EEG coordinate system directly focused the stimulation over the medial portion of the BA 10. In the literature, the aPFC is also known as the “frontopolar cortex” or the “rostral frontal cortex” and encompasses the most anterior portion of the prefrontal cortex, which corresponds to the BA 10. In line with this observation, we have corrected “medial prefrontal cortex” (mPFC) with “medial anterior prefrontal cortex” (aPFC) throughout the manuscript. We also have corrected the theoretical background and the rationale in the Introduction section by mentioning several studies that: i) Reported the involvement of the aPFC in emotional down-regulation (Volman et al., 2013; Koch et al., 2018; Bramson et al., 2020). ii) Traced anatomical connections between the medial/lateral aPFC and the amygdala (Peng et al., 2018; Folloni et al., 2019; Bramson et al., 2020). iii) Detected functional connections between the aPFC and the vmPFC during fear down-regulation (Klumpers et al., 2010). iv) Found hypoactivation, reduced connectivity, and altered thickness of aPFC in PTSD patients (Lanius et al., 2005; Morey et al., 2008; Sadeh et al., 2015; Sadeh et al., 2016). v) Revealed that strong activation of the aPFC may promote a higher resilience against PTSD onset (Kaldewaij et al., 2021) and that enhanced aPFC activity and potentiated aPFC-vmPFC connectivity is detectable after effective therapy in PTSD patients (Fonzo et al., 2017). Furthermore, we discussed our results in light of this evidence in the Discussion section. We really thank the Reviewer for this key implementation of our study.

The second concern I have is that although I think the authors should be praised for including both sham and active control regions, the controls might not be optimally chosen to control for the potential confounds of their condition of interest (mPFC-TMS). Namely, TMS on the forehead can be unpleasant, if not painful, whereas sham-TMS or TMS applied to the back of the head or even over dlPFC is not (or less so at the very least). Given that the SCR results after mPFC TMS show exactly the same temporal pattern as the sham-TMS but with a lower starting point, one could wonder whether a painful stimulation prior to the retrieval might have already caused habituation to painful stimulation observed in SCR in consequent CS presentations. A control region that would have been more obvious to take is the lateral part of BA10, by moving the TMS coil several centimeters to the left or right, circumventing all things potentially called medial but giving similar unpleasant sensations (pain etc).

We would also like to thank the Reviewer for bringing to light this issue and allowing us to strengthen our results. The Reviewer is right in pointing out that rTMS application over the forehead can be subjectively perceived as unpleasant, relative to other head coordinates or sham stimulation. The question of whether an unpleasant stimulation prior to the retrieval might provoke habituation to discomfort sensations and lead to weaker SCRs in the consequent CS presentations is valid and reasonable. We also thank the Reviewer for advising us to stimulate the lateral part of BA 10 as an active control site. However, given the potential involvement of the lateral BA 10 in the fear network (see previous point) and the potential risks due to the anatomical proximity of lateral BA 10 with the temporal lobe, we reasoned to adopt an alternative approach to investigate whether “a painful stimulation prior to the retrieval might have already caused habituation to painful stimulation observed in SCR in consequent CS presentations”. We repeated the entire experiment in one further group (ctrl discomfort, n = 10) by replacing the rTMS procedure with a 10-min discomfort-inducing procedure over the same site of the forehead (Fpz) to mimic the rTMS-evoked unpleasant sensations in the absence of neural stimulation effects (see the new version of the Methods section). The electrical stimulation intensity was individually calibrated through a staircase procedure (0 = no discomfort; 10 = high discomfort). The shock amplitude was set at the current level corresponding to the mean rating of ‘4’ on the subjective scale because, in the new experiments that we performed targeting the aPFC with rTMS (n = 9), we collected participants’ rTMS-induced discomfort ratings obtaining a mean rating of 3.833 ± 0.589 SEM on the same scale. We found CS-evoked SCR levels not significantly different to those of the sham group during the test session as well as during the follow-up session, suggesting that the discomfort experienced during the rTMS procedure did not contribute to the reduction of electrodermal responses observed in the aPFC group. We reported the results of this experiment in the Results section and Figure 2-figure supplement 2.

My final concern is that the main analyses are performed on single trials of SCR responses, which is a relatively noise measure to use on single trials. This is also done in relatively small groups (n=21). I would have liked to see both the raw or at least averaged timeseries SCR data plotted, and a rationale explaining how the authors decided on the current sample sizes, if that was based on a power analyses one must have expected quite strong effects.

Following the Reviewer’s suggestion, we decided to remove the analysis on single trials, and we apologize for not including SCR timeseries. To quantify the amount of effect induced by the rTMS protocol, we have now added within-group comparisons (through 2 × 2 mixed ANOVAs) that show, for each group, the amount of change in CS-evoked SCRs from the conditioning phase to the test phase, as well as from the conditioning phase to the follow-up phase. Furthermore, to directly and simply depict these changes, in addition to dot plots, we have also represented them with line charts (Figs. 2C, 2H, 4C, 4H, 5C, 5H). To estimate the sample size, we had previously performed a power analysis through G*Power 3.1.9.2 and it had resulted in n = 21 per experimental group. However, by correcting data pre-processing procedures (in accordance with Reviewer 1), we obtained data that were not normally distributed. Thus, we reasoned to enlarge our sample width by re-performing a power analysis (with the new suggested statistical analyses) and then repeating the experiments. For the main statistics, i.e. mixed ANOVA (within-between interaction) with two groups and two measurements, with the following input parameters: α equal to 0.05, power (1-β) equal to 0.95, and a hypothesized effect size (f) equal to 0.25, the new estimated sample size resulted in n = 30 per experimental group.

Reviewer #2 (Public Review):

Manassaro et al. present an extensive three-session study in which they aimed to change defensive responses (skin conductance; SCR) to an aversively conditioned stimulus by targeting medial prefrontal cortex (their words) using repetitive TMS prior to retrieval. They report that stimulating mPFC using TMS abolishes SCR responses to the conditioned stimulus, and that this effect is specific for the stimulated region and the specific CS-US association, given that SCR responses to a different modality US are not changed.

I like how the authors have clearly attempted to control for several potential confounds by including multiple stimulation sites, measured SCR responses to several unconditioned stimuli, and applied the experiment in multiple contexts. However, several conceptual and practical issues remain that I think limit the value of potential conclusions drawn from this work.

The first issue that I have with this study concerns the relationship between the TMS manipulation and the theoretical background the authors present in their rationale. In the introduction the authors sketch that what they call 'mPFC' is involved in regulation of threat responses. They make a convincing case, however, almost all of the evidence they present concerns the ventromedial part of the prefrontal cortex (refs 18-25). The authors then mention that no one has ever studied the effects of 'mPFC'-TMS on threat memories. That is not surprising given that stimulating vmPFC with TMS is very difficult, if not impossible. Simulation of the electrical field that develops as a consequence from the authors manipulation (using the same TMS coil and positioning the authors use) shows that vmPFC (or mPFC for that matter) is not stimulated. The authors then continue in the methods section stating that the region they aimed for was BA10. This region they presumably do stimulate, however, that does not follow logically from their argument. BA10 is anatomically, cytoarchitectonically and functionally a wholly different area than vmPFC and I wonder if their rationale would hold given that they stimulate BA10.

The second concern I have is that although I think the authors should be praised for including both sham and active control regions, the controls might not be optimally chosen to control for the potential confounds of their condition of interest (mPFC-TMS). Namely, TMS on the forehead can be unpleasant, if not painful, whereas sham-TMS or TMS applied to the back of the head or even over dlPFC is not (or less so at the very least). Given that the SCR results after mPFC TMS show exactly the same temporal pattern as the sham-TMS but with a lower starting point, one could wonder whether a painful stimulation prior to the retrieval might have already caused habituation to painful stimulation observed in SCR in consequent CS presentations. A control region that would have been more obvious to take is the lateral part of BA10, by moving the TMS coil several centimeters to the left or right, circumventing all things potentially called medial but giving similar unpleasant sensations (pain etc).

My final concern is that the main analyses are performed on single trials of SCR responses, which is a relatively noise measure to use on single trials. This is also done in relatively small groups (n=21). I would have liked to see both the raw or at least averaged timeseries SCR data plotted, and a rationale explaining how the authors decided on the current sample sizes, if that was based on a power analyses one must have expected quite strong effects.

Author Response

Reviewer #1 (Public Review):

Payne et al. have investigated the neural basis of VOR adaptation with the goal of constraining sites and mechanisms of plasticity supporting cerebellar learning. This has been an area of intense debate for decades; previous competing models have argued extensively about the sites of plasticity and the strength of eye velocity feedback/ efference copy signals to Purkinje cells has been central to the debate. This paper nicely explores the consequences of varying the strength of this feedback and in so doing, provides a potential explanation for why Purkinje cell responses during VOR cancellation could exhibit stronger responses following learning, despite net depression of the strength of their vestibular inputs. In that sense it provides some reconciliation of existing models. The work appears to be well done and the paper is well written. The manuscript could be improved and the significance of the work clarified and enhanced by contextualizing the work more appropriately within the existing literature in this area.

We thank the reviewer for the nice summary of this work’s contribution to the long-standing debate regarding sites and mechanisms of plasticity underlying cerebellar learning.

We have revised the manuscript to address several key points raised by the reviewer. We now emphasize that the main evidence for weak feedback arises from interpreting our model in the context of the existing experimental evidence for plasticity rules in the cerebellar cortex, and we have clarified the commonalities and differences from the Miles-Lisberger model. Several missing references are now included. Additionally, we clarify the comparison of our model to data after learning, and explain how altered signaling through the visual pathways drives paradoxical changes in neural activity without requiring plasticity in the visual pathways. We hope that these changes better situate the work to be interpreted appropriately in the context of the existing literature.

Reviewer #2 (Public Review):

Payne et al. use a computational approach to predict the sites and directions of plasticity within the vestibular cerebellum that explain an unresolved controversy regarding the basis of VOR learning. Specifically, the conclusion by Miles and Lisberger (1981) that vestibular inputs onto Purkinje cells (PCs) must potentiate, rather than depress (as in the Marr/Albus/Ito model), following gain-increase learning because when the VOR is cancelled, PC firing increases rather than decreases. Payne et al. provide a novel model solution that recapitulates the results of Miles and Lisberger but, paradoxically, uses plasticity in the cerebellar cortex that weakens PC output rather than strengthens it. However, the model only succeeds when efference copy feedback to the cerebellar cortex is relatively weak thereby allowing a second feedback pathway to drive PC activity during VOR cancellation to counteract the learned change in gain. Because the model is biologically constrained, the findings are well supported. This work will likely benefit the field by providing a number of potentially experimentally testable conclusions. The findings will be of interest to a wider audience if the results can be extrapolated to other cerebellar-dependent learning behaviors rather then just VOR gain-increase learning. Overall, the manuscript is very well written with clearly delineated results and conclusions.

We appreciate the reviewer’s comments that the model is well-constrained and provides a solution to the long-standing debate surrounding sites and directions of plasticity underlying VOR learning.

The reviewer raises an important question: do our results generalize across the cerebellum? We note first that we are studying the cerebellum to illustrate a core problem in modeling systems throughout the brain, namely, how to disambiguate plasticity in the face of ubiquitous feedback loops, both within the brain and between the brain and the environment. Within the cerebellum, we focused on VOR learning due to the wealth of experimental data available. While the specific effect of feedback strength on plasticity will depend on the details of the relevant cerebellar circuit, our general approach can be applied to other areas, given sufficient data, in order to determine how plasticity is distributed in the face of potential feedback loops. Importantly, error-driven LTD of the parallel fiber-Purkinje cell synapse is a fundamental hypothesized mechanism for cerebellar learning which has been generally accepted elsewhere in the cerebellum, but was called into question for VOR learning in the flocculus by the Miles-Lisberger model. Thus, our study of VOR learning has broad implications for reconciling plasticity mechanisms across the cerebellum.

We also note that, even within the VOR circuit, the direction of plasticity and the relative dependence on plasticity at each site may depend on the timescale of learning. On longer timescales, there is thought to be consolidation of learning from a cerebellar cortical site to a brainstem site. Such consolidation from a faster-learning site to a slower-learning site is known as systems consolidation and has been shown theoretically to mitigate the ‘plasticity-stability dilemma’ of having fast learning without over-writing longer-term learning. Our model is compatible with both error-driven plasticity in the cerebellar cortex and a site of plasticity in the brainstem, with brainstem plasticity potentially mediating consolidation of earlier learned changes in the cerebellar cortex. We have now updated the text significantly to discuss the broader implications of the results and to address the reviewer’s specific comments.

Reviewer #3 (Public Review):

Summary: In this study, the authors attempt to determine what is the role (and strength) of feedback in a closed-loop (cerebellar) system.

Strengths:

1) By combining extensive data fitting of cerebellar experimental observations this study provides deep insights into existing questions and more broadly on the role of feedback and what are the limitations when inferring feedback in (plastic) neural circuits.

2) Another strength of this study is the gradual build-up of evidence by using models of different complexities to help build the argument that weak feedback is sufficient to explain experimental observations.

3) The paper is well-written and structured.

Weaknesses:

1) In principle feedback can (i) drive dynamics or/and (ii) drive learning directly. Throughout the paper, the authors refer to only the first case (i.e. dynamics). However, the role of feedback in learning is already implicitly assumed by the authors when jointly fitting the model before and after learning. Note that the general conclusion that feedback (in general) is weak may be to the first view (i.e. dynamics), but not the second. Given that a key conclusion of the paper is that no feedback is sufficient to explain the data, this suggests that feedback may instead be used for learning/plasticity.

We fully agree with the reviewer that our conclusions do not preclude an important role for many other types of feedback, including as an instructive signal for learning. Instead of explicitly considering feedback for learning in our model, we consider static snapshots before and after learning to infer plasticity, while remaining agnostic to the neural algorithm used to achieve such plasticity. A widely held hypothesis is that motor error signals carried by climbing fibers instruct LTD at co-active parallel fiber inputs to Purkinje cells; this is indeed a form of feedback, operating on a slower timescale than “feedback for dynamics.” This “feedback for learning” is not modeled here but is fully consistent with our results, as discussed in a new paragraph of our Discussion (end of Section 3.4.1 “Pathways undergoing plasticity”).

2) There are some potential limitations of the conclusions drawn due to the model inference methods used. The methods used (fmincon) can easily get stuck in local minima and more importantly they do not provide an overview of the likelihood of parameters given the data. A few studies have now shown that it is important to apply more powerful inference techniques both to infer plasticity (Bykowska et al. Frontiers 2019) and neural dynamics (Gonçalves et al. eLife 2020). As highlighted by Costa et al. Frontiers 2013 using more standard fitting methods can lead to misleading interpretations. Given the large range of experimental data used to constrain the model, this may not be an issue, but it is not explicitly shown.

The reviewer correctly points out that we used a deterministic model-fitting procedure. To address this concern, we complemented the full dynamic model with a simple analytic model ( Figure 5 ) for which we could fully derive the cost function landscape and analytically show that there is a line of parameters corresponding to a perfect degeneracy in the model. Thus, the challenge in the model we analyze is that there are too many solutions, rather than it being difficult to find a solution. Given this degeneracy, we chose to fix the level of efference copy feedback and then find the (now non-degenerate) solutions, and to then compare these different solutions with regards to their implications for the correlated strengths and changes in strengths of different pathways. We have edited the relevant section of the Discussion for clarity on this topic, and have added references to the additional strategies for model inference mentioned above, in Section 3.3 “Relation to other sloppy models”.

3) There is some lack of clarity on how the feedback pathways as currently presented should be interpreted in the brain.

We interpret this comment as referring to the questions of (1) whether our model includes a pathway for learning through feedback, (2) what is the anatomical implementation of the efference copy feedback pathway and visual pathways, and (3) how should the positive weights on the efference copy feedback pathway k PE be interpreted. We address these below.

(1) Feedback for learning was discussed in point 1 above.

(2) Anatomical implementation of efference copy pathway: We have edited the Discussion to clarify that there is anatomical evidence for efference copy input to the cerebellum, but that a key aspect of ‘feedback’ is that activity functionally loops back onto itself. Instead, neurons carrying eye movement commands (such as in the vestibular nucleus) could send signals to the cerebellum, without receiving output from the same cerebellar neurons – this would correspond to a ‘spiraling’ pathway that does not form a closed feedback loop (Figure 8). Thus we argue that the existence of the gross anatomical pathways does not necessitate a role for strong, functional, efference copy feedback (Discussion, Section 3.1, lines 481-491).

Anatomical implementation of visual pathway: The visual feedback pathways considered here are those that would receive visual motion information from the environment. This visual feedback is itself changed by eye movements, thus providing a net overall negative feedback loop that helps to stabilize gaze. This pathway has been proposed to involve cortical regions such as MST (discussed in Materials and Methods, Model Implementation, lines 769-774).

(3) Interpretation of positive feedback loop: In our model, the efference copy feedback filter, k PE , has positive weight. This corresponds to the positive net sign of the Purkinje cell to brainstem to Purkinje cell feedback loop. Specifically, the Purkinje cell to brainstem pathway is inhibitory (because Purkinje cells are inhibitory), the brainstem to eye velocity command pathway is inhibitory (to achieve counter-rotation of the eyes in response to head turns), and the feedback of this eye velocity command back to Purkinje cells (k PE ) is positive. Thus this loop in our model represents positive feedback. This is now clarified in Materials and Methods, Model Implementation, lines 748.

4) The functional benefits of having (or not) feedback could be better discussed (related to point 1 above).

Related to point 1 above, it is certainly the case that feedback is necessary for learning. We do not explicitly model the climbing fiber feedback thought to be involved in learning/plasticity of the parallel fiber pathway.

We instead focus on the role of efference copy feedback, and how it functionally impacts the required sites and signs of plasticity in the circuit. As shown in the paper, if the efference copy pathway is strong, then this is most consistent with learned changes in eye movements being driven primarily by plasticity in the brainstem pathway (as in the Miles-Lisberger hypothesis), whereas if the efference copy pathway is weak, then this is most consistent with learned changes in eye movements being driven by net depression in the parallel fiber to Purkinje cell pathway (as in the classic Marr-Albus-Ito model and as suggested by most cellular and molecular studies of parallel fiber-Purkinje cell plasticity), in addition to a role of plasticity in the brainstem pathway. We also note that, in the ‘Strong Feedback’ model, the feedback is so strong that the system is on the brink of instability – this has been argued to have the functional benefit of providing ‘inertia’ to eye movements that could help to maintain eye movements during smooth pursuit when a target goes behind an occluder, but it also has the disadvantage of placing the system at a level of positive feedback near the brink of instability. We also note that the visual feedback pathway through the environment, emphasized in this work, serves as a negative feedback loop that reduces deviations between the eye and target velocity. We have extensively re-written the first section of the Discussion (Section 3.1), in order to more clearly lay out the implications of each model for circuit plasticity and feedback.

