On the Estonian Independence Day I do not work and Usually I am at home. I wake property and I go to Toompea in the morning. I sing the Estonian anthem there. Go In the cafe and I eat pea soup. Then I walk in the city and look at the parade. Then I take the bus home. Go in the grocery store and buying food. At home I am cooking. I do not clean up because it is Festive day! In the afternoon I listen to A little classic music, I sit I read on the computer and read the news. Perhaps I'm sleeping a little. In the evening I watch TV and listen to the President's speech. A line of champagne And I eat sprat sandwiches. I call my mother and wish "good anniversary of the Republic!"

• Hallo! Merike hears!
• Hi here Margus, your new Neighbor. What are you doing? Do you you're at work?
• Ah Margus! How are you lan- Heb? Today I am not working, I am At home and I wash the laundry.
• I have a question. Whether You know who is Martha? He is born in the Kaarup and is Currently an old woman.
• Marta? I don't know that. Nobody whose name is Marta. Why are you asking that?
• I read my grandfather's letters. Marta was a little girl in 1943. He was mine Grandfather's friend.
• Very interesting. I'm going to work tomorrow and then asking everyone who comes shop.
• Thank you! Call me then.
• Let's do so. All the best!
• Will hear from you!

.

Tik-tok democracy has the power to turn this on its head

Author Response

We are grateful for the constructive feedback and the possibility of further improving our manuscript in terms of quality and clarity. Below, we have prepared a brief answer to the points raised in the reviewers’ feedback. We plan to address all these issues fully in the revised version of the manuscript.

We agree that some of our claims were overly enthusiastic. We will rewrite parts of the manuscript to tame our statements. Additionally, we are thankful for the comments on the use of language, which we will certainly apply while editing the manuscript. Below, we focus on the main comments.

Both reviewers: We appreciate advice on possible confounding factors. We should note here that there is substantial evidence on the effects of alpha rhythm amplitude on the excitability of a neuronal network and, as a consequence, on the amplitude of evoked responses (Baumgarten et al., 2016 Cerebral Cortex; Iemi et al., 2017 eLife; Stephani et al., 2021 eLife). This effect is due to changing the gain for evoked responses, and it is quite different compared to the baseline-shift mechanism (BSM). In BSM, the changes in the amplitude of evoked responses occur due to the generation of an additional evoked response component, which we tried to reveal in our current work. Still, we agree with suggestions to test additional factors, such as earlier evoked responses, baseline window, and head size, and we will test those.

Reviewer #2 Comment 2: Certainly, for low-density recordings, some method of data transformation is required. Here we would like to show our reasoning for why we did not use current-source density (CSD) but rather utilised other approaches. First, the CSD transform performs well for spatially localised activities since it is a spatial high-pass filter. In our case, P300 and alpha amplitude dynamics are fairly widespread with low spatial frequency, and we believe we would not benefit from applying CSD. Second, CSD has been shown to be more sensitive to surface sources in the crowns of gyri. For activity in the P300 window, we have no reason to believe that this is the case. Third, as we completely agree that low density montage is a limitation, we used source reconstruction with eLoreta (Fig. 5) to refine the spatial localisation of potential sources of P300 and alpha amplitude change.

Reviewer #1 Comment 4: Our study is indeed based on a sample of older participants. However, in our previous work (Studenova et al., 2022), we compared young and elderly participants using resting-state data. There, we measured the baseline-shift index (BSI). We found that BSIs for elderly participants were lower in comparison to those for young participants. Therefore, despite these limitations, in the current study, we were still able to detect a correspondence between BSIs and evoked responses in elderly participants. Therefore, we believe that for a sample of young participants, the results should not be different.

Reviewer #2 Comment 4: We agree that mediation analysis will provide additional insights, and we will add it to the revised version of the manuscript.

Overall, we found the reviewer's comments very helpful. We will update the manuscript accordingly.

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Reply to the reviewers

We thank the reviewers for their comments and insights, we feel the manuscript is now greatly improved. Please find below our answers to the reviewer’s queries

Reviewer #1 (Evidence, reproducibility and clarity):

The manuscript by Niccoli et al. describes the identification of a novel modifier of C9orf72-derived toxicity based on the manipulation of the brain metabolic pathways. The premise for this work is supported by strong literature describing the aberrant glucose metabolism in FTD, AD and other degenerative disorders. The idea tested here is whether increasing the import of pyruvate produced in glia into neurons. They test three different types of importers and find that one of them, Bumpel, the orthologue of human SLC5A12, suppresses toxicity and reduces the accumulation of arginine-containing repeats, GP and PR. The authors investigate several potential mechanisms mediating this reduction of toxic DPRs, but do not find strong evidence linking pyruvate import and increase autophagy or mitochondria metabolism.

Overall, this is an interesting discovery based on a candidate approach that shows the power of Drosophila to efficiently identify novel mediators of neurodegeneration. The article is well written, although more detailed explanations of some experiments would be helpful. The weaknesses of the manuscript are the lack of a clear mechanism mediating the protective activity of pyruvate, the incomplete experiments lacking relevant controls, and the presentation of western blots.

Specific comments:

1. The reduced levels of DPRs require that the expression of C9 mRNA or the GR and PR constructs is examined by qPCR. In figure 3E, GP is not even detectable_

We agree with the reviewer, ideally we would have measured the RNA by qPCR. However, the C9 repeats and the DPR constructs are highly repetitive, it is therefore impossible to do a qPCR for them. The upstream and downstream sequence is identical for the C9 and the bumpel constructs, there isn’t, to our knowledge any unique sequence we can use to measure levels of expression in the presence of bumpel.

We did run a GFP control (Fig 2D) and did not see any difference and we have now carried out a qPCR for Gal4-GeneSwitch (Fig S3) to show that the levels of the driver do not change.

1. I wonder if there are constructs available to silence Bumpel or overexpress the human orthologues of bumpel. These would be nice controls for the effects observed with the Bumpel overexpression

This would be an extremely interesting experiment, however bumpel is normally only expressed in glia, therefore we can’t down-regulated it in glia whilst upregulating 36R in neurons, as we are limited to one driver (since everything is driven by the Gal4/UAS system). Expression of C9 in glia does not have a clear phenotype (our observation), so we can’t drive both in glia. We tried over-expressing the human homologue SLC5A12 , but it did not rescue the C9 phenotype (data not shown), possibly because it requires (like other human SLC5A type transporters) PDZK1 as extra co-factor (Srivastava S. et al, 2019), and this is not present in flies.

1. The argument about bumpel modulating autophagy downstream of Atg1 is not supported by the experimental data

We now have imaging data showing that bumpel modulates the formation of lysosomes, downstream of Atg1 (Fig 5). We also show that bumpel and Atg1 can act synergistically, leading to a much stronger rescue of C9 expression (See Fig 5I.), which also suggests that the two are acting at different points in the same pathway. We also show that bumpel rescues the downregulation of TFEB targets (Fig 5J)

1. Western blots throughout show no control lanes and in several occasions are created with cutout bands. The standard for this type of experiments should be more stringent, with entire gels showing all experimental conditions, which requires consistent methods and results vs selecting the best bands from different gels.

We apologise if this was mis-understood, the lanes shows are all from the same blot, where other samples were run too, and it would be confusing for the reader to include them. We have re-run samples where we had remaining sample from our quantifications, so that the lanes are now contiguous and we provide original blot images in the supplemental information for those we could not re-run. The control for all experiments are the C9 expressing line without bumpel, and this is always present, if the reviewer means we are missing -RU controls, these do not produce any DPRs so are not included in western blot or ELISA quantifications as the signal is not above back-ground.

1. For figures 2B and 5C, please, show representative WBs

These are ELISA quantifications, not western blots, we choose to run these when possible, as they are more quantitative.

1. Figure 5D describes the survival curve as significantly rescued. Statistical tests can indicate differences, but that is in no way convincing. The test may show the curves are different, but the abeta Atg1 flies also seem to start falling early, so an argument could be made in both directions, as a suppressor or an enhancer.

We agree the rescue is not strong enough, we have now removed this lifespan.

1. It is unclear why several results are placed in the supplemental materials. In general, all this material seems highly relevant and related to what is shown in the main figures

We are happy to include them in the main manuscript if this would help the reader, and we have now placed all mitochondrial data in Fig 4.

Minor comments:

Please, define several abbreviations throughout

We apologise for this over-sight, we have now does this.

A couple of sections could be improved by carefully sequencing human vs Drosophila background to advance the argument rather than going in circles. There is also a section on mitophagy in between two sections related to autophagy that could be sequenced better.

We have re-structured the sections, we think this has improved the flow.

There is a sentence at the end of page 6 that seems misplaced

We apologise for the over-sight, and we have removed this

Reviewer #1 (Significance):

Overall, this is an interesting discovery based on a candidate approach that shows the power of Drosophila to efficiently identify novel mediators of neurodegeneration. The article is well written, although more detailed explanations of some experiments would be helpful. The weaknesses of the manuscript are the lack of a clear mechanism mediating the protective activity of pyruvate, the incomplete experiments lacking relevant controls, and the presentation of western blots.

We thank the reviewer for the helpful comments, we have added some details in the methods section, we apologise for not having made it clear that the westerns were all derived from the same blot (we have now placed the originals in the supplemental materials). Regarding mechanism, we now show that bumpel over-expression increases clearance of late stage autolysosomes, possibly by increasing transcription of TFEB target lysosomal genes.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary:<br /> Project investigates the role in dementias of glial glucose uptake, conversion to lactate and shuttling via transporters to neurons to produce pyruvate to fuel TCA cycle production of ATG. The experiments are conducted in Drosophila melanogaster, which have become a powerful model system for understanding neurodegeneration mechanisms associated with ALS/FTD associated C9orf72 pathology. Bumple misexpression is shown to rescue early death phenotype in flies expressing a C9orf72 expansion and flies expressing arginine containing di-peptide repeat proteins. The report describes novel insight into the function of bumpel, demonstrating that this conserved orthologue of human SLC14A functions as a sodium exchange transporter for monocarboxylates pyruvate and lactate. These findings conclude that increased neuronal pyruvate, but not its metabolites, rescues C9orf72 associated pathology.<br /> The authors next set out to describe the mechanism by which increase pyruvate rescues survival in C9orf72 expressing flies. Levels of autolysosomes were increased in C9orf72 expressing flies, and stimulation of autophagy by overexpression of atg1 shown to decrease levels of DPRs (though not to same extent as bumple expression). Expression of bumple in C9orf72 flies led to a modest increase in LC3-II, indicating increased autophagy. Co-overexpression of bumple and atg1 did not have an additive effect, suggesting bumple activates autophagy downstream or independent of atg1 activity. Finally the author extend their findings to amyloid models, suggest a common protective mechanism for elevating neuronal pyruvate levels in neurodegenerative disease.

Major comments

Prior data suggests that bumpel is expressed in glia (for example Yildirim et al 2022). In their study the authors do not present any data to demonstrate that the transporter is normally expressed in neurons in flies. This calls into questions the physiological relevance of their findings, that neuronal upregulation of bumpel is protective against C9orf72 associated pathology in neurons, from which it is reasonable for a reader to conclude that bumpel may be a neuronal target for therapeutic intervention. However, the report well demonstrates that regardless of whether the transporter in native to neurons, the increase in monocarboxylates it facilitates is projective against C9orf72 pathology and thus the overall conclusion of the project is supported by experimental evidence. The point of upregulation of a natively expressed gene versus misexpression of a glial enriched transporter should be considered in a bit more detail in the discussion text. The authors may consider speculating the identify of members of the sodium coupled monocarboxylate transporters that are enriched in neurons. Are any of the bumple human orthologues expressed in neurons?_

We thank the reviewer for this comment and suggestion. The reviewer correctly points out that we do not show whether there is a defect in pyruvate import in C9 expressing flies. We could not identify a validated sodium coupled pyruvate transporter in flies with a strong neuronal expression, we have added a comment in the discussion about this. There are a number of human homologues, some, such as SLC5A8, are expressed in neurons, thus providing a possible therapeutic target. We have added a sentence to this regard in the discussion.

[_OPTIONAL] cDNA overexpression of neuron specific sodium coupled monocarboxylate transporters in C9orf72 fly models would strengthen the conclusion their physiological relevance for ALS/FTD. Fly lines for these are not available in repositories, but could be generated and tested at reasonable cost (<£700, ~3 month duration).

This would be an ideal experiment, however, we could not find a neuronal sodium coupled transporter which is known to import monocarboxylates. There are a number of sodium coupled neuronal transporters, but they are mostly homologous to SLC5A6, which is a glucose coupled transporter. Going forward, we will screen a number of transporters to identify if there are any which import pyruvate.

The role of bumple expression in survival (Figure 1) could be a technical artifact due to dilution of Gal4 between C9orf72 and bumple-ORF transgenes. No expression control is shown (for example GFP, LacZ etc). This theory is unlikely as no improvement in survival was seen for the SLC14A class of transporters which have a matching site directed transgene insertion. For clarity this point relating to controls should be commented on in the text.

The reviewer is correct, there could be a dilution of the Gal4. We don’t like using GFP as a control as we have often seen a worsening when expressing other highly stable proteins at high levels. We have generated an “empty” flyORF line (generated by injecting the empty plasmid into the identical attP site), and used it as a control to check for dilution effects, bumpel still rescued relative to this control, we now include this is the supplementary (Fig S1B).

Reduced Mito-GFP levels are used to support a role for bumple in increasing mitophagy. As mito-GFP is a marker for mitochondria but not specifically mitophagy, an alternative explanation for decreased levels could be reduced mitochondria biogenesis. The text should be amended to clarify this point.<br /> The role of Pink1 RNAi in modifying mitophagy is a bit overstated. Whilst Pink1 is involved in stress associated mitophagy, its role in basal mitochondria turnover is less well defined. Text should be adapted.

We have added qualifying statements regarding the possibility of reduced mitochondrial biogenesis, and the fact that Pink1’s role in basal mitophagy is not very clear. The use of the mitophagy inducer drug, Kaempferol, however, suggests that mitophagy is unlikely to be a cause of the DRP reduction.

Minor comments

Introduction well describes current state of C9orf72 fly models. Introduction would benefit from a few comparable lines for AD models. The first paragraph of reports may also be better placed in the introduction._

We thank the reviewer for the suggestion, and have added a more in depth introduction to Aß and have moved the first paragraph of the results section to the introduction

Figure 1 presents survival for three SLC16A transporters and bumple. The C9 control curve appears to be consistent between charts, likely indicating the same control used across experiments, rather than independent controls for each chart. The authors should considered showing either all SLC16A and bumple data on a single chart, or clarify in the figure legend that a common control dataset is used. GFP control is used in later experiments (Figure 2).

We have now indicated that the SLC16A transporters were run together in the figure legend.

Choice of amyloid model needs a line of explanation, particularly with regard to extra/intracellular deposition of amyloid in this model.

We have now added a few sentences describing this when the model is introduced

Fruit Fly Injection method section needs a bit more detail to describe site of injection (head, body etc). This is not clear in the result section either.

We have now added this, the injection was done in the abdomen.

How were bumple orthologues identified? What degree of conservation (sequence homology etc?)

The bumpel orthologues are those identified as most similar by flybase. We have now added the degree of conservation in the text

The speculative mechanism for C9 pathology modification involves interaction of neurons and glia, monocarboxylate transporters and changes in autophagy activity. For clarity a diagram showing the model may be a helpful addition.

We have now added a diagram explaining how we think the rescue is achieved

Typos:<br /> Figure 1 Legend - "p values of ona way ANOVA "

We apologise for the error, and have now corrected it

Figure S2 Legend - Atg1 RNAi genotypes from S2 legend are mentioned erroneously

We apologise for the error, and have now corrected it

Repetition of text in results: "Bumpel, together with its paralogues kumpel and rumpel, is expressed in glia in flies, where it is thought to promote transport of substrates across the brain (31)."

We apologise and have rectified this

"Modulation of Atg1 when bumpel was co-overexpressed, however, did not affect GP<br /> levels (Fig 4E, F)" - Should be refering to Fig 4D, E)

We apologise and have rectified this

Reviewer #2 (Significance):

The study will be of broadly of interest to researcher working in the fields of neurodegeneration and metabolism, providing evidence for a protective role of elevated pyruvate in neuron that provide new understand relating to pathology in C9orf72 associated motor neuron disease and frontotemporal dementia.

Strengths:<br /> The study presents novel data to demonstrate that overexpression of fly monocarboxylate transporter bumple rescues an early death phenotype associate with ALS/FTD gene C9orf72. Any novel therapeutic strategies of ALS are of interest to the field, and the strategy demonstrated here may be readily translated to human cell culture systems for proof of principle translational studies to a more physiologically relevant system. This study further demonstrates the utility of invertebrate models to generate novel understanding of C9orf72 pathology.

Limitations:<br /> The study speculates that there is a link between pyruvate levels and increased autophagy, however the mechanisms by which this occurs is not defined in present study. This is a limitation of the experiment, though opens up an interesting question for future studies._

We thank the reviewer for their comments, and we have now added experiments characterising the role of bumpel in autophagy, particularly showing its rescue of a late autolysosomal block.

Reviewer expertise: The reviewer researches ALS and dementia associated neurodegeneration, utilising Drosophila, rodent and stem cell derived model systems.

Reviewer #3 (Evidence, reproducibility and clarity):

This is an interesting manuscript in which the authors provide evidence that elevated neuronal expression of the pyruvate transporter bumpel can partially rescue shortened lifespan in fly models of frontotemporal dementia and Alzheimer's disease. In addition, elevated neuronal bumpel expression can reduce accumulation of arginine containing FTD-linked dipeptide repeat proteins. Some evidence is presented that elevated neuronal bumpel expression may activate autophagy. These findings are novel and may have implications for therapeutic interventions based on pyruvate import/metabolism to treat neurodegenerative disorders. However, I have several concerns as follows:

Major Comments:

1. The authors provide no explanation as to why they targeted bumpel overexpression in neurons. Endogenous bumpel appears to be predominately expressed in glia cells so why not target these cells instead?

We wanted to increase pyruvate import in neurons, so we over-expressed a number of pyruvate transporter that were available in the fly ORF stock centre (so that they would all be inserted into the same site and therefore directly comparable), we were mainly interested in cell autonomous effects of importing glycolytic metabolites. Over-expressing bumpel in glia would be indeed an extremely interesting experiment, unfortunately we do not have the ability to express C9 in neurons while over-expressing bumpel in glia as we only have one over-expression system that works. We are working towards generating a new C9 model so we can then use the Gal 4 system to over-express bumpel in glia, but this is currently not available yet. Over-expression of C9 in glia is not toxic and not a good model of disease.

1. Data is shown that overexpressed bumpel can suppress GR and PR dipeptide repeat toxicity when these peptides are translated using an ATG start codon (Fig 2D,E). Does bumpel mediated neuroprotection also correlate with a reduction in DPR levels driven with an ATG start codon?

This would be a very interesting question, unfortunately, whist the Isaacs lab kindly made available the GR antibody for the initial ELISA experiment, we no longer have that antibody available and we do not have a working PR antibody. GR and PR westerns are not possible to carry out as the proteins are too positively charged to run. We do show that bumpel can down-regulate Aß from a UAS promoter, so its effect is not specific to RAN translation.

1. The authors provide some evidence suggesting that overexpression of bumpel increases autophagy in the fly brain. However, knockdown of Atg1 while co-expressing bumpel (Fig 4E) did not result in increased GP protein levels. In addition, Atg1 knockdown did not attenuate the protective effects of bumpel overexpression (Fig 4I), suggesting that bumpel is working through a pathway independent of autophagy to promote DPR clearance and protection against toxic peptide accumulation. The authors need to modify the interpretation of their data and temper their claim that autophagy contributes to bumpel-mediated protective effects in the CNS.

We apologise the data was not strong enough. We have now added evidence that bumpel acts downstream of Atg1, on late stage autolysosomal clearance. We also show that bumpel and Atg1 can act synergistically to improve the C9 phenotype when over-expressed, this is now described in Fig 5.

1. Although the authors present evidence that increased bumpel expression can activate autophagy, the data is not convincing that the neuroprotective effects associated with bumpel are mediated through autophagy. Pyruvate, in some circumstances, can non-enzymatically scavenge hydrogen peroxide or in other cases trigger oxidative stress resistance through hormetic ROS signaling. The authors should consider these alternative possibilities.

These are indeed possibilities, we have added a sentence to that effect in the discussion, we have now also showed that bumpel is affecting late clearance of autolysosomes, and is leading to an increase in TFEB targets.

1. The authors rely on overexpressing bumpel to attenuate C9 toxicity in flies. They should perform the opposite experiment and knockdown bumpel to demonstrate that reduced bumpel expression results in potentiation of C9 and amyloid beta neurotoxicity. In addition, then should show that knockdown of bumpel expression has some effect on autophagy.

This would be a very interesting experiment, unfortunately bumpel is expressed only in a few glia subtypes in a wild type fly, and we can’t downregulate it in glia while over-expressing toxic proteins in neurons, because of limitations of our expression system, both genes need to be over-expressed in the same cell type. We have tried downregulating bumpel in neurons, and don’t get an effect on phenotype, and no effect on DPR levels, but bumpel expression in neurons is extremely low. Moreover, bumpel has 2 paralogs, rumpel and kumpel,(also only present in glia) and all three need to be knocked out for phenotypes to become visible in glia (Yildirim et al, 2022). These experiments would be interesting but outside out scope.

We are in the process of generating new C9 models to be able to do these experiments, but these are currently outside the scope of this work.

Minor Comments:

1. Neuronal overexpression of bumpel appears to shorten lifespan of wild type flies (Fig 2A). It is possible that neuronal import of pyruvate may drive mitochondrial oxidative phosphorylation and ROS formation. The authors should comment on this possibility in the discussion._

This is a very good point, we have added a point to that effect.

1. In Fig 3 the authors used a mixture of sodium pyruvate and ethyl pyruvate to demonstrate the import properties of bumpel. The rationale for using ethyl pyruvate is unclear as this membrane-permeable metabolite can by-pass any transporters.

The ethyl pyruvate was only used in the injection of flies, not for the FRET experiments looking at the import properties of bumpel. Since we were not over-expressing bumpel, we needed the pyruvate to by-pass the requirement for a transporter. We were showing that delivery of pyruvate by another methods (other than by a transporter) was able to phenocopy the over-expression of bumpel, thus showing the effect is mediated by pyruvate entrance into the cell.

1. In the introduction several acronyms are used (i.e. GRN, MAPT, TREM2) that are not defined.

We apologise and have now rectified this.

Reviewer #3 (Significance):

To my knowledge, this is the first study to identify that bumpel can permit the import of pyruvate and lactate into neurons when ectopically expressed in the fly brain. The fact that increased neuronal pyruvate import can partially protect against toxic peptide accumulation is unexpected and quite novel. Although some evidence is presented that bumpel can trigger autophagy, it is not clear if autophagy is mediating bumpel neuroprotective effects. Alternative mechanisms related to pyruvate effects on ROS and oxidative stress resistance should be considered.

We thank the reviewer for their comments, and have added clarifying statements regarding the potential role of ROS.

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Referee #2

Evidence, reproducibility and clarity

Summary:

Project investigates the role in dementias of glial glucose uptake, conversion to lactate and shuttling via transporters to neurons to produce pyruvate to fuel TCA cycle production of ATG. The experiments are conducted in Drosophila melanogaster, which have become a powerful model system for understanding neurodegeneration mechanisms associated with ALS/FTD associated C9orf72 pathology. Bumple misexpression is shown to rescue early death phenotype in flies expressing a C9orf72 expansion and flies expressing arginine containing di-peptide repeat proteins. The report describes novel insight into the function of bumpel, demonstrating that this conserved orthologue of human SLC14A functions as a sodium exchange transporter for monocarboxylates pyruvate and lactate. These findings conclude that increased neuronal pyruvate, but not its metabolites, rescues C9orf72 associated pathology.

The authors next set out to describe the mechanism by which increase pyruvate rescues survival in C9orf72 expressing flies. Levels of autolysosomes were increased in C9orf72 expressing flies, and stimulation of autophagy by overexpression of atg1 shown to decrease levels of DPRs (though not to same extent as bumple expression). Expression of bumple in C9orf72 flies led to a modest increase in LC3-II, indicating increased autophagy. Co-overexpression of bumple and atg1 did not have an additive effect, suggesting bumple activates autophagy downstream or independent of atg1 activity. Finally the author extend their findings to amyloid models, suggest a common protective mechanism for elevating neuronal pyruvate levels in neurodegenerative disease.

Major comments

Prior data suggests that bumpel is expressed in glia (for example Yildirim et al 2022). In their study the authors do not present any data to demonstrate that the transporter is normally expressed in neurons in flies. This calls into questions the physiological relevance of their findings, that neuronal upregulation of bumpel is protective against C9orf72 associated pathology in neurons, from which it is reasonable for a reader to conclude that bumpel may be a neuronal target for therapeutic intervention. However, the report well demonstrates that regardless of whether the transporter in native to neurons, the increase in monocarboxylates it facilitates is projective against C9orf72 pathology and thus the overall conclusion of the project is supported by experimental evidence. The point of upregulation of a natively expressed gene versus misexpression of a glial enriched transporter should be considered in a bit more detail in the discussion text. The authors may consider speculating the identify of members of the sodium coupled monocarboxylate transporters that are enriched in neurons. Are any of the bumple human orthologues expressed in neurons?<br /> [OPTIONAL] cDNA overexpression of neuron specific sodium coupled monocarboxylate transporters in C9orf72 fly models would strengthen the conclusion their physiological relevance for ALS/FTD. Fly lines for these are not available in repositories, but could be generated and tested at reasonable cost (<£700, ~3 month duration).<br /> The role of bumple expression in survival (Figure 1) could be a technical artifact due to dilution of Gal4 between C9orf72 and bumple-ORF transgenes. No expression control is shown (for example GFP, LacZ etc). This theory is unlikely as no improvement in survival was seen for the SLC14A class of transporters which have a matching site directed transgene insertion. For clarity this point relating to controls should be commented on in the text.<br /> Reduced Mito-GFP levels are used to support a role for bumple in increasing mitophagy. As mito-GFP is a marker for mitochondria but not specifically mitophagy, an alternative explanation for decreased levels could be reduced mitochondria biogenesis. The text should be amended to clarify this point.<br /> The role of Pink1 RNAi in modifying mitophagy is a bit overstated. Whilst Pink1 is involved in stress associated mitophagy, its role in basal mitochondria turnover is less well defined. Text should be adapted.

Minor comments

Introduction well describes current state of C9orf72 fly models. Introduction would benefit from a few comparable lines for AD models. The first paragraph of reports may also be better placed in the introduction.

Figure 1 presents survival for three SLC16A transporters and bumple. The C9 control curve appears to be consistent between charts, likely indicating the same control used across experiments, rather than independent controls for each chart. The authors should considered showing either all SLC16A and bumple data on a single chart, or clarify in the figure legend that a common control dataset is used. GFP control is used in later experiments (Figure 2).

Choice of amyloid model needs a line of explanation, particularly with regard to extra/intracellular deposition of amyloid in this model.

Fruit Fly Injection method section needs a bit more detail to describe site of injection (head, body etc). This is not clear in the result section either.

How were bumple orthologues identified? What degree of conservation (sequence homology etc?)

The speculative mechanism for C9 pathology modification involves interaction of neurons and glia, monocarboxylate transporters and changes in autophagy activity. For clarity a diagram showing the model may be a helpful addition.

Typos:

Figure 1 Legend - "p values of ona way ANOVA "

Figure S2 Legend - Atg1 RNAi genotypes from S2 legend are mentioned erroneously

Repetition of text in results: "Bumpel, together with its paralogues kumpel and rumpel, is expressed in glia in flies, where it is thought to promote transport of substrates across the brain (31)."

