Syngap1 regulates the synaptic drive and membrane excitability of Parvalbumin-positive interneurons in mouse auditory cortex
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eLife assessment
This study provides valuable evidence indicating that SynGap1 regulates the synaptic drive and membrane excitability of parvalbumin- and somatostatin-positive interneurons in the auditory cortex. Since haplo-insufficiency of SynGap1 has been linked to intellectual disabilities without a well-defined underlying cause, the central question of this study is timely. However, the support for the authors' conclusions is incomplete in general and some parts of the experimental evidence are inadequate. Specifically, the manuscript requires further work to properly evaluate the impact on synaptic currents, intrinsic excitability parameters, and morphological features.
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Abstract
SYNGAP1 haploinsufficiency-related intellectual disability (SYNGAP1-ID) is characterized by moderate to severe ID, generalized epilepsy, autism spectrum disorder, sensory processing dysfunction and other behavioral abnormalities. While most studies, so far, have focussed on the role of Syngap1 in cortical excitatory neurons, recent studies suggest that Syngap1 plays a role in GABAergic inhibitory neuron development as well. However, the molecular pathways by which Syngap1 acts on GABAergic neurons, and whether they are similar or different from the mechanisms underlying its effects in excitatory neurons, is unknown. Here we examined whether, and how, embryonic-onset Syngap1 haploinsufficiency restricted to GABAergic interneurons derived from the medial ganglionic eminence (MGE) impacts their synaptic and intrinsic properties in adulthood. We found that Syngap1 haploinsufficiency affects the intrinsic properties, overall leading to increased firing threshold, and decreased excitatory synaptic drive of Parvalbumin (PV)+ neurons from Layer IV auditory cortex in adult mice, whilst Somatostatin (SST)+ interneurons were mostly resistant to Syngap1 haploinsufficiency. Further, the AMPA component of thalamocortical evoked-EPSC was decreased in PV+ cells from mutant mice. Finally, we found that targeting the Kv1 family of voltage-gated potassium channels was sufficient to rescue PV+ mutant cell-intrinsic properties to wild-type levels. Together, these data suggest that Syngap1 plays a specific role in the maturation of PV+ cell intrinsic properties and synaptic drive, and its haploinsufficiency may lead to reduced PV cell recruitment in the adult auditory cortex, which could thus underlie the auditory processing alterations found in SYNGAP1-ID preclinical models and patients.
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Author response:
eLife assessment
This study provides valuable evidence indicating that Syngap1 regulates the synaptic drive and membrane excitability of parvalbumin- and somatostatin-positive interneurons in the auditory cortex. Since haplo-insufficiency of Syngap1 has been linked to intellectual disabilities without a well-defined underlying cause, the central question of this study is timely. However, the support for the authors' conclusions is incomplete in general and some parts of the experimental evidence are inadequate. Specifically, the manuscript requires further work to properly evaluate the impact on synaptic currents, intrinsic excitability parameters, and morphological features.
We are happy that the editors found that our study provides valuable evidence and that the central question is timely. We thank the reviewers …
Author response:
eLife assessment
This study provides valuable evidence indicating that Syngap1 regulates the synaptic drive and membrane excitability of parvalbumin- and somatostatin-positive interneurons in the auditory cortex. Since haplo-insufficiency of Syngap1 has been linked to intellectual disabilities without a well-defined underlying cause, the central question of this study is timely. However, the support for the authors' conclusions is incomplete in general and some parts of the experimental evidence are inadequate. Specifically, the manuscript requires further work to properly evaluate the impact on synaptic currents, intrinsic excitability parameters, and morphological features.
We are happy that the editors found that our study provides valuable evidence and that the central question is timely. We thank the reviewers for their detailed comments and suggestions. Below, we provide a point-by-point answer (in blue) to the specific comments and indicate the changes to the manuscript and the additional experiments we plan to perform to answer these comments.
Public Reviews:
Reviewer #1 (Public Review):
The study is designed to assess the role of Syngap1 in regulating the physiology of the MGE-derived PV+ and SST+ interneurons. Syngap1 is associated with some mental health disorders, and PV+ and SST+ cells are the focus of many previous and likely future reports from studies of interneuron biology, highlighting the translational and basic neuroscience relevance of the authors' work.
Strengths of the study are using well-established electrophysiology methods and the highly controlled conditions of ex vivo brain slice experiments combined with a novel intersectional mouse line, to assess the role of Syngap1 in regulating PV+ and SST+ cell properties. The findings revealed that in the mature auditory cortex, Syngap1 haploinsufficiency decreases both the intrinsic excitability and the excitatory synaptic drive onto PV+ neurons from Layer 4. In contrast, SST+ interneurons were mostly unaffected by Syngap1 haploinsufficiency. Pharmacologically manipulating the activity of voltage-gated potassium channels of the Kv1 family suggested that these channels contributed to the decreased PV+ neuron excitability by Syngap insufficiency. These results therefore suggest that normal Syngap1 expression levels are necessary to produce normal PV+ cell intrinsic properties and excitatory synaptic drive, albeit, perhaps surprisingly, inhibitory synaptic transmission was not affected by Syngap1 haploinsufficiency.
