eLife Assessment

In this detailed study, Cohen and Ben-Shaul characterized Accessory Olfactory Bulb (AOB) cell responses to various conspecific urine samples in female mice across the estrous cycle. The authors found that AOB cell responses varied depending on the strain and sex of the sample, but no clear differences were observed between estrous and non-estrous females. These findings provide convincing evidence that the AOB functions as a stable sensory relay, without directly modulating responses based on reproductive state, which supports the role of downstream brain regions in integrating reproductive state. Overall, this study provides valuable insights for researchers in the fields of olfaction and social neuroscience.

Reviewer #1 (Public review):

Summary:

In this detailed study, Cohen and Ben-Shaul characterized the AOB cell responses to various conspecific urine samples in female mice across the estrous cycle. The authors found that AOB cell responses vary with the strains and sexes of the samples. Between estrous and non-estrous females, no clear or consistent difference in responses was found. The cell response patterns, as measured by the distance between pairs of stimuli, are largely stable. When some changes do occur, they are not consistent across strains or male status. The authors concluded that AOB detects the signals without interpreting them. Overall, this study will provide useful information for scientists in the field of olfaction.

Strengths:

The study uses electrophysiological recording to characterize the responses of AOB cells to various urines in female mice. AOB recording is not trivial as it requires activation of VNO pump. The team uses a unique preparation to activate the VNO pump with electric stimulation, allowing them to record AOB cell responses to urines in anesthetized animals. The study comprehensively described the AOB cell responses to social stimuli and how the responses vary (or not) with features of the urine source and the reproductive state of the recording females. The dataset could be a valuable resource for scientists in the field of olfaction.

Weaknesses:

(1) The figures could be better labeled.

(2) For Figure 2E, please plot the error bar. Are there any statistics performed to compare the mean responses?

(3) For Figure 2D, it will be more informative to plot the percentage of responsive units.

(4) Could the similarity in response be explained by the similarity in urine composition? The study will be significantly strengthened by understanding the "distance" of chemical composition in different urine.

(5) If it is not possible for the authors to obtain these data first-hand, published data on MUPs and chemicals found in these urines may provide some clues.

(6) It is not very clear to me whether the female overrepresentation is because there are truly more AOB cells that respond to females than males or because there are only two female samples but 9 male samples.

(7) If the authors only select two male samples, let's say ICR Naïve and ICR DOM, combine them with responses to two female samples, and do the same analysis as in Figure 3, will the female response still be overrepresented?

(8) In Figure 4B and 4C, the pairwise distance during non-estrus is generally higher than that during estrus, although they are highly correlated. Does it mean that the cells respond to different urines more distinctively during diestrus than in estrus?

(9) The correlation analysis is not entirely intuitive when just looking at the figures. Some sample heatmaps showing the response differences between estrous states will be helpful.

Reviewer #2 (Public review):

Summary:

Many aspects of the study are carefully done, and in the grand scheme this is a solid contribution. I have no "big-picture" concerns about the approach or methodology. However, in numerous places the manuscript is unnecessarily vague, ambiguous, or confusing. Tightening up the presentation will magnify their impact.

Strengths:

(1) The study includes urine donors from males of three strains each with three social states, as well as females in two states. This diversity significantly enhances their ability to interpret their results.

(2) Several distinct analyses are used to explore the question of whether AOB MCs are biased towards specific states or different between estrus and non-estrus females. The results of these different analyses are self-reinforcing about the main conclusions of the study.

(3) The presentation maintains a neutral perspective throughout while touching on topics of widespread interest.

Weaknesses:

(1) Introduction:<br /> The discussion of the role of the VNS and preferences for different male stimuli should perhaps include Wysocki and Lepri 1991

(2) Results:<br /> a) Given the 20s gap between them, the distinction between sample application and sympathetic nerve trunk stimulation needs to be made crystal clear; in many places, "stimulus application" is used in places where this reviewer suspects they actually mean sympathetic nerve trunk stimulation.<br /> b) There appears to be a mismatch between the discussion of Figure 3 and its contents. Specifically, there is an example of an "adjusted" pattern in 3A, not 3B.<br /> c) The discussion of patterns neglects to mention whether it's possible for a neuron to belong to more than one pattern. For example, it would seem possible for a neuron to simultaneously fit the "ICR pattern" and the "dominant adjusted pattern" if, e.g., all ICR responses are stronger than all others, but if simultaneously within each strain the dominant male causes the largest response.

(3) Discussion:<br /> a) The discussion of chemical specificity in urine focuses on volatiles and MUPs (citation #47), but many important molecules for the VNS are small, nonvolatile ligands. For such molecules, the corresponding study is Fu et al 2015.<br /> b) "Following our line of reasoning, this scarcity may represent an optimal allocation of resources to separate dominant from naïve males": 1 unit out of 215 is roughly consistent with a single receptor. Surely little would be lost if there could be more computational capacity devoted to this important axis than that? It seems more likely that dominance is computed from multiple neuronal types with mixed encoding.

(4) Methods:<br /> a) Male status, "were unambiguous in most cases": is it possible to put numerical estimates on this? 55% and 99% are both "most," yet they differ substantially in interpretive uncertainty.<br /> b) Surgical procedures and electrode positioning: important details of probes are missing (electrode recording area, spacing, etc).<br /> c) Stimulus presentation procedure: Are stimuli manually pipetted or delivered by apparatus with precise timing?<br /> d) Data analysis, "we applied more permissive criteria involving response magnitude": it's not clear whether this is what's spelled out in the next paragraph, or whether that's left unspecified. In either case, the next paragraph appears to be about establishing a noise floor on pattern membership, not a "permissive criterion."<br /> e) Data analysis, method for assessing significance: there's a lot to like about the use of pooling to estimate the baseline and the use of an ANOVA-like test to assess unit responsiveness.<br /> But:<br /> i) for a specific stimulus, at 4 trials (the minimum specified in "Stimulus presentation procedure") kruskalwallis is questionable. They state that most trials use 5, however, and that should be okay.<br /> ii) the methods statement suggests they are running kruskalwallis individually for each neuron/stimulus, rather than once per neuron across all stimuli. With 11 stimuli, there is a substantial chance of a false-positive if they used p < 0.05 to assess significance. (The actual threshold was unstated.) Were there any multiple comparison corrections performed? Or did they run kruskalwallis on the neuron, and then if significant assess individual stimuli? (Which is a form of multiple-comparisons correction.)

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this detailed study, Cohen and Ben-Shaul characterized the AOB cell responses to various conspecific urine samples in female mice across the estrous cycle. The authors found that AOB cell responses vary with the strains and sexes of the samples. Between estrous and non-estrous females, no clear or consistent difference in responses was found. The cell response patterns, as measured by the distance between pairs of stimuli, are largely stable. When some changes do occur, they are not consistent across strains or male status. The authors concluded that AOB detects the signals without interpreting them. Overall, this study will provide useful information for scientists in the field of olfaction.

Strengths:

The study uses electrophysiological recording to characterize the responses of AOB cells to various urines in female mice. AOB recording is not trivial as it requires activation of VNO pump. The team uses a unique preparation to activate the VNO pump with electric stimulation, allowing them to record AOB cell responses to urines in anesthetized animals. The study comprehensively described the AOB cell responses to social stimuli and how the responses vary (or not) with features of the urine source and the reproductive state of the recording females. The dataset could be a valuable resource for scientists in the field of olfaction.

Weaknesses:

(1) The figures could be better labeled.

Figures will be revised to provide more detailed labeling.

(2) For Figure 2E, please plot the error bar. Are there any statistics performed to compare the mean responses?

We did not perform statistical comparisons (between the mean rates across the population). We will add this analysis and the corresponding error bars. 

(3) For Figure 2D, it will be more informative to plot the percentage of responsive units.

We will do it.

(4) Could the similarity in response be explained by the similarity in urine composition? The study will be significantly strengthened by understanding the "distance" of chemical composition in different urine.

We agree. As we wrote in the Discussion: “Ultimately, lacking knowledge of the chemical space associated with each of the stimuli, this and all the other ideas developed here remain speculative.”

A better understanding of the chemical distance is an important aspect that we aim to include in our future studies. However, this is far from trivial, as it is not chemical distance per se (which in itself is hard to define), but rather the “projection” of chemical space on the vomeronasal receptor neurons array. That is, knowledge of the chemical composition of the stimuli, lacking full knowledge of which molecules are vomeronasal system ligands, will only provide a partial picture. Despite these limitations, this is an important analysis which we would have done had we access to this data.

(5) If it is not possible for the authors to obtain these data first-hand, published data on MUPs and chemicals found in these urines may provide some clues.

Measurements about some classes of molecules may be found for some of the stimuli that we used here, but not for all. We are not aware of any single dataset that contains this information for any type of molecules (e.g., MUPs) across the entire stimulus set that we have used. More generally, pooling results from different studies has limited validity because of the biological and technical variability across studies. In order to reliably interpret our current recordings, it would be necessary to measure the urinary content of the very same samples that were used for stimulation. Unfortunately, we are not able to conduct this analysis at this stage.

(6) It is not very clear to me whether the female overrepresentation is because there are truly more AOB cells that respond to females than males or because there are only two female samples but 9 male samples.

It is true that the number of neurons fulfilling each of the patterns depends on the number of individual stimuli that define it. However, our measure of “over-representation” aims to overcome this bias, by using bootstrapping to reveal if the observed number of patterns is larger than expected by chance. We also note that more generally, the higher frequency of responses to female, as compared to male stimuli, is obtained in other studies by others and by us, also when the number of male and female stimuli is matched (e.g., Bansal et al BMC Biol 2021, Ben-Shaul et al, PNAS 2010, Hendrickson et al, JNS, 2008).

(7) If the authors only select two male samples, let's say ICR Naïve and ICR DOM, combine them with responses to two female samples, and do the same analysis as in Figure 3, will the female response still be overrepresented?

We believe that the answer is positive, but we can, and will perform this analysis to check.

(8) In Figure 4B and 4C, the pairwise distance during non-estrus is generally higher than that during estrus, although they are highly correlated. Does it mean that the cells respond to different urines more distinctively during diestrus than in estrus?

This is an important observation. For the Euclidean distance there might be a simple explanation as the distance depends on the number of units (and there are more units recorded in non-estrus females). However, this simple explanation does not hold for the correlation distance. A higher distance implies higher discrimination during the non-estrus stage, but our other analyses of sparseness and the selectivity indices do not support this idea. We note that absolute values of distance measures should generally be interpreted cautiously, as they may depend on multiple factors including sample size. Also, a small number of non-selective units could increase the correlation in responses among stimuli, and thus globally shift the distances. For these reasons, we focus on comparisons, rather than the absolute values of the correlation distances. In the revised manuscript, we will note and discuss this important observation.

(9) The correlation analysis is not entirely intuitive when just looking at the figures. Some sample heatmaps showing the response differences between estrous states will be helpful.

If we understand correctly, the idea is to show the correlation matrices from which the values in 4B and 4C are taken. We can and will do this, probably as a supplementary figure.

Reviewer #2 (Public review):

Summary:

Many aspects of the study are carefully done, and in the grand scheme this is a solid contribution. I have no "big-picture" concerns about the approach or methodology. However, in numerous places the manuscript is unnecessarily vague, ambiguous, or confusing. Tightening up the presentation will magnify their impact.

We will revise the text with the aim of tightening the presentation.

Strengths:

(1) The study includes urine donors from males of three strains each with three social states, as well as females in two states. This diversity significantly enhances their ability to interpret their results.

(2) Several distinct analyses are used to explore the question of whether AOB MCs are biased towards specific states or different between estrus and non-estrus females. The results of these different analyses are self-reinforcing about the main conclusions of the study.

(3) The presentation maintains a neutral perspective throughout while touching on topics of widespread interest.

Weaknesses:

(1) Introduction:

The discussion of the role of the VNS and preferences for different male stimuli should perhaps include Wysocki and Lepri 1991

Agreed. we will refer to this work in our discussion.

(2) Results:

a) Given the 20s gap between them, the distinction between sample application and sympathetic nerve trunk stimulation needs to be made crystal clear; in many places, "stimulus application" is used in places where this reviewer suspects they actually mean sympathetic nerve trunk stimulation.

In this study, we have considered both responses that are triggered by sympathetic trunk activation, and those that occur (as happens in some preparations) immediately following stimulus application (and prior to nerve trunk stimulation). An example of the latter Is provided in the second unit shown in Figure 1D (and this is indicated also in the figure legend). In our revision, we will further clarify this confusing point.

b) There appears to be a mismatch between the discussion of Figure 3 and its contents. Specifically, there is an example of an "adjusted" pattern in 3A, not 3B.

True. Thanks for catching this error. We will correct this.

c) The discussion of patterns neglects to mention whether it's possible for a neuron to belong to more than one pattern. For example, it would seem possible for a neuron to simultaneously fit the "ICR pattern" and the "dominant adjusted pattern" if, e.g., all ICR responses are stronger than all others, but if simultaneously within each strain the dominant male causes the largest response.

This is true. In the legend to Figure 3B, we actually write: “A neuron may fulfill more than one pattern and thus may appear in more than one row.”, but we will discuss this point in the main text as well.

(3) Discussion:

a) The discussion of chemical specificity in urine focuses on volatiles and MUPs (citation #47), but many important molecules for the VNS are small, nonvolatile ligands. For such molecules, the corresponding study is Fu et al 2015.

We fully agree. We will expand our discussion and refer to Fu et al.

b) "Following our line of reasoning, this scarcity may represent an optimal allocation of resources to separate dominant from naïve males": 1 unit out of 215 is roughly consistent with a single receptor. Surely little would be lost if there could be more computational capacity devoted to this important axis than that? It seems more likely that dominance is computed from multiple neuronal types with mixed encoding.

We agree, and we are not claiming that dominance, nor any other feature, is derived using dedicated feature selective neurons.  Our discussion of resource allocation is inevitably speculative. Our main point in this context is that a lack of overrepresentation does not imply that a feature is not important. We will revise our discussion to better clarify our view of this issue.

(4) Methods:

a) Male status, "were unambiguous in most cases": is it possible to put numerical estimates on this? 55% and 99% are both "most," yet they differ substantially in interpretive uncertainty.

This sentence is actually misleading and irrelevant. Ambiguous cases were not considered as dominant for urine collection. We only classified mice as dominant if they were “won” in the tube test and exhibited dominant behavior in the subsequent observation period in the cage. We will correct the wording in the revised manuscript.

b) Surgical procedures and electrode positioning: important details of probes are missing (electrode recording area, spacing, etc).

True. We will add these details.

c) Stimulus presentation procedure: Are stimuli manually pipetted or delivered by apparatus with precise timing?