"Modulation of Atg1 when bumpel was co-overexpressed, however, did not affect GP<br /> levels (Fig 4E, F)" - Should be refering to Fig 4D, E)

Significance

The study will be of broadly of interest to researcher working in the fields of neurodegeneration and metabolism, providing evidence for a protective role of elevated pyruvate in neuron that provide new understand relating to pathology in C9orf72 associated motor neuron disease and frontotemporal dementia.

Strengths:

The study presents novel data to demonstrate that overexpression of fly monocarboxylate transporter bumple rescues an early death phenotype associate with ALS/FTD gene C9orf72. Any novel therapeutic strategies of ALS are of interest to the field, and the strategy demonstrated here may be readily translated to human cell culture systems for proof of principle translational studies to a more physiologically relevant system. This study further demonstrates the utility of invertebrate models to generate novel understanding of C9orf72 pathology.

Limitations:

The study speculates that there is a link between pyruvate levels and increased autophagy, however the mechanisms by which this occurs is not defined in present study. This is a limitation of the experiment, though opens up an interesting question for future studies.

Reviewer expertise: The reviewer researches ALS and dementia associated neurodegeneration, utilising Drosophila, rodent and stem cell derived model systems.

Author Response

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

This important work reports the identification of a list of proteins that may participate in the clearance of paternal mitochondria during fertilization, which is known as essential for normal fertilization and embryonic and fetal development. While the main method used is state of the art and the supporting data are solid, the vigor of the biochemical assays and function validation is inadequate. This work will be of interest to developmental and reproductive biologists working on fertilization. Key revisions (for the authors) include 1) Use a mitochondria-enriched fraction instead of whole sperm for the assays, and add more control samples to monitor what got lost during sperm and oocyte treatments before the coincubation step. 2) Functional validation of the key proteins identified.

We thank Editors of eLife, as well as Special Issue Guest-Editors and Reviewers for a favorable assessment and helpful recommendations for key revisions. Provisional revisions included in our revised article are detailed below. We agree with Editors’ comment about the use of mitochondrion enriched fractions and additional functional validation of key proteins. In fact, we are developing experimental protocols for oocyte extract coincubation with isolated sperm heads and tails, and eventually with purified mitochondrial sheaths, to separate the ooplasmic sperm nucleus remodeling factors from the mitophagic ones. Such experiments, as well as functional validations using porcine zygotes are contingent upon anticipated post-pandemic rebound in the availability of porcine oocytes, obtained from ovaries harvested on slaughterhouse floors, requiring currently unavailable workforce which has hampered our access to this necessary resource.

Reviewer #1 (Peer Review):

Could the authors make clear how much the presented pictures reflect the described localisation? There is no information on the number of spermatozoa and embryos observed nor the fraction of these embryos showing the presented pattern of localisation. This must be included.

Two hundred spermatozoa were counted per replicate of the cell-free system co-incubation and 20 zygotes per replicate, with 3 replicates of immunolabelling for each phase/picture which were examined to establish the typical localization patterns that were observed. The displayed patterns were observed in 65 to 88% of examined spermatozoa/zygotes; varying dependent on protein, replicate, and phase of immunolabelling. In all cases, the signal displayed is the typical pattern that was displayed in most cells. This information has been added to the Materials and Methods section for clarification.

It is not clear if the authors also examined the localization of other proteins and obtained a different pattern than anticipated from the proteomic approach or if they only tested these 6 proteins and got a 100% of correlation.

These are the 6 proteins which were selected based on extensive literature review into known functions of all identified proteins, as well as extensive research into available and reliable antibodies to detect such proteins within our porcine systems. Even so, no particular localization patterns were anticipated; instead, we presented the patterns actually observed and even some patterns which defied our expectations (i.e., the localization of BAG5 in the sperm acrosome).

The authors use "MS" in the text to indicate "mitochondrial Sheath" and "Mass spectrometry". this is confusing.

The authors agree and the usage of MS as an acronym for either has been removed entirely to avoid confusion.

In the introduction the author refers to Ankel-Simons and Cummins, 1996 as a reference for the number of sperm mitochondria in mammalian species, this is incorrect since the quoted paper is about the number of mtDNA molecules and mentioned an earlier publication.

This has been revised and the appropriate citation has been used.

Reviewer #2 (Peer Review):

Major:

1) It has been proved from the earlier studies from this group that the porcine cell-free system is useful to observe spermatozoa interacting with ooplasmic proteins in a single trial and could recapitulate fertilization sperm mitophagy events that take place in a zygote without affecting later cell-division process. However, the post-fertilization sperm mitophagy process is a complex time-associated event that many processes that occur sequentially and interactively, which means ooplasmic proteins might be involved in this process but may not directly interact with sperm or may associate with sperm-ooplasmic protein complex at different time points. It is certainly a great advance already in knowledge to identify "the candidate players" from the list of 185 proteins; however, with the time-resolution (4 and 24hr) in the current study and without functional validation experiments at this stage, it is still difficult to postulate the importance of these identified proteins. The functional validation experimental designs, in my opinion, is critically important for better interpretation of the data.

The authors agree with this reviewer’s sentiments and do plan to conduct further functional analysis. This project was able to generate a list of candidate, sperm-mitophagy promoting proteins and we were further able to show that many of these proteins were detectable both via mass spectrometry and via immunocytochemistry in spermatozoa exposed to our cell-free system. Furthermore, similar localization patterns were found in spermatozoa that were detected within newly fertilized zygotes. These results boost our confidence in our cell-free system and show that our list of candidate proteins is truly a useful list for future localization and functional analyses. We are certainly aware that we have not captured every protein that may play a role in post-fertilization sperm mitophagy and that the proteins captured are just candidates until proven otherwise. Likewise, we have almost certainly captured multiple proteins that are currently candidates that will likely not be shown to play a role in postfertilization sperm mitophagy, while it is plausible that at least some of these candidate proteins do play a role in mitophagy and some of them likely participate (perhaps have yet to be described roles) in other fertilization events, in which we would be extremely interested in as well.

2) As shown in Figure 1, whole sperm was used in the co-incubation and the later MS analysis; thus, proteins identified in the current study might be relevant in fertilization processes other than postfertilization sperm mitophagy, as proteins identified in the current study may be associated with other parts of the sperm (e.g. sticky sperm head, e.g. PSMG2 associated with sperm midpieces, tail at 4hr coincubation, but then only associate with sperm head at 24hr co-incubation) rather than sperm midpiece, despite the fact that authors applied immunohistochemistry to show the localization of this protein, but the evidence is indirect, so how authors functionally differentiate these 6 identified proteins from sperm mitophagy process with other processes and to confirm (or to associate) the relevance of these proteins with sperm mitophagy process?

The authors agree that the 6 proteins which were further studied by using immunocytochemistry may be playing roles in other processes such as pronuclear formation. We discussed some potential roles including and beyond post-fertilization mitophagy, in the Supplemental Discussion. After reviewer comments, we moved the Supplemental Discussion back in the main Discussion section. Thus, this section now considers additional putative pathways in which the said 6 proteins cold participate, though we concede that thorough functional studies must still be performed.

3) Class 3 proteins were present in both the gametes or only the primed control spermatozoa, but are decreased in the spermatozoa after co-incubation, which authors interpreted as sperm-borne mitophagy determinants and/or sperm-borne proteolytic substrates of the oocyte autophagic system, this data categorization may need to be revised as sperm-borne proteolytic substrates of the oocyte autophagic system only, not for sperm borne mitophagy determinants. The argument for this disagreement is due to the fact that if the protein is a sperm-borne mitophagy determinant, after coincubation, to execute the mitophagy process, this protein should still be associated with the sperm at least at the early stage (of 4hr) (constant under MS detection when comparing control with 4hr treated) rather than being released from the sperm. Or alternatively, they could result in class 3 proteins (but not all those 6 were in class 3). Nevertheless, if these proteins serve as substrates, they can be used (consumed) and show decreased under MS detection.

This argument for redefining the Class 3 proteins more accurately is understood and we agree. The definition is revised in the paper.

4) Of particular interest among the 6 proteins that were further investigated. Unlike other proteins, MVP was highly significant (p<0.001) after 4hr incubation, but the significance became less after 24hr (p=0.19). Interpretation of this dynamic change in the relevance of the mitophagy process would facilitate the readers to understand the relevance and the role of MVP.

The differences in significance are likely influenced by the abundance of MVP detectable by mass spectrometry. As the time of cell-free system incubation increases, the variability between replicates also seemed to increase, likely due to the sustained proteolytic activity taking place in our system. This work was based on three replicates of mass spectrometry for each time point; additional replicates likely would have reduced the p-value for the 24hr cell-free data set, for MVP and potentially other proteins also. At both time points, MVP was only detectable in spermatozoa after they had been exposed to the cell-free system treatment which is the criteria that truly interested us more than the actual differences in content between the timepoints and is why it was added to our list of candidate proteins.

5) In figure 3, the association of ooplasmic MVP to sperm midpiece is not convincing enough as sperm midpiece and tail often show some levels of non-specific signals under fluorescent microscopy. And the dynamic association of ooplasmic MVP to sperm midpiece in Fig. 3F-G is difficult to reach a conclusion solely based on data presented in the manuscript. Additional negative control of sperm MVP staining from the primed and treated sperm would be helpful. Additionally, a quantitative comparison (15 vs 25hr) of sperm-associated MVP signals from the fertilized embryo or a stack image from different angles would clarify the doubts raised here.

For all images and all replicates, serum controls were also generated. These controls were then viewed under fluorescent microscope, and light intensities and exposures thresholds for each fluorescent light channel were set based on the background intensity that came from these nonimmune serum-treated control samples. We set our light intensity/acquisition time below a threshold where the non-specific signal began to appear. All the presented patterns are based on setting this peak intensity threshold and as such the signal we see should be the true signal. Furthermore, 200 spermatozoa were counted per treatment per replicate of the cell-free system co-incubation and 20 zygotes per replicate, with 3 replicates of immunolabelling for each protein and data point, which was used to represent the typical localization patterns that were observed. The displayed patterns were observed between in 65- 88% of examined spermatozoa/zygotes. Invariably, the signal displayed in the manuscript is the typical pattern that was seen in a majority of cells. This information has now been added to the Materials & Methods section for clarification.

6) Same concerns for the other 5 proteins (PSMG2, PSMA3, FUNDC2, SAMM50, BAG5) as indicated above.

See response to Question 5.

7) The patterns of these 6 proteins under the immunofluorescent study are confusing as the pattern varies after co-incubation (treated), and mostly, the signal of these proteins observed from the fertilized embryos is not really associated with sperm midpieces. Therefore, the evidence of these proteins involving in post-fertilization sperm mitophagy is, at this moment, weak based on the data presented. But the relevance of these proteins in events post-fertilization or early embryo development is certainly (evidence did not strong enough to support "sperm mitophagy," in my opinion).

The authors agree that some of these proteins seem to be playing roles beyond postfertilization sperm mitophagy and that there is a need for true functional studies before the authors can state with certainty that these proteins play a role in any of the discussed fertilization events. We state this in the discussion: “Considering the dynamic proteomic remodeling of both the oocyte and spermatozoa which takes place during early fertilization, these 185 proteins which have been identified likely play roles in processes beyond sperm mitophagy.” It should be noted that the authors went into greater detail about potential alternative protein functions based on the present data and literature review in the Supplemental Discussion. Based on this comment and other reviewer comments we have now included the Supplemental Discussion as part of the main Discussion section, and this will hopefully help clarify some of the authors’ thoughts about the 6 candidate proteins which were further analyzed during this study.

Minor:

1) To my understanding, statistical significance (relevance) is normally set at a p-value of either <0.1 or 0.05. The reason for loosening the p-value of 0.2 in the current study needs to be justified as this was not a common statistical criterium, and the interpretation of those candidates from this loosened criterium should also be careful.

The loosening of statistical relevance in this study to 0.2, only applied to our Class 1 proteins. This is because for a protein to fall into the Class 1 proteins it was a protein that was only present in samples after they were exposed to the cell-free system. In the case of these Class 1 proteins, this happened for all 3 replicates at each stated timepoint. We found this pattern of detection to be important whether the p-value fell under 0.1 or 0.2. As such, we loosened our statistical threshold for our Class 1 proteins. Any proteins added to our candidate list will be subject to further investigation before definitive conclusions can be drawn, and as such we think that capturing more proteins was more important for the goals of this study than limiting the number of proteins captured, especially for those Class 1 proteins. An explanation of this has been added to the Materials & Methods section Mass Spectrometry Data Statistical Analysis.

2) First cell cleavage of porcine embryo normally occurs within 48hr post-insemination or activation; therefore, the 4 and the 24hr time points used in the current study require justification included in the discussion or methods and material section.

First cleavage of porcine embryos normally occurs around 24 - 28 hours post-insemination. Thus, for both the cell-free system and the embryo studies we were capturing an advanced 1 cell stage zygote/zygote like system with our 24 hour and 25-hour time points.

3) In figure 2, colors used in different time points and in two different classes represent (sometimes) different protein categories, would be easier for the readers for quick comparisons if the same color could be used to represent the same protein category throughout the graph. (E.g, proteins for early zygote development are shown in red in "A", but blue in "B")

This has been corrected and the color scheme for Figure 2 has been revised for easier comparisons.

Reviewer #3 (Peer Review):

I am not used to seeing a supplementary discussion in a manuscript. I also believe it should be incorporated into normal discussion.

The Supplemental Discussion has been incorporated into the main Discussion now.

It would be very helpful to make an additional figure in which the proposed interactome of identified factors with the sperm mitochondria before and after incubation are drawn schematically and also which factors are not IDed in both cases (when comparing to somatic mito- or autophagy). This eases to get through the discussion and will beautifully summarize and illustrate the importance and progress that the authors have made with this assay.

We made a diagram that depicts the changes in protein localization patterns overtime within our cell-free system. This diagram has been added to the manuscript as Figure 9.

Reviewer #1 (Public Review):

In this manuscript, the authors used an unbiased method to identify proteins from porcine oocyte extracts associated with permeabilised boar spermatozoa in vitro. The identification of the proteins is done by mass spectrometry. A previous publication of this lab validated the cell-free extract purification methods as recapitulating early events after sperm entry in the oocyte. This novel method with mammalian gametes has the advantage that it can be done with many spermatozoa at the time and allows the identification of proteins associated with many permeabilised boar spermatozoa at the time. This allowed the authors to establish a list of proteins either enriched or depleted after incubation with the oocytes extract or even only associated with spermatozoa after incubation for 4h or 24h. The total number of proteins identified in their test is around 2 hundred and with very few present in the sample only when spermatozoa were incubated with the extracts. The list of proteins identified using this approach and these criteria provide a list of proteins likely associated with spermatozoa remnants after their entry and either removed or recruited for the transformation of spermatozoa-derived structures. Using WB and histochemistry labelling of spermatozoa and early embryos using specific antibodies the authors confirmed the association/dissociation of 6 proteins suspected to be involved in autophagy.

While this unique approach provides a list of potential proteins involved in sperm mitochondria clearance it's (only) a starting point for many future studies and does not provide the demonstration that any of these proteins has indeed a role in the processes leading to sperm mitochondria clearance since the protein identified may also be involved in other processes going-on in the oocyte at this time of early development.

We thank reviewer 1 for positive comments. We added a sentence in Discussion addressing the obvious shortcoming of present study, as further functional validations of candidate mitophagy factors are planned.

Concerning the localisation of the 6 proteins further analysed, the authors must add how much the presented picture represents the observed patterns. They must include the details on the fraction of spermatozoa and embryos displaying the presented pattern.

We now specify that the patterns depicted in manuscript are typical and representative of data from at least three replicates of immunolabeling in spermatozoa and zygotes. For each of these replicates, 200 spermatozoa were examined per replicate of the cell-free system co-incubation or 20 zygotes per replicate. The displayed patterns were observed between 65-88% in examined spermatozoa/zygotes. Invariably, the signal displayed in manuscript is the typical pattern that was seen in a majority of cells. This information has now been added to the Materials & Methods section for clarification.

Reviewer #2 (Public Review):

Mitochondria are essential cellular organelles that generate ATPs as the energy source for maintaining regular cellular functions. However, the degradation of sperm-borne mitochondria after fertilization is a conserved event known as mitophagy to ensure the exclusively maternal inheritance of the mitochondrial DNA genome. Defects on post-fertilization sperm mitophagy will lead to fatal consequences in patients. Therefore, understanding the cellular and molecular regulation of the postfertilization sperm mitophagy process is critically important. In this study, Zuidema et. al applied mass spectrometry in conjunction with a porcine cell-free system to identify potential autophagic cofactors involved in post-fertilization sperm mitophagy. They identified a list of 185 proteins that might be candidates for mitophagy determinants (or their co-factors). Despite the fact that 6 (out of 185) proteins were further studied, based on their known functions, using a porcine cell-free system in conjunction with immunocytochemistry and Western blotting, to characterize the localization and modification changes these proteins, no further functional validation experiments were performed. Nevertheless, the data presented in the current study is of great interest and could be important for future studies in this field.

We thank reviewer 2 for positive comments. As we explain in our response to Editors and Reviewer 1, further validation studies will be resumed once the availability of slaughterhouse ovaries for such studies improves. Examples of such functional validation of pro-mitophagic proteins SQSTM1 and VCP are included in our previous studies (DOI: 10.1073/pnas.1605844113 and DOI: 10.3390/cells10092450) that led to the development of cell-free system reported here, and are cited in present study.

Reviewer #3 (Public Review):

In this manuscript, a cytosolic extract of porcine oocytes is prepared. To this end, the authors have aspirated follicles from ovaries obtained from by first maturing oocytes to meiose 2 metaphase stage (one polar body) from the slaughterhouse. Cumulus cells (hyaluronidase treatment) and the zona pellucida (pronase treatment) were removed and the resulting naked mature oocytes (1000 per portion) were extracted in a buffer containing divalent cation chelator, beta-mercaptoethanol, protease inhibitors, and a creatine kinase phosphocreatine cocktail for energy regeneration which was subsequently triple frozen/thawed in liquid nitrogen and crushed by 16 kG centrifugation. The supernatant (1.5 mL) was harvested and 10 microliters of it (used for interaction with 10,000 permeabilized boar sperm per 10 microliter extract (which thus represents the cytosol fraction of 6.67 oocytes). The sperm were in this assay treated with DTT and lysoPC to prime the sperm's mitochondrial sheath. After incubation and washing these preps were used for Western blot (see point 2) for Fluorescence microscopy and for proteomic identification of proteins.

Points for consideration:

1) The treatment of sperm cells with DTT and lysoPC will permeabilize sperm cells but will also cause the liberation of soluble proteins as well as proteins that may interact with sperm structures via oxidized cysteine groups (disulfide bridges between proteins that will be reduced by DTT).

This is certainly a possibility, the lysoPC and DTT permeabilization steps were designed to mimic natural processing (plasma membrane removal and sperm protein disulfide bond reduction), which the spermatozoa would undergo during fertilization. However, we do realize that this is a chemically induced processing and thus is not a perfect recapitulation of fertilization processes. However, in this study and in previous studies with this system, we were able to show alignment between proteomic interactions taking place in the cell-free system and within the zygotes.

2) Figure 3: Did the authors really make Western blots with the amount of sperm cells and oocyte extracts as the description in the figures is not clear? This point relates to point 1. The proteins should also be detected in the following preparations (1) for the oocyte extract only (done) (2) for unextracted nude oocytes to see what is lost by the extraction procedure in proteins that may be relevant (not done) (3) for the permeabilized (LPC and DTT treated and washed) sperm only (not done) (4) For sperm that were intact (done) (5) After the assay was 10,000 permeabilized sperm and the equivalent of 6.67 oocyte extracts were incubated and were washed 3 times (or higher amounts after this incubation; not done). Note that the amount of sperm from one assay (10,000) likely will give insufficient protein for proper Western blotting and or Coomassie staining. In the materials and methods, I cannot find how after incubation material was subjected to western blotting the permeabilized sperm. I only see how 50 oocyte extracts and 100 million sperm were processed separately for Western blot.

The authors did make Western blots with the number of spermatozoa and oocytes stated in the materials and methods, a total protein equivalent of 10 to 20 million spermatozoa (equivalent to ~20-40 µg of total protein load) and 100 MII oocytes (equivalent to ~20 µg of total protein load). These numbers have been corrected in the Materials & Methods. Also, we did find in the Materials & Methods section that the Co-Incubation of Permeabilized Mammalian Spermatozoa with Porcine Oocyte Extracts section refers to using cell-free exposed spermatozoa for electrophoresis; however, for none of the presented Western blot work was this true. Rather, all of the presented Western blots as per their descriptions are utilizing ejaculated or capacitated sperm or oocytes. This line has been removed from the Materials & Methods to reduce confusion.

Regarding preparation (2), we have previously assessed the difference between oocyte extract and intact oocytes in this manner internally and we are certainly losing proteins due to the oocyte extraction process. We make caveats in this vein throughout the article such as: “Furthermore, this cell-free system while useful does not perfectly capture all the events which take place during in vivo fertilization. The cell-free system is intended to mimic early fertilization events but is presumably not the exact same as in vitro fertilization.”

3) Figures 4, 5, 6, 7, and 8 see point 2. I do miss beyond these conditions also condition 1 despite the fact that the imaged ooplasm does show positive staining.

For all the presented Western blots, the tissue type is stated in the image description and the protocol which was used to prepare these samples is stated in the Materials & Methods.

4) These points 1-3 are all required for understanding what is lost in the sperm and oocyte treatments prior to the incubation step as well as the putative origin of proteins that were shown to interact with the mitochondrial sheath of the oocyte extract incubated permeabilized sperm cells after triple washing. Is the origin from sperm only (Figs 5-8) or also from the oocyte? Is the sperm treatment prior to incubation losing factors of interest (denaturation by DTT or dissolving of interacting proteins preincubation Figs 3-8)?

The authors understand that there are proteins and interactions lost on both sides of the cellfree system equation and we have added a sentence to the Discussion to caveat this limitation in the system.

5) Mass spectrometry of the permeabilized sperm incubated with oocyte extracts and subsequent washing has been chosen to identify proteins involved in the autophagy (or cofactors thereof). The interaction of a number of such factors with the mitochondrial sheath of sperm has been shown in some cases from sperm and others for an oocyte origin. Therefore, it is surprising that the authors have not sub-fractionated the sperm after this incubation to work with a mitochondrial-enriched subfraction. I am very positive about the porcine cell-free assay approach and the results presented here. However, I feel that the shortcomings of the assay are not well discussed (see points 1-5) and some of these points could easily be experimentally implemented in a revised version of this manuscript while others should at least be discussed.

We agree that the use of a mitochondrial-enriched subfraction for further analysis would be interesting and useful. We are actively developing experimental protocols for oocyte extract coincubation with isolated sperm heads and tails, and eventually with purified mitochondrial sheaths. However, such experiments are contingent upon our access to porcine oocytes, which has continued to be a struggle since the COVID-19 pandemic compromised our ability to attain oocytes in large, cheap, and reliable quantities. This was a continuous problem with preparing materials for this very paper and has continued to be an issue for our laboratory as well as many others at our university and across the country. We continue to maximize oocytes every time we can get access to them, but the unfortunate reality is that this access has become sparce and unreliable over the past three years.

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Author Response

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

Reviewer 1 (Recommendations For The Authors):

1) The strikingly different conclusion from the previous Bourane study seems to stem from the experimental approaches. Rather than using genetic crosses that target all neurons from the hindbrain and spinal cord that express Npy at any point in development, Boyle et al target their manipulations specifically to the lumbar region of the superficial dorsal horn in adult mice using direct viral injections. Thus, Boyle is almost certainly manipulating much fewer neurons that the original study. How then is their behavioral effects so much greater? At the minimum, the authors need to discuss this discrepancy head on. Better would be a direct molecular/anatomical comparison of the neurons targeted by each approach. This could be done using Nyp-Cre mice crossed to a Rosa-LSL-reporter strain and quantifying the overlap with the same markers used here. Perhaps, the intersectional approach with Lbx1 resulted in labeling of a different population of neurons than the adult AAV injections? Although likely outside the scope, given this work directly questions the main conclusion of the Bourane paper, it will be important to see a replication of the original finding of selectivity to mechanical itch.

We agree that our approach should be manipulating a smaller population of neurons, and that it is therefore suprising that we see greater behavioural effects. Please see our response to "Weakness 1" of Reviewer 2 for consideration of this point. We have already provided a direct molecular comparison as requested by the reviewer, and this appears in Figure 1 supplement 1. Here we used tissue from NPY::Cre that had been crossed with Ai9 mice (i.e. a Rosa-LSL-reporter) and had received intraspinal injections of AAV.flex.GFP. We then characterised the neurochemistry of tdTomato+ cells that were GFP+ or GFP-negative.

2) The authors state that, "91.6% ± 0.3% of cells classed as Cre-positive cells were also Npy-positive, and these accounted for 62.1% ± 0.6% of Npy-positive cells" If I am reading this correctly, does that mean that 40% of the Npy+ cells are Cre negative? If so, how is this possible?

This interpretation is correct. For quantification of RNAscope data we used a cut-off level of 4 transcripts, and cells with fewer than 4 transcripts were classed as negative. It is likely that some of the NPY cells classified as negative for Cre would have had some Cre mRNA (sufficient to cause recombination), but at a level below this threshold. It is also possible that some NPY+ cells would fail to express Cre, since this is a BAC transgenic mouse, rather than a knock-in.

3) Similarly, the authors state that "great majority of FP-expressing neurons in laminae I-III were immunoreactive (IR) for NPY (78.5% ± 3.6%), and these accounted for 74.6% ± 109 1.9% of the NPY-IR neurons in this area". So does this mean 20% of the recombination is non-specific/in other cell types that could be involved in pain/itch sensation?

Our finding that 91.6% of cells with Cre mRNA were also positive for Npy mRNA (see above) indicates that Cre expression was largely restricted to NPY cells. The failure to detect NPY peptide in some of these cells probably results from the relatively low level of peptide seen in the cell bodies of peptidergic neurons, which results from the rapid transport of peptides into their axons.

4) Comparing Fig 3B and Fig4B it seems the control baseline von Frey responses are different. In fact, baseline response in Fig4b is quite like the CNO effect in Fig 3B. Unless I'm misunderstanding something, this seems quite odd?

We agree that there is a difference between the baseline responses. We are not aware of any particular reason for this, and we think that it reflects a degree of variability that is seen with the von Frey test. Interestingly, the baseline values for the SNI cohort (Fig 4E) lies between the values in Fig 3B and Fig 4B.

5) In Fig 4E, the behavior of the CNO treated mice is quite variable. Can the authors comment as to how this might be happening? Does the effect correlate with viral transduction?

We did not see any obvious correlation between the extent of viral transduction and the behaviour of individual mice.

6) Fig6, the PDyn-Cre experiment, is a bit of a non sequitur?

Please see our response to "Weakness 2" of Reviewer 2 for consideration of this point.

7) The conclusion is unusually long. I recommend trimming it to make it more concise.

We presume that this refers to the Discussion. However, this was ~1550 words, and we do not feel that that is unusually long.

Reviewer 2 (Public Review):

Weaknesses

1) There is inadequate discussion about previous studies of NPY interneurons. Specifically, the authors should address why a more restricted subset of these neurons (this study) have broader effects than seen previously.

We have expanded the discussion on the discrepancies between our findings and those reported previously. We state at the outset that we are targeting a more restricted population (lines 509-10), and we now go into more detail concerning both similarities and differences between our findings and the reasons that we think may underlie any discrepancies (various changes between lines 522-575).