Since the electrophysiology experiments were performed in the adult auditory cortex, while Syngap1 expression was potentially affected since embryonic stages in the MGE, future studies should address two important points that were not tackled in the present study. First, what is the developmental time window in which Syngap1 insufficiency disrupted PV+ neuron properties? Albeit the embryonic Syngap1 deletion most likely affected PV+ neuron maturation, the properties of Syngap-insufficient PV+ neurons do not resemble those of immature PV+ neurons. Second, whereas the observation that Syngap1 haploinsufficiency affected PV+ neurons in auditory cortex layer 4 suggests auditory processing alterations, MGE-derived PV+ neurons populate every cortical area. Therefore, without information on whether Syngap1 expression levels are cortical area-specific, the data in this study would predict that by regulating PV+ neuron electrophysiology, Syngap1 normally controls circuit function in a wide range of cortical areas, and therefore a range of sensory, motor and cognitive functions. These are relatively minor weaknesses regarding interpretation of the data in the present study that the authors could discuss.
We agree with the reviewer on the proposed open questions, which we will certainly discuss in the revised manuscript we are preparing. We do have experimental evidence suggesting that Syngap1 mRNA is expressed by PV+ and SST+ neurons in different cortical areas, during early postnatal development and in adulthood; therefore, we agree that it will be important, in future experiments, to tackle the question of when the observed phenotypes arise.
Reviewer #2 (Public Review):
Summary:
In this manuscript, the authors investigated how partial loss of SynGap1 affects inhibitory neurons derived from the MGE in the auditory cortex, focusing on their synaptic inputs and excitability. While haplo-insufficiently of SynGap1 is known to lead to intellectual disabilities, the underlying mechanisms remain unclear.
Strengths:
The questions are novel
Weaknesses:
Despite the interesting and novel questions, there are significant concerns regarding the experimental design and data quality, as well as potential misinterpretations of key findings. Consequently, the current manuscript fails to contribute substantially to our understanding of SynGap1 loss mechanisms and may even provoke unnecessary controversies.
Major issues:
(1) One major concern is the inconsistency and confusion in the intermediate conclusions drawn from the results. For instance, while the sEPSC data indicates decreased amplitude in PV+ and SOM+ cells in cHet animals, the frequency of events remains unchanged. In contrast, the mEPSC data shows no change in amplitudes in PV+ cells, but a significant decrease in event frequency. The authors conclude that the former observation implies decreased excitability. However, traditionally, such observations on mEPSC parameters are considered indicative of presynaptic mechanisms rather than changes of network activity. The subsequent synapse counting experiments align more closely with the traditional conclusions. This issue can be resolved by rephrasing the text. However, it would remain unexplained why the sEPSC frequency shows no significant difference. If the majority of sEPSC events were indeed mediated by spiking (which is blocked by TTX), the average amplitudes and frequency of mEPSCs should be substantially lower than those of sEPSCs. Yet, they fall within a very similar range, suggesting that most sEPSCs may actually be independent of action potentials. But if that was indeed the case, the changes of purported sEPSC and mEPSC results should have been similar.
We understand the reviewer’s perspective; indeed, we asked ourselves the very same question regarding why the sEPSC and mEPSC frequency fall within a similar range when we analysed neuron means (bar graphs). We have already recorded sEPSCs followed by mEPSCs from several PV neurons (control and cHet) and are in the process of analyzing the data. We will add this data to the revised version of the manuscript. We will also rephrase the manuscript to present multiple potential interpretations of the data.
We hope that we have correctly interpreted the reviewer's concern. However, if the question is why sEPSC amplitude but not frequency is affected in cHet vs ctrl then the reviewer’s comment is perhaps based on the assumption that the amplitude and frequency of miniature events should be lower for all events compared to those observed for spontaneous events. However, it's essential to note that changes in the mean amplitude of sEPSCs are primarily driven by alterations in large sEPSCs (>9-10pA, as shown in cumulative probability in Fig. 1b right), with smaller ones being relatively unaffected. Consequently, a reduction in sEPSC amplitude may not necessarily result in a significant decrease in frequency since their values likely remain above the detection threshold of 3 pA. This could explain the lack of a significant decrease in average inter-interval event of sEPSCs (as depicted in Fig. 1b left).
If the question is whether we should see the same parameters affected by the genetic manipulation in both sEPSC and mEPSC, then another critical consideration is the involvement of the releasable pool in mEPSCs versus sEPSCs. Current knowledge suggests that activity-dependent and -independent release may not necessarily engage the same pool of vesicles or target the same postsynaptic sites. This concept has been extensively explored (reviewed in Kavalali, 2015). Consequently, while we may have traditionally interpreted activity-dependent and -independent data assuming they utilize the same pool, this is no longer accurate. The current discussion in the field revolves around understanding the mechanisms underlying such phenomena. Therefore, comparisons between sEPSCs and mEPSCs may not yield conclusive data but rather speculative interpretations. For a rigorous analysis, particularly in this context involving thousands of events, it is essential to assess these data sets (mEPSCs vs sEPSCs) separately and provide cumulative probability curves. This approach allows for a more comprehensive understanding of the underlying distributions and helps to elucidate any potential differences between the two types of events. We will rephrase the text, and as mentioned above, add additional data, to better reflect these considerations.