They are delivered manually. We will clarify this as well.

d) Data analysis, "we applied more permissive criteria involving response magnitude": it's not clear whether this is what's spelled out in the next paragraph, or whether that's left unspecified. In either case, the next paragraph appears to be about establishing a noise floor on pattern membership, not a "permissive criterion."

True, the next paragraph is not the explanation for the more permissive criteria. The more permissive criteria involving response magnitude are actually those described in Figure 3A and 3B. The sentence that was quoted above merely states that before applying those criteria, we had also searched for patterns defined by binary designation of neurons as responsive, or not responsive, to each of the stimuli (this is directly related to the next comment below). Using those binary definitions, we obtained a very small number of neurons for each pattern and thus decided to apply the approach actually used and described in the manuscript.

e) Data analysis, method for assessing significance: there's a lot to like about the use of pooling to estimate the baseline and the use of an ANOVA-like test to assess unit responsiveness.

But:

i) for a specific stimulus, at 4 trials (the minimum specified in "Stimulus presentation procedure") kruskalwallis is questionable. They state that most trials use 5, however, and that should be okay.

The number of cases with 4 trials is truly a minority, and we will provide the exact numbers in our revision.

ii) the methods statement suggests they are running kruskalwallis individually for each neuron/stimulus, rather than once per neuron across all stimuli. With 11 stimuli, there is a substantial chance of a false-positive if they used p < 0.05 to assess significance. (The actual threshold was unstated.) Were there any multiple comparison corrections performed? Or did they run kruskalwallis on the neuron, and then if significant assess individual stimuli? (Which is a form of multiple-comparisons correction.)

First, we indeed failed to mention that our criterion was 0.05. We will correct that in our revision. We did not apply any multiple comparison measures. We consider each neuron-stimulus pair as an independent entity, and we are aware that this leads to a higher false positive rate. On the other hand, applying multiple comparisons would be problematic, as we do not always use the same number of stimuli in different studies. Applying multiple comparison corrections would lead to different response criteria across different studies. Notably, most, if not all, of our conclusions involve comparisons across conditions, and for this purpose we think that our procedure is valid. We do not attach any special meaning to the significance threshold, but rather think of it as a basic criterion that allows us to exclude non-responsive neurons, and to compare frequencies of neurons that fulfill this criterion.

duty to obey law comes from fairness: different from promise-keeping, because it's extended to all citizens, as a moral ground to everyone, not just to those who choose to participate in the politics SO, you should obey the law, because it would be unfair not to; you owe your fellow citizens "if they all comply and you benefit, it is unfair if you benefit without complying"

duty to obey the law comes from promise-keeping: citizens promise to obey the law in exchange for protection & other benefits (kind of a "social contract" like in Rawls' theory)

eLife Assessment

Pinho et al use in vivo calcium imaging and chemogenetic approaches to examine the involvement of hippocampal sub-regions across the different stages of a sensory preconditioning task in mice. They find convincing evidence for sensory preconditioning in male mice. They also find that, in these mice, CaMKII-positive neurons in the dorsal hippocampus: (1) encode the audio-visual association that forms in stage 1 of the task, and (2) retrieve/express sensory preconditioned fear to the auditory stimulus at test. These findings are supported by evidence that ranges from incomplete to convincing. The study will be valuable to researchers in the field of learning and memory.

Reviewer #1 (Public review):

Summary:

The study by Pinho et al. presents a novel behavioral paradigm for investigating higher-order conditioning in mice. The authors developed a task that creates associations between light and tone sensory cues, driving mediated learning. They observed sex differences in task acquisition, with females demonstrating faster-mediated learning compared to males. Using fiber photometry and chemogenetic tools, the study reveals that the dorsal hippocampus (dHPC) plays a central role in encoding mediated learning. These findings are crucial for understanding how environmental cues, which are not directly linked to positive/negative outcomes, contribute to associative learning. Overall, the study is well-designed, with robust results, and the experimental approach aligns with the study's objectives.

Strengths:

(1) The authors develop a robust behavioral paradigm to examine higher-order associative learning in mice.

(2) They discover a sex-specific component influencing mediated learning, with females exhibiting enhanced learning abilities.

(3) Using fiber photometry and chemogenetic techniques, the authors identify the dorsal hippocampus but not the ventral hippocampus, which plays a crucial for encoding mediated learning.

Weaknesses:

(1) The study would be strengthened by further elaboration on the rationale for investigating specific cell types within the hippocampus.

(2) The analysis of photometry data could be improved by distinguishing between early and late responses, as well as enhancing the overall presentation of the data.

(3) The manuscript would benefit from revisions to improve clarity and readability.

Reviewer #2 (Public review):

Summary:

Pinho et al. developed a new auditory-visual sensory preconditioning procedure in mice and examined the contribution of the dorsal and ventral hippocampus to learning in this task. Using photometry they observed activation of the dorsal and ventral hippocampus during sensory preconditioning and conditioning. Finally, the authors combined their sensory preconditioning task with DREADDs to examine the effect of inhibiting specific cell populations (CaMKII and PV) in the DH on the formation and retrieval/expression of mediated learning.

Strengths:

The authors provide one of the first demonstrations of auditory-visual sensory preconditioning in male mice. Research on the neurobiology of sensory preconditioning has primarily used rats as subjects. The development of a robust protocol in mice will be beneficial to the field, allowing researchers to take advantage of the many transgenic mouse lines. Indeed, in this study, the authors take advantage of a PV-Cre mouse line to examine the role of hippocampal PV cells in sensory preconditioning.

Weaknesses:

(1) The authors report that sensory preconditioning was observed in both male and female mice. However, their data only supports sensory preconditioning in male mice. In female mice, both paired and unpaired presentations of the light and tone in stage 1 led to increased freezing to the tone at test. In this case, fear to the tone could be attributed to factors other than sensory preconditioning, for example, generalization of fear between the auditory and visual stimulus.

(2) In the photometry experiment, the authors report an increase in neural activity in the hippocampus during both phase 1 (sensory preconditioning) and phase 2 (conditioning). In the subsequent experiment, they inhibit neural activity in the DH during phase 1 (sensory preconditioning) and the probe test, but do not include inhibition during phase 2 (conditioning). It was not clear why they didn't carry forward investigating the role of the hippocampus during phase 2 conditioning. Sensory preconditioning could occur due to the integration of the tone and shock during phase two, or retrieval and chaining of the tone-light-shock memories at test. These two possibilities cannot be differentiated based on the data. Given that we do not know at which stage the mediate learning is occurring, it would have been beneficial to additionally include inhibition of the DH during phase 2.

(3) In the final experiment, the authors report that inhibition of the dorsal hippocampus during the sensory preconditioning phase blocked mediated learning. While this may be the case, the failure to observe sensory preconditioning at test appears to be due more to an increase in baseline freezing (during the stimulus off period), rather than a decrease in freezing to the conditioned stimulus. Given the small effect, this study would benefit from an experiment validating that administration of J60 inhibited DH cells. Further, given that the authors did not observe any effect of DREADD inhibition in PV cells, it would also be important to validate successful cellular silencing in this protocol.

Reviewer #3 (Public review):

Summary:

Pinho et al. investigated the role of the dorsal vs ventral hippocampus and the gender differences in mediated learning. While previous studies already established the engagement of the hippocampus in sensory preconditioning, the authors here took advantage of freely-moving fiber photometry recording and chemogenetics to observe and manipulate sub-regions of the hippocampus (dorsal vs. ventral) in a cell-specific manner. The authors first found sex differences in the preconditioning phase of a sensory preconditioning procedure, where males required more preconditioning training than females for mediating learning to manifest, and where females displayed evidence of mediated learning even when neutral stimuli were never presented together within the session.

After validation of a sensory preconditioning procedure in mice using light and tone neutral stimuli and a mild foot shock as the unconditioned stimulus, the authors used fiber photometry to record from all neurons vs. parvalbumin_positive_only neurons in the dorsal hippocampus or ventral hippocampus of male mice during both preconditioning and conditioning phases. They found increased activity of all neurons, as well as PV+_only neurons in both sub-regions of the hippocampus during both preconditioning and conditioning phases. Finally, the authors found that chemogenetic inhibition of CaMKII+ neurons in the dorsal, but not ventral, hippocampus specifically prevented the formation of an association between the two neutral stimuli (i.e., light and tone cues), but not the direct association between the light cue and the mild foot shock. This set of data: (1) validates the mediated learning in mice using a sensory preconditioning protocol, and stresses the importance of taking sex effect into account; (2) validates the recruitment of dorsal and ventral hippocampi during preconditioning and conditioning phases; and (3) further establishes the specific role of CaMKII+ neurons in the dorsal but not ventral hippocampus in the formation of an association between two neutral stimuli, but not between a neutral-stimulus and a mild foot shock.

Strengths:

The authors developed a sensory preconditioning procedure in mice to investigate mediated learning using light and tone cues as neutral stimuli, and a mild foot shock as the unconditioned stimulus. They provide evidence of a sex effect in the formation of light-cue association. The authors took advantage of fiber-photometry and chemogenetics to target sub-regions of the hippocampus, in a cell-specific manner and investigate their role during different phases of a sensory conditioning procedure.

Weaknesses:

The authors went further than previous studies by investigating the role of sub-regions of the hippocampus in mediated learning, however, there are several weaknesses that should be noted:

(1) This work first validates mediated learning in a sensory preconditioning procedure using light and tone cues as neutral stimuli and a mild foot shock as the unconditioned stimulus, in both males and females. They found interesting sex differences at the behavioral level, but then only focused on male mice when recording and manipulating the hippocampus. The authors do not address sex differences at the neural level.

(2) As expected in fear conditioning, the range of inter-individual differences is quite high. Mice that didn't develop a strong light-->shock association, as evidenced by a lower percentage of freezing during the Probe Test Light phase, should manifest a low percentage of freezing during the Probe Test Tone phase. It would interesting to test for a correlation between the level of freezing during mediated vs test phases.

(3) The use of a synapsin promoter to transfect neurons in a non-specific manner does not bring much information. The authors applied a more specific approach to target PV+ neurons only, and it would have been more informative to keep with this cell-specific approach, for example by looking also at somatostatin+ inter-neurons.

(4) The authors observed event-related Ca2+ transients on hippocampal pan-neurons and PV+ inter-neurons using fiber photometry. They then used chemogenetics to inhibit CaMKII+ hippocampal neurons, which does not logically follow. It does not undermine the main finding of CaMKII+ neurons of the dorsal, but not ventral, hippocampus being involved in the preconditioning, but not conditioning, phase. However, observing CaMKII+ neurons (using fiber photometry) in mice running the same task would be more informative, as it would indicate when these neurons are recruited during different phases of sensory preconditioning. Applying then optogenetics to cancel the observed event-related transients (e.g., during the presentation of light and tone cues, or during the foot shock presentation) would be more appropriate.

(5) Probe tests always start with the "Probe Test Tone", followed by the "Probe Test Light". "Probe Test Tone" consists of an extinction session, which could affect the freezing response during "Probe Test Light" (e.g., Polack et al. (http://dx.doi.org/10.3758/s13420-013-0119-5)). Preferably, adding a group of mice with a Probe Test Light with no Probe Test Tone could help clarify this potential issue. The authors should at least discuss the possibility that the tone extinction session prior to the "Probe Test Light" could have affected the freezing response to the light cue.

Reviewer #4 (Public review):

Summary

Pinho et al use in vivo calcium imaging and chemogenetic approaches to examine the involvement of hippocampal sub-regions across the different stages of a sensory preconditioning task in mice. They find clear evidence for sensory preconditioning in male but not female mice. They also find that, in the male mice, CaMKII-positive neurons in the dorsal hippocampus: (1) encode the audio-visual association that forms in stage 1 of the task, and (2) retrieve/express sensory preconditioned fear to the auditory stimulus at test. These findings are supported by evidence that ranges from incomplete to convincing. They will be valuable to researchers in the field of learning and memory.

Abstract

Please note that sensory preconditioning doesn't require the stage 1 stimuli to be presented repeatedly or simultaneously.

"Finally, we combined our sensory preconditioning task with chemogenetic approaches to assess the role of these two hippocampal subregions in mediated learning."<br /> This implies some form of inhibition of hippocampal neurons in stage 2 of the protocol, as this is the only stage of the protocol that permits one to make statements about mediated learning. However, it is clear from what follows that the authors interrogate the involvement of hippocampal sub-regions in stages 1 and 3 of the protocol - not stage 2. As such, most statements about mediated learning throughout the paper are potentially misleading (see below for a further elaboration of this point). If the authors persist in using the term mediated learning to describe the response to a sensory preconditioned stimulus, they should clarify what they mean by mediated learning at some point in the introduction. Alternatively, they might consider using a different phrase such as "sensory preconditioned responding".

Introduction

"Low-salience" is used to describe stimuli such as tone, light, or odour that do not typically elicit responses that are of interest to experimenters. However, a tone, light, or odour can be very salient even though they don't elicit these particular responses. As such, it would be worth redescribing the "low-salience" stimuli in some other terms.

"These higher-order conditioning processes, also known as mediated learning, can be captured in laboratory settings through sensory preconditioning procedures2,6-11."<br /> Higher-order conditioning and mediated learning are not interchangeable terms: e.g., some forms of second-order conditioning are not due to mediated learning. More generally, the use of mediated learning is not necessary for the story that the authors develop in the paper and could be replaced for accuracy and clarity. E.g., "These higher-order conditioning processes can be studied in the laboratory using sensory preconditioning procedures2,6-11."

In reference to Experiment 2, it is stated that: "However, when light and tone were separated on time (Unpaired group), male mice were not able to exhibit mediated learning response (Figure 2B) whereas their response to the light (direct learning) was not affected (Figure 2D). On the other hand, female mice still present a lower but significant mediated learning response (Figure 2C) and normal direct learning (Figure 2E). Finally, in the No-Shock group, both male (Figure 2B and 2D) and female mice (Figure 2C and 2E) did not present either mediated or direct learning, which also confirmed that the exposure to the tone or light during Probe Tests do not elicit any behavioral change by themselves as the presence of the electric footshock is required to obtain a reliable mediated and direct learning responses."<br /> The absence of a difference between the paired and unpaired female mice should not be described as "significant mediated learning" in the latter. It should be taken to indicate that performance in the females is due to generalization between the tone and light. That is, there is no sensory preconditioning in the female mice. The description of performance in the No-shock group really shouldn't be in terms of mediated or direct learning: that is, this group is another control for assessing the presence of sensory preconditioning in the group of interest. As a control, there is no potential for them to exhibit sensory preconditioning, so their performance should not be described in a way that suggests this potential.

