Oxytocin facilitates social behavior of female rats via selective modulation of interneurons in the medial prefrontal cortex

Domain: chemistry
Published: 2026-02-20
Editorial outcome: major_revision

Abstract
The hypothalamic neuropeptide oxytocin is best known for its prosocial behavioral effects. However, the precise anatomical and cellular targets for oxytocin in the cortex during social behavior remain elusive. Here we show that oxytocin neurons project directly to the medial prefrontal cortex where evoked axonal oxytocin release facilitates social behaviors in adult female rats. In conjunction, we report that local oxytocin receptor-expressing (OTR+) cells are predominantly interneurons, whose activation promotes social interaction. Notably, this prosocial effect persists even under physiological challenge (hunger), pointing to a dedicated prosocial circuit capable of overriding primary survival drives. We further demonstrate that activation of these OTR+ interneurons inhibits principal cells specifically projecting to the basolateral amygdala, thus providing a putative mechanism of selective oxytocin action in this sociability-promoting cortical network.

Introduction
The neuropeptide oxytocin (OT) plays a central role in modulating various types of social behavior and is primarily synthesized in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory nuclei1,2. Hypothalamic OT is subsequently transported to the posterior lobe of the pituitary gland and released into the blood where it elicits milk ejection during lactation and uterine contractions during childbirth3. Beyond its peripheral effects, OT is actively involved in modulating neural circuits within the brain. Particularly, direct social interaction strongly activates the OT system via OT neurons in the PVN4. Axonal collaterals of OT neurons extend to various forebrain regions5, which coincides with the expression of OT receptors (OTRs) at the site of putative neuropeptide release6. These areas, including the central nucleus of the amygdala, nucleus accumbens, ventromedial hypothalamic nucleus, lateral septum, and hippocampus7,8, are pivotal in processing social information, emotional responses, and stress regulation9. The infusion of OT, evoked OT release, or activation of OTR neurons in these regions trigger a broad spectrum of region-specific social behaviors, such as social fear5,10,11, social transmission and contingency12, social memory13, social avoidance14, and aggression15. While most studies show that OT’s behavioral effects arise from subcortical circuits, emerging work has started to uncover its role in cortical signaling. OT signaling in the murine auditory cortex and its role in nursing, has been extensively studied by Froemke and colleagues16–18. Moreover, OT has been shown to drive top-down modulation of early sensory processing in the olfactory bulb to enhance social recognition19. Additionally, several studies have directly implicated cortical OT signaling in the regulation of socio-sexual behaviors: in the medial prefrontal cortex (mPFC), Heintz and colleagues identified OTR-expressing (OTR+) interneurons that gate female sociosexual behavior and revealed a sex-dimorphic OTR–CRH interaction shaping social and anxiety-related responses20,21. Moreover, OTR-expressing glutamatergic neurons in the mPFC have been shown to selectively regulate social recognition memory22.

The mPFC is instrumental in orchestrating various higher order and computational aspects of social behavior in mammals, including but not limited to social motivation23, recognition of social cues24,25, and decision-making26. The connections of the mPFC to other brain areas like the amygdala27 and hypothalamus28 suggest its role in top-down executive control, such as guiding goal-directed social behaviors. In rodents, the mPFC can be divided into three distinct brain subregions, each with unique functions and connectivity: the infralimbic cortex (ILC), the prelimbic cortex (PrL) and the cingulate cortex (Cgt)29. Considering the widespread influence of OT across various brain regions and its known input to the mPFC5, it is likely that OT modulates mPFC functions. Therefore, we injected a recombinant adeno-associated virus (rAAV) under the control of the OT promoter (rAAV-OTp-Venus) into either the PVN or SON in separate animals, with respective injection sites. Despite the inspiring demonstrations that OT action and/or OTR+ neurons in the mPFC modulate paternal30 and sexual behavior20 in voles and mice, respectively, the OT-modulated circuit that mediates same-sex affiliative (non-sexual) social behavior in the mPFC remains largely unexplored. To address this gap, we aimed to describe the pattern of OT innervation in the mPFC (1), the composition of the local OT receptive cells (2), and their downstream target structures (3), which might promote social behavior in adult female rats.