(2) Another significant concern is the quality of synapse counting experiments. The authors attempted to colocalize pre- and postsynaptic markers Vglut1 and PSD95 with PV labelling. However, several issues arise. Firstly, the PV labelling seems confined to soma regions, with no visible dendrites. Given that the perisomatic region only receives a minor fraction of excitatory synapses, this labeling might not accurately represent the input coverage of PV cells. Secondly, the resolution of the images is insufficient to support clear colocalization of the synaptic markers. Thirdly, the staining patterns are peculiar, with PSD95 puncta appearing within regions clearly identified as somas by Vglut1, hinting at possible intracellular signals. Furthermore, PSD95 seems to delineate potential apical dendrites of pyramidal cells passing through the region, yet Vglut1+ partners are absent in these segments, which are expected to be the marker of these synapses here. Additionally, the cumulative density of Vglut2 and Vglut1 puncta exceeds expectations, and it's surprising that subcortical fibers labeled by Vglut2 are comparable in number to intracortical Vglut1+ axon terminals. Ideally, N(Vglut1)+N(Vglut2) should be equal or less than N(PSD95), but this is not the case here. Consequently, these results cannot be considered reliable due to these issues.
We apologize, as it appears that the images we provided have caused confusion. The selected images represent a single focal plane of a confocal stack, which was visually centered on the PV cell somata. We chose just one confocal plane because we thought it showed more clearly the apposition of presynaptic and postsynaptic immunolabeling around the somata. In the revised version of the manuscript, we will provide higher magnification images, which will clearly show how we identified and selected the region of interest for the quantification of colocalized synaptic markers. In our confocal stacks, we can also identify PV immunolabeled dendrites and colocalized vGlut1/PSD95 or vGlut2/PSD95 puncta on them; but these do not appear in the selected images because, as explained, only one focal plane, centered on the PV cell somata, was shown.
We acknowledge the reviewer's point that in PV+ cells the majority of excitatory inputs are formed onto dendrites; however, we focused on the somatic excitatory inputs to PV cells, because despite their lower number, they produce much stronger depolarization in PV neurons than dendritic excitatory inputs (Hu et al., 2010; Norenberg et al., 2010). Further, quantification of perisomatic putative excitatory synapses is more reliable since by using PV immunostaining, we can visualize the soma and larger primary dendrites, but smaller, higher order dendrites are not be always detectable. Of note, PV positive somata receive more excitatory synapses than SST positive and pyramidal neuron somata as found by electron microscopy studies in the visual cortex (Hwang et al., 2021; Elabbady et al., 2024).
Regarding the comment on the density of vGlut1 and vGlut2 puncta, the reason that the numbers appear high and similar between the two markers is because we present normalized data (cHet normalized to their control values for each set of immunolabelling) to clearly represent the differences between genotypes. This information is present in the legends but we apologize for not clearly explaining it the methods section. We will provide a more detailed explanation of our methods in the revised manuscript.
Briefly, immunostained sections were imaged using a Leica SP8-STED confocal microscope, with a 63x (NA 1.4) at 1024 X 1024, z-step =0.3 μm, stack size of ~15 μm. Images were acquired from the auditory cortex from at least 3 coronal sections per animal. All the confocal parameters were maintained constant throughout the acquisition of an experiment. All images shown in the figures are from a single confocal plane. To quantify the number of vGlut1/PSD95 or vGlut2/PSD95 putative synapses, images were exported as TIFF files and analyzed using Fiji (Image J) software. We first manually outlined the profile of each PV cell soma (identified by PV immunolabeling). At least 4 innervated somata were selected in each confocal stack. We then used a series of custom-made macros in Fiji as previously described (Chehrazi et al, 2023). After subtracting background (rolling value = 10) and Gaussian blur (σ value = 2) filters, the stacks were binarized and vGlut1/PSD95 or vGlut2/PSD95 puncta were independently identified around the perimeter of a targeted soma in the focal plane with the highest soma circumference. Puncta were quantified after filtering particles for size (included between 0-2μm2) and circularity (included between 0-1). Data quantification was done by investigators blind to the genotype, and presented as normalized data over control values for each experiment.
(3) One observation from the minimal stimulation experiment was concluded by an unsupported statement. Namely, the change in the onset delay cannot be attributed to a deficit in the recruitment of PV+ cells, but it may suggest a change in the excitability of TC axons.
We agree with the reviewer, please see answer to point below.
(4) The conclusions drawn from the stimulation experiments are also disconnected from the actual data. To make conclusions about TC release, the authors should have tested release probability using established methods, such as paired-pulse changes. Instead, the only observation here is a change in the AMPA components, which remained unexplained.
We agree with the reviewer and we will perform additional paired-pulse ratio experiments at different intervals. We will rephrase the discussion and our interpretation and potential hypothesis according to the data obtained from this new experiment.
(5) The sampling rate of CC recordings is insufficient to resolve the temporal properties of the APs. Therefore, the phase-plots cannot be interpreted (e.g. axonal and somatic AP components are not clearly separated), raising questions about how AP threshold and peak were measured. The low sampling rate also masks the real derivative of the AP signals, making them apparently faster.