Methods - Behavior

I appreciate the reasons for testing the animals in a new context. This does, however, raise other issues that complicate the interpretation of any hippocampal engagement: e.g., exposure to a novel context may engage the hippocampus for exploration/encoding of its features - hence, it is engaged for retrieving/expressing sensory preconditioned fear to the tone. This should be noted somewhere in the paper given that one of its aims is to shed light on the broader functioning of the hippocampus in associative processes.

This general issue - that the conditions of testing were such as to force engagement of the hippocampus - is amplified by two further features of testing with the tone. The first is the presence of background noise in the training context and its absence in the test context. The second is the fact that the tone was presented for 30 s in stage 1 and then continuously for 180s at test. Both changes could have contributed to the engagement of the hippocampus as they introduce the potential for discrimination between the tone that was trained and tested.

Results - Behavior

The suggestion of sex differences based on differences in the parameters needed to generate sensory preconditioning is interesting. Perhaps it could be supported through some set of formal analyses. That is, the data in supplementary materials may well show that the parameters needed to generate sensory preconditioning in males and females are not the same. However, there needs to be some form of statistical comparison to support this point. As part of this comparison, it would be neat if the authors included body weight as a covariate to determine whether any interactions with sex are moderated by body weight.

What is the value of the data shown in Figure 1 given that there are no controls for unpaired presentations of the sound and light? In the absence of these controls, the experiment cannot have shown that "Female and male mice show mediated learning using an auditory-visual sensory preconditioning task" as implied by its title. Minimally, this experiment should be relabelled.

"Altogether, this data confirmed that we successfully set up an LTSPC protocol in mice and that this behavioral paradigm can be used to further study the brain circuits involved in higher-order conditioning."<br /> Please insert the qualifier that LTSPC was successfully established in male mice. There is no evidence of LTSPC in female mice.

Results - Brain

"Notably, the inhibition of CaMKII-positive neurons in the dHPC (i.e. J60 administration in DREADD-Gi mice) during preconditioning (Figure 4B), but not before the Probe Test 1 (Figure 4B), fully blocked mediated, but not direct learning (Figure 4D)."<br /> The right panel of Figure 4B indicates no difference between the controls and Group DPC in the percent change in freezing from OFF to ON periods of the tone. How does this fit with the claim that CaMKII-positive neurons in the dorsal hippocampus regulate associative formation during the session of tone-light exposures in stage 1 of sensory preconditioning?

Discussion

"When low salience stimuli were presented separated on time or when the electric footshock was absent, mediated and direct learning were abolished in male mice. In female mice, although light and tone were presented separately during the preconditioning phase, mediated learning was reduced but still present, which implies that female mice are still able to associate the two low-salience stimuli."<br /> This doesn't quite follow from the results. The failure of the female unpaired mice to withhold their freezing to the tone should not be taken to indicate the formation of a light-tone association across the very long interval that was interpolated between these stimulus presentations. It could and should be taken to indicate that, in female mice, freezing conditioned to the light simply generalized to the tone (i.e., these mice could not discriminate well between the tone and light).

"Indeed, our data suggests that when hippocampal activity is modulated by the specific manipulation of hippocampal subregions, this brain region is not involved during retrieval."<br /> Does this relate to the results that are shown in the right panel of Figure 4B, where there is no significant difference between the different groups? If so, how does it fit with the results shown in the left panel of this figure, where differences between the groups are observed?

"In line with this, the inhibition of CaMKII-positive neurons from the dorsal hippocampus, which has been shown to project to the restrosplenial cortex56, blocked the formation of mediated learning."<br /> Is this a reference to the findings shown in Figure 4B and, if so, which of the panels exactly? That is, one panel appears to support the claim made here while the other doesn't. In general, what should the reader make of data showing the percent change in freezing from stimulus OFF to stimulus ON periods?

Author response:

Reviewer #1 (Public review):

Summary:

The study by Pinho et al. presents a novel behavioral paradigm for investigating higher-order conditioning in mice. The authors developed a task that creates associations between light and tone sensory cues, driving mediated learning. They observed sex differences in task acquisition, with females demonstrating faster-mediated learning compared to males. Using fiber photometry and chemogenetic tools, the study reveals that the dorsal hippocampus (dHPC) plays a central role in encoding mediated learning. These findings are crucial for understanding how environmental cues, which are not directly linked to positive/negative outcomes, contribute to associative learning. Overall, the study is well-designed, with robust results, and the experimental approach aligns with the study's objectives.

Strengths:

(1) The authors develop a robust behavioral paradigm to examine higher-order associative learning in mice.

(2) They discover a sex-specific component influencing mediated learning, with females exhibiting enhanced learning abilities.

(3) Using fiber photometry and chemogenetic techniques, the authors identify the dorsal hippocampus but not the ventral hippocampus, which plays a crucial for encoding mediated learning.

Weaknesses:

(1) The study would be strengthened by further elaboration on the rationale for investigating specific cell types within the hippocampus.

We will add more information to better explain the rationale of our experiments and/or manipulations.

(2) The analysis of photometry data could be improved by distinguishing between early and late responses, as well as enhancing the overall presentation of the data.

We will provide new photometry analysis to differentiate between early and late responses during stimuli presentations.

(3) The manuscript would benefit from revisions to improve clarity and readability.

We will improve the clarity and readability of our manuscript.

Reviewer #2 (Public review):

Summary:

Pinho et al. developed a new auditory-visual sensory preconditioning procedure in mice and examined the contribution of the dorsal and ventral hippocampus to learning in this task. Using photometry they observed activation of the dorsal and ventral hippocampus during sensory preconditioning and conditioning. Finally, the authors combined their sensory preconditioning task with DREADDs to examine the effect of inhibiting specific cell populations (CaMKII and PV) in the DH on the formation and retrieval/expression of mediated learning.

Strengths:

The authors provide one of the first demonstrations of auditory-visual sensory preconditioning in male mice. Research on the neurobiology of sensory preconditioning has primarily used rats as subjects. The development of a robust protocol in mice will be beneficial to the field, allowing researchers to take advantage of the many transgenic mouse lines. Indeed, in this study, the authors take advantage of a PV-Cre mouse line to examine the role of hippocampal PV cells in sensory preconditioning.

Weaknesses:

(1) The authors report that sensory preconditioning was observed in both male and female mice. However, their data only supports sensory preconditioning in male mice. In female mice, both paired and unpaired presentations of the light and tone in stage 1 led to increased freezing to the tone at test. In this case, fear to the tone could be attributed to factors other than sensory preconditioning, for example, generalization of fear between the auditory and visual stimulus.

To address the pertinent doubt raised by the reviewer, we will perform new experiments to generate a new unpaired group in female mice through the increase of the temporal interval between light and tone exposure during the preconditioning phase. We believe this new results will bring additional information to better understand the performance of female mice in sensory preconditioning.

(2) In the photometry experiment, the authors report an increase in neural activity in the hippocampus during both phase 1 (sensory preconditioning) and phase 2 (conditioning). In the subsequent experiment, they inhibit neural activity in the DH during phase 1 (sensory preconditioning) and the probe test, but do not include inhibition during phase 2 (conditioning). It was not clear why they didn't carry forward investigating the role of the hippocampus during phase 2 conditioning. Sensory preconditioning could occur due to the integration of the tone and shock during phase two, or retrieval and chaining of the tone-light-shock memories at test. These two possibilities cannot be differentiated based on the data. Given that we do not know at which stage the mediate learning is occurring, it would have been beneficial to additionally include inhibition of the DH during phase 2.

We will perform new experiments to generate novel data by inhibiting the CamK-positive neurons of the dorsal hippocampus during the conditioning phase.

(3) In the final experiment, the authors report that inhibition of the dorsal hippocampus during the sensory preconditioning phase blocked mediated learning. While this may be the case, the failure to observe sensory preconditioning at test appears to be due more to an increase in baseline freezing (during the stimulus off period), rather than a decrease in freezing to the conditioned stimulus. Given the small effect, this study would benefit from an experiment validating that administration of J60 inhibited DH cells. Further, given that the authors did not observe any effect of DREADD inhibition in PV cells, it would also be important to validate successful cellular silencing in this protocol.

By combining chemogenetic and fiber photometry approaches, we will perform a control experiments to demonstrate that our chemogenetic experiments are decreasing CAMK- or PV-dependent activity in dorsal and ventral hippocampus.

Reviewer #3 (Public review):

Summary:

Pinho et al. investigated the role of the dorsal vs ventral hippocampus and the gender differences in mediated learning. While previous studies already established the engagement of the hippocampus in sensory preconditioning, the authors here took advantage of freely-moving fiber photometry recording and chemogenetics to observe and manipulate sub-regions of the hippocampus (dorsal vs. ventral) in a cell-specific manner. The authors first found sex differences in the preconditioning phase of a sensory preconditioning procedure, where males required more preconditioning training than females for mediating learning to manifest, and where females displayed evidence of mediated learning even when neutral stimuli were never presented together within the session.

After validation of a sensory preconditioning procedure in mice using light and tone neutral stimuli and a mild foot shock as the unconditioned stimulus, the authors used fiber photometry to record from all neurons vs. parvalbumin_positive_only neurons in the dorsal hippocampus or ventral hippocampus of male mice during both preconditioning and conditioning phases. They found increased activity of all neurons, as well as PV+_only neurons in both sub-regions of the hippocampus during both preconditioning and conditioning phases. Finally, the authors found that chemogenetic inhibition of CaMKII+ neurons in the dorsal, but not ventral, hippocampus specifically prevented the formation of an association between the two neutral stimuli (i.e., light and tone cues), but not the direct association between the light cue and the mild foot shock. This set of data: (1) validates the mediated learning in mice using a sensory preconditioning protocol, and stresses the importance of taking sex effect into account; (2) validates the recruitment of dorsal and ventral hippocampi during preconditioning and conditioning phases; and (3) further establishes the specific role of CaMKII+ neurons in the dorsal but not ventral hippocampus in the formation of an association between two neutral stimuli, but not between a neutral-stimulus and a mild foot shock.

Strengths:

The authors developed a sensory preconditioning procedure in mice to investigate mediated learning using light and tone cues as neutral stimuli, and a mild foot shock as the unconditioned stimulus. They provide evidence of a sex effect in the formation of light-cue association. The authors took advantage of fiber-photometry and chemogenetics to target sub-regions of the hippocampus, in a cell-specific manner and investigate their role during different phases of a sensory conditioning procedure.

Weaknesses:

The authors went further than previous studies by investigating the role of sub-regions of the hippocampus in mediated learning, however, there are several weaknesses that should be noted:

(1) This work first validates mediated learning in a sensory preconditioning procedure using light and tone cues as neutral stimuli and a mild foot shock as the unconditioned stimulus, in both males and females. They found interesting sex differences at the behavioral level, but then only focused on male mice when recording and manipulating the hippocampus. The authors do not address sex differences at the neural level.

As discussed above, we will perform additional experiment to evaluate the presence of a reliable sensory preconditioning in female mice. In addition, although observing sex differences at the neural level can be very interesting, we think that it is out of the scope of the present work. However, we will mention this issue/limitation in the Discussion in the new version of the manuscript.

(2) As expected in fear conditioning, the range of inter-individual differences is quite high. Mice that didn't develop a strong light-->shock association, as evidenced by a lower percentage of freezing during the Probe Test Light phase, should manifest a low percentage of freezing during the Probe Test Tone phase. It would interesting to test for a correlation between the level of freezing during mediated vs test phases.

We will provide correlations between the behavioral responses in both probe tests.

(3) The use of a synapsin promoter to transfect neurons in a non-specific manner does not bring much information. The authors applied a more specific approach to target PV+ neurons only, and it would have been more informative to keep with this cell-specific approach, for example by looking also at somatostatin+ inter-neurons.

We will better justify the use of specific promoters and the targeting of PV-positive neurons. We will also add discussion on potential interesting future experiments such as the targeting of other GABAergic subtypes.

(4) The authors observed event-related Ca2+ transients on hippocampal pan-neurons and PV+ inter-neurons using fiber photometry. They then used chemogenetics to inhibit CaMKII+ hippocampal neurons, which does not logically follow. It does not undermine the main finding of CaMKII+ neurons of the dorsal, but not ventral, hippocampus being involved in the preconditioning, but not conditioning, phase. However, observing CaMKII+ neurons (using fiber photometry) in mice running the same task would be more informative, as it would indicate when these neurons are recruited during different phases of sensory preconditioning. Applying then optogenetics to cancel the observed event-related transients (e.g., during the presentation of light and tone cues, or during the foot shock presentation) would be more appropriate.

We will perform new experiments to analyze the activity of CAMK-positive neurons during light-tone associations during the preconditioning phase in male mice.

(5) Probe tests always start with the "Probe Test Tone", followed by the "Probe Test Light". "Probe Test Tone" consists of an extinction session, which could affect the freezing response during "Probe Test Light" (e.g., Polack et al. (http://dx.doi.org/10.3758/s13420-013-0119-5)). Preferably, adding a group of mice with a Probe Test Light with no Probe Test Tone could help clarify this potential issue. The authors should at least discuss the possibility that the tone extinction session prior to the "Probe Test Light" could have affected the freezing response to the light cue.

We will add discussion on this issue raised by the reviewer.

Reviewer #4 (Public review):

Summary

Pinho et al use in vivo calcium imaging and chemogenetic approaches to examine the involvement of hippocampal sub-regions across the different stages of a sensory preconditioning task in mice. They find clear evidence for sensory preconditioning in male but not female mice. They also find that, in the male mice, CaMKII-positive neurons in the dorsal hippocampus: (1) encode the audio-visual association that forms in stage 1 of the task, and (2) retrieve/express sensory preconditioned fear to the auditory stimulus at test. These findings are supported by evidence that ranges from incomplete to convincing. They will be valuable to researchers in the field of learning and memory.

Abstract

Please note that sensory preconditioning doesn't require the stage 1 stimuli to be presented repeatedly or simultaneously.

We will correct this wrong sentence in the abstract.

"Finally, we combined our sensory preconditioning task with chemogenetic approaches to assess the role of these two hippocampal subregions in mediated learning."

This implies some form of inhibition of hippocampal neurons in stage 2 of the protocol, as this is the only stage of the protocol that permits one to make statements about mediated learning. However, it is clear from what follows that the authors interrogate the involvement of hippocampal sub-regions in stages 1 and 3 of the protocol - not stage 2. As such, most statements about mediated learning throughout the paper are potentially misleading (see below for a further elaboration of this point). If the authors persist in using the term mediated learning to describe the response to a sensory preconditioned stimulus, they should clarify what they mean by mediated learning at some point in the introduction. Alternatively, they might consider using a different phrase such as "sensory preconditioned responding".

Through the text, we will avoid the term “mediated learning” and we will replace it with more accurate terms. In addition, we will interrogate the role of dHPC in Stage 2 as commented above.