2) I cannot see the reason for including results from manipulation of Dyn+ interneurons in this paper. First, the title does not reflect roles of spinal Dyn+ population. In addition, without further experiments characterizing relationships between NPY and Dyn interneurons in modulating itch and/or nociception, Dyn datasets seem to deviate from the main theme.

We had previously shown that activating Dyn-INs suppressed pruritogen-evoked itch (Huang et al 2018), but it was important to test whether silencing these cells would have the opposite effect. Our finding of overlap in function (i.e. both NPY-INs and Dyn-INs suppress itch, and that both innervate GRPR cells) provides strong evidence against the idea that neurochemically-defined interneuron populations have highly specific functions, and we now state this in the Discussion. The anatomical experiments (which follow on from the functional studies) provide important new information concerning synaptic circuitry of the dorsal horn, by showing that NPY-INs preferentially innervate GRPR cells, and provide around twice as many synapses on these cells, compared to the Dyn-INs. Interestingly, this correlates with the relatively large optogenetically-evoked IPSCs that we saw when NPY-INs were activated, compared to those reported by Liu et al (2019) when galanin-expressing (which largely correspond to Dyn-INs) were activated. By including these findings in the paper, we are able to make comparisons between these two populations.

3) While the authors provided convincing evidence that GRPR+ neurons serve as a downstream effector of NPY+ neuron evoked itch, the relationship between GRPR and NPY neurons in modulating pain is not examined. Therefore, Fig. 7B is pure speculation and should be removed.

We feel that our recent findings that GRPR neurons correspond to vertical cells, that they respond to noxious stimuli, and that activating them results in pain-related behaviours, makes it reasonable to speculate that the NPY/GRPR circuit may also be involved in the anti-nociceptive action of NPY cells. The legend for Fig 7B already refers to this as a "potential circuit", and we have toned down the corresponding part of the discussion to say that our findings "raise the possibility" that this is the case (lines 605-7). We feel that this part of the figure is important, as otherwise our summary diagram ignores some of the main findings of the paper, and we hope that this is now acceptable.

Recommendations For The Authors

1) Fig. 1G: the "misexpression" of tdTomato neurons was much more prominent in deep dorsal horn laminae but not in the superficial ones. Was this representative? Can the authors perform a laminae specific characterization?

We did test for this possibility in 2 NPY::Cre;Ai9 mice that had received intraspinal injections of AAV.flex.GFP, and found that there was a modest difference - 62% of tdTomato+ cells in laminae I-II, but only 39% of those in lamina III, were GFP+. This suggests that "misexpression" may have differed slightly between these regions. However, since the difference was quite modest, and we were only able to analyse tissue from two mice in this way, we did not include these findings in the paper.

2) I have a lot of problems interpreting the c-Fos data in Fig. 2 E and F. For the mCherry- population, how was the quantification performed? From the image, it does not look like 2030% of cells express c-Fos; at a minimum a clear stain of neurons would be needed. Similarly, the identification of NPY cells is not particularly convincing (e.g., middle arrowhead lower 2 panels in C).

We have provided further details on how the analysis was performed (changes made to lines 1016-29). NeuN staining was used to reveal all neurons, and a modified optical disector method was performed from somatotopically appropriate regions of the dorsal horn. As noted by the Reviewer, NeuN staining was required to allow identification of mCherrynegative cells. However, we have not included the NeuN immunoreactivity in the image, as this would add considerably to the complexity. These images are from single optical sections, and therefore the overall numbers of cells are low (in comparison to what would be seen in a projected image). The intensity of mCherry staining varied between cells. However, for all mCherry-positive cells (including the example referred to by the Reviewer), there was clear staining in the membrane, which could be followed in serial sections.

3) Please add individual data points for all quantifications.

These have been added.

Reviewer 3 Recommendations For The Authors:

1) It is somewhat surprising that there is no effect on CPP after activating spinal NPY neurons in neuropathic mice, given the almost complete rescue of hypersensitivity to baseline values in the nociceptive tests. Based on the methods, it appears that conditioning was carried out already 5 min after CNO injection. Yet, suppression of c-fos activity in excitatory spinal dh neurons was observed 30min after CNO injection. Also, it is not clear to me when CNO was injected prior to the nociceptive or CQ testing?

Have the authors considered that conditioning from 5-35 min after CNO injection might be too short after CNO injection to achieve a profound analgetic effect?

In a previous study (Polgár et al 2023), we had observed the timecourse of CNO-evoked itch and pain behaviours in mice in which GRPR cells expressed hM3Dq. We found that these started within 5 minutes of i.p. CNO injection (e.g. Fig S2 in that paper). In addition, the timecourse of action of gabapentin and CNO (both given i.p.) are likely to be similar, and there was a preference for the chamber paired with gabapentin. We are therefore confident that the conditioning period with CNO was adequate. We now explain this in the Methods section (lines 846-52). The timing of CNO injections for the nociceptive and CQ tests is now described (lines 749-55).

2) The authors claim that tonic pain was not affected based on the conditioned place preference test. Efficacy in withdrawal response tests and in the CPP differ by more than duration of the stimulus. I'd suggest using more cautious wording here.

We agree that caution is needed in interpreting the results of the CPP experiments. We have therefore replaced "does" with "may" in the Results section (line 336) and "did" with "may" in the Discussion (line 620).

3) On page 9 the authors state "...suggesting that they suppress the transmission of pain- and itch-related information in the dorsal horn." However, pain is not affected in the loss of function experiments suggesting some qualitative differences in the role of the NPY neurons in itch and pain. This should also be reflected more clearly in this statement and in the discussion e.g. "suppress itch" and "can suppress pain".

We accept the point made by the Reviewer. We have slightly altered the wording in lines 249-51 and 610 to reflect this.

This is an example comment.

Reviewer #1 (Public Review):

Genetic, physiological, and environmental manipulations that increase roaming increase leaving rates. The connection between increased roaming and increased leaving is lost when tax4-expressing sensory neurons are inactivated. This study is conceptually important in its characterization of worm behaviors as time-series of discrete states, a promising framework for understanding behavioral decisions as algorithms that govern state transitions. This framework is well-established in other animals, thanks to Berman and others, but relatively new to worms.

A key discovery is that lawn leaving behavior is probabilistically favored in states of behavioral arousal. I like the use of response-triggered averages (triggered on leaving events) that illustrate a "state-dependent receptive field" of the behavioral response. Response-triggered averages are common in sensory neuroscience, used, for example, to characterize the diverse "stimulus-dependent receptive fields" of different retinal ganglion cell types. It's nice to adapt the idea to illustrate the state-dependence of behavioral state transitions.

The simplest metric of arousal state is crawling speed. When animals crawl faster, they are more likely to leave lawns. A more sophisticated metric of behavioral context is whether the animal is in a "roaming" or "dwelling" state, two-state HMM modeling from previous work (Flavell et al., 2013). Roaming animals are more likely to leave lawns than dwelling animals. Different autoregressive HMM tools can segment worm behavior into 4-states. Also with ARHMMs, the most aroused state is again the state that promotes lawn-leaving.

(With the AR-HMM, I have a small quibble in its characterization as "orthogonal" to the 2-state HMM. Orthogonal has a precise mathematical meaning, but here orthogonal is taken loosely to only mean "very different". I'd prefer the authors just call them "very different" and not use mathematical terms so loosely.)

HMM analysis seems to disentangle effects that were lumped by the simpler metric of overall speed. Crawling speed before lawn leaving events, when analyzed only within roaming periods, is only higher for ¡1 min before the event. I presume that the higher speed that is observed for several minutes before lawn leaving when all states are taken into account (e.g., Fig 1J, Fig 2A, and others) reflects the tendency to be in the faster roaming state than the slower dwelling state for several minutes before lawn leaving? If this is correct, it would be nice for the authors to be explicit about this interpretation, to help the reader understand what is going on.

My principal worry is about the possible artifact if worms are more likely to be at lawn boundaries when moving quickly or in an arousal state (roaming in the 2-state HMM or in state 3 in the AR-HMM)? Lawn-leaving events only occur when the animal is at lawn boundaries. If animals are more likely to be at lawn boundaries when aroused, this should artificially increase the likelihood that these states precede lawn-leaving behaviors for a trivial environment-dependent reason instead of their interesting internal state-dependent reason. The authors might consider trying to disentangle the state-dependent statistics of lawn edge proximity when assessing by how much arousal states precede lawn-leaving events. I realize this is could be a formidable analytical challenge.

One recourse is to align speed, HMM, and AR-HMM states to the other behavioral events that only happen at lawn boundaries. When they do this for head poke-reversals in Figure 2-supplement 3, they also observe an (albeit modest) increase in arousal states before head poke-reversals. It should be easy to also compare what happens with head poke-forward and head poke-pause to better understand potential artifacts in quantifying edge-associated events. In any case, this concern and their strategies to address it should be discussed for clarity and transparency.

The authors use diverse environmental, genetic, and optogenetic perturbations to regulate the roaming state, thereby regulating the statistics of leaving in the expected manner. One surprise is that feeding inhibition evokes roaming and lawn-leaving in both pdfr-1 and tph-1 mutants, even though the tph-1-expressing NSM neurons have been shown to sense bacterial ingestion and food availability. I'm curious, is there anything in these new results that is inconsistent with previous claims by Rhoades et al., 2019, or did Rhoades et al. simply not do these tests?

Another surprise is that evoking roaming does not evoke leaving in tax-4 mutants (which is something of an internal control that argues against the worry that roaming artificially increases the likelihood of leaving, see above). Without sensory neuron activity, worms are only more likely to roam for a minute before leaving rather than roaming for several minutes before leaving like wild-type (Figure 6C). ASJ seems to be the most important sensory neuron in this coupling between roaming and leaving (which is uncoupled when sensory neurons are inactivated).

I'm a little puzzled why the wild-type animals shown in Figure 6C show elevated roaming for several minutes before leaving events, whereas the wild-type animals shown in Figures 4I,J,K show elevated roaming for only about a minute, not much different than tax-4 mutants. Am I missing something? What is different about these different wild-type animals?

Mary cannot faint in "pretence". She explains that before, when she was surrounding by screaming girls and judges who seemed to believe in the spirits, she thought she actually saw spirits and so could faint.

Now, she realises that she never saw the spirits and cannot faint.

This shows how hysteria changes people's perception of reality. Mary was not lying before. When she said she saw spirits, she really thought she did because of the hysteria and reactions of the people around her.

This description of Leo feels full of reference to nature. So not exactly an explicit description of the natural world itself, however Leo's characteristics feel as if they highlight qualities of nature. Leo is described as faun-like, is sensitive, playful/mischievous yet easily hurt. The description of his eyes as "green-gold" reminds me of the line of poetry in S.E. Hinton's nature's 'nature's first green is gold", although written a 50 years later or so, is part of what drew me to this passage as a description of nature.

Above supraglenoid tubercle= biceps long head Below Infraglenoid - triceps

Like this.

Coler as the head of the HRS grew the annual budget from $3.7 billion to $7.5 Billion. This shows he was incredibly successful in networking and expanding the agency. Are there clear lines when a public official oversteps or wades into impropriety?

While the death of Bradley should never had happened; this is one man who is overseeing a massive and underemployed organization that has done more good within his short time than his predecessors. Using a GSA/ state owned vehicle for personals\ use isn't allowed nowadays. Back in the 1980/1990's it was frowned upon. However, Rep. Tobin (Coler's superior) agreed for this usage since "he should be accessible 7 days a week, 24 hours a day".

From the beginning of his tenure as head of the HRS, Coler was extremely successful as a manager and implemented certain traits that fit into Mintzberg's assessment of managerial roles. Coler, from the start, used the role of liaison to further expand his network to work with both elected officials as well as members of the press to further advance the HRS mission.

Author Response

Reviewer #1 (Public Review):

This study combines in vitro somatic and dendritic recordings and computational modeling to study how cholinergic agonists modulate the response of CA1 pyramidal neurons to triangular current injections. The authors have previously used a similar approach (Upchurch, 2022, JNeuroscience) to show that CA1 neurons exhibit asymmetric AP firing (more firing on the upward ramp) in response to such current injections and that this effect is due to Na channel inactivation. The present work builds on these results by showing that cholinergic modulation changes this response, i.e., there is more firing on the downward part of the ramp. This change appears to require an intracellular Ca2+ concentration increase (mediated via IP3 and voltage-gated Ca2+ channels), which activates TRPM4 channels. In this scheme, cholinergic activity increases IP3, and the depolarizing current injection opens voltage-gated Ca2+ channels. This study will be of some interest to cellular neurophysiology experts working on the hippocampus.

1) This study claims that the triangular current injections recapitulate hippocampal place cell activity. However, it has been shown recently that the asymmetric firing of CA1 place cells is due to synaptic weight changes resulting from synaptic plasticity (e.g., Bittner et al., 2017). This suggests that the asymmetric firing of place cells is primarily the result of asymmetric synaptic input. Therefore, the authors should test whether carbachol similarly affects a synaptically driven membrane potential ramp. If this is not the case, the strong claim that this work has implications for place cell firing is not justified, in my opinion.

We have added the results showing the effects of cholinergic modulation on a synaptically-driven membrane potential ramp, obtained by electrically stimulating the Schaffer collaterals with a stimulation frequency that was adjusted according to a linear, symmetric ramp (see also Hsu et al, Neuron 99,147-162, 2018). These results have been added to the manuscript in the Results section for new Figure 2 (lines 169-197) and in the Methods section (lines 716-726).

2) Along the same lines, it has been shown before that the precision of spike timing depends on the stimulation pattern in vitro (Mainen and Sejnowski, 1995). Constant stimuli led to imprecise AP firing trains, whereas current injections that included fluctuations resembling synaptic input generated spike trains that were more reliable and reproducible in terms of timing. This study concluded that a low intrinsic noise level in spike generation was essential in generating informative spike sequences. Following this pivotal work, the authors could add noise to their current stimulus and observe the effect on the AP firing patterns. If this is not possible, the authors should at least report the sweep-to-sweep variability for the data shown, e.g., in panels 1A2, 1B2, 1D2, and 1E2.

We thank the reviewer for this suggestion to acknowledge the variability in the data across trials and we have added the Mainen and Sejnowski, 1995 citation to the manuscript (see Results lines 128-134). We addressed sweep-to-sweep variability among the various trials.

3) In most of the data presented in this manuscript, Carbachol appears to induce a 3 mV hyperpolarization and increase input resistance. As a result, the amount of current injected during Carbachol is drastically lower than during the controls. This should be emphasized more, and the input resistance should be quantified for each experimental condition. It should also be discussed whether this change in input resistance can account for the changes in the firing pattern observed. Finally, it should be clearly stated how the amount of the current injected was chosen for each cell, and data from a range of injected current ramps should be shown for each cell.

We thank the reviewers for this comment, which made us realize that our initial presentation was not clear, in particular with regard to the traces that were chosen as examples in the initial submission of the paper. We now clarify on page 5 (lines 113-125) of the manuscript as follows:

“In some trials, under control conditions, we applied a baseline depolarization prior to the ramp, in order to capture the variability observed in vivo (Harvey et al Nature 461:941–946, 2009; Epsztein et al. Neuron 70:109–120, 2011). Application of the cholinergic agonist carbachol (CCh, 2 µM) caused a depolarization of 2-6 mV. We compensated for this depolarization by injecting tonic hyperpolarizing current to reestablish the original membrane potential (see also Losonczy, et al., Nature 452, 436-442, 2008), as indicated by an offset from the 0 pA current level in the traces of the injected current ramps. The amplitude of background fluctuations in the resting membrane potential increased from a few tenths of a mV in control to 2-4 mV in CCh. Moreover, the threshold for action potential generation became more hyperpolarized. For all these reasons, we were not able to consistently vary the membrane potential using baseline depolarizations in the presence of CCh, because baseline depolarization alone frequently evoked spiking.”

For this reason, many of the carbachol example traces in the initial submission had more hyperpolarized Vm than their control counterparts. Acetylcholine also caused a depolarization in a dose-dependent manner, that was compensated for in the same way. In this new version of the manuscript, we systematically report the effects of cholinergic agonists on membrane potential and neuronal excitability. Further, we show example traces with resting membrane potentials within 1 mV for each pharmacological comparison, therefore removing this variable and hopefully making results clearer. We also now state how the amount of injected current was chosen for each condition, and that the amount of injected current was generally lower in the presence of cholinergic agonists. Both the tonic hyperpolarizing current and the amplitude of the injected ramp for each example can now be appreciated in each figure.

Finally, the reviewers’ comment also made us realize that, in principle, the center of mass of firing could be systematically skewed by the initial membrane potential, the amplitude of the current ramp injection and/or the input resistance. For this reason, we added a supplementary figure (1-2) where the adaptation index was plotted as a function of each these variables. In all cases, it is apparent that the main factor determining whether the center of mass of firing is shifted earlier or later in the ramp is the presence or absence of carbachol rather than initial membrane potential, current injection amplitude, or input resistance.

4) It remains unclear how the current result that TRPM4 channels can mediate the firing pattern change relates to the previous finding that the current injection evoked CA1 neuronal firing pattern is due to long-term Na channel inactivation.

We thank the reviewers for this suggestion, which helps to clarify our initial results. New Figure 8 addresses the connection between long-term inactivation of Na+ channels and the activation of TRPM4 channels, as characterized by the model (see Results lines 375-391). Furthermore, the model was instrumental in assessing how the Ca2+ and voltage-dependence of TRPM4 channels synergize to contribute to the shift in the center of mass of firing (Figure 9). Figure 9 illustrates the positive feedback loop between Ca2+ entry and the additional depolarization produced by Ca2+ activation of TRPM4 channels that can potentially accelerate firing (see Results lines 392-427).

5) Figure 8: Panel C is supposed to confirm the prediction from the model that the carbachol-mediated change of firing activity is related to intracellular Ca2+ domains. However, the example cell shown is depolarized to -52 mV, and there is no hyperpolarization following Carbachol. Is this an effect of the high concentration of BAPTA? Again, what was the current injected under this experimental condition?

Again, we thank the reviewer for pointing out the lack of clarity in the presentation of our results. We have now rewritten the results section for former Figure 8 (now Figure 10) to more clearly present these findings. The reviewer is correct that with the combination of 30 mM BAPTA + 10 nM free Ca2+ added to the intracellular solution (panel C of current Figure 10) the addition of carbachol did not change the membrane potential, as there were no changes in the holding current. Also, the amplitude of the ramp is comparable in control conditions and in the presence of carbachol under these conditions.

We have now added all these details in the Results section for figure 10C.

Reviewer #2 (Public Review):

The manuscript focuses on the cholinergic modulation of TRPM4 channels in the CA1 pyramidal neurons. The authors presented solid convincing evidence that TRPM4 but not TRPC channels are the Ca2+-activated nonselective cation channel in CA1 pyramidal neurons being modulated by activation of muscarinic receptors. Using bi-directional ramp protocol, the authors revealed that ACh modulation could lead to forward shifts in place field center of mass, whereas decreased ACh modulation could contribute to backward shifts. This represents a significant molecular/cellular finding that links neuromodulation of intrinsic properties to place field shifts, a phenomenon seen in vivo. The authors used a computational approach to model this CA1 neuron spiking to further reveal the mechanism.

To further improve the manuscript, I have the following suggestions/questions:

1) The triangular ramp stimulation (introduced by the same group; Upchurch et al., 2022) makes it possible to emulate the hill-shaped depolarization during place field firing. However, one concern is the time scale/duration of the ramp (2 sec) compared to the physiological pattern (100ms~200ms in the in vivo recording in freely moving rat, Epsztein et al., 2011). Using a longer ramp to generate more spikes for calculating the adaptation index is understandable. However, considering the Ca entry/accumulation during prolonged depolarization, repeating one set of experiments with a shorter ramp is crucial to verify the major findings.

When determining the duration of the current injections for our ramps, we relied on the data recorded in vivo in freely moving rats (Epsztein et al. Neuron 70:109–120, 2011) or in head-fixed mice running on spherical a treadmill immersed in virtual reality (Harvey et al Nature 461:941–946, 2009). In those papers, the voltage deflections are shown as a function of time, and gray bars or boxes represent the time the animals spend traversing the place field. We interpret those figures as showing that the hill-shaped depolarizations have variable durations, on the order of 1-20 s; we therefore think that our experiments with 2 and 10 second-long ramps cover a fair range of these durations. The place fields in Epsztein et al., 2011 were 4 cm long, and the authors give an example in Figure 3, in which the 2 meter track is traversed 1.5 times in 3 minutes. At that rate, the rat spent on average 2.4 seconds in each place field. We interpret the numerous shorter epochs of firing on the order of 100-200 ms shown Figure 2 in Epsztein et al. as the result of ongoing theta modulation within one overall depolarization during a single place field traversal. The following quote from that paper supports our interpretation “Some (Figure 2E, trace 1), but not all (trace 2), passes revealed spiking associated with a series of large (to ~-25 mV), long-lasting (~100 ms) depolarizations (Kandel and Spencer, 1961; Wong and Prince, 1978; Traub and Llinás, 1979; Takahashi and Magee, 2009) occurring rhythmically at ~4–5 Hz (theta frequency).” We thank the reviewer for pointing out these traces; our results are more directly applicable to the traces without theta modulation. Adding theta modulation is beyond the scope of this study but will be considered in future studies. Our average results in Figure 1 show that carbachol similarly affects 2 s and 10 s ramps, therefore we decided to present only the data on 2 second ramps for all the subsequent figures (see Results lines 156-157).

2) Strictly speaking, the term "Ca2+-induced Ca2+ release (CICR)" is only used in ER Ca2+ release via ryanodine receptors (RyR) rather than IP3Rs. The author should be careful since it is used in the abstract (Line 36). In addition, pharmacology inhibition experiments should be incorporated to further dissect the role of RyR-induced CICR.

We thank the reviewer for pointing out the possible confusion regarding the use of the term Ca2+-induced Ca2+ release (CICR) and we removed it from the text. Further, for this resubmission, we have pharmacologically dissected the role of IP3 vs ryanodine receptors in the cholinergic shift in the center of mass of firing due to the activation of TRPM4 channels, as suggested by the reviewer (see new Figure 6). To our surprise, neither the IP3R antagonist, Xestospongin C (1-2 µM), nor the RyR antagonist ryanodine (40 µM) were effective in preventing the cholinergic shift of the center of mass of firing when added to the intracellular solution (see Results lines 310-340).

3) Applying strong buffering BAPTA not only removed the IP3R-TRPM nanodomain but also hindered Ca entry via VGCC. To validate the role of ER Ca2+ release in regulating TRPM, depletion of ER Ca2+ pool with SERCA inhibitor (e.g. thapsigargin) would be a more direct way to test the model (also make sure to add TRPC inhibitor to avoid the store-operated Ca2+ entry).

We agree with the reviewer that 30 mM BAPTA also disrupts intracellular Ca2+ elevation via voltage-dependent Ca2+ channels on the neuronal membrane. Given that our experiments excluded a role of Ca2+ release from the intracellular stores (see below), our new model includes a nanodomain where, during cholinergic activation, the Ca2+ entry through VGCC is amplified to reach micromolar concentrations, through a currently unknown mechanism. As pointed out by the reviewer, the experimental results with 30 mM BAPTA support the existence of a nanodomain for the activation of TRPM4 channels, regardless of the nature of the calcium source.

We have also addressed the role of ER Ca2+ release in our experiments.

4) How does the TRPM current overcome the long-term inactivation of Nav? A channel state model should be added to the manuscript to make it easier to understand.

Figure 11C now shows the Markov model of the NaV channel and new Figure 8 is devoted to explaining the mechanism by which current through the TRPM4 channels overcomes the long-term inactivation of the NaV channel.

Reviewer #3 (Public Review):

Combining slice physiology and simulation, Combe and colleagues discovered that TRPM4 channels activated by Ca2+ in nanodomains mediate ICAN currents in CA1 pyramidal neurons that drive the cholinergic modulation of firing rate. The finding is novel and interesting.

Strengths:

1) Identification of TRPM4 channels as the carrier of ICAN currents with independent pharmacological inhibitors and other supporting evidence.

2) Physiological and simulational verification of physically closely located Ca2+ source and TRPM4 channels required for ICAN activation.

Weaknesses:

1) The conclusion of the cholinergic role in down-ramp or backward firing shifts is not convincing.

We agree with the reviewer that our interpretation is somewhat speculative, and we have now included disclaimers throughout the manuscript as well as placed most of these interpretations in a portion of the discussion titled “Ideas and speculations: Implications of our results for place fields in intact rodents”. In addition, we added the word “potential” in the title.

Reviewer #2 (Public Review):

The authors examine the transport of chemical compounds from a surrounding fluid environment to the surface of the polyp Hydra. They propose that spontaneous contractions of the body, which are known to occur roughly three times per hour, provide a new fluid environment near the body surface and thereby increase the total rate of compound uptake. Experimental measurements and a mathematical model are used to support the main claim. Active probes of the system involve the use of ion channel inhibitors, which can affect the frequency of contractions. But there is a useful feature of Hydra already present which the authors also use for a comparative study, namely the different microbial environments near the Hydra's motionless foot and near its moving head. The evidence which is provided puts the claim on solid footing. The main result represents an important observation about the role of hydrodynamics on organism behavior, in particular in its relation to diffusive chemical transport processes.

Author Response

Reviewer #1 (Public Review):

Weaknesses

1) I was curious as to how novel this setup is. Although I do not do head-fixed research myself, I thought there were already some open-source, relatively cheap systems available. I'm not sure how the current setup differs from those already available. Personally, even if this system involves only the wheel turning, as this is a truly operant response, that is novel enough for my liking.

The novelty of the system stems from the synergistic combination of functionality, the low-cost open source nature of the design, and the breadth of behavioral procedures the system is able to support. The use of a wheel as an operant response was adapted from the International Brain Laboratory rig which has been used extensively for visual discrimination tasks. We adapted this wheel design to make the response closer to lever pressing through the use of the wheel brake, which ensures that subjects have to rotate the wheel in discrete rotational bouts rather than continuously spinning the wheel and potentially disengaging and allowing the wheel to rotate independently. There are no examples of systems capable of delivering 5+ solutions within a behavioral session or conducting valence testing with a modification of real-time place preference without the cost and complexity associated with virtual reality. We believe that the combination of factors, the flexibility and scalability of the system makes OHRBETS a novel and useful system for diverse motivation and consumption behaviors in head-fixed mice.

2) It would be useful to have a bit more detail in the manuscript (not just on the GitHub link - in supplemental material perhaps?) on how to build such a system, just to get a sense of how difficult building such a system might be and how many components it has.

With this submission we have included detailed assembly instructions as a supplement to the main manuscript and added reference to the file within the methods section. We have also added details, including time estimates, to the methods section.

3) I wasn't sure how to feel about the comparisons across experimental set-ups in Figures 2 and 3. Usually, these sorts of comparisons are not considered statistically valid due to the many variables that differ between set-ups. However, I do see that the intent here is a bit different - i.e. is to show that despite all these alterations in variables the behavioural outputs are still highly correlated. However, without commenting on this intent, I did find these comparisons a little jarring to read.

Thank you for highlighting this. We have added in a justification for why we measured the consistency in behavior measured with each head-fixed system.

4) The only dataset I was not wholly convinced by was that in Figure 3 (real-time place preference and aversion). I think the authors have done the best job that they can of replicating such a procedure in a head-fixed mouse, but the head-fixed version is going to necessarily differ from the freely moving version in a fundamental way when the contextual cues and spatial navigation form part of the RTPT task. Giving a discrete cue, such as a tone, just is not a sufficient substitute for contextual cues, and I think the two types of task would engage fundamentally different brain cells and circuits (e.g. only the free-moving version is likely to engage place cells in the hippocampus).