We acknowledge that a higher sampling rate could offer a more detailed analysis of the action potential waveform. However, in the context of action potential analysis, it is acceptable to use sampling rates ranging from 10 kHz to 20 kHz (Golomb et al., 2007; Stevens et al., 2021; Zhang et al., 2023), which are considered adequate in the context of the present study. Indeed, our study aims to evaluate "relative" differences in the electrophysiological phenotype when comparing groups following a specific genetic manipulation. A sampling rate of 10 kHz is commonly employed in similar studies, including those conducted by our collaborator and co-author S. Kourrich (e.g., Kourrich and Thomas 2009, Kourrich et al., 2013), as well as others (Russo et al., 2013; Ünal et al., 2020; Chamberland et al., 2023).
Despite being acquired at a lower sampling rate than potentially preferred by the reviewer, our data clearly demonstrate significant differences between the experimental groups, especially for parameters that are negligibly or not affected by the sampling rate used here (e.g., #spikes/input, RMP, Rin, Cm, Tm, AP amplitude, AP latency, AP rheobase).
Regarding the phase-plots, we agree that a higher sampling rate would have resulted in smoother curves and more accurate absolute values. However, the differences were sufficiently pronounced to discern the relative variations in action potential waveforms between the experimental groups.
A related issue is that the Methods section lacks essential details about the recording conditions, such as bridge balance and capacitance neutralization.
We indeed performed bridge balance and neutralized the capacitance before starting every recording. We will add the information in the methods.
(6) Interpretation issue: One of the most fundamental measures of cellular excitability, the rheobase, was differentially affected by cHet in BCshort and BCbroad. Yet, the authors concluded that the cHet-induced changes in the two subpopulations are common.
We are uncertain if we have correctly interpreted the reviewer's comment. While we observed distinct impacts on the rheobase (Fig. 7d and 7i), there seems to be a common effect on the AP threshold (Fig. 7c and 7h), as interpreted and indicated in the final sentence of the results section for Figure 7 (page 12). If our response does not address the reviewer's comment adequately, we would greatly appreciate it if the reviewer could rephrase their feedback.
(7) Design issue:
The Kv1 blockade experiments are disconnected from the main manuscript. There is no experiment that shows the causal relationship between changes in DTX and cHet cells. It is only an interesting observation on AP halfwidth and threshold. However, how they affect rheobase, EPSCs, and other topics of the manuscript are not addressed in DTX experiments.
Furthermore, Kv1 currents were never measured in this work, nor was the channel density tested. Thus, the DTX effects are not necessarily related to changes in PV cells, which can potentially generate controversies.
While we acknowledge the reviewer's point that Kv1 currents and density weren't specifically tested, an important insight provided by Fig. 5 is the prolonged action potential latency. This delay is significantly influenced by slowly inactivating subthreshold potassium currents, namely the D-type K+ current. It's worth noting that D-type current is primarily mediated by members of the Kv1 family. The literature supports a role for Kv1.1-containing channels in modulating responses to near-threshold stimuli in PV cells (Wang et al., 1994; Goldberg et al., 2008; Zurita et al., 2018). However, we recognize that besides the Kv1 family, other families may also contribute to the observed changes.
To address this concern, we will revise our interpretation. We will opt for the more accurate term "D-type K+ current" and only speculate about the involved channel family in the discussion. It is not our intention to open unnecessary controversy, but present the data we obtained. We believe this approach and rephrasing the discussion as proposed will prevent unnecessary controversy and instead foster fruitful discussions.
(8) Writing issues:
Abstract:
The auditory system is not mentioned in the abstract.
One statement in the abstract is unclear. What is meant by "targeting Kv1 family of voltage-gated potassium channels was sufficient..."? "Targeting" could refer to altered subcellular targeting of the channels, simple overexpression/deletion in the target cell population, or targeted mutation of the channel, etc. Only the final part of the Results revealed that none of the above, but these channels were blocked selectively.
We agree with the reviewer and we will rephrase the abstract accordingly.
Introduction:
There is a contradiction in the introduction. The second paragraph describes in detail the distinct contribution of PV and SST neurons to auditory processing. But at the end, the authors state that "relatively few reports on PV+ and SST+ cell-intrinsic and synaptic properties in adult auditory cortex". Please be more specific about the unknown properties.
We agree with the reviewer and we will rephrase more specifically.
(9) The introduction emphasizes the heterogeneity of PV neurons, which certainly influences the interpretation of the results of the current manuscript. However, the initial experiments did not consider this and handled all PV cell data as a pooled population.
In the initial experiments, we handled all PV cell data together because we wanted to be rigorous and not make assumptions/biases on the different PV cells, which in later experiments we were to distinguish based on the intrinsic properties alone. We will make this point clear in the revised manuscript.
(10) The interpretation of the results strongly depends on unpublished work, which potentially provide the physiological and behavioral contexts about the role of GABAergic neurons in SynGap-haploinsufficiency. The authors cite their own unpublished work, without explaining the specific findings and relation to this manuscript.