Introduction

"Low-salience" is used to describe stimuli such as tone, light, or odour that do not typically elicit responses that are of interest to experimenters. However, a tone, light, or odour can be very salient even though they don't elicit these particular responses. As such, it would be worth redescribing the "low-salience" stimuli in some other terms.

We will substitute “low-salience” for “innocuous”.

"These higher-order conditioning processes, also known as mediated learning, can be captured in laboratory settings through sensory preconditioning procedures2,6-11."

Higher-order conditioning and mediated learning are not interchangeable terms: e.g., some forms of second-order conditioning are not due to mediated learning. More generally, the use of mediated learning is not necessary for the story that the authors develop in the paper and could be replaced for accuracy and clarity. E.g., "These higher-order conditioning processes can be studied in the laboratory using sensory preconditioning procedures2,6-11."

Through the text, we will avoid the term “mediated learning” and we will replace it with more accurate terms.

In reference to Experiment 2, it is stated that: "However, when light and tone were separated on time (Unpaired group), male mice were not able to exhibit mediated learning response (Figure 2B) whereas their response to the light (direct learning) was not affected (Figure 2D). On the other hand, female mice still present a lower but significant mediated learning response (Figure 2C) and normal direct learning (Figure 2E). Finally, in the No-Shock group, both male (Figure 2B and 2D) and female mice (Figure 2C and 2E) did not present either mediated or direct learning, which also confirmed that the exposure to the tone or light during Probe Tests do not elicit any behavioral change by themselves as the presence of the electric footshock is required to obtain a reliable mediated and direct learning responses."<br /> The absence of a difference between the paired and unpaired female mice should not be described as "significant mediated learning" in the latter. It should be taken to indicate that performance in the females is due to generalization between the tone and light. That is, there is no sensory preconditioning in the female mice. The description of performance in the No-shock group really shouldn't be in terms of mediated or direct learning: that is, this group is another control for assessing the presence of sensory preconditioning in the group of interest. As a control, there is no potential for them to exhibit sensory preconditioning, so their performance should not be described in a way that suggests this potential.

We will re-write the text to clarify the right comments raised by the Reviewer.

Methods - Behavior

I appreciate the reasons for testing the animals in a new context. This does, however, raise other issues that complicate the interpretation of any hippocampal engagement: e.g., exposure to a novel context may engage the hippocampus for exploration/encoding of its features - hence, it is engaged for retrieving/expressing sensory preconditioned fear to the tone. This should be noted somewhere in the paper given that one of its aims is to shed light on the broader functioning of the hippocampus in associative processes.

We will further discuss this aspect on the manuscript.

This general issue - that the conditions of testing were such as to force engagement of the hippocampus - is amplified by two further features of testing with the tone. The first is the presence of background noise in the training context and its absence in the test context. The second is the fact that the tone was presented for 30 s in stage 1 and then continuously for 180s at test. Both changes could have contributed to the engagement of the hippocampus as they introduce the potential for discrimination between the tone that was trained and tested.

We will consider the aspect raised by the reviewer on the manuscript.

Results - Behavior

The suggestion of sex differences based on differences in the parameters needed to generate sensory preconditioning is interesting. Perhaps it could be supported through some set of formal analyses. That is, the data in supplementary materials may well show that the parameters needed to generate sensory preconditioning in males and females are not the same. However, there needs to be some form of statistical comparison to support this point. As part of this comparison, it would be neat if the authors included body weight as a covariate to determine whether any interactions with sex are moderated by body weight.

We will add statistical comparisons between male and female mice.

What is the value of the data shown in Figure 1 given that there are no controls for unpaired presentations of the sound and light? In the absence of these controls, the experiment cannot have shown that "Female and male mice show mediated learning using an auditory-visual sensory preconditioning task" as implied by its title. Minimally, this experiment should be relabelled.

We will relabel Figure 1.

"Altogether, this data confirmed that we successfully set up an LTSPC protocol in mice and that this behavioral paradigm can be used to further study the brain circuits involved in higher-order conditioning."

Please insert the qualifier that LTSPC was successfully established in male mice. There is no evidence of LTSPC in female mice.

We will generate new experiments to try to demonstrate that SPC can be also observed in female mice.

Results - Brain

"Notably, the inhibition of CaMKII-positive neurons in the dHPC (i.e. J60 administration in DREADD-Gi mice) during preconditioning (Figure 4B), but not before the Probe Test 1 (Figure 4B), fully blocked mediated, but not direct learning (Figure 4D)."

The right panel of Figure 4B indicates no difference between the controls and Group DPC in the percent change in freezing from OFF to ON periods of the tone. How does this fit with the claim that CaMKII-positive neurons in the dorsal hippocampus regulate associative formation during the session of tone-light exposures in stage 1 of sensory preconditioning?

We will rephrase and add more Discussion regarding this section of the results to stick to what the graphs are showing. We will clarify that the group where dHPC activity is inhibited during preconditioning is the only one where the % of change is not significantly different from 0 (compared to the control or the group where the dHPC activity was modulated during the test).

Discussion

"When low salience stimuli were presented separated on time or when the electric footshock was absent, mediated and direct learning were abolished in male mice. In female mice, although light and tone were presented separately during the preconditioning phase, mediated learning was reduced but still present, which implies that female mice are still able to associate the two low-salience stimuli."

This doesn't quite follow from the results. The failure of the female unpaired mice to withhold their freezing to the tone should not be taken to indicate the formation of a light-tone association across the very long interval that was interpolated between these stimulus presentations. It could and should be taken to indicate that, in female mice, freezing conditioned to the light simply generalized to the tone (i.e., these mice could not discriminate well between the tone and light).

We will rewrite this part depending on the results observed in female mice.

"Indeed, our data suggests that when hippocampal activity is modulated by the specific manipulation of hippocampal subregions, this brain region is not involved during retrieval."

Does this relate to the results that are shown in the right panel of Figure 4B, where there is no significant difference between the different groups? If so, how does it fit with the results shown in the left panel of this figure, where differences between the groups are observed?

We will re-write it to clearly describe our results and we will also revise all the statistical analysis.

"In line with this, the inhibition of CaMKII-positive neurons from the dorsal hippocampus, which has been shown to project to the restrosplenial cortex56, blocked the formation of mediated learning."

Is this a reference to the findings shown in Figure 4B and, if so, which of the panels exactly? That is, one panel appears to support the claim made here while the other doesn't. In general, what should the reader make of data showing the percent change in freezing from stimulus OFF to stimulus ON periods?

We will rewrite the text to clearly describe our results, and we will also revise all the statistical analysis. In addition, we will better explain the data showing the % of change.

eLife Assessment

In this paper, the authors report important structural and functional findings on the interaction of how the group A streptococci (GAS) M3 protein (expressed on GAS strains emm3, which are associated with invasive disease) binds to human collagens. They demonstrate an unusual T-shaped structure within the N-terminal hypervariable region of M3 protein that can bind two copies of collagen triple helix in parallel. These solid data advance understanding of how GAS M3 interacts with human collagen, information relevant to understanding and developing treatments for GAS infection. A major limitation of the work is the lack of mutational work to test if the T-shaped structure is necessary for binding collagen.

Reviewer #1 (Public review):

Summary:

Wojnowska et al. report structural and functional studies of the interaction of Streptococcus pyogenes M3 protein with collagen. They show through X-ray crystallographic studies that the N-terminal hypervariable region of M3 protein forms a T-like structure and that the T-like structure binds a three-stranded collagen-mimetic peptide. They indicate that the T-like structure is predicted by AlphaFold3 (with varying confidence level) in other M proteins that have sequence similarity to M3 protein and M-like proteins from group C and G streptococci. For some, but not all, of these related M and M-like proteins, AlphaFold3 predicts complexes similar to the one observed for M3-collagen. Functionally, the authors show that emm3 strains form biofilms with more mass when surfaces are coated with collagen, and this effect can be blocked by an M3 protein fragment that contains the T-structure. They also show the co-occurrence of emm3 strains and collagen in patient biopsies and a skin tissue organoid.

Strengths:

The paper is well-written and the data presented is mostly sound.

Weaknesses:

However, a major limitation of the paper is that it is almost entirely observational and fails to draw a causal relationship. This is mainly due to the near-total absence of mutational studies.

Reviewer #2 (Public review):

Streptococcus pyogenes, or group A streptococci (GAS) can cause diseases ranging from skin and mucosal infections, to plasma invasion, and post-infection autoimmune syndromes. M proteins are essential GAS virulence factors that include an N-terminal hypervariable region (HVR). M proteins are known to bind to numerous human proteins; a small subset of M proteins were reported to bind collagen, which is thought to promote tissue adherence. In this paper, the authors characterize M3 interactions with collagen and its role in biofilm formation. Specifically, they screened different collagen type II and III variants for full-length M3 protein binding using an ELISA-like method, detecting anti-GST antibody signal. By statistical analysis, hydrophobic amino acids and hydroxyproline were found to positively support binding, whereas acidic residues and proline negatively impacted binding (Table 1). The authors applied X-ray crystallography to determine the structure of the N-terminal domain (42-151 amino acids) of M3 protein (M3-NTD). M3-NTD dimmer (PDB 8P6K) forms a T-shaped structure with three helices (H1, H2, H3), which are stabilized by a hydrophobic core, inter-chain salt bridges and hydrogen bonds on H1, H2 helices, and H3 coiled coil. The conserved Gly113 serves as the turning point between H2 and H3 (Figure 5). The M3-NTD is co-crystalized with a 24-residue peptide, JDM238, to determine the structure of M3-collagen binding. The structure (PDB 8P6J) shows that two copies of collagen in parallel bind to H1 and H2 of M3-NTD. Among the residues involved in binding, conserved Try96 is shown to play a critical role supported by structure and isothermal titration calorimetry (ITC). The authors also apply a crystal-violet assay and fluorescence microscopy to determine that M3 is involved in collagen type I binding, but not M1 or M28 (Figure 9). Tissue biopsy staining indicates that M3 strains co-localize with collagen IV-containing tissue, while M1 strains do not. The authors provide generally compelling evidence to show that GAS M3 protein binds to collagen, and plays a critical role in forming biofilms, which contribute to disease pathology. This is a very well-executed study and a well-written report relevant to understanding GAS pathogenesis and approaches to combatting disease; data are also applicable to emerging human pathogen Streptococcus dysgalactiae. One caveat that was not entirely resolved is if/how different collagen types might impact M3 binding and function. Due to the technical constraints, the in vitro structure and other binding assays use type II collagen whereas in vivo, biofilm formation assays and tissue biopsy staining use type I and IV collagen; it was unclear if this difference is significant. One possibility is that M3 has an unbiased binding to all types of collagens, only the distribution of collagens leads to the finding that M3 binds to type IV (basement membrane) and type I (varies of tissue including skin), rather than type II (cartilage).

Author response:

Many thanks for assessing our submission. We are grateful for the reviews and recommendations that will inform a revised version of the paper, which will include additional data and modified text to take into account the reviewers’ comments.

We appreciate Reviewer #1’s suggestion regarding the use of mutational work to demonstrate that collagen binding is indeed dependent on the T-shaped fold. However, we believe that this approach is neither feasible nor necessary for our study. Instead, we propose to measure collagen binding to a monomeric form of M3, which preserves all residues including the ones involved in binding, but cannot form the T-shaped structure. This will achieve the same as unravelling the T fold through mutations, but at the same time removes the risk of directly affecting binding through altering residues that are involved in both binding and definition of the T fold.

Structural biology is by its nature observational, which is not a limitation but the very purpose of this approach. Our study goes beyond observing structures. We identify a critical residue within a previously mapped binding site, and demonstrate through mutagenesis a causal link between presence of this residue on a tertiary fold and collagen binding activity. We will firm up our mutational experiments with a characterisation of the M3 Tyr96 variants to confirm that these mutations did not affect the overall fold. We further demonstrate that the interaction between M3 and collagen promotes biofilm formation as observed in patient biopsies and a tissue model of infection. We show that other streptococci, that do not possess a surface protein presenting collagen binding sites like M3, do not form collagen-dependent biofilm. We therefore do not think that criticising our study for being almost entirely observational is justified. 

We thank Reviewer #2 for the thorough analysis of our reported findings. The main criticism here concerns the question if binding of emm3 streptococci would differ for different types of collagen. We will address this point in the revised manuscript. Our collagen peptide binding assays together with the structural data identify the collagen triple helix as the binding site for M3. While collagen types differ in their functions and morphology in various tissues, they all have in common triple-helical tropocollagen regions (with very high sequence similarity) that are non-specifically recognised by M3. Therefore, our data in conjunction with the body of published work showing binding of M3 to collagens I, II, III and IV suggest it is highly likely that emm3 streptococci will indeed bind to many if not all types of collagen in the same manner. Whether this means all collagen types, in the various tissues where they occur, are targeted by emm3 streptococci is a very interesting question, however one that goes beyond the scope of our study.

difficult to see how preventive war can be seen as a last-resort

so, normally 315 of Reunification Treaty prevents acts commited on East German soil prior to reunification from being punished, if they were not punishable under East German Law (kinda like our warunek podwójnej karalaności)

BUT, here: - immunity does not apply where there was already West German Law (west german law applied to crimes on foreign soil if): 1. the acts are commited against German 2. the person that committed them becomes a resident of WG or comes to WG

prof. Samson argues that EG became part of WG, so their law is applicable 7(2) (similarly, the people who were shot, were Germans, so 7(1))

BUT it's a stretch, because EGs were considered foreigners by WG

eLife Assessment

This important theoretical study introduces an extension to the commonly used SIR model for infectious disease dynamics, to explicitly consider the role of larger group sizes. Instead of the commonly used individual-based network models, the authors developed a simplified approach based on group sampling, with discrete high- and low-risk groups, which makes the results easier to produce and interpret, at the cost of less detail in the model. The evidence is convincing in terms of the soundness of the theoretical projections and the impact that accounting for group sizes may have on inferences from surveillance data. However, it has not yet been demonstrated that the predictions provide more realistic projections when based on real-world data.

Reviewer #1 (Public review):

Summary:

This work considers the biases introduced into pathogen surveillance due to congregation effects, and also models homophily and variants/clades. The results are primarily quantitative assessments of this bias but some qualitative insights are gained e.g. that initial variant transmission tends to be biased upwards due to this effect, which is closely related to classical founder effects.

Strengths:

The model considered involves a simplification of the process of congregation using multinomial sampling that allows for a simpler and more easily interpretable analysis.

Weaknesses:

This simplification removes some realism, for example, detailed temporal transmission dynamics of congregations.