To avoid confusion regarding the place component of the real-time place preference assay name, we have renamed the head-fixed assay for assessing valence to Wheel-Time Preference (WTP). We have also added a full paragraph to the discussion where we outline the differences in the task requirements and relevant neuronal circuits between the freely-moving RTPP and head-fixed WTP. We understand that the head-fixed task is not a perfect analog of the RTPP task, however based on the similarity in the resulting time spent in the stimulation chamber/zone we believe that the WTP is able to replicate the valence assessment that many in the field uses RTPP to measure. We believe that the WTP with OHRBETS opens up new possibilities for assessing preference in head-fixed mice and this justifies keeping the figure within the main manuscript.

To thoroughly address the potential confound of spatial information during the multi-spout experiment, we have added an additional supplemental figure (Figure 4- figure supplement 5) that depicts the proportion of trials with licking and added a paragraph to the discussion centered on the potential confound associated with learning the solution identity.

5) Personally, I found having the statistics in a separate file confusing.

Thank you for raising this concern. With our initial submission, we were concerned that including all of the statistics within the main text would make the paper difficult to read due to the extensive amount of statistics. With this submission, in addition to the statistics table, we have included statistics within the figure legends and main text where applicable.

6) Line 589-594. Suggesting the medial/lateral shell recording results mean that the medial shell 'tracks value, and the range of values during the multi-spout consumption of gradients of NaCl is greater than the range of values during multi-spout consumption of gradients of sucrose" seems to engage in circular logic to me. That is, the authors should use behavioural data to infer what the animal is experiencing and whether it is a change in value, and/or a greater change in value during NaCl vs. sucrose consumption, and only then should they make an inference about what the larger medial shell response means.

Thank you for identifying this potential site of confusion. To address this concern we have modified the language to better communicate our interpretation of the data.

“If we assume that the range of values is greater during multi-spout consumption of gradients of NaCl compared to gradients of sucrose, as indicated by a greater range in licking behavior (Figure 8- Figure Supplement 4), then the greater range of dopamine release in the NacShM could imply that dopamine release in this structure tracks value.”

By nodding your head, making sure they know you understand or agree with what they are saying. Have a good posture makes them feel comfortable. Eye contact is essential to make them feel seen, payed attention to. If the audience is leaning in to the speaker, it helps the speaker know that the audience is engaged and attentive.

this sounds cool #trips

Reviewer #1 (Public Review):

In this paper, Scholz and colleagues introduce a new paradigm aimed to bridge the gap between two domains that rely on hierarchical processing: language and memory. They find that, generally in line with their hypotheses, hierarchical processing is associated with activation in hippocampus (especially anterior), medial prefrontal cortex (mPFC), posterior superior temporal sulcus (pSTS), and inferior frontal gyrus (IFG). They also report that these effects in IFG are particularly strong late in the task, once participants have had a lot of experience and processing is presumably more automatic.

This work has many strengths. The goal to bridge these literatures by developing a new task is commendable. I appreciate also that the authors separately validated their new task behaviorally by comparing it to another accepted as tapping hierarchical processing. I also liked that the authors were transparent about their hypotheses, and certain analyses like the grid coding one that was planned but did not work out. I do however have a number of concerns about the interpretations of the findings, such as whether some patterns are ambiguous as to the true underlying effects. I also have a number of clarification questions. All concerns are described below.

1. Broadly, I would like to see the authors provide more information and logic on why hierarchical processing should be associated with a big reduction in univariate activation between P1 and P2-why would this signify item in contexts binding? How does this relate to existing work using other methods (e.g., like animal studies, which seem to make predictions more about representational structures)?

2. There are many differences between what kind of information participants are processing between Position 1 and Position 2 for the HIER but not ITER conditions, and these may not be related to the hierarchical structure specifically. Related to but I think distinct from some of the limitations mentioned in the Discussion is the fact that in the HIER condition, what is happening cognitively between Position 1 and Position 2 items is more distinct (attending to color for position 1, and shape for position 2), whereas the two positions are equivalent in the ITER condition. This is a bit different from the authors' intended manipulation of hierarchy, because it involves a specific dimension. A stronger design might have been to flip the dimensions with respect to position specifically, to make shape sometimes important for position 1, and color for position 2 (perhaps by counterbalancing across subjects, so half would see the current P1=color and P2=shape rules, and the other half P1=shape and P2=color rules). Another important difference between color and shape is that while color is a simple binary distinction that participants can make based on their preexisting knowledge of red versus green, and to which they can assign a verbal label; whereas, the shape distinction was something novel they acquired during the experiment, has no real-world validity or meaning, and would presumably rely more on visuospatial processing. The shape dimension was also much more variable, I believe. I should say that I do find comfort in a few things - (1) that behavior on this task is correlated with another one that also indexes hierarchy processing, and (2) that the results show regional specificity in a pattern at least not easily explained by this distinction. However, I do think future work will be needed to ask whether it is hierarchy processing per se or rather something to do with the particular cognitive states engaged during each phase in this particular task that is eliciting activation in this set of regions. It would strengthen the paper to discuss this issue directly so readers are alerted to the caveat.

3. I did not understand what data went into creating the schematic in Figure 2E. First, I think this depiction of a gradient might be easily misinterpreted because it seems to imply that the authors have a higher resolution analysis than they actually do. I believe the data were just analyzed in three subregions of hippocampus - head, body, and tail. Variability within each subregion (as seems to be implied by certain parts of a region being more grey and others more red/orange), is not something that could be assessed in this analysis. For example, why does the medial part of the head seem to be more "unspecific" whereas lateral regions look more HIER Pos1 specific? This type of depiction would only make sense in my mind if the authors had performed something like a voxelwise analysis to determine where specifically the interaction "peaks." I would recommend this visualization be cut or significantly changed to do away with the gradient.

4. I believe the authors have not reported enough information for us to know that hippocampus involvement indeed does not change with experience. It is interesting that hippocampus in the task x experience ROI analysis shows, if anything, bigger differentiation between the two tasks (numerically) for the late trials. This seems to go against the authors' hypothesis, and a lot of existing data, that hippocampus is preferentially involved in early (vs. late) learning. Given that the key signature in this region, though, is that it differentiates between position 1 and position 2 in HIER but not ITER, and doesn't show a big difference in magnitude across the two tasks, it makes me wonder whether the task x experience interaction collapsing across the two positions makes sense for this region. Did the authors consider a similar task x experience interaction within hippocampus, but additionally considering position? I think there are multiple ways to look at this question (e.g., either looking for a task x experience x position interaction, a task x experience within position 1, a task x position interaction separately in early vs. late portions of the task, or even a position x experience interaction only within the HIER task), and I'm sure the authors would be in a better place to decide on a specific path forward. The same logic might go for mPFC, which shows an interaction but no main effect of task. This relates to claims in the discussion as well, such as that "hippocampus was equally active in early and late trials," but given this analysis is collapsing across the dimension hippocampus (and mPFC) seem to be sensitive to (position), it seems like this could be masking an underlying effect in which hippocampus/mPFC might still be differentially involved early vs. late (i.e., they might show the task x position interaction preferentially during some task phases).

5. For the IFG regions, the task x experience interaction seems to be driven mainly by change (decrease in activation) for the ITER, rather than change in the HIER. The authors are at times careful to talk about this as "sustained" activity in IFG, which I appreciated, but other times talk about a "relative increase." I am not sure how I feel about that. I see the compelling evidence that there are task differences by experience, and that there is reduction for ITER that is interestingly not present for HIER, but I think I am still feeling uncomfortable with the term "increase" or even "relative increase" for HIER. For example, couldn't it simply be that the ITER task is requiring less processing with experience, whereas the HIER does not (perhaps because it requires more processing to begin with)? i.e., we do not know whether the reduction for ITER is simply a neural signal thing (i.e., activations diminish over time/experience) or a cognitive thing, specific to the ITER task. I think the authors are wanting to interpret the reductions as the former, but perhaps it would be more powerful to demonstrate if there was a baseline task that also showed reductions but for which not much would be expected in the way of cognitive change. Can the authors provide more justification for their choice of terminology (through either more logic or analyses), or if not, simply talk about it as sustained activity for HIER-which is especially interesting in the face of reductions for the ITER task?

6. Please define what is meant by the term "automaticity" in the introduction. A clearer definition of the concept would make the paper generally easier to follow, and it would also help foreshadow the hypotheses about mPFC activity in the introduction. To this end, it could be useful to elaborate on how learning takes place in this task, how it could foster increasing automaticity, and how automaticity maps onto behaviour (e.g., is it RT decrease alone, which happens for both conditions in this task?) the brain regions discussed.

7. There was no association between brain and behavior, which the authors interpret as a positive (as therefore task difficulty differences could not explain the effects). However in light of these null findings, it is on the flip side hard to know whether this neural engagement carries any behavioral significance. It seems to me as though the authors' framework makes predictions about brain-behavior correlations that were not tested in the manuscript. For example, I believe the authors asked whether behavior overall was correlated with activation. However, wouldn't the automaticity in IFG explanation for example predict that more engagement or an increase in engagement from early to late should be associated with e.g., faster RTs-not necessarily a relationship overall?

8. On p. 8, it is stated that "In the hippocampus, this effect is driven by higher betas for the presentation of the first object (H1 > I1) and lower betas for the second object (H2 < I2) when comparing across tasks." Can the authors confirm whether the pairwise comparisons following up on the interaction here are significant, or rather if they are referring to a numerical difference in the betas? It looked like the same (numerically) would be true for mPFC; is there a reason why the same information is not included for the mPFC ROI? Also, might the authors provide more speculation as to why one might see both enhanced and reduced activation for P1 and P2, respectively?

9. I was expecting some discussion of how hippocampus does not seem to show preferential involvement early, given that its potential role being restricted to early in learning (i.e., during acquisition only) was one of the primary motivators for using this task. As noted in my above comment (#4), I am not quite sure that I think there is evidence that the hippocampal role remains constant over this task, given the analyses provided (i.e., that they did not look at the position effect for early vs. late). However upon further analysis if it does seem to be more stable, and/or if it even increases over experience, the authors might want to talk about that in the Discussion.

10. The fact that the hierarchies in this paradigm unfolded over time makes them distinct on some level from the hierarchies present in the VRT task that was used to validate the HIER task's hierarchical processing demands. For example, there might be additional computations required to processes these temporally ordered structures, support online maintenance, and so on. It may be worth considering this aspect of the task, and whether/to what extent the results could be related to it, in the paper.

11. I also have many methodological and analytic clarification questions, which I detail in the recommendations for authors.

Reviewer #2 (Public Review):

In this manuscript, Scholz et al., adopt a set of tasks to study how brain regions are differentially activated with temporal context clues. In one task, the first item in a two item sequence will dictate the value of the second. In another task, there is no hierarchy in temporal order, though subjects must still maintain information across the delay to add the value of the two presented items. Using univariate analyses, the authors found many regions that showed an interaction between item position and task, including: the mPFC, anterior hippocampus and the left prefrontal and posterior temporal cortices. The results are interpreted as evidence for a dedicated system for understanding hierarchical relationships across domains as various as spatial cognition, music, and language.

The question raised by the authors is important and fMRI may be an appropriate means of studying the neural basis for hierarchical computations. The main limitation of the manuscript, and one that is briefly mentioned and dismissed in the discussion is the task design, which confounds whether or not a hierarchical relationship must be formed, and the content of the information that must be held across working memory (color in the hierarchy task and number in the iterative task).

The authors also report an interesting difference between the activation observed in the head and tail of the hippocampus during the different tasks. However, the authors compare each region independently, show one is significant and the other is not, and then conclude "the effect of hierarchical context representation in the hippocampus is specific to its anterior regions." Such a conclusion requires direct comparison of the regions.

Finally, it isn't clear if the motivating prior work makes a simple univariate prediction. A strong prediction however is that the representational similarity should be very different for objects in the first versus second position in the hierarchy task and much less so in the iterative task. Such a representational similarity analysis would better connect this study to prior research and to the hypothesis that hierarchical processing affects the coding of items in sequence.

interesting- not sure which scenario is more likely off the top of my head

‘The Canto of Ulysses’ records a truly remarkable feat of cultural memory, as Levi recalls and recites Dante amidst the ruin of Auschwitz. He does so to teach Italian to his campmate Jean. But as he recites the words of Dante, Levi also feels his past come back to him. He begins to recover something of his identity and his humanity in the degraded world of the Lager, to feel again that he is ‘a man’.

Dante is not, however, the only canonical writer to appear in ‘The Canto of Ulysses’. So too does Shakespeare. <br /> Levi admits to ‘gaps’, ‘holes’, and ‘lacuna[e]’ in his memory as he recites Dante, some of which would seem to be ‘irreparable’. Unable to recall his Dante, Levi is forced to confront ‘silence’:

I would give today’s soup to know how to connect ‘the like on any day’ to the last lines. I try to reconstruct it through rhymes, I close my eyes, I bite my fingers – but it is no use, the rest is silence [il resto è silenzio]. Other verses dance in my head: ‘…The sodden ground belched wind …’, no, it is something else. [Woolf’s translation.]

‘[T]he rest is silence’: Levi is quoting the last words that are spoken by Hamlet, before he dies:

O, I die, Horatio!

The potent poison quite o’ercrows my spirit.

I cannot live to hear the news from England.

But I do prophesy th’election lights

On Fortinbras; he has my dying voice.

So tell him, with th’occurrents more and less

Which have solicited – the rest is silence.

With his ‘spirit’ succumbing to the ‘potent poison’ Claudius and Laertes have used to kill him, Hamlet undertakes the impossible: to testify to his own death, to ‘tell’ in words his fall into deathly silence. This is his ‘dying voice’.

What is Hamlet doing in ‘The Canto of Ulysses’? Why does Levi place it where he does, in ‘the caesura’, the abyssal gap between Auschwitz and his cultural identity and memory, embodied by Dante? It is certainly ironic that Levi draws once again on the Western canonical literary tradition to record the moment of its ostensible breakdown. What emerges from the lapse, the silence that Levi testifies to, is another tie to his compromised past, and the literary culture that would seem to have been obliterated by the Holocaust – even if Levi does not choose to bring obvious notice to his allusion by using quotation marks or by writing, ‘as Hamlet says’. The poetry of Hamlet appears to be among the ‘other verses’ Levi has confusedly ‘dancing in his head’ while he tries to fill the gaps in his memory.

By quoting Hamlet, Levi would appear to again testify to the power of literature as a mainstay of culture and humanity, evincing his commitment to humanist ideals. This is certainly the positive interpretation of Hamlet in ‘The Canto of Ulysses’. I would make the case, however, that Levi is using Shakespeare for an altogether more challenging purpose. By appropriating the ‘dying voice’ of Hamlet, Levi records the place where his memory and his identity collapse, testifying to the reduction of the camp inmate to silence and oblivion. Bryan Cheyette writes that ‘even when his memory self-consciously fails him’, Levi is always ‘at pains to bear witness to those moments of failure’ (Cheyette 1999, 64). Levi seeks to testify to the silence, to show that ‘something has occurred even if it cannot be understood’ (Druker 2009, 64). This is the role played by Hamlet in ‘The Canto of Ulysses’. Levi does not use the play to testify to the endurance of the human spirit in the camps, but to silence, to a ‘world of negation’ (OC I, 235). Hamlet, perhaps ironically given its near-unrivalled canonical and cultural status, marks the space beyond language and culture, into which Levi is at risk of falling. This, as Levi called it, is the ‘black hole’ of Auschwitz (OC II, 1663).

Through his allusion to Hamlet, Levi rewrites Shakespeare from the perspective of Auschwitz, endowing the play with new meanings as he confronts the lacunae and voids produced by the world of the concentration camp. Levi uses the play to record the disintegration of humane values in Auschwitz, as memory and language are brought to the point of collapse. Jacques Derrida does much the same in his own work on the play. He draws Hamlet into conversation with Holocaust testimony when he compares the play to the poetry of survivor Paul Celan in his 1995 piece ‘The Time is Out of Joint’. Derrida contends that the paradox of testimony is that the witness must uncannily ‘outlive his life’ (3.2.117) – or ‘survive’ that which is not ‘survivable’: the collapse of all meaning and death (Derrida 1995). This is the sense in which Hamlet is a play about the ‘impossible possibility of testimony’, as Derrida calls it. Hamlet has ‘seen the worst’ and is ‘the witness of the worst disorder, of absolute injustice’, writes Derrida. Hamlet has witnessed too much for words – but testimony, ‘though it hath no tongue, will speak’ (2.2.546).

Levi must also confront and testify to the painful death of memory, the destruction of human identity and culture, before the event of physical death itself – or as Jacques Lacan would call it, ‘symbolic’ before ‘actual’ death, the death of the self before physical death. Lawrence Langer uses the phrase ‘deathlife’ to name the same phenomenon, of ‘dying while one is living’ (Langer 2021, 13). Not unlike the melancholic prince, Levi attempts the impossible of testifying to his own demise. He deploys Hamlet to record his fall into a place beyond humanity and beyond culture – even beyond language. It is, to adopt the words of Jean Améry, an act of both resignation and revolt: resignation to silence, and a determined revolt against oblivion, by testifying to it. It is an astonishing moment.

RA

Reviewer #1 (Public Review):

There are a number of outstanding questions concerning how cohesin turnover on DNA is controlled by various accessory factors and how such turnover is controlled by post-translational modification. In this paper, Nasmyth et al. perform a series of AlphaFold structure predictions that aim to address several of these outstanding questions. Their structure predictions suggest that the release factor WAPL forms a ternary complex with PDS5 and SA/SCC3. This ternary complex appears to be able to bind the N-terminal end of SCC1, suggesting how formation of such a complex could stabilize an open state of the cohesin ring. Additional calculations suggest how the Eco/ESCO acetyltransferases and Sororin engage the SMC3 head domain and thereby protect against WAPL-mediated release.

This work thus demonstrates the power of AF prediction methods and how they can lead to a number of interesting and testable hypotheses that can transform our understanding of cohesin regulation. These findings require orthogonal experimental validation, but the authors argue convincingly that such validation should not be a pre-requisite to publication.

As the authors did not systematically include model confidence scores it is difficult for the reader to evaluate the reliability of the models obtained. The caveat is that many readers will by default assume that the presented models are correct, when in fact, some of them may score poorly and require careful assessment. As numerous readers will not be very familiar with the AF confidence scoring mechanisms, it would be important to include such metrics and indicate what these scores mean for the different interfaces (Acceptable, Medium and High confidence?). pLDDT and PAE plots should be included. When they report on a key interaction (E.g. WAPL-SCC1) they should indicate the key region (SCC1 N-terminus) on the PAE plot. False positives are always possible even with good scores, especially when many protein pairs are tried. It would therefore be important to also include a table showing the global scores for pTM and ipTM to summarise the confidence scores of interfaces.

It is exciting to see AF-multimer predictions being applied to cohesin. As some of the reported interactions are not universally conserved and some involve relatively small interfaces the possibility arises that these interfaces show poor or borderline confidence scores. As some of these interfaces map to mutants that have previously been obtained by hypothesis-free genetic screens and mutational analyses they appear nevertheless valid. Thus, an important point to make is that even interfaces that show modest confidence scores may turn out to be valid while others may be not. The authors therefore should emphasize that the proposed models are just predictions and that additional orthogonal validations are required.

Reviewer #2 (Public Review):

This paper addresses an important computational problem in learning and memory. Why do related memory representations sometimes become more similar to each other (integration) and sometimes more distinct (differentiation)? Classic supervised learning models predict that shared associations should cause memories to integrate, but these models have recently been challenged by empirical data showing that shared associations can sometimes cause differentiation. The authors have previously proposed that unsupervised learning may account for these unintuitive data. Here, they follow up on this idea by actually implementing an unsupervised neural network model that updates the connections between memories based on the amount of coactivity between them. The goal of the authors' paper is to assess whether such a model can account for recent empirical data at odds with supervised learning accounts. For each empirical finding they wish to explain, the authors built a neural network model with a very simple architecture (two inputs layers, one hidden layer, and one output layer) and with prewired stimulus representations and associations. On each trial, a stimulus is presented to the model, and inhibitory oscillations allow competing memories to pop up. Pre-specified u-shaped learning rules are used to update the weights in the model, such that low coactivity leaves model connections unchanged, moderate coactivity weakens connections, and high coactivity strengthens connections. In each of the three models, the authors manipulate stimulus similarity (following Chanales et al), shared vs distinct associations (following Favila et al), or learning strength (a stand in for blocked versus interleaved learning schedule; following Schlichting et al) and evaluate how the model representations evolve over trials.

As a proof of principle, the authors succeed in demonstrating that unsupervised learning with a simple u-shaped rule can produce qualitative results in line with the empirical reports. For instance, they show that pairing two stimuli with a common associate (as in Favila et al) can lead to *differentiation* of the model representations. Demonstrating these effects isn't trivial and a formal modeling framework for doing so is a valuable contribution. Overall, the authors do a good job of both formally describing their model and giving readers a high level sense of how their critical model components work, though there are some places where the robustness of the model to different parameter choices is unclear. In some cases, the authors are very clear about this (e.g. the fast learning rate required to observe differentiation). However, in other instances, the paper would be strengthened by a clearer reporting of the critical parameter ranges. For instance, it's clear from the manipulation of oscillation strength in the model of Schlichting et al that this parameter can dramatically change the direction of the results. The authors do report the oscillation strength parameter values that they used in the other two models, but it is not clear how sensitive these models are to small changes in this value. Similarly, it's not clear whether the 2/6 hidden layer overlap (only explicitly manipulated in the model of Chanales et al) is required for the other two models to work. Finally, though the u-shaped learning rule is essential to this framework, the paper does little formal investigation of this learning rule. It seems obvious that allowing the u-shape to collapse too much toward a horizontal line would reduce the model's ability to account for empirical results, but there may be other more interesting features of the learning rule parameterization that are essential for the model to function properly.

There are a few other points that may limit the model's ability to clearly map onto or make predictions about empirical data. The model(s) seems very keen to integrate and do so more completely than the available empirical data suggest. For instance, there is a complete collapse of representations in half of the simulations in the Chanales et al model and the blocked simulation in the Schlichting et al model also seems to produce nearly complete integration. Even if the Chanales et al paper had observed some modest behavioral attraction effects, this model would seem to over-predict integration. The author's somewhat implicitly acknowledge this when they discuss the difficulty of producing differentiation ("Practical Advice for Getting the Model to Show Differentiation") and not of producing integration, but don't address it head on. Second, the authors choice of strongly prewiring associations in the Chanales and Favila models makes it difficult to think about how their model maps onto experimental contexts where competition is presumably occurring while associations are only weakly learned. In the Chanales et al paper, for example, the object-face associations are not well learned in initial rounds of the color memory test. While the authors do justify their modeling choice and their reasons have merit, the manipulation of AX association strength in the Schlichting et al model also makes it clear that the association strength has a substantial effect on the model output. Given the effect of this manipulation, more clarity around this assumption for the other two models is needed.

Overall, this is strong and clearly described work that is likely to have a positive impact on computational and empirical work in learning and memory. While the authors have written about some of the ideas discussed in this paper previously, a fully implemented and openly available model is a clear advance that will benefit the field. It is not easy to translate a high-level description of a learning rule into a model that actually runs and behaves as expected. The fact that the authors have made all their code available makes it likely that other researchers will extend the model in numerous interesting ways, many of which the authors have discussed and highlighted in their paper.

Author Response

Reviewer #1 (Public Review):

The Introduction starts by setting up a straw-man argument, claiming that the assumption is that gene expression is set up as stable expression domains that undergo little or no subsequent change. I don't think that any current developmental biologist thinks this is true. The references used to support this claim are from the 1990s up to the early 2000s. There are numerous examples since then that show that developmental gene expression is dynamic as a rule.

Our argument might seem like a strawman for certain sector of developmental biologists who work in the field of pattern formation, or aware of the latest advances in the field. However, a look at current publications on developmental enhancers reveals that the dominant model with which enhancer biologists interpret their data is still the French Flag model (specifically, the eve-stripe-2 model of enhancer function). We meant to address this audience, and attempted to clarify this from the very beginning by stating that “Much of our models of how enhancers work during development relies on the assumption that …”. Please, note here that we are talking about “models of how enhancers work”, not models of pattern formation in general.

The Introduction then continues as a rather detailed review of enhancers, Tribolium methodology, tools for identifying enhancers, and more. The Introduction cites 99 references, which seems excessive for what is essentially an experimental paper. Significant parts of the Introduction can be trimmed or removed. There is no need to mention all the tools available for Tribolium if they are not used in the described experiments. A thorough analysis of the advantages and disadvantages of different modes of ATAC-seq is also beyond the scope of the Introduction. The authors should explain why they chose the tools they chose without excessive background.

In the revised manuscript, we shortened the discussion of Tribolium methodologies and imaging techniques. However, we think that the paragraph discussing ATAC-seq strategies are important to justify our choices as why we took the effort to cut the embryos to perform tissue-specific ATAC-seq analysis, instead of performing whole-embryo ATAC-seq.

Having said that, the Introduction actually overlooks a lot of significant work that is relevant to the subject of the paper. Specifically, the authors completely ignore all of the work on development in hemimetabolous insects such as Oncopeltus and Gryllus - the omission is glaring. There has been a lot of relevant work on dynamic gene expression patterns coming out of these species.

You are right indeed. We apologize for that. We added now citations to relevant works from those to insect to the manuscript.

The experimental setup involves cutting embryos into three sections at two time points. The results then discuss differences in "space" and "time" but there is no discussion of the embryological meaning of these terms. What is happening at the two time points from a developmental perspective? What is the difference between the three sections? There is a lot of relevant development going on at these stages and important regional differences, which have been well-studied in Tribolium and in other insects but are not even mentioned.

A good point. Correlating chromatin landscape changes with embryological events is an interesting point that needs further analysis and the application of ATAC-seq to further timepoints. We chose leaving this to future work (possibly using single cell ATAC-seq). In this work, we restricted our analysis to the benefits of applying time- and tissue-specific ATAC-seq in predicting active enhancers. We added a note on this point in the discussion.

In the preliminary results of the ATAC-seq analysis, it is clear that there are significant differences between the sections, which should come as no surprise, but fairly minor differences between the same section at the two time points. This could be because the two time points are pretty close together at a stage when there is a lot of repetitive patterning going on. A possible interpretation, which the authors don't mention because it goes against their main thesis, is that maybe most of the processes that are taking place at this stage are not dynamic enough to show up at the temporal resolution they have applied. This is worth at least a mention.

We agree with this observation. We would like to draw the reviewer’s attention to our statement “Together, our findings indicate that changes in chromatin accessibility in Tribolium at this developmental stage are primarily associated with space rather than time…””. Detailed analysis of the chromatin dynamics across time would need taking more datapoints, which is something we plan to do in future work.

The authors link each accessible site to the nearest gene when looking at putative enhancer function. This is a risky assumption since there are many examples of enhancer sites that are far upstream or downstream of the target gene and often closer to an unrelated gene than to the target gene. The authors should at least acknowledge this problem with their functional annotation.

The reviewer is correct in that, in particular for large eukaryotic genomes, enhancers are often located far away from their target genes. We have no comprehensive enhancer-target data that would enable us to perform a more accurate analysis. Furthermore, the assumption that at least for some of the enhancers the nearest genes will also be their targets, and hence, provide insight into the function of the enhancers themselves seems reasonable given the relatively compact organization of the Tribolium genome. In any case, the analysis was just presented as one of several sanity checks for our ATAC-seq data; for the sake of streamlining the manuscript we no longer include this analysis in the current version of the manuscript.