We agree with the reviewer and apologize for the lack of clarity. Our unpublished work is in revision right now. We will provide more information and update references in the revised version of this manuscript.
(11) The introduction of Scholl analysis experiments mentions SOM staining, however, there is no such data about this cell type in the manuscript.
We apologize for the error, we will change SOM with SST (SOM and SST are two commonly used acronyms for Somatostatin expressing interneurons).
Reviewer #3 (Public Review):
This paper compares the synaptic and membrane properties of two main subtypes of interneurons (PV+, SST+) in the auditory cortex of control mice vs mutants with Syngap1 haploinsufficiency. The authors find differences at both levels, although predominantly in PV+ cells. These results suggest that altered PV-interneuron functions in the auditory cortex may contribute to the network dysfunction observed in Syngap1 haploinsufficiency-related intellectual disability. The subject of the work is interesting, and most of the approach is direct and quantitative, which are major strengths. There are also some weaknesses that reduce its impact for a broader field.
(1) The choice of mice with conditional (rather than global) haploinsufficiency makes the link between the findings and Syngap1 relatively easy to interpret, which is a strength. However, it also remains unclear whether an entire network with the same mutation at a global level (affecting also excitatory neurons) would react similarly.
The reviewer raises an interesting and pertinent open question which we will address in the discussion of the revised paper.
(2) There are some (apparent?) inconsistencies between the text and the figures. Although the authors appear to have used a sophisticated statistical analysis, some datasets in the illustrations do not seem to match the statistical results. For example, neither Fig 1g nor Fig 3f (eNMDA) reach significance despite large differences.
We respectfully disagree, we do not think the text and figures are inconsistent. In the cited example, large apparent difference in mean values does not show significance due to the large variability in the data; further, we did not exclude any data points, because we wanted to be rigorous. In particular, for Fig.1g, statistical analysis shows a significant increase in the inter-mEPSC interval (*p=0.027, LMM) when all events are considered (cumulative probability plots), while there is no significant difference in the inter-mEPSCs interval for inter-cell mean comparison (inset, p=0.354, LMM). Inter-cell mean comparison does not show difference with Mann-Whitney test either (p=0.101, the data are not normally distributed, hence the choice of the Mann-Whitney test). For Fig. 3f (eNMDA), the higher mean value for the cHet versus the control is driven by two data points which are particularly high, while the other data points overlap with the control values. The Mann-Whitney test show also no statistical difference (p=0.174).
In the manuscript, discussion of the data is based on the results of the LMM analysis, which takes in account both the number of cells and the numbers of mice from which these cells are recorded. We chose this statistical approach because it does not rely on the assumption that cells recorded from same mouse are independent variables. In the supplemental tables, we provided the results of the statistical analysis done with both LMM and the most commonly used Mann Whitney (for not normally distributed) or t-test (for normally distributed), for each data set.
Also, the legend to Fig 9 indicates the presence of "a significant decrease in AP half-width from cHet in absence or presence of a-DTX", but the bar graph does not seem to show that.
We apologize for our lack of clarity. In legend 9, we reported the statistical comparisons between 1) cHET mice in absence of a-DTX and control mice and 2) cHET mice in presence of a-DTX and control mice. We will rephrase result description and the legend of the figure to avoid confusion.
(3) The authors mention that the lack of differences in synaptic current kinetics is evidence against a change in subunit composition. However, in some Figures, for example, 3a, the kinetics of the recorded currents appear dramatically different. It would be important to know and compare the values of the series resistance between control and mutant animals.
We agree with the reviewer that there appears to be a qualitative difference in eNMDA decay between conditions, although quantified eNMDA decay itself is similar between groups. We have used a cutoff of 15 % for the series resistance (Rs), which is significantly more stringent as compared to the cutoff typically used in electrophysiology, which are for the vast majority between 20 and 30%. To answer this concern, we re-examined the Rs, we compared Rs between groups and found no difference for Rs in eAMPA (13.2±0.5 in WT n=16 cells, 7 mice vs 13.7±0.3 in cHet n=14 cells, 7 mice, p=0.432 LMM) and eNMDA (12.7±0.7 in WT n=6 cells, 3 mice vs 13.8±0.7 in cHet n=6 cells, 5 mice, p=0.231, LMM). Thus, the apparent qualitative difference in eNMDA decay stems from inter-cell variability rather than inter-group differences. Notably, this discrepancy between the trace (Fig. 3a) and the data (Fig. 3f, right) is largely due to inter-cell variability, particularly in eNMDA, where a higher but non-significant decay rate is driven by a couple of very high values (Fig. 3f, right). In the revised manuscript, we will show traces that better represent our findings.
(4) A significant unexplained variability is present in several datasets. For example, the AP threshold for PV+ includes points between -50-40 mV, but also values at around -20/-15 mV, which seems too depolarized to generate healthy APs (Fig 5c, Fig7c).
We acknowledge the variability in AP threshold data, with some APs appearing too depolarized to generate healthy spikes. However, we meticulously examined each AP that spiked at these depolarized thresholds and found that other intrinsic properties (such as Rin, Vrest, AP overshoot, etc.) all indicate that these cells are healthy. Therefore, to maintain objectivity and provide unbiased data to the community, we opted to include them in our analysis. It's worth noting that similar variability has been observed in other studies (Bengtsson Gonzales et al., 2020; Bertero et al., 2020).
