Local Inhibitory Dynamics Underpin Temporal Integration and Functional Segregation between Barrels and Septa in the Mouse Barrel Cortex

Reviewer #1 (Public review):

Summary:

Argunşah et al. describe and investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains of the whisker-related primary somatosensory cortex (S1). Upon repeated stimulation, the authors report that the response ratio between multi- and single-whisker stimulation increases in layer (L) 4 neurons of the septal domain, while remaining constant in barrel L4 neurons. This difference is attributed to the short-term plasticity properties of interneurons, particularly somatostatin-expressing (SST+) neurons. This claim is supported by the increased density of SST+ neurons found in L4 of the septa compared to barrels, along with a stronger response of (L2/3) SST+ neurons to repeated multi- vs single-whisker stimulation. The role of the synaptic protein Elfn1 is then examined. Elfn1 KO mice exhibited little to no functional domain separation between barrel and septa, with no significant difference in single- versus multi-whisker response ratios across barrel and septal domains. Consistently, a decoder trained on WT data fails to generalize to Elfn1 KO responses. Finally, the authors report a relative enrichment of S2- and M1-projecting cell densities in L4 of the septal domain compared to the barrel domain.

Strengths:

This paper describes and aims to study a circuit underlying differential response between barrel columns and septal domains of the primary somatosensory cortex. This work supports the view that barrel and septal domains contribute differently to processing single versus multi-whisker inputs, suggesting that the barrel cortex multiplexes sensory information coming from the whiskers in different domains.

Weaknesses:

While the observed divergence in responses to repeated SWS vs MWS between the barrel and septal domains is intriguing, the presented evidence falls short of demonstrating that short-term plasticity in SST+ neurons critically underpins this difference. The absence of a mechanistic explanation for this observation limits the work's significance. The measurement of SST neurons' response is not specific to a particular domain, and the Elfn1 manipulation does not seem to be specific to either stimulus type or a particular domain. The study's reach is further constrained by the fact that results were obtained in anesthetized animals, which may not generalize to awake states. The statistical analysis appears inappropriate, with the use of repeated independent tests, dramatically boosting the false positive error rate. Furthermore, the manuscript suffers from imprecision; its conclusions are occasionally vague or overstated.

The authors suggest a role for SST+ neurons in the observed divergence in SWS/MWS responses between barrel and septal domains. However, this remains speculative, and some findings appear inconsistent. For instance, the increased response of SST+ neurons to MWS versus SWS is not confined to a specific domain. Why, then, would preferential recruitment of SST+ neurons lead to divergent dynamics between barrel and septal regions? The higher density of SST+ neurons in septal versus barrel L4 is not a sufficient explanation, particularly since the SWS/MWS response divergence is also observed in layers 2/3, where no difference in SST+ neuron density is found. Moreover, SST+ neuron-mediated inhibition is not necessarily restricted to the layer in which the cell body resides. It remains unclear through which differential microcircuits (barrel vs septum) the enhanced recruitment of SST+ neurons could account for the divergent responses to repeated SWS versus MWS stimulation.

The Elfn1 KO mouse model seems too unspecific to suggest the role of the short-term plasticity in SST+ neurons in the differential response to repeated SWS vs MWS stimulation across domains. Why would Elfn1-dependent short-term plasticity in SST+ neurons be specific to a pathway, or a stimulation type (SWS vs MWS)? Moreover, the authors report that Elfn1 knockout alters synapses onto VIP+ as well as SST+ neurons (Stachniak et al., 2021; previous version of this paper)-so why attribute the phenotype solely to SST+ circuitry? In fact, the functional distinctions between barrel and septal domains appear largely abolished in the Elfn1 KO.

Reviewer #2 (Public review):

Summary:

Argunsah and colleagues demonstrate that SST-expressing interneurons are concentrated in the mouse septa and differentially respond to repetitive multi-whisker inputs. Identifying how a specific neuronal phenotype impacts responses is an advance.

Strengths:

(1) Careful physiological and imaging studies.

(2) Novel result showing the role of SST+ neurons in shaping responses.

(3) Good use of a knockout animal to further the main hypothesis.

(4) Clear analytical techniques.