5) Some of the key conclusions of the work are not described in the abstract, namely that feedback is weak in the cerebellar system.

Thank you for raising this point, we have added this key conclusion to the end of the abstract: “Our results address a long-standing debate regarding cerebellum-dependent motor learning, suggesting a reconciliation in which error-driven plasticity of synaptic inputs to Purkinje cells is compatible with seemingly oppositely directed changes in Purkinje cell activity. More broadly, the results demonstrate how learning-related changes in neural activity can appear to contradict the sign of the underlying plasticity when either internal feedback or feedback through the environment is present.”

Claims:

The argument is well-built throughout the paper, but there are some potential caveats with the general interpretation (see weaknesses).

Impact:

This work has the potential to bring important messages on how best to interpret and infer the role of feedback in neural systems. For the field of the cerebellum, it also proposes solutions to long-standing problems.

Spanish to English translation by Google with some grammar/readability adjustments by Keith Taylor.

Hortensia, True Story

In the city of Cumaná, Sucre state, Venezuela. Faithful witness to the story of Hortensia, a woman who was held captive for 40 years in a house by her husband, Fernando Inserny.

Hortensia Maestrucci, a young woman who lived in the state of Bolívar, where she was born and grew up for a time, arrived in the city of Manzanares with her boyfriend Fernandito Inserny. Once she arrived in the city, her stay was comfortable and good for the first few months. It is said that she was a very pretty young woman who attracted the attention of many men, due to her beauty and innocence. Her boyfriend, Fernando, was the owner, at that time, of many businesses. Owner of luxurious cars at that time, an icon in the city of Cumaná. He was a descendant of a militant family of Christian democracy, a man with many things around him, among those women. Some time later, she began to feel jealous and put things in Hortensia's head, saying that if she went out on the street, the communists would take her. Things that were a lie but since she was an innocent young woman she believed her and agreed to her confinement. That's how the young woman spent her days, months, and even years. To be more exact 40 years.

This event occurred, especially near Plaza Ribero, on Úrica Street in Cumaná. During that time, she was fed through the slots in the door of the room where she was. There she was tied the entire time without making any noise so that the communists would not hear her and take her away. After 40 years locked up, she was discovered. There are two versions: - It is said that there were some children playing on the street, and they heard Hortensia crying. - And another that the neighbors warned that thieves had broken into that house.

It was at that moment when the community looks out and realizes it. They immediately call the competent authorities to investigate the case. At the time of her departure, Hortensia looked very bad. She was extremely skinny, with super long nails, and hair that reached almost her size. Very old, she had a bad smell. She was taken out of the house around 5 in the afternoon, covered and in the arms of a police commissioner. The people and everything caused her some discomfort and hurt Hortensia's eyesight, since she had been without sunlight for 40 years.

After 2 years from her release, Hortensia dies. The story of this woman who moved the city of Cumaná transcended through the years and will remain in the memory of those who lived that time. How someone manages to harm another person of that magnitude, all their years of life lost due to be locked-up.

What name could we give to this story?

I can definitely agree with this. I have seen ideas come down the administrative pipe that left me scratching my head and wondering why they though it was a good idea.

I understand what the author is trying to say here. Homosexuality has existed for centuries and gay men and lesbians are often group together, despite not sharing a gender or a member of the preferred sex. So when we group them together, they often do face different levels of oppression. Lesbians belong to a group of women who have been oppressed in this country for a long time. On the other hand gay men are still men and men have been at the head of this patriarchal society, so they can be argued that they face different levels of oppression, despite belonging to the same group. Which is why you have to examine the individuals of said group instead of looking at the group as a whole. In society, so many people can be put in so many different groups, because we have allowed for freedom of expression in this modern society.

"the central insight expressed in the concept of exploitation is that this oppression occurs through a steady process of the transfer of the results of this labor of one social group to benefit another " - I agree with this as oppression can result from exploitation. there are many shows that show this type of exploitation when maids and assistants are treated horribly from the people with higher powers. This is because once an individual knows they have power, it gets to their head. Wealth is the root of a lot problems

"Violence is systemic because it is directed at members of a group simply because they are members of that group." This statement is so simply stated yet so significant at the same time because of how easy it is to understand what Young is trying to say. Additionally, when she is talking about violence, what came to my head as well is how stereotypes could play a role in violence that people experience

The author adding "including women" at the end of the sentence proves that it was truly surprising to see women outside of a house environment during the colonial era. Watching this performance just required being on street to view it, thus proving how odd it was to see a women not doing house work during this time.

This situation is something that I hope no one else has to go through. The teacher is teaching other students that it is okay to say that word and is not validating his feelings. Removing her "treatment" towards him just because of his disagreement is just so not okay. Her job as a teacher is to guide her students but she is showing so much discrimination towards him.

Great that you find out, it was a mistake from my side as they changed the file-format of the data recently.

Head of School, Dr. Caren Walker Gregory, has been an educator and leader in Boston Public Schools for 32 years, 14 of those leading EMK. Dr. Walker's experience in math and science has perfectly prepared her to foster a robust college preparatory environment that prepares students to excel in health focused careers and higher education after graduation.

– Rebecca Grainger, Senior Advisor for Youth and Schools

We're excited about the partnership between Roxbury Community College and the following schools: Boston International Newcomers Academy, Margarita Muniz Academy, and English High School. This early college program is specifically designed for multilingual students. The first cohort will be starting this month.

English High School is the oldest public school in the Nation, established in 1821, just 45 years after our Nation was founded. There are four technical pathways where students earn industry-recognized credentials and certifications: Business, Finance & Entrepreneurship; Design & Visual Communications; Health Assisting & Wellness; and Programming & Web Development. Caitlin Murphy is the Head of English High School, continuing the legacy of honor, achievement, and service to humanity.

Margarita Muniz Academy is Boston’s only two-way bilingual high school, with a rigorous college preparatory curriculum instructed in English and Spanish. Their mission, led by Dania Vazquez, is that every graduate will be accepted to a 4-year college.

Tony King is the Head of Schools of Boston International Newcomers Academy (BINcA). Their mission is to embrace new immigrant adolescent English language learners and their families by providing a curriculum designed to teach students English while also teaching rigorous content and ensuring students will be college and career-ready with motivation to pursue a life of learning and civic engagement.

– Jisca Philippe, Director of Youth and Schools

Charlestown High School is a diverse school that has been serving students since 1845. Charlestown's Head of School, Ajay Trivedi's strong belief in all students having access to challenging and inspiring curriculum has been foundational to expanding opportunities for advanced learning such as early college at Bunker Hill. Charlestown High School currently offers three career pathways - C-Town Business, Technology and Health.

– Rebecca Grainger, Senior Advisor for Youth and Schools

This post discusses the responsibilities and challenges faced by a school head, including tasks such as purchasing supplies, distributing resources, and providing technical assistance to teachers. Despite the challenges, the author finds inspiration and happiness in the smiles of the students and parents.

• Who: The author, a school head, teachers, learners, school personnel, stakeholders, parents, and other stakeholders.
• What: The responsibility of being a school head and the tasks involved, such as providing technical assistance to teachers, supervising them, conducting meetings, purchasing supplies, distributing rice, hauling armchairs, and preparing reports.
• Where: The school and the author's house.
• Why: The author discusses the challenges and responsibilities of being a school head, such as the need to adjust to learners' different capacities, the lack of representation and travel allowance, and the financial burden of personal expenses.
• When: The post does not specify a specific time frame, but it mentions the impact of the pandemic on teaching and learning tasks and the need for office evaluation.
• How: The author describes the tasks and responsibilities they undertake, such as providing technical assistance, attending meetings, purchasing supplies, distributing rice, and hauling armchairs. They also mention using their own personal finances for expenses and finding inspiration and motivation from the smiles of the school children and their parents.

Head of School, Dr. Caren Walker Gregory, has been an educator and leader in Boston Public Schools for 32 years, 14 of those leading EMK. Dr. Walker's experience in math and science has perfectly prepared her to foster a robust college preparatory environment that prepares students to excel in health focused careers and higher education after graduation.

– Rebecca Grainger, Senior Advisor for Youth and Schools

We're excited about the partnership between Roxbury Community College and the following schools: Boston International Newcomers Academy, Margarita Muniz Academy, and English High School. This early college program is specifically designed for multilingual students. The first cohort will be starting this month.

English High School is the oldest public school in the Nation, established in 1821, just 45 years after our Nation was founded. There are four technical pathways where students earn industry-recognized credentials and certifications: Business, Finance & Entrepreneurship; Design & Visual Communications; Health Assisting & Wellness; and Programming & Web Development. Caitlin Murphy is the Head of English High School, continuing the legacy of honor, achievement, and service to humanity.

Margarita Muniz Academy is Boston’s only two-way bilingual high school, with a rigorous college preparatory curriculum instructed in English and Spanish. Their mission, led by Dania Vazquez, is that every graduate will be accepted to a 4-year college.

Tony King is the Head of Schools of Boston International Newcomers Academy (BINcA). Their mission is to embrace new immigrant adolescent English language learners and their families by providing a curriculum designed to teach students English while also teaching rigorous content and ensuring students will be college and career-ready with motivation to pursue a life of learning and civic engagement.

– Jisca Philippe, Director of Youth and Schools

Charlestown High School is a diverse school that has been serving students since 1845. Charlestown's Head of School, Ajay Trivedi's strong belief in all students having access to challenging and inspiring curriculum has been foundational to expanding opportunities for advanced learning such as early college at Bunker Hill. Charlestown High School currently offers three career pathways - C-Town Business, Technology and Health.

– Rebecca Grainger, Senior Advisor for Youth and Schools

This attitude is still prevelant and shows its head in many ways. The "Tom" or "Coon", self hate, or in my opinion the attitude that many black people have about our lack of cohesion as community is derived from this

Spending my fall term at the university of Oregon, it became a habit to observe everyone else around me. Walking to class I would see all different kinds of people and made assumptions about their lifestyles and career paths based off of the way they dressed. When you see people with wired head phones riding a skateboard or scooter vs people dressed in suits or scrubs you are bound to take one more seriously than the other.

What comes to my head when I think of the Middle Ages are knights, literally cutthroat politics, plagues, and wealth disparity. I assume most of these associations come from tv shows or books I've read set in vaguely medieval europe settings

Was Stengers bei Whitehead findet: einen Weg die Agency nichtmenschlicher Entitäten zu erfassen und das Wissen von diesen Entitäten als „sozial“—im Sinne ihrer Mitwirkung an diesem Wissen—zu denken.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, Janssens et al. addressed the challenge of mapping the location of transcriptionally unique cell types identified by single nuclei sequencing (snRNA-seq) data available through the Fly Cell Atlas. They identified 100 transcripts for head samples and 50 transcripts for fly body samples allowing the identification of every unique cell type discovered through the Fly Cell Atlas. To map all of these cell types, the authors divided the fly body into head and body samples and used the Molecular Cartography (Resolve Biosciences) method to visualize these transcripts. This approach allowed them to build spatial tissue atlases of the fly head and body, to identify the location of previously unknown cell types and the subcellular localization of different transcripts. By combining snRNA-seq data from the Fly Cell Atlas with their spatially resolved transcriptomics (SRT) data, they demonstrated an automated cell type annotation strategy to identify unknown clusters and infer their location in the fly body. This manuscript constitutes a proof-of-principle study to map the location of the cells identified by ever-growing single-cell transcriptomic datasets generated by others.

Strengths:<br /> The authors used the Molecular Cartography (Resolve Biosciences) method to visualize 100 transcripts for head samples and 50 transcripts for fly body samples in high resolution. This method achieves high resolution by multiplexing a large number of transcript visualization steps and allows the authors to map the location of unique cell types identified by the Fly Cell Atlas.

Weaknesses:<br /> Combining single-nuclei sequencing (snRNA-seq) data with spatially resolved transcriptomics (SRT) data is challenging, and the methods used by the authors in this study cannot reliably distinguish between cells, especially in brain regions where the processes of different neurons are clustered, such as in neuropils. This means that a grid that the authors mark as a unique cell may actually be composed of processes from multiple cells.

Reviewer #2 (Public Review):

Summary:<br /> The landmark publication of the "Fly Atlas" in 2022 provided a single cell/nuclear transcriptomic dataset from 15 individually dissected tissues, the entire head, and the body of male and female flies. These data led to the annotation of more than 250 cell types. While certainly a powerful and data-rich approach, a significant step forward relies on mapping these data back to the organism in time and space. The goal of this manuscript is to map 150 transcripts defined by the Fly Atlas by FISH and in doing so, provide, for the first time, a spatial transcriptomic dataset of the adult fly. Using this approach (Molecular Cartography with Resolve Biosciences), the authors, furthermore, distinguish different RNA localizations within a cell type. In addition, they seek to use this approach to define previously unannotated clusters found in the Fly Atlas. As a resource for the community at large interested in the computational aspects of their pipeline, the authors compare the strengths and weaknesses of their approach to others currently being performed in the field.

Strengths:<br /> 1. The authors use Resolve Biosciences and a novel bioinformatics approach to generate a FISH-based spatial transcriptomics map. To achieve this map, they selected 150 genes (50 body; 100 head) that were highly expressed in the single nuclear RNA sequencing dataset and were used in the 2022 paper to annotate specific cell types; moreover, the authors chose several highly expressed genes characteristic of unannotated cell types. Together, the approach and generated data are important next steps in translating the transcriptomic data to spatial data in the organism.<br /> 2. Working with Resolve, the authors developed a relatively high throughput approach to analyze the location of transcripts in Drosophila adults. This approach confirmed the identification of particular cell types suggested by the FlyAtlas as well as revealed interesting subcellular locations of the transcripts within the cell/tissue type. In addition, the authors used co-expression of different RNAs to unbiasedly identify "new cell types". This pipeline and data provide a roadmap for additional analyses of other time points, female flies, specific mutants, etc.<br /> 3. The authors show that their approach reveals interesting patterns of mRNA distribution (e.g alpha- and beta-Trypsin in apical and basal regions of gut enterocytes or striped patterns of different sarcomeric proteins in body muscle). These observations are novel and reveal unexpected patterns. Likewise, the authors use their more extensive head database to identify the location of cells in the brain. They report the resolution of 23 clusters suggested by the single-cell sequencing data, given their unsupervised clustering approach. This identification supports the use of spatial cell transcriptomics to characterize cell types (or cell states).<br /> 4. Lastly, the authors compare three different approaches --- their own described in this manuscript, Tangram, and SpaGE - which allow integration of single cell/nuclear RNA-seq data with spatial localization FISH. This was a very helpful section as the authors compared the advantages and disadvantages (including practical issues, like computational time).

Weaknesses:<br /> 1. Experimental setup. It is not clear how many and, for some of the data, the sex of the flies that were analyzed. It appears that for the body data, only one male was analyzed. For the heads, methods say male and female heads, but nothing is annotated in the figures. As such, it remains unclear how robust these data are, given such a limited sample from one sex. As such, the claims of a spatial atlas of the entire fly body and its head ("a rosetta stone") are overstated. Also, the authors should clearly state in the main text and figure legends the sex, the age, how many flies, and how many replicates contributed to the data presented (not just the methods). What also adds to the confusion is the use of "n" in para 2 of the results. " ... we performed coronal sections at different depths in the head (n=13)..." 13 sections in total from 1 head or sections from 13 heads? Based on the body and what is shown in the figure, one assumes 13 sections from one head. Please clarify.<br /> 2. Probes selected: Information from the methods section should be put into the main text so that it is clear what and why the gene lists were selected. The current main text is confusing. If the authors want others to use their approach, then some testing or, at the very least, some discussion of lower expressed genes should be added. How useful will this approach be if only highly expressed genes can be resolved? In addition, while it is understood that the company has a propriety design algorithm for the probes, the authors should comment on whether the probes for individual genes detect all isoforms or subsets (exons and introns?), given the high level of splicing in tissues such as muscle.<br /> 3. Imaging: it isn't clear from the text whether the repeated rounds of imaging impacted data collection. In many of what appear to be "stitched" images, there are gradients of signal (eg, figure 2F); please comment. Also, since this a new technique, could a before and after comparison of the original images and the segmented images be shown in the supplemental data so that the reader can better appreciate how the authors assessed/chose/thresholded their data? More discussion of the accuracy of spot detection would be helpful.<br /> 4. The authors comment on how many RNAs they detected (first paragraph of results). How do these numbers compare to the total mRNA present as detected by single-cell or single-nuclear sequencing?<br /> 5. Using this higher throughput method of spatial transcriptomics, the authors discern different cell types and different localization patterns within a tissue/cell type.<br /> a. The authors should comment on the resolution provided by this approach, in terms of the detection of populations of mRNAs detected by low throughput methods, for example, in glia, motor neuron axons, and trachea that populate muscle tissue. Are these found in the images? Please show.<br /> b. The authors show interesting localization patterns in muscle tissue for different sarcomere protein-coding mRNAs, including enrichment of sls in muscle nuclei located near the muscle-tendon attachment sites. As this high throughput approach is newly being applied to the adult fly, it would increase confidence in these data, if the authors would confirm these data using a low throughput FISH technique. For example, do the authors detect such alternating "stripes" ( Act 88F, TpnC4, and Mhc) or enriched localization (sls) using FISH that doesn't rely on the repeated colorization, imaging, decolorization of the probes?<br /> 6. The authors developed an unbiased method to identify "new cell types" which relies on co-expression of different transcripts. Are these new cell types or a cell state? While expression is a helpful first step, without any functional data, the significance of what the authors found is diminished. The authors need to soften their statements.

Appraisal:<br /> The authors' goal is to map single cell/nuclear RNAseq data described in the 2022 Fly Atlas paper spatially within an organism to achieve a spatial transcriptomic map of the adult fly; no doubt, this is a critical next step in our use of 'omics approaches. While this manuscript does the hard work of trying to take this next step, including developing and testing a new pipeline for high throughput FISH and its analysis, it falls short, in its present form, in achieving this goal. The authors discuss creating a robust spatial map, based on one male fly. Moreover, they do not reveal principles of mRNA localization, as stated in the abstract; they show us patterns, but nothing about the logic or function of these patterns. This same criticism can be said of the identification of "new cell types, just based on RNA colocalization. In both cases (mRNA subcellular localization or cell type identification), further data in the form of validation with traditional low throughput FISH and genetic manipulations to assess the relation to cell function are required for the authors to make such claims.

Discussion of likely impact:<br /> If revised, these data, and importantly the approach, would impact those working on Drosophila adults as well as those working in other model systems where single cell/nuclear sequencing is being translated to the spatial localization within the organism. The subcellular localization data - for example, the size of transcripts and how that relates to localization or the patterns of sarcomeric protein localization in muscle - are intriguing, and would likely impact our thinking on RNA localization, transport, etc if confirmed. Lastly, the authors compare their computational approaches to those available in the field; this is valuable as this is a rapidly evolving field and such considerations are critical for those wishing to use this type of approach.

