In vitro work revealed that excitatory synaptic inputs to hippocampal inhibitory interneurons could undergo Hebbian, associative, or non-associative plasticity. Both behavioral and learning-dependent reorganization of these connections has also been demonstrated by measuring spike transmission probabilities in pyramidal cell-interneuron spike cross-correlations that indicate monosynaptic connections. Here we investigated the activity-dependent modification of these connections during exploratory behavior in rats by optogenetically inhibiting pyramidal cell and interneuron subpopulations. Light application and associated firing alteration of pyramidal and interneuron populations led to lasting changes in pyramidal-interneuron connection weights as indicated by spike transmission changes. Spike transmission alterations were predicted by the light-mediated changes in the number of pre- and postsynaptic spike pairing events and by firing rate changes of interneurons but not pyramidal cells. This work demonstrates the presence of activity-dependent associative and non-associative reorganization of pyramidal-interneuron connections triggered by the optogenetic modification of the firing rate and spike synchrony of cells. It is increasingly recognized that plastic reorganization of brain circuits needed for learning and other cognitive functions involves not only principal cells but also local inhibitory interneurons . A largeIn vivo work, primarily during anesthesia, has also demonstrated that plastic alterations can occur between afferent excitatory fibers and CA1 interneurons . This woIn this study, we optogenetically interfered with the circuit function by activating Halorhodopsin or Archaerhodopsin in a subpopulation of pyramidal cells and interneurons. This manipulation led to the inhibition of a subset of pyramidal cells and interneurons and also light-triggered disinhibition of many pyramidal cells . Here, wWe recorded multiple unit and field potential activities from the dorsal hippocampus in five rats, during exploration and quiet immobility sessions. In these rats, Halorhodopsin  or Archaerhodopsin  was expressed in the dorsal CA1 region of the hippocampus under the control of the CaMKIIα promoter using an adeno-associated virus (AAV2/1). In four rats, fifteen independently-movable tetrodes and one 200 µm optic fiber centered in the middle of the tetrode bundle targeted the dorsal CA1 region while, in the remaining animal, 24 tetrodes and four optic fibers were used . We recorded during four 25 min exploration sessions in which first a familiar environment (FAM1) was explored followed by a novel environment (NOV), and finally, the animals were returned to the familiar environment for the remaining two sessions. During the second familiar exploration session laser stimulation was applied (FAML) in a fixed part of the environment but not in the last exploration , indicating that a more lasting change had also occurred. We set out to determine the factors that accounted for the connection strength observed in FAM2 by testing at the population level whether spike transmission in FAM2 was predicted by that observed in the previous sessions. In addition, we controlled for the possible variability across animals by including animal identity as a variable into the analysis . Spike tThe significant influence of the FAML session on FAM2 suggests that the light-induced alterations of the network activity led to lasting changes in the spike transmission probabilities even in the absence of light in the same environmental context. Direct optogenetic inhibition of some cells and the indirect firing increase of others led to either an increase or a decrease of spike transmission probabilities during the light application. Therefore, next, we tested whether the direction of change in spike transmission in FAML relative to FAM1 predicted similar change from FAM1 to FAM2 . Indeed,The light application can change the firing rate of the presynaptic pyramidal cell or the postsynaptic interneuron, which may, in turn, account for the observed plastic changes in transmission probability. Therefore, to address whether our effects reflected a pre- or postsynaptic mechanism, we assessed the relationship between firing rate changes within connected pairs and the modification of their monosynaptic connection. To do this, we calculated the relative firing rate changes between FAML-FAM1 for both pyramidal cells and interneurons, a measure that reflects the influence of light on the baseline firing of these cells. We then analyzed whether this measure predicted changes in transmission probabilities between FAM1 and FAM2, which reflects the longer-lasting change in synaptic strength. As a control, we also measured relative firing rate changes between FAM2-FAM1 because rate alterations reflecting the alterations in the average excitatory inputs cells received. Such changes in excitability may have influenced spike transmission beyond the rate alteration-mediated changes of the chance joint firing probability, which later were already compensated for by normalizing the histograms . We founThe finding that interneuron rate change between FAM2-FAM1 itself independently influenced FAM2-FAM1 spike transmission changes suggests that the excitability of the postsynaptic interneuron has a strong influence on the strength of spike transmission. Spike transmission changes we detected in the NOV session may, in part, have been caused by the excitability change of the interneurons. To test whether rate changes of pyramidal cells or interneurons in NOV influenced spike transmission in FAM2, we calculated normalized (difference divided by the sum) spike transmission and rate changes, in order to predict weight change separately for both cell types . As befoNext, we examined whether the direction of firing rate change during light application influenced the spike transmission change between FAM1 and FAM2 . As befo2>0.859) by pyramidal and interneuron firing rates, explaining why spike transmission changes could not be predicted independently from the combined interneuron and pyramidal rates by spike pairing.We found that the modulation of interneuron activity during light stimulation or exposure to a novel environment directly influenced changes in transmission probabilities between putative monosynaptic connections of pyramidal-interneuron cell pairs. This raises the possibility that such changes are activity-dependent. To examine this, we measured the number of instances in which pyramidal cell firing occurred in 10 ms, 20 ms, 50 ms, and 100 ms time windows before or after the interneuron spike as well as their sum for all spike-pairing events . The relHere we showed that light-induced, optogenetic alterations of the CA1 network activity can trigger lasting alterations of the monosynaptic spike transmission probability of pyramidal-cell interneuron pairs. During the light session, both changes in the postsynaptic interneuron rates and the pairing probability of cell firing predicted the changes in monosynaptic spike transmission within the same familiar environment, when sessions before and after the light interference were compared. In addition, we observed spike transmission changes in the novel environment relative to the familiar environment, which took place before the light application. These changes were specific to the novel environment, however, and were not maintained during the subsequent familiar sessions. This suggests that altered interneuron firing rate and the activity-dependent alterations of the pyramidal-interneuron spike pairing can modify pyramidal-interneuron connection weights during exploratory behavior. Our study did not use control animals in which only YFP was expressed. Therefore, we cannot exclude the possibility that optogenetic channel expression, or, perhaps, light application enhanced the plasticity on pyramidal-interneuron synapses. Yet, we observed similar activity-dependent changes during spatial learning before . So, it We used a measure of spike transmission probability that compensated for the changes in the chance probability that the two cells fire together as a result of firing rate alterations. However, the average depolarization level of the cell, as reflected by its mean firing rate, can lead to more efficient spike transmission, even without changes in the synaptic weight. Indeed, the spike transmission changes from FAM1 to FAM2 were influenced by the rate changes of the postsynaptic interneuron between these sessions. In turn, this suggests that the postsynaptic interneuron’s general level of depolarization can influence spike transmission. However, in a similar manner, changes in FAML-FAM1 interneuron rate also predicted FAM2-FAM1 spike transmission changes. Indeed, when FAML interneuron rate increased, a