Further, we conducted a significance test on AP threshold excluding these potentially unhealthy cells and found that the significant differences persist. After removing two outliers from the cHet group with values of -16.5 and 20.6 mV, we obtain: -42.6±1.01 mV in control, n=33, 15 mice vs -36.2±1.1 mV in cHet, n=38 cells, 17 mice, ***p<0.001, LMM. Thus, whether these cells are included or excluded, our interpretations and conclusions remain unchanged.
We would like to clarify that these data have not been corrected with the junction potential. We will add this info in the revised version.
(5) I am unclear as to how the authors quantified colocalization between VGluts and PSD95 at the low magnification shown in Supplementary Figure 2.
We apologize for our lack of clarity. Although the analysis was done at high resolution, the figures were focused on showing multiple PV somata receiving excitatory inputs. We will add higher magnification figures and more detailed information in the methods of the revised version. Please also see our response to reviewer #2.
(6) The authors claim that "cHet SST+ cells showed no significant changes in active and passive membrane properties", but this claim would seem to be directly refused by the data of Fig 8f. In the absence of changes in either active or passive membrane properties shouldn't the current/#AP plot remain unchanged?
While we acknowledge the theoretical expectation that changes in intrinsic parameters should correlate with alterations in neuronal firing, the absence of differences in the parameters analyzed in this study should not overshadow the clear and significant decrease in firing rate observed in cHet SST+ cells. This decrease serves as a compelling indication of reduced intrinsic neuronal excitability. It's certainly possible that other intrinsic factors, not assessed in this study, may have contributed to this effect. However, exploring these mechanisms is beyond the scope of our current investigation. We will rephrase the discussion and add this limitation of our study in the revised version.
(7) The plots used for the determination of AP threshold (Figs 5c, 7c, and 7h) suggest that the frequency of acquisition of current-clamp signals may not have been sufficient, this value is not included in the Methods section.
This study utilized a sampling rate of 10 kHz, which is a standard rate for action potential analysis in the present context. We will describe more extensively the technical details in the method section of the revised manuscript we are preparing. While we acknowledge that a higher sampling rate could have enhanced the clarity of the phase plot, our recording conditions, as detailed in our response to Rev#2/comment#5, were suitable for the objectives of this study.
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eLife assessment
This study provides valuable evidence indicating that SynGap1 regulates the synaptic drive and membrane excitability of parvalbumin- and somatostatin-positive interneurons in the auditory cortex. Since haplo-insufficiency of SynGap1 has been linked to intellectual disabilities without a well-defined underlying cause, the central question of this study is timely. However, the support for the authors' conclusions is incomplete in general and some parts of the experimental evidence are inadequate. Specifically, the manuscript requires further work to properly evaluate the impact on synaptic currents, intrinsic excitability parameters, and morphological features.
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Reviewer #1 (Public Review):
The study is designed to assess the role of Syngap1 in regulating the physiology of the MGE-derived PV+ and SST+ interneurons. Syngap1 is associated with some mental health disorders, and PV+ and SST+ cells are the focus of many previous and likely future reports from studies of interneuron biology, highlighting the translational and basic neuroscience relevance of the authors' work.
Strengths of the study are using well-established electrophysiology methods and the highly controlled conditions of ex vivo brain slice experiments combined with a novel intersectional mouse line, to assess the role of Syngap1 in regulating PV+ and SST+ cell properties. The findings revealed that in the mature auditory cortex, Syngap1 haploinsufficiency decreases both the intrinsic excitability and the excitatory synaptic drive …
Reviewer #1 (Public Review):
The study is designed to assess the role of Syngap1 in regulating the physiology of the MGE-derived PV+ and SST+ interneurons. Syngap1 is associated with some mental health disorders, and PV+ and SST+ cells are the focus of many previous and likely future reports from studies of interneuron biology, highlighting the translational and basic neuroscience relevance of the authors' work.
Strengths of the study are using well-established electrophysiology methods and the highly controlled conditions of ex vivo brain slice experiments combined with a novel intersectional mouse line, to assess the role of Syngap1 in regulating PV+ and SST+ cell properties. The findings revealed that in the mature auditory cortex, Syngap1 haploinsufficiency decreases both the intrinsic excitability and the excitatory synaptic drive onto PV+ neurons from Layer 4. In contrast, SST+ interneurons were mostly unaffected by Syngap1 haploinsufficiency. Pharmacologically manipulating the activity of voltage-gated potassium channels of the Kv1 family suggested that these channels contributed to the decreased PV+ neuron excitability by Syngap insufficiency. These results therefore suggest that normal Syngap1 expression levels are necessary to produce normal PV+ cell intrinsic properties and excitatory synaptic drive, albeit, perhaps surprisingly, inhibitory synaptic transmission was not affected by Syngap1 haploinsufficiency.