Reviewer #2 (Public review):

Summary:

In "Founder effects arising from gathering dynamics systematically bias emerging pathogen surveillance" Bradford and Hang present an extension to the SIR model to account for the role of larger than pairwise interactions in infectious disease dynamics. They explore the impact of accounting for group interactions on the progression of infection through the various sub-populations that make up the population as a whole. Further, they explore the extent to which interaction heterogeneity can bias epidemiological inference from surveillance data in the form of IFR and variant growth rate dynamics. This work advances the theoretical formulation of the SIR model and may allow for more realistic modeling of infectious disease outbreaks in the future.

Strengths:

(1) This work addresses an important limitation of standard SIR models. While this limitation has been addressed previously in the form of network-based models, those are, as the authors argue, difficult to parameterize to real-world scenarios. Further, this work highlights critical biases that may appear in real-world epidemiological surveillance data. Particularly, over-estimation of variant growth rates shortly after emergence has led to a number of "false alarms" about new variants over the past five years (although also to some true alarms).

(2) While the results presented here generally confirm my intuitions on this topic, I think it is really useful for the field to have it presented in such a clear manner with a corresponding mathematical framework. This will be a helpful piece of work to point to to temper concerns about rapid increases in the frequency of rare variants.

(3) The authors provide a succinct derivation of their model that helps the reader understand how they arrived at their formulation starting from the standard SIR model.

(4) The visualizations throughout are generally easy to interpret and communicate the key points of the authors' work.

(5) I thank the authors for providing detailed code to reproduce manuscript figures in the associated GitHub repo.

Weaknesses:

(1) The authors argue that network-based SIR models are difficult to parameterize (line 66), however, the model presented here also has a key parameter, mainly P_n, or the distribution of risk groups in the population. I think it is important to explore the extent to which this parameter can be inferred from real-world data to assess whether this model is, in practice, any easier to parameterize.

(2) The authors explore only up to four different risk groups, accounting for only four-wise interactions. But, clearly, in real-world settings, there can be much larger gatherings that promote transmission. What was the justification for setting such a low limit on the maximum group size? I presume it's due to computational efficiency, which is understandable, but it should be discussed as a limitation.

(3) Another key limitation that isn't addressed by the authors is that there may be population structure beyond just risk heterogeneity. For example, there may be two separate (or, weakly connected) high-risk sub-groups. This will introduce temporal correlation in interactions that are not (and can not easily be) captured in this model. My instinct is that this would dampen the difference between risk groups shown in Figure 2A. While I appreciate the authors's desire to keep their model relatively simple, I think this limitation should be explicitly discussed as it is, in my opinion, relatively significant.

Author response:

Reviewer #1 (Public review):

Summary:

This work considers the biases introduced into pathogen surveillance due to congregation effects, and also models homophily and variants/clades. The results are primarily quantitative assessments of this bias but some qualitative insights are gained e.g. that initial variant transmission tends to be biased upwards due to this effect, which is closely related to classical founder effects.

Strengths:

The model considered involves a simplification of the process of congregation using multinomial sampling that allows for a simpler and more easily interpretable analysis.

Weaknesses:

This simplification removes some realism, for example, detailed temporal transmission dynamics of congregations.

We appreciate Reviewer #1's comments. We hope our framework, like the classic SIR model, can be adapted in the future to build more complex and realistic models.

Reviewer #2 (Public review):

Summary:

In "Founder effects arising from gathering dynamics systematically bias emerging pathogen surveillance" Bradford and Hang present an extension to the SIR model to account for the role of larger than pairwise interactions in infectious disease dynamics. They explore the impact of accounting for group interactions on the progression of infection through the various sub-populations that make up the population as a whole. Further, they explore the extent to which interaction heterogeneity can bias epidemiological inference from surveillance data in the form of IFR and variant growth rate dynamics. This work advances the theoretical formulation of the SIR model and may allow for more realistic modeling of infectious disease outbreaks in the future.

Strengths:

(1) This work addresses an important limitation of standard SIR models. While this limitation has been addressed previously in the form of network-based models, those are, as the authors argue, difficult to parameterize to real-world scenarios. Further, this work highlights critical biases that may appear in real-world epidemiological surveillance data. Particularly, over-estimation of variant growth rates shortly after emergence has led to a number of "false alarms" about new variants over the past five years (although also to some true alarms).

(2) While the results presented here generally confirm my intuitions on this topic, I think it is really useful for the field to have it presented in such a clear manner with a corresponding mathematical framework. This will be a helpful piece of work to point to to temper concerns about rapid increases in the frequency of rare variants.

(3) The authors provide a succinct derivation of their model that helps the reader understand how they arrived at their formulation starting from the standard SIR model.

(4) The visualizations throughout are generally easy to interpret and communicate the key points of the authors' work.

(5) I thank the authors for providing detailed code to reproduce manuscript figures in the associated GitHub repo.

Weaknesses:

(1) The authors argue that network-based SIR models are difficult to parameterize (line 66), however, the model presented here also has a key parameter, mainly P_n, or the distribution of risk groups in the population. I think it is important to explore the extent to which this parameter can be inferred from real-world data to assess whether this model is, in practice, any easier to parameterize.

(2) The authors explore only up to four different risk groups, accounting for only four-wise interactions. But, clearly, in real-world settings, there can be much larger gatherings that promote transmission. What was the justification for setting such a low limit on the maximum group size? I presume it's due to computational efficiency, which is understandable, but it should be discussed as a limitation.

(3) Another key limitation that isn't addressed by the authors is that there may be population structure beyond just risk heterogeneity. For example, there may be two separate (or, weakly connected) high-risk sub-groups. This will introduce temporal correlation in interactions that are not (and can not easily be) captured in this model. My instinct is that this would dampen the difference between risk groups shown in Figure 2A. While I appreciate the authors's desire to keep their model relatively simple, I think this limitation should be explicitly discussed as it is, in my opinion, relatively significant.

We appreciate Reviewer 2's thoughtful comments and wish to address some of the weaknesses:

We agree that inferring P_n from real data will be challenging, but think this is an important direction for future research. Further, we’d like to reframe our claim that our approach is "easier to parameterize" than network models. Rather, P_n has fewer degrees of freedom than analogous network models, just as many different networks can share the same degree distribution. Fewer degrees of freedom mean that we expect our model to suffer from fewer identifiability issues when fitting to data, though non-identifiability is often inescapable in models of this nature (e.g., \beta and \gamma in the SIR model are not uniquely identifiable during exponential growth). Whether this is more or less accurate is another question. Classic bias-variance tradeoffs argue that a model with a moderate complexity trained on one data set can better fit future data than overly simple or overly complex models.

We chose four risk groups for purposes of illustration, but this can be increased arbitrarily. It should be noted that the simulation bottleneck when increasing the numbers of risk groups is numerical due the stiffness of the ODEs. This arises because the nonlinearity of infection terms scales with the number of risk groups (e.g., ~ \beta * S * I^3 for 4 risk groups). As such, a careful choice of numerical solvers may be required when integrating the ODEs. Meanwhile, this is not an issue for stochastic, individual based implementation (e.g., Gillespie). As for how well this captures super-spreading, we believe choosing smaller risk groups does not hinder modeling disease spread at large gatherings. Consider a statistical interpretation, where individuals at a large gathering engage in a series of smaller interactions over time (e.g., 2/3/4/etc person conversations). The key determinants of the resulting gathering size distribution at any one large gathering are the number of individuals within some shared proximity over time and the infectiousness/dispersal of the pathogen. Of course, whether this interpretation is a sufficient approximation for classic super-spreading events (e.g., funerals during 2014-2015 West Africa Ebola outbreak) is a matter of debate. Our framework is best interpreted at a population level where the effects of any single gathering are washed out by the overall gathering distribution, P_n. As the prior weakness highlighted, establishing P_n is challenging, but we believe empirically measuring proxies of it may provide future insight in how behavior impacts disease spread. For example, prior work has combined contact tracing and co-location data from connection to WiFi networks to estimate the distribution of contacts per individual, and its degree of overdispersion (Petros et al. Med 2022).

We chose to introduce our framework in a simple SIR context familiar to many readers. This decision does not in any way limit applying it to settings with more population structure. Rather, we believe our framework is easily adaptable and that our presentation (hopefully) makes it clear how to do this. For example, two weakly connected groups could be easily achieved by (for each gathering) first sampling the preferred group and then sampling from the population in a biased manner. The biased sampling could even be a function of gathering sizes, time, etc. The resulting infection terms are still (sums of) multinomials. More generally, the sampling probabilities for an individual of some type need not be its frequency (e.g., S/N, I/N). Indeed, we believe generating models with complex social interactions is both simplified and made more robust by focusing on modeling the generative process of attending gatherings.

eLife Assessment

The study introduces new tools for measuring the intracellular calcium concentration close to transmitter release sites, which may be relevant for synaptic vesicle fusion and replenishment. This approach yields important new information about the spatial and temporal profile of calcium concentrations near the site of entry at the plasma membrane. This experimental work is complemented by a coherent, open-source, computational model that successfully describes changes in calcium domains. Key gaps in the data presented mean that the evidence for the main conclusions is currently incomplete.

Reviewer #1 (Public review):

This paper describes technically-impressive measurements of calcium signals near synaptic ribbons in goldfish bipolar cells. The data presented provides high spatial and temporal resolution information about calcium concentrations along the ribbon at various distances from the site of entry at the plasma membrane. This is important information. Important gaps in the data presented mean that the evidence for the main conclusions is currently inadequate.

Strengths

(1) The technical aspects of the measurements are impressive. The authors use calcium indicators bound to the ribbon and high-speed line scans to resolve changes with a spatial resolution of ~250 nm and a temporal resolution of less than 10 ms. These spatial and temporal scales are much closer to those relevant for vesicle release than previous measurements.

(2) The use of calcium indicators with very different affinities and different intracellular calcium buffers helps provide confirmation of key results.

Weaknesses

(1) Multiple key points of the paper lack statistical tests or summary data from populations of cells. For example, the text states that the proximal and distal calcium kinetics in Figure 2A differ. This is not clear from the inset to Figure 2A - where the traces look like scaled versions of each other. Values for time to half-maximal peak fluorescence are given for one example cell but no statistics or summary are provided. Figure 8 shows examples from one cell with no summary data. This issue comes up in other places as well.

(2) Figure 5 is confusing. The figure caption describes red, green, and blue traces, but the figure itself has only two traces in each panel and none are red, green, or blue. It's not possible currently to evaluate this figure.

(3) The rise time measurements in Figure 2 are very different for low and high-affinity indicators, but no explanation is given for this difference. Similarly, the measurements of peak calcium concentration in Figure 4 are very different from the two indicators. That might suggest that the high-affinity indicator is strongly saturated, which raises concerns about whether that is impacting the kinetic measurements.

Reviewer #2 (Public review):

Summary:

The study introduces new tools for measuring intracellular Ca2+ concentration gradients around retinal rod bipolar cell (rbc) synaptic ribbons. This is done by comparing the Ca2+ profiles measured with mobile Ca2+ indicator dyes versus ribbon-tethered (immobile) Ca2+ indicator dyes. The Ca2+ imaging results provide a straightforward demonstration of Ca2+ gradients around the ribbon and validate their experimental strategy. This experimental work is complemented by a coherent, open-source, computational model that successfully describes changes in Ca2+ domains as a function of Ca2+ buffering. In addition, the authors try to demonstrate that there is heterogeneity among synaptic ribbons within an individual rbc terminal.

Strengths:

The study introduces a new set of tools for estimating Ca2+ concentration gradients at ribbon AZs, and the experimental results are accompanied by an open-source, computational model that nicely describes Ca2+ buffering at the rbc synaptic ribbon. In addition, the dissociated retinal preparation remains a valuable approach for studying ribbon synapses. Lastly, excellent EM.

Weaknesses:

Heterogeneity in the spatiotemporal dynamics of Ca2+ influx was not convincingly related to ribbon size, nor was the functional relevance of Ca2+ dynamics to rod bipolars demonstrated (e.g., exocytosis to different postsynaptic targets). In addition, the study would benefit from the inclusion of the Ca2+ currents that were recorded in parallel with the Ca2+ imaging.

Reviewer #3 (Public review):

Summary:

In this study, the authors have developed a new Ca indicator conjugated to the peptide, which likely recognizes synaptic ribbons, and have measured microdomain Ca near synaptic ribbons at retinal bipolar cells. This interesting approach allows one to measure Ca close to transmitter release sites, which may be relevant for synaptic vesicle fusion and replenishment. Though microdomain Ca at the active zone of ribbon synapses has been measured by Hudspeth and Moser, the new study uses the peptide recognizing synaptic ribbons, potentially measuring the Ca concentration relatively proximal to the release sites.

Strengths:

The study is in principle technically well done, and the peptide approach is technically interesting, which allows one to image Ca near the particular protein complexes. The approach is potentially applicable to other types of imaging.

Weaknesses:

Peptides may not be entirely specific, and the genetic approach tagging particular active zone proteins with fluorescent Ca indicator proteins may well be more specific. I also feel that "Nano-physiology" is overselling, because the measured Ca is most likely the local average surrounding synaptic ribbons. With this approach, nobody knows about the real release site Ca or the Ca relevant for synaptic vesicle replenishment. It is rather "microdomain physiology" which measures the local Ca near synaptic ribbons, relatively large structures responsible for fusion, replenishment, and recycling of synaptic vesicles.

Author response:

Public Reviews:

Reviewer #1 (Public review):

This paper describes technically-impressive measurements of calcium signals near synaptic ribbons in goldfish bipolar cells. The data presented provides high spatial and temporal resolution information about calcium concentrations along the ribbon at various distances from the site of entry at the plasma membrane. This is important information. Important gaps in the data presented mean that the evidence for the main conclusions is currently inadequate.

Strengths

(1) The technical aspects of the measurements are impressive. The authors use calcium indicators bound to the ribbon and high-speed line scans to resolve changes with a spatial resolution of ~250 nm and a temporal resolution of less than 10 ms. These spatial and temporal scales are much closer to those relevant for vesicle release than previous measurements.

(2) The use of calcium indicators with very different affinities and different intracellular calcium buffers helps provide confirmation of key results.

Thank you very much for this positive evaluation of our work.

Weaknesses

(1) Multiple key points of the paper lack statistical tests or summary data from populations of cells. For example, the text states that the proximal and distal calcium kinetics in Figure 2A differ. This is not clear from the inset to Figure 2A - where the traces look like scaled versions of each other. Values for time to half-maximal peak fluorescence are given for one example cell but no statistics or summary are provided. Figure 8 shows examples from one cell with no summary data. This issue comes up in other places as well.

Thank you for this feedback. We will address this in our revised manuscript.