stronger spike transmission was seen subsequently in FAM2, while reduced spike transmission was associated with reduced interneuron rate. Considering that the interneuron rate increase in FAML was not directly mediated by the light, we cannot exclude the possibility that stronger pyramidal inputs caused the interneuron rate increase in FAML that is caused by the plastic strengthening of these connections. Nevertheless, the excitability of interneurons in FAM2 alone, which may be indicative of plasticity-mediated input to interneurons, did not explain the spike transmission changes. Indeed, rate changes in FAML could predict spike transmission changes in FAM2-FAM1 independent of FAM2-FAM1 rate changes. That is, FAML-FAM1 rate changes predicted FAM2-FAM1 spike transmission changes even when the FAM2-FAM1 interneuron rate changes were accounted for. Therefore, FAML-FAM1 interneuron rate changes had further predictive value beyond those observed by FAM2-FAM1 rates changes and consequently excitability/depolarization alterations in FAML that were no longer present in FAM2 still predicted FAM2-FAM1 spike transmission changes. This finding indicates that interneuronal excitability changes during light application session caused lasting changes of the pyramidal interneuron connections, beyond any lasting nonspecific excitability changes that occurred between FAM2-FAM1.We observed changes in spike transmission between FAM1 and the novel environment, which were larger in amplitude than those across the familiar environment before and after the light application. Exposure to the novel environment nevertheless did not influence changes in the familiar environment. Can we expect that pyramidal-interneuron weights change from one environment to another but they revert to the previous configuration when the animal is returned to the first environment? Although we cannot exclude this possibility, it is more likely that the average depolarization level of each interneuron is different across different environments, which in turn reveals different monosynaptic connections and connection strengths. Interneuron rates reorganize across the familiar and novel environment, which may reflect the influence of different nonspecific neurotransmitters . Moreove++ permeable AMPA receptors mediate the primary form of LTP and LTD. This form of plasticity requires the stimulation of their synaptic inputs and the hyperpolarization or a non-depolarized state of the interneuron  and the remaining animal was injected with a recombinant adeno-associated virus to express Archaerhodopsin (ArchT) in the dorsal CA1 area (AAV2/1.CaMKII::ArchT.GFP.WPRE.SV40 (12 genome copies per mL). The virus was injected at four sites into dorsal CA1 of the right hemisphere in four rats and bilaterally in one rat: site 1: −3.0 AP, ±2.2 L, 2.1 DV; site 2: −3.7 AP, ±2.9 L, 2.0 DV; site 3: −4.3 AP, ±3.5 L, 2.0 DV; site 4: −5.0 AP, ±4.2 L, 2.2 DV. 3.5 weeks after virus injection, animals were implanted with 15 (28 in one rat) independently movable wire-tetrodes under deep anesthesia using isoflurane (0.5–2%), oxygen (1–2 L/min) and an initial dose of buprenorphine (0.1 mg/kg). Tetrodes were attached to the 15-tetrode (24-tetrode and 4-octrode in one rat) microdrive assemblies, enabling their independent movement. The tetrodes were constructed from four individual tungsten wires, 12 µm in diameter , twisted and then heated to bind them into a single bundle. The tips were then gold plated to reduce their impedance to 200–300 kΩ.Four male adult rats  were injected with a recombinant adeno-associated virus to express Halorhodopsin-YFP in the dorsal CA1 area  located in the center of the tetrode array was used to apply laser light directly to the dorsal CA1 area. During surgery, a craniotomy was prepared above the dorsal hippocampus centered at AP = −4.0; ML = ± 3.0. Two stainless steel screws inserted through the skull above the cerebellum served as ground and reference electrodes, and six additional screws were used to permanently attach the microdrive assembly to the skull. Implantation was performed such as to position the tip of the optic fiber at a depth of 1.7 mm. The paraffin-wax coated electrodes and microdrives were then daubed with bone cement to encase the electrode-microdrive assembly and anchor it to the screws in the skull. Following a recovery period of 7 days, the tetrodes were lowered to their target locations over a further period of around 7 d. Tetrode locations were identified by electrophysiological markers such as theta band power, sharp wave polarity, and the presence of ripple oscillations, and by extrapolating the location of the electrodes by tracing the distances back along each electrode tract according to the daily advancement of the recorded electrodes. Implanted animals were housed individually in a separate room under a 12 hr light/12 hr dark cycle with ad libitum access to water, and they were maintained in a food-deprived state between 85–90%  of their post-operative weight. Experiments were performed during the light phase. All rats used in this study were naïve and not used for additional procedures before surgery.All procedures involving experimental animals were carried out in accordance with Austrian  animal law under a project license  approved by the Austrian Federal Science Ministry (BMWFW).32-channel unity-gain preamplifier panels  were used to reduce cable movement artifacts. Wide-band (0.1/1 Hz – 5 kHz) recordings were taken, and the amplified local field potential and multiple-unit activity were continuously digitized at 24 kHz using a 128-channel data acquisition system . Two red LEDs mounted on the preamplifier headstage were used to track the location of the animal.Green/yellow laser light for NpHR activation was provided by a 561 nm DPSS laser system equipped with an acousto-optic modulator (Omicron). The light was coupled into an optic fiber (four optic fibers in one rat) connected to a fiber-optic rotary joint (Doric lenses) from where a 200 μm/0.48 NA patch cord transmitted the light to the microdrive. Laser intensity was set to reach 25 mW total power at the tip of every implanted fiber stub. Data were recorded 6–7 weeks after AAV injection to ensure sufficient NpHR-YFP/ArchT GFP expression levels.Data was recorded while the animals explored different arenas or rested in a sleep box. The sleep box was small (20 cm × 27 cm) with 60 cm high walls and cushioned with a terry towel for the animal to sleep/rest comfortably. During training and electrode positioning, the animals were familiarized with a 120 cm circular environment with 20 cm high walls (minimum of 60 min of exposure per day for at least seven days) that served as the familiar arena in all experiments. Curtains were used to enclose this arena and provide a stable set of external cues. In all exploration sessions, small food pellets were dropped at random from an automated overhead system (2–3/min) to motivate the animals to explore the entire arena. For recordings in a novel environment, several other arenas with different sizes, shapes, and textures were used. In addition, curtains were opened to provide novel distal room cues.Typical recording days consisted of 10 sessions: four exploration sessions flanked by five sleep sessions and a final test session where brief laser pulses were applied while the animal still rested in the sleep box. Typically, sleep and exploration sessions lasted 25 min, whereas the laser test session lasted 18 min. The animals first explored the familiar arena. After visiting a different novel arena, the familiar arena was explored again, but laser illumination was automatically triggered when the animal entered a specific part of the arena (light zone). Finally, the same arena was explored again. All exploration sessions were flanked by sleep. The light zone was defined by a center position and an angle between 120° and 180° such that it covered one-third to half of the arena. The initial angle defining the illumination zone was random and thus random also with respect to the hippocampal place fields. Every day, a novel illumination zone that had about 50% overlap with the previous day’s zone was defined. During the course of the project and also within individual animals, the angle defining the size of the illumination zone was increased to include more place fields in the light zone. After completion of the experiments, the rats were deeply anesthetized and perfused through the heart first with PBS followed by a 4% buffered formalin phosphate solution for the histological verification of electrode tracks and optic fiber position. Furthermore, NpHR-YFP/ArchT-GFP expression in dorsal CA1 was verified in each animal by checking the fluorescence of the YFP/GFP tag.http://klustakwik.sourceforge.net/). Finally, the generated clusters were