In the Discussion, the authors claim that contrary to how it may seem, the question they are addressing is not a "fringe problem". Once again, I think this is a straw man. No active researcher thinks that the question of dynamic regulation of gene expression during development is a fringe problem. On the contrary, most researchers will accept that this is one of the most interesting and important questions in current developmental biology.

This whole argument was removed from the Discussion in the revised manuscript.

Perhaps the most significant problem with the manuscript is that it is all built around the premise of enhancer switching between dynamic enhancers and static enhancers. The authors find one site that is consistent with their prediction for a dynamic enhancer and one site - regulating a different gene - that is consistent with their prediction for a static enhancer and claim that they have provided support for their model. I think this claim is grossly exaggerated. They present data that can be seen as consistent with their model but are a long way from providing evidence for it.

We actually thought we were cautious enough about this. Nowhere in our text did we mention that our data “support” the enhancer switching model. We stated quite early (in the abstract, actually) that:

“We found our data consistent with a model in which the timing of gene expression during embryonic pattern formation is mediated by a balancing act between enhancers that induce rapid changes in gene expressions (that we call ‘dynamic enhancers’) and enhancers that stabilizes gene expressions (that we call ‘static enhancers’).”

To make this message clearer, we added the following sentence to the abstract of the revised manuscript: “However, more data is needed for a strong support for this or any other alternative models.” And again at the end of the Introductions: “While these data are in line with our Enhancer Switching model, more data is needed as a strong support for the model.” Also, at the end of the Results section examining runB enhancer dynamics, we stated: “However, this merely shows that runB activity dynamics are consistent with our model, but is still far from strongly supporting the model (more on that in the Discussion).” Also for the Results section on enhancer hbA dynamics: “Again, this merely shows that hbA activity dynamics are consistent with our model, but is still far from strongly supporting it.”.

Moreover, in the opening paragraph of the Discussion, we explicitly and quite openly addressed this point, and suggested what kind of observations and experiments needed in the future to qualify as a “strong support” for the model. We even ran simulations for what kind of observation should one expect in enhancer deletion experiments if the model is correct (Figure 7).

But it seems like discussing the enhancer switching model in detail gives the impression of its central importance to the paper. In our view, our experimental system is quite general and does not depend on that model, but the point of mentioning it is that it is an example of how could an alternative model of enhancer regulation be of relevance to the problem of dynamic gene expression. This wouldn’t be obvious without this or a similar model that is showing this, even if it is hypothetical. But since our presentation is obviously giving the impression that our claims are stronger that they really are, we altered our phrasing in the introduction of the revised manuscript to make our point clearer:

“Despite its potential inaccuracies, the Enhancer Switching model exemplifies the type of alternative frameworks we need to explore in order to elucidate the mechanisms driving the generation of gene expression waves during development. Consequently, an appropriate model system is required, allowing us to test not only the Enhancer Switching model but also any other prospective model that provides a satisfactory explanation for the initiation of gene expression waves at the enhancer level.”

We hope that this addresses the reviewer’s quite legitimate concerns.

Like the Introduction, the Discussion includes long paragraphs (lines 450-480) that are more suitable for a review/hypothesis paper. The data presented in this manuscript has little relevance to the question of kinematic vs. trigger waves, and therefore there is no real reason for the question to be discussed here.

We have now significantly shortened the discussion.

Reviewer #2 (Public Review):

Open questions:

What happens with the runB enhancer at later stages of embryogenesis? With what kind of dynamics do the anterior-most stripes fade and does that agree with the model? Do they show the same dynamics throughout segmentation? I think later stages need to be shown because the prediction from the model would be that the dynamics are repeated with each wave. I am not so sure about the prediction for ageing stripes – yet it would have been interesting to see the model prediction and the activity of the static enhancer.

Yes, the dynamics repeats in the germband. This is shown in Supplementary Figure 8. The dynamics in germband were shown by visualizing yellow mRNA and intronic probes. MS2 imaging was not possible to be used because the embryo dive into the yolk for a while, and then it becomes difficult to capture the germband in the right orientation for imaging. We are currently working to use light sheet microscopy for imaging germband stages.

I understand that the mRNA of the reporter gene yellow is more stable than the runt mRNA. This might interfere with the possibility to test your prediction for static enhancers: The criterion is that the stripes should increase in strength as the wave migrates towards the anterior. You show this for runB – but given that yellow has a more stable transcript – could this lead to a “false positive” increase in intensity with the slower migration and accumulation of transcripts? I would feel more comfortable with the statement that this is a static enhancer if you could exclude that the signal is blurred by an artifact based on different mRNA stability. What about re-running the simulation (with the p–rameters that have shown to well reflect endogenous –unt mRNA levels) but i“creasing the parameter for the stability of the mRNA? Are static and dynamic enhancers still distinguishable? The claim of having found a static enhancer rests on this increase in signal, hence, other explanations need to be excluded carefully.

Good questions. Note that runB reporter dynamics were examined not only by visualizing yellow mRNAs (which indeed seem to be more stable than endogenous run mRNA; see Supplementary Figure 10), but also using MS2 (with virtually zero mRNA stability; although stability was simulated in the shown movies to show virtual mRNA dynamics), and intronic yellow mRNA (showing de novo transcription; Supplementary Figure 10; you will need to zoom in to see intronic de novo transcripts). The expected dynamics of a static enhancer reporter is quite unique: it progressively increases initially as it propagates from posterior to anterior, then it progressively decreases as it slows down and stabilizes at the anterior. Then they eventually fade. These full range of dynamics is obvious in germband embryos stained for intronic yellow to show de novo transcription of runB enhancer reporter (Supplementary Figure 10; you will need to zoom in to see intronic de novo transcripts).

Running the simulation for the model using different degradation rates for the enhancer reporter made the static enhancer’s expression either less or more persistent, but gave the same overall result: the static enhancer expression has diminished expression at the very posterior, but high expression as its expression wave exiting the growth-zone/SAZ. This is consistent with not only yellow mRNA expressions of runB, but with its intronic expression as well (Supplementary Figure 10; you will need to zoom in to see intronic de novo transcripts).

What about the head domain of the runB enhancer (e.g. Fig. 6A lowest row): This seems to be different from endogenous expression in your work and in Choe et al. Is that aspect different from endogenous expression and can this be reconciled with your model?

Yes, indeed this aspect cannot be explained by our model. We believe that head patterning in insects is regulated by a different regulatory network. This network might be (de)-activated by missing repressors in the selected DNA segment for runB enhancer. We mentioned this issue in the revised manuscript.

The claim of similar dynamics of expression visualized by in situ and MS2 in vivo relies on comparing Fig. 6C with 6A. To compare these two panels, I would need to know to what stage in A the embryo in C should be compared. Actually, the stripe in 6C appears more crisp than the stripes in 6A.

Were the enhancer dynamics tested in vivo at later stages as well? I would appreciate a clear statement on what stages can be visualized and where the technical boundaries are because this will influence any considerations by others using this system.

One really cannot be that super-precise about the timing of a very dynamic process in space and time like this one we are studying. We believe that Figure 6D shows clearly that runB activity dynamics are similar to endogenous run expression.

How do the reported accessibility dynamics of runA enhancer correlate with the activity of the reporter: E.g. is the enhancer open in the middle body region but closed at the posterior part of the embryo? Or is it closed at the anterior – and if so: why is there a signal of the reporter in the head?

You show that chromatin accessibility dynamics help in identifying active enhancers. Is this idea new or is it based on previous experience with Drosophila (e.g. PMID: 29539636 or works cited in https://doi.org/10.1002/bies.201900188)? Or in what respect is this novel?

Our manuscript contributes to the growing body of evidence confirming that accessibility per se does not imply activity. Of course, this is not a new idea, but given the widely use of accessibility as a proxy for enhancer activity in the genomics community, we do feel it is important to reiterate the message. As the reviewer correctly indicates, several published findings point to a correlation between accessibility dynamics and enhancer activity. However, to our knowledge, this is the first example in Tribolium. It is important to point out that what “dynamic” means strongly depends on the experimental design. Even in Drosophila, not enough studies have been conducted to fully understand the relationship (e.g., ideally, this should be done on a continuous time scale and at single cell level). We acknowledge in the manuscript that this relationship has been observed before in other species (and have added the references suggested by the reviewer, for which we are very grateful), but still believe that our observations are highly significant to the Tribolium community.

Reviewer #3 (Public Review):

I have two major concerns: First, the claim about differential accessibility being related to enhancer activity is not really established from the presented data, in my view. This needs to be clarified. (I do believe in the claim to some extent, but not based on presented evidence.)

We agree with the reviewer that more data – and, more importantly, independent replication – are necessary to confirm this finding. Please, refer to our response to your comment regarding the statistical significance of the findings.

Second, the evidence in support of the Enhancer Switching model for runt should be accompanied by identification of and spatiotemporal profiling of the “speed regulator”, if this is not established yet.

Experiments supporting the role of Cad as a speed regulator for both pair-rule and gap genes have been published in El-Sherif et al PLOS Genetics 2014 and Zhu et al PNAS 2017. We added a comment stressing this fact.

In addition to these two concerns, the simulations of the Enhancer Switching model need to be described, at least in the outline, in the Methods section.

Done

Reviewer #2 (Public Review):

Embryonic development requires differential gene expression, which is regulated by enhancer elements. Regulatory proteins bind to these DNA elements to regulate close-by promoters. Key insights into the molecular mechanisms of enhancer function have been gained by studying fly segmentation, where a hierarchical cascade of gene regulation subdivides the embryo into ever smaller units. However, segmentation in other insects and arthropods as well as in vertebrates relies on a much more dynamic process where repetitive gene expression patterns appear to migrate across tissues similar to waves. Only recently, models have been proposed that make predictions on the underlying gene regulatory networks (GRN) and the properties of the respective enhancers. Specifically, the previously suggested model of the authors, the enhancer switching model, predicted that each gene expression wave should actually be regulated by two GRNS - one based on a "dynamic enhancer", which directs the early wave-like pattern and the other involving a "static enhancer", which directs the more stable expression defining the segment anlagen at the end of each cycle. However, these predicted enhancer types have not been described so far. In flies, where the respective methodology would be available, the segmentation does not show prominent wave-like patterns. In beetles, where pronounced wave-like patterns have been described, the respective methodology has been missing.

With this work, the authors establish a genomic resource and a transgenic line in the red flour beetle in order to establish it as a model system to tackle questions on enhancers driving dynamic expressions during development. First, they determine the open chromatin at early embryonic stages thereby generating a valuable resource for enhancer detection. They did so by dissecting the embryos of two temporal stages into three parts (head, middle part, and growth zone) and then determined open chromatin via ATAC-seq. This setup allowed for a comparison across tissues and stages to identify dynamically regulated chromatin. Indeed, Mau et al. find that dynamic chromatin regulation is a good criterion to enrich for active enhancers.

Second, they established an enhancer reporter system, which allows for visualization of de novo transcription by both in situ hybridization and in vivo. This MS2 system has for the first time been implemented in this beetle and the authors convincingly show its functionality. Indeed, the expression dynamics can be very nicely visualized in vivo at blastoderm stages.

Combing these two resources, they predicted enhancers based on the criterion of dynamic chromatin regulation (from their ATAC-seq resource) and tested them using their novel MS2 system. Out of 9 tested enhancers located close to the gap genes hunchback and Krüppel and the pair-rule gene runt, 4 drove expression. Combining these data with previously published beetle enhancers, they show that DNA regions with differential accessibility were indeed enriched in active enhancers (appr. 60%), providing a good selection criterion.

Finally, they characterize two of the newly identified enhancers that reflect wave-like expression patterns in fixed embryos and in vivo by using the MS2 system to test predictions of the enhancer switching model. The results are compared with an elaboration of their previously suggested enhancer-switching model, which predicts different patterns for static vs. dynamic enhancers. Indeed, they think that the runB enhancer fits the predictions of a static enhancer.

The authors have generated a genomic resource that will be of very high value to the community in the future. The fact that they dissected the embryos for that purpose makes it even more precise and valuable. Likewise, the transgenic system that allows for testing enhancer activity in vivo will be very valuable for the highly active research field dealing with the prediction and analyses of enhancers.

The analysis of the identified enhancers provides partial confirmation for their model. As the authors state in the discussion, finding at least one pair of such enhancers for one gene would be a great test of their hypothesis - finding pairs of static and dynamic enhancers in several genes would be strong support. Unfortunately, they found only one of the two enhancer types in runt and one in hunchback, respectively. Hence, the prediction of the model remains to be tested in the future.

The authors provide a lot of high-quality data visualized nicely in the figures. The text would profit from some re-formulation, re-structuring, and shortening.

Open questions:<br /> What happens with the runB enhancer at later stages of embryogenesis? With what kind of dynamics do the anterior-most stripes fade and does that agree with the model? Do they show the same dynamics throughout segmentation? I think later stages need to be shown because the prediction from the model would be that the dynamics are repeated with each wave. I am not so sure about the prediction for ageing stripes - yet it would have been interesting to see the model prediction and the activity of the static enhancer.<br /> I understand that the mRNA of the reporter gene yellow is more stable than the runt mRNA. This might interfere with the possibility to test your prediction for static enhancers: The criterion is that the stripes should increase in strength as the wave migrates towards the anterior. You show this for runB - but given that yellow has a more stable transcript - could this lead to a "false positive" increase in intensity with the slower migration and accumulation of transcripts? I would feel more comfortable with the statement that this is a static enhancer if you could exclude that the signal is blurred by an artifact based on different mRNA stability. What about re-running the simulation (with the parameters that have shown to well reflect endogenous runt mRNA levels) but increasing the parameter for the stability of the mRNA? Are static and dynamic enhancers still distinguishable? The claim of having found a static enhancer rests on this increase in signal, hence, other explanations need to be excluded carefully.<br /> What about the head domain of the runB enhancer (e.g. Fig. 6A lowest row): This seems to be different from endogenous expression in your work and in Choe et al. Is that aspect different from endogenous expression and can this be reconciled with your model?<br /> The claim of similar dynamics of expression visualized by in situ and MS2 in vivo relies on comparing Fig. 6C with 6A. To compare these two panels, I would need to know to what stage in A the embryo in C should be compared. Actually, the stripe in 6C appears more crisp than the stripes in 6A.<br /> Were the enhancer dynamics tested in vivo at later stages as well? I would appreciate a clear statement on what stages can be visualized and where the technical boundaries are because this will influence any considerations by others using this system.<br /> How do the reported accessibility dynamics of runA enhancer correlate with the activity of the reporter: E.g. is the enhancer open in the middle body region but closed at the posterior part of the embryo? Or is it closed at the anterior - and if so: why is there a signal of the reporter in the head?<br /> You show that chromatin accessibility dynamics help in identifying active enhancers. Is this idea new or is it based on previous experience with Drosophila (e.g. PMID: 29539636 or works cited in https://doi.org/10.1002/bies.201900188)? Or in what respect is this novel?

Author Response

eLife assessment

Mizukami et al. propose a scenario for the evolutionary origin of the coronary artery in amniotes by comparing the morphologies of the vasculatures across several species and developmental timepoints. They show that the coronary arteries of non-amniotes most closely resemble embryonic amniote aortic subepicardial vessels (ASVs), which are replaced by the true coronary arteries during amniote development. While the identification of common vascular structures in diverse taxa is a valuable contribution, additional developmental evidence is needed to confirm that such vessels are truly homologous.

We have extensively revised our paper by including additional animal data and references. While we were unable to obtain useful data on lungfish or coelacanth, we have obtained new data related to the physiology of coronary artery, which has been added to Fig. 7. We have also attempted to compare blood vessels at the molecular level, but found that gene expression patterns in blood vessels throughout the body were not always conserved between lineages, making it difficult to make comparisons between amphibians and amniotes. However, based on comparative morphological analysis using newly added three-dimensional data, it is reasonable to consider the amniotes' ASVs and amphibians' ASV-like vessels to be homologous.

Reviewer #3 (Public Review):

Mizukami et al. compare the structure of the coronary arteries in multiple species of amniotes, amphibians, and fish. By selecting species from each of these taxa, the authors were able to evaluate modifications to the coronary arteries during key evolutionary transitions. In mice and quail, they show two populations of vessels that are visible on the developing heart-true coronary arteries on the ventricle and a second population of vessels on the outflow tract known as the ASV., They found that in amphibians, outflow tract vessels were present but ventricular coronary arteries were completely absent. In zebrafish (a more ancestral species) an arterial branch off the rostral section of the hypobranchial artery was shown to have similar anatomical features to outflow tract vessels found in higher organisms. These zebrafish outflow tract arteries also appeared conserved in several chondriichthyes specimens. The authors conclude that rearrangement of the outflow tract vasculature or hypobranchial arteries in fish during evolution, could be homologous to the ASV population of coronary arteries in amphibians and amniotes. These data give new insight into the evolutionary origins of the coronary vasculature.　

Major Points

1) The manuscript presents important data on the coronary vascular structure of several different species. However, these data alone do not conclusively demonstrate whether the developmental origins of ASV like vessels are homologous. Therefore, care should be taken when concluding that the outflow tract vessels found in all different species are conserved features. While this is a reasonable hypothesis and should be presented, the manuscript could be improved by also discussing alternate explanations. For example, ASVs in mice originate during embryonic development, while in fish and amphibians outflow tract vessels are formed only in mature animals.

We have added data on mice and amphibians (e.g., Fig. 2) and substantially revised the overall development and discussion of the paper. Morphological homology is evident for ASVs and amphibian ASV-like vessels, but the homologous relationship with the hypobranchial artery only suggests a similarity in the embryonic region.

Comparisons of developmental timings of the various structures among diferent lineages of vertebrates reveal that heterochronical shifts are not uncommon. For example, ossification of the head skeleton and vertebrae occurs during the fetal stage in amniotes, but after hatching in larval amphibians and teleosts. A similar trend is observed in the development of the limb bud (paired fins). Overall, the larval stages of amphibians and teleosts are comparable to the fetal stages of amniotes for many structures. We did not suppose this to be particularly unusual, and we did not include it in the text.

2) Figure 3 A-D: The authors state that "the ASV ran through the outflow tract, then entered the aortic root before reaching the ventricle to form a secondary orifice". Do the authors have serial sections to conclude that the vessel branching off the carotid runs the length of the aorta and is continuous with an orifice at the aortic root? The endothelial projection off the aorta in panel C could reasonably be an independent projection. For example, Chen et al., described similar looking projections in the base of the aorta that were not attached to external vessels. A whole mount approach would be the most convincing to show the attachments of the ASV vessel.

We added the data of the whole-mount immunohistochemistry. Please refer Figs. 2 and S2.

3) Figure 3E: Similar as above, how is it concluded that the orifice is continuous with the ASV and that this projection is not the coronary artery stem?

As for quail, we could not achieve as a clear whole-mount staining as in mice. It was also difficult to trace the route in sections because in quail, ASVs are not restricted to a few lines as in mice, but are the plexus of small vessels. Thus, we added the detailed data from mice (Fig. 2, S2) and we emphasized that the position of orifice in quail is exactly same as that in mice.

4) The discussion section could be improved by making some statements more consistent, using more precise or appropriate terminology accepted in the field, and being more cognizant of how the authors' findings fit within the history of the field. For example, when referring to coronary arteries, please clarify whether this refers to ASV/ outflow tract coronary arteries, or true ventricular coronary arteries. In addition, the first sentence of the discussion makes it seem like the origins of coronary arteries were unknown prior to this study, however, their origins have been described in multiple papers previously. The authors could revise their statement to acknowledge these previous findings.

We rewrote the entire text to clarify what each "coronary artery" refers to. We also changed the first section of the discussion as suggested by the reviewer.

https://web.archive.org/web/20230612101920/https://thefugue.space/thoughts/the-glimmer

Spatial computing

what of early insights wrt [[Ambient Findability by Peter Morville]] 2006, and my conclusion 2008 that though adding an info layer while interacting in the physical world was key, we put it all in our pocket. I doubt it will end up as ski goggles on our head much.

Via [[Boris Mann]] https://blog.bmannconsulting.com/2023/06/08/kharis-oconnell-has.html

Everyone has their own world and their own problems to deal with. Even with our self centered thinking, most of the time we think about others and how the slightest thing could make someone's day such as a small smile. We don't know what a person goes through on a daily basis, but when we interact with each other even for a brief second, we understand that we don't know this person and have to give them the benefit of the doubt even for a brief moment. Using the mom example exemplifies how sometimes we just think about ourselves and have to try to make those brief interactions smooth and easy because we truly don't know what is going on in someone else's head.

I disagree with Wallace because there's a lot of people that think differently and think about the present. Different thoughts can be running in a person's head when they are in a difficult situation such as what is going on right now? How can it be fixed? Is there something can be done? Some people will try to figure something out a solution to a problem right there and then rather than waiting to solve it. Not everyone will think this way and will overthink a situation or problem the way that Wallace is describing.

This is a very powerful statement. In this day and age it is so easy to fill our minds with an immense amount of information because its all right at our fingertips. Parents are often admonished to filter the content that accessible to our children on electronic devices; but its equally important for adults to do the same for ourselves. It is so imperative to protect our minds so as not to become overwhelmed with information. That could mean sometimes foregoing watching the evening news or taking a hiatus form social media. These platforms have proved to be double-edged swords. On one end beneficial in a lot of ways, but also harmful.

Reviewer #3 (Public Review):

My general assessment of the paper is that the analyses done after they find the model are exemplary and show some interesting results. However, the method they use to find the number of states (Calinski-Harabasz score instead of log-likelihood), the model they use generally (HMM), and the fact that they don't show how they find the number of states on HCP, with the Schaeffer atlas, and do not report their R^2 on a test set is a little concerning. I don't think this perse impedes their results, but it is something that they can improve. They argue that the states they find align with long-standing ideas about the functional organization of the brain and align with other research, but they can improve their selection for their model.

Strengths:

- Use multiple datasets, multiple ROIs, and multiple analyses to validate their results<br /> - Figures are convincing in the sense that patterns clearly synchronize between participants<br /> - Authors select the number of states using the optimal model fit (although this turns out to be a little more questionable due to what they quantify as 'optimal model fit')<br /> - Replication with Schaeffer atlas makes results more convincing<br /> - The analyses around the fact that the base state acts as a flexible hub are well done and well explained<br /> - Their comparison of synchrony is well-done and comparing it to resting-state, which does not have any significant synchrony among participants is obvious, but still good to compare against.<br /> - Their results with respect to similar narrative engagement being correlated with similar neural state dynamics are well done and interesting.<br /> - Their results on event boundaries are compelling and well done. However, I do not find their Chang et al. results convincing (Figure 4B), it could just be because it is a different medium that explains differences in DMN response, but to me, it seems like these are just altogether different patterns that can not 100% be explained by their method/results.<br /> - Their results that when there is no event, transition into the DMN state comes from the base state is 50% is interesting and a strong result. However, it is unclear if this is just for the sitcom or also for Chang et al.'s data.<br /> - The involvement of the base state as being highly engaged during the comedy sitcom and the movie are interesting results that warrant further study into the base state theory they pose in this work.<br /> - It is good that they make sure SM states are not just because of head motion (P 12).<br /> - Their comparison between functional gradient and neural states is good, and their results are generally well-supported, intuitive, and interesting enough to warrant further research into them. Their findings on the context-specificity of their DMN and DAN state are interesting and relate well to the antagonistic relationship in resting-state data.

Weaknesses:

- Authors should train the model on part of the data and validate on another<br /> - Comparison with just PCA/functional gradients is weak in establishing whether HMMs are good models of the timeseries. Especially given that the HMM does not explain a lot of variance in the signal (~0.5 R^2 for only 27 brain regions) for PCA. I think they don't report their own R^2 of the timeseries<br /> - Authors do not specify whether they also did cross-validation for the HCP dataset to find 4 clusters<br /> - One of their main contributions is the base state but the correlation between the base state in their Song dataset and the HCP dataset is only 0.399<br /> - Figure 1B: Parcellation is quite big but there seems to be a gradient within regions<br /> - Figure 1D: Why are the DMNs further apart between SONG and HCP than the other states<br /> - Page 5 paragraph starting at L25: Their hypothesis that functional gradients explain large variance in neural dynamics needs to be explained more, is non-trivial especially because their R^2 scores are so low (Fig 1. Supplement 8) for PCA<br /> - Generally, I do not find the PCA analysis convincing and believe they should also compare to something like ICA or a different model of dynamics. They do not explain their reasoning behind assuming an HMM, which is an extremely simplified idea of brain dynamics meaning they only change based on the previous state.<br /> - For the 25- ROI replication it seems like they again do not try multiple K values for the number of states to validate that 4 states are in fact the correct number.<br /> - Fig 2B: Colorbar goes from -0.05 to 0.05 but values are up to 0.87<br /> - P 16 L4 near-critical, authors need to be more specific in their terminology here especially since they talk about dynamic systems, where near-criticality has a specific definition. It is unclear which definition they are looking for here.<br /> - P16 L13-L17 unnecessary<br /> - I think this paper is solid, but my main issue is with using an HMM, never explaining why, not showing inference results on test data, not reporting an R^2 score for it, and not comparing it to other models. Secondly, they use the Calinski-Harabasz score to determine the number of states, but not the log-likelihood of the fit. This clearly creates a bias in what types of states you will find, namely states that are far away from each other, which likely also leads to the functional gradient and PCA results they have. Where they specifically talk about how their states are far away from each other in the functional gradient space and correlated to (orthogonal) components. It is completely unclear to me why they used this measure because it also seems to be one of many scores you could use with respect to clustering (with potentially different results), and even odd in the presence of a log-likelihood fit to the data and with the model they use (which does not perform clustering).<br /> - Grammatical error: P24 L29 rendering seems to have gone wrong

Questions:

- Comment on subject differences, it seems like they potentially found group dynamics based on stimuli, but interesting to see individual differences in large-scale dynamics, and do they believe the states they find mostly explain global linear dynamics?<br /> - P19 L40 why did the authors interpolate incorrect or no-responses for the gradCPT runs? It seems more logical to correct their results for these responses or to throw them out since interpolation can induce huge biases in these cases because the data is likely not missing at completely random.

Reviewer #1 (Public Review):

Qin et al. set out to investigate the role of mechanosensory feedback during swallowing and identify neural circuits that generate ingestion rhythms. They use Drosophila melanogaster swallowing as a model system, focusing their study on the neural mechanisms that control cibarium filling and emptying in vivo. They find that pump frequency is decreased in mutants of three mechanotransduction genes (nompC, piezo, and Tmc), and conclude that mechanosensation mainly contributes to the emptying phase of swallowing. Furthermore, they find that double mutants of nompC and Tmc have more pronounced cibarium pumping defects than either single mutants or Tmc/piezo double mutants. They discover that the expression patterns of nompC and Tmc overlap in two classes of neurons, md-C and md-L neurons. The dendrites of md-C neurons warp the cibarium and project their axons to the subesophageal zone of the brain. Silencing neurons that express both nompC and Tmc leads to severe ingestion defects, with decreased cibarium emptying. Optogenetic activation of the same population of neurons inhibited filling of the cibarium and accelerated cibarium emptying. In the brain, the axons of nompC∩Tmc cell types respond during ingestion of sugar but do not respond when the entire fly head is passively exposed to sucrose. Finally, the authors show that nompC∩Tmc cell types arborize close to the dendrites of motor neurons that are required for swallowing, and that swallowing motor neurons respond to the activation of the entire Tmc-GAL4 pattern.