This is a striking take on why MLK Jr. was assassinated. I agree that MLK Jr. put a target on his head when he began challenging government actions in Vietnam while showing communist inclinations at the height of the Cold War.

Authors' response (22 December 2023)

GENERAL ASSESSMENT

KATP is a remarkably important potassium-selective ion channel that is inhibited by intracellular ATP, allowing it to serve key roles throughout the body, including regulation of insulin release from the pancreas. Driggers et al. describe an important study that reports the structure of an open KATP channel complex formed from the Q52R diabetes mutation of Kir 6.2, SUR1, and long-chain PIP2. Although earlier structures have been determined in closed, open and inactivated conformations, none have resolved where PIP2 binds. This has been an important limitation of the available structures given the key role of this membrane component in promoting the open state and influencing the inhibitory actions of intracellular ATP. The open channel structure reported here resembles a previous structure from the MacKinnon laboratory of a mutant channel that exhibits a very high open probability (G334D, C166S). The authors also show that channel opening is accompanied by a dilation of the Kir6.2 inner helix gate, in particular rotation of L164 and F168 away from the channel's pore, and by a conformational rearrangement at the interface between Kir6.2 and the TMD0 domain of SUR1.

The key advance that allowed the authors to resolve PIP2 bound to KATP appears to be the use of brain-derived long-chain PIP2, which was incubated with membranes prior to extraction of the channel protein with digitonin and purification, as well as the use of the Q52R mutant that promotes the open state. Remarkably, the structures of PIP2 bound to KATP reveals two PIP2 binding sites: one related to previously resolved sites in other Kir channels; and a new, unanticipated one where the lipid snuggles into the interface between Kir6.2 and the TMD0 helix of SUR1, a region of SUR1 previously implicated in promoting the open state of KATP. To examine the functional impact of the two PIP2 binding sites, the authors identify two positively-charged residues – one in each binding site – to neutralise by mutation to alanine, in an effort to disrupt the two putative PIP2 interactions. Indeed, they find that KATP channel currents on patch excision are very small and require much longer exposure to PIP2 to fully activate, compared to wild-type channels. Although one could question the physiological relevance of the new PIP2 binding site, that PIP2 remains bound throughout extraction and purification in digitonin solutions, and can be readily resolved in the structure, suggests that interactions of longer-chain PIP2 with both sites is quite favourable and likely to be occupied under biological conditions. One of the remarkable features of the new structures is that PIP2 binding to KATP causes a conformation change in the inhibitory ATP binding site, providing a mechanistic explanation for how PIP2 and ATP antagonistically engage to promote open or closed states, respectively. The structure also reveals how the Q52R mutant likely forms a cation-π interaction with W51 of SUR1, explaining how the diabetes mutation promotes the open state of KATP. Ultimately, further experiments will help unravel the physiological impact of the newly identified PIP2 site, as the electrophysiology presented in this structural study is understandably limited. However, that does not diminish the impact of the study.

Overall, this is an important study that helps to explain how PIP2 exerts such a profound regulatory influence over KATP, which will be valuable for future studies on KATP and of general interest to scientists investigating how PIP2 regulates other membrane proteins. The preprint is well-written, the work appears to have been carried out with rigor and attention to detail, and the authors present new conclusions and discuss them in the context of previous findings, some of which have been enigmatic until now.

We thank the reviewers for the positive evaluation of our work and helpful suggestions. We have revised the manuscript to address the essential revisions as well as additional suggestions from the reviewers. Moreover, since the preprint was posted in bioRxiv, we have conducted additional analysis of our cryoEM data and identified a closed channel conformation in which the new PIP2 binding site is occluded and the cytoplasmic domain of Kir6.2Q52R is rotated and translated away from the plasma membrane--a conformation that corresponds to an apo SUR1/Kir6.2Q52R and is similar to our previously published WT apo (no PIP2 added) closed structure. We have also performed additional mutational and functional studies to show the dependence of the gain-of-function phenotype of Kir6.2Q52R on intact PIP2 binding sites. The additional data included in the revised manuscript further strengthens the conclusions of our study.

Below we provide detailed response to the reviewers' essential revision requests and additional suggestions.

RECOMMENDATIONS

Essential revisions:

1. Most of the figures focus on a comparison of the new PIP2-bound open state for the Q52R mutant with a closed state of KATP. A more extensive structural comparison between the PIP2-bound open structure and other open structures solved in absence of PIP2 (e.g. ref 27 where Kir6.2 mutations C166S and G334D were used) would help clarify the functional roles of the putative PIP2 binding sites. A structural comparison of the PIP2 binding sites between the two open structures (apo and holo) would reveal whether the PIP2 binding sites are conserved in the absence of PIP2, for example. Such a comparison would help the general reader to understand which of the structural changes observed in the new structure have been seen before and those that have not. Another example would be to clarify the extent to which structural changes around the inhibitory ATP binding site seen here are related to those in previous structures thought to be open.

We thank the reviewers for the excellent suggestion to compare the PIP2-bound SUR1/Kir6.2Q52R structure with previous open KATP channel structures.. We have now included a comparison of the PIP2-bound SUR1/Kir6.2Q52R structure (holo) and the previously published pre-open structure (rodent SUR1-39aa-Kir6.2H175K fusion channel; PMID: 35589716) in the revised Figure S4. Note that while the open structure of human SUR1/Kir6.2C166S, G334D (PMID: 34815345) is also very similar to our PIP2-bound SUR1/Kir6.2Q52R structure, due to sequence variations in the different species of SUR1 used and an additional amino acid present in the construct (glycine at aa position 2), we have chosen to compare our structure with the pre-open rodent structure to avoid confusion in key SUR1 residues shown. In revised Fig.S4a,b, we also compared lipid density in the PIP2 binding pocket of all three open structures to highlight the difference between the long-chain PIP2 in this study, and the unassigned density in the pocket observed in the open structure of human SUR1/Kir6.2C166S, G334D (no exogenous PIP2 added) and the pre-open structure of rodent SUR1-39aa-Kir6.2H175K fusion (short-chain diC8-PIP2 added post purification of channel-containing detergent micelles).

1. It would be valuable for readers if the authors could explain their thinking about why the new PIP2 binding site is likely to be physiologically relevant. In its current form, some readers may be unconvinced about whether the new site is occupied under physiological conditions. For example, in the last paragraph of page 15, the authors acknowledge that in their previously published closed structures without exogenous PIP2, they saw lipid densities in the novel PIP2 site which they modelled as phosphatidylserines. Similar lipid densities were also seen near this site in the other published open state (see Fig. S6 in DOI:10.1073/pnas.2112267118). In the next paragraph on page 16, they also comment on the unusually low specificity of Kir6.2 towards phosphoinositides and other lipids, and the ability of purified KATP channels to open in the absence of PIP2. Given these findings, and the potentially high concentration of PIP2 incubated with the sample, is it conceivable that the new PIP2 site is not occupied under physiological conditions? What do the authors know about the molar fraction of PIP2 achieved in the final sample and how this might compare to the estimates of PIP2 abundance in native human cell membranes (0.2-1%)? Would PIP2 ever reach high enough concentrations in the membrane for this site to be bound? The authors might also emphasize that the channel was only exposed to brain PIP2 for a short time before being extracted and purified in the detergent solution, indicating that the interaction between PIP2 and the channel at both sites is quite strong and likely to be occupied physiologically.

We thank the reviewers for asking an important question about the physiological relevance of the two PIP2 binding sites. We addressed this question by introducing PIP2 binding site mutations in KATP channels expressed in native membranes, and tested the consequences of these mutations on channel activity (results shown in the revised Fig. 3). Mutating key residues involved in PIP2 binding, at either sites or both sites, markedly reduced channel activity in native membranes without exogenous PIP2. Adding exogenous PIP2 reversed the effects of these PIP2 binding-site mutations, providing strong evidence that these PIP2 binding sites have physiological roles in channel gating.

Regarding the question of whether PIP2 binds at the novel binding site under physiological conditions, our structural analysis suggests it is likely state-dependent. When the channel is in the open conformation, the new site accommodates PIP2 as shown in our PIP2-bound structure. Although other phosphoinositides also stimulate channel activity when applied to isolated membrane patches containing channels, given PI(4,5)P2 is the most abundant phosphoinositide in the plasma membrane, we suspect the site is likely bound to PI(4,5)P2 under physiological conditions. To address the frequency of PIP2 occupancy at the new PIP2 binding site under different physiological conditions (high ATP/ADP resembling high glucose, versus low ATP/ADP resembling low glucose) experimentally, alternative approaches will be needed, for example, by crosslinking channel subunits with bound lipids followed by identification with mass spectrometry, or solving channel structures in native membranes. These are studies we are very interested in pursuing.

In the current study, we were interested in maximizing the chance of capturing channels in the PIP2-bound conformation to understand the structural basis of channel activation by PIP2. Even under the experimental conditions of exogenous PIP2, we did observe conformations that likely correspond to apo structure at the new PIP2 binding site (from our additional analysis of the cryoEM data; see revised Figure S2 and S8), indicating the new PIP2 binding site is dynamic and can bind PIP2 when the channel is open, but not when the channel is closed.Although we did not attempt to compare PIP2 abundance (molar fraction) before and after incubation with exogenous PIP2 in the current study, we have previously quantified PIP2 in cell membranes from control INS-1 (a rat insulinoma) cells and INS-1 cells overexpressing PIP5 kinase and labelled with 32P using thin-layer chromatography (Lin et al., Diabetes, 2005; PMID: 16186385). The study showed a ~30-fold increase in PIP2, which was accompanied by a ~26-fold decrease in ATP sensitivity of KATP channels measured by inside-out patch-clamp recording, and a decrease in glucose-stimulated insulin secretion (GSIS) response. While the study does not directly answer whether physiological PIP2 concentrations would ever reach high enough as our in vitro experimental system, it does show that increasing PIP2 levels by manipulating an enzyme involved in PIP2 synthesis can induce abnormally high channel activity (i.e. ATP-insensitive) and reduce GSIS, resembling phenotype caused by the neonatal diabetes mutation Kir6.2 Q52R that stabilizes the second PIP2 binding site.

1. The electrophysiological data presented in Fig. 2, while corroborating the existence of a second PIP2 site, is not definitive. On page 9 of the text, the authors mention striking differences between wild-type and mutant channels in terms of "the initial currents upon membrane excision, the extent of current increase upon PIP2 stimulation, and the PIP2 exposure time required for currents to reach maximum." The extent of current increase is shown for multiple patches in Fig. 2E and the other differences are inferred from representative traces. The authors may wish to include some form of quantification for the amount of initial current and time course for data from multiple patches. For example, the authors mention "barely detectable currents" for the SUR1-K134A/Kir6.2-R176A mutant. Taking into account the difference in scale bars, the currents in the example provided don't look any smaller than the currents from Kir6.2-R176A/SUR1 channels. Given the proximity between the two sites, it seems possible that a mutation in one site could allosterically affect PIP2 binding at the other site. In principle, two mutations could independently affect PIP2 binding at the same functional site and have additive effects. Perhaps the strongest arguments in favour of two distinct functional sites come from the mutation map in Fig. 7, which nicely matches the two bound PIP2 molecules, and previous studies showing that KATP is less sensitive to PIP2 in the absence of SUR1, which forms part of the second binding site.

We thank the reviewers for this great suggestion to include quantification of initial currents and response time to maximum currents after PIP2 exposure, which is now included in the revised Figure 3.

1. The increase in current in PIP2 in Fig. 2E may represent the extent of the increase in probability of opening. However, calculating the fold increase in current depends on accurate measurements of the very small currents at the beginning of the experiment, which will be heavily affected by residual leak or noise. In the absence of any direct measurements of open probability (for example with single channels), the authors may wish to discuss these limitations in the text.

The initial currents were calculated as currents observed in K-INT solution upon patch excision minus currents measured in 1mM ATP (KATP currents are inhibited >99% at 1 mM ATP). As such, any leak currents were accounted for. We have now stated this in the electrophysiology section in Methods (page 15). For PIP2 stimulation experiments, PIP2 effects plateau with regard to channel P__o (i.e. total currents) and any further stimulatory effects are reflected by a gradual loss of ATP inhibition (which we check by exposure to 0.1mM or 1mM ATP as shown in revised Fig. 3). Patches that do not show the PIP2 plateauing effects on currents indicate potential leak, which we can confirm by exposing channels to the high affinity inhibitor glibenclamide. Patches showing significant leaks (gradual shift in baseline) were not included in the analysis.

Optional suggestions:

We appreciate the many excellent suggestions from the reviewers, and have made substantial changes in the revised manuscript based on these suggestions. They are detailed below:

1. The Kir6.2-Q52R mutation, which stabilizes the open channel, mediates its effects by interacting with W51 on SUR1 (Fig. 6). Q52R is also very close to the PIP2 headgroup in the novel binding site and thus could help stabilize PIP2. Hence, it would be interesting to test the PIP2 sensitivity of this mutant in excised patches, as in Fig. 2D,E. If PIP2 binding at the second site is favoured by the mutation, the PIP2-induced increase of KATP current should be lower than that observed in WT channels.

From the angle shown in the original Fig.6A, Q52R does appear very close to the headgroup of PIP2 in the novel binding site. However, Q52R is actually very far away from the second bound PIP2. We have now provided additional viewing angle in the revised Fig. 7a that offers better visualization of the distance to avoid confusion.

1. It would be helpful for the authors to provide a biochemical interpretation for the functional results in Fig. 2 D, E. It would seem that the two mutations, Kir6.2-H176A and SUR1-K134A, diminish PIP2 binding affinity but do not prevent PIP2 binding, as the low basal currents seen in the mutants can be rescued by increasing PIP2 concentration.

The text has been revised to better reflect the functional data (original Fig.2D,E, now shown in the revised Figure 3). It now reads (page 6 top): "To probe the functional role of the two PIP2 binding sites, we compared the PIP2 response of WT channels to channels containing the following mutations: Kir6.2R176A , which is predicted to weaken the first PIP2 binding site; SUR1K134A, which is expected to weaken the second PIP2 binding site; Kir6.2R176A and SUR1K134A, which weaken both PIP2 binding sites (Fig.3a)."

1. The discussion on the role of H175 on pH regulation is interesting, but speculative. As the H175K mutant still undergoes acid-induced inhibition (ref 39), it does not seem appropriate to state that the H175K mutant "abolishes channel sensitivity to pH" (p7). A positive charge at position H175 increases basal activity, suggesting that the cationic form of H175 mediates acid-induced activation. The structure shows two H175 rotamers but does not clarify which of these rotamers are populated at different pH values. In addition, there is no evidence to suggest that the cationic form of H175 preferentially interacts with PIP2 and that its neutral form interacts with E179 (p8). On the contrary, it seems more logical to predict that the cationic form of H175, which is positively charged, interacts with E179, which is negatively charged. It would be helpful to clarify several of these points.

We thank the reviewers for the constructive criticisms and agree our statement is too speculative in the absence of experimental evidence that the cationic form of H175 preferentially interacts with PIP2 while its neutral form interacts with E179. Accordingly, we have removed discussion on the role of H175 and instead, simply describe what we observe in our structure.

The revised paragraph now reads (page 5 top):

Interestingly, the Kir6.2-H175 sidechain exhibited two rotameric positions in the PIP2-bound SUR1/Kir6.2Q52R channel cryoEM density map: one oriented towards PIP2 in the conserved binding site and the other towards E179 in the same Kir6.2 subunit (Fig.2b). Kir6.2-H175 has been previously implicated in acid-induced activation of KATP channels between pH 7.4 and 6.8 with a pK of 7.16. Mutating Kir6.2-H175 to lysine, which mimics protonated state of H175, increased basal channel activity and abolished channel activation by pH, suggesting protonation of H175 favors channel opening. Moreover, mutating Kir6.2-E179 to Q greatly attenuated pH-induced channel activation, suggesting Kir6.2-E179 also has a role in acid activation of KATP channels. Our structural observation that H175 sidechain has interaction with PIP2 head group in the conserved site and with Kir6.2-E179 is consistent with the aforementioned mutation-function correlation studies. Since our protein sample was prepared at pH~7.5 (see Methods), which predicts H175 to be largely unprotonated based on the pK of acid activation of the channel of 7.16, it remains to be determined how protonation of H175 at lower pH that activates the channel alters interaction with PIP2 and E179 to stabilize channel opening.

1. There are many questions that come to mind that might be interesting topics to add to the discussion. What is the relative affinity of this novel site for PIP2 over other PI's and even other lipids? Given that previous attempts to establish this (e.g. cited in reference 38) may have been measuring the summed contribution of both sites, if both are functionally relevant (as the present results suggest), do the sites differ in their selectivity? Could this be a lipid binding pocket, which has been displaced by high levels of PIP2 and a locked-open channel? These are not trivial questions to answer, but they are important for understanding the relative importance of the two PIP2 binding sites for the function of KATP and it would be useful to discuss the limits of what can be reasonably concluded at this time. Some of these points might be addressed in the results while other could add to the discussion. In thinking about the roles of the two PIP2 binding sites, have the authors considered the possibility that the PIP2 site found in other Kir channels might act as a reservoir for PIP2 and that PIP2 moves to the new site at the interface with SUR1 once the channel opens?

We agree that the many topics raised by the reviewers are all very interesting and worth pursuing in the future. We have tried to include these and point out limitations of our data in relation to these questions in the results and discussion. It is our hope that the reviewers' comments will spark interest for further research related to these topics. For example, we plan to use MD simulation studies to address whether PIP2 bound at the conserved site may migrate to the novel site.

1. Page 9, lines 6-10: The authors suggest that the slower washout of long-chain PIP2 activation from excised patches compared to that of short-chain synthetic PIP2 is due to hydrophobic interactions between the longer acyl chains and KATP. However, this observation has been previously explained by the differences in the solubility of short- and long-chain PIP2 and therefore their rate of partition into and out of the plasma membrane. Is any data available to distinguish these possibilities?

We do not currently have data to distinguish these possibilities, but based on the structure it would be possible in the future to design mutations that perturb channel interaction with the acyl chains to dissect these possibilities.

1. Can the authors provide higher quality micrographs in Fig. S1 along with a scale bar. Why are three different micrographs shown? Also, this figure would probably benefit from moving some of the text embedded in the figure to a traditional legend along with a somewhat expanded description of what is shown graphically in the figure.

The different micrographs with KATP particles are included to show the different areas of the grid coated with graphene oxide. In one area, the folds of the graphene oxide layer are clearly visible.

1. In the main text when describing the results in Fig. 2D, it would be helpful for the general reader to first explain the protocol employing both low and high ATP concentrations and what value this has for assessing the impact of mutations. As it currently stands, the reader is left guessing why this expertly devised protocol was used.

We have revised the text to better explain the rationale of the recording protocol in both the Results section and Methods. Specifically, the alternating brief exposures to low and high ATP were designed to monitor the gradual decrease of ATP sensitivity during PIP2 exposure.