Results
Evoked OT release in the infralimbic cortex facilitates social behavior in female rats
Immunohistochemical (IHC) labeling revealed the presence of OT projections through the entire mPFC (Figs. 1A, B1–2). The highest density of OT axons was found in the infralimbic cortex (ILC) compared to the cingulate (Cgt) and prelimbic (PrL) cortices (49.9 ± 9.2 vs. 1.5 ± 0.8 axonal segments per mm2 in 50µm coronal slices; this and following data are presented as mean ± sd; p = 0.0002, details of statistical tests are provided in Supplementary Data 1; sample sizes are reported in the figure legends) (Fig. 1B3). Based on this finding, we focused specifically on the ILC in all follow-up experiments. Considering that the IHC approach labeled all OT fibers independent of origin, we next employed a viral approach to label specific nuclei in the hypothalamus to determine the source of OT inputs to the ILC. Therefore, we injected a recombinant adeno-associated virus (rAAV) equipped with the OT promoter (rAAV-OTp-Venus) either in the PVN or the SON of female rats (Fig. 1C1), and analyzed fiber density in the ILC with a semi-automated Imaris approach. We found significantly denser innervation originating from the SON than from the PVN (135.4 ± 65.6 vs. 51.8 ± 32.1 103 × µm3 sum volume of fibers, p = 0.0253) (Fig. 1C2). For both hypothalamic nuclei we found that they mostly innervate layer 5/6, with additional substantial innervation in layer 1, while the least innervation was found in layer 2/3 (Fig. 1C3) (p = 0.0007 for layer 1 vs. layer 5/6 SON projections, p < 0.0001 for layer 2/3 vs. layer 5/6 SON projections; significance of post hoc tests of ANOVA and mixed effect models are indicated with # in figures).

Next, we investigated whether OT axons release the neuropeptide in the ILC. To this end, we injected a rAAV expressing an opsin under the control of the OT promoter into the PVN and SON (rAAV-OTp-ChrimsonR-mScarlet, Supplementary Fig. S1A, right: patch-clamp verification of opsin functionality, Supplementary Fig. S1B1: representative injection site). In parallel, we injected a rAAV expressing the GRAB OT 1.9 sensor (rAAV-hSyn-OT1.9-GRAB) [kindly provided by Prof. Yulong Li, GRAB OT 1.0 published in:31 in the ILC, and performed imaging experiments in acute ILC slice preparations. We observed a significant increase of GRAB OT 1.9 fluorescence emission upon stimulation of OT fibers (625 nm light, 30 Hz, 10 ms light on, for 30 s) (Fig. 2A1), consistent with OT release and consecutive binding32. This effect was blocked by incubation of the tissue with the OT receptor antagonist (Atosiban) (Fig. 2A2–3, Supplementary Fig. S1B2–3) (123.4 ± 110.5 vs. −23.9 ± 155.8 AUC, p = 0.0232).

To functionally probe the hypothalamic-ILC OT pathway, we next expressed channelrhodopsin 2 (ChR2) in OT neurons of the PVN and SON (rAAV-OTp-ChR2-mCherry, Supplementary Fig. S1A left for patch-clamp verification of opsin functionality) and implanted an optic fiber above the ILC (Fig. 2B1-2). Next, rats were exposed to an unknown, age-, strain- and sex-matched conspecific (Fig. 2B3) in an open field arena (OF) for 5 min, where we analyzed the social behavior throughout the session upon light stimulation of OT fibers in the ILC for the first 2 min of the session. In keeping with previous studies4,5, we employed adult female rats to exclude the aggressive component of social behavior often observed between unfamiliar males33. Social interaction time was significantly increased (total interaction time 47.1 ± 11.8 s vs. 95.9 ± 11.9 s, p < 0.0001) during and immediately after optogenetic stimulation with blue light (BL, 470 nm, 30 Hz, 10 ms pulses, laser power ~10 mW, 120 s) compared to controls expressing Venus fluorescent protein instead of ChR2. This effect was mainly due to significantly increased non-anogenital sniffing and following behaviors (25.0 ± 8.5 s vs. 46.8 ± 14.7 s, p = 0.0103 and 10.7 ± 5.9 s vs. 27.2 ± 8.6 s, p = 0.0019, respectively) (Fig. 2B4). Importantly, no difference was observed between the two groups when comparing the time they spent investigating a toy rat (25.6 ± 12.0 s vs. 25.4 ± 10.8 s, p = 0.9826) (Fig. 2B5), indicating that the behavior reflects a social component rather than general interest and novelty seeking. No changes in locomotor activity, exploratory, or anxiety-related behaviors were found (Supplementary Fig. S2). Altogether, these results indicate that optogenetic activation of the OTergic hypothalamic-ILC projections are sufficient to enhance social behavior in female rats.