Strengths:<br /> -The authors rigorously quantify ingestion behavior to convincingly demonstrate the importance of mechanosensory genes in the control of swallowing rhythms and cibarium filling and emptying<br /> -The authors demonstrate that a small population of neurons that express both nompC and Tmc oppositely regulate cibarium emptying and filling when inhibited or activated, respectively<br /> -They provide evidence that the action of multiple mechanotransduction genes may converge in common cell types

Weaknesses:<br /> -A major weakness of the paper is that the authors use reagents that are expressed in both md-C and md-L but describe the results as though only md-C is manipulated<br /> -Severing the labellum will not prevent optogenetic activation of md-L from triggering neural responses downstream of md-L. Optogenetic activation is strong enough to trigger action potentials in the remaining axons. Therefore, Qin et al. do not present convincing evidence that the defects they see in pumping can be specifically attributed to md-C.<br /> -GRASP is known to be non-specific and prone to false positives when neurons are in close proximity but not synaptically connected. A positive GRASP signal supports but does not confirm direct synaptic connectivity between md-C/md-L axons and MN11/MN12.<br /> -As seen in Figure Supplement 2, the expression pattern of Tmc-GAL4 is broader than md-C alone. Therefore, the functional connectivity the authors observe between Tmc expressing neurons and MN11 and 12 cannot be traced to md-C alone

Overall, this work convincingly shows that swallowing and swallowing rhythms are dependent on several mechanosensory genes. Qin et al. also characterize a candidate neuron, md-C, that is likely to provide mechanosensory feedback to pumping motor neurons, but the results they present here are not sufficient to assign this function to md-C alone. This work will have a positive impact on the field by demonstrating the importance of mechanosensory feedback to swallowing rhythms and providing a potential entry point for future investigation of the identity and mechanisms of swallowing central pattern generators.

Reviewer #2 (Public Review):

In this study, the authors design a study to examine how place cell representations in the hippocampus change when the rules of a navigational task change. In one group of animals (group 1), the rules change in the same environment as the initial task was performed, and in the second group of animals (group 2), the task with the new rules is presented in a different environment, and then the animals are returned to the first environment with the original rule. (Briefly, on a cross maze, animals first learned to turn right, then the task rule changed to require turning east, and then the rule changed back to turning right). Broadly, using one photon calcium imaging with head mounted mini microscopes, the authors show that, at both the single cell and population level, more remapping occurs in group 1 animals in the initial environment than in group 2 animals.

This work is bolstered by the unique and rigorous way in which the authors track cells across days, in which they compare the rotation angles of crossed-registered groups of cells-I will definitely be using this in the future! The work also benefits from the extensive analysis of both temporal and spatial correlations of cellular activity. However, there are several shortcomings of the behavioral setup and learning conditions that need to be addressed in order to fully support the conclusions of the authors:

First, group 1 animals spend significantly more time in maze 1 than group 2 animals, since group two animals were switched to a different maze when the rule was changed. It is thus difficult to make direct comparison between the two groups, particularly in the last phase of experimentation when although both groups are in the first environment with the task rule, group 1 has experienced maze one for 6 days while group 2 has only experienced in for 3 days. It is therefore potentially difficult to disentangle differences caused by task changes versus length of environmental exposure.

Secondly, and similarly, during the task period, group 1 animals only have exposure to one environment while group 2 animals have exposure to 2 environments. Ideally, group 1 animals would also be exposed to environment 2, to rule out any potential effects of experiencing a novel environment may have on place cell representations, otherwise this cannot be disentangled from the effect of a task rule change.

Third, two concerns about how the animals are trained: First, if I am interpreting the methods correctly, both Group 1 and Group 2 animals are trained so turn-right is on one maze and turn east is on another way. As such, both groups thus have an "original understanding" that different rules are associated with different mazes. This seems potentially confounding given that it is consistent with the future training of Group 2 but not Group 1 mice. Additionally confounding is the fact that, because of the pretraining, group 1 mice have actually experienced the task in 3 different environments; I am unclear if and how this might be expected to affect results. Additionally, it is methodically unclear why pre-training occurs in a different environment than testing does, and what the criterion is for switching the animals from pre-training to training.

It would additionally be useful to discuss the results of this study in the context of spatial and non-spatial tasks. The authors, usefully, spend a significant portion of the paper comparing their results to results seen during fear extinction. It might be worth contextualizing the differences in how fear conditioning has a contextual "background" (i.e., the animals are conditioned to the context) while in their experiment the entire task is based entirely on navigation.

Overall, this is an interesting manuscript that attempts to address how contextual representations change as task parameters change. While the paper contains thorough statistical analysis but could benefit from more discussion of behavior in the context of learning as well as more rigorous behavioral controls. This work will be of interest to researchers studying hippocampus, navigation, and learning.

Reviewer #2 (Public Review):

This study reports a novel role of thalamic activity in the late components of a cortical event related potential (ERP). To show this association, the authors used high-density EEG together with multiple deep electrophysiological recordings combined with electrical stimulation of superficial and deep cortical layers. Stimulation of deep layers elicits a late ERP component that is closely related to bursts of thalamic activity during quiet wakefulness. This relationship is quite noticeable when deep layers of the cortex are stimulated, and it does depend on arousal state, being maximal during quiet wakefulness, diminished during active wakefulness, and absent during anesthesia.

The study is very well performed, with a high number of subjects and appropriate methodology. Performing simultaneous recording of EEG and several neuropixels probes together with cortical microstimulation is no small feat considering the size of the mouse head and the fact that mice are freely behaving in many of the experiments. It is also noticeable how the authors use a seemingly outdated technique (electrical microstimulation) to produce compelling and significant research. The conclusions regarding the thalamic contributions to the ERP components are strongly supported by the data.

The spatiotemporal complexity is almost a side point compared to what seems to me the most important point of the paper: showing the contribution of thalamic activity to some components of the cortical ERP. Scalp ERP's have long been regarded as purely cortical phenomena, just like most of EEG, and this study shows convincing evidence to the contrary.

The data presented seemingly contradicts the results presented in Histed et al. (2009), who asserts that cortical microstimulation only affects passing fibers near the tip of the electrodes, and results in distant, sparse, and somewhat random neural activation. In this study, it is clear that the maximum effect happens near the electrodes, decays with distance, and it is not sparse at all, suggesting that not only passing fibers are activated but that also neuronal elements might be activated by antidromic propagation from the axonal hillock. This appears to offer proof that microstimulation might be much more effective than it was thought after the publication of Histed 2009, as the uber-successful use of DBS to treat Parkinson disease has also shown.

Not likely.

Reviewer #1 (Public Review):

This manuscript by Bohannon et al. continues a line of work from the Larsson laboratory with fundamental contributions describing the effects of polyunsaturated fatty acids (PUFAs) on the cardiac delayed rectifier potassium channel (IKs) formed by Kv7.1 and KCNE1 heteromers. Although the activating effect of PUFAs on these specific channels has been previously described, the authors now present a novel finding related to PUFAs containing large aromatic tyrosine head groups, showing significant activation effects on IKs, larger than other PUFAs previously studied. A combination of site-directed mutagenesis, electrophysiological and pharmacological approaches are used to dissect the different molecular mechanisms and sites involved in the functional interactions. The main conclusions are: 1) PUFA analogues with Tyr head groups are strong activators of the cardiac IKs channel by action on two previously described mechanisms: left-shift of the voltage-activation curve (by interaction with the voltage-sensor region of Kv7.1); and increased Gmax (by interacting with the pore region). 2) the underlying molecular interactions between PUFA and Kv7.1 are not cation-pi, as shown by the lack of effect of different chemical variations that disrupt the electrostatic surface potential. 3) the presence of electronegative groups on the aromatic ring favors increases in the maximal conductance. 4) the generation of a hydrogen bond with the -OH on the Tyr group seems to selectively impact on IKs voltage dependence of activation. 4) Kv7.1 sites involved in interactions with aromatic PUFAs are similar to the ones previously described for non-aromatic PUFAS, that is: R231 in S4 and K326 in S6. 5) residue T224 is newly identified as a potential site forming a hydrogen bond between the Tyr in the aromatic PUFA and Kv7.1.

The manuscript is very well written and structured. The experiments are solid and lead to mostly well-grounded conclusions. There are some aspects that would benefit from some clarification, which are mainly related to the different effects of the aromatic PUFA variants on IKs voltage dependence and/or conductance.

Reviewer #2 (Public Review):

In the present study, Briana M. Bohannon et al. expand on the study of the effect of Polyunsaturated fatty acids (PUFAS) on Iks (KV7.1 + KCNE1), a delayed rectifier potassium channel of critical relevance in cardiac physiology. PUFAs are amphipathic molecules that activate IKs channels by interacting with positively charged residues on the voltage sensor domain and in the channel's pore. The authors aim to characterize the molecular mechanisms behind the Iks activation by PUFA analogs that contains a tyrosine head group instead of the carboxyl or sulfonyl group present in other PUFAs.

The authors present a well-written manuscript with clear data and well-presented figures. The authors describe the effects of various tyrosine-PUFA analogs and unveil the mechanistic nature of their interactions with the channel. The focus is the N -(alpha-linolenoyl) Tyrosine (NALT), a potent activator by shifting the channel G-V by more than 50mV facilitating the opening of the channel, although the authors tested other tyrosine-PUFA analogs. Remarkably, the hydroxyl group in the tyrosine head is essential to shift the voltage-dependence of activation due to an H-bond with a threonine from the S3-S4 linker that helps coordinate the PUFA together with an electrostatic interaction with arginine in the S4. Furthermore, to test whether the aromatic ring from the tyrosine had a role in the interaction, the authors took a fascinating and exciting approach by modifying it and making the ring more electronegative by adding negatively charged atoms. Interestingly, they discovered that an electronegative-modified aromatic PUFA could increase the channel's conductance, an effect mediated by a specific interaction with a Lysine at the top of the S6 helix.

Although the question addressed in the manuscript is fascinating due to the possible use of these tyrosine-PUFA analogs as IKs modulators, the presented work is very mechanistic and specialized. While the effect of tyrosine-PUFA analogs is robust, the authors could improve the story by highlighting their interest in them and discussing whether they have potential therapeutic uses.

Due to the relevance of IKs currents in cardiac physiology and Long QT syndrome, the discovery and characterization of activators are highly relevant. The present manuscript presents a group of potent IKs channel activators that have the potential to impact the cardiac physiology field dramatically if they can perform under pathophysiological conditions or in the presence of disease-causing mutations.

interesting point! some other things in my head:

1) in addition to our increased writing endeavors, we've also been engaging in extensive reading as well, but our reading material has evolved beyond books, encompassing the plethora of content available in the vast expanse of cyberspace

2) and while the quantity of reading has expanded significantly, it is equally intriguing to recognize that the nature of these texts has shifted towards shorter formats—tweets, ig post captions, microblogs, etc

3) AND lastly, the act of reading has swiftly evolved into the realm of listening, with the emergence of podcasts, audiobooks, listenable videos, and similar forms of content consumption

This expression pattern is interesting, but it's not immediately obvious to me how the magnitude of tissue expression relates to heat sensing. For example, is it known that Rhodnius preferentially sense heat with their head far more than their antennae? Profiling TRPA52 expression in tissues unrelated to sensation, showing specific localization or enrichment to sensory neurons, or linking the highly expressed tissues with known behavior might all help clarify how this suggestive tissue expression pattern is linked to heat sensing!

to manufacture more anxiety in the form of buildings

This piece is fantastic because there are specific little details that will sit with you, but no central animating move-it-along-now thesis that would malform, crush the vignettes it needs to present. It's a dynamic, a mood, a tone – get your head into the space correctly and you'll find your own theses.

Via Ethan Marcotte

Remarkably low level of funding, 30& or 27.8 mil out of 91.3

Coupled with the t-SNE visualization, it is clear that deltas for a particular PC are clustered, and what if we just trained a model for that? These results do puzzle me a bit because I am having a hard time wrapping my head around it.

‘The Canto of Ulysses’ records a truly remarkable feat of cultural memory, as Levi recalls and recites Dante amidst the ruin of Auschwitz. He does so to teach Italian to his campmate Jean. But as he recites the words of Dante, Levi also feels his past come back to him. He begins to recover something of his identity and his humanity in the degraded world of the Lager, to feel again that he is ‘a man’.

Dante is not, however, the only canonical writer to appear in ‘The Canto of Ulysses’. So too does Shakespeare. <br /> Levi admits to ‘gaps’, ‘holes’, and ‘lacuna[e]’ in his memory as he recites Dante, some of which would seem to be ‘irreparable’. Unable to recall his Dante, Levi is forced to confront ‘silence’:

I would give today’s soup to know how to connect ‘the like on any day’ to the last lines. I try to reconstruct it through rhymes, I close my eyes, I bite my fingers – but it is no use, the rest is silence [il resto è silenzio]. Other verses dance in my head: ‘…The sodden ground belched wind …’, no, it is something else. [Woolf’s translation.]

‘[T]he rest is silence’: Levi is quoting the last words that are spoken by Hamlet, before he dies:

O, I die, Horatio!

The potent poison quite o’ercrows my spirit.

I cannot live to hear the news from England.

But I do prophesy th’election lights

On Fortinbras; he has my dying voice.

So tell him, with th’occurrents more and less

Which have solicited – the rest is silence.

With his ‘spirit’ succumbing to the ‘potent poison’ Claudius and Laertes have used to kill him, Hamlet undertakes the impossible: to testify to his own death, to ‘tell’ in words his fall into deathly silence. This is his ‘dying voice’.

What is Hamlet doing in ‘The Canto of Ulysses’? Why does Levi place it where he does, in ‘the caesura’, the abyssal gap between Auschwitz and his cultural identity and memory, embodied by Dante? It is certainly ironic that Levi draws once again on the Western canonical literary tradition to record the moment of its ostensible breakdown. What emerges from the lapse, the silence that Levi testifies to, is another tie to his compromised past, and the literary culture that would seem to have been obliterated by the Holocaust – even if Levi does not choose to bring obvious notice to his allusion by using quotation marks or by writing, ‘as Hamlet says’. The poetry of Hamlet appears to be among the ‘other verses’ Levi has confusedly ‘dancing in his head’ while he tries to fill the gaps in his memory.

By quoting Hamlet, Levi would appear to again testify to the power of literature as a mainstay of culture and humanity, evincing his commitment to humanist ideals. This is certainly the positive interpretation of Hamlet in ‘The Canto of Ulysses’. I would make the case, however, that Levi is using Shakespeare for an altogether more challenging purpose. By appropriating the ‘dying voice’ of Hamlet, Levi records the place where his memory and his identity collapse, testifying to the reduction of the camp inmate to silence and oblivion. Bryan Cheyette writes that ‘even when his memory self-consciously fails him’, Levi is always ‘at pains to bear witness to those moments of failure’ (Cheyette 1999, 64). Levi seeks to testify to the silence, to show that ‘something has occurred even if it cannot be understood’ (Druker 2009, 64). This is the role played by Hamlet in ‘The Canto of Ulysses’. Levi does not use the play to testify to the endurance of the human spirit in the camps, but to silence, to a ‘world of negation’ (OC I, 235). Hamlet, perhaps ironically given its near-unrivalled canonical and cultural status, marks the space beyond language and culture, into which Levi is at risk of falling. This, as Levi called it, is the ‘black hole’ of Auschwitz (OC II, 1663).

Through his allusion to Hamlet, Levi rewrites Shakespeare from the perspective of Auschwitz, endowing the play with new meanings as he confronts the lacunae and voids produced by the world of the concentration camp. Levi uses the play to record the disintegration of humane values in Auschwitz, as memory and language are brought to the point of collapse. Jacques Derrida does much the same in his own work on the play. He draws Hamlet into conversation with Holocaust testimony when he compares the play to the poetry of survivor Paul Celan in his 1995 piece ‘The Time is Out of Joint’. Derrida contends that the paradox of testimony is that the witness must uncannily ‘outlive his life’ (3.2.117) – or ‘survive’ that which is not ‘survivable’: the collapse of all meaning and death (Derrida 1995). This is the sense in which Hamlet is a play about the ‘impossible possibility of testimony’, as Derrida calls it. Hamlet has ‘seen the worst’ and is ‘the witness of the worst disorder, of absolute injustice’, writes Derrida. Hamlet has witnessed too much for words – but testimony, ‘though it hath no tongue, will speak’ (2.2.546).

Levi must also confront and testify to the painful death of memory, the destruction of human identity and culture, before the event of physical death itself – or as Jacques Lacan would call it, ‘symbolic’ before ‘actual’ death, the death of the self before physical death. Lawrence Langer uses the phrase ‘deathlife’ to name the same phenomenon, of ‘dying while one is living’ (Langer 2021, 13). Not unlike the melancholic prince, Levi attempts the impossible of testifying to his own demise. He deploys Hamlet to record his fall into a place beyond humanity and beyond culture – even beyond language. It is, to adopt the words of Jean Améry, an act of both resignation and revolt: resignation to silence, and a determined revolt against oblivion, by testifying to it. It is an astonishing moment.

RA

Much like motherhood being tied to femininity, it seems like being a father/head of the household is tied to masculinity.

Reviewer #2 (Public Review):

The article presents 'Mesotrode,' a technique that integrates chronic widefield calcium imaging and electrophysiology recordings using tetrodes in head-fixed mice. This approach allows recording the activity of a few single neurons in multiple cortical/subcortical structures, in which the tetrodes are implanted, in combination with widefield imaging of dorsal cortex activity on the mesoscale level, albeit without cellular resolution. The authors claim that Mesotrode can be used to sample different combinations of cortico-subcortical networks over prolonged periods of time, up to 60 days post-implantation. The results demonstrate that the activity of neurons recorded from distinct cortical and subcortical structures are coupled to diverse but segregated cortical functional maps, suggesting that neurons of different origins participate in distinct cortico-subcortical pathways. The study also extends the capability of Mesotrode by conducting electrophysiological recordings from the facial motor nerve. It demonstrates that facial nerve spiking is functionally associated with several cortical areas( PTA, RSP, and M2), and optogenetic inhibition of the PTA area significantly reduced the facial movement of the mice.

Studying the relationship between widefield cortical activity patterns and the activity of individual neurons in cortical and subcortical areas is very important, and Murphy's lab has been a pioneer in the field. However, the choice of low-yield recording methods (tetrode) instead of more high-yield recording techniques, such as silicon probes, makes the approach presented in this study somewhat less appealing. Also, the authors claim that a tetrode-based approach can allow chronic recordings of single neural activity over days - a topic that is very controversial. In terms of results, I was under the impression that most of the conclusions presented in the bulk of the paper ( Figures 1-5) are very similar to what previous work from Murphy's lab and other labs has shown using acute preparation. In this respect, the paper can benefit from a more in-depth analysis of the heterogeneity of single-neuron functional coupling. The last part of the facial nerve recording is interesting (Figure 6), but I think it can be integrated better into the rest of the paper.

eLife assessment

This valuable work provides a near-complete description of the mechanosensory bristles on the Drosophila melanogaster head and the anatomy and projection patterns of the bristle mechanosensory neurons that innervate them. The data presented are solid. The study has generated numerous invaluable resources for the community that will be of interest to neuroscientists in the field of circuits and behaviour, particularly those interested in mechanosensation and behavioural sequence generation.

Reviewer #1 (Public Review):

Sensory neurons of the mechanosensory bristles on the head of the fly project to the sub esophageal ganglion (SEZ). In this manuscript, the authors have built on a large body of previous work to comprehensively classify and quantify the head bristles. They broadly identify the nerves that various bristles use to project to the SEZ and describe their region-specific innervation in the SEZ. They use dye-fills, clonal labelling, and electron microscopic reconstructions to describe in detail the phenomenon of somatotopy - conserved peripheral representations within the central brain - within the innervation of these neurons. In the process they develop novel tools to access subsets of these neurons. They use these to demostrate that groups of bristles in different parts of the head control different aspects of the grooming sequence.

Reviewer #2 (Public Review):

The authors combine genetic tools, dye fills and connectome analysis techniques to generate a "first-of-its-kind", near complete, synaptic resolution map of the head bristle neurons of Drosophila. While some of the BMN anatomy was already known based on previous work by the authors and other researchers, this is the first time a near complete map has been created for the head BMNs at electron microscopy resolution.

Strengths:<br /> 1. The authors cleverly use techniques that allow moving back and forth between periphery (head bristle location) and brain, as well as moving between light microscopy and electron microscopy data. This allows them to first characterize the pathways taken by different head BMNs to project to the brain and also characterize anatomical differences among individual neurons at the level of morphology and connectivity.<br /> 2. The work is very comprehensive and results in a near complete map of all head BMNs.<br /> 3. Authors also complement this anatomical characterization with a first-level functional analysis using optogenetic activation of BMNs that results in expected directed grooming behavior.

Weaknesses:<br /> 1. The clustering analysis is compelling but cluster numbers seem to be arbitrarily chosen instead of by using some informed metrics.<br /> 2. It could help provide context if authors revealed some of the important downstream pathways that could explain optogenetics behavioral phenotypes and previously shown hierarchical organization of grooming sequences.<br /> 3. In contrast to the rigorous quantitative analysis of the anatomical data, the behavioral data is analyzed using much more subjective methods. While I do not think it is necessary to perform a rigorous analysis of behaviors in this anatomy focused manuscript, the conclusions based on behavioral analysis should be treated as speculative in the current form e.g. calling "nodding + backward walking" as an avoidance response is not justified as it currently stands. Strong optogenetic activation could lead to sudden postural changes that due to purely biomechanical constraints could lead to a couple of backward steps as seen in the example videos. Moreover since the quantification is manual, it is not clear what the analyst interprets as backward walking or nodding. Interpretation is also concerning because controls show backward walking (although in fewer instances based on subjective quantification).

Summary:<br /> The authors end up generating a near-complete map of head BMNs that will serve as a long-standing resource to the Drosophila research community. This will directly shape future experiments aimed at modeling or functionally analyzing the head grooming circuit to understand how somatotopy guides behaviors.

Reviewer #3 (Public Review):

Eichler et al. set out to map the locations of the mechanosensory bristles on the fly head, examine the axonal morphology of the bristle mechanosensory neurons (BMNs) that innervate them, and match these to electron microscopy reconstructions of the same BMNs in a previously published EM volume of the female adult fly brain. They used BMN synaptic connectivity information to create clusters of BMNs that they show occupy different regions of the subesophageal zone brain region and use optogenetic activation of subsets of BMNs to support the claim that the morphological projections and connectivity of defined groups of BMNs are consistent with the parallel model for behavioral sequence generation.

The authors have beautifully cataloged the mechanosensory bristles and the projection paths and patterns of the corresponding BMN axons in the brain using detailed and painstaking methods. The result is a neuroanatomy resource that will be an important community resource. To match BMNs reconstructed in an electron microscopy volume of the adult fly brain, the authors matched clustered reconstructed BMNs with light-level BMN classes using a variety of methods, but evidence for matching is only summarized and not demonstrated in a way that allows the reader to evaluate the strength of the evidence. The authors then switch from morphology-based categorization to non-BMN connectivity as a clustering method, which they claim demonstrates that BMNs form a somatotopic map in the brain. This map is not easily appreciated, and although contralateral projections in some populations are clear, the distinct projection zones that are mentioned by the authors are not readily apparent. Because of the extensive morphological overlap between connectivity-based clusters, it is not clear that small projection differences at the projection level are what determines the post-synaptic connectivity of a given BMN cluster or their functional role during behavior. The claim the somatotopic organization of BMN projections is preserved among their postsynaptic partners to form parallel sensory pathways is not supported by the result that different connectivity clusters still have high cosine similarity in a number of cases (i.e. Clusters 1 and 3, or Clusters 1 and 2). Finally, the authors use tools that were generated during the light-level characterization of BMN projections to show that specifically activating BMNs that innervate different areas of the head triggers different grooming behaviors. In one case, activation of a single population of sensory bristles (lnOm) triggers two different behaviors, both eye and dorsal head grooming. This result does not seem consistent with the parallel model, which suggests that these behaviors should be mutually exclusive and rely on parallel downstream circuitry.

This work will have a positive impact on the field by contributing a complete accounting of the mechanosensory bristles of the fruit fly head, describing the brain projection patterns of the BMNs that innervate them, and linking them to BMN sensory projections in an electron microscopy volume of the adult fly brain. It will also have a positive impact on the field by providing genetic tools to help functionally subdivide the contributions of different BMN populations to circuit computations and behavior. This contribution will pave the way for further mechanistic study of central circuits that subserve grooming circuits.

Here's how the number of heads affects the transformer architecture:

Increased Capacity: Adding more attention heads increases the model's capacity to capture diverse patterns and dependencies in the input data. Each head attends to different aspects of the input sequence, allowing the model to attend to multiple positions simultaneously.

Enhanced Representation: With more heads, the model can learn more fine-grained representations. Each head specializes in attending to different parts of the sequence, enabling the model to capture both local and global dependencies more effectively.

Parallelization: The self-attention mechanism can be computed in parallel for different heads, which speeds up training and inference. As a result, increasing the number of heads can lead to improved computational efficiency.

restaurant and person at fron taking long time to decide.

Head of line blocking happens when a particular packet or set of packets gets delayed for some reason, like congestion or errors. These delayed packets act as a "blockage" for the subsequent packets that are waiting to be processed. Just like in the restaurant example, the packets behind the delayed ones have to wait until the delay is resolved and the blocked packets are processed.

reply to u/muhlfriedl by way of reply to u/chounosumuheya at https://www.reddit.com/r/Zettelkasten/comments/13s6dsg/comment/jlpt8ai/?utm_source=reddit&utm_medium=web2x&context=3

Examples of zettelkasten users

S.D. Goitein, Beatrice Webb, Ludwig Wittgenstein, Harold Innis, Victor Margolin, Eminem, Aby Warburg, Antonin Sertillanges, Jacques Barzun, C. Wright Mills, Gotthard Deutsch, Roland Barthes, Umberto Eco, Vladimir Nabokov, Gerald Weinberg, Michael Ende, Twyla Tharp, Hans Blumenberg, Keith Thomas, Arno Schmidt, Mario Bunge, Sönke Ahrens, Dan Allosso for a few more. If you go with those who used commonplace books and waste books, which are notebook-based instead of index card-based, there are thousands upon thousands more.

Historically the easier question might be: what creators didn't use one of these systems and was successful?!? The broad outlines of these methods go back much, much farther than Niklas Luhmann. These patterns are not new...

Personally, I've used my own slip box to write large portions of the articles on my website. I also queried it to compile this reply.

Author Response:

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

In brief, we incorporated all wording and clarity suggestions into the manuscript. We also updated figure legends to include additional details, including replicate numbers. New data have been added in response to requests from the reviewers. Volumetric intake data are included as a supplemental figure (Figure 1–Figure Supplement 1A) and we will include movies of the confocal stacks from our CaMPARI imaging. We worked hard to address all the reviewers’ concerns and provide a detailed response below to the reviewers’ public comments as well as their author-specific comments.

Reviewer #1 (Public Review):

1) All feeding data presented in the manuscript are from the interactions of individual flies with a source of liquid food, where interaction is defined as 'physical contact of a specific duration.' It would be helpful to approach the measurement of feeding from multiple angles to form the notion of hedonic feeding since the debate around hedonic feeding in Drosophila has been ongoing for some time and remains controversial. One possibility would be to measure food intake volumetrically in addition to food interaction patterns and durations (e.g. via the modified CAFE assay used by Ja).

We acknowledge that our FLIC assays address only one dimension of feeding behavior, physical interaction with liquid food. However, there is clear evidence that interactions are strongly predictive of consumption, and it would be technically difficult to measure feeding durations at the resolution of milliseconds using a Café assay.  Nevertheless, we appreciate the spirit of this comment and agree that expanding our inference to other measures of feeding, as well as feeding environments, is an important next step. To this end, we now include measures of feeding on more traditional solid food, using the ConEx assay, and find that flies in the hedonic environment consume twice as much sucrose volume compared to flies in the control environment. These have been added as supplemental data (Figure 1 – Figure Supplement 1A), and the text has been updated to reflect our findings.