1. In Fig. 3 it would be helpful to align the three panels so the reader can appreciate how the structure gives rise to the pore radius plot in panel C. Also, the point made about the G-loop not changing appreciably between closed and opens states would be good to show in the structures.

We have revised this figure (now Fig. 4) according to the suggestion.

1. The G-loop was previously proposed to aid in preventing the leakage of K ions into the internal solution as polyamines block Kir channels (Xu et al, 2009 NSMB). It might be worth commenting on this as it seems compatible with what is found here in that region.

Since we do not have any direct experiments to test this, we have decided to leave out the reference in the current study.

1. Fig. 4A could be improved. The superimposition of open and closed structures in panel A takes some time for the reader to grasp. Maybe showing structures side by side with key distance measurements highlighting regions where there is movement between open and closed states would help, and then showing superimposition for a more limited view of where PIP2 binds? In panels B and C, it is not easy to appreciate how the structure in the open state disrupts the binding of ATP to the inhibitory site. Perhaps some use of space-filling models like those in Fig. S6 would help to illuminate the space occupied by ATP in the closed state, along with a zoomed-in view of all the residues coordinating ATP, and also similar views for how the conformational change during opening would interfere with ATP binding or move key coordinating residues. Fig. 4 contains a lot of information but it is not presented in a way that is easy for the reader to comprehend.

We have revised this figure (revised Fig. 5) as suggested and hope it is now improved.

1. In the figures, the authors focus their comparisons between the structure solved in this manuscript (open, PIP2 bound) and previous structures solved in the same lab (closed, ATP and/or inhibitors bound). While comparisons are made in the text to the open and 'pre-open' structures solved by other investigators, it might be clearer if visual comparisons were offered as well – especially of the interaction between the SUR1-W51 residue and the wild-type Kir6.2-Q52 residue in both other structures, the similarity of which offers support for the authors arguments about common structural rearrangements on page 17.

We have added a supplement figure (Fig. S6) to make this comparison.

1. Could the authors comment on how the Rb efflux assay results in Fig. 6 panel D add to the electrophysiological results shown in that figure in panels B and C? Differences in data from the flux assay in Fig. 6D may reflect changes in channel function, but they may simply reflect different expression levels for mutant channels.

The Rb efflux data shown in the original Fig. 6 (now Fig. 7) complement the electrophysiology data and show channel behaviour in intact cells. For all mutants we confirmed comparable expression levels in transfected cells by Western blots.

1. The map in Fig. 7 corresponds to both loss-of-function mutations, that cause diabetes, and gain-of-function mutations, that cause hyperinsulinism. Is it the opinion of the authors that these mutations mediate their effects by modulating PIP2 binding? LOF mutations could reduce PIP2 binding whereas GOF mutations could strengthen PIP2 binding.

We believe the reviewers meant loss-of-function mutations that cause hyperinsulinism and gain-of-function mutations that cause neonatal diabetes. As stated in the Discussion section, these mutations could exert their effects by modulating PIP2 binding or by affecting PIP2 gating allosterically.

1. As referred to above, Fig. S6 in DOI:10.1073/pnas.2112267118 shows lipid densities near the new PIP2 site – how do they compare to the location of the PIP2 densities resolved in this manuscript?

Please see the comparison we have now included in the revised Fig.S4a, b.

1. The idea advanced in the discussion and Fig. S6 that PIP2 binds to the new site only after the channel opens is interesting and seems conceptually related to what was recently proposed for PIP2 modulation of KCNQ by Mandala and MacKinnon (PNAS 2023). It might be helpful for the reader to see those dots connected.

We thank the reviewers for the suggestion and have now cited the paper by Mandala and MacKinnon (page 10 top, reference 49).

1. The allosteric models of ligand regulation of the KATP channel have been predicated on the existence of four PIP2 binding sites across the molecule – how does the existence of eight potential PIP2 binding sites alter previous attempts to quantitively model KATP activity (e.g. reviewed in DOI:10.1085/jgp.200308878 and DOI:10.1085/jgp.201711978)? Perhaps this deserves a comment.

This is an interesting question that would be an excellent topic for researchers who are interested in kinetic modeling.

1. The experiments described on pages 13-14 and ion Fig. 6 that explore the Kir6.2-Q52 and SUR1-W51 interaction are convincing, but the dose-response curves (especially for WT and the W51C-Q52R) would benefit from some lower concentrations of ATP.

We have conducted additional experiments to obtain data at lower ATP concentrations (see revised Fig. 7).

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

Reviewer #2 (Public Review):

Summary:

Jeong & Choi (2023) use a semi-naturalistic paradigm to tackle the question of how the activity of neurons in the mPFC might continuously encode different functions. They offer two possibilities: either there are separate dedicated populations encoding each function, or cells alter their activity depending on the current goal of the animal. In a threat-avoidance task rats procured sucrose in an area of a chamber where, after remaining there for some amount of time, a 'Lobsterbot' robot attacked. To initiate the next trial rats had to move through the arena to another area before returning to the robot encounter zone. Therefore the task has two key components: threat avoidance and navigating through space. Recordings in the IL and PL of the mPFC revealed encoding that depended on what stage of the task the animal was currently engaged in. When animals were navigating, neuronal ensembles in these regions encoded distance from the threat. However, whilst animals were directly engaged with the threat and simultaneously consuming reward, it was possible to decode from a subset of the population whether animals would evade the threat. Therefore the authors claim that neurons in the mPFC switched between two functional modes: representing allocentric spatial information, and representing egocentric information pertaining to the reward and threat.

Strengths:

As the authors point out, whilst these multiple functions of activity in the mPFC have generally been observed in tasks dedicated to the study of a singular function, less work has been done in contexts where animals continuously switch between different modes of behaviour in a more natural way. Being able to assess whether previous findings of mPFC function apply in natural contexts is very valuable to the field, even outside of those interested in the mPFC directly. This also speaks to the novelty of the work; although mixed selectivity encoding of threat assessment and action selection has been demonstrated in some contexts (e.g. Grunfeld & Likhtik, 2018) understanding the way in which encoding changes on-the-fly in a self-paced task is valuable for verifying whether current understanding holds true.

The authors are also generally thoughtful in their analyses and use a variety of approaches to probe the information encoded in the recorded activity. In particular, they also use relatively close analysis of behaviour as well as manipulating the task itself by removing the threat to verify their own results. The use of such a rich task also allows them to draw comparisons, e.g. in different zones of the arena or different types of responses to threats, that a more reduced task would not otherwise allow.

Weaknesses:

The central question the paper seeks to answer is whether 'individual cells are dedicated to spatial representation and emotional stimuli processing or if they adapt their function to the current goal'. However, there does not seem to be a direct analysis that answers this question. It is not clear what proportion of each of the ensembles recorded is necessary for decoding distance from the threat, and whether it is these same neurons that directly 'switch' to responding to head entry or withdrawal in the encounter phase within the total population. The PCA gets closest to answering this question by demonstrating that activity during the encounter is different from activity in the nesting or foraging zones, but in principle this could be achieved by neurons or ensembles that did not encode spatial parameters. The population analyses are focused on neurons sensitive to behaviours relating to the threat encounter, but even before dividing into subtypes etc., this is at most half of the recorded population. And again it is difficult to ascertain how the final ensemble analysis of the avoidance response relates to the prior spatial encoding. As a result, the model of the results proposed in Fig. 7 cannot be validated by the data as is.

A second concern is also illustrated by Fig. 7: in the data presented, separate reward and threat encoding neurons were not shown - in the current study design, it is not possible to dissociate reward and threat responses as the data without the threat present were only used to study spatial encoding integrity. To be able to claim this working model, a key additional analysis is to compare PETHs around head entry and withdrawal for sucrose without attack. Alternatively, a small proportion of probe trials could have been added where rats did not receive any reward for being in the encounter zone. This would allow the authors to ascertain whether the elevated response of the Type 2 neurons in particular is partially driven by reward receipt.

Thirdly, the findings of this work are not mechanistic or functional but are purely correlational. For example, it is claimed that analysing activity around the withdrawal period allows for ascertaining their functional contributions to decisions. But without a direct manipulation of this activity, it is difficult to make such a claim. The authors later discuss whether the elevated response of Type 2 neurons might simply represent fear or anxiety motivation or threat level, or whether they directly contribute to the decision-making process. As is implicit in the discussion, the current study cannot differentiate between these possibilities. However, the language used throughout does not reflect this.

Fourthly, the authors mention the representation of different functions in 'distinct spatiotemporal regions' but the bulk of the analyses, particularly in terms of response to the threat, do not compare recordings from PL and IL although - as the authors mention in the introduction - there is prior evidence of functional separation between these regions.

This vivid imagery alludes to lynching. Emmett Till was beaten, mutilated, shot in the head, and then thrown into the river with a cotton gin fan tied around his neck with barbed wire.

This vivid imagery alludes to lynching. Emmett Till was beaten, mutilated, shot in the head, and then thrown into the river with a cotton gin fan tied around his neck with barbed wire.

Dr. Ross - the head of Black studies; active in activism w students * miltant

I am most familiar with the term, "bots," in this way. In the sneaker head community, it is common for resellers to use bots that are programmed to instantaneously buy sneakers that have just been released. These bots are used by humans to get the cheapest retail price they possibly can so that they could resell to other people for a profit.

1988 - Soviet Active Measures in the Era of Glasnost publication date.

Author Response

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

eLife assessment

The manuscript describes the synergy among PI3Kbeta activators, providing compelling results concerning the mechanism of their activation. The particular strengths of the work arise to a great extent from the reconstitution system better mimicking the natural environment of the plasma membrane than previous setups have. The study will be a landmark contribution to the signaling field.

Public Reviews:

Reviewer #1 (Public Review):

The manuscript aims to provide mechanistic insight into the activation of PI3Kbeta by its known regulators tyrosine phosphorylated peptides, GTP-loaded Rac1 and G-protein beta-gamma subunits. To achieve this the authors have used supported lipid bilayers, engineered recombinant peptides and proteins (often tagged with fluorophores) and TIRF microscopy to enable bulk (averages of many molecules) and single molecule quantitation. The great strength of this approach is the precision and clarity of mechanistic insight. Although the study does not use "in transfecto" or in vivo models the experiments are performed using "physiologically-based" conditions and provide a powerful insight into core regulatory principles that will be relevant in vivo.

The results are beautiful, high quality, well controlled and internally consistent (and with other published work that overlaps on some points) and as a result are compelling. The primary conclusion is that the primary regulator of PI3Kbeta are tyrosine phosphorylated peptides (and by inference tyrosine phosphorylated receptors/adaptors) and that the other activators can synergise with that input but have relatively weak impacts on their own.

Although the methodology is not easily imported, for reasons of both cost and the experience needed to execute them well, the results have broad importance for the field and reverse an impression that had built in large parts of the broader signalling and PI3K communities that all of the inputs to PI3Kbeta were relatively equivalent, however, these conclusions were based on "in cell" or in vivo studies that were very difficult to interpret clearly.

Reviewer #2 (Public Review):

The manuscript of Duewell et al has made critical observations that help to understand the mechanisms of activation of the class IA PI3Ks. By using single-molecule kinetic measurements, the authors have made outstanding progress toward understanding how PI3Kbeta is uniquely activated by phosphorylated tyrosine kinase receptors, Gbeta/gamma heterodimers and the small G protein Rac1. While previous studies have defined these as activators of PI3Kbeta, the current manuscript makes clear the quantitative limitations of these previous observations. Most previous quantitative in vitro studies of PI3Kbeta activation have used soluble peptides derived from bis-phosphorylated receptors to stimulate the enzyme. These soluble peptides stimulate the enzyme, and even stimulate membrane interaction. Although these previous studies showed that the release of p85-mediated autoinhibition unmasks an intrinsic affinity of the enzyme for lipid membranes, they ignored what would be the consequence of these peptide sequences being present in the context of intrinsic membrane proteins. The current manuscript shows that the effect of membrane-conjugated peptides on the enzyme activity is profound, in terms of recruiting the enzyme to membranes. In this context, the authors show that G proteins associated with the membranes have an important contribution to membrane recruitment, but they also have a profound allosteric effect on the activity on the membrane, These are observations that would not have been possible with bulk measurements, and they do not simply recapitulate observations that were made for other class IA PI3Ks.

An important observation that the authors have made is that Gbeta/gamma heterodimers and RAc1 alone have almost no ability to recruit PI3Kbeta to the membranes that they are using, and this is central to one of the most profoundly novel activation mechanisms offered by the manuscript. The authors propose that the nSH2- and Gbeta/gamma binding sites partially overlap, so that Gbeta/gamma can only bind once the nSH2 domain releases the p110beta subunit. This mechanism would mean that once the nSH2 is engaged by membrane-conjugated pY, the Gbg heterodimer can bind and increase the association of the enzyme with membranes. Indeed, this increased membrane association is observed by the authors. However, the authors also show that this increased recruitment to membranes accounts for relatively little increase in activity, and that the far greater component of activation is due to an allosteric effect of the membrane association on the activity of the enzyme. The proposal for competition between Gbg binding and the nSH2 is consistent with the behavior of an nSH2 mutant that cannot bind to pY and which, consequently, does not vacate the Gbg-binding site. In addition to the outstanding contribution to understanding the kinetics of activation of PI3Kbeta, the authors have offered the first structural interpretation for the kinetics of Gbg activation in synergy with pY activation. The proposal for an overlapping nSH2/Gbg binding site is supported by predictions made by John Burke, using alphafold multimer. Although there is no experimental structure to support this structural model, it is consistent with HDX-MS analyses that were published previously.

Reviewer #1 (Recommendations For The Authors):

1. The approx relative concentrations (surface densities ) of Rac1-GTP, GBetagammas and PY-peptides used in experiments in Fig 1 are not easy to understand and useful to give an intuitive feel for the relative sensitivity of the PI3Kbeta reporter to those inputs.

In our revised manuscript, we provide densities of the individual signaling inputs used to reconstitute Dy647-PI3Kβ membrane recruitment (see Figure legend 1). We provide a more detailed explanation about our quantification method in subsequent figures where the membrane surface density of signaling inputs is varied to modulate the strength of PI3Kβ membrane localization and activity.

Building off the quantification of Rac1-GTP and pY membrane density measurements presented in our initial manuscript submission, we now include an estimate of the GβGγ membrane density. For these new measurements, we recombinantly expressed and purified additional SNAP-GβGγ protein, which we fluorescently labeled with AlexaFluor 555. The membrane surface density of GβGγ was quantified at equilibrium using a combination of AF488-SNAP-GβGγ (bulk signal) and dilute AF555-SNAP-GβGγ (0.0025%), which allowed us to resolve and count the single molecule density (Figure 3A). We calculate the total surface density of GβGγ based on the AF555-SNAP-GβGγ dilution factor. In the methods section titled, “surface density calibration,” we describe our protocol.

1. The estimates of the PIP3 concentrations/densities measured using the BTK reporter seem good but its unclear (to me) how they were derived.

The density of PI(3,4,5)P3 lipids in our supported lipid bilayers was calculated based on the incorporation of a define molar ratio of PI(3,4,5)P3 in our small unilamellar vesicles. Based on the average footprint of 0.72 nm2 for a single lipid, we calculated the density of lipids per µm2. In the methods section titled, “kinetic measurements of PI(3,4,5)P3 lipid production,” we include the following description:

“Assuming an average footprint of 0.72 nm2 for phosphatidylcholine (Carnie et al., 1979; Hansen et al., 2019), we calculated a density of 2.8 × 104 PI(3,4,5)P3 lipids/μm2 for supported membranes that contain an initial concentrations of 2% PI(4,5)P2. We assume that the plateau fluorescence intensity of the AF488-SNAP-Btk sensor following reaction completion in the presence of PI3Kβ represents the production of 2% PI(3,4,5)P3. The bulk membrane intensity of AF488-SNAP-Btk was normalized from 0 to 1, and then multiplied times the total density of PI(3,4,5)P3 lipids to generate kinetic traces that report the kinetics of PI(3,4,5)P3 production.”

Minor points

l164; Rac1(GTP) AND GBeta gammas. In this context it should be OR. Or have I misunderstood?

l1093; kineticS measurementS.

Thank you for pointing out these typos. We made the appropriate edits.

The paper of Suire etal (Suire, S., Lécureuil, C., Anderson, K. E., Damoulakis, G., Niewczas, I., Davidson, K., Guillou, H., Pan, D., Jonathan Clark, Phillip T Hawkins, & Stephens, L. (2012). GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4. The EMBO journal, 31(14), 3118-3129. https://doi.org/10.1038/emboj.2012.167) make the point that in vivo it appears that although Ras-activation is required for full activation of PI3Kgamma (and can activate PI3Kgamma in vitro directly) if you use tools to activate Ras in the absence of receptor and Gbetagamma signalling, it has no affect on PIP3 . This directly supports the authors conclusions.

Thank you for sharing this citation. We incorporated the reviewer’s insight into our discussion section to broaden the significance of our work.

Reviewer #2 (Recommendations For The Authors):

There are only a few relatively minor points that could be addressed to improve the paper:

1. Why is the density still going up after 10 minutes in Figure 1 Figure supplement 2? Doesn't this seem like a very long time? Are we seeing fast on/off combined with fast on/slow off? Are the particles eventually becoming stuck in odd places or are they slowly denaturing?

Our movies do not indicate a slow accumulation of immobilized or stuck Dy647-PI3Kβ particles on the membrane surface. On the long timescale, we believe that a small fraction of Dy647-PI3Kβ molecular do exhibit longer dwell times on membranes containing a high density of pY (>6,000 molecules/µm2). This is likely due to membrane hopping of Dy647-PI3Kβ. In other words, rather than Dy647-PI3Kβ dissociating from the membrane surface directly into the solution, the Dy647-PI3Kβ molecule immediately rebinds to another membrane conjugated pY peptide. This type of behavior of a peripheral membrane binding protein is generally correlated with there being a higher surface density of the binding partner (Yasui et al., 2014). Characterization of potential Dy647-PI3Kβ membrane hopping will require additional experimentation (e.g. PI3Kβ mutants) and quantitative analysis that goes beyond the scope of this study.

1. Lines 188-189. "By quantifying the average number of Alexa488-pY particles per unit area of supported membrane we calculated the absolute density of pY per μm2 (Figure 2D). I think this should be Figure 2C, right hand y-axis.

Thank you for identifying our typo. We’ve corrected the text for clarity.

1. Lines 102-193. "When Dy647-PI3Kβ was flowed over a membrane containing a low density of {less than or equal to} 500 pY/μm2, we observed rapid equilibration kinetics consistent with a 1:1 binding stoichiometry (Figure 2E).” There is no density shown in Fig. 2E. There is only "membrane intensity." Perhaps it was their intent to include a right-hand axis with density (number of particles/area), as they did in Figure 2C. However, they did not, so Figure 2E does not support the text. The value of Intensity/#py/um**2 does not appear to be the same for Figure 2C as for Figure 2E, assuming that the statement in the text is correct. The authors should include the density as a right-hand axis in 2E.