Since the electrophysiology experiments were performed in the adult auditory cortex, while Syngap1 expression was potentially affected since embryonic stages in the MGE, future studies should address two important points that were not tackled in the present study. First, what is the developmental time window in which Syngap1 insufficiency disrupted PV+ neuron properties? Albeit the embryonic Syngap1 deletion most likely affected PV+ neuron maturation, the properties of Syngap-insufficient PV+ neurons do not resemble those of immature PV+ neurons. Second, whereas the observation that Syngap1 haploinsufficiency affected PV+ neurons in auditory cortex layer 4 suggests auditory processing alterations, MGE-derived PV+ neurons populate every cortical area. Therefore, without information on whether Syngap1 expression levels are cortical area-specific, the data in this study would predict that by regulating PV+ neuron electrophysiology, Syngap1 normally controls circuit function in a wide range of cortical areas, and therefore a range of sensory, motor and cognitive functions. These are relatively minor weaknesses regarding interpretation of the data in the present study that the authors could discuss.
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Reviewer #2 (Public Review):
Summary:
In this manuscript, the authors investigated how partial loss of SynGap1 affects inhibitory neurons derived from the MGE in the auditory cortex, focusing on their synaptic inputs and excitability. While haplo-insufficiently of SynGap1 is known to lead to intellectual disabilities, the underlying mechanisms remain unclear.
Strengths:
The questions are novel
Weaknesses:
Despite the interesting and novel questions, there are significant concerns regarding the experimental design and data quality, as well as potential misinterpretations of key findings. Consequently, the current manuscript fails to contribute substantially to our understanding of SynGap1 loss mechanisms and may even provoke unnecessary controversies.
Major issues:
(1) One major concern is the inconsistency and confusion in the …
Reviewer #2 (Public Review):
Summary:
In this manuscript, the authors investigated how partial loss of SynGap1 affects inhibitory neurons derived from the MGE in the auditory cortex, focusing on their synaptic inputs and excitability. While haplo-insufficiently of SynGap1 is known to lead to intellectual disabilities, the underlying mechanisms remain unclear.
Strengths:
The questions are novel
Weaknesses:
Despite the interesting and novel questions, there are significant concerns regarding the experimental design and data quality, as well as potential misinterpretations of key findings. Consequently, the current manuscript fails to contribute substantially to our understanding of SynGap1 loss mechanisms and may even provoke unnecessary controversies.
Major issues:
(1) One major concern is the inconsistency and confusion in the intermediate conclusions drawn from the results. For instance, while the sEPSC data indicates decreased amplitude in PV+ and SOM+ cells in cHet animals, the frequency of events remains unchanged. In contrast, the mEPSC data shows no change in amplitudes in PV+ cells, but a significant decrease in event frequency. The authors conclude that the former observation implies decreased excitability. However, traditionally, such observations on mEPSC parameters are considered indicative of presynaptic mechanisms rather than changes of network activity. The subsequent synapse counting experiments align more closely with the traditional conclusions. This issue can be resolved by rephrasing the text. However, it would remain unexplained why the sEPSC frequency shows no significant difference. If the majority of sEPSC events were indeed mediated by spiking (which is blocked by TTX), the average amplitudes and frequency of mEPSCs should be substantially lower than those of sEPSCs. Yet, they fall within a very similar range, suggesting that most sEPSCs may actually be independent of action potentials. But if that was indeed the case, the changes of purported sEPSC and mEPSC results should have been similar.
(2) Another significant concern is the quality of synapse counting experiments. The authors attempted to colocalize pre- and postsynaptic markers Vglut1 and PSD95 with PV labelling. However, several issues arise. Firstly, the PV labelling seems confined to soma regions, with no visible dendrites. Given that the perisomatic region only receives a minor fraction of excitatory synapses, this labeling might not accurately represent the input coverage of PV cells.
Secondly, the resolution of the images is insufficient to support clear colocalization of the synaptic markers. Thirdly, the staining patterns are peculiar, with PSD95 puncta appearing within regions clearly identified as somas by Vglut1, hinting at possible intracellular signals. Furthermore, PSD95 seems to delineate potential apical dendrites of pyramidal cells passing through the region, yet Vglut1+ partners are absent in these segments, which are expected to be the marker of these synapses here.
Additionally, the cumulative density of Vglut2 and Vglut1 puncta exceeds expectations, and it's surprising that subcortical fibers labeled by Vglut2 are comparable in number to intracortical Vglut1+ axon terminals. Ideally, N(Vglut1)+N(Vglut2) should be equal or less than N(PSD95), but this is not the case here. Consequently, these results cannot be considered reliable due to these issues.(3) One observation from the minimal stimulation experiment was concluded by an unsupported statement. Namely, the change in the onset delay cannot be attributed to a deficit in the recruitment of PV+ cells, but it may suggest a change in the excitability of TC axons.
(4) The conclusions drawn from the stimulation experiments are also disconnected from the actual data. To make conclusions about TC release, the authors should have tested release probability using established methods, such as paired-pulse changes. Instead, the only observation here is a change in the AMPA components, which remained unexplained.
(5) The sampling rate of CC recordings is insufficient to resolve the temporal properties of the APs. Therefore, the phase-plots cannot be interpreted (e.g. axonal and somatic AP components are not clearly separated), raising questions about how AP threshold and peak were measured. The low sampling rate also masks the real derivative of the AP signals, making them apparently faster.