manually refined by a graphical cluster cutting program. Only units with clear refractory periods in their autocorrelation and well-defined cluster boundaries were used for further analysis. Periods of waking spatial exploration, immobility, and sleep were clustered together. The stability of the cells was verified by plotting spike features over time. In addition, an isolation distance  was calIsolation of monosynaptically-connected pyramidal cell-interneuron pairs was performed as described previously by identifying cross-correlograms between pyramidal cells and interneurons that exhibited a large, sharp peak in the 0.5–2.5 ms bins  . BecauseTo compare firing rates between two sessions, we calculated the relative firing rate change by dividing the signed difference between the mean firing rates by the sum of the mean rates (i.e. c=(r2-r1)/(r2+r1)), where r1 and r2 denote the mean firing rates in the two sessions that are compared . This scWe used linear mixed models and ANOVA to determine the significance of variables in predicting spike transmission probabilities and their changes. We used a mixed model comparison to test whether predictions were independent of other variables and displayed corresponding partial correlations in the figures. We added the animal variable as a random effect to all linear mixed models to account for variability across animals and used the comparison of linear mixed model and linear model without an animal variable to test whether an animal variable contributed significantly to the prediction of spike probability and their changes. We used Holm-Bonferroni multiple testing correction to account for comparisons in multiple time windows in the analysis with prediction through pairing. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study investigates into changes in pyramidal cell and interneuron spike transmission probability in pyramidal cell-interneuron monosynaptic pairs in hippocampal area CA1 following optogenetic inhibition and disinhibition of some of these cells in behaving rats exploring novel and familiar environments. Insights regarding how cell-cell correlations change with experience in freely behaving animals add to our understanding of how hippocampal circuits support learning and memory. The work also changes the current thinking about processes of plasticity in hippocampal networks and has relevance for interpretation of datasets in which optical inhibition is applied.Decision letter after peer review:eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Michael Frank as the Senior Editor.Thank you for sending your article entitled "Optogenetic inhibition-mediated activity-dependent modification of CA1 pyramidal-interneuron connections during behavior" for peer review at The reviewers were all generally positive about the manuscript but also felt that it has a number of major issues in its current form. The lack of a YFP only control group was a major concern. Without an appropriate control group, the authors are unable to definitively state that their effects are due to optogenetic manipulations . However, the reviewers agreed in the consultation and discussion that they would not require the authors to include this optimal control group, assuming that the authors are able to demonstrate in some other way that the reported effects are actually due to light stimulation. One suggestion was offered by reviewer 2 (point 4). Another suggestion that came up in the Discussion was to compare the first and second halves of light-free sessions . Another major concern was the lack of cell-type specificity of the CaMKII promoter (see reviewer 1's point 1 and reviewer 3's major point 2). Another essential revision is to demonstrate that the optogenetic manipulations did not cause behavioral changes that could potentially explain the differences in neuronal activity and co-activity patterns. Issues were also raised regarding statistical analyses (see reviewer 1's point 4 and other statistics-related points from reviewer 3). Lastly, the authors have previously shown the effects of these optogenetic manipulations on place cell responses. However, this was not entirely clear, and the authors should not assume that all readers have read their earlier paper (see points about place cells from reviewer 2 below). The authors should address the points raised by reviewer 2 by explicitly stating the effects that they have already shown and citing their earlier work. In addition, the authors should also discuss how the current plasticity results may be involved in the place cell changes reported in their earlier paper. The reviews are printed in their entirety below.Reviewer #1:The manuscript by Schoenenberger et al. all investigates into changes in pyramidal cell interneuron spike transmission probability in pyramidal cell-interneuron monosynaptic pairs in the rat hippocampal area CA1 following direct optogenetic inhibition and indirect disinhibition of some of these cells. The manuscript is a clever follow-up and re-analysis of the dataset collected for a paper that has already been published . The current analysis is focused at lasting changes of CA1 pyramidal cell-interneuron monosynaptic pairs following optogenetic inhibition. The authors report that these lasting changes are primarily predicted by changes in interneuron excitability during optogenetic modifications of firing rate and synchrony of hippocampal CA1 neurons.This is an interesting study from one of the leading labs in the field and the reported changes in spike transmission probability between pyramidal cells and interneurons following optogenetic circuit manipulation are of potential importance for our understanding of activity-dependent reorganization of hippocampal circuits during learning.There are some questions related to the analysis and to the interpretation of the data that should be addressed by the authors at this stage.1) Similar to their previous paper , the authors' efforts here are somewhat also stymied by the apparent lack of cell-type specificity of the CaMKII viral promoter. This limitation should be explicitly stated and discussed in the current manuscript, especially since there are still conflicting results out there suggesting that these promoters could indeed be reliably used for cell-type specific targeting of principal cells, which is clearly not the case.2) Relatedly, it is somewhat unfortunate that the optogenetic manipulation approach and the resulting dataset did not really allow the authors to directly test their main finding in the manuscript – that changes in interneuron excitably primarily dictates plasticity in their afferent inputs. This would have ideally required to selectively excite and/or inhibit interneurons in CA1. Of note, specific rAAV promoters actually do exist for selective manipulations of GABAergic interneurons .3) The authors should analyze and report if the optogenetic manipulation has caused any acute (during FAML) or chronic (during FAM2) effects on the animals' behavior and to what extent these behavioral changes may contribute to the observed changes in pyramidal cell-interneuron spike transmission probability. On another note, the proper control for optogenetic experiments should have been the use of rAAV with a static fluorophore .4) Statistical analysis should also be performed and reported on animal as a unit in order to better account for inter-animal variance.Reviewer #2:Schoenenberger et al. examine spike timing dynamics of pyramidal cell interneuron pairs in familiar and novel environments and following perturbation of pyramidal cell sub-populations in a familiar environment. The main findings address spike transmission probabilities for pyramidal cell interneuron pairs that are likely to have synaptic connections (based on short-latency cross-correlation peaks). Optogenetic inhibition of sub-populations of pyramidal neurons decreases activity in some neurons (as expected) and increases activity in others. Interneurons are largely inhibited in their activity during inhibition of pyramidal cells. Presumably, this is a result of reduced pyramidal cell excitation of feedback interneurons. Alterations in firing appear to produce longer-lasting changes in spike transmission probabilities. This suggests that spike timing relationships between pyramidal cells and interneurons are significant features of network dynamics that impact representation in hippocampus. In general, the findings are solidly backed up by the analytical approach and statistics and are clearly presented. There are some aspects of the data that seem absent from the report that might well make well place the results in a larger context and might make for greater impact.1) The authors show that altered spike transmission probabilities in a novel environment are observed, but that these alterations