(2) Figure 5 is confusing. The figure caption describes red, green, and blue traces, but the figure itself has only two traces in each panel and none are red, green, or blue. It's not possible currently to evaluate this figure.

Thank you for pointing out this oversight. The figure indeed only shows the proximal and distal calcium signals, but not the cytoplasmic ones. The figure will be corrected in our revised manuscript.

(3) The rise time measurements in Figure 2 are very different for low and high-affinity indicators, but no explanation is given for this difference. Similarly, the measurements of peak calcium concentration in Figure 4 are very different from the two indicators. That might suggest that the high-affinity indicator is strongly saturated, which raises concerns about whether that is impacting the kinetic measurements.

As we had mentioned in the text, we do believe that the high-affinity version is partially saturated. This will be a problem for strong depolarizations and signals near the membrane. The higher affinity indicators are more useful for reporting calcium levels on the ribbon after the depolarization when the signal from the low affinity indicators is small. We will address this in the discussion of the revision.

Reviewer #2 (Public review):

Summary:

The study introduces new tools for measuring intracellular Ca2+ concentration gradients around retinal rod bipolar cell (rbc) synaptic ribbons. This is done by comparing the Ca2+ profiles measured with mobile Ca2+ indicator dyes versus ribbon-tethered (immobile) Ca2+ indicator dyes. The Ca2+ imaging results provide a straightforward demonstration of Ca2+ gradients around the ribbon and validate their experimental strategy. This experimental work is complemented by a coherent, open-source, computational model that successfully describes changes in Ca2+ domains as a function of Ca2+ buffering. In addition, the authors try to demonstrate that there is heterogeneity among synaptic ribbons within an individual rbc terminal.

Strengths:

The study introduces a new set of tools for estimating Ca2+ concentration gradients at ribbon AZs, and the experimental results are accompanied by an open-source, computational model that nicely describes Ca2+ buffering at the rbc synaptic ribbon. In addition, the dissociated retinal preparation remains a valuable approach for studying ribbon synapses. Lastly, excellent EM.

Thank you very much for this appreciation.

Weaknesses:

Heterogeneity in the spatiotemporal dynamics of Ca2+ influx was not convincingly related to ribbon size, nor was the functional relevance of Ca2+ dynamics to rod bipolars demonstrated (e.g., exocytosis to different postsynaptic targets). In addition, the study would benefit from the inclusion of the Ca2+ currents that were recorded in parallel with the Ca2+ imaging.

Thank you for this critique. We agree that the relationship between size and Ca2+ signal is not established by our recordings. By analogy to the hair cell literature, we believe that it is a reasonable hypothesis, but more studies will be necessary to definitively determine whether the signal relates to the ribbon size or synaptic signaling. This will be addressed in future experiments.

We will include the Ca²⁺ currents in the revision.

Reviewer #3 (Public review):

Summary:

In this study, the authors have developed a new Ca indicator conjugated to the peptide, which likely recognizes synaptic ribbons, and have measured microdomain Ca near synaptic ribbons at retinal bipolar cells. This interesting approach allows one to measure Ca close to transmitter release sites, which may be relevant for synaptic vesicle fusion and replenishment. Though microdomain Ca at the active zone of ribbon synapses has been measured by Hudspeth and Moser, the new study uses the peptide recognizing synaptic ribbons, potentially measuring the Ca concentration relatively proximal to the release sites.

Strengths:

The study is in principle technically well done, and the peptide approach is technically interesting, which allows one to image Ca near the particular protein complexes. The approach is potentially applicable to other types of imaging.

Thank you very much for this appreciation.

Weaknesses:

Peptides may not be entirely specific, and the genetic approach tagging particular active zone proteins with fluorescent Ca indicator proteins may well be more specific. I also feel that "Nano-physiology" is overselling, because the measured Ca is most likely the local average surrounding synaptic ribbons. With this approach, nobody knows about the real release site Ca or the Ca relevant for synaptic vesicle replenishment. It is rather "microdomain physiology" which measures the local Ca near synaptic ribbons, relatively large structures responsible for fusion, replenishment, and recycling of synaptic vesicles.

The peptide approach has been used fairly extensively in the ribbon synapse field and the evidence that it efficiently labels the ribbon is well established, however, we do acknowledge that the peptide is in equilibrium with a cytoplasmic pool. Thus, some of the signal arises from this cytoplasmic pool. The alternative of a genetically encoded Ca-indicator concatenated to a ribbon protein would not have this problem, but would be more limited in flexibility in changing calcium indicators. We believe both approaches have their merits, each with separate advantages and disadvantages.

As for the nano vs. micro argument, we certainly do not want to suggest that we are measuring the same nano-domains, in the 10s of nanometers, that drive neurotransmitter release, but we do believe we are in the sub-micrometer--100s of nm—range. We chose the term based on the usage by other authors to describe similar measurements (Neef et al., 2018; https://doi.org/10.1038/s41467-017-02612-y), but we see the reviewer’s point. To avoid confusion, we will change the title in the revision.

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eLife Assessment

This study presents valuable findings on the increased prevalence of pain in women with polycystic ovary syndrome and its relationship to health outcomes. The evidence supporting the conclusions is compelling with a large number of patients and sound methodology, and can be used as a starting point for studies of etiology and mechanisms of pain in women with polycystic ovary syndrome and comorbidities. The work will be of interest to medical biologists working on polycystic ovary syndrome pathophysiology and clinicians.

Reviewer #1 (Public review):

Summary:

This retrospective study provides new data regarding the prevalence of pain in women with PCOS and its relationship with health outcomes. Using data from electronic health records (EHR), the authors found a significantly higher prevalence of pain among women with PCOS compared to those without the condition: 19.21% of women with PCOS versus 15.8% in non-PCOS women. The highest prevalence of pain was conducted among Black or African American (32.11%) and White (30.75%) populations. Besides, women with PCOS and pain have at least a 2-fold increased prevalence of obesity (34.68%) at baseline compared to women with PCOS in general (16.11%). Also, women with PCOS had the highest risk for infertility and T2D, but women with PCOS and pain had higher risks for ovarian cysts and liver disease. Regarding these results, the authors suggested the critical need to address pain in the diagnosis and management of PCOS due to its significant impact on patient health outcomes.

Strengths:

(1) The problem of pain assessment in PCOS patients is well described and the authors provided a clear rationale selection of the retrospective design to investigate this problem.

(2) A large number of analyzed patient records (76,859,666 women) and their uniformity increases the power of the study. Using the Propensity Score Matching makes it possible to reduce the heterogeneity of the compared cohorts and the influence of comorbid conditions.

(3) Analysis in different ethnic cohorts provides actual and necessary data regarding the prevalence of pain and its relationship with different health conditions that will be helpful for clinicians to make a diagnosis and manage PCOS in women of different ethnicities.

(4) Assessment of the risk of different health conditions including PCOS-associated pathology as other common groups of diseases in PCOS women with or without pain allows to differentiate the risk of comorbid conditions depending on the presence of one symptom (pelvic or abdominal pain, dysmenorrhea).

Weaknesses:

(1) Although the paper has strengths in methodology and data analysis, it also has some weaknesses. The lack of a hypothesis doesn't allow us to evaluate the aim and significance of this study.

(2) The exclusion criteria don't include conditions, that can lead to symptoms similar to PCOS: thyroid diseases, hyperprolactinemia, and congenital adrenal hyperplasia. Thyroid status is not being taken into account in the criteria for matching. All these conditions could occur as on prevalence results as on risk assessment.

(3) The significant weakness of the study is the absence of a Latin American cohort. Probably the White cohort includes Latin Americans or others, but the results of the study cannot be extrapolated to particular White ethnicities.

(4) The authors didn't provide sufficient rationale for future health outcomes and this list didn't include diseases of the digestive system or disorders of thyroid glands, which can also cause abdominal pain.

Reviewer #2 (Public review):

Summary:

The study offers a thorough analysis of the prevalence of pain in women with polycystic ovary syndrome (PCOS) and its associations with health outcomes across various racial groups. Furthermore, the research investigates the prevalence of PCOS and pain among different racial demographics, as well as the increased risk of developing various conditions in comparison to individuals who have PCOS alone.

Strengths:

The study emphasizes pain as a significant comorbidity of PCOS, an area that is critically underexplored in existing literature. The findings regarding the increased prevalence of some of the diseases in the PCOS + pain group provide valuable direction for future research and clinical care. I believe physicians should incorporate pain score assessments into their clinical practice to improve patient's quality of life and raise awareness about pain management. If future research focuses on the mechanisms of pain, it would provide a better understanding of pain and allow for a focus on the underlying causes rather than just symptomatic management. The study also highlights the association between PCOS+pain and various comorbidities, such as obesity, hypertension, and type 2 diabetes, as well as conditions like infertility and ovarian cysts, offering a holistic view of the burden of PCOS.

Weaknesses:

Due to the nature of the retrospective study, some data may not be readily available in the system. Instead of simply categorizing participants based on whether they experience pain, it would be more useful to employ a pain scale or questionnaire to better understand the severity and type of patients' pain. This approach would allow for a more thorough analysis of pain improvement following treatment with the three widely used medications for PCOS. Additionally, it would be beneficial for the authors to specify subtypes of the disease rather than generalizing conditions, such as mentioning specific digestive system disorders or mental health disorders. The lack of detailed analysis of specific disorders limits the depth of the findings. This may cause authors to make incorrect conclusions.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This retrospective study provides new data regarding the prevalence of pain in women with PCOS and its relationship with health outcomes. Using data from electronic health records (EHR), the authors found a significantly higher prevalence of pain among women with PCOS compared to those without the condition: 19.21% of women with PCOS versus 15.8% in non-PCOS women. The highest prevalence of pain was conducted among Black or African American (32.11%) and White (30.75%) populations. Besides, women with PCOS and pain have at least a 2-fold increased prevalence of obesity (34.68%) at baseline compared to women with PCOS in general (16.11%). Also, women with PCOS had the highest risk for infertility and T2D, but women with PCOS and pain had higher risks for ovarian cysts and liver disease. Regarding these results, the authors suggested the critical need to address pain in the diagnosis and management of PCOS due to its significant impact on patient health outcomes.

Strengths:

(1) The problem of pain assessment in PCOS patients is well described and the authors provided a clear rationale selection of the retrospective design to investigate this problem.(2) A large number of analyzed patient records (76,859,666 women) and their uniformity increases the power of the study. Using the Propensity Score Matching makes it possible to reduce the heterogeneity of the compared cohorts and the influence of comorbid conditions.(3) Analysis in different ethnic cohorts provides actual and necessary data regarding the prevalence of pain and its relationship with different health conditions that will be helpful for clinicians to make a diagnosis and manage PCOS in women of different ethnicities. (4) Assessment of the risk of different health conditions including PCOS-associated pathology as other common groups of diseases in PCOS women with or without pain allows to differentiate the risk of comorbid conditions depending on the presence of one symptom (pelvic or abdominal pain, dysmenorrhea).

We appreciate the positive feedback on this manuscript. Pain assessment in women with PCOS is of paramount interest and because of a gap in this research area, we are trying to address it.

Weaknesses:

(1) Although the paper has strengths in methodology and data analysis, it also has some weaknesses.

The lack of a hypothesis doesn't allow us to evaluate the aim and significance of this study.

We would like to thank the Reviewer for their valuable feedback regarding the hypothesis of this study. We understand that the hypothesis may not have been written clearly under the objectives and we will correct this in the formal revision.

The primary hypothesis of this study is that women with PCOS experience a higher prevalence to pain (including dysmenorrhea, abdominal pain and pelvic pain) compared to women without PCOS, and this prevalence varies by racial groups. Our hypothesis aims to explore the relationship between PCOS and pain, the associated health risks, and the potential racial disparities in pain prevalence and long-term health outcomes. Additionally, we seek to assess the effect of treatment on reducing pain symptoms in women with PCOS. This study not only examines the immediate burden of pain but also investigates its long-term consequences, including risks of infertility, obesity, and type 2 diabetes.

To enhance clarity for readers, we will explicitly state this hypothesis in the revised manuscript and ensure that its connection to the study’s objectives is clearly articulated. We appreciate the Reviewer’s insights and will incorporate these refinements to strengthen the manuscript.

(2) The exclusion criteria don't include conditions, that can lead to symptoms similar to PCOS: thyroid diseases, hyperprolactinemia, and congenital adrenal hyperplasia. Thyroid status is not being taken into account in the criteria for matching. All these conditions could occur as on prevalence results as on risk assessment.

We would like to thank the Reviewer for highlighting the need to include these additional conditions that mimic PCOS. After excluding hypothyroidism, hyperprolactinemia, and adrenal hyperplasia from the PCOS and PCOS and pain cohorts, we observed that 7,690 patients (1.65%) with PCOS and 1,854 patients (1.36%) with PCOS were removed. Based on this observation, we plan to add these three conditions to our exclusion criteria and rerun our analysis for disease prevalence and relative risk for our resubmission.

We will update the manuscript accordingly to reflect these exclusions and ensure clarity in our methodology. Additionally, we will discuss the rationale for excluding these conditions to improve transparency and provide a more precise interpretation of our findings.

(3) The significant weakness of the study is the absence of a Latin American cohort. Probably the White cohort includes Latin Americans or others, but the results of the study cannot be extrapolated to particular White ethnicities.

We appreciate the Reviewer’s suggestion to include Latin American cohorts in studies. In this paper we only used race as a variable and did not incorporate ethnicity. However, for our resubmission we plan to include self-reported ethnicity in our analysis which will capture the Latin American cohort stratified by self-reported race groups. This addition will provide a more comprehensive understanding of racial and ethnic differences in our study population, and we will update the manuscript accordingly to reflect this expansion.

(4) The authors didn't provide sufficient rationale for future health outcomes and this list didn't include diseases of the digestive system or disorders of thyroid glands, which can also cause abdominal pain.

We appreciate the Reviewer comment and understand their concern. Our current results highlight the prevalence of disorders of the digestive system in Figure 2 and in the results section. To further strengthen our analysis, we plan to include disorders of the digestive system in our relative risk (RR) assessment. However, we will not be able to include the same analysis for thyroid dysfunctions as they will be considered as an exclusion criterion. These updates will be incorporated into the revised manuscript to ensure clarity and completeness.

Reviewer #2 (Public review):

Summary:

The study offers a thorough analysis of the prevalence of pain in women with polycystic ovary syndrome (PCOS) and its associations with health outcomes across various racial groups. Furthermore, the research investigates the prevalence of PCOS and pain among different racial demographics, as well as the increased risk of developing various conditions in comparison to individuals who have PCOS alone.