OTR+ neurons in the ILC are intrinsically active during social behavior
Next, to target and manipulate OTR+ neurons in the ILC we utilized newly generated OTR-Cre knock-in rats32. The rats were injected with a rAAV expressing GFP in a Cre-dependent (double-floxed-inverted-orientation: DIO) manner into the ILC (rAAV-Ef1a-DIO-GFP) (Fig. 3A1, see also Supplementary Fig. S3B for further characterization of the rat line and protein expression). Semi-automated quantification revealed that among the entire ILC neuronal population, visualized by NeuN (Fig. 3A2), 1.2% of neurons expressed OTRs with a higher occurrence in superficial layers 2/3 compared to the deeper layers 5/6 (1.6% and 0.9%, respectively, p < 0.0001) (Fig. 3A3, and Supplementary Fig. S3A). To monitor OTR+ neuronal intrinsic activity, we performed in vivo fiber photometry during social interaction utilizing a rAAV expressing a Cre-dependent Ca2+ sensor (rAAV-Ef1a-DIO-GCaMP6s) and implanted optic fibers (Fig. 3B1–2). During repeated 5-min social interaction sessions, multiple behavioral events (non-anogenital and anogenital sniffing, and investigation of a toy) were scored and the aligned calcium activity of OTR+ neurons was compared (Fig. 3B3). Social sniffing behaviors performed by the test rat were accompanied by a significant increase of calcium signal in OTR+ neurons compared to baseline (z score for anogenital: 0.12 ± 0.01, non-anogenital: 0.18 ± 0.01), while toy investigation did not lead to a significant change in calcium signal (0.04 ± 0.01) (mean ± sem, p < 0.0001 for anogenital and non-anogenital sniffing compared to baseline, p = 0.4707 for toy) (Fig. 3B4). Furthermore, we also compared the amplitude of calcium signals during social events that were initiated by the test rat (active social behavior) with the amplitude of calcium signals during social events performed by the stimulus rat (received social behavior) (Fig. 3B5). Only active social behavior increased calcium-dependent fluorescence in OTR+ neurons in the ILC (z score for active: 0.12 ± 0.006, p < 0.0001, received: −0,01 ± 0.008, p = 0.9035, mean ± sem) compared to baseline (Fig. 3B6). Therefore, we conclude that OTR+ neurons in the ILC are highly and specifically activated during active social behavior.

OTR+ neurons in the ILC facilitate social behavior
After demonstrating that OTR+ neurons in the ILC are activated during social interaction, we aimed to artificially activate them using optogenetic stimulation, in order to test whether activation of ILC OTR+ neurons affects social behavior. For that, we injected a rAAV expressing ChR2 in a Cre-dependent manner (rAAV-Ef1a-DIO-ChR2) into the ILC of OTR-Cre-rats and implanted optic fibers above the injection sites (Fig. 3C1–2). During the first 2 min of a 5-min social interaction session, OTR+ neurons were either activated by BL (30 Hz, 10 ms pulses), or no light was applied for a baseline social behavior readout. Compared to the baseline, BL activation significantly increased social interaction times (total interaction time 45.6 ± 15.8 s vs. 87.4 ± 30.6 s, p = 0.0124), and more specifically increased anogenital and non-anogenital sniffing (9.8 ± 5.9 s vs. 24.4 ± 11.6 s, p = 0.0426 and 26.4 ± 9.7 s vs. 49.4 ± 21.2 s, p = 0.0036, respectively) (Fig. 3C3). To verify that the endogenous OT/OTR+ signaling in the ILC modulates sociability, we introduced a local and discreet mutation in the OTR gene34 (Supplementary Fig. S3D) by injecting a mixture of two rAAVs expressing Cas9 (rAAV-hSyn-spCas9) and a guide RNA specific for the OTR-sequence (rAAV-u6-gRNA(OTR)-hSyn-mCherry) or LacZ-sequence which served as a control (rAAV-u6-gRNA(lacZ)-hSyn-mCherry) (Fig. 3D1). Injection sites and viral expression were verified postmortem (Supplementary Fig. S3C). We first confirmed the depletion of OTRs in the ILC functionally by bath application of the selective OTR agonist, TGOT, and ex vivo patch-clamp electrophysiological recordings of OTR+ neurons in both groups (Supplementary Fig. S3E). TGOT-induced membrane depolarization was significantly abolished by the viral OTR depletion compared to the control group (Supplementary Fig. S3F). Notably, the ILC OTR depletion was characterized by a significantly decreased social interaction time (total social interaction time 71.5 ± 31.1 s vs. 41.3 ± 22.2 s, p = 0.0259) with an unfamiliar conspecific. This effect was driven by a significant decrease in anogenital sniffing (36.2 ± 26.6 vs. 21.2 ± 13.0 s, p = 0.0493), while non-anogenital sniffing also trended towards a decrease in time (Fig. 3D2). Importantly, no difference was observed regarding the investigation time of a toy rat (37.7 ± 18.0 s vs. 41.7 ± 22.5 s, p = 0.6656) (Fig. 3D3).