do not impact dynamics in subsequent visits to the familiar environment. The authors do not reach far enough to attempt to explain this. Alterations induced by inhibition in the familiar environment do persist. These differences are interpreted as reflecting learning in the form of changes in pyramidal cell-interneuron synapses. This seems inconsistent with the lack of effect of novel environment dynamics. What might constitute an explanation for this? Perhaps there is some interaction with other inputs to interneurons that is critical? Furthermore, it would be of interest to determine whether it is the novelty of the environment that precludes persistence of spike transmission probabilities or simply the fact that the animal is in a different environment with a largely different set of pyramidal cell ensemble activity patterns.2) The optogenetic inhibition was applied only in certain regions of the environment, yet the authors make no use of this design feature. Are the observed effects limited to pyramidal cell-interneuron pairs for which the pyramidal cell has a place field in the region of the environment where inhibition was applied? In general, a major limitation of the work is that it does not consider the effects of alterations in pyramidal cell-interneuron spike transmission on representation of place. Do the observed changes yield rate-remapping, global remapping, partial remapping, etc.? In the absence of such analyses, it is unclear whether one should consider the observed changes in interaction to be impactful on network function or not.3) When considering the effects of co-activity, the authors should expand beyond the somewhat arbitrary time window of 20ms. It would be more informative to test a range of intervals and determine at what point paired spikes have no impact on subsequent transmission.4) To place the results in context, the authors might include an analysis of odd versus even minutes of one or all of the inhibition-free sessions. This will provide somewhat of a baseline for spike transmission changes.5) Do pyramidal cells released with increased firing in response to optogenetic inhibition of other pyramidal cells exhibit place-specific firing?Reviewer #3:In this manuscript, the authors intended to demonstrate that plastic changes occurred at the hippocampal pyramidal to interneuron connections in response to optogenetic manipulation during a spatial task in rats. To do so, the authors applied light stimulations as rats explored a familiar open arena (FAML session), after and before the rats explored the same arena without light in two sessions . The authors then compared spike transmission probability between identified putative pyramidal-interneuron pairs, as a measure of their connections, among these sessions. The authors made two key conclusions: (i) Optogenetic light stimulations led to lasting plasticity in spike transmission between connected pyramidal neurons and interneurons. (ii) The plastic changes were caused by firing rate changes in the postsynaptic interneurons. What and where synaptic plasticity occurs during behavior and how it leads to a particular learning behavior in vivo are important questions. However, identifying and manipulating synaptically connected neuron pairs is difficult in behaving animals. I applaud the authors' effort in this study and its outcome will be valuable to our understanding of learning and memory. However, as much as I like the study, I also have major concerns, which need to be resolved to make sure the key conclusions in the manuscript are valid.1) Regarding the first conclusion, a major concern is that it is difficult to tell whether the observed changes had anything to do with the light stimulations. Judging from Figure 1C, D and Figure 2E, the changes in spike transmission and firing rate were small. One possibility is that these changes could arise just passively with time or other unrelated experience. What is lacking is a control experiment that includes the same recording procedure, but without the light stimulation or even better, with a control light stimulation session when neurons are designed not to respond (like a different color of light). I understand that this takes a lot of effort. However, at least in the existing data, the authors should analyze how spike transmissions within FAM1 or FAM2 fluctuate and how the fluctuation level was compared to the changes between FAM1 and FAM2.2) I am confused by the authors' interpretation of the direct effect of light on firing rates of interneurons. The authors used halorhodopsin to inhibit neurons under the control of CaMKIIα. First, isn't it true that the promotor would restrict the halorhodopsin expression to pyramidal neurons, not much in interneurons? If this is not true, the authors need to provide references or histological evidence for this. Second, even if this is not true, how can the authors make sure the inhibition is caused directly by light, but not by the inhibition of pyramidal neurons? The evidence for a direct inhibition of interneurons  seems not strong, because the light was on for a long time. Third, some interneurons even increased their firing rates during FAML, which cannot be a direct effect of light. The picture of the light's direct effect is not clear in my mind, given a potential mixture of potential direct inhibition from light, if true, and the indirect inhibition from pyramidal neurons. The authors should clearly describe and discuss this issue, since this is important to their other key conclusion (see below).3) The authors concluded that the plasticity in spike transmission was mainly caused by the light-induced changes in the firing rates of postsynaptic interneurons. First, how can the authors be sure that this is not the other way around? That is, plasticity could occur first, then led to reduced firing rates in interneurons? Second, the authors need to clearly state the direction of spike transmission plasticity caused by the interneuron rate change: lower/higher firing rates of interneuron lead to lower/higher spike transmissions. The authors touched the issue in the Discussion but seemed vague about this. One concern is that the enhanced firing rates in some interneurons were clearly not a direct effect of light stimulations, but they were important to the correlations the authors used to make the conclusion. I believe the authors should be straightforward about this and modify the conclusion accordingly.4) One key result is Figure 3A. Here the FAML/FAM2 partial correlation was only reduced slightly from FAML alone, even though FAM2 alone was quite high. Can the authors verify and explain this?5) The authors described the number of animals, the number of cells, and the number of pairs analyzed. It is unclear how many days were recorded, how many cells or pairs were obtained from each animal per day, whether at least the key results can be seen in multiple animals, and whether the same pairs were repeatedly used in the analysis.6) What are the N's in Figure 2B-D, Figure 3A-B, Figure 4? What does each sample mean in these plots? If each sample was a day and there were 63 pairs in a number of days/animals, were there sufficient number of pairs for computing correlation or regression on a given day? What is the prediction in the labels? There is no description about this key analysis in the method.7) What is the y-axis in Figure 1B?eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Laura Colgin as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Optogenetic inhibition-mediated activity-dependent modification of CA1 pyramidal-interneuron connections during behavior" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions would benefit from additional new data or new experiments, as reviewers feel is the case with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions would benefit from additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.In the latter case, our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:This study investigates into changes in pyramidal cell interneuron spike transmission probability in pyramidal cell-interneuron monosynaptic pairs in hippocampal area CA1 following optogenetic inhibition and disinhibition of some of these cells in behaving rats exploring novel and familiar environments. Insights regarding how cell-cell correlations change with experience in freely behaving animals add to our understanding of how hippocampal circuits support learning and memory. The work also changes the current thinking about processes of plasticity in hippocampal networks and has relevance for interpretation of datasets in which optical inhibition is applied. However, the paper does include technical confounds, as noted below.Revisions for this paper:Figure 1C, D: the description of these two figure panels was not