We have reworded this portion of the results section for clarity. In reading the reviewers comment, we recognize that a more convincing way to support our claim of a 1:1 binding stoichiometry would be to show that there are ~500 Dy647-PI3Kβ/μm2 membrane bound complexes when the pY surface density equals ~500 pY/μm2. For us to make this connection, we would need to perform experiments using a Dy647-PI3Kβ concentration that fully saturates all the binding pY binding sites. However, at this elevated Dy647-PI3Kβ solution concentration, individual Dy647-PI3Kβ complexes can start to bind to a single phosphotyrosine of the dually phosphorylated peptide due to competition for pY binding sites. As an alternative to performing the experiment described above, we can infer binding stoichiometry from the shape of the membrane absorption kinetic traces. For example, a simple bimolecular interaction exhibits rapid equilibration kinetics with a hyperbolic shaped kinetic trace. Systems that have more complex binding equilibria, however, generally take longer to equilibrate (due to the change in KOFF) and can often be broken down into 2 or 3 distinct dissociation constants (KD). This type of kinetic analysis has previously been used to describe multivalent membrane binding interactions for the Btk-PI(3,4,5)P3 (Chung et al., 2019) and PI3Kγ-GβGγ (Rathinaswamy et al., 2021) complexes. Considering that there are multiple interpretations of the Dy647-PI3Kβ membrane absorption traces show in Figure 2E, we refrain from saying that our results explicitly reveal a 1:1 binding stoichiometry. Instead, we provide several possible explanations for the results. Ultimately, additional experiments and kinetic modeling of wild type and mutant PI3Kβ is necessary to define the binding stoichiometry under different conditions.

1. Table 1. The authors have analysed the data to extract two dwell times and two diffusion coefficients. The legend should make this clear, referring to D1 as the slow diffusion component and D2 as fast diffusion, similarly, there are short and long dell times. This should be stated in the legend. There are two columns labelled "alpha". This presumably should be alpha1 and alpha2, the fractions of particles with short and long dwell times. The table legend should clarify this.

In our revision, additional text has been added to the figure legends and Table 1.

Text from Table 1: “Alpha (α) equals the fraction of molecules with the characteristic dwell time, τ1 (DT = dwell time). The fraction of molecules with the characteristic dwell time, τ2, equals 1-α. Alpha (αD) equals the fraction of molecules with the characteristic diffusion coefficient, D1. The fraction of molecules with diffusion coefficient, D2, equals 1-αD.”

1. In the legend for Figure 5 figure supplement 1, for part D, the "Cumulative membrane of binding events..." The "of" should be deleted.

Thank you for identifying this typo.

1. Lines 423-426: "We found that PI3Kβ kinase activity is also relatively insensitive to either Rac1(GTP) or GβGγ alone. This is in contrast to previous reports that showed Rho-GTPases (Fritsch et al. 2013) and GβGγ (Katada et al. 1999; Hashem A. Dbouk et al. 2012; Maier, Babich, and Nürnberg 1999) can activate PI3Kβ, albeit modest, compared to synergistic activation with pY peptides plus Rac1(GTP) or GβGγ." It is not clear what this statement means. On the surface, it might be interpreted as saying that these previous studies had some flaw that led the authors to conclude that there is some activation caused by Rac1 or Gbeta/gamma on their own. The current manuscript is an important contribution to understanding the mechanism of synergistic activation, but it is also true that the Hansen and his colleagues have not used the same membranes as were used previously. The authors state that they have used a wide range of membrane compositions, but the only ones that have appeared in the manuscript are nearly pure PC (with 2% PIP2) or PC with 20% PS. Extensive studies with varying membrane compositions are beyond the scope of the current study, since the current manuscript concisely makes important observations regarding mechanism. However, it would be helpful for readers if the authors at least mention the differences in membrane compositions among the studies.

The reviewer raises an important point concerning our interpretation of PI3Kβ activation data in relationship to existing literature. In our original submission, we made conclusions concerning how individual signaling inputs modulate PI3Kβ activity, without showing all our data or providing sufficient explanation. In our revised manuscript, we include PI3Kβ kinase activity measurements performed in the presence of either pY, Rac1(GTP), or GβGγ alone (Figure 5B-5C). These experiments were reconstituted on supported membranes in the absence or presence of 20% PS lipids. We found that increasing the density of anionic lipids increased the overall activity of PI3Kβ in the presence of pY or GβGγ alone. This is consistent with a subtle increase in PI3Kβ membrane affinity due to the negatively charged PS lipids. Mutations that disrupt the direct interaction between PI3Kβ and GβGγ eliminated the observed lipid kinase activity. We were unable to detect PI3Kβ activity in the presence of Rac1(GTP) alone. In conclusion, we’re able to detect some PI3Kβ activity in the presence of GβGγ alone, which is consistent with previous reports (Dbouk et al., 2010; Katada et al., 1999; Maier et al., 2000). In the future, a more comprehensive analysis will be required to map the relationship between PI3Kβ activity, membrane localization, and lipid composition. For example, previous reconstitutions have revealed differential activation of PI3Kα that depends on the most abundant lipid being phosphatidylethanolamine (PE) rather than phosphatidylcholine (PC) (Hon et al., 2012; Ziemba et al., 2016). PE lipids comprise 25-30% of the cellular plasma membrane (Yang et al., 2018) and have been used in previous studies to measure PI3K lipid kinase activity on small unilamellar vesicles (Dbouk et al., 2010; Hon et al., 2012).

In this study, we elected to use a simplified membrane composition that minimized non-specific membrane localization of fluorescently labeled PI3Kβ. This allowed us to more clearly define the strength of individual and combinations of protein-protein interactions that regulate PI3Kβ localization and kinase activity. When reconstituting amphiphilic molecules (i.e. lipids) in aqueous solution a variety of structures, including micelles, inverted micelles, and planar bilayers can form based on the lipid composition (Kulkarni, 2019). The organization of these membrane structures is related to the molecular packing parameter of the individual phospholipids (Israelachvili et al., 1976). The packing parameter (P=v⁄((a•l_c))) depends on the volume of the hydrocarbon (v), area of the lipid head group (a), and the lipid tail length (l_c). When generating supported lipid bilayers on a flat two-dimensional glass surface, we aim to create a fluid lamellar membrane. We find that phosphatidylcholine (PC) lipids are ideal for making supported lipid bilayers because they have a packing parameter of ~1 (Costigan et al., 2000). In other words, PC lipids are cylindrical like a paper towel roll. In contrast, cholesterol and phosphatidylethanolamine (PE) lipids have packing parameters of 1.22 and 1.11, respectively (Angelov et al., 1999; Carnie et al., 1979). This gives cholesterol and PE lipids an inverted truncated cone shape, which prefers to adopt a non-lamellar phase structure. Due to the intrinsic negative curvature of PE lipids, they can spontaneously form inverted micelles (i.e. hexagonal II phase) in aqueous solution when they are the predominant lipid species (Israelachvili et al., 1980; Kobierski et al., 2022; Wnętrzak et al., 2013). In the methods section of our manuscript, we note that from our experience incorporation of PE lipids dramatically reduces the protein-maleimide coupling efficiency, displayed more membrane defects, and resulted in a larger fraction of surface immobilized Dy647-PI3Kβ. This could be related to the intrinsic negative curvature of PE membranes. However, further investigation is needed to decipher these issues.

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I think that racism is prominent within these types of educators because they see themselves as "great white men" not only that but because they view whiteness as being the norm. Other cultures and people are viewed as wrong or wierd. This is also true with heteronormativity and the long history the US has had with the nuclear family where there is a white normative family with the father as the head of the house hold. These ideas need to be challenged because it doesn't help students of different cultures learn or succeed in school if their professor does not want them to.

I see the paradox shows a fundamental tension in the American dream. The dream starts with an egalitarian idea, but in real life, success often rests on the advantages one gets from one's parents. This keeps differences going and calls into question the idea that all generations should have the same chances. People want to be successful on their own, but they also want society to be fair, which leads to conflict. These ideas are inherently at odds with each other, which makes it hard to make decisions about education policy. The need for individual success and fairness for everyone seems impossible to combine.

Author Response

eLife assessment

This study provides direct evidence showing that Kv1.8 channels underly several potassium currents in the two types of sensory hair cells found in the mouse vestibular system. This is an important finding because the nature of the channels underpinning the unusual potassium conductance gK,L in type I hair cells has been under scrutiny for many years. Although most of the experimental evidence is compelling and the analysis is rigorous, the evidence supporting some of the claims related to Kv1.4 channels is incomplete. The study will be of interest to cell and molecular biologists and auditory neuroscientists.

We are thankful to the editor and reviewers for their thorough assessment of our work and insightful feedback. Our responses to the comments and suggestions are below.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage-activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:

The experiments are comprehensive and clearly described, both in the text and in the figures. Statistical analyses are provided throughout.

Weaknesses:

None.

Reviewer #2 (Public Review):

The focus of this manuscript was to investigate whether Kv1.8 channels, which have previously been suggested to be expressed in type I hair cells of the mammalian vestibular system, are responsible for the potassium conductance gK,L. This is an important study because gK,L is known to be crucial for the function of type I hair cells, but the channel identity has been a matter of debate for the past 20 years. The authors have addressed this research topic by primarily investigating the electrophysiological properties of the vestibular hair cells from Kv1.8 knockout mice. Interestingly, gK,L was completely abolished in Kv1.8-deficient mice, in agreement with the hypothesis put forward by the authors based on the literature. The surprising observation was that in the absence of Kv1.8 potassium channels, the outward potassium current in type II hair cells was also largely reduced. Type II hair cells express the largely inactivating potassium conductance gK,A, but not gK,L. The authors concluded that heteromultimerization of non-inactivating Kv1.8 and the inactivating Kv1.4 subunits could be responsible for the inactivating gK,A. Overall, the manuscript is very well written and most of the conclusions are supported by the experimental work. The figures are well described, and the statistical analysis is robust.

My only comment relates to the statement regarding the results providing "evidence" that Kv1.4 form heteromultimers with Kv1.8 channels (see Discussion). The only data I can see from the results is that Kv1.4 channels are expressed in the membrane of type II hair cells, which is not sufficient evidence for the above claim. Is the distribution of Kv1.8 and Kv1.4 overlapping in type II hair cells? Have the authors attempted to perform some pharmacological studies on Kv1.4? For example, would gK,A be completely blocked by a Kv1.4 antagonist? Addressing at least some of these questions would strengthen your argument.

Author response: With respect to the “evidence” for heteromultimerization of Kv1.4 and Kv1.8: We agree that there is not conclusive evidence but have pulled together reasons to suggest that the fast inactivation of Kv1.8-dependent gA in type II hair cells reflects a contribution from Kv1.4 subunits. The reasons we note are mostly from other sources: 1) Kv1.4 subunits are the only Kv1 alpha subunits known to make channels with intrinsic rapid inactivation (Bertoli et al., 1994); 2) Kv1.4 is highly expressed in type II hair cells, but not type I hair cells, in mouse utricle (McInturff et al., Biol. Open., 2018; Jan et al., Cell Reports, 2021; Orvis et al., Nat. Methods, 2021); 3) previous work from M. Correia and colleagues suggested Kv1.4 as the likely source of A-current in pigeon vestibular hair cells; 4) some rat type II hair cells show comparatively strong Kv1.4-like immunoreactivity (our Fig. 5). While we consider heteromultimerization of Kv1.4 and Kv1.8 alpha subunits a plausible explanation consistent with available data from different sources, we agree that the question is not at all settled, and indeed raise the possibility that KV beta subunits, which are also differentially expressed by type I and II hair cells, play a role. Experiments to definitively advance or refute this hypothesis are beyond the scope of this paper.

Reviewer #3 (Public Review):

Summary:

This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular, amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts, and heterozygotes) are examined here and whole-cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in the current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition, immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that the removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

1. From previous work it is known that GK,L in type I hair cells has unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+, and modulated by cyclic GMP. The question arises, if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells and immunostaining for Kv1.4 channels in rat utricle presented here in Fig. 6. However, hair cell-specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

Author response: Our results raise the question of how Kv1.8/Kcna10 is regulated to produce gK,L in type I hair cells, which has different properties from the Kv1.8 conductance expressed heterologously (Lang et al., Am. J. Physiol. Renal Physiol., 2000; Ranjan et al., Front. Cell. Neurosci., 2019; Dierich et al., Cell Reports, 2020) and the Kv1.8 conductance inferred in inner hair cells (Dierich et al., 2020). We lay out several possibilities in the Discussion, but testing these suggestions is beyond the scope of the present paper.

The relatively high Cs+ permeability of gK,L (0.31 pCs/pK, Rüsch & Eatock, J. Neurophysiol., 1996; Rennie & Correia, J. Membr. Biol., 2000) suggests there is something different about the selectivity filter and pore region of gK,L relative to most Kv1 family members. Although the intrinsic Cs+ permeability of heterologously expressed Kv1.8 is not reported. While we note that the pore region in Kv1.8 differs from other Kv1 subunits by a single amino acid (a glycine instead of alanine at position 411 – placed by AlphaFold in the pore helix of hKCNA10, Jumper et al., Nature, 2021), the effect of this difference is not known. A separate study is needed to determine why gK,L has a high Cs+ permeability relative to other Kv channels.

For type II hair cells, the Cs+ permeability of Kv currents has not been fully characterized. Internal Cs+ does appear to reduce outward current more effectively in type II hair cells (Lang & Correia, J. Neurophysiol., 1989; Sokolowski et al., Dev. Biol., 1993) than in type I hair cells (Rüsch & Eatock, J. Neurophysiol., 1996; Rennie & Correia, J. Membr. Biol., 2000).

With respect to cochlear inner hair cells, note that the assignment of Kv1.8 by Dierich et al. (2021) to a delayed rectifier in cochlear inner hair cells (IHCs) was based on inference – that is, existing inner ear expression databases show that Kv1.8 is expressed in IHCs, and heterologous Kv1.8 channels have a current resembling that observed in IHCs after block of multiple other K channels. We agree with Dierich et al. that Kv1.8 is an attractive candidate for the residual conductance in cochlear IHCs based on comparison with its properties in heterologous expression data. Together their study and our study suggest that Kv1.8 takes on quite different voltage dependence depending on the hair cell environment, and it will be an interesting challenge to sort out the reasons.

1. Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

Author response: Our pharmacology with XE991 suggests a small but significant population of Kv7 channels in type I and II hair cells (Fig 7). With the immunogold technique, Kharkovets et al. (PNAS, 2000) and Hurley et al. (J. Neurosci., 2006) counted significant Kv7.4 particles in type I hair cells, although the particles occurred at much greater density in the postsynaptic calyx membrane facing the hair cell. These results lead us to propose that the Kv7 channel we identified pharmacologically includes the Kv7.4 subunit, possibly in combination with other Kv7 subunits (Lysakowski et al., J. Neurosci., 2011). By this argument, the absence of clear hair cell staining in the confocal images of Fig. 5A is likely to reflect differences in methods, which include the use of different mouse strains, different sensitivities of immunogold vs. confocal imaging, and different antibodies.

Holt et al. (J. Neurosci., 2007) indeed saw inhibition of gK,L in hair cells grown in organotypic cultures of the neonatal mouse utricle after viral expression of a dominant negative Kv7.4 construct. However, other studies show that Kv7 antagonists do not block gK,L (Hurley et al., J. Neurosci., 2006), and the Jentsch group, which first proposed Kv7.4 as a likely candidate for gK,L (Kharkovets et al., PNAS, 2000), ultimately showed that knocking out Kv7.4 and Kv7.5 expression failed to eliminate gK,L (Spitzmaul et al., J. Biol. Chem., 2013). Together, these results suggest that in Holt et al. (2007), the inhibition of gK,L by transfection with the dominant negative KCNQ4 construct may have occurred through unintended interactions with native gK,L channels. The young age of the neonatal cultured and transfected utricles raises the possibility of a developmental effect – that functional Kv7 channels are needed for the developmental transition to a Kv1.8 conductance. Consistent with this idea is the observation that Kv7 current is present in neonatal hair cells, where it is a relatively large proportion of Kv current in type I HCs before they acquire gK,L (Hurley et al., J. Neurosci., 2006). Alternatively, the overexpression of nonfunctional Kv7.4 channels in virally-transfected hair cells may have inhibited or delayed gK,L acquisition through a more general effect on membrane proteins.

1. Strong immunosignal appears in the cuticle plates of hair cells in addition to signal in basal regions of hair cells and supporting cells. Please provide a possible explanation for this.

Author response: There is significant non-specific staining of apical cell surfaces and supporting cell membranes in addition to specific staining of hair cell basolateral membranes. We infer non-specific staining when immunolabeling is present in the knockout tissue, as it is for the apical surfaces and supporting cell membranes—compare Fig. 5B.3 (control tissue) with Fig. 5B.4 (Kv1.8 null mutant). Non-specific immunostaining can occur with polyclonal antibodies (specific to several epitopes) if the antibodies are not affinity-purified, but we used an affinity-purified antibody. The apical surfaces are reputed to be “sticky” (susceptible to non-specific staining) but the non-specific labeling in the basal parts of supporting cells is more puzzling. One possibility is that the Kv1.8 antibody weakly recognized closely related Kv1.1 channels, which are more strongly expressed in supporting cells than hair cells (Scheffer et al., J. Neurosci., 2015).

1. A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be very interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Author response: We agree; some of these questions are the subject of another paper in preparation.

Reviewer #1 (Public Review):

Summary:<br /> In this paper, the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage-activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:<br /> The experiments are comprehensive and clearly described, both in the text and in the figures. Statistical analyses are provided throughout.

Weaknesses:<br /> None.

Reviewer #2 (Public Review):

Summary:<br /> Tian et al. explore the developmental organs of cortical reorganization in blindness. Previous work has found that a set of regions in the occipital cortex show different functional responses and patterns of functional correlations in blind vs. sighted adults. In this paper, Tian et al. ask: how does this organization arise over development? Is the "starting state" more like the blind pattern, or more like the adult pattern? Their analyses reveal that the answer depends on the particular networks investigated; some functional connections in infants look more like blind than sighted adults; other functional connections look more like sighted than blind adults; and others fall somewhere in the middle, or show an altogether different pattern in infants compared with both sighted and blind adults.

Strengths:<br /> The question raised in this paper is extremely important: what is the starting state in development for visual cortical regions, and how is this organization shaped by experience? This paper is among the first to examine this question, particularly by comparing infants not only with sighted adults but also blind adults, which sheds new light on the role of visual (and cross-modal) experience. Another clear strength lies in the unequivocal nature of many results. Many results have very large effect sizes, critical interactions between regions and groups are tested and found, and infant analyses are replicated in split halves of the data.