OTRs are predominantly expressed in GABAergic neurons of the ILC
A multiplex fluorescent in situ hybridization (RNAscope) approach was used to characterize the types of ILC OTR+ neurons (Fig. 4A1–3). Probes for both OTR as well as Cre were used to verify the specificity of our OTR-Cre rat line; whereas probes for vGlut1 (vesicular glutamate transporter), vGlut2 and vGAT1 (vesicular GABA transporter) were utilised to identify both glutamatergic and GABAergic neurons. We observed that 92.6 ± 8.6% of Cre-positive neurons were also OTR-positive, and vice versa 82.9 ± 8.5% of OTR-positive neurons were also Cre-positive, confirming high specificity of the transgenic OTR-Cre line (Fig. 4A4). Notably, the vast majority of OTR-positive neurons co-expressed vGAT1 (87.63%), while only a small fraction co-expressed vGlut1 (9.14%), and none co-expressed vGlut2 (Fig. 4A5). Moreover, we observed differences between the superficial and deeper layers of the ILC: OTR+ neurons in layer 2/3 were predominantly GABAergic (96.84% vGAT1-positive), while OTR+ neurons in layer 5/6 were prevalently GABAergic (78.02% vGAT1-positive), but also included a substantial number of glutamatergic neurons (17.58% vGlut1-positive) (Fig. 4A5). In line with the viral approach (Fig. 3A), OTR+ neurons constitute only a small population of 1.35% of all neurons). Amongst the entire GABAergic and glutamatergic population, OTR+ neurons make up 6.82% and 0.16% respectively (Fig. 4A6).

To complementarily characterize the distinct glutamatergic and GABAergic OTR+ populations in the ILC, we injected both: a rAAV labeling all OTR+ neurons in red (rAAV-Ef1a-DIO-mCherry) as well as a second rAAV labeling all OTR+ GABAergic interneurons (specificity achieved by DLX-enhancer35) in green (rAAV-DLX-DIO-GFP, as all OTR+ interneurons will also be OTR+ neurons, they will appear yellow) into the ILC of female OTR-Cre rats (Fig. 4B1). Similar to our findings with the RNAscope approach, we could identify an OTR+ interneuron population in layer 2/3 (yellow) and a mixed OTR+ pyramidal (red) and interneuron population in layer 5/6 (Fig. 4B2, and Supplementary Fig. S4A). The results of semi-automated quantification of OTR+ neurons in distinct cortical layers (2/3 and 5/6) and along the rostro-caudal axis (Bregma 2.5, 3.0, 3.5), resembled the findings of in situ hybridization: in layer 2/3, 92.93% of OTR+ neurons are interneurons, while layer 5/6 also has a larger population of 37.04% pyramidal neurons (Fig. 4C). No significant differences of OTR+ neuron distribution were found along the rostro-caudal axis. Together, these experiments demonstrate that the largest population of OTR+ neurons in the ILC consists of GABAergic interneurons located in layer 2/3.

Histological quantification after viral labeling of OTR+ cell types confirmed that the majority of OTR+ neurons were GAD-expressing interneurons immunopositive for parvalbumin and/or calbindin (Supplementary Fig. S4B). Detailed anatomical imaging of cell bodies of OTR+ neurons and OT fibers revealed the localization of OT-immunoreactive axons in close proximity to OTR+ neurons in different layers, pointing to axonal release as a probable source of OT acting on ILC OTR+ neurons and corroborating our aforementioned optogenetic experiments (Supplementary Fig. S4C). Notably, OT fibers were found to be significantly closer to OTR+ neurons in layer 2/3 compared to layer 5/6 (0.04 ± 0.07 µm vs. 0.91 ± 0.55 µm, p < 0.0001) (Supplementary Fig. S4D).