quantitative in both the main text and the figure legend. The authors stated that the plots "showed changes" and "altered the ability…", although the data points were highly correlated. It is better to quantify whether the changes were more than those in a control condition . The authors' statements could be supported by the lower FAML-FAM1 correlations than those of the control condition. Also, it may be worth quantifying the overall changes (mean or median differences) in rate and transmission probability to understand whether a significant net effect of the stimulation occurred in this experiment.Revisions expected in follow-up work:1) Ideally, the proper controls for optogenetic experiments  should have been included.2) Follow-up experiments will include specific rAAV promoters for selective manipulations of GABAergic interneurons. Reviewer #1:[…]There are some questions related to the analysis and to the interpretation of the data that should be addressed by the authors at this stage.1) Similar to their previous paper , the authors' efforts here are somewhat also stymied by the apparent lack of cell-type specificity of the CaMKII viral promoter. This limitation should be explicitly stated and discussed in the current manuscript, especially since there are still conflicting results out there suggesting that these promoters could indeed be reliably used for cell-type specific targeting of principal cells, which is clearly not the case.Now we discuss the lack of cell specificity in the manuscript. Cell specificity of viruses using CaMKII promoter may depend on the region where the virus is expressed, and type of virus used. An earlier study has observed similar effects as well, e.g., Nathanson et al., 2009. In the Schoenenberger et al., 2016 paper we presented immunolabeling results showing that, in addition to pyramidal cells, our virus indeed expressed in both parvalbumin and somatostatin immunopositive cells and the light response delay of 1-2ms was similar for both the inhibited pyramidal cells and interneurons.2) Relatedly, it is somewhat unfortunate that the optogenetic manipulation approach and the resulting dataset did not really allow the authors to directly test their main finding in the manuscript – that changes in interneuron excitably primarily dictates plasticity in their afferent inputs. This would have ideally required to selectively excite and/or inhibit interneurons in CA1. Of note, specific rAAV promoters actually do exist for selective manipulations of GABAergic interneurons .Yes, unfortunately, in this manuscript, we were not able to selectively manipulate interneuron activity using new tools that have recently emerged. This finding was unexpected and selectively manipulating interneurons would require a collection of a dataset in size and effort similar to the one we use in this study. Note also that even in cases in which interneuron firing is specifically altered using optogenetics, such manipulations will indirectly influence the firing of pyramidal cells. For example, suppressing interneuron activity will lead to the disinhibition of pyramidal cells. We see such disinhibitory effect even when we selectively inhibit a smaller subset of CCK interneurons in transgenic mice.3) The authors should analyze and report if the optogenetic manipulation has caused any acute (during FAML) or chronic (during FAM2) effects on the animals' behavior and to what extent these behavioral changes may contribute to the observed changes in pyramidal cell-interneuron spike transmission probability. On another note, the proper control for optogenetic experiments should have been the use of rAAV with a static fluorophore .In the Schoenenberger et al., 2016 paper, we compared the speed across different sessions of the animal and possible occupancy differences in the light zone and outside. Now, we performed the same analysis in our extended data as well . No significant differences were seen either in occupancy or speed, inside and outside the light zone across FAM1, FAML and FAM2.In relation to the control experiments, as suggested by the Editor’s letter, we assessed the within-session variability of spike transmission probabilities in FAM1 and showed that these are smaller than those seen across FAM1-FAM2 .4) Statistical analysis should also be performed and reported on animal as a unit in order to better account for inter-animal variance.As suggested, in all analyses, we incorporated animal as a random effect in our linear mixed models and ANOVA and show that this did not influence our results.Reviewer #2:[…]1) The authors show that altered spike transmission probabilities in a novel environment are observed, but that these alterations do not impact dynamics in subsequent visits to the familiar environment. The authors do not reach far enough to attempt to explain this. Alterations induced by inhibition in the familiar environment do persist. These differences are interpreted as reflecting learning in the form of changes in pyramidal cell-interneuron synapses. This seems inconsistent with the lack of effect of novel environment dynamics. What might constitute an explanation for this? Perhaps there is some interaction with other inputs to interneurons that is critical? Furthermore, it would be of interest to determine whether it is the novelty of the environment that precludes persistence of spike transmission probabilities or simply the fact that the animal is in a different environment with a largely different set of pyramidal cell ensemble activity patterns.In the revision, we discuss further the possible mechanism behind the altered spike transmission in the novel environment. We show in Figure 4A that spike transmission changes from familiar to novel environments were predicted by the change of the interneuron rates but not by the pyramidal rates. This suggests a postsynaptic effect, which, at least in part, may be related to the change of the depolarization/excitability of interneurons. We agree with the reviewer that other inputs to the interneurons may be a cause for the spike transmission changes from the familiar to the novel environment. Such inputs may include other place cell or non-specific neuromodulation, e.g., acetylcholine levels are higher in a novel environment. Unfortunately, we do not have data in which two different familiar environments and a novel environment are all simultaneously recorded, which would be required if pyramidal-interneuron spike transmissions reorganize across familiar environments.2) The optogenetic inhibition was applied only in certain regions of the environment, yet the authors make no use of this design feature. Are the observed effects limited to pyramidal cell-interneuron pairs for which the pyramidal cell has a place field in the region of the environment where inhibition was applied? In general, a major limitation of the work is that it does not consider the effects of alterations in pyramidal cell-interneuron spike transmission on representation of place. Do the observed changes yield rate-remapping, global remapping, partial remapping, etc.? In the absence of such analyses, it is unclear whether one should consider the observed changes in interaction to be impactful on network function or not.In the previous Schoenenberger et al., 2016 paper, we showed that a fraction of place cells that were inhibited by the light remapped their place fields as a result of light-mediated inhibition. In a new analysis, we attempted to relate place field remapping to the changes of monosynaptic connections, but we did not see a relationship.We report this in the revision by saying that “In addition to the changing the firing rate, light application can cause remapping in a subpopulation of cells . However, a change in spike transmission in FAM2 did not predict the degree of pyramidal place field remapping ”.We may not have been able to see such a relationship because only a subpopulation of light-inhibited cells exhibited place field remapping.3) When considering the effects of co-activity, the authors should expand beyond the somewhat arbitrary time window of 20ms. It would be more informative to test a range of intervals and determine at what point paired spikes have no impact on subsequent transmission.2=0.849) predicted by the combined rate changes . We comment on these results in the Discussion now:We checked the effect in additional time windows of 10ms, 20ms, 50ms and 100ms. When we included animal and used different intervals (leading to multiple comparisons) as additional factors, only the 50ms time window was significant. Moreover, spike paring no longer predicted the spike transmission changes when pyramidal and interneuron rate changes together were accounted for. This is possibly due to the fact that spike pairing alone during FAML is very strongly  To place the