Weaknesses:

No major weaknesses were identified by this reviewer. Overall I appreciated the paper but feel it overlooked a few issues and had some recommendations on how additional clarifications could strengthen the paper. These include:

(1) Significant work from Jerry Chen on how S1 neurons that project to M1 versus S2 respond in a variety of behavioral tasks should be included (e.g. PMID: 26098757). Similarly, work from Barry Connor's lab on intracortical versus thalamocortical inputs to SST neurons, as well as excitatory inputs onto these neurons (e.g. PMID: 12815025) should be included.

(2) Using Layer 2/3 as a proxy to what is happening in layer 4 (~line 234). Given that layer 2/3 cells integrate information from multiple barrels, as well as receiving direct VPm thalamocortical input, and given the time window that is being looked at can receive input from other cortical locations, it is not clear that layer 2/3 is a proxy for what is happening in layer 4.

(3) Line 267, when discussing distinct temporal response, it is not well defined what this is referring to. Are the neurons no longer showing peaks to whisker stimulation, or are the responses lasting a longer time? It is unclear why PV+ interneurons which may not be impacted by the Elfn1 KO and receive strong thalamocortical inputs, are not constraining activity.

(4) Line 585 "the earliest CSD sink was identified as layer 4..." were post-hoc measurements made to determine where the different shank leads were based on the post-hoc histology?

(5) For the retrograde tracing studies, how were the M1 and S2 injections targeted (stereotaxically or physiologically)? How was it determined that the injections were in the whisker region (or not)?

(6) Were there any baseline differences in spontaneous activity in the speta versus barrel regions, and did this change in the KO animals?

Reviewer #3 (Public review):

Summary:

This study investigates the functional differences between barrel and septal columns in the mouse somatosensory cortex, focusing on how local inhibitory dynamics, particularly involving Elfn1-expressing SST⁺ interneurons, may mediate temporal integration of multi-whisker (MW) stimuli in septa. Using a combination of in vivo multi-unit recordings, calcium imaging, and anatomical tracing, the authors propose that septa integrate MW input in an Elfn1-dependent manner, enabling functional segregation from barrel columns.

Strengths:

The core hypothesis is interesting and potentially impactful. While barrels have been extensively characterized, septa remain less understood, especially in mice, and this study's focus on septal integration of MW stimuli offers valuable insights into this underexplored area. If septa indeed act as selective integrators of distributed sensory input, this would add a novel computational role to cortical microcircuits beyond what is currently attributed to barrels alone. The narrative of this paper is intellectually stimulating.

Weaknesses:

The methods used in the current study lack the spatial and cellular resolution needed to conclusively support the central claims. The main physiological findings are based on unsorted multi-unit activity (MUA) recorded via low-channel-count silicon probes. MUA inherently pools signals from multiple neurons across different distances and cell types, making it difficult to assign activity to specific columns (barrel vs. septa) or neuron classes (e.g., SST⁺ vs. excitatory). The recording radius (~50-100 µm or more) and the narrow width of septa (~50-100 µm or less) make it likely that MUA from "septal" electrodes includes spikes from adjacent barrel neurons. The authors do not provide spike sorting, unit isolation, or anatomical validation that would strengthen spatial attribution. Calcium imaging is restricted to SST⁺ and VIP⁺ interneurons in superficial layers (L2/3), while the main MUA recordings are from layer 4, creating a mismatch in laminar relevance.

Furthermore, while the role of Elfn1 in mediating short-term facilitation is supported by prior studies, no new evidence is presented in this paper to confirm that this synaptic mechanism is indeed disrupted in the knockout mice used here. Additionally, since Elfn1 is constitutively knocked out from development, the possibility of altered circuit formation-including changes in barrel structure and interneuron distribution, cannot be excluded and is not addressed.

Author response:

Reviewer #1 (Public review):

Summary:

Argunşah et al. describe and investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains of the whisker-related primary somatosensory cortex (S1). Upon repeated stimulation, the authors report that the response ratio between multi- and single-whisker stimulation increases in layer (L) 4 neurons of the septal domain, while remaining constant in barrel L4 neurons. This difference is attributed to the short-term plasticity properties of interneurons, particularly somatostatin-expressing (SST+) neurons. This claim is supported by the increased density of SST+ neurons found in L4 of the septa compared to barrels, along with a stronger response of (L2/3) SST+ neurons to repeated multi- vs single-whisker stimulation. The role of the synaptic protein Elfn1 is then examined. Elfn1 KO mice exhibited little to no functional domain separation between barrel and septa, with no significant difference in single- versus multi-whisker response ratios across barrel and septal domains. Consistently, a decoder trained on WT data fails to generalize to Elfn1 KO responses. Finally, the authors report a relative enrichment of S2- and M1-projecting cell densities in L4 of the septal domain compared to the barrel domain.