2) Some of the statistical analyses were presented in a way that may make understanding the data unnecessarily difficult for readers. Examples include:

a) In Table I the authors present food interaction classifications based on direct observation. These are helpful. However, the classification system is updated or incompletely used as the manuscript progresses, most importantly changing from four categories with seven total subcategories to three categories and no subcategories. In subsequent data analyses, only one or two of these categories are assessed. It would be helpful, especially when moving from direct observation to automated categorization, to quantify the exact correspondences between all of the prior and new classifications, as well as elaborate on the types of data that are being excluded.

We appreciate the feedback on our usage of the behavioral classification system and have made several adjustments to improve it. We renamed some of the behaviors to make them more intuitive (see Reviewer #2, comment #1), and updated the main text and Table 1 to reflect these changes. We updated the text and figures to be more transparent about when we group subcategories into main categories for quantification and when we quantify all subcategories separately. Because these videos required manual scoring by an experimenter, after our initial characterizations we opted to score only main categories (which contain subcategories). We agree that it would be useful to quantify correspondence between subcategories and the automated FLIC signal. However, we believe this task is better suited for more advanced and automated video tracking software, and, incidentally, more sophisticated analysis of FLIC data, which has a very high-dimensional character that has yet to be properly exploited. At the moment, therefore, we are not confident in the ability to understand the data at the desired resolution.

b) The authors switch between a variety of biological and physiological conditions with varying assays, which makes following the train of reasoning nearly impossible to follow. For example, the authors introduce us to circadian aspects of feeding behavior to introduce the concept of 'meal' and 'non-meal' periods of the day. It is then not clear in which of the subsequent experiments this paradigm is used to measure food interactions. Is it the majority of the subsequent figure panels? However, the authors also use starved flies for some assays, which would be incompatible with circadian-locked meals. The somewhat random and incompletely reported use of males and females, which the authors show behave differently, also makes the results more difficult to parse. Finally, the authors are comparing within-fly for the 'control environment' and between flies for their 'hedonic environment' (Figure 3A and subsequent panels), which I believe is not a good thing to do.

We apologize for our difficulties conveying our inference, which was also noted by Reviewer #2.  We have worked hard to improve this component in the revision. With respect to the confusion about circadian feeding, we introduced circadian meal-times to complement starvation as a second (perhaps more natural) way to measure behaviors associated with hunger. Importantly, we do not use circadian meal-times beyond Figure 1; all subsequent FLIC experiments were conducted during non-meal times of day for 6 hours, which avoids confounding our data with circadian-locked meals even when we use starved flies. We have clarified this point in the revision.

The reviewer also points out that we make both within-fly and between-fly comparisons, which is a point that we note. Perhaps some concern arises, again, from the challenges that we faced in properly delineating our inferences about different types of feeding measures (and motivations). Inference about homeostatic feeding was made using within-fly measures, comparing events on sucrose vs. those on yeast.  Inference about hedonic feeding was made using between fly measures (average durations of different flies on 2% vs. 20% sucrose).  Treatment comparisons to control always used measures of the same type, such that inference was not made using between-fly measures for treatment and within-fly for control (i.e., all of our figure panels were either within-fly or between fly). We have worked to clarify this in the revision.

Importantly, our approach to all experiments avoided confounding by used randomized design at multiple levels (e.g., randomizing control and hedonic environments to FLIC DFMs, alternating food choice sidedness in the DFMs), by ensuring that flies in both environments are sibling flies that came from the same vial environment before being tested, and by performing each experiment multiple times.

c) Statistical analyses are not always used consistently. For example, in Figures 3B and C, post hoc test results are shown for sucrose vs. yeast interactions, but no such statistics are given for 3E and 3F, preventing readers from assessing if the assay design is measuring what the authors tell us it is measuring.

We report p-values for two-way ANOVA interaction terms for all appropriate experiments. If (and only if) the interaction term is significant, we conduct post-hoc tests for more detailed statistical analysis and report the p-values. The reviewer points out that we do not perform post-hoc tests in figures 3E and 3F. These figures had a non-significant interaction term, and thus, we did not feel a post-hoc test was warranted.

Reviewer #2 (Public Review):

1) The dissection of feeding into distinct behavioral elements and its correlation with electrical FLIC signals that allow interpreting feeding types is a fundamental new method to dissect feeding in flies. However, the categories of micro-behaviors in Table 1 are not intuitive.

We agree and have updated the Table, figures, and main text. Please see also our response to Reviewer #1, comment #1.

2) The details for the behavioral data analysis are not clear and should be made more obvious. For example, how many males and females were used in each experiment? Were any of the females mated or were they all virgins? If all virgins, why not use mated females? Mating status may have an effect on the feeding drive. If mated and virgin females were used, are there any differences between them? Similarly, for diurnal feeding experiments, it is not immediately clear from the graphs how many animals were used and how the frequencies were obtained (Fig. 1F, presumably averages for each category per fly but that is inconsistent with the legend in the supplement for this figure). Why does the transition heat map not include all micro-behaviors (Fig. 1E, no LQ data which are significant in diurnal feeding)?

We have clarified the number of flies and events for each behavioral experiment in Figure 1, and we updated the figure legend appropriately. We note that these behavioral datasets are non-overlapping, and each time we mention the number of events scored in the text, that number includes only “new” videos. Female and male flies for all experiments were mated, and we have clarified this in the main text and methods.

For the diurnal experiment in Figure 1F, we scored over 700 events from new (non-overlapping) video compilations and updated the number of flies and event number in the figure legend. The diurnal data we present in the supplement for this figure is a separate experiment conducted on 38 flies, intended only to demonstrate the circadian nature of fly feeding.

For the transition heat map, analysis of this sort seems to require a large amount of data to have sufficient power to return a transition matrix. LQ events are relatively low in frequency, so we opted to combine them with L events for this analysis. We have updated the figure and figure legend to reflect this.

3) The CaMPARI images do not look great, particularly in the pan-neuronal condition (Fig. 5A). It would be useful to include the movie of the stack. Did any other brain regions show activity differences, such as SEZ or PI? These regions are known to be involved in feeding so it seems surprising they show no effect.

We find that CaMPARI imaging is subject to high levels of noise and background, especially when using a broad driver as the reviewer has pointed out. This is why we opted to follow-up our pan-neuronal CaMPARI experiment using a more specific mushroom body driver and to test our correlational findings of increased MB activity in hedonic environments with genetic approaches in the remainder of Figure 5. We have included movies of the confocal stacks for both CaMPARI experiments, as requested. 

Reviewer #1 (Recommendations For The Authors): 

Main concern: 

No measurements of intake, either in volume or in caloric value. Hence, 'hedonic' feeding is only indirectly supported. 

I would like to suggest to the authors that they measure intake volumetrically in addition to food interaction patterns and durations. For example, William Ja developed a modified CAFE assay that measures consumption volume in real-time in freely behaving flies (http://dx.doi.org/10.1038/nprot.2017.096). Liming Wang has another capable assay. Additional values of expanding measurement methods for feeding are that it helps tie the research more directly to that of others, and it helps remove the concern that any one assay may introduce unknown biases. 

For the CaMPARI, it would be helpful to provide a demonstration of its effectiveness by recapitulating a deep brain neural pathway known to be engaged by a stimulus by GCaMP or electrophysiology. 

Additional concern: 

The authors assume satiety states during different circadian periods (line 253, for example). It seems critical to directly measure the satiety state. 

Technical concerns: 

Figure 5 A, B: there is reported near zero UV transmission through the head: https://doi.org/10.1364%2FBOE.6.000514, hence the CaMPARI measurements are suspect. It appears that there may be an effect in the optic lobes that may receive greater UV illumination by being more peripheral. A positive control to demonstrate deep brain access by UV is needed. 

Y-axes vary for the same measurement types within figures, for example, Figure 5 C-G. Also Figures 3F, G, I, K, M and Figures 3D, E, H, J, L. This hinders direct comparisons. 

Figure 2: why are there no statistics to distinguish interaction (I) events from F and L? Why are the example graphs presented using different scale x-axes? For A-C, why no averaged response graphs for the classifications? Were there other events that did not fit these classifications? 

In lines 224-226, the claim of statistical significance at p=0.061 makes the reader suspicious of the statistical interpretations throughout the manuscript. 

Figure 3B starved looks the same as Figure 3C sated for females, using the same assay and conditions. This implies a huge amount of variance in behavior between experiments. 

We appreciate the recommendations from Reviewer #1 and have done our best to address many of their concerns. Regarding their main concerns, we have added volumetric feeding data to the manuscript, included movies of the confocal stacks for the CaMPARI experiments, and clarified the circadian timing of our behavioral experiments. These details are outlined in our public response to both reviewers. The reviewer also expressed a few technical concerns, mostly regarding statistical analyses. We agree that there seems to be a large amount of biological variability between experiments, which we do indeed find to be the case with behavioral experiments of this sort. For this reason, we avoid making direct comparisons on absolute values between experiments, as the reviewer suggests, and thus allow our Y-axes to vary for each figure to better facilitate within-experiment comparisons. The reviewer also points out that, in one instance, we refer to a p-value of 0.061 as statistically significant in the text. While we have changed our language to reflect the perceived convention, we note that there is little inferential difference between these values, and we report exact p-values to allow the reader to make an informed decision.

Reviewer #2 (Recommendations For The Authors): The writing and data presentation in this paper is somewhat dense and confusing at times. Comments and questions below are intended to help improve data presentation and resolve questions that will help the reader navigate and understand the data to better appreciate the significance of the findings. 

Comments and questions: 

Line 160 cites Chen et al, 2002 as an example of behavioral characterization that is useful for read-outs of neural states, but no neural states were defined in that work. A better example where a circuit was linked to a specific behavioral category is PMID30415997 (Duistermars et al., 2018). 

Line 171: were the females mated or virgin or was it variable? 

The classification system in Table 1 is a bit confusing. For example, the distinction is made between Fast and Long feeding events as well as interactions with food and other events. FH meet the requirements of F and H, presumably meaning that flies are fast feeding and touching the food with their front legs. Why are front legs and hind legs touching food abbreviated H and FF respectively instead of something more obvious like IF and IH (referring to Interaction with Front legs or Interaction with Hind legs)? 

Also was there never any tasting with the middle legs? In Fig1B, all the I events are grouped. Are most of these H or FF events? The frequency in Fig. 1B is shown as normalized as a frequency of all events. The statistical analyses are all parametric. Are these data normally distributed? 

Lines 224-229: the relative frequency of L-type feeding is increased in starved flies and the relative frequency of F feeding is decreased. Is the relative L- or F-type feeding frequency considered on total behavior or just the sum of long and fast feeding or the sum of all types of feeding? 

The events that are analyzed vary throughout the paper. Line 173 mentions 300 events, line 222, 500 events, and line 257, 700 feeding events. Are these all independent experiments, or are these overlapping data sets analyzed for different parameters? 

For diurnal feeding behavior, the authors analyzed 700 events and found significantly more LQ events during meal time (i.e. at the beginning and end of the day). Based on the figure legend in the supplement to Figure 1, it appears that these data were collected on 38 female flies. But in Fig 1F, there are ~8 points per feeding type (F, L, and LQ) during meal and non-meal conditions. Shouldn't all 38 flies have an average frequency for each type of feeding during meal and non-meal times? Were these females mated or not? Is this effect also true for males? To help the reader understand the data better, it would be helpful to note the number of flies used in each experiment or in each analysis in the different figures and wherever the data are mentioned in the manuscript. It also seems likely that the mating state may have an effect on feeding so knowing the result in mated versus unmated would be a useful analysis. 

It is interesting that there is a difference in feeding in starved flies versus diurnal feeding in the presumably hungry versus sated phase (meal versus non-meal phases). As mentioned by the authors earlier in the manuscript, starved flies have a relative increase in L-type feeding. However, they perform less LQ feeding than sated flies, and yet LQ feeding is the only significantly different type of feeding in the hungry state of diurnal feeding. In the morning, the transition to feeding is very abrupt compared to the gradual increase in the evening. Is there any difference between the type of feeding or the transition matrix in the evening versus morning meal times? Also, why is LQ feeding not included as a category in the transition matrix in Fig 1E? 

In Fig 2, the authors examine FLIC signals with video data to identify feeding types from FLIC signals. Why are there signal durations for F-type feeding that are longer than 3 seconds when it is defined as 1-3 sec of the proboscis contact with food and conversely signals of L-type feeding shorter than 4 seconds when it is defined as >4 seconds of continuous proboscis contact? Does this mean that signal can be longer or shorter than the actual time the proboscis is in the food? 

With these parameters, the authors develop an assay to identify homeostatic and hedonic feeding by applying the signal analysis to food choices representing homeostatic (2% sucrose versus yeast) and hedonic (2% sucrose versus 20%) conditions. In Fig 3C, they show that fully-fed females show a stronger preference for yeast food than sugar food compared to males (line 335). Is this in fully fed animals? The yeast preference in females looks almost the same as in the starved females in Fig 3B. 

The CaMPARI images shown in Fig 5A (and to a lesser extent Fig 5B) are not particularly convincing although the quantification looks clear. Providing the movies of the stacks may help the reader better appreciate the difference in MB red signal in the hedonic state. It would also help to show the number of flies that were tested in these experiments as well as the sex and mating status. Provide the n in the figure legend and in the relevant sections in the text. 

Were the mushroom bodies the only brain region with significant, measurable activity changes? One might expect changes in other feeding areas, such as the subesophageal zone (SEZ) and the peptidergic regions of the brain (PI), which are both known to affect feeding in flies. This may also be a useful method to examine differences in mated versus unmated flies. 

In Fig 5C the caption reads MB lambda lobe inhibition. Shouldn't this be gamma lobe inhibition as suggested in the figure legend? 

The paper largely distinguishes homeostatic from hedonic feeding only. It may be useful to discuss other non-homeostatic mechanisms as well or at least make the distinction in the introduction and or discussion.

We thank reviewer #2 for their thoughtful suggestions to improve the clarity of the manuscript. They suggest several improvements, which we implemented, including that we improve the classification system in Table 1 to make it more intuitive, state how we normalized observed behavioral frequencies, clarify that the number of events we cite for each experiment are non-overlapping, and explain the use of circadian meal vs. non-meal times. We also noticed, as did this reviewer, that the usage of L vs. LQ events differs between starved flies and flies observed during meal-time. We agree that it may be interesting to sort out the nuances of why and how these differences occur, as it suggests that starvation may in some ways be different from physiological hunger. However, our method of manually observing flies would make this difficult at present. We hope to utilize more advanced video tracking software in the future to investigate this question. The reviewer also posed several questions about the hunger/satiety state of flies that we used for each experiment, which we clarified throughout the main text, figure legends, and methods.

This reviewer points out two technical concerns, which we have addressed. The concerns about our CaMPARI imaging are noted, and we have discussed them in response to reviewer #1 and in our public response. We now include movies of the confocal stacks, as requested. There was also a question about FLIC durations of F and L events in Figure 2, with some visually identified F events producing FLIC signals longer than 4 seconds and some L events producing FLIC signals shorter than 4 seconds. Although we show that population averages from the FLIC can reliably recapitulate our visual metrics, there is occasional noise at the individual level. For example, although a fly may have contact of its proboscis with the food for less than 4 seconds, the FLIC signal may persist slightly beyond that interaction due to sustained contact with a non-proboscis body part or due to liquid food contacting the signal pad. We also occasionally observed L events that we visually identified to last longer than 4 seconds, but nevertheless did not produce a FLIC signal of equal length. This can occur when a fly feeds on the liquid food but transiently loses contact with the signal pad. Although there is some noted technical noise, we show that population-level data is sufficient to reflect our visual observations.

Locking: Locking protects web team members from overwriting each other’s changes. When two or more people are working on the same file, WebDAV ensures that they compare or merge changes before writing to the file.

Git solved this problem in a much cleaner and respectable way: it's an append-only log. The only thing left is to "flatten" the two namespaces into a single one. On first inspection, this seems like it would re-introduce the need for rewrites (and locking?), but this can be cleverly solved by preregistration of forward references. Locating the current "head" can be resolved in log2(n) time wrt number of "revisions" (and can also be avoided in many cases, besides).

Reviewer #2 (Public Review):

This is a very dense and thorough analysis of the role of Uso1 in Aspergillus using genetics, pulldown assays, and modelling.<br /> Uso1 has been established as an essential tethering factor that acts in conjunction with Rab1 to deliver ER-derived vesicles to the Golgi. The current picture is that Uso1 is a Rab1 effector, but the authors challenge this interpretation using a combination of genetics experiments, biochemical analysis of protein-protein interactions, and alphafold2 prediction.

While Rab1 is essential, they identify strains of Aspergillus that bypass the need for Rab1, which carry two mutations in Uso1. They go on to show that Uso1 binds directly to the Bos1 and Bet1 components of the SNARE complex and that the rescue mutations cause tighter binding of the Uso1 globular head domain to Bos1 and (hypothetically) to the membrane. They support their genetics and biochemical analysis by doing structure predictions with alphafold2 and suggesting how these mutants might act. They also show that an overexpressed mutated monomeric globular domain of Uso1 (without the coiled-coil 'tether' that causes dimerisation) rescues growth defects of delta Uso1, suggesting the essential activity of Uso1 is not the tethering but its being part of the SNARE complex.

The data is solid, and the interpretation is convincing, showing Uso1 is not 'merely' a tethering factor. It has multiple roles, and this study opens up new questions regarding what exactly is Uso1's function as part of the SNARE bundle, and also in which way the Rab1-mediated tethering and the SNARE complex aspects of Uso1 are linked and/or regulated.

However, there are some aspects of this work that need to be strengthened/clarified including some of the modelling and the interpretation of the role of Uso1 dimerisation. Also, given the availability of models for all homologues, it would be interesting to test whether analogous Uso1 mutant in S.cerevisiae can also rescue rab1- lethality. This would suggest the new proposed role of Uso1 is a general feature, at least for fungi, rather than a particularity of Aspergillus.

Reviewer #3 (Public Review):

The manuscript by Bravo-Plaza et al. identifies and characterizes new mutations (E6K and G540S) in the Uso1 globular head domain that suppress the loss of function mutations in Rab1. Further experiments show that the combined E6K/G540S mutant restores apparent Golgi-localization of Uso1 in Rab1 deficient cells, that this mutant preferentially co-purifies with ER/Golgi SNARE proteins, that monomeric E6K/G540S globular head-domain binds more avidly to purified Bos1 SNARE protein than wild type head-domain, and that overexpression of E6K/G540S or wild type head-domain alone is sufficient for viability. Based on these findings the authors propose that long-distance tethering by Uso1 is dispensable and that the head domain provides an essential function to directly regulate ER/Golgi SNARE-dependent membrane fusion.

Strengths of the study are that an unbiased screen was used to identify new Rab1 suppresser mutations that land in the Uso1 globular head domain. Characterization of these suppressor mutants reveals that SNARE binding activity of Uso1 resides in the head domain and that elevated expression of the Uso1 head domain is sufficient for viability. Imaging experiments document the localization and dynamics of Uso1 on Golgi compartments and biochemical studies show the properties and binding activity of Uso1 domain mutants. These are new findings and the conclusion that monomeric globular head-domain interacts with specific SNAREs to maintain viability is justified.

Weaknesses are that it is well documented that both Rab1 and Uso1 activity can be bypassed by activation of ER/Golgi SNARE machinery either by overexpression of SNARE proteins or by the single copy SLY1-20 allele. Therefore, it was not surprising that tethering by the Uso1 coiled-coil domain is dispensable. The proposal that the E6K mutation in the head domain of Uso1 promotes membrane targeting was not well supported by experimental evidence. And while the AlphaFold modeling of Uso1 with the ER/Golgi fusion machinery was intriguing, the proposed molecular models remain speculative until further tested.

Here, Hewitt corrects his employer based upon the technicality of the word "significant." He is meant to appear particularly clever, but may come across as cocky or even rude. Audiences, therefore, may have difficulties deciding how to feel about him.

Judith refers to the deuterocanonical "Book of Judith" that is excluded from the Hebrew canon. Judith, a beautiful widow, is perturbed by her Jewish countrymen's lack of faith in God to save them from the Assyrians. Judith slowly integrates herself into the camp, promising the general Holofernes that she will give him information on the Israelites. Later, she decapitates him, brings his head back to her countrymen, and the Assyrians disperse.

forcefully pulled the head toward the author

Here, Stokes is described as possessing physical traits that derive from normal beauty standards, particularly by being a short and larger man, almost squared. Allen chooses to place him as the hero of the story and therefore actively prescribes to the dismantling of these structures. This could speak to a wider-ranging audience compared to pieces with detectives that are chronically and conventionally attractive.

I think the justification of crowd harassment can be situational. The most recent example off the top of my head was when the queen of England died and people in the comments would make jokes and have a lack of sympathy for the royals. In this context, I also feel less sympathy due to thee money and power that the royals hold, as well as scandals like the queen crown having stolen jewels from India, and well colonialism.

Author Response

Reviewer #1 (Public Review):

This manuscript is interesting because of the exploration of a novel model organisms utilizing next-generation sequencing approaches, such as single-cell-RNA-seq. Despite the authors' efforts the manuscript lacks a cohesive narrative and suffers from being extremely preliminary in nature. For example, most of the figures are cut and pasted directly from the computational programs with very little formatting or thought to creating new knowledge from the data generated. Essentially the manuscript consists of 2-3 experiments where the authors performed single-cell-RNA-seq on different anatomical locations in the pig and also on a couple of different pig types (The Chenghua and Large White). The authors used standard computational pipelines consisting of Seurat, Monocle, Cell Chat, and others to characterize differences in their data.

There is potential in this manuscript but the authors should improve upon the manuscript by mining the data better and generating a better understanding of anatomical positions of pig skin by evaluating the Hox genes.

(1) Thanks for the reviewer's positive evaluation for our article and providing valuable feedback to improve the quality of our manuscript. To provide a more cohesive narrative, we have edited throughout the manuscript.

(2) Meanwhile, we also modified and formatted some figures including Figures 2-6, Figure 4—figure supplement 1 and 2, Figure 5—figure supplement 1 and 2, and Figure 6—figure supplement 1.

(3) We have analyzed these data of regional- or species-based differences more extensively, and the added content are in Result Section of “Heterogeneity of skin FBs in different anatomic sites” and “Heterogeneity of skin cells in different pig populations”.

(4) However, in our study, we did not identify any Hox gene among these differentially expressed genes in skin fibroblasts from both different anatomical sites and different pig populations. The differences of Hox code expression patterns might come from the heterogeneity of different species.

Reviewer #2 (Public Review):

The authors aimed to analyze different dermal compositions of various skin regions, focusing on fibroblast, endothelium and smooth muscle cells. They collect skin samples from six different skin regions of adult pig skin including the head, ear, shoulder, back, abdomen, and leg skins. After dissociating the tissues into single cells, they perform single-cell RNA analyses. A total of 215 thousand cells were analyzed. The authors identified distinct cell clusters, enriched molecules within each cell cluster, and the dynamic of cell cluster transition and interactions. Based on their findings, they conclude that tenascin N, collagen 11A1, and inhibin A are candidate genes for facilitating extracellular matrix accumulation.

Strength:

The methodology they used to prepare scRNA data is appropriate. Bioinformatic analyses are solid. The authors emphasize the heterogeneous phenotypes and composition ratios of smooth muscle cells, endothelial cells and fibroblasts in each skin region. They identify potential cell communication pathways among cell clusters. Expression of selective molecules on tissue sections were done.

Weakness:

While tenascin, collagen and inhibin are highlighted as genes important for ECM accumulation, there is no functional evaluation data. The discussion section is a compilation of comparisons, and is somewhat fragmentary. More significance from this dataset could have been extracted.

(1) We appreciate the reviewer's suggestions for evaluating the functional significance further. In our next research, we will perform some experiments in vitro and in vivo to explore the functions of these identified key genes.

(2) The discussion section have been greatly modified and it shall be more logical and readable.

Reviewer #2 (Public Review):

The authors aimed to analyze different dermal compositions of various skin regions, focusing on fibroblast, endothelium and smooth muscle cells. They collect skin samples from six different skin regions of adult pig skin including the head, ear, shoulder, back, abdomen, and leg skins. After dissociating the tissues into single cells, they perform single-cell RNA analyses. A total of 215 thousand cells were analyzed. The authors identified distinct cell clusters, enriched molecules within each cell cluster, and the dynamic of cell cluster transition and interactions. Based on their findings, they conclude that tenascin N, collagen 11A1, and inhibin A are candidate genes for facilitating extracellular matrix accumulation.

Strength:

The methodology they used to prepare scRNA data is appropriate. Bioinformatic analyses are solid. The authors emphasize the heterogeneous phenotypes and composition ratios of smooth muscle cells, endothelial cells and fibroblasts in each skin region. They identify potential cell communication pathways among cell clusters. Expression of selective molecules on tissue sections were done.

Weakness:

While tenascin, collagen and inhibin are highlighted as genes important for ECM accumulation, there is no functional evaluation data. The discussion section is a compilation of comparisons, and is somewhat fragmentary. More significance from this dataset could have been extracted.

Summary:

The manuscript has the potential to be a useful cellular atlas. The direct impact of this paper on skin biology is limited because of the lack of evaluation data. But the database can be useful to many future studies using the pig skin model.

YES ABSOLUTELY!!!!

CC Licensing: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)

Valmiki starts by asking Narada about the identity of a virtuous and heroic individual, someone who embodies qualities such as gratitude, righteousness, and protection of the people. Narada responds by describing the characteristics of Rama, the hero of the Ramayana. He praises Rama's physical appearance, his virtues, skills, and his commitment to justice and righteousness. Rama is depicted as the epitome of a hero, with divine-like qualities that make him comparable to gods such as Indra and Vishnu.

In analyzing the gender aspect of the text, we can observe the traditional gender roles and definitions prevalent in the culture from which the text originates. The passage portrays Rama as the ideal hero, upholding the patriarchal norms of the society. He is described as a strong and powerful warrior, with physical attributes that emphasize his masculinity. Rama's qualities are praised, and he is depicted as the protector and savior of mankind.

The text also portrays Sita, Rama's wife, as a devoted and obedient wife. She is described as Rama's "darling wife" who clings to him and follows him even in exile. Sita's role is primarily that of a supportive and loyal wife, whose virtue and purity are highlighted. However, it is important to note that in later parts of the Ramayana, Sita's agency and strength are further explored, challenging traditional gender roles.

In terms of linguistic value, the text is composed in a poetic and descriptive manner, employing vivid imagery and metaphors to depict the qualities of the hero and his spouse. The language used is rich and evocative, contributing to the aesthetic appeal of the work.

Note: This annotation is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) license. This means that others are free to share and adapt this work for non-commercial purposes as long as they attribute the original author and use the same CC license for their derivative works.

antagonism between local government and MFIs

epic simile

Uses a source that also leads to another link. This proves their credibility to the reader regarding the topic of civil liberties in New York.

a man who is the head of an abbey of monks. 男修道院長

Once again, the author uses some vivid imagery and detail in order to paint a clear picture of the story and "set the scene" in the courtroom. The quality of story-telling of this article in general seems to stem from the specific moments and details that are covered and the in depth analysis that the author provides.

Learning as a Network LaaN and Personal Knowledge Network PKN , do these labels give me anything new?