results in context, the authors might include an analysis of odd versus even minutes of one or all of the inhibition-free sessions. This will provide somewhat of a baseline for spike transmission changes.We thank the reviewer for this suggestion, and we perform such an analysis as well to show stability. Indeed, in the difference in the spike transmission measured in alternating time windows within FAM1 was significantly less than across FAM1-FAM2 .5) Do pyramidal cells released with increased firing in response to optogenetic inhibition of other pyramidal cells exhibit place-specific firing?Yes, in the Schoenenberger et al., 2016 paper, we showed that both disinhibited and inhibited pyramidal cells exhibited place-related firing. Interestingly, disinhibition did not alter the place fields, whereas inhibition triggered place field remapping.Reviewer #3:[…]1) Regarding the first conclusion, a major concern is that it is difficult to tell whether the observed changes had anything to do with the light stimulations. Judging from Figure 1C, D and Figure 2E, the changes in spike transmission and firing rate were small. One possibility is that these changes could arise just passively with time or other unrelated experience. What is lacking is a control experiment that includes the same recording procedure, but without the light stimulation or even better, with a control light stimulation session when neurons are designed not to respond (like a different color of light). I understand that this takes a lot of effort. However, at least in the existing data, the authors should analyze how spike transmissions within FAM1 or FAM2 fluctuate and how the fluctuation level was compared to the changes between FAM1 and FAM2.In the revision, as suggested by reviewer 2 as well, we compare spike transmissions within FAM1 using alternating time windows and compare it to the changes occurring across FAM1-FAM2 sessions. The within-session variability of spike transmission was significantly less than those across the FAM1-FAM2 sessions . Note, however, that some additional findings of the manuscript also argue against the possibility that our effects are simply due to random changes that may occur over time passed. First, we showed that changes in the light session but not those in the novel session predict the changes across FAM1-to-FAM2 sessions. Second, we show that other factors such interneuron rate changes will also predict spike transmission changes. We do not see how random fluctuations of spike transmission could still lead to predictions that involve factors of the light session but not the other “control” intervening session of the novel environment.2) I am confused by the authors' interpretation of the direct effect of light on firing rates of interneurons. The authors used halorhodopsin to inhibit neurons under the control of CaMKIIα. First, isn't it true that the promotor would restrict the halorhodopsin expression to pyramidal neurons, not much in interneurons? If this is not true, the authors need to provide references or histological evidence for this. Second, even if this is not true, how can the authors make sure the inhibition is caused directly by light, but not by the inhibition of pyramidal neurons? The evidence for a direct inhibition of interneurons  seems not strong, because the light was on for a long time. Third, some interneurons even increased their firing rates during FAML, which cannot be a direct effect of light. The picture of the light's direct effect is not clear in my mind, given a potential mixture of potential direct inhibition from light, if true, and the indirect inhibition from pyramidal neurons. The authors should clearly describe and discuss this issue, since this is important to their other key conclusion (see below).We further discussed these issues in the revised manuscript. In the Schoenenberger et al., 2016 paper, we quantified these effects. We showed that both the light-inhibited pyramidal cells and interneurons suppressed their firing within a short 1-2ms time delay relative to the light onset. The light responses were tested using 500ms light pulses in the end of the recordings while the animal was rested. We also performed immunolabeling in the Schoenenberger et al., 2016 study and showed that both parvalbumin and somatostatin immunopositive cells expressed halorhodopsin. Note also that an earlier paper reported a similar effect Nathanson et al., 2009. Of course, we cannot exclude that this virus is more specific in other brain regions or other virus serotypes or constructs may be more specific.3) The authors concluded that the plasticity in spike transmission was mainly caused by the light-induced changes in the firing rates of postsynaptic interneurons. First, how can the authors be sure that this is not the other way around? That is, plasticity could occur first, then led to reduced firing rates in interneurons? Second, the authors need to clearly state the direction of spike transmission plasticity caused by the interneuron rate change: lower/higher firing rates of interneuron lead to lower/higher spike transmissions. The authors touched the issue in the Discussion but seemed vague about this. One concern is that the enhanced firing rates in some interneurons were clearly not a direct effect of light stimulations, but they were important to the correlations the authors used to make the conclusion. I believe the authors should be straightforward about this and modify the conclusion accordingly.Indeed, Figure 3C shows that the interneuron rate increase in FAML is associated with a stronger spike transmission, whereas in cases of reduced spike transmission, FAM2 interneuron rate is weaker. As asked, in the revision, we spelled out this relationship in the Discussion. We agree that the rate increase of some interneurons may be caused by strengthened pyramidal connections triggered by complex network effects mediated by light-induced inhibition of a subgroup of pyramidal cells and interneurons. We discussed this scenario in revision, as suggested by the reviewer.4) One key result is Figure 3A. Here the FAML/FAM2 partial correlation was only reduced slightly from FAML alone, even though FAM2 alone was quite high. Can the authors verify and explain this?This finding showed that, although FAM2-FAM1 interneuron rate changes predicted the corresponding spike transmission changes, similar rate changes between FAML-FAM1 have further predictive value. The relationship between FAM2-FAM1 rate and spike transmission changes may solely suggest that excitability changes of the interneuron led to the spike transmission changes. However, with the partial correlation (and the associated ANOVA model comparisons), we were able to show that rate  alterations in the light session were able to independently influence the FAM1-FAM2 spike transmission changes. That is that excitability/depolarization alterations in FAML that were no longer present in FAM2 still influenced FAM1-FAM2 spike transmission changes. We explain this better in the revision.5) The authors described the number of animals, the number of cells, and the number of pairs analyzed. It is unclear how many days were recorded, how many cells or pairs were obtained from each animal per day, whether at least the key results can be seen in multiple animals, and whether the same pairs were repeatedly used in the analysis.In the original work in four animals, we recorded from n=31, 23, 5, 4 detected cell pairs. In the revision, we added one additional animal with n=16 cell pairs to ensure that all the results could be replicated independently of the critical contribution of a single animal. In all analyses, we included animal as a random effect and showed that this did not influence our results. In three animals we recorded in two recording days, while four recording days were used in the remaining two animals. The electrodes were moved between recording days to ensure that a different set of cells are recorded across days. Therefore, we think that only very few cell pairs may have recorded across the two recordings days. Yet, even if we did record from the same cell pairs across days, the light zone location was changed daily; therefore, the cells experienced an entirely different network activity background in our manipulations with different potential outcomes.6) What are the N's in Figure 2B-D, Figure 3A-B, Figure 4? What does each sample mean in these plots? If each sample was a day and there were 63 pairs in a number of days/animals, were there sufficient number of pairs for computing correlation or regression on a given day? What is the prediction in the labels? There is no description about this key analysis in the method.In each plot, each dot represents the value related to a single cell pair so n=78 (extended dataset) always. With a few