Weaknesses:<br /> A central claim is that "infant secondary visual cortices functionally resemble those of blind more than sighted adults" (abstract, last paragraph of intro). I see two potential issues with this claim. First, a minor change: given the approaches used here, no claims should be made about the "function" of these regions, but rather their "functional correlations". Second (and more importantly), the claim that the secondary visual cortex in general resembles blind more than sighted adults is still not fully supported by the data. In fact, this claim is only true for one aspect of secondary visual area functional correlations (i.e., their connectivity to A1/M1/S1 vs. PFC). In other analyses, the infant secondary visual cortex looks more like sighted adults than blind adults (i.e., in within vs. across hemisphere correlations), or shows a different pattern from both sighted and blind adults (i.e., in occipito-frontal subregion functional connectivity). It is not clear from the manuscript why the comparison to PFC vs. non-visual sensory cortex is more theoretically important than hemispheric changes or within-PFC correlations (in fact, if anything, the within-PFC correlations strike me as the most important for understanding the development and reorganization of these secondary visual regions). It seems then that a more accurate conclusion is that the secondary visual cortex shows a mix of instructive effects of vision and reorganizing effects of blindness, albeit to a different extent than the primary visual cortex.

Relatedly, group differences in overall secondary visual cortex connectivity are particularly striking as visualized in the connectivity matrices shown in Figure S1. In the results (lines 105-112), it is noted that while the infant FC matrix is strongly correlated with both adult groups, the infant group is nonetheless more strongly correlated with the blind than sighted adults. I am concerned that these results might be at least partially explained by distance (i.e., local spread of the bold signal), since a huge portion of the variance in these FC matrices is driven by stronger correlations between regions within the same system (e.g., secondary-secondary visual cortex, frontal-frontal cortex), which are inherently closer together, relative to those between different systems (e.g., visual to frontal cortex). How do results change if only comparisons between secondary visual regions and non-visual regions are included (i.e., just the pairs of regions within the bold black rectangle on the figure), which limits the analysis to long-rang connections only? Indeed, looking at the off-diagonal comparisons, it seems that in fact there are three altogether different patterns here in the three groups. Even if the correlation between the infant pattern and blind adult pattern survives, it might be more accurate to claim that infants are different from both adult groups, suggesting both instructive effects of vision and reorganizing effects of blindness. It might help to show the correlation between each group and itself (across independent sets of subjects) to better contextualize the relative strength of correlations between the groups.

It is not clear that differences between groups should be attributed to visual experience only. For example, despite the title of the paper, the authors note elsewhere that cross-modal experience might also drive changes between groups. Another factor, which I do not see discussed, is possible ongoing experience-independent maturation. The infants scanned are extremely young, only 2 weeks old. Although no effects of age are detected, it is possible that cortex is still undergoing experience-independent maturation at this very early stage of development. For example, consider Figure 2; perhaps V1 connectivity is not established at 2 weeks, but eventually achieves the adult pattern later in infancy or childhood. Further, consider the possibility that this same developmental progression would be found in infants and children born blind. In that case, the blind adult pattern may depend on blindness-related experience only (which may or may not reflect "visual" experience per se). To deal with these issues, the authors should add a discussion of the role of maturation vs. experience and temper claims about the role of visual experience specifically (particularly in the title).

The authors measure functional correlations in three very different groups of participants and find three different patterns of functional correlations. Although these three groups differ in critical, theoretically interesting ways (i.e., in age and visual/cross-modal experience), they also differ in many uninteresting ways, including at least the following: sampling rate (TR), scan duration, multi-band acceleration, denoising procedures (CompCor vs. ICA), head motion, ROI registration accuracy, and wakefulness (I assume the infants are asleep).

Addressing all of these issues is beyond the scope of this paper, but I do feel the authors should acknowledge these confounds and discuss the extent to which they are likely (or not) to explain their results. The authors would strengthen their conclusions with analyses directly comparing data quality between groups (e.g., measures of head motion and split-half reliability would be particularly effective).

I really love this concept of celebrating and encouraging students and educators to speak up against injustices. It seems highly different to the "keep your head down and the traditions strong" thought process that dominated most of my primary and secondary schooling.

We just finished watching as well and I agree, it really sucked me in! I did have to turn my head/walk away quite a bit during the last episode though!

Reviewer #2 (Public Review):

The authors succeed in generalizing the pre-alignment procedure for their cell identification method to allow it to work effectively on data with only small subsets of cells labeled. They convincingly show that their extension accurately identifies head angle, based on finding auto florescent tissue and looking for a symmetric l/r axis. They demonstrate method works to allow the identification of a particular subset of neurons. Their approach should be a useful one for researchers wishing to identify subsets of head neurons in C. elegans, and the ideas might be useful elsewhere.

The authors also assess the relative usefulness of several atlases for making identity predictions. They attempt to give some additional general insights on what makes a good atlas, but here insights seem less clear as available data does not allow for experiments that cleanly decouple: 1. the number of examples in the atlas 2. the completeness of the atlas. and 3. the match in strain and imaging modality discussed. In the presented experiments the custom atlas, besides the strain and imaging modality mismatches discussed is also the only complete atlas with more than one example. The neuroPAL atlas, is an imperfect stand in, since a significant fraction of cells could not be identified in these data sets, making it a 60/40 mix of Openworm and a hypothetical perfect neuroPAL comparison. This waters down general insights since it is unclear if the performance is driven by strain/imaging modality or these difficulties creating a complete neuroPal atlas. The experiments do usefully explore the volume of data needed. Though generalization remains to be shown the insight is useful for future atlas building that for the specific (small) set of cells labeled in the experiments 5-10 examples is sufficient to build a accurate atlas.

I should be able to name all the greek gods and dieties like this off the top of my head

Author Response

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

Reviewer #1 (Public Review):

In this study, single neurons were recorded, using tetrodes, from the parahippocampal cortex of 5 rats navigating a double-Y maze (in which each arm of a Y-maze forks again). The goal was located at any one of the 4 branch terminations, and rats were given partial information in the form of a light cue that indicated whether the reward was on the right or left side of the maze. The second decision point was uncued and the rat had no way of knowing which of the two branches was correct, so this phase of the task was more akin to foraging. Following the outbound journey, with or without reward, the rat had to return (inbound journey) to the maze and start to begin again.

Neuronal activity was assessed for correlations with multiple navigation-relevant variables including location, head direction, speed, reward side, and goal location. The main finding is that a high proportion of neurons showed an increase in firing rate when the animal made a wrong turn at the first branch point (the one in which the correct decision was signalled). This increase, which the authors call rate remapping, persisted throughout the inbound journey as well. It was also found that head direction neurons (assessed by recording in an open field arena) in the same location in the room were more likely to show the rate change. The overall conclusion is that "during goal-directed navigation, parahippocampal neurons encode error information reflective of an animal's behavioral performance" or are "nodes in the transmission of behaviorally relevant variables during goal-directed navigation."

Overall I think this is a well-conducted study investigating an important class of neural representation: namely, the substrate for spatial orientation and navigation. The analyses are very sophisticated - possibly a little too much so, as the basic findings are relatively straightforward and the analyses take quite a bit of work to understand. A difficulty with the study is that it was exploratory (observational) rather than hypothesis-driven. Thus, the findings reveal correlations in the data but do not allow us to infer causal relationships.

We would like to clarify that this report consists of hypothesis-driven experiments, with post-hoc exploratory analyses. We have now made hypotheses more explicit in the text, and pointed out that follow-up analyses were to understand how these effects came to be. We thank the reviewer for pointing out that our hypotheses were not explicit in the introduction. We believe our results open the door for investigating the causal role of these regions in the propagation or generation of error signals during navigational behavior. Those types of experiments are however, outside the scope of the current work.

That said, the observation of increased firing in a subset of neurons following an erroneous choice is potentially interesting. However, the effect seems small. What were the actual firing rate values in Hz, and what was the effect size?

We thank the reviewer for the opportunity to clarify the effect size question. As there are multiple neurons in the analyses, differences in firing rate need necessarily to be normalized by a neuron's mean activity. For example, a difference of 1 spk/s is less meaningful when a neuron's base rate is 50 spk/s than when it is 10spks/s. Furthermore, our reports are for population level analyses, at which point comparing raw firing rate values and differences becomes more challenging. Nonetheless, we are including these raw metrics in two new supplemental figures (Figure 2 - figure supplement 4,5), where differences in individual neurons change can be up to 15 spks/s. Additionally, the patterns and statistical results observed in the main text are preserved, with outbound Right Cue minus Left Cue showing a left>stem>right (indicating error coding), and RW minus NRW showing negative values across all segments, indicating NRW>RW or higher activity following on inbound unrewarded trials. Statistics follow the corresponding main text results (Cue: segment LRT = 71.70; RW: segment LRT=45.80).

I also feel we are lacking information about the underlying behavior that accompanies these firing rate effects. The authors say "one possibility is that the head-direction signal in the parahippocampal region reflects a behavioral state related to the navigational choice or the lack of commitment to a particular navigational route" which is a good thought and raises the possibility that on error trials, rats are more uncertain and turn their heads more (vicarious trial and error) and thus sample the preferred firing direction more thoroughly. Another possibility is that they run more slowly, which is associated with a higher firing rate in these cells. I think we, therefore, need a better understanding of how behavior differed between error trials in terms of running speed, directional sampling, etc.

In terms of running speed, there was a small effect of mean running speed between correct and incorrect trials (across subjects LMEM: Cue correct>incorrect Z=2.3, p=0.02; RW Z=2.15, p=0.03). In most neurons, increases in speed will be accompanied by increases in firing rate. Thus, the differences in running speed cannot explain the observed results, as higher speed during correct trials would predict higher activity, which is the opposite of what we found.

A few good, convincing raw-data plots showing a remapping neuron on an error trial and a correct trial on the same arm would also be helpful (the spike plots were too tiny to get a good sense of this: fewer, larger ones would be more helpful).

Additional plots for individual units have been added, Figure 2 - figure supplement 3.

It would be useful to know at what point the elevated response returned to baseline, how - was it when the next trial began, and was the drop gradual (suggesting perhaps a more neurohumoral response) or sudden.

Due to the experimental design, this question cannot be addressed fully. Concretely, error trials incur a time-penalty in which the rats need to wait an additional 10 seconds before the next trial, while a new trial would start immediately when the animal nose-poked the home well after a correct trial. Nonetheless, the data on Reward provides insight into this question. The magnitude of the responses on left and right segments of the maze were larger than on the stem for Unrewarded (NRW) vs Rewarded (RW) trials on inbound trajectories, Fig. 4c. This suggests that while activity is still elevated post-incorrect throughout the maze, across units, this effect is smaller on the stem segment. Additionally, the analyses indicate that in the transition between outbound vs inbound trajectories (Figure 4 - figure supplement 3), activity patterns are sustained (incorrect>correct). Together, these results indicate that elevated "error-like" signal are slow in returning to baseline.  

Reviewer #2 (Public Review):

This work recorded neurons in the parahippocampal regions of the medial entorhinal cortex (MEC) and pre- and para-subiculum (PrS, PaS) during a visually guided navigation task on a 'tree maze'. They found that many of the neurons reflected in their firing the visual cue (or the associated correct behavioral choice of the animal) and also the absence of reward in inbound passes (with increased firing rate). Rate remapping explained best these firing rate changes in both conditions for those cells that exhibited place-related firing. This work used a novel task, and the increased firing rate at error trials in these regions is also novel. The limitation is that cells in these regions were analyzed together.

We acknowledge this limitation on our study, and we believe there might be interesting differences between these regions. Unfortunately, the post-mortem extraction of the recording implant micro-drive used for these experiments generated too much tissue damage for exact localization of the tetrodes. Nonetheless, given that the patterns were observed in all subjects, we are confident that at least the major findings of "error-like" signaling is present across the parahippocampal regions. At the same time, the distributions of functional cell types as defined in the open field are different across the PaS, PrS and MEC, leaving the possibility of a more nuanced error coding scheme by region.

Reviewer #3 (Public Review):

The authors set out to explore how neurons in the rodent parahippocampal area code for environmental and behavioral variables in a complex goal-directed task. The task required animals to learn the association between a cue and a spatial response and to use this information to guide behavior flexibly on a trial-by-trial basis. The authors then used a series of sophisticated analytical techniques to examine how neurons in this area encode spatial location, task-relevant cues, and correct vs. incorrect responding. While these questions have been addressed in studies of hippocampal place cells, these questions have not been addressed in these upstream parahippocampal areas.

Strengths:

1) The study presents data from ensembles of simultaneously recorded neurons in the parahippocampal region. The authors use a sophisticated method for ensuring they are not recording from the same neurons in multiple sessions and yet still report impressive sample sizes.

2) The use of the complex behavioral task guards against stereotyped behavior as rats need to continually pay attention to the relevant cue to guide behavior. The task is also quite difficult ensuring rats do not reach a ceiling level of performance which allows the authors to examine correct and incorrect trials and how spatial representations differ between them.

3) The authors take the unusual approach of not pre-processing the data to group neurons into categories based on the type of spatial information that they represent. This guards against preconceived assumptions as to how certain populations of neurons encode information.

4) The sophisticated analytical tools used throughout the manuscript allow the authors to examine spatial representations relative to a series of models of information processing.

5) The most interesting finding is that neurons in this region respond to situations where rewards are not received by increasing their firing rates. This error or mismatch signal is most commonly associated with regions of the basal ganglia and so this finding will be of particular interest to the field.

Weaknesses:

1) The histological verification of electrode position is poor and while this is acknowledged by the authors it does limit the ability to interpret these data. Recent advances have enabled researchers to look at very specific classes of neurons within traditionally defined anatomical regions and examine their interactions with well-defined targets in other parts of the brain. The lack of specificity here means that the authors have had to group MEC, PaS, and PrS into a functional group; the parahippocampus. Their primary aim is then to examine these neurons as a functional group. Given that we know that neurons in these areas differ in significant ways, there is not a strong argument for doing this.

See response to Reviewer 2.

2) The analytical/statistical tools used are very impressive but beyond the understanding of many readers. This limits the reader's ability to understand these data in reference to the rest of the literature. There are lots of places where this applies but I will describe one specific example. As noted above the authors use a complex method to examine whether neurons are recorded on multiple consecutive occasions. This is commendable as many studies in the field do not address this issue at all and it can have a major impact as analyses of multiple samples of the same neurons are often treated as if they were independent. However, there is no illustration of the outputs of this method. It would be good to see some examples of recordings that this method classifies as clearly different across days and those which are not. Some reference to previously used methods would also help the reader understand how this new method relates to those used previously.

We have added an additional Supplemental Figure (Figure 7 - figure supplement 1, that showcases the matching waveform approach. In the original manuscript, Fig. 7a provided an example output of the method.

3) The effects reported are often subtle, especially at the level of the single neuron. Examples in the figures do not support the interpretations from the population-level analysis very convincingly.

Additional plots for individual units have been added, Figure 2 - figure supplement 3. However, the effects, though small by unit, are consistent across neurons and subjects.

The authors largely achieve their aims with an interesting behavioral task that rats perform well but not too well. This allows them to examine memory on a trial-by-trial basis and have sufficient numbers of error trials to examine how spatial representations support memory-guided behavior. They report ensemble recordings from the parahippocampus which allows them to make conclusions about information processing within this region. This aim is relatively weak though given that this collection of areas would not usually be grouped together and treated as a single unitary area. They largely achieve their aim of examining the mechanisms underlying how these neurons code task-relevant factors such as spatial location, cue, and presence of reward. The mismatch or error-induced rate remapping will be a particularly interesting target for future research. It is also likely that the analytical tools used in this study could be used in future studies.

Reviewer #1 (Recommendations For The Authors):

1) Typo: "attempted to addresses these challenges"

We thank the reviewer for pointing out, this has been fixed.

2) "classified using tuning curve based metrics" - what does "tuning curve" mean in this context?

We have clarified this sentence in the main text.

3) "MEC neurons encode time-elapsed" should be "MEC neurons encode time elapsed" (no hyphen)

We thank the reviewer for pointing out, this has been fixed.

4) "a phenomenon referred to as 'global remapping'." - I dislike this term because it has two meanings in the literature. On the one hand, it is used to contrast with rate remapping: that is, it refers to a change in the location of place fields. On the other hand, it refers to the remapping of the whole population of cells at once, as contrasted with partial remapping. I suggest calling them location remapping (vs. rate) and complete remapping (vs. partial)

We agree that this is an overloaded term in the field. We have added 'location remapping' in the intro as a more specific term for global remapping.

5) " tasks with no trial-to-trial predictability or experimenter-controlled cues and goals in the same environment." - ambiguously worded as it isn't clear whether the "no" refers to one or both of what follows. Also needs a hyphen after experimenter.

We thank the reviewer for pointing out, this sentence has been reworded for clarity.

6) " neurons changed their firing activity as a function of cue identity" - this is confounded by behavior and reward contingency, both linked to cue identity, so the statement is slightly misleading.

We thank the reviewer for pointing this out, however, we disagree that this wording is misleading. Neurons changed their activity as a function cue identity and reward contingencies. Why neurons change their activity in such a manner is a different, more nuanced question, that we addressed through our analyses by converging on the "error" like signal that these signals seem to carry.

7) "remapping" - I am not fully comfortable with the use of this term in this context. It derives from the original reports of change in the firing location of place cells, and the proposal that these cells form a "map" with the change in activity reflecting recruitment of a new map. With observations of rate changes in some place cells, the new term "rate remapping" was introduced, and now the authors are using "rate remapping" to mean firing rate changes in non-spatial cells. The meaning is thus losing its value. "Re-coding" might be slightly better, although we can argue about whether "code" is much better than "map"

While we agree with the reviewer that "remapping" has been coerced into a grab-all term, these are the accepted semantics in the field. Thus, we are disinclined to change the language.

8) Figure 1 - it would be useful to indicate somehow that one of the decision points was cued and once free choice with the random outcome - it took me a while to work this out. Also, the choice of colors for the cues needs explaining - my understanding is that rats are very insensitive to these wavelengths. And what does Pse mean? I didn't understand those scatterplots at all.

The section "Tree-Maze behavior and electrophysiological recordings" under Results go into the details of the task. A reference and additional context for the selection of cues is now included in the "Behavioral Training" methods section. Rats possess dichromatic vision systems. Caption of Figure 1, 2 includes what Pse means, the performance of a subject for a given session. The scatter plots relate remapping to performance.

9) Also on Figure 1 - in the examples shown, it looks like the animals always checked the two end arms in the same order. Was this position habit typical?

We have added additional context in "Behavioral Training" methods section. Well trained rats do exhibit stereotyped behaviors (eg. going to one well then the other).

10) "...we hypothesized that the cue remapping score would be related to a subject's performance in the task." I am struggling to see why this doesn't follow trivially from the observation that remapping occurred on error trials.