To verify the presence of functional OTRs, we performed an ex vivo patch-clamp experiment, where OTR+ neurons were virally labeled by rAAV-Ef1a-DIO-GFP (Supplementary Fig. S4E1), and classified based on their firing pattern, localization, and biocytin-based morphology visualization (Supplementary Fig. S4E2). All tested cells (interneurons n = 9; pyramids n = 5) were depolarized in response to bath application of selective OTR agonist (20-100 nM TGOT) (Supplementary Fig. S4E3).

Activity of ILC OTR+ interneurons promotes both social interaction and social preference
Given that the major population of ILC OTR-expressing neurons consists of inhibitory cells (primarily in layers 2/3), we aimed to investigate whether modulation of their activity induces similar behavioral effects as recruiting all ILC OTR+ neurons (Fig. 3C). To achieve this, we employed a rAAV equipped with the interneuron-specific enhancer DLX35 to express Cre-dependent DREADDS (designer receptor exclusively activated by designer drugs, either hm3Dq (Gq) for activation or hM4Di (Gi) for inhibition) in OTR+ interneurons (rAAV-DLX-DIO-hm3Dq-GFP or rAAV-DLX-DIO-hm4Di-GFP) of the ILC (Fig. 5A1). Post-hoc IHC analysis verified the main population of OTR+ interneurons being located in layer 2/3 (Fig. 5A2). The two groups of rats (Gq: activation or Gi: inhibition) received CNO (3 mg/kg) or saline i.p. injections 40 min before being introduced into a free social interaction session with an unknown conspecific. Chemogenetic activation of OTR+ interneurons significantly increased social interaction time (61.5 ± 17.7 s saline vs. 77.0 ± 16.4 s CNO, p = 0.0346), while chemogenetic inhibition significantly decreased social interaction time (61.0 ± 16.6 s saline vs. 42.7 ± 13.3 s CNO, p = 0.0197) (Fig. 5B). Investigation of a toy rat was not altered by either activation or inhibition of OTR+ interneurons (activation: 35.7 ± 15.6 s vs. 40.8 ± 19.7 s, p = 0.4381; inhibition: 39.2 ± 16.0 s vs. 49.0 ± 28.5 s, p = 0.4372). Given the previously indicated anxiolytic effect of OT action in OTR+ interneurons of male mice21, we performed OF and elevated zero maze (EZM) tests to investigate the putative effect of ILC OTR+ interneuron modulation on anxiety levels in female rats. Notably, chemogenetic activation of these cells changed neither anxiety nor locomotion parameters in our experiments (Supplementary Fig. S5A, B). Moreover, we conducted a social novelty preference test and observed no difference induced by chemogenetic activation of ILC OTR+ interneurons (corner: p = 0.0100, treatment: p = 0.1474, corner x treatment: p = 0.1937, post-hoc tests all ns) (Supplementary Fig. S5C). Taken together, these experiments showed that recruitment of ILC OTR+ interneurons is sufficient to promote social behavior without alterations of social novelty preference or anxiety.

To further elucidate the social motivational aspect of the observed behavior, we analyzed whether manipulation of the OTR+ interneuron population has an effect on social preference over another rewarding stimuli. Therefore, we subjected animals to an adapted social vs. food preference paradigm36 (Supplementary Fig. S5E1–2), where in a satiated state animals typically prefer social interaction, while food restriction for 24 h increases the interest in food. Rats were injected either with the chemogenetic excitatory rAAV system (rAAV-DLX-DIO-hm3Dq-GFP, ‘Gq’) previously used (Fig. 5A1), or with a rAAV lacking the Gq sequence as a control (rAAV-DLX-DIO-GFP, ‘GFP’). In the food vs. social preference experiment we observed that animals spent significantly more time at the social compared to the food corner (main effect corner, p < 0.0001), and that food deprivation altered allocation across corners as expected (more time spent investigating the food corner after 24 hrs of food restriction; satiety × corner, p = 0.0004). Most importantly, chemogenetic activation influenced the interaction time with the corners in a group specific manner (virus × corner, p = 0.0431), pointing to a significant increase in social corner interaction time independently of satiety state (no interaction of virus × corner × satiety state p = 0.2387). Additional post-hoc tests showed greater social corner time in Gq vs. GFP condition under deprivation (p = 0.0155), with no group difference in food corner time (adjusted p = 0.9766) (Fig. 5C1). Notably, this effect was driven by an increased average length of bouts (average length per visit of the social corner, virus × corner, p = 0.0475) after CNO injection (Fig. 5C2), while the number of bouts (number of visits to the social corner) (Supplementary Fig. S5E3) remained unaltered. To further investigate the effect of ILC OTR+ interneurons on non-social interest in a non-competing context we performed a similar test where only food was offered in one of the corners, with the second corner being empty. In this experiment, food deprivation had a significant effect on food investigation time (main effect food restriction, p = 0.0448), but chemogenetic activation of OTR+ interneurons did not alter it (main effect virus, p = 0.6628). The number of bouts of food investigation, as well as distance traveled and speed during the test were also not affected (Supplementary Fig. S5D1–4). Together, these findings indicate that when a rat is presented with conflicting interests, ILC OTR⁺ interneurons promote social engagement in the hungry state, overriding competing homeostatic demands while not exerting a direct anorexigenic influence.