Strengths:

This paper describes and aims to study a circuit underlying differential response between barrel columns and septal domains of the primary somatosensory cortex. This work supports the view that barrel and septal domains contribute differently to processing single versus multi-whisker inputs, suggesting that the barrel cortex multiplexes sensory information coming from the whiskers in different domains.

We thank the reviewer for the very neat summary of our findings that barrel cortex multiplexes converging information in separate domains.

Weaknesses:

While the observed divergence in responses to repeated SWS vs MWS between the barrel and septal domains is intriguing, the presented evidence falls short of demonstrating that short-term plasticity in SST+ neurons critically underpins this difference. The absence of a mechanistic explanation for this observation limits the work's significance. The measurement of SST neurons' response is not specific to a particular domain, and the Elfn1 manipulation does not seem to be specific to either stimulus type or a particular domain.

We appreciate the reviewer’s perspective. Although further research is needed to understand the circuit mechanisms underlying the observed phenomenon, we believe our data suggest that altering the short-term dynamics of excitatory inputs onto SST neurons reduces the divergent spiking dynamics in barrels versus septa during repetitive single- and multi-whisker stimulation. Future work could examine how SST neurons, whose somata reside in barrels and septa, respond to different whisker stimuli and the circuits in which they are embedded. At this time, however, the authors believe there is no alternative way to test how the short-term dynamics of excitatory inputs onto SST neurons, as a whole, contribute to the temporal aspects of barrel versus septa spiking.

The study's reach is further constrained by the fact that results were obtained in anesthetized animals, which may not generalize to awake states.

We appreciate the reviewer’s concern regarding the generalizability of our findings from anesthetized animals to awake states. Anesthesia was employed to ensure precise individual whisker stimulation (and multi-whisker in the same animal), which is challenging in awake rodents due to active whisking. While anesthesia may alter higher-order processing, core mechanisms, such as short and long term plasticity in the barrel cortex, are preserved under anesthesia (Martin-Cortecero et al., 2014; Mégevand et al., 2009).

The statistical analysis appears inappropriate, with the use of repeated independent tests, dramatically boosting the false positive error rate.

Thank you for your feedback on our analysis using independent rank-based tests for each time point in wild-type (WT) animals. To address concerns regarding multiple comparisons and temporal dependencies (for Figure 1F and 4D for now but we will add more in our revision), we performed a repeated measures ANOVA for WT animals (13 Barrel, 8 Septa, 20 time points), which revealed a significant main effect of Condition (F(1,19) = 16.33, p < 0.001) and a significant Condition-Time interaction (F(19,361) = 2.37, p = 0.001). Post-hoc tests confirmed significant differences between Barrel and Septa at multiple time points (e.g., p < 0.0025 at times 3, 4, 6, 7, 8, 10, 11, 12, 16, 19 after Bonferroni posthoc correction), supporting a differential multi-whisker vs. single-whisker ratio response in WT animals. In contrast, a repeated measures ANOVA for knock-out (KO) animals (11 Barrel, 7 Septa, 20 time points) showed no significant main effect of Condition (F(1,14) = 0.17, p = 0.684) or Condition-Time interaction (F(19,266) = 0.73, p = 0.791), indicating that the Barrel-Septa difference observed in WT animals is absent in KO animals.

Furthermore, the manuscript suffers from imprecision; its conclusions are occasionally vague or overstated. The authors suggest a role for SST+ neurons in the observed divergence in SWS/MWS responses between barrel and septal domains. However, this remains speculative, and some findings appear inconsistent. For instance, the increased response of SST+ neurons to MWS versus SWS is not confined to a specific domain. Why, then, would preferential recruitment of SST+ neurons lead to divergent dynamics between barrel and septal regions? The higher density of SST+ neurons in septal versus barrel L4 is not a sufficient explanation, particularly since the SWS/MWS response divergence is also observed in layers 2/3, where no difference in SST+ neuron density is found.