Mohamed Amine Chatti: The LaaN Theory. In: Personalization in Technology Enhanced Learning: A Social Software Perspective. Aachen, Germany: Shaker Verlag, 2010, pp. 19-42. http://mohamedaminechatti.blogspot.de/2013/01/the-laan-theory.html I've followed Chatti's blog in the past I think. Prof. Dr. Mohamed Amine Chatti is professor of computer science and head of the Social Computing Group in the Department of Computer Science and Applied Cognitive Science at the University of Duisburg-Essen. (did his PhD at RWTH in 2010, which is presumably how I came across him, through Ralf Klamma)

The problem with bemoaning this trope is that, in reality, there are myriad pockets of the North American population that literally correlate to many of these espoused characteristics - I personally come from a family rooted in rural New Brunswick, with ancestral connections to settlers that landed at various points along the Eastern seaboard and all drawing descent from the hills and rural areas of mostly Scotland but Ireland, Wales and norther England too. There is a distinct vernacular, an identifiable accent, a commonality of folklore and values packed into the steamer trunks that escorted their progenitors' passage to the New World, wherein the vast and various isolated, yet periphorally connected, diaspora gradually customized (basterdized) to suit their more specially environmental niches.

While I was raised external to the heart of my particular ancesteral perculiarities, I am well aware that I still am steeped in and am the direct product of this isolated cauldron, that in my tired or excitable moments, a discernaible accent sleps into my vocal cadence and a subtle peppering of quaint and geographically iconic jargon creeps into my syntax.

And yes, there is a degree of 'ignorance' - not in a perjorative sense but rather in the sense that most choose to remain connected to the geogrpahically homestead and limit their formal or further education accordingly. There are even slight whispers of the more antipodal position of 'suspicion' against those who separate from the familial 'roots' to pursue life in the wider landscape, encapuslated in folksy pronouncements 'Her head is so far in the books, she's lost her common sense' or "Now he's got soft hands"

So many people have their interest in school diminished because they aren't recognized enough. They are meant to feel unimportant and therefore don't want to participate anymore.

This is very accurate especially now, in less black and white ways. If someone hears that an individual was SA'd by coercion, they may be more likely to dismiss their story. This also connects to the idea that a victim is untrustworthy due to their relationship with the victim. The trust aspect furthers the idea that they are less worthy of support and belief.

This to me smells a little fishy?

Without seq or any sort of indication of causal dependencies (e.g. origin/originLeft/originRight), how do we know how to properly order remote operations?

I'm guessing we encode some of this information in the ID field but this is not obvious

I'm going to keep reading but this raised a flag in my head

had to read this twice because in my head, this second column is usually an arbitrary datastructure that the CRDT implementation chooses (e.g. a semilattice, tree, list, etc.)

thought keeping it as list for consistency with examples given so far also makes sense

evidence for mercury to treat ectoparasites

I don't understand - are we both praising Holmes and discouraging ambition? What happened to questioning the stigma about female founders? It's okay to believe you can do things.

i literally said "vicarious" in my head before i read the term oh god

Reviewer #3 (Public Review):

This manuscript describes interesting experiments on how information from the two eyes is combined in cortical areas, sub-cortical areas, and perception. The experimental techniques are strong and the results are potentially quite interesting. But the manuscript is poorly written and tries to do too much in too little space. I had a lot of difficulty understanding the various experimental conditions, the complicated results, and the interpretations of those results. I think this is an interesting and useful project so I hope the authors will put in the time to revise the manuscript so that regular readers like myself can better understand what it all means.

Now for my concerns and suggestions:

The experimental conditions are novel and complicated, so readers will not readily grasp what the various conditions are and why they were chosen. For example, in one condition different flicker frequencies were presented to the two eyes (2Hz to one and 1.6Hz to the other) with the flicker amplitude fixed in the eye presented to the lower frequency and the flicker amplitude varied in the eye presented to the higher frequency. This is just one of several conditions that the reader has to understand in order to follow the experimental design. I have a few suggestions to make it easier to follow. First, create a figure showing graphically the various conditions. Second, come up with better names for the various conditions and use those names in clear labels in the data figures and in the appropriate captions. Third, combine the specific methods and results sections for each experiment so that one will have just gone through the relevant methods before moving forward into the results. The authors can keep a general methods section separate, but only for the methods that are general to the whole set of experiments.

I wondered why the authors chose the temporal frequencies they did. Barrionuevo et al (2014) showed that the human pupil response is greatest at 1Hz and is nearly a log unit lower at 2Hz (i.e., the change in diameter is nearly a log unit lower; the change in area is nearly 2 log units lower). So why did the authors choose 2Hz for their primary frequency? And why did the authors choose 1.6Hz which is quite close to 2Hz for their off frequency? The rationale behind these important decisions should be made explicit.

By the way, I wondered if we know what happens when you present the same flicker frequencies to the two eyes but in counter-phase. The average luminance seen binocularly would always be the same, so if the pupil system is linear, there should be no pupil response to this stimulus. An experiment like this has been done by Flitcroft et al (1992) on accommodation where the two eyes are presented stimuli moving oppositely in optical distance and indeed there was no accommodative response, which strongly suggests linearity.

Figures 1 and 2 are important figures because they show the pupil and EEG results, respectively. But it's really hard to get your head around what's being shown in the lower row of each figure. The labeling for the conditions is one problem. You have to remember how "binocular" in panel c differs from "binocular cross" in panel d. And how "monocular" in panel d is different than "monocular 1.6Hz" in panel e. Additionally, the colors of the data symbols are not very distinct so it makes it hard to determine which one is which condition. These results are interesting. But they are difficult to digest.

The authors make a strong claim that they have found substantial differences in binocular interaction between cortical and sub-cortical circuits. But when I look at Figures 1 and 2, which are meant to convey this conclusion, I'm struck by how similar the results are. If the authors want to continue to make their claim, they need to spend more time making the case.

Figure 5 is thankfully easy to understand and shows a very clear result. These perceptual results deviate dramatically from the essentially winner-take-all results for spatial sinewaves shown by Legge & Rubin (1981); whom they should cite by the way. Thus, very interestingly the binocular combination of temporal variation is quite different than the binocular combination of spatial variation. Can the pupil and EEG results also be plotted in the fashion of Figure 5? You'd pick a criterion pupil (or EEG) change and use it to make such plots.

My main suggestion is that the authors need to devote more space to explaining what they've done, what they've found, and how they interpret the data. I suggest therefore that they drop the computational model altogether so that they can concentrate on the experiments. The model could be presented in a future paper.

Well that truly was the impetus of the issue. Those who are fighting against minimum increase defentily should not be the ones at the head, at least not more than they already are

I actually remember seeing videos from this popping up on my TikTok feed. In the moment I found it funny, but I realize now that it could have been harmful. As the book said " All of this is almost certainly not the impression the original TikToker was trying to convey."..

I remember seeing this on TikTok for the first time probably like a year or two ago and I thought it was really funny. I really commend the first persons duet because he is really the one who started it.

I wonder who painted it

• Indigestion can be treated with lifestyle and dietary changes.
• Patients with mild indigestion may not need any intervention, but drugs that cause gastroesophageal reflux or dyspepsia should be stopped if possible.
• GERD patients should limit ethanol, caffeine, chocolate, and tobacco use and can ingest a low-fat diet, avoid snacks before bedtime, and elevate the head of the bed.
• Functional dyspepsia patients can be advised to reduce intake of fat, spicy foods, caffeine, and alcohol. Dietary lactose restriction is appropriate for lactase deficiency, while gluten exclusion is indicated for celiac disease. Low FODMAP diets are effective for gaseous symptoms in IBS.
• Drugs that reduce or neutralize gastric acid are often prescribed for GERD, such as histamine H2 antagonists and PPIs. Up to one-third of GERD patients do not respond to standard PPI doses, and complications of long-term PPI therapy include diarrhea, small-intestinal bacterial overgrowth, nutrient deficiency, hypomagnesemia, bone demineralization, interstitial nephritis, and impaired medication absorption.
• Antacids are useful for short-term control of mild GERD but have less benefit in severe cases.
• H. pylori eradication is indicated for peptic ulcer and mucosa-associated lymphoid tissue gastric lymphoma. The benefits of eradication therapy in functional dyspepsia are limited but statistically significant.
• • Agents that modify gastrointestinal motor activity, such as baclofen, can be used in patients with refractory acid or nonacid reflux. Prokinetic drugs may be effective for functional dyspepsia, but publication bias and small sample sizes raise questions about reported benefits. 5-HT1A agonists and acotiamide may improve some functional dyspepsia symptoms.
• The GABA-B agonist baclofen reduces esophageal exposure to acid and nonacidic fluids by reducing TLESRs by 40%, making it useful in patients with refractory acid or nonacid reflux.
• Agents that stimulate gastric emptying have shown a 33% relative risk reduction in functional dyspepsia, but publication bias and small sample sizes raise questions about their reported benefits.
• The newer 5-HT4 agonist prucalopride was reported to reduce symptoms in patients with idiopathic gastroparesis, but no similar studies have been conducted in functional dyspepsia.
• The 5-HT1A agonists buspirone and tandospirone may improve some functional dyspepsia symptoms by enhancing meal-induced gastric accommodation.
• Acotiamide stimulates gastric emptying and augments accommodation by enhancing acetylcholine release via muscarinic receptor antagonism and acetylcholinesterase inhibition, and it is approved for functional dyspepsia in Japan and India.
• Some patients with refractory functional heartburn may respond to antidepressants in the tricyclic and selective serotonin reuptake inhibitor (SSRI) classes, although studies are limited.
• In a controlled trial in functional dyspepsia, the tricyclic drug amitriptyline produced symptom reductions, whereas the SSRI escitalopram had no benefit in a three-way comparison with placebo. In another controlled trial in functional dyspepsia, the antidepressant mirtazapine produced superior symptom reductions versus placebo.
• Antireflux surgery (fundoplication) to enhance the barrier function of the LES may be offered to GERD patients who are young and require lifelong therapy, have typical heartburn, are responsive to PPIs, and show acid reflux on pH monitoring. Surgery also is effective for some cases of nonacidic reflux.
• Gas and bloating are bothersome in some patients with indigestion and are difficult to treat. Simethicone, activated charcoal, and alpha-galactosidase provide benefits in some cases. One trial suggested possible benefits of the nonabsorbable antibiotic rifaximin in functional dyspepsia, while another reported improvement with the probiotic Lactobacillus gasseri.
• Herbal remedies like STW 5 (Iberogast, a mixture of nine herbal agents) and formulations of caraway oil and menthol are useful in some dyspeptic patients.
• Psychological treatments (e.g., behavioral therapy, psychotherapy, hypnotherapy) may be offered for refractory functional dyspepsia; a meta-analysis of four trials reported benefits in patients with persistent dyspepsia.

[Start: History and Physical Examination]

• Does the patient complain of heartburn?
  • Yes:
    • Does heartburn worsen after meals or awaken the patient?
      • Yes: GERD is likely. Proceed to treatment empirically.
      • No: Consider atypical GERD. Check for pharyngeal erythema, wheezing, and poor dentition.
  • No:
    • Does the patient have upper abdominal pain?
      • Yes:
        • Is the pain meal-related (postprandial distress syndrome) or independent of food ingestion (epigastric pain syndrome)?
          • Postprandial distress syndrome: Likely functional dyspepsia. Check for GERD, IBS, or idiopathic gastroparesis.
          • Epigastric pain syndrome: Further evaluation needed.
      • No: Proceed to next question.
• Does the patient exhibit any alarm features? (unexplained weight loss, recurrent vomiting, dysphagia, occult or gross bleeding, nocturnal symptoms, jaundice, palpable mass or adenopathy, family history of gastrointestinal neoplasm)
  • Yes: Further evaluation needed.
  • No: Proceed to next question.
• Does the patient exhibit any historic or exam features concerning for organic causes of indigestion?
  • Yes: Further evaluation needed.
  • No: No further evaluation needed. Treat empirically for GERD or functional dyspepsia.

[End: History and Physical Examination]

[Start: Diagnostic Testing]

• Typical GERD:
  • No further evaluation needed. Treat empirically.
• Atypical symptoms or alarm factors present:
  • Upper endoscopy is indicated.
• Heartburn >5 years in duration, especially in patients >50 years old:
  • Endoscopy is advocated to screen for Barrett's metaplasia.
• Low-risk patients who respond to acid suppressants:
  • Endoscopy is not needed.
• Drug-refractory symptoms and atypical symptoms like unexplained chest pain:
  • Ambulatory esophageal pH testing using a catheter method or a wireless capsule endoscopically attached to the esophageal wall is considered.
• Surgical treatment of GERD is considered:
  • High-resolution esophageal manometry is ordered.
    • Low LES pressure predicts failure of drug therapy.
    • Poor esophageal body peristalsis raises concern about postoperative dysphagia and directs the choice of surgical technique.
• Nonacidic reflux may be detected by combined esophageal impedance-pH testing in medication-unresponsive patients.

[End: Diagnostic Testing]

summary Oropharyngeal Dysphagia: - Poor bolus formation and control, drooling, and difficulty initiating swallowing are characteristic signs. - May result in premature spillage of food into the hypopharynx, aspiration into the trachea, or regurgitation into the nasal cavity. - Causes include neurologic, muscular, structural, iatrogenic, infectious, and metabolic factors, with iatrogenic, neurologic, and structural pathologies being the most common. - Iatrogenic causes include head and neck cancer treatments such as surgery and radiation. - Neurogenic dysphagia resulting from cerebrovascular accidents, Parkinson’s disease, and amyotrophic lateral sclerosis is a major cause of morbidity related to aspiration and malnutrition. - Asymmetry in the cortical representation of the pharynx provides an explanation for the dysphagia that occurs as a consequence of unilateral cortical cerebrovascular accidents. - Structural lesions causing dysphagia include Zenker’s diverticulum, cricopharyngeal bar, and neoplasia. - Rapid-sequence fluoroscopy is necessary to evaluate for functional abnormalities. - Adequate fluoroscopic examination requires that the patient be conscious and cooperative. - Timing and integrity of pharyngeal contraction and opening of the UES with a swallow are analyzed to assess both aspiration risk and the potential for swallow therapy. - Structural abnormalities of the oropharynx should be assessed by direct laryngoscopic examination.

Esophageal Dysphagia: - The adult esophagus measures 18-26 cm in length and is anatomically divided into the cervical esophagus and the thoracic esophagus. - Solid food dysphagia becomes common when the lumen is narrowed to <13 mm. - The most common structural causes of dysphagia are Schatzki’s rings, eosinophilic esophagitis, and peptic strictures. - Propulsive disorders leading to esophageal dysphagia result from abnormalities of peristalsis and/or deglutitive inhibition, potentially affecting the cervical or thoracic esophagus. - Rapid-sequence fluoroscopy is necessary to evaluate for functional abnormalities. - Adequate fluoroscopic examination requires that the patient be conscious and cooperative. - High-resolution manometry is used to measure pressure changes along the length of the esophagus during swallowing. - Structural abnormalities of the esophagus should be assessed by endoscopic examination.

Here's a table summarizing the information:

| Category | Oropharyngeal Dysphagia | Esophageal Dysphagia | | --- | --- | --- | | Signs and Symptoms | Poor bolus formation and control, drooling, difficulty initiating swallowing | Solid food dysphagia, potentially accompanied by altered esophageal sensation, reduced distensibility, or motor dysfunction | | Causes | Neurologic, muscular, structural, iatrogenic, infectious, metabolic | Structural causes include Schatzki’s rings, eosinophilic esophagitis, and peptic strictures; propulsive disorders due to abnormalities of peristalsis and/or deglutitive inhibition | | Iatrogenic Causes | Head and neck cancer treatments such as surgery and radiation | N/A | | Diagnosis | Rapid-sequence fluoroscopy, direct laryngoscopic examination | Rapid-sequence fluoroscopy, high-resolution manometry, endoscopic examination |

There are two cons here. One is the high risk of body rejection. The other is the the complexity of the brain's connections to the body are much more complicated than we imagine.

I find interesting how the idea of women to provide for their family is unthinkable. Without the head of the family (man) the rest of the family is presented as helpless. Also the relation of crime an nutrition sounds very extreme to me. Cook to your family and cook well otherwise your family might end up helpless and your partner in jail?!

Poor white people often make the comment that they don't benefit from white privilege as evidence by the fact that they are poor or are disadvantaged in some other way. Privilege isn't about being given a head start, its about not being in the race at all.

This is yet another example of Watson praising Holmes and another example of his perspective shifting into tight, sharp words in order to give Holmes a layer of sophistication and intelligence beyond the average person.

This is yet another example of Watson praising Holmes and another example of his perspective shifting into tight, sharp words in order to give Holmes a layer of sophistication and intelligence beyond the average person.

Not objecting to rule, but to rule by parasites

That theorized division between those who tell the computers what to do and those who are told by computers what to do

The difference between the "robot" and the man is only what is left out, what has been optimized away

Here, Stokes is described possessing physical traits that derive from normal beauty standards, particularly by being a short and larger man, almost squared. Allen chooses to place him as the hero of the story and therefore actively prescribes to the dismantling of these structures. This could speak to a wider-ranging audience compared to pieces with detectives that are chronically conventionally attractive.

Deeper significance of naming of a bone

Author Response

Reviewer 1 (Public Review):

In this paper, Reato, Steinfeld et al. investigate a question that has long puzzled neuroscientists: what features of ongoing brain activity predict trial-to-trial variability in responding to the same sensory stimuli? They record spiking activity in the auditory cortex of head-fixed mice as the animals performed a tone frequency discrimination task. They then measure both overall activity and the synchronization between neurons, and link this ’baseline state’ (after removing slow drifts) of cortex to decision accuracy. They find that cortical state fluctuations only affect subsequent evoked responses and choice behavior after errors. This indicates that it’s important to take into account the behavioral context when examining the effects of neural state on behavior.

Strengths of this work are the clear and beautiful presentation of the figures, and the careful consideration of the temporal properties of behavioral and neural signals. Indeed, slowly drifting signals are tricky as many authors have recently addressed (e.g. Ashwood, Gupta, Harris). The authors are well aware of the difficulties in correlating different signals with temporal and cross-correlation (such as in their ’epoch hypothesis’). To disentangle such slow trends from more short-lived state fluctuations, they remove the impact of the past 10 trials and continue their analyses with so-called ’innovations’ (a term that is unusual, and may more simply be replaced with ’residuals’).

The terms ‘innovations’ and ‘residuals’ are sometimes used interchangeably. We used innovations because that’s how they were introduced in the signal processing literature (i.e., Kailath, T (1968). ”An innovations approach to least-squares estimation–Part I: Linear filtering in additive white noise.” IEEE transactions on automatic control). We try to be explicit in the text about the formal definition of this quantity, to avoid problems with terminology.

I do wonder if this throws out the baby with the bathwater. If the concern is statistical confound, the ’session permutation’ method (Harris) may be better suited. If the concern is that short-term state fluctuations are more behaviorally relevant (and obscured by slow drifts), then why are the results with raw signals in the supplement (Suppfig 8) so similar?

The concern was statistical confound, although this concern is ameliorated when using a mixed model approach and focusing on fixed effects. However, our approach allowed us to assess the relative importance of slow versus single-trial timescales in the predictive relationship between cortical state (and arousal) and behavior, revealing that, in the conditions of our experiment, only the fast timescales are relevant. Because of this, we think that the baby wasn’t thrown out with the bathwater as, qualitatively, no new phenomenology was revealed when the slow components of the signals were included. In hindsight, it is true that the results we obtained suggest that maybe the effort we made to isolate the fast component of the signals was unjustified. However, this can only be known after both options have been tried, as we did. Moreover, we started using innovations based on the results in Figure 2 where, as we show, the use of innovations does make a difference, even at the level of fixed effects in a mixed model. We agree that we could have used the ‘session permutation’ method, but given the depth at which we have explored this issue in the manuscript already, and the clarity of the results, we think that adding a third method would only make reading the manuscript more difficult without adding any substantially new content.

While the authors are correct that go-nogo tasks have drawbacks in dissociating sensitivity from response bias, they only cursorily review the literature on 2AFC tasks and cortical state. In particular, it would be good to discuss how the specific method - spikes, EEG (Waschke), widefield (Jacobs) and algorithm for quantifying synchronization may affect outcomes. How do these population-based measures of cortical state relate to those described extensively with slightly different signals, notably LFP or EEG in humans (e.g. work by Saskia Haegens, Niko Busch, reviewed in https://doi.org/10.1016/j.tics.2020.05.004)? This review also points out the importance of moving beyond simple measures of accuracy and using SDT, which would be an interesting improvement for this paper too.

We thank the reviewer for pointing us towards the oscillation-based brain-state literature in humans. We have expanded the paragraph in the discussion where we compare our results with previous work in order to (i) elaborate on the literature on 2AFC tasks, (ii) specifically address the literature linking alpha power in the pre-stimulus baseline and psychophysical performance, and (iii) mention different methods for assessing desynchronization. Our view is that absence of lowfrequency power is a robust measure which can be assessed using different types of signals (spikes, imaging, LFP, EEG). That said, the relationship between desynchronization and behavior appears subtle and variable, specially within discrimination paradigms. These issues are discussed in the paragraph starting in line 527 in the text.

Regarding the use of SDT, we had already established that our main finding could be expressed as a significant interaction between FR/Synch and the stimulus-strength regressor, when predicting choice after errors (Supplementary Fig. 4A in original manuscript), which is equivalent to a cortical state-dependent increase in d′ after the mice made a mistake. In order to consider a possible effect of cortical state on the ‘criterion’ (i.e., an effect on the bias of the mice towards either response spout), we re-run this GLMM but adding the cortical state regressors as main effects. The results show that the FR-Synch predictor is only significantly greater than zero as an interaction after errors (p = 0.0025). As a main effect, it’s not significantly different from zero neither after errors (p = 0.28), nor after correct trials (p = 0.97). We have included this analysis as Figure 3-figure supplement 1B (replacing the previous Supplementary Fig. 4A) and commented on them in the text (lines 222-225).

Reviewer 2 (Public Review):

The relationship between measures of brain state, behavioral state, and performance has long been speculated to be relatively simple - with arousal and engagement reflecting EEG desynchronization and improved performance associated with increases in engagement and attention. The present study demonstrates that the outcome of the previous trial, specifically a miss, allows these associations to be seen - while a correct response appears less likely to do so. This is an interesting advance in our understanding of the relationship between brain state, behavioral state, and performance.

This is probably just a typo, but we would like to clarify that the relevant outcome in the previous trial is not a miss, but an incorrect choice in an otherwise valid trial (i.e., a trial with a response within the allowed response window).

While the study is well done, the results are likely to be specific to their trial structure and states exhibited by the mice. To examine the full range of arousal states, it needs to be demonstrated that animals are varying between near-sleep (e.g. drowsiness) and high-alertness such as in rapid running. The fact that the trials occurred rapidly means that the physiological and neural variables associated with each trial will overlap with upcoming trials - it takes a mouse more than a few seconds to relax from a previous miss or hit, for example. Spreading the rapidity of the trials out would allow for a broader range of states to be examined, and perhaps less cross-talk between adjacent trials. The interpretation of the results, therefore, must be taken in light of the trial structure and the states exhibited by the mice.

We thank the reviewer for the positive assessment of our work and also for raising this point in particular. This motivated us to look more carefully at this issue, with results that, we believe, strengthen our study.

Reviewer #1 (Public Review):

In this paper, Reato, Steinfeld et al. investigate a question that has long puzzled neuroscientists: what features of ongoing brain activity predict trial-to-trial variability in responding to the same sensory stimuli? They record spiking activity in the auditory cortex of head-fixed mice as the animals performed a tone frequency discrimination task. They then measure both overall activity and the synchronization between neurons, and link this 'baseline state' (after removing slow drifts) of cortex to decision accuracy. They find that cortical state fluctuations only affect subsequent evoked responses and choice behavior after errors. This indicates that it's important to take into account the behavioral context when examining the effects of neural state on behavior.

Strengths of this work are the clear and beautiful presentation of the figures, and the careful consideration of the temporal properties of behavioral and neural signals. Indeed, slowly drifting signals are tricky as many authors have recently addressed (e.g. Ashwood, Gupta, Harris). The authors are well aware of the difficulties in correlating different signals with temporal and cross-correlation (such as in their 'epoch hypothesis'). To disentangle such slow trends from more short-lived state fluctuations, they remove the impact of the past 10 trials and continue their analyses with so-called 'innovations' (a term that is unusual, and may more simply be replaced with 'residuals').

I do wonder if this throws out the baby with the bathwater. If the concern is statistical confound, the 'session permutation' method (Harris) may be better suited. If the concern is that short-term state fluctuations are more behaviorally relevant (and obscured by slow drifts), then why are the results with raw signals in the supplement (Suppfig 8) so similar?

While the authors are correct that go-nogo tasks have drawbacks in dissociating sensitivity from response bias, they only cursorily review the literature on 2AFC tasks and cortical state. In particular, it would be good to discuss how the specific method - spikes, EEG (Waschke), widefield (Jacobs) and algorithm for quantifying synchronization may affect outcomes. How do these population-based measures of cortical state relate to those described extensively with slightly different signals, notably LFP or EEG in humans (e.g. work by Saskia Haegens, Niko Busch, reviewed in https://doi.org/10.1016/j.tics.2020.05.004)? This review also points out the importance of moving beyond simple measures of accuracy and using SDT, which would be an interesting improvement for this paper too.

From LAWLER 251: Wilde added this paragraph on two handwritten pages to add to the typescript.

this is a straw man scratching their head and sneezing some words..

From LAWLER 190: Following this sentence, Wilde originally had the following passage: Most modern portrait painting comes under the head of elegant fiction or if it aims at realism, gives one something between a caricature and a photograph. But this was different. It had all the mystery of life, and all the mystery of beauty. Within the world, as men know it, there is a finer world that only artists know of--artists or those to whom the temperament of the artist has been given. Creation within creation--that is what Basil Hallward named it, that is what he had attained to."

From LAWLER 178: Wilde made several stylistic changes in the preceding eight lines in the 1891 text.

ZABROUSKI: Changed to "Hallward shook his head" in 1891.

instead, consider; The updated ...

starting the sentence with New felt awkward and head to read it a couple times for it to flow

Uses the CEO and general manager of KSPDCL as an interview source. This source is credible as they are the head of the organization and can give reliable information about future goals as well as reasons.

How does it work? It seems like the trailing sheild would cause the magnetic field lines to be less perpendicular, and hence less effective. Maybe it's because the increase in field strength outweighs the decrease in perpendicular projection.

If the reason for PMA being better is simply that the write head is better, then there would be no advantage for out of plane spin torques.

The quote raises questions about the nature of freedom and when it will truly arrive for the woman, as she wonders if she will ever be able to answer the question, "Can I live?" This uncertainty and the longing for a more genuine sense of freedom underscore the complex nature of American womanhood, as women throughout history have grappled with questions of freedom, rights, and equality.

For alteplase, a CT of the head should be completed, as brain tumor, and hemorrhagic stroke are contraindications.

Also first quotes from World Leaders that I have found in articles. I find it interesting that other articles didn't do this because it gives more info about how other people feel and how many people are affected.

Why did he not do this earlier or before the crash? The Titanic was too huge to turn in that quick of a time. They also should have taken the hit head-on because the bow of a ship is made to take hits like that. But, I guess that they wanted to try to avoid the hit fully if possible. But knowing what happens, that did not happen.

Having a question that you are focused on rather than choosing one from the top of your head can make it easier for you to do tons of research

This depicts the value of conflict. This article states these comments because the issue on the table is a large conflict. They show evidence of this with the prominence of another celebrity mentioning it, and with the academy not allowing reporters to ask questions about it.

To write the best story, sometimes you must get out of your own head by Dale Keiger

PESOS example: https://niemanstoryboard.org/stories/longform-narrative-structure-writing-process-profiles/