A related issue is that the Methods section lacks essential details about the recording conditions, such as bridge balance and capacitance neutralization.(6) Interpretation issue: One of the most fundamental measures of cellular excitability, the rheobase, was differentially affected by cHet in BCshort and BCbroad. Yet, the authors concluded that the cHet-induced changes in the two subpopulations are common.
(7) Design issue:
The Kv1 blockade experiments are disconnected from the main manuscript. There is no experiment that shows the causal relationship between changes in DTX and cHet cells. It is only an interesting observation on AP halfwidth and threshold. However, how they affect rheobase, EPSCs, and other topics of the manuscript are not addressed in DTX experiments.
Furthermore, Kv1 currents were never measured in this work, nor was the channel density tested. Thus, the DTX effects are not necessarily related to changes in PV cells, which can potentially generate controversies.(8) Writing issues:
Abstract:
The auditory system is not mentioned in the abstract.
One statement in the abstract is unclear. What is meant by "targeting Kv1 family of voltage-gated potassium channels was sufficient..."? "Targeting" could refer to altered subcellular targeting of the channels, simple overexpression/deletion in the target cell population, or targeted mutation of the channel, etc. Only the final part of the Results revealed that none of the above, but these channels were blocked selectively.
Introduction:
There is a contradiction in the introduction. The second paragraph describes in detail the distinct contribution of PV and SST neurons to auditory processing. But at the end, the authors state that "relatively few reports on PV+ and SST+ cell-intrinsic and synaptic properties in adult auditory cortex". Please be more specific about the unknown properties.(9) The introduction emphasizes the heterogeneity of PV neurons, which certainly influences the interpretation of the results of the current manuscript. However, the initial experiments did not consider this and handled all PV cell data as a pooled population.
(10) The interpretation of the results strongly depends on unpublished work, which potentially provide the physiological and behavioral contexts about the role of GABAergic neurons in SynGap-haploinsufficiency. The authors cite their own unpublished work, without explaining the specific findings and relation to this manuscript.
(11) The introduction of Scholl analysis experiments mentions SOM staining, however, there is no such data about this cell type in the manuscript.
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Reviewer #3 (Public Review):
This paper compares the synaptic and membrane properties of two main subtypes of interneurons (PV+, SST+) in the auditory cortex of control mice vs mutants with Syngap1 haploinsufficiency. The authors find differences at both levels, although predominantly in PV+ cells. These results suggest that altered PV-interneuron functions in the auditory cortex may contribute to the network dysfunction observed in Syngap1 haploinsufficiency-related intellectual disability. The subject of the work is interesting, and most of the approach is direct and quantitative, which are major strengths. There are also some weaknesses that reduce its impact for a broader field.
(1) The choice of mice with conditional (rather than global) haploinsufficiency makes the link between the findings and Syngap1 relatively easy to …
Reviewer #3 (Public Review):
This paper compares the synaptic and membrane properties of two main subtypes of interneurons (PV+, SST+) in the auditory cortex of control mice vs mutants with Syngap1 haploinsufficiency. The authors find differences at both levels, although predominantly in PV+ cells. These results suggest that altered PV-interneuron functions in the auditory cortex may contribute to the network dysfunction observed in Syngap1 haploinsufficiency-related intellectual disability. The subject of the work is interesting, and most of the approach is direct and quantitative, which are major strengths. There are also some weaknesses that reduce its impact for a broader field.
(1) The choice of mice with conditional (rather than global) haploinsufficiency makes the link between the findings and Syngap1 relatively easy to interpret, which is a strength. However, it also remains unclear whether an entire network with the same mutation at a global level (affecting also excitatory neurons) would react similarly.
(2) There are some (apparent?) inconsistencies between the text and the figures. Although the authors appear to have used a sophisticated statistical analysis, some datasets in the illustrations do not seem to match the statistical results. For example, neither Fig 1g nor Fig 3f (eNMDA) reach significance despite large differences. Also, the legend to Fig 9 indicates the presence of "a significant decrease in AP half-width from cHet in absence or presence of a-DTX", but the bar graph does not seem to show that.
(3) The authors mention that the lack of differences in synaptic current kinetics is evidence against a change in subunit composition. However, in some Figures, for example, 3a, the kinetics of the recorded currents appear dramatically different. It would be important to know and compare the values of the series resistance between control and mutant animals.
(4) A significant unexplained variability is present in several datasets. For example, the AP threshold for PV+ includes points between -50-40 mV, but also values at around -20/-15 mV, which seems too depolarized to generate healthy APs (Fig 5c, Fig7c).
(5) I am unclear as to how the authors quantified colocalization between VGluts and PSD95 at the low magnification shown in Supplementary Figure 2.
(6) The authors claim that "cHet SST+ cells showed no significant changes in active and passive membrane properties", but this claim would seem to be directly refused by the data of Fig 8f. In the absence of changes in either active or passive membrane properties shouldn't the current/#AP plot remain unchanged?
(7) The plots used for the determination of AP threshold (Figs 5c, 7c, and 7h) suggest that the frequency of acquisition of current-clamp signals may not have been sufficient, this value is not included in the Methods section.
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