Moreover, SST+ neuron-mediated inhibition is not necessarily restricted to the layer in which the cell body resides. It remains unclear through which differential microcircuits (barrel vs septum) the enhanced recruitment of SST+ neurons could account for the divergent responses to repeated SWS versus MWS stimulation.

We fully appreciate the reviewer’s comment. We currently do not provide any evidence on the contribution of SST neurons in the barrels versus septa in layer 4 on the response divergence of spiking observed in SWS versus MWS. We only show that these neurons differentially distribute in the two domains in this layer. It is certainly known that there is molecular and circuit-based diversity of SST-positive neurons in different layers of the cortex, so it is plausible that this includes cells located in the two domains of vS1, something which has not been examined so far. Our data on their distribution are one piece of information that SST neurons may have a differential role in inhibiting barrel stellate cells versus septa ones. Morphological reconstructions of SST neurons in L4 of the somatosensory barrel cortex has shown that their dendrites and axons project locally and may confine to individual domains, even though not specifically examined (Fig. 3 of Scala F et al., 2019). The same study also showed that L4 SST cells receive excitatory input from local stellate cells) and is known that they are also directly excited by thalamocortical fibers (Beierlein et al., 2003; Tan et al., 2008), both of which facilitate.

As shown in our supplementary figure, the divergence is also observed in L2/3 where, as the reviewer also points out, where we do not have a differential distribution of SST cells, at least based on a columnar analysis extending from L4. There are multiple scenarios that could explain this “discrepancy” that one would need to examine further in future studies. One straightforward one is that the divergence in spiking in L2/3 domains may be inherited from L4 domains, where L4 SST act on. Another is that even though L2/3 SST neurons are not biased in their distribution their input-output function is, something which one would need to examine by detailed in vitro electrophysiological and perhaps optogenetic approaches in S1. Despite the distinctive differences that have been found between the L4 circuitry in S1 and V1 (Scala F et al., 2019), recent observations indicate that small but regular patches of V1 marked by the absence of muscarinic receptor 2 (M2) have high temporal acuity (Ji et al., 2015), and selectively receive input from SST interneurons (Meier et al., 2025). Regions lacking M2 have distinct input and output connectivity patterns from those that express M2 (Meier et al., 2021; Burkhalter et al., 2023). These findings, together with ours, suggest that SST cells preferentially innervate and regulate specific domains -columns- in sensory cortices.

Regardless of the mechanism, the Elfn1 knock-out mouse line almost exclusively affects the incoming excitability onto SST neurons (see also reply to comment below), hence what can be supported by our data is that changing the incoming short-term synaptic plasticity onto these neurons brings the spiking dynamics between barrels and septa closer together.

The Elfn1 KO mouse model seems too unspecific to suggest the role of the short-term plasticity in SST+ neurons in the differential response to repeated SWS vs MWS stimulation across domains. Why would Elfn1-dependent short-term plasticity in SST+ neurons be specific to a pathway, or a stimulation type (SWS vs MWS)? Moreover, the authors report that Elfn1 knockout alters synapses onto VIP+ as well as SST+ neurons (Stachniak et al., 2021; previous version of this paper)-so why attribute the phenotype solely to SST+ circuitry? In fact, the functional distinctions between barrel and septal domains appear largely abolished in the Elfn1 KO.

Previous work by others and us has shown that globally removing Elfn1 selectively removes a synaptic process from the brain without altering brain anatomy or structure. This allows us to study how the temporal dynamics of inhibition shape activity, as opposed to inhibition from particular cell types. We will nevertheless update the text to discuss more global implications for SST interneuron dynamics and include a reference to VIP interneurons that contain Elfn1.

When comparing SWS to MWS, we find that MWS replaces the neighboring excitation which would normally be preferentially removed by short-term plasticity in SST interneurons, thus providing a stable control comparison across animals and genotypes. On average, VIP interneurons failed to show modulation by MWS. We were unable to measure a substantial contribution of VIP cells to this process and also note that the Elfn1 expressing multipolar neurons comprise only ~5% of VIP neurons (Connor and Peters, 1984; Stachniak et al., 2021), a fraction that may be lost when averaging from 138 VIP cells. Moreover, the effect of Elfn1 loss on VIP neurons is quite different and marginal compared to that of SST cells, suggesting that the primary impact of Elfn1 knockout is mediated through SST+ interneuron circuitry. Therefore, even if we cannot rule out that these 5% of VIP neurons contribute to barrel domain segregation, we are of the opinion that their influence would be very limited if any.