sessions, we were able to record >10 pairs yet the yield of detecting these monosynaptic pairs was relatively low. Prediction refers to the linear regression and the correlation coefficient (r). We added correlation to the legend. We thank the reviewer for pointing out the confusion. However, we kept prediction on the axes because prediction is easier to understand (according to our opinion) than using “correlation with” on these labels.7) What is the y-axis in Figure 1B?It shows the probability of a spike occurring within the 20ms time bins. Now we also explain it in the legend.Revisions for this paper:Figure 1C, D: the description of these two figure panels was not quantitative in both the main text and the figure legend. The authors stated that the plots "showed changes" and "altered the ability…", although the data points were highly correlated. It is better to quantify whether the changes were more than those in a control condition . The authors' statements could be supported by the lower FAML-FAM1 correlations than those of the control condition. Also, it may be worth quantifying the overall changes (mean or median differences) in rate and transmission probability to understand whether a significant net effect of the stimulation occurred in this experiment.We performed the requested quantification and indeed the within session (FAM1) correlations we higher that across session (FAM1-FAML) ones, both for firing rate and spike transmission. Median rates were not significantly different however because cells either reduced or increased their rate during the light application. But there was a significant reduction in the spike transmission probabilities from FAM1 to FAML.Revisions expected in follow-up work:1) Ideally, the proper controls for optogenetic experiments  should have been included.We acknowledge this in the beginning of the Discussion saying that “Or study did not use control animals in which only YFP was expressed. Therefore, we cannot exclude the possibility that optogenetic channel expression, or, perhaps, light application enhanced the plasticity on pyramidal-interneuron synapses. Yet, we observed similar activity-dependent changes during spatial learning before . So, it is likely that the optogenetic, light-mediated rate alteration was a primary driver of the activity-dependent, lasting connection-weight changes. “2) Follow-up experiments will include specific rAAV promoters for selective manipulations of GABAergic interneurons.We state these follow-up experiments in the Discussion but we also point out that the disinhibition of pyramidal cells might hinder these experiments: “Future work in which interneuron (or a certain genetic type) firing rate is selectively altered by optogenetics may provide further evidence for the independent contribution of postsynaptic interneuron depolarization in plasticity. Nevertheless, even in such manipulations, indirect alteration of pyramidal rates  is expected to occur.”


HOMER1 gene have been associated with phenotypes related to glutamate signaling dysregulation. In this study, we report the association of a single nucleotide polymorphism (SNP) in the HOMER1 gene (rs3822568) with specific aspects of sleep in a sample of the Brazilian population. To accomplish this, 1,042 individuals were subjected to a full-night polysomnography, and a subset of 983 subjects had rs3822568 genotyping data available. When compared with the A allele carriers, GG genotyped individuals showed higher sleep latency, lower sleep efficiency, reduced number of arousals per hour, lower apnea-hypopnea index (AHI) and lower theta spectral power. In summary, the present findings suggest that the rs3822568 polymorphism in the HOMER1 gene is associated with sleep EEG profiles and might have an impact on sleep quality and sleep structure, with potential to explain inter-individual variation in sleep homeostasis.Glutamate is the most excitatory neurotransmitter in the central nervous system and it is involved in the initiation and maintaining of waking and rapid-eye-movement (REM) sleep. Homer proteins act in the trafficking and/or clustering of metabotropic glutamate receptors, and polymorphisms in the  The sleep-wake cycle depends on a complex and well-orchestrated neuronal circuitry involving diverse neurotransmitter signaling that induce and maintain sleep and wakefulness. Glutamate, the most excitatory neurotransmitter in the central nervous system, shows dynamic changes in its levels throughout the sleep-wake states . In addiHomer1, Homer2 and Homer3 ‒ predominantly expressed at the nervous system as several isoforms resulting from alternative splicing events ) than AG genotype carriers  and lower sleep efficiency (80.56 [13.95]) than AG genotype carriers . In addition, GG carriers showed lower arousals per hour index (13.27 [9.12]) and AHI (6.63 [11.13]) than AA genotype carriers . We also found a trend for association between HOMER1 rs3822568 polymorphism and the percentage of stages N3, but no significant associations were found after the post hoc test .We verified the association of hoc test . Also, dHOMER1 rs3822568 genotypes as well as sex, age, BMI and European ancestry proportion derived from ancestry informative markers as independent variables and the z-score of each significantly associated parameter in univariate analyses as dependent variables in each model. After adjustment for the studied confounders, HOMER1 rs3822568 polymorphism was significantly and independently associated only with sleep latency, regardless of other variables in the model (p = 0.005).To verify the effect of potential confounders on the identified associations, we fitted GLMs using HOMER1 rs3822568 polymorphism and theta spectral power in all EEG derivations, even after adjustment for sex, age, BMI and European ancestry proportion. Overall, GG genotype carriers showed lower theta spectral power in stage 1, stage 2 and REM sleep; however, after adjustment for potential confounders, associations remained significant only in the occipital derivations and in sleep stage 1 and REM sleep  of the HOMER1 gene, for which the ancestral allele is a guanine (G) and the alternative allele is an adenine (A).In the present study, we report the association between rs3822568 in the Here, we found that homozygous individuals for the G allele, on average, experience higher sleep latency times, lower sleep efficiency, less arousals per hour and lower AHI than subjects carrying the A allele. This suggests that the presence of the G allele is associated with changes in sleep onset ‒ leading to the higher latency times ‒ but not the sleep maintenance ‒ since no associations with arousals per hour were found. In this sense, the observed lower sleep efficiency might be attributed to the long sleep latency rather than to a higher sleep fragmentation caused by many arousals during the sleep time ,25.On the other hand, even if AHI is a common measure for the diagnosis and severity determination of OSAS, rs3822568 did not show any association with the presence of the syndrome according to the definitions we used . This coIt is known that oscillations in the sleep EEG reflect the homeostatic regulation of sleep . Common Homer1 upregulation during wakefulness and its down regulation at sleep is not a simple correlate, but a matter of cause and effect. Using Drosophila and mouse models, these authors showed that Homer1a/homer1a knockout leads to reduced and fragmented sleep in flies, while it causes inability to sustain the wake state in the rodents. Thus, it is possible that genetic variation in the Homer genes could modulate the subtle balance of HOMER proteins in the regulation of glutamatergic neurotransmission and yield different states of neuronal excitability in the nervous system, affecting sleep.Naidoo and colleagues  demonstrHomer genes could, therefore, modulate the interactions among HOMER proteins in regulating glutamatergic neurotransmission and yield different states of neuronal excitability in the nervous system affecting sleep state. Considering that the 3’UTR is a key post-transcriptional regulation site, figuring a target for non-coding RNA based regulatory mechanisms such as microRNAs Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this study, the authors evaluated the association of a specific SNP in the HOMER1 gene with specific aspects of sleep in a representative sample of the Brazilian population. They in fact hypothesize that, given the importance of the glutamatergic system regulating sleep and prolonged wakefulness, genetic variation in the HOMER1 gene might explain inter-individual variability in several sleep traits measured using polysomnography.Even if they studied only a gene and only one SNP of this gene, the study is well conducted and the sample size of studied subjects is consistentThe