Strengths:

The study emphasizes pain as a significant comorbidity of PCOS, an area that is critically underexplored in existing literature. The findings regarding the increased prevalence of some of the diseases in the PCOS + pain group provide valuable direction for future research and clinical care. I believe physicians should incorporate pain score assessments into their clinical practice to improve patient's quality of life and raise awareness about pain management. If future research focuses on the mechanisms of pain, it would provide a better understanding of pain and allow for a focus on the underlying causes rather than just symptomatic management. The study also highlights the association between PCOS+pain and various comorbidities, such as obesity, hypertension, and type 2 diabetes, as well as conditions like infertility and ovarian cysts, offering a holistic view of the burden of PCOS.

We sincerely appreciate the Reviewer’s insightful comments. We hope that our findings will encourage further research on the occurrence of pain in women with PCOS and that others will replicate our results to strengthen the evidence in this area. As noted in our introduction, there are currently no standardized abdominal pain score assessments specifically for women with PCOS. We hope that the findings from this study will contribute to efforts toward developing a standardized pain assessment for the PCOS community. In the meantime, further research across more diverse populations will be essential to build a more comprehensive understanding of this issue.

Weaknesses:

Due to the nature of the retrospective study, some data may not be readily available in the system. Instead of simply categorizing participants based on whether they experience pain, it would be more useful to employ a pain scale or questionnaire to better understand the severity and type of patients' pain. This approach would allow for a more thorough analysis of pain improvement following treatment with the three widely used medications for PCOS. Additionally, it would be beneficial for the authors to specify subtypes of the disease rather than generalizing conditions, such as mentioning specific digestive system disorders or mental health disorders. The lack of detailed analysis of specific disorders limits the depth of the findings. This may cause authors to make incorrect conclusions.

We appreciate the Reviewer for highlighting the importance of categorizing pain levels experienced by women with PCOS. However, there is currently no standardized pain assessment for abdominal pain, and therefore more research is required before such a classification can be made. Additionally, the electronic health record data we leveraged via the TriNextX platform does not include any pain scale data from unstructured notes. Despite these limitations, this study is an important step toward recognizing abdominal and pelvic pain in women with PCOS. Our findings indicate that women with PCOS report abdominal pain independent of digestive conditions such as irritable bowel syndrome— a condition often associated with pain in this population.

We would like to thank the Reviewer for their thoughtful comment with respect to subtyping the future health outcomes. To address this, we plan to include the most common diseases associated with PCOS for each general disease group as a supplemental figure in the revised manuscript.

This comic cleverly illustrates that this sentiment has been a recurring theme throughout history, with quotes from various eras echoing the same concerns about the accelerating pace of life. From a sociological perspective, the comic provides a lighthearted yet insightful look into the human perception of time and progress. It challenges the notion that the feeling of life speeding up is unique to the current age, suggesting instead that it is a perennial human experience. The comic invites readers to consider how technological change impacts our sense of time and our reactions to societal advancements. Discussing this comic in a course could enhance our understanding of cultural perceptions of technology and progress. It encourages a broader reflection on how each generation views the tools and technologies of its time and how these views shape societal expectations and narratives.

The history of the Luddite movement provides an interesting perspective on the ethical dilemmas of technological progress. While Luddites are often mischaracterized as simply anti-technology, their protests were actually about how industrial automation was displacing skilled workers and worsening labor conditions. This connects to modern debates about AI and automation—are we facing a new wave of "digital Luddites" as workers fear job loss due to AI-driven systems?

从你提供的文章 "The most important computer you’ve never heard of: How SAGE jumpstarted today’s technology and built IBM into a powerhouse" 中，我提取了以下关键点：

什么是 SAGE？

• SAGE (Semi-Automatic Ground Environment): 一个于 1958 年实施的，用于在冷战期间防御北美洲免受苏联轰炸机袭击的空防系统。
• 目的: 建立一个横跨美国和加拿大的大规模互联雷达阵列，连接到分布式计算机和雷达显示网络，以探测、识别和拦截潜在的敌机。

历史背景和动因

• 冷战威胁: 苏联原子弹试验和快速轰炸机的发展，使美国感到需要建立中央化的防御战略。
• 空防薄弱: 二战后美国空防力量分散，缺乏统一协调，无法有效应对高速轰炸机威胁。
• 技术挑战: 当时的技术水平难以满足构建有效中央空防系统的需求。

技术特点与创新

• Whirlwind I 计算机: SAGE 的技术基础，最初由 MIT 为海军开发的飞行模拟器，后被空军评估用于空防。
  • 实时计算机: Whirlwind I 是世界首台真正的实时计算机，能够即时响应用户操作。
  • 关键技术: 高可靠性真空管和磁芯存储器的发展使 Whirlwind I 成为可能，并提高了其速度和可靠性。
• SAGE 系统架构:
  • 区域划分: 北美分为 23 个扇区，每个扇区设有一个方向中心。
  • 层级结构: 多个方向中心数据汇集到作战指挥中心。
  • 冗余设计: 通过冗余通信链路和“交叉互告”同步协议确保系统在部分中心被摧毁后仍能运行。
• AN/FSQ-7 计算机: SAGE 系统的核心计算机，是有史以来建造的最大的计算机。
  • 双处理器: 采用双处理器结构，一主一备，提高可靠性。
  • 硬件规模: 使用 49,000 个真空管，68K 32 位磁芯内存，运行速度约为 75,000 指令/秒。
  • 存储: 使用磁鼓内存进行永久存储。
  • 实时系统: AN/FSQ-7 是真正的实时系统，能够快速处理雷达数据并做出反应。
• 人机交互界面: 每个方向中心配备约 90 个控制台，操作员通过控制台监控空域、识别不明飞行器、指挥拦截任务。
  • 图形显示: 大型屏幕用于显示空情态势。
  • 直接指令传输: 拦截指令和目标数据可以直接无线电传输到战斗机，无需语音通信。

SAGE 的运行场景 (拦截过程)

1. 雷达探测: 雷达网络探测到不明飞行器。
2. 信息传递: 雷达数据高速传输到 SAGE 方向中心。
3. 目标识别: 空军人员在 SAGE 控制台扫描空域，识别不明飞行器，判断威胁等级。
4. 拦截指令: 武器指挥员评估威胁后，武器指挥员下达拦截指令。
5. 任务分配: 拦截指挥员将目标分配给战斗机，系统自动计算最佳拦截航线和高度。
6. 数据上传: 关键目标数据无线电传输到战斗机计算机。
7. 自动驾驶拦截: 战斗机飞行员启动自动驾驶，飞机根据地面控制中心数据自动调整航向进行拦截。
8. 飞行员接管: 接近目标后，飞行员接管控制，进行武器发射。
9. 返回基地: 完成拦截后，自动驾驶系统控制战斗机返回基地。

SAGE 的影响与意义

• 技术革命的跳板: SAGE 推动了许多现代科技的发展，如：
  • 实时计算: SAGE 是最早的大规模实时计算系统之一，为后续的实时系统发展奠定了基础。
  • 计算机网络: SAGE 的互联架构预示了计算机网络的雏形。
  • 人机交互: SAGE 的控制台设计是早期人机交互的尝试。
  • 大规模软件工程: SAGE 的开发是早期大规模软件工程的实践。
• IBM 的崛起: IBM 是 SAGE 计算机 AN/FSQ-7 的主要承包商，SAGE 项目极大地提升了 IBM 的技术实力和行业地位，使其成为计算机行业的巨头。
• 对现代科技的深远影响: 文章认为 SAGE 是“你从未听说过的最重要的计算机”，它为今天的技术奠定了基础，尽管其本身已成为“古代历史”。

总结

SAGE 不仅仅是一个军事项目，更是一项技术奇迹，它在冷战背景下诞生，为了应对当时的空防威胁而建立，却意外地推动了计算机技术和相关领域的飞速发展，为我们 आज के 技术世界 奠定了重要的基石。

从你提供的文章 "J. C. R. Licklider and the Birth of Cloud Computing: How His Vision Changed the World" 中，我提取了以下关键点：

• J.C.R. Licklider 是云计算的先驱： 文章强调 Licklider 是一个有远见的计算机科学家和心理学家，他在云计算的早期概念发展中扮演了重要角色，尽管当时的云计算尚未被命名。

• “星际计算机网络” (Intergalactic Computer Network) 是云计算的早期构想： Licklider 在 1960 年代在 MIT 提出了 "星际计算机网络" 的概念。这个概念设想了一个全球性的计算机网络，允许世界各地的人们从任何地点访问计算能力和信息。

• 云计算的民主化愿景： Licklider 设想的系统允许多台计算机联网，用户无需拥有自己的计算机，即可从任何地点访问计算资源和信息。他认为这能实现计算的民主化，使任何需要的人都能使用计算能力。

• 云计算概念的超前性： Licklider 的想法非常超前，他的云计算愿景在几十年后才成为现实。然而，他的工作为互联网的发展奠定了基础。

• 云计算的普及和重要性： 如今，云计算已成为一项普遍使用的技术，被个人、企业和政府广泛采用。它允许用户将数据和应用程序存储在远程服务器上，并通过互联网连接的任何设备进行访问，这极大地降低了计算能力和信息访问的门槛，并促进了新技术和商业模式的出现。

• Licklider 对互联网和现代计算的贡献： Licklider 在云计算方面的工作是互联网和现代计算发展中的关键贡献。他的想法持续影响着我们对技术及其社会角色的思考。

• Licklider 的遗产： 文章赞扬 Licklider 的遗产，认为他的远见卓识、创新精神以及对互联互通、民主世界的愿景在今天比以往任何时候都更加重要。他的故事证明了想象力和创新的力量。

总而言之，这篇文章的核心论点是： J.C.R. Licklider 通过他提出的 “星际计算机网络” 概念，成为了云计算的先驱，他的超前 vision 为互联网和现代云计算奠定了重要的思想基础，并深远地影响了我们今天数字世界的发展。

This statement underscores the need for users to understand not only the functionality of social media platforms but also the psychological impacts these platforms can have, how personal data is utilized, and the tactics behind user interactions, including negative phenomena like harassment and the presence of spam bots. From a practical perspective, the demand for such a high level of digital literacy highlights a gap in public education regarding technology. Most users are not naturally equipped with the knowledge of how algorithms influence what they see online or how their data can be harvested and used—often without explicit consent. This issue ties into broader societal and educational discussions about the responsibilities of social media platforms to ensure their users are well-informed. It challenges us to consider whether enough is being done to educate the public about the digital spaces they inhabit daily. By integrating this topic into course discussions, we can explore how education systems and policy reforms can better prepare individuals to engage with social media responsibly and safely.

O requerimento de limitação de litisconsórcio é cabível em qualquer fase do processo.

Bien sûr. Voici un sommaire des principaux points abordés dans la vidéo "Y a-t-il un nombre de personnes idéal pour une réunion ?". Veuillez noter que le transcript ne contient pas d'indications de timestamps.

• La fréquence et la perception négative des réunions en France sont introduites, soulignant qu'on les organise et y assiste souvent sans en percevoir clairement l'utilité.
• La question de la taille idéale pour une réunion est posée, avec l'idée qu'au-delà d'un certain nombre, elle se transforme en spectacle où peu de personnes participent activement.
• Il est suggéré qu'un groupe de 4 à 7 personnes est généralement plus efficace pour une réunion, permettant une meilleure participation de chacun et bénéficiant de "la sagesse des fous". Au-delà de ce nombre, les participants peuvent devenir passifs, et en dessous, la puissance du groupe est limitée.
• L'importance de l'ordre du jour est soulignée, qui doit être pertinent pour toutes les personnes présentes. Un grand nombre de participants peut rendre l'ordre du jour plus lent et mener à une succession de "mini-réunions" au sein de la même séance.
• L'état de la réunion influence la prise de parole. Des facteurs comme l'identité commune, la sécurité psychologique pour exprimer des opinions négatives, le partage des responsabilités, du stress et des ressources, ainsi que la justesse (fairness) sont cruciaux.
• L'autocensure, particulièrement en présence de supérieurs hiérarchiques, est identifiée comme un obstacle majeur à l'efficacité des réunions. Les personnes ayant une anxiété sociale peuvent préférer ne rien dire en réunion, même si elles ont des idées pertinentes.
• Des conseils pour améliorer les réunions sont donnés : laisser les personnes les plus susceptibles de s'autocensurer parler en premier, faire parler les supérieurs hiérarchiques en second, et diviser les grands groupes en plus petits pour des discussions séparées avant de mettre en commun. Il est également important de considérer les conséquences de l'expression libre.
• En conclusion, la meilleure réunion est potentiellement celle qui n'a pas lieu. Les réunions devraient être utilisées avec parcimonie pour préparer l'action, en évitant de confondre le "mouvement" (réunions) avec l'action concrète. Il est crucial de concrétiser les décisions prises en réunion et de passer à la pratique.

I don't see the Overton window moving in a straight line. Sometimes, changes happen abruptly, which means that social changes don't always occur gradually but in bursts, depending on the circumstances.

I think this shows the ability of media and think tanks to shift the Overton window by reframing concepts in ways that make them more palatable to accept. It does not seem to be merely a matter of shifting the window by attempting to push the ends in, but of shifting perceptions of certain questions.

Briefing Document : Les Pièges de l'Objectivation Excessive et des KPI Source : Excerpts from "Et si vos objectifs... vous empêchaient de réussir ? | Psycho-Boulot"

Date : (Date de publication non spécifiée dans l'extrait)

Thèmes Principaux :

La prolifération des KPIs (Key Performance Indicators) : Leur omniprésence dans divers secteurs et l'intention de mesurer objectivement la performance, le rendement, et les interactions.

La Loi de Goodhart : Le danger de transformer une mesure en cible, ce qui la rend inefficace et peut entraîner des comportements contre-productifs. La Loi de Campbell : Les difficultés et les conséquences négatives de vouloir objectiver quantitativement des aspects qualitatifs.

La dégradation de la qualité et des processus : Comment la focalisation excessive sur les KPIs peut nuire à la qualité du service, aux relations et à l'atteinte des objectifs réels.

L'importance du qualitatif et de la subjectivité : La nécessité de compléter les mesures quantitatives par des approches qualitatives et de reconnaître la valeur du jugement subjectif.

Analogie avec le système éducatif : Comment l'obsession de la note peut nuire à l'apprentissage réel. Idées et Faits Importants :

Définition et omniprésence des KPIs : Les KPIs sont présentés comme des mesures destinées à quantifier la performance et divers aspects au sein des entreprises et organisations, touchant des secteurs variés comme la restauration, les hôpitaux et les ressources humaines. L'objectif initial est d'améliorer l'efficience et l'efficacité, et potentiellement de récompenser ou punir en fonction des résultats.