We thank the reviewer for pointing out that this could use further clarity. We have added that the magnitude of remapping is what should relate to performance. To further clarify, remapping does not occur on error trials, remapping as operationally defined in this work, is the difference of spatial maps as a function of Cue identity or Reward contingency. Thus, as a difference metric, remapping occurs because there is a difference in activity between correct and incorrect trials. The magnitude of that difference need not relate to how the subject performed on the task.

11) "With this approach, found that incorrect coding units were more likely to overlap between cue and RW coding units than correct." Missing "we". I didn't understand this sentence - what does "overlap" mean?

We have added a sentence to further clarify this point.

12) "We found that incorrect>correct activity levels on outbound trajectories predicted incorrect>correct activity levels on inbound trajectories" - I don't understand how this can be the case given that many of these units were head direction tuned and therefore shouldn't even have been active in both directions.

As seen in Figure 7b, we were able to match 217 units across tasks. Of those, "Cluster 0" with 98 units showed strong head-direction coding. While "Cluster 0" units showed strong remapping effects, there were a lot of other units that could have contributed to the "incorrect>correct" across (out/in)-bound segments. Further, head-direction coding is defined in the Open-field environment, and there's no constraint on what these neurons could do on the Tree Maze task.

13). " Error or mismatch signals conform a fundamental computation" - should be "perform"

Wording slightly changed, but "conform" as in "act in accordance to" is what we intend here.

14) " provides it with the required stiffness and chemical resistivity"- what does "chemical resistivity" mean? To what chemicals?

This is mostly in reference to rat waste and cleaning products (alcohol). We changed the wording to durability for simplicity.

15) Supp Fig 1 shows that behavioral performance was very distinctly different for one of the animals. Was its neural data any different? What happens to the overall effect if this animal is removed from the analysis?

Unless otherwise stated, all analyses are performed through linear mixed effects with "subject" as a random effect. Thus, the effects of individual subjects are accounted for.

16) Histology - it would be useful to have a line drawing from the atlas alongside the micrographs to enable easier anatomical understanding.

There was variability in the medial lateral location of the tetrodes across animals and in the histological images provided and thus, we felt this would not be useful information as a single line drawing will not encompass/apply to all histology photos.

17) Supp. Fig. 5/6 I didn't understand what Left, Stem, and Right mean at the top. Also, the color keys are too tiny to be noticed

An additional sentence has been added to the caption to clarify that left, stem, right refer to what segment was selected via the ranking of scores.

Reviewer #2 (Recommendations For The Authors):

Was there a particular reason why cells in these regions were analyzed together? Can some of the results be tested for cells of a particular region, especially the MEC? One major limitation of these results is that it is unclear which regions it applies to, e.g., one cannot be certain that data shows here that MEC cells have these firing properties.

Damage due to the extraction of the recording tetrode bundle was extensive and we were not able to parcelate out individual regions. We have added additional details on this in the "Histology" section of the methods.

It is unclear how many cells in each region are included in each analysis. There is supplementary fig 3 but not sure how many of these met the criteria to be included in a certain analysis and it does not differentiate regions. Also, was any of the MUA included in the analyses?

Isolated single units were included in all analyses, but we did not differentiate from what region each unit came from. Analyses that include MUA are separate from the main findings, and are included in supplemental figures as reference.

Was the error trial defined as a trial when the animal did not make the right light-guided choice or did it also include cases in which the light-related arm choice was correct, but the animal first went to the unrewarded end arm? Nomenclature in the results is not explained well - what is an unrewarded trial or unrewarded trajectory or an error trial?

We have added a new paragraph in the methods under Behavioral Training that address trial nomenclature. This methods section is now referenced twice in the initial paragraphs of the results section.

Were any grid cells included in the data, especially could any cross-matched across the open field and the maze runs?

This was indeed a question of interest to us, however, the number of grid-cells recorded was not adequate for meaningful statistical inference. We further sought to avoid tuning curve based functional classifications of units.

In general, the results section is difficult to read, and its accessibility could be improved.

We thank the reviewer for this accessibility point. We hope that the small tweaks as a product of this revision will improve the readability of the manuscript. We tried to have major takeaways for each result, but the nature of the analyses necessarily make the text somewhat dense.

Minor:

One of the Figure 3f references should be Figure 3g and later, Figure 44 should be corrected.

We thank the reviewer for noting this, it has been fixed.

Reviewer #3 (Recommendations For The Authors):

There are a number of issues that I think could be addressed to improve the manuscript:

1) The figure could make it clearer where the LED panel is. Are the authors confident the rats see the cue on each trial?

We have added a new supplemental figure to address this question (Figure 1 - figure supplement 1). The new figures show the 3D geometry of the maze and the location of the Cue panel. The rats were able to see the cue, otherwise task performance would have remained at chance levels.

2) The same maze has been used in a series of studies of hippocampal place cells by Paul Dudchenko's group. They also went on to examine how these representations are affected in a very similar cued spatial response task. These studies should be acknowledged.

We thank the reviewer for pointing out this oversight. We have added the Ainge et al. citation ( https://doi.org/10.1523/JNEUROSCI.2011-07.2007) when first introducing the maze and in the methods.

3) In a number of supplementary figures, the authors present neurons that are selective for different properties such as segment, cue, reward, and direction. However, the number of spatially and cue-selective cells and the criteria by which cells are designated as selective are not reported. The analyses of spatial remapping and response to cues are done at the population level so I'm not sure how these cells are classified or selected for the figures.

The procedure for selection is included in the figure captions. Each unit is ranked based on the Uz score by segment as originally shown in Figures 2 and 4.

4) Related to this, the example cells on the figures do not clearly represent the effects presented. For example, given the title of Figure 2, I assume that the cells in 2B significantly remap. However, they don't look like they remap - the cells in the top row show rate remapping in one segment of the maze while the cells in the bottom do not show clear rate remapping responses. I suspect that traditional rate map-based analyses using maps based on consistently sized pixels rather than large segments would show only very modest changes in correlations or rates across these different types of trials. It is important to report the findings in this way as the authors interpret their data relative to the rate-remapping studies which have used these analyses. Readers who do not have the time or expertise to examine the methods in detail will conclude that the effects reported here are the same as previous rate remapping studies which the examples suggest is not the case.

Additional plots for individual units have been added to the supplement, Figure 2 - figure supplement 3. However, the effects, though small by unit, are consistent across neurons and subjects (Figure 2 - figure supplement 5).

5) Why is there a bias on the stem in 2C? This is of similar size to the effect on the right size and so deserves discussion.

The analysis in question is the across unit level bias in cue-coding by maze segment. The left segment shows elevated Right Cue coding, while the right segment shows elevated Left Cue coding. There was one reported statistical result, the main effect of segment in the Linear Mixed Effects model. We expand this result in the following two ways:

1. Individual statistical results by segment

a. Left Segment (Uz Coef. Estimate = 0.5, CI95%=[0.26, 0.75; p<1e-4])

b. Stem Segment (Uz Coef. Estimate = 0.22, CI95%=[-0.01, 0.47]; p=0.06)

c. Right Segment (Uz Coef. Estimate = -0.27, CI95%=[-0.51, -0.03], p=0.03)

1. Reporting the joint hypothesis test of left > stem > right by unit.

a. X2=90.45, p=2.28e-20

b. The comparison of left>stem by unit:

i. coefficient estimate = 0.28, CI95%=[0.11, 0.44], p=0.0008

Although the reviewer is correct in pointing out the effect size similarity, the appropriate statistical comparisons within and across units support the stated conclusions. In terms of systematic coding bias, there is a small bias across units (60% of units) and animals (4 out 5) for the Right Cue. Although interesting, this effect is orthogonal to the comparisons of interests (within unit differences). In order to highlight this point we have added the statistics of the joint hypothesis test of left>stem>right to the main manuscript.

eLife assessment

In this study, neurons were recorded and combined across the parahippocampal area while rats performed a memory-guided spatial navigation task. Sophisticated analytical tools were used to provide convincing evidence that neuronal populations in these areas show behavior-related changes that might indicate the encoding of errors by the system. The valuable results suggest that rate remapping is a likely mechanism to support changes in representations that support memory-guided behavior in these regions, most interestingly in neurons that code head direction.

Reviewer #1 (Public Review):

In this study, single neurons were recorded, using tetrodes, from the parahippocampal cortex of 5 rats navigating a double-Y maze (in which each arm of a Y-maze forks again). The goal was located at any one of the 4 branch terminations, and rats were given partial information in the form of a light cue that indicated whether the reward was on the right or left side of the maze. The second decision point was un-cued and the rat had no way of knowing which of the two branches was correct, so this phase of the task was more akin to foraging. Following the outbound journey, with or without reward, the rat had to return (inbound journey) to the maze start, to begin again.

Neuronal activity was assessed for correlations with multiple navigation-relevant variables including location, head direction, speed, reward side, and goal location. The main finding is that a high proportion of neurons showed an increase in firing rate when the animal made a wrong turn at the first branch point (the one in which the correct decision was signalled). This increase, which the authors call rate remapping, persisted throughout the inbound journey as well. It was also found that head direction neurons (assessed by recording in an open field arena) in the same location in the room were more likely to show the rate change. The overall conclusion is that "during goal-directed navigation, parahippocampal neurons encode error information reflective of an animal's behavioral performance" or are "nodes in the transmission of behaviorally relevant variables during goal-directed navigation."

Overall I think this is a well-conducted study investigating an important class of neural representation: namely, the substrate for spatial orientation and navigation. The analyses are very sophisticated - possibly a little too much so, as the basic findings are relatively straightforward and the analyses take quite a bit of work to understand. A difficulty with the study is that it was exploratory (observational) rather than hypothesis-driven. Thus, the findings reveal correlations in the data but do not allow us to infer causal relationships. That said, the observation of increased firing in a subset of neurons following an erroneous choice is potentially interesting. However, the effect seems small. What were the actual firing rate values in Hz, and what was the effect size?

I also feel we are lacking information about the underlying behavior that accompanies these firing rate effects. The authors say "one possibility is that the head-direction signal in the parahippocampal region reflects a behavioral state related to navigational choice or the lack of commitment to a particular navigational route" which is a good thought and raises the possibility that on error trials, rats are more uncertain and turn their heads more (vicarious trial and error) and thus sample the preferred firing direction more thoroughly. Another possibility is that they run more slowly, which is associated with a higher firing rate in these cells. I think we therefore need a better understanding of how behaviour differed between error trials in terms of running speed, directional sampling, etc. A few good, convincing raw-data plots showing a remapping neuron on an error trial and a correct trial on the same arm would also be helpful (the spike plots were too tiny to get a good sense of this: fewer, larger ones would be more helpful). It would be useful to know at what point the elevated response returned to baseline, how - was it when the next trial began, and was the drop gradual (suggesting perhaps a more neurohumoral response) or sudden?

Comments on the revised submission:

The authors have clarified a number of points arising from my original review but some remain.

On the issue of hypotheses: I was really referring, and apologies that I was unclear on this, to the hypothesis about the neural responses predicted in this experiment. The authors aimed to "examine whether spatial representations flexibly adapt to behaviorally relevant factors" but this is not really a hypothesis as such, in the true mechanistic sense so much as "let's see what we can find" which is not an invalid reason to do this type of study. However, no manipulations were made that test causal relationships arising from the study. It thus remains observational. It does however raise testable hypotheses which is valuable. The strongest in my mind is that the rise in firing rates is a catecholamine response to frustration, a conclusion supported by the slow temporal dynamics of the changes.

On the issue of running speed: it needs to be ruled out that this might have been the cause of the altered firing rates since running speeds were different. More generally, the lack of other concurrent behavioral data means we cannot rule out other possible behavioral bases to this effect that are unrelated to error but are related to the motor correlates of the error.

Reviewer #3 (Public Review):

Summary & Strengths:

This study is useful in revealing the neural correlates of goal-directed navigation in the rodent parahippocampal regions, including the medial entorhinal cortex, presubiculum, and parasubiculum. It shows that task-relevant information represented by the parahippocampus is strongly related to task performance. It also reports the relationships of navigational factors (e.g., head direction signal) recorded during foraging in an open field with task variables.

Gonzalez and Giocomo investigated the neural activities in the parahippocampal cortex modulated by visual cues and error signals while the animal performed a goal-directed navigation task on the tree maze. They confirmed that the firing rates and spatial firing patterns in the parahippocampus were significantly correlated with the animal's task performance and the general navigational coding in the open field arena. The authors have concluded that the parahippocampal neurons encode mismatch-like signals, suggesting the functional role of the parahippocampus as a feedback system in a goal-directed task. However, a few major concerns should be addressed more closely to support the conclusion.

1) Due to the limitations of histological verification, the neural responses in the medial entorhinal cortex, presubiculum, and parasubiculum are analyzed together, and this limits the study from understanding the differential information processing across these regions. Because the medial entorhinal cortex and the pre/parasubiculum are believed to be located in very different positions in the information flow within the rodent medial temporal lobe with different anatomical connections, it would have been more convincing if the distinctive functions between the regions could be identified.

2) The authors should carefully differentiate rate remapping and global remapping in their analysis. Rate remapping generally indicates firing rate modulation with little or no shift of spatial firing fields (Leutgeb et al., 2005; Colgin et al., 2008). Therefore, the neurons exhibiting global remapping should not be included in the analysis suited for rate remapping (e.g., the encoding model that considers the cue-dependent rate-remapping effect).

3) One of the major findings in this study is that the parahippocampal neural responses to a visual cue or reward were correlated with task performance. One can expect that cue representation before the decision point is likely to have a greater impact on task performance. Although the Uz score between the left cue and right cue seemed not significantly different from zero on the stem, it would be beneficial if the authors verify whether the remapping score based on the firing rate maps will still be correlated with the task performance when examined only before the decision point, not for the entire maze.

4) There is a need to set the analytic epoch in more detail. The boundary between outbound and inbound journeys was set as 'last goal well visit.' However, even in a correct trial, if the reward was not received in the first goal well, an error signal could occur before the animal triggered the second goal well which was rewarding. This might have caused the rate remapping between two cue conditions, specifically on the arms. To eliminate this possibility, it is recommended to set the outbound journey from the home well trigger to the first goal well approach or to select only trials where the animal received rewards from the first goal well triggering.

Weaknesses:

Incomplete results could limit support for the arguments of the study and may require more rigorous analytical methods.

Basically as the subtitle says the war on taking over the movie industry ended because of the lack of people to head into the movies, although still common to see them not as busy as they used to be.

case in point: nobody does this anymore.

This vivid imagery alludes to lynching. Emmett Till was beaten, mutilated, shot in the head, and then thrown into the river with a cotton gin fan tied around his neck with barbed wire.

📌

and it's all inside the head therefore my thoughts can't possibly influence you hundreds of kilometers away

Nelson Mandela, South African anti-apartheid revolutionary, political leader, and philanthropist who served as President of South Africa from 1994 to 1999. He was the country's first black head of state and the first elected in a fully representative democratic election.

Redmond may be turning the very American image of "barbeque", often associated with the Fourth of July, on its head here. The word emphasizes how the massacre was a product of American ideals and intervention. Additionally, many lynchings of African Americans were cruelly and flippantly referred to as "barbeques" by their perpetrators; in this word choice, Redmond begins to establish a shared experience between the victims at My Lai and the Black victims of racial violence throughout American history.

classic old head doesn't understand new thing

In this verse, Kendrick compares the drowning of Peggy and her family to a baptism - the boat, intending to take them back to slavery, instead leads them to glory through death.

reply to Andy at https://forum.zettelkasten.de/discussion/comment/19108/#Comment_19108

I've got no evidence for nor against the presence of a box for these or any idea what the earlier portion of his process looks like at present. The bulletin board slips pictured were held up with pins and those on the table appear to be taped down, ostensibly to prevent accidental movement. Given their temporary nature and placement in this context, and the fact that they were highly portable for at least the span of the five shows he was preparing for in the documentary, there was certainly some container (even if it was as simple as a binder clip or a simple rubber band). Having seen shows like this roll in and out of venues before, I'm reasonably sure it was in a box at some point, so only a pedant would worry about it.

Box aside, the point here is that it shows a version of how he manufactures his output and manages his arrangement—portions of an overall process which are less frequently discussed and incredibly rarely visualized or pictured within the general community, much less in mass popular culture.

Many have argued that Eminem didn't have a zettelkasten either, and he definitely had both slips and a box. There's obviously no winning here... I won't worry too much about it until the naysayers' own Zettelkasten can manage to help them sell out Jones Beach Theater, The Prudential Center, PNC Bank Arts Center, Barclays Center, and Madison Square Garden.

Caveat pedanticus: Anyone talking about "Chris Rock's box" in public, might be held up to ridicule in his next sold-out tour. After Headliners Only and the Will Smith incident, I'm not taking any chances. 😜🃏🗃️

You can read more about them here: https://giuliaandlaraproject.wordpress.com/description-on-people-events-and-placed-cited-in-the-book/

"The Dream" is a very important concept in the book. What he means by it is his own design of the American Dream, which usually refers to the idea that equality and freedom stand as the foundations of the American's nation, and therefore anyone can prosper in the U.S. as long as they work hard for it. The author argues that the foundations are not quality and freedom, but rather the exploitation of Black People. Additionally, he believes that the dream cannot exist without racist injustice, and is therefore incompatible with African American's empowerment.

It is important to note that the concept of race, according to modern science, is merely a social construct. When the author talks about race, he means a categorization of humans based on shared physical or social qualities into groups generally viewed as distinct within a given society. Here's a more detailed definition of the term: Sense 1a of this entry describes the word race as it is most frequently used: to refer to the various groups that humans are often divided into based on physical traits, these traits being regarded as common among people of a shared ancestry. This use of race dates to the late 18th century, and was for many years applied in scientific fields such as physical anthropology, with race differentiation being based on such qualities as skin color, hair form, head shape, and particular sets of cranial dimensions. Advances in the field of genetics in the late 20th century determined no biological basis for races in this sense of the word, as all humans alive today share 99.99% of their genetic material. For this reason, the concept of distinct human races today has little scientific standing, and is instead understood as primarily a sociological designation, identifying a group sharing some outward physical characteristics and some commonalities of culture and history. “Race.” Merriam-Webster.com Dictionary, Merriam-Webster, https://www.merriam-webster.com/dictionary/race. Accessed 24 Dec. 2023.

Just casually mentioning "emergency squade" "trained to help in moments of crisis" and "could not [get their guns" and "SOME of them were left unarmed." This is an armed security team they're talking about. Obviously that makes them targets.

This is an allusion to Peacock's poem, specifically the lines "That head/between your legs so long."

Another example of the young woman reacting to the grief

Counting back, 99,98,97 is a pattern intrreruption.

eLife assessment

George et al. present a convincing new Python toolbox ("RatInABox") that allows researchers to generate synthetic behavior and neural data specifically focusing on hippocampal functional cell types (place cells, grid cells, boundary vector cells, head direction cells).