research is quite original, the experiments and the statistical analyses were conducted in a proper way and the paper is written in a clear and intelligible way.Some minor issues are to be considered:The author should better discuss that this study involve only one gene (and one SNP) but the phenotypes they are discussing is multifactorial and the genetic factors involved can be numerous. The gene they are studying may give a contribution but it does not explain the whole genetic variability of these phenotypes. They should also discuss about the possibility of other SNPs (and HAPLOTYPES) of this gene involved Are already known other SNPs? Could they sequence the entire gene in a group of subjects in order to study this variability and the existence of other SNPs? They should at least discuss itThey should also discuss about the functional role and meaning of the SNP they analyzedReviewer #2: In the current manuscript Pedrazzoli et al., examine the association between overnight sleep parameters and a polymorphism in the Homer1 gene in a Brazilian population. The authors report significant genotype interactions in the Homer1 3'UTR and sleep latency, efficiency and other parameters, as well as an interaction with Theta power in the EEG recordings. Recent studies have revealed genetic links with the Homer1 gene and psychiatric conditions. Basic research has also shown an important role for the Homer1 gene in sleep behavior in rodents and insects. The current work is presented in a straight forward manner and the data collection has been done rigorously and appropriately.Considering the material covered in this manuscript it would be highly appropriate to include in the bibliography a recent high profile paper on the role of Homer1a in synaptic plasticity during sleep. . This paper shows that Homer1a protein is important to regulate mGluR1/5 signaling during sleep and for sleep-dependent remodeling of synapses. A second paper Holst et al., 2017 eLife, PMID: 28980941 shows that mGluR5 is upregulated during sleep deprivation in humans and is required for appropriate response to sleep deprivation in mice.I have no other concerns about the current manuscript.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  17 Apr 2020http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols# EDITOR: To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: RESPONSE: The majority of our protocols involve standard methods such as the scores of polysomnography data and SNP genotyping. We have specified the thermal conditions in Material and Methods section (“rs3822568 Genotyping” subsection). We do not think it is necessary to specify other conventional protocols involved in our study.# REVIEWER 1: The author should better discuss that this study involve only one gene (and one SNP) but the phenotypes they are discussing is multifactorial and the genetic factors involved can be numerous. The gene they are studying may give a contribution but it does not explain the whole genetic variability of these phenotypes.RESPONSE: Thank you for pointing it out. We have added a paragraph (L229-234) in Discussion section arguing about this matter:“When considering that the relationship between disease susceptibility and genetic variation is complex, and for the most phenotypes more than one gene is generally involved, care has to be taken when considering just one SNP in one gene to draw conclusions about a complex and multifactorial phenotype as sleep. Nevertheless, when considering the physiological mechanisms related to glutamate neurotransmission and previous associations with sleep, HOMER1 sounds like a good candidate gene for sleep phenotypes related to extended wake like insomnia.”# REVIEWER 1: They should also discuss about the possibility of other SNPs (and HAPLOTYPES) of this gene involved Are already known other SNPs? Could they sequence the entire gene in a group of subjects in order to study this variability and the existence of other SNPs? They should at least discuss it.RESPONSE: We appreciate your suggestion. However, it is not possible at this time to sequence the samples. Therefore, we have added a supplemental material informing about the linkage disequilibrium  and also a paragraph (L235-240) discussing the possibilities for other SNPs in HOMER1 gene that might have a role in the phenotype:“HOMER1 has many single nucleotide polymorphisms along its sequence. According to the 1000 Genomes Project data, in American populations rs3822568 is in strong linkage disequilibrium with at least five other SNPs , although all of them are localized in intergenic regions and therefore have low potential to yield genetic consequences. Also, neither of them has been associated with any relevant clinical phenotype. This fact does not exclude the possibility of other SNPs and even phased haplotypes play important roles in the genetic control of the sleep parameters analyzed in this study.”# REVIEWER 1: They should also discuss about the functional role and meaning of the SNP they analyzed.RESPONSE: Great point. We have added a new paragraph (L220-L228) in Discussion, which we cautionary suggest (L226-228) the role of rs3822568 in a physiological context:“In the view of recent studies about glutamatergic neurotransmission, sleep homeostasis and synaptic plasticity, Homer1 is strictly linked to the (mGluR5) function. It selectively uncouples mGluR5 from the effector targets in the postsynaptic density (33) that results in attenuation of mGluR5-mediated activation of phospholipase C and downstream signaling (34). In humans, increased mGluR5 availability after sleep deprivation was tightly associated with increased propensity to fall asleep (35) and considering the interplay between Homer1 and mGluR5, it has been suggested that Homer1a serves as a molecular integrator of arousal and sleep need (36). In that way, we may cautionary propose the SNP described here could mediates the integration Homer1/mGluR5 through 3’UTR mechanisms cited above, and therefore would mediates a compensatory mechanism to promote wakefulness in the sleep-deprived state like insomnia states.”#REVIEWER 2: In the current manuscript Pedrazzoli et al., examine the association between overnight sleep parameters and a polymorphism in the Homer1 gene in a Brazilian population. The authors report significant genotype interactions in the Homer1 3'UTR and sleep latency, efficiency and other parameters, as well as an interaction with Theta power in the EEG recordings. Recent studies have revealed genetic links with the Homer1 gene and psychiatric conditions. Basic research has also shown an important role for the Homer1 gene in sleep behavior in rodents and insects. The current work is presented in a straight forward manner and the data collection has been done rigorously and appropriately.Considering the material covered in this manuscript it would be highly appropriate to include in the bibliography a recent high profile paper on the role of Homer1a in synaptic plasticity during sleep. . This paper shows that Homer1a protein is important to regulate mGluR1/5 signaling during sleep and for sleep-dependent remodeling of synapses. A second paper Holst et al., 2017 eLife, PMID: 28980941 shows that mGluR5 is upregulated during sleep deprivation in humans and is required for appropriate response to sleep deprivation in mice.I have no other concerns about the current manuscript.RESPONSE: Thank you for the suggestion. We have added a new paragraph (L220-L228) in Discussion with references suggested above and other:“In the view of recent studies about glutamatergic neurotransmission, sleep homeostasis and synaptic plasticity, Homer1 is strictly linked to the (mGluR5) function. It selectively uncouples mGluR5 from the effector targets in the postsynaptic density (33) that results in attenuation of mGluR5-mediated activation of phospholipase C and downstream signaling (34). In humans, increased mGluR5 availability after sleep deprivation was tightly associated with increased propensity to fall asleep (35) and considering the interplay between Homer1 and mGluR5, it has been suggested that Homer1a serves as a molecular integrator of arousal and sleep need (36). In that way, we may cautionary propose the SNP described here could mediates the integration Homer1/mGluR5 through 3’UTR mechanisms cited above, and therefore would mediates a compensatory mechanism to promote wakefulness in the sleep-deprived state like insomnia states.”AttachmentResponse to Reviewers.pdfSubmitted filename: Click here for additional data file. 17 Jun 2020A single nucleotide polymorphism in the HOMER1 gene is associated with sleep latency and theta power in sleep electroencephalogramPONE-D-19-26778R1Dear Dr. Pedrazzoli,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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