Reviewer #2 (Public review):

Summary:

Argunsah and colleagues demonstrate that SST-expressing interneurons are concentrated in the mouse septa and differentially respond to repetitive multi-whisker inputs. Identifying how a specific neuronal phenotype impacts responses is an advance.

Strengths:

(1) Careful physiological and imaging studies.

(2) Novel result showing the role of SST+ neurons in shaping responses.

(3) Good use of a knockout animal to further the main hypothesis.

(4) Clear analytical techniques.

We thank the reviewer for their appreciation of the study.

Weaknesses:

No major weaknesses were identified by this reviewer. Overall, I appreciated the paper but feel it overlooked a few issues and had some recommendations on how additional clarifications could strengthen the paper. These include:

(1) Significant work from Jerry Chen on how S1 neurons that project to M1 versus S2 respond in a variety of behavioral tasks should be included (e.g. PMID: 26098757). Similarly, work from Barry Connor's lab on intracortical versus thalamocortical inputs to SST neurons, as well as excitatory inputs onto these neurons (e.g. PMID: 12815025) should be included.

We thank the reviewer for these valuable resources that we overlooked. We will include Chen et al. (2015), Cruikshank et al. (2007) and Gibson et al. (1999) to contextualize S1 projections and SST+ inputs, strengthening the study’s foundation as well as Beierlein et al. (2003) which nicely show both local and thalamocortical facilitation of excitatory inputs onto L4 SST neurons, in contrast to PV cells. The paper also shows the gradual recruitment of SST neurons by thalamocortical inputs to provide feed-forward inhibition onto stellate cells (regular spiking) of the barrel cortex L4 in rat.

(2) Using Layer 2/3 as a proxy to what is happening in layer 4 (~line 234). Given that layer 2/3 cells integrate information from multiple barrels, as well as receiving direct VPm thalamocortical input, and given the time window that is being looked at can receive input from other cortical locations, it is not clear that layer 2/3 is a proxy for what is happening in layer 4.

We agree with the reviewer that what we observe in L2/3 is not necessarily what is taking place in L4 SST-positive cells. The data on L2/3 was included to show that these cells, as a population, can show divergent responses when it comes to SWS vs MWS, which is not seen in L2/3 VIP neurons. Regardless of the mechanisms underlying it, our overall data support that SST-positive neurons can change their activation based on the type of whisker stimulus and when the excitatory input dynamics onto these neurons change due to the removal of Elfn1 the recruitment of barrels vs septa spiking changes at the temporal domain. Having said that, the data shown in Supplementary Figure 3 on the response properties of L2/3 neurons above the septa vs above the barrels (one would say in the respective columns) do show the same divergence as in L4. This suggests that a circuit motif may exist that is common to both layers, involving SST neurons that sit in L4, L5 or even L2/3. This implies that despite the differences in the distribution of SST neurons in septa vs barrels of L4 there is an unidentified input-output spatial connectivity motif that engages in both L2/3 and L4. Please also see our response to a similar point raised by reviewer 1.

(3) Line 267, when discussing distinct temporal response, it is not well defined what this is referring to. Are the neurons no longer showing peaks to whisker stimulation, or are the responses lasting a longer time? It is unclear why PV+ interneurons which may not be impacted by the Elfn1 KO and receive strong thalamocortical inputs, are not constraining activity.

We thank the reviewer for their comment and will clarify the statement.

This convergence of response profiles was further clear in stimulus-aligned stacked images, where the emergent differences between barrels and septa under SWS were largely abolished in the KO (Figure 4B). A distinction between directly stimulated barrels and neighboring barrels persisted in the KO. In addition, the initial response continued to differ between barrel and septa and also septa and neighbor (Figure 4B). This initial stimulus selectivity potentially represents distinct feedforward thalamocortical activity, which includes PV+ interneuron recruitment that is not directly impacted by the Elfn1 KO (Sun et al., 2006; Tan et al., 2008). PV+ cells are strongly excited by thalamocortical inputs, but these exhibit short-term depression, as does their output, contrasting with the sustained facilitation observed in SST+ neurons. These findings suggest that in WT animals, activity spillover from principal barrels is normally constrained by the progressive engagement of SST+ interneurons in septal regions, driven by Elfn1-dependent facilitation at their excitatory synapses. In the absence of Elfn1, this local inhibitory mechanism is disrupted, leading to longer responses in barrels, delayed but stronger responses in septa, and persistently stronger responses in unstimulated neighbors, resulting in a loss of distinction between the responses of barrel and septa domains that normally diverge over time (see Author response image 1 below).