This is valuable for theory-driven research where synthetic benchmarks should be used. Beyond just navigation, it can be highly useful for novel tool development that requires jointly modeling behavior and neural data. The authors provide convincing evidence of its utility with well documented and easy to use code and the corresponding manuscript.

Reviewer #3 (Public Review):

George et al. present a convincing new Python toolbox that allows researchers to generate synthetic behavior and neural data specifically focusing on hippocampal functional cell types (place cells, grid cells, boundary vector cells, head direction cells). This is highly useful for theory-driven research where synthetic benchmarks should be used. Beyond just navigation, it can be highly useful for novel tool development that requires jointly modeling behavior and neural data. The code is well organized and written and it was easy for us to test.

We have a few constructive points that they might want to consider.

- Right now the code only supports X,Y movements, but Z is also critical and opens new questions in 3D coding of space (such as grid cells in bats, etc). Many animals effectively navigate in 2D, as a whole, but they certainly make a large number of 3D head movements, and modeling this will become increasingly important and the authors should consider how to support this.

- What about other environments that are not "Boxes" as in the name - can the environment only be a Box, what about a circular environment? Or Bat flight? This also has implications for the velocity of the agent, etc. What are the parameters for the motion model to simulate a bat, which likely has a higher velocity than a rat?

- Semi-related, the name suggests limitations: why Rat? Why Not Agent? (But its a personal choice)

- A future extension (or now) could be the ability to interface with common trajectory estimation tools; for example, taking in the (X, Y, (Z), time) outputs of animal pose estimation tools (like DeepLabCut or such) would also allow experimentalists to generate neural synthetic data from other sources of real-behavior.

- What if a place cell is not encoding place but is influenced by reward or encodes a more abstract concept? Should a PlaceCell class inherit from an AbstractPlaceCell class, which could be used for encoding more conceptual spaces? How could their tool support this?

- This a bit odd in the Discussion: "If there is a small contribution you would like to make, please open a pull request. If there is a larger contribution you are considering, please contact the corresponding author3" This should be left to the repo contribution guide, which ideally shows people how to contribute and your expectations (code formatting guide, how to use git, etc). Also this can be very off-putting to new contributors: what is small? What is big? we suggest use more inclusive language.

- Could you expand on the run time for BoundaryVectorCells, namely, for how long of an exploration period? We found it was on the order of 1 min to simulate 30 min of exploration (which is of course fast, but mentioning relative times would be useful).

- Regarding the Geometry and Boundary conditions, would supporting hyperbolic distance might be useful, given the interest in alternative geometry of representations (ie, https://www.nature.com/articles/s41593-022-01212-4)?

- In general, the set of default parameters might want to be included in the main text (vs in the supplement).

- It still says you can only simulate 4 velocity or head directions, which might be limiting.

- The code license should be mentioned in the Methods.

literally turning away from faith and ignoring the idea of innocence. Complete corruption and sin takes over Salem

Now this is a nice visual example I can understand

Reviewer #2 (Public Review):

This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however the biological insight gained is rather limited.

Reviewer #3 (Public Review):

Complex behavior requires complex neural control involving multiple brain regions. The currently available tools to measure neural activity in multiple brain regions in small animals are limited and often involve obligatory head-fixation. The latter, obviously, impacts the behaviors under study. Hur and colleagues present a novel recording device, the E-Scope, that combines optical imaging of fluorescent calcium imaging in one brain region with high-density electrodes in another. Importantly, the E-Scope can be implanted and is, therefore, compatible with usage in freely moving mice. The authors used their new E-Scope to study neural activity during social interactions in mice. They demonstrate the presence of neural correlates of social interaction that happen simultaneously in the cerebellum and the anterior cingulate cortex.

The major accomplishment of this study is the development and introduction of the E-Scope. The evaluation of this part can be short: it works, so the authors succeeded.

The authors managed to reduce the weight of the implant to 4.5 g, which is - given all functionality - quite an accomplishment in my view. However, a mouse weighs between 20 and 40 g, so that an implant of 4.5 g is still quite considerable. It can be expected that this has an impact on the behavior and, possibly, the well-being of the animals. Whether this is the case or not, is not really addressed in this study. The authors suffice with the statement that "Recorded animals made more contact with the other mouse than with the object (Figure 2A), suggesting a normal preference for social contact with the E-Scope attached." A direct comparison between mice before and after implant, or between mice with and without an implant would provide more insight into the putative impact of the E-Scope on (social) behavior.

In Figure 1 D-G, the authors present raw data from the neurophysiological recordings. In panel D, we see events with vastly different amplitudes. It would be very insightful if the authors would describe which events they considered to be action potentials, and which not. Similarly, indicating the detected complex spikes in the raw traces of Figure 1E would provide more insight into the interpretation of the data. Although the authors mention to consider the occurrence of complex spikes and simple spikes, a clear definition of what is considered a single unit recording is lacking. As there is quite a wide range in reported firing rates in Figure 2 - figure supplement 3, more clarity on this aspect would be insightful. Furthermore, in their text, the authors state that the pause in simple spike firing following a complex spike normally lasts until around 40 ms, and for this statement they refer to Figure 1G that shows a pause of less than 10 ms.

The number of Purkinje cells recorded during social interactions is quite low: only 11 cells showed a modulation in their spiking activity (unclear whether in complex spikes, simple spikes or both. During object interaction, only 4 cells showed a significant modulation. Unclear is whether the latter 4 are a subset of the former 11, or whether "social cells" and "object cells" are different categories. Having so few cells, and with these having different types of modulation, the group of cells for each type of modulation is really small, going down to 2 cells/group. The small group sizes complicate the interpretation of the data - in particular also on the analysis of movement-related activity that is now very noisy (Figure 2 - figure supplement 4).

In conclusion, the authors present a novel method to record neural activity with single cell-resolution in two brain regions in freely moving mice. Given the challenges associated with understanding of complex behaviors, this approach can be useful for many neuroscientists. The authors demonstrate the potential of their approach by studying social interactions in mice. Clearly, there are correlations in activity of neurons in the anterior cingulate cortex and the cerebellum related to social interactions. To bring our understanding of these patterns to a higher level, more detailed analyses (and probably also larger group sizes of cerebellar neurons) are required, though.

Reviewer #1 (Public Review):

Summary: Using a cross-modal sensory selection task in head-fixed mice, the authors attempted to characterize how different rules reconfigured representations of sensory stimuli and behavioral reports in sensory (S1, S2) and premotor cortical areas (medial motor cortex or MM, and ALM). They used silicon probe recordings during behavior, a combination of single-cell and population-level analyses of neural data, and optogenetic inhibition during the task.

Strengths: A major strength of the manuscript was the clarity of the writing and motivation for experiments and analyses. The behavioral paradigm is somewhat simple but well-designed and well-controlled. The neural analyses were sophisticated, clearly presented, and generally supported the authors' interpretations. The statistics are clearly reported and easy to interpret. In general, my view is that the authors achieved their aims. They found that different rules affected preparatory activity in premotor areas, but not sensory areas, consistent with dynamical systems perspectives in the field that hold that initial conditions are important for determining trial-based dynamics.

Weaknesses: The manuscript was generally strong. The main weakness in my view was in interpreting the optogenetic results. While the simplicity of the task was helpful for analyzing the neural data, I think it limited the informativeness of the perturbation experiments. The behavioral read-out was low dimensional -a change in hit rate or false alarm rate- but it was unclear what perceptual or cognitive process was disrupted that led to changes in these read-outs. This is a challenge for the field, and not just this paper, but was the main weakness in my view. I have some minor technical comments in the recommendations for authors that might address other minor weaknesses.

I think this is a well-performed, well-written, and interesting study that shows differences in rule representations in sensory and premotor areas and finds that rules reconfigure preparatory activity in the motor cortex to support flexible behavior.

intake kekek

Author Response

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

Thank you very much for forwarding these two important reviews on our paper. Please find hereby our point-by-point responses addressing the ideas, arguments and points of concern raised by the reviewers. We provide explanation of how these points have been incorporated in the paper.

We feel the review process has been a useful exercise and that the paper has greatly benefited in terms of clarity and accessibility. It is our hope that our findings may ignite renewed interest on unexplored and “unexpected” aspects of great ape vocal communication, inspire novel research, and invite bold new advances on the long-standing puzzle of language origins and evolution. In several relevant sections, we have also sought to explicitly address the point of doubt raised in eLife’s editorial assessment, published alongside the reviewed preprint of our paper. The editorial assessment stated that “…However the evidence provided to support the major claims of the paper is currently incomplete. Specifically, it is not yet clear how the rhythmic structuring found in these long calls is more similar to human language recursion per se rather than isochrony as a broader, more common phenomenon.” To directly clarify this point, we provide now various examples of how recursion is distinct from repetition, using everyday objects for an intuitive understanding (e.g., lines 43-51). We have also expanded the discussion to better contextualise and clarify the implications of our findings on language evolution theory. We hope this will help addressing the implicit request for clarification in the previous editorial assessment.

Thank you very much for your kind and dedicated attention in the processing of our study.

Public Reviews:

Reviewer #1 (Public Review):

This study investigates the structuring of long calls in orangutans. The authors demonstrate long calls are structured around full pulses, repeated following a regular tempo (isochronic rhythm). These full pulses are themselves structured around different sub-pulses, themselves repeated following an isochronic rhythm. The authors argue this patterning is evidence for self-embedded, recursive structuring in orangutang long calls.

The analyses conducted are robust and compelling and they support the rhythmicity the authors argue is present in the long calls. Furthermore, the authors went above and beyond and confirmed acoustically the sub-categories identified were accurate.

We thank the reviewer for this important support regarding our methods and findings.

However, I believe the manuscript would benefit from a formal analysis of the specific recursive patterning occurring in the long call. Indeed, as of now, it is difficult for the reader to identify what the authors argue to be recursion and distinguish it from simple repetitions of motifs, which is essential.

We agree with the reviewer that the distinction between repetition and recursion is very important for the adequate interpretation of our findings. Following the reviewer’s point (and the Editorial Assessement), we have now rephrased several passages in the initial paragraph of the paper for added clarity, where recursion is introduced and explained. We now also provide various new examples of recursion in everyday life and popular culture to better illustrate in an easy and accessible way the fundamental nature of recursion. We then use two of these common examples (computer folders and Russian dolls) to specifically distinguish repetition from recursion.

Although the authors already discuss briefly why linear patterning is unlikely, the reader would benefit from expanding on this discussion section and clarifying the argument here (a lay terminology might help).

Corrected accordingly.

I believe an illustration here might help. In the same logic, I believe a tree similar to the trees used in linguistics to illustrate hierarchical structuring would help the reader understand the recursive patterning in place here. This would also help get the "big picture", as Fig 1A is depicting a frustratingly small portion of the long call.

We completely understand the reviewer’s concern here. As proposed by the reviewer, and in addition to changes in the Introduction (see above) and Discussion (see below), we have now added a new figure in the Discussion to help the reader get the “big picture” of our findings.

We have also made revisions throughout the Introduction and Discussion to simplify the text, clarify our exposition and facilitate the reader better and intuitively understand the nature and relevance of our results.

Notwithstanding these comments, this paper would provide crucial evidence for recursion in the vocal production of a non-human ape species. The implication it would have would represent a key shift in the field of language evolution. The study is very elegant and well-constructed. The paper is extremely well written, and the point of view adopted is original, well-argued and compelling.

We are humbled by the reviewer’s words, and we thank the reviewer for attributing these qualities to our paper. This feedback reassures us of the disruptive potential that these and similar future findings may have on our understanding of language evolution.

Reviewer #2 (Public Review):

I am not qualified to judge the narrow claim that certain units of the long calls are isochronous at various levels of the pulse hierarchy. I will assume that the modelling was done properly. I can however say that the broad claims that (i) this constitutes evidence for recursion in non-human primates, (ii) this sheds light on the evolution of recursion and/or language in humans are, when not made trivially true by a semantic shift, unsupported by the narrow claims. In addition, this paper contains errors in the interpretation of previous literature.

We report the first confirmed case of “vocal sequences within vocal sequences” in a wild nonhuman primate, namely a great ape. The currently prevailing models of language evolution often rest on the (purely theorical) premise that such structures do not exist in any animal bar humans. We find the discovery of such structures in a wild great ape exciting, remarkable, and promising. We regret that the reviewer does not share this sentiment with us. We feel that the statement that these findings are trivial and narrow is unfounded.

In order to clarify and better communicate the significance of our findings, we now explain in more detail in the Introduction and Discussion how the discovery of nested isochrony in wild orangutans promises to stimulate new series of studies in nature and captivity. Our findings dovetail nicely with previous captive studies that have shown that animals can learn how to recognise recursive patterns and invite new research efforts for the investigation of recursive abilities in the wild and in the absence of human priming and in nonhuman primates.

The main difficulty when making claims about recursion is to understand precisely what is meant by "recursion" (arguably a broader problem with the literature that the authors engage with). The authors offer some characterization of the concept which is vague enough that it can include anything from "celestial and planetary movement to the splitting of tree branches and river deltas, and the morphology of bacteria colonies". With this appropriately broad understanding, the authors are able to show "recursion" in orangutans' long calls. But they are, in fact, able to find it everywhere.

The reviewer is correct in highlighting that recursion is ubiquitous in nature and this is something that we explicitly state in the paper. This only makes it the more surprising that, when it comes to vocal combinatorics, recursion has only been described in human language and music, but in no other animals. If studies providing such evidence are known to reviewer, we kindly request their corresponding references.

In the new revised version, we have paid attention to this aspect raised by the reviewer, and we have sought to disambiguate that our observations pertain to temporal recursion. This clarification will hopefully allow a better understanding of our results.

The sound of a plucked guitar string, which is a sum of self-similar periodic patterns, count as recursive under their definition as well.

The example pointed out here by reviewer is factually correct; sound harmonics represent a recursive pattern of a fundamental frequency. (In fact, we explain this phenomenon in the Discussion.) The reviewer’s comment seems to offer an analogy to oscillatory phenomena in the physiology of the vocal folds, and so, it is misplaced with regards to our present study, which focused vocal sequences. Admittedly, this misinterpretation may have been implicitly caused by our wording and we apologise for this. We now refer to “vocal combinatorics” instead of “vocal production” throughout the paper to avoid the reader considering that our findings pertain to the physiology of the vocal folds.

One can only pick one's definition of recursion, within the context of the question of interest: evolution of language in humans. One must try to name a property which is somewhat specific to human language, and not a ubiquitous feature of the universe we live in, like self-similarity. Only after having carved out a sufficiently distinctive feature of human language, can we start the work of trying to find it in a related species and tracing its evolutionary history. When linguists speak of recursion, they speak of in principle unbounded nested structure (as in e.g., "the doctor's mother's mother's mother's mother ..."). The author seems to acknowledge this in the first line of the introduction: "the capacity to iterate a signal within a self-similar signal" (emphasis added). In formal language theory, which provides a formal and precise definition of one notion of recursivity appropriate for human language, unbounded iteration makes a critical difference: bounded "nested structures" are regular (can be parsed and generated using finite-state machines), unbounded ones are (often) context-free (require more sophisticated automaton). The hierarchy of pulses and sub-pulses only has a fixed amount of layers, moreover the same in all productions; it does not "iterate".

The reviewer explains here how recursion, in its fully fledged form in modern language(s), is defined by linguistics. We fully agree and do not contest such descriptions and definitions in any way. These descriptions and definitions aim to describe how recursion operates today, not how it evolved. Nor do these descriptions and definitions generate data-driven, testable predictions about precursors or proto-states of recursion as used by modern language-able humans. This is scientifically problematic and heuristically unsatisfying regarding the open question of language evolution.

Following human-specific definitions for recursion, as proposed by the reviewer, cannot per se be used to undertake a comparative approach to evolution because they leave nothing to compare recursion with in other (wild) species. Using human-specific definitions unavoidably leads to black-and-white notions that language is always absolutely present in humans and always absolutely absent in other animals, regardless of their degree of relatedness to humans. It is unpreventable that these descriptions flout foundational principles of evolution, such as descent with modification and shared ancestry.

This conceptual problem is not new. Less than a century ago, it was believed that humans were the only tool-user (thousands of examples are known today in nonhuman animals, including fish and invertebrates), and later, that humans were the only cultural animal (today it is known that migrating caribou and fruit flies can establish traditions based on social learning). We must follow in the footsteps of those who have helped redefine human nature in the past. As famously stated by Louis Leakey when presented with evidence for chimpanzee tool-use collected by Jane Goodall, “Now we must redefine tool, redefine man, or accept chimpanzees as human”. Therefore, as a matter of course, we must redefine recursion, embracing empirically (other than purely theoretically) definitions that allow recursion to take on forms and functions different from that of modern language-able humans.

Another point is that the authors don't show that the constraints that govern the shape of orangutans long calls are due to cognitive processes.

The reviewer is indeed correct. This does not, however, refute our findings. We do not directly show that cognitive processes govern recursion in orangutan long calls. Instead, we show that the observed patterns cannot be explained by simple bodily or motoric processes, excluding therefore low-level explanations. With more than 50 years of accumulated field experience in primatology, this was the only possible way that our team found to go about conducting research and analyses on natural behaviour, in the wild, with a critically endangered primate. We would be very interested in learning from the reviewer what ethical and non-invasive methods, specific locations in the wild, and type of behavioural or socio-ecological data could be otherwise viably used to demonstrate what the reviewer requests. If other scientists believe that the patterns observed in wild orangutan long calls – three independent, but simultaneously-occurring recursive motifs – can be generated based on low-level physiological mechanisms alone, the burden of proof resides with them.

Any oscillating system will, by definition, exhibit isochrony.

We disagree with this statement. The example provided above by the reviewer him/her-self disproves the statement: a guitar string when struck is an oscillating system but it is not isochronic nor is it combinatorial. Isochrony cannot be established with single events, only with event sequences (in practice, ideally >3).

For instance, human trills produce isochronouns or near isochronous pulses. No cognitive process is needed to explain this; this is merely the physics of the articulators. Do we know that the rhythm of the pulses and sub-pulses in orangutans is dictated by cognition as opposed to the physics of the articulators?

The reviewer seems to misinterpret our results here. Our focus is on vocal combinatorics, not vocal fold oscillation (see previous response). We have now reworded all instances where the text could be unclear.

Even granting the authors' unjustified conclusion that wild orangutans have "recursive" structures and that these are the result of cognition, the conclusions drawn by the authors are too often fantastic leaps of induction. Here is a cherry-picked list of some of the far-fetched conclusions: - "our findings indicate that ancient vocal patterns organized across nested structural strata were likely present in ancestral hominids". Does finding "vocal patterns organized across nested structural strata" in wild orangutans suggest that the same were present in ancestral hominids?

Following the reviewer’s comment, we have now rephrased and toned down this passage, stating that such structures “may have been present” in ancestral hominids. We are grateful to the reviewer for this comment.