"est-ce que vous connaissez les KPI les KPI les key performance indicator si vous connaissez pas c'est pas grave je connaissais pas non plus je viens de chercher sur google c'est quoi KPI mais c'est quelque chose qu'on a vu apparaître dans plein d'entreprises dans plein de secteurs [...] c'est des sortes de mesures pour mesurer des choses dans une entreprise la performance combien est-ce que les gens s'entendent entre eux le rendement les pertes tout est mesuré tout est objectivé tout est calculé pour essayer de maximiser l'efficience l'efficacité et pouvoir récompenser ceux qui ont de bons KPI et potentiellement punir ceux qui en ont de mauvais"

La Loi de Goodhart : Quand la mesure devient la cible, elle cesse d'être une bonne mesure. L'auteur introduit cette loi fondamentale pour expliquer pourquoi l'objectivation à outrance peut être contre-productive. "la loi de goodhart qui dit que quand une mesure devient la cible elle arrête d'être une bonne mesure"

Exemple concret dans un café : Pour illustrer la Loi de Goodhart, l'auteur prend l'exemple d'un café où les employés sont récompensés pour la rapidité du service. Cette mesure, initialement destinée à améliorer l'efficacité, conduit à une dégradation de la qualité du service (erreurs, café moins bien infusé, manque d'amabilité) car l'objectif devient de maximiser le score de rapidité plutôt que de fournir un bon service.

"problème c'est que automatiquement la loi de goodhart est venu se meler à cette histoire et il y a eu une sorte de course non pas à avoir un bon service avec les clients mais une course à servir aussi vite que possible pour avoir le meilleur score pour être compenser à la fin du mois et de manière quasi mécanique si moi je suis en train de servir aussi vite que possible je vais faire plus d'erreur je vais peut-être laisser le café infuser un peu moins de temps nettoyer la machine un peu moins bien passer un peu moins de temps à être souriant parce que je veux juste passer à la personne d'après et le simple fait d'avoir implémenté ces mesures de KPI dans ce dans cette cafette ou dans ce café a fait que le service s'est dégradé"

Exemple de la pandémie et des tests COVID : L'auteur cite l'exemple du gouvernement britannique fixant un objectif de 100 000 tests COVID par jour. Bien que l'objectif ait été atteint en termes de nombre, la qualité des tests a pu être compromise, rendant la mesure moins fiable pour évaluer la situation réelle.

"par exemple un exemple qui est maintenant souvent cité sur la loi de goodhart c'est pendant la pandémie le gouvernement britannique avait annoncé de vouloir faire 100000 tests covid par jour pour essayer de faire une sorte de diagnosticque généralisé c'était ça la cible et là aussi dès qu'on met une mesure c'est la mesure qui devient la cible et non pas la tâche qu'on veut mesurer et dans ce cas-là beaucoup de tests étaient fait à l'arrache à la va vite sans vraiment bien les faire et donc on avait des fut positifs des fois négatifs même si le gouvernement était très fier de dire on a atteint notre cible en fait en réalité c'était pas les 100000 tests qui étaient bien faits"

La Loi de Campbell : Objectiver le qualitatif mène à la détérioration. Cette loi complémentaire met en lumière les difficultés et les conséquences négatives de vouloir mesurer de manière quantitative des aspects intrinsèquement qualitatifs comme le bonheur au travail ou la relation client.

"la loi de Campbell dit que quand on veut objectiver de manière quantitative des problématiques qualitatives et ben les deux se détériore quand vous voulez évaluer le bonheur au travail et vous voulez essayer de l'objectiver quand vous voulez évaluer votre relation client et voulez l'objectiver c'est des mesures qui ne sont pas aussi simples à capturer en statistique brut et vous exposer à dégrader aussi bien vos processus que les relations que vous avez avec ces personnes"

Recommandations : Privilégier le qualitatif et la prudence dans la mesure. L'auteur ne préconise pas l'abandon total de la mesure, mais insiste sur la nécessité d'être prudent et de ne pas être obsédé par les chiffres. Il suggère de renforcer les approches qualitatives et de solliciter le feedback direct des personnes concernées.

"la première chose que vous pouvez faire au lieu d'être obsédé un peu par les chiffres par les States en sachant que les States peuvent mentir aussi très bien c'est de faire plus de qualitatif au lieu de vouloir tout le temps faire du quantitatif de demander aux gens comment est-ce que ça va pas tout doit être mesuré cette cette obsession de la mesure n'est pas nécessairement une bonne chose et c'est pas parce que vous avez des données que vous allez pouvoir mieux décider"

Analogie avec l'éducation : La note comme cible au détriment de l'apprentissage. L'auteur établit un parallèle avec le système éducatif où la note devient souvent l'objectif principal, encourageant des comportements comme le bachotage au détriment d'un apprentissage profond et durable. Cela explique potentiellement pourquoi beaucoup de connaissances acquises à l'école sont rapidement oubliées.

"on retrouve la loi de goodhart d'ailleurs même dans notre enfance on a tous été étudiant à un moment ou un autre de notre vie et un des gros problèmes de l'école c'est qu'au lieu de récompenser l'apprentissage on veut récompenser les notes et donc qu'est-ce qui se passe j'ai l'examen d'histoire géo qui arrive dans dans 2 jours je suis pas vraiment à jour dans mon histoire géo c'est pas je suis pas à jour dans mon apprentissage de l'histoire géo parce que je veux l'apprendre si je suis pas à jour dans l'ingurgitation des chapitres pour pouvoir avoir une bonne note et donc je vais faire du bachotage je vais aller regarder les examens de l'année passée et me dire ah le prof a mis la guerre de 14 18 l'année passée donc si ça se trouve cette année ça va être la deuxième guerre mondiale donc je peux ne pas étudier ça parce que c'est la mesure qui devient l'objet le but c'est d'avoir une bonne note indépendamment de si j'ai appris ou pas"

Conclusion : Éviter d'infantiliser et reconnaître la valeur de la subjectivité. L'auteur encourage à traiter les collaborateurs comme des professionnels et à ne pas les réduire à de simples scores. Il plaide pour une reconnaissance de la valeur du jugement subjectif et pour un éloignement de la volonté de tout objectiver.

"essayez de ne pas infantiliser les gens avec qui vous travaillez de ne pas les traiter un peu comme des écoliers à l'école qui doivent juste avoir une bonne note parce que dans le monde réel c'est pas la note qui compte mais c'est le travail qu'on fait et donc éloignez-vous un peu de cette volonté de vouloir tout objectiver et dites-vous que peut-être un monde subjectif n'est pas si mauvais que ça" Implications et Points de Vigilance :

Lors de la mise en place de systèmes de mesure de la performance (KPIs), il est crucial de réfléchir aux comportements induits et de s'assurer que la mesure ne devienne pas l'objectif au détriment de la qualité et des objectifs réels.

L'objectivation excessive des aspects qualitatifs peut entraîner une perte de sens et une dégradation des relations humaines et des processus.

Une approche équilibrée, combinant des données quantitatives et des informations qualitatives (feedback, observations), est essentielle pour une évaluation plus juste et efficace.

Il est important de communiquer clairement les objectifs et les valeurs de l'organisation pour éviter que les individus ne se concentrent uniquement sur l'atteinte des KPIs au détriment d'autres aspects importants.

S'inspirer de l'analogie avec l'éducation rappelle l'importance de favoriser un apprentissage et un engagement réels plutôt qu'une simple course aux "bonnes notes" ou aux bons scores.

Voici un sommaire des principaux points abordés dans la vidéo "Et si vos objectifs... vous empêchaient de réussir ? | Psycho-Boulot" :

• Introduction des KPI (Key Performance Indicators) comme mesures de performance utilisées dans de nombreuses entreprises et secteurs. Les KPI visent à objectiver et calculer des aspects comme la performance, l'entente entre les personnes, le rendement et les pertes pour maximiser l'efficience et récompenser ou punir en conséquence.

• Présentation de la loi de Goodhart qui stipule que lorsqu'une mesure devient la cible, elle cesse d'être une bonne mesure.

• Illustration de la loi de Goodhart avec l'exemple concret d'un magasin de café où la mesure de l'efficacité des employés basée sur la rapidité du service a entraîné une dégradation de la qualité du service. Les employés se sont concentrés sur la vitesse au détriment de la qualité (préparation du café, nettoyage, relation client) pour obtenir de meilleurs scores.

• Autre exemple de l'application de la loi de Goodhart pendant la pandémie, où l'objectif du gouvernement britannique d'effectuer 100 000 tests COVID par jour a conduit à la réalisation de tests parfois bâclés, la cible (le nombre de tests) devenant plus importante que la qualité des tests.

• Introduction de la loi de Campbell, qui explique que lorsque l'on cherche à objectiver quantitativement des problématiques qualitatives, les deux se détériorent. Tenter de mesurer objectivement le bonheur au travail ou la relation client peut ainsi dégrader ces aspects.

• Suggestion de ne pas être obsédé par les chiffres et de privilégier davantage le qualitatif en demandant aux gens comment ils vont. L'obsession de la mesure n'est pas toujours bénéfique, et les données ne garantissent pas toujours de meilleures décisions.

• Application de la loi de Goodhart au système éducatif, où l'accent mis sur les notes plutôt que sur l'apprentissage réel conduit les étudiants à bachoter pour obtenir de bonnes notes sans forcément acquérir une compréhension profonde. Ce manque d'apprentissage pour le contenu pourrait expliquer l'oubli des connaissances acquises à l'école.

• Recommandation de ne pas infantiliser les employés en les traitant comme des écoliers focalisés sur les notes, car dans le monde du travail, c'est la qualité du travail qui compte.

• Conclusion encourageant à s'éloigner de la volonté de tout objectiver et à accepter qu'un monde plus subjectif n'est pas nécessairement négatif.

how do you define the trl?

https://www.theguardian.com/environment/2025/mar/12/global-weirding-climate-whiplash-hitting-worlds-biggest-cities-study-reveals

Some student asked why it is w.h.p. here. The proof only shows with constant probability

This passage highlights how even well-intentioned technological advancements can have unintended and often harmful consequences. It raises an important ethical question: should inventors be held responsible for the negative impacts of their creations, or is it impossible to predict how technology will evolve? This dilemma is especially relevant in modern tech, where innovations like AI and social media algorithms are shaping society in ways their creators may not have fully anticipated.

DOI: 10.4162/nrp.2018.12.3.208

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.3389/fmicb.2018.01052

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.18632/oncotarget.24834

Resource: RRID:BDSC_35835

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_35835

What is this?

DOI: 10.1534/genetics.118.300935

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/genetics.118.300808

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1534/g3.118.200260

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.15252/embj.201797741

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_67065

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_67065

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_60120

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_60120

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_57316

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_57316

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_57314

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_57314

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_36304

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_36304

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_55866

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_55866

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_55359

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_55359

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_67334

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_67334

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_54461

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_54461

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_53884

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_53884

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_67231

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_67231

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_53321

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_53321

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_66341

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_66341

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_41944

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_41944

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_65175

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_65175

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_40859

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_40859

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_34329

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_34329

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_65014

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_65014

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_31766

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_31766

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_29453

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_29453

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_28355

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28355

What is this?

DOI: 10.1371/journal.ppat.1007050

Resource: RRID:BDSC_67067

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_67067

What is this?

DOI: 10.1371/journal.pone.0198161

Resource: RRID:BDSC_57300

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_57300

What is this?

DOI: 10.1371/journal.pone.0198161

Resource: RRID:BDSC_7415

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_7415

What is this?

DOI: 10.1371/journal.pone.0198161

Resource: RRID:BDSC_31393

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_31393

What is this?

DOI: 10.1371/journal.pone.0197822

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.204266

Resource: (Zebrafish International Resource Center Cat# zn-5, RRID:AB_10013770)

Curator: @bandrow

SciCrunch record: RRID:AB_10013770

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1242/dev.165985

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s12864-018-4755-1

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s12864-018-4755-1

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1155/2018/5065190

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1111/cge.14732

Resource: Zebrafish International Resource Center (RRID:SCR_005065)

Curator: @bandrow

SciCrunch record: RRID:SCR_005065

What is this?

DOI: 10.1101/2025.02.18.638841

Resource: None

Curator: @bandrow

SciCrunch record: RRID:ZIRC_ZL10486.01

What is this?

DOI: 10.1101/2025.02.18.638841

Resource: (ZIRC Cat# ZL1,RRID:ZIRC_ZL1)

Curator: @bandrow

SciCrunch record: RRID:ZIRC_ZL1

What is this?

DOI: 10.1101/2025.02.15.638407

Resource: (ZIRC Cat# ZL1,RRID:ZIRC_ZL1)

Curator: @bandrow

SciCrunch record: RRID:ZIRC_ZL1

What is this?

DOI: 10.1101/2025.02.13.638191

Resource: Zebrafish International Resource Center (RRID:SCR_005065)

Curator: @bandrow

SciCrunch record: RRID:SCR_005065

What is this?

DOI: 10.1096/fj.202401875R

Resource: Zebrafish International Resource Center (RRID:SCR_005065)

Curator: @bandrow

SciCrunch record: RRID:SCR_005065

What is this?

DOI: 10.1093/hmg/ddy200

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/hmg/ddy200

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/hmg/ddy200

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/hmg/ddy190

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/hmg/ddy190

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/hmg/ddy190

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1093/hmg/ddy190

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1083/jcb.201708096

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-025-88866-9

Resource: Zebrafish International Resource Center (RRID:SCR_005065)

Curator: @bandrow

SciCrunch record: RRID:SCR_005065

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_63041

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_63041

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_55135

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_55135

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_9972

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_9972

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_9970

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_9970

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_23129

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_23129

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_42747

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_42747

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_30026

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_30026

What is this?

DOI: 10.1038/s41598-018-26825-3

Resource: RRID:BDSC_26818

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_26818

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26364-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-26294-8

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-018-25382-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28260

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28260

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25187

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25187

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_55031

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_55031

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28243

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28243

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28224

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28224

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_55026

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_55026

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28222

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28222

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_29652

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_29652

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28206

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28206

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_29651

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_29651

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28202

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28202

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28275

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28275

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28265

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28265

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28153

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28153

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28123

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28123

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_28262

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_28262

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25208

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25208

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25199

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25199

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25194

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25194

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25193

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25193

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25186

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25186

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25185

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25185

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_25174

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_25174

What is this?

DOI: 10.1038/s41588-018-0140-x

Resource: RRID:BDSC_42272

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_42272

What is this?

DOI: 10.1038/s41556-018-0106-3

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41556-018-0106-3

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41556-018-0106-3

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1016/j.neuron.2018.05.014

Resource: RRID:BDSC_34393

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_34393

What is this?

DOI: 10.1016/j.jneumeth.2018.05.014

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1016/j.devcel.2018.04.020

Resource: RRID:BDSC_33971

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_33971

What is this?