Author response image 1.

A) Barrel responses are longer following whisker stimulation in KO. B) Septal responses are slightly delayed but stronger in KO. C) Unstimulated neighbors show longer persistent responses in KO.

(4) Line 585 "the earliest CSD sink was identified as layer 4..." were post-hoc measurements made to determine where the different shank leads were based on the post-hoc histology?

Post hoc histology was performed on plane-aligned brain sections which would allow us to detect barrels and septa, so as to confirm the insertion domains of each recorded shank. Layer specificity of each electrode therefore could therefore not be confirmed by histology as we did not have coronal sections in which to measure electrode depth.

(5) For the retrograde tracing studies, how were the M1 and S2 injections targeted (stereotaxically or physiologically)? How was it determined that the injections were in the whisker region (or not)?

During the retrograde virus injection, the location of M1 and S2 injections was determined by stereotaxic coordinates (Yamashita et al., 2018). After acquiring the light-sheet images, we were able to post hoc examine the injection site in 3D and confirm that the injections were successful in targeting the regions intended. Although it would have been informative to do so, we did not functionally determine the whisker-related M1 and whisker-related S2 region in this experiment.

(6) Were there any baseline differences in spontaneous activity in the septa versus barrel regions, and did this change in the KO animals?

Thank you for this interesting question. Our previous study found that there was a reduction in baseline activity in L4 barrel cortex of KO animals at postnatal day (P)12, but no differences were found at P21 (Stachniak et al., 2023).

Reviewer #3 (Public review):

Summary:

This study investigates the functional differences between barrel and septal columns in the mouse somatosensory cortex, focusing on how local inhibitory dynamics, particularly involving Elfn1-expressing SST⁺ interneurons, may mediate temporal integration of multi-whisker (MW) stimuli in septa. Using a combination of in vivo multi-unit recordings, calcium imaging, and anatomical tracing, the authors propose that septa integrate MW input in an Elfn1-dependent manner, enabling functional segregation from barrel columns.

Strengths:

The core hypothesis is interesting and potentially impactful. While barrels have been extensively characterized, septa remain less understood, especially in mice, and this study's focus on septal integration of MW stimuli offers valuable insights into this underexplored area. If septa indeed act as selective integrators of distributed sensory input, this would add a novel computational role to cortical microcircuits beyond what is currently attributed to barrels alone. The narrative of this paper is intellectually stimulating.

We thank the reviewer for finding the study intellectually stimulating.

Weaknesses:

The methods used in the current study lack the spatial and cellular resolution needed to conclusively support the central claims. The main physiological findings are based on unsorted multi-unit activity (MUA) recorded via low-channel-count silicon probes. MUA inherently pools signals from multiple neurons across different distances and cell types, making it difficult to assign activity to specific columns (barrel vs. septa) or neuron classes (e.g., SST⁺ vs. excitatory).

The recording radius (~50-100 µm or more) and the narrow width of septa (~50-100 µm or less) make it likely that MUA from "septal" electrodes includes spikes from adjacent barrel neurons.

The authors do not provide spike sorting, unit isolation, or anatomical validation that would strengthen spatial attribution. Calcium imaging is restricted to SST⁺ and VIP⁺ interneurons in superficial layers (L2/3), while the main MUA recordings are from layer 4, creating a mismatch in laminar relevance.

We thank the reviewer for pointing out the possibility of contamination in septal electrodes. Importantly, it may not have been highlighted, although reported in the methods, but we used an extremely high threshold (7.5 std, in methods, line 583) for spike detection in order to overcome the issue raised here, which restricts such spatial contaminations. Since the spike amplitude decays rapidly with distance, at high thresholds, only nearby neurons contribute to our analysis, potentially one or two. We believe that this approach provides a very close approximation of single unit activity (SUA) in our reported data. We will include a sentence earlier in the manuscript to make this explicit and prevent further confusion.

Regarding the point on calcium imaging being performed on L2/3 SST and VIP cells instead of L4. Both reviewer 1 and 2 brought up the same issue and we responded as follows. As shown in our supplementary figure, the divergence is also observed in L2/3 where we do not have a differential distribution of SST cells, at least based on a columnar analysis extending from L4. There are multiple scenarios that could explain this “discrepancy” that one would need to examine further in future studies. One straightforward one is that the divergence in spiking in L2/3 domains may be inherited from L4 domains, where L4 SST act on. Another is that even though L2/3 SST neurons are not biased in their distribution their input-output function is, something which one would need to examine by detailed in vitro electrophysiological and perhaps optogenetic approaches in S1. Despite the distinctive differences that have been found between the L4 circuitry in S1 and V1 (Scala F et al., 2019), recent observations indicate that small but regular patches of V1 marked by the absence of muscarinic receptor 2 (M2) have high temporal acuity (Ji et al., 2015), and selectively receive input from SST interneurons (Meier et al., 2025). Regions lacking M2 have distinct input and output connectivity patterns from those that express M2 (Meier et al., 2021; Burkhalter et al., 2023). These findings, together with ours, suggest that SST cells preferentially innervate and regulate specific domains -columns- in sensory cortices.

Furthermore, while the role of Elfn1 in mediating short-term facilitation is supported by prior studies, no new evidence is presented in this paper to confirm that this synaptic mechanism is indeed disrupted in the knockout mice used here.

We thank Reviewer #3 for noting the absence of new evidence confirming Elfn1’s disruption of short-term facilitation in our knockout mice. We acknowledge that our study relies on previously strong published data demonstrating that Elfn1 mediates short-term synaptic facilitation of excitatory inputs onto SST+ interneurons (Sylwestrak and Ghosh, 2012; Tomioka et al., 2014; Stachniak et al., 2019, 2023). These studies consistently show that Elfn1 knockout abolishes facilitation in SST+ synapses, leading to altered temporal dynamics, which we hypothesize underlies the observed loss of barrel-septa response divergence in our Elfn1 KO mice (Figure 4). Nevertheless, to address the point raised, we will clarify in the revised manuscript (around lines 245-247 and 271-272) that our conclusions are based on these established findings, stating: “Building on prior evidence that Elfn1 knockout disrupts short-term facilitation in SST+ interneurons (Sylwestrak and Ghosh, 2012; Tomioka et al., 2014; Stachniak et al., 2019, 2023), we attribute the abolished barrel-septa divergence in Elfn1 KO mice to altered SST+ synaptic dynamics, though direct synaptic measurements were not performed here.”

Additionally, since Elfn1 is constitutively knocked out from development, the possibility of altered circuit formation-including changes in barrel structure and interneuron distribution, cannot be excluded and is not addressed.

We thank Reviewer #3 for raising the valid concern that constitutive Elfn1 knockout could potentially alter circuit formation, including barrel structure and interneuron distribution. To address this, we will clarify in the revised manuscript (around line ~271 and in the Discussion) that in our previous studies that included both whole-cell patch-clamp in acute brain slices ranging from postnatal day 11 to 22 (P11 - P21) and in vivo recordings from barrel cortex at P12 and P21, we saw no gross abnormalities in barrel structure, with Layer 4 barrels maintaining their characteristic size and organization, consistent with wild-type (WT) mice (Stachniak et al., 2019, 2023). While we cannot fully exclude subtle developmental changes, prior studies indicate that Elfn1 primarily modulates synaptic function rather than cortical cytoarchitecture (Tomioka et al., 2014). Elfn1 KO mice show no gross morphological or connectivity differences and the pattern and abundance of Elfn1 expressing cells (assessed by LacZ knock in) appears normal (Dolan and Mitchell, 2013).

We will add the following to the Discussion: “Although Elfn1 is constitutively knocked out, we find here and in previous studies that barrel structure is preserved (Stachniak et al., 2019, 2023). Further, the distribution of Elfn1 expressing interneurons is not different in KO mice, suggesting minimal developmental disruption (Dolan and Mitchell, 2013). Nonetheless, we acknowledge that subtle circuit changes cannot be ruled out without the usage of time-depended conditional knockout of the gene.”

References

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