OTR+ fast-spiking interneurons inhibit pyramidal neurons in the ILC
The aforementioned ex vivo patch-clamp experiments and consecutive biocytin-based morphological analysis revealed that most layer 2/3 OTR+ cells in the ILC display fast-spiking firing patterns (83%, n = 24 patched cells, slices obtained from 5 rats) and exhibits axonal arborization characteristic for axo-axonic interneurons (Chandelier cells) (85.7%, n = 14)37,38 (Fig. 5D). Bath application of TGOT at both low and high concentrations (20 nM and 100 nM, respectively) during ex vivo patch-clamp recordings reliably and significantly increased their firing activity. Note that although the percentage of responding cells did not depend on the TGOT concentration, the average firing rate was twice as high at higher agonist concentrations (Supplementary Fig. S5F). Furthermore, IHC staining for ankyrin (a marker for the axon initial segment) revealed that the terminals of axon arbors of the OTR+ neurons form distinct arrays (so-called “cartridges”) along the axon initial segments of OTR-negative (OTR-) pyramids (Fig. 5E). Based on these morphological and functional features we could identify the vast majority of OTR+ interneurons in layer 2/3 of the ILC as Chandelier cells.

To functionally test whether OTR+ interneurons in layer 2/3 provide an inhibitory synaptic input to the neighboring pyramidal neurons, we performed ex vivo paired patch-clamp recordings in brain slices. Three weeks following an injection of a rAAV expressing GFP in a Cre-dependent manner into the ILC (rAAV-Ef1a-DIO-GFP), we searched for synaptic connections between fluorescently labeled OTR+ neurons and non-fluorescent OTR- pyramidal cells in layer 2/3 (Fig. 6A1). We observed that stimulation of putatively presynaptic OTR+ interneurons led to generation of a post-synaptic potential in 43% of recorded pairs with latency indicative of monosynaptic connections (Fig. 6A2–3). Additionally, we applied a population-based ex vivo approach, utilizing optogenetic stimulation of ChR2-expressing OTR+ neurons (in rAAV-Ef1a-DIO-ChR2-mCherry ILC injected OTR-Cre rats) while simultaneously recording layer 2/3 OTR- pyramidal neurons (Fig. 6B1). Consistent with the paired-patch recordings, BL stimulation resulted in a significant (p < 0.0001) decrease of action potential frequency in all recorded local pyramidal neurons (Fig. 6B2–3). In a follow-up ex vivo patch-clamp experiment we recorded spontaneous excitatory (EPSC) and inhibitory (IPSC) post-synaptic currents in layer 2/3 OTR- pyramidal neurons. The bath application of TGOT significantly increased the IPSC amplitude, with no effect on EPSC amplitude or EPSC and IPSC frequency (Supplementary Fig. S6), further corroborating the inhibitory nature of the synaptic connection of OTR+ neurons in layer 2/3 targeting OTR-negative pyramidal neurons. Together, these experiments suggest that endogenous OT release in the ILC activates layer 2/3 OTR+ interneurons, which inhibit neighboring OTR- downstream principal cells.

OTR+ interneurons inhibit the ILC-basolateral amygdala pathway
Having established the targeted inhibitory input from OTR+ interneurons to layer 2/3 OTR- pyramidal neurons, our next objective was to investigate their potential downstream projection sites. Therefore, we analyzed the neuronal activity marker cFos expression in seven pre-selected brain regions downstream to the ILC39, which were also reported to be functionally involved in modulation of social behaviors40, upon the chemogenetic activation (rAAV-DLX-DIO-Gq-GFP) of ILC OTR+ interneurons. While control animals receiving saline injections showed no specific cFos expression in OTR+ interneurons (0.69 ± 0.37 % cFos/GFP double-positive cells, n = 375 GFP+ neurons from 3 animals), animals receiving CNO showed cFos signal in these GFP+ cells (99.91 ± 0.15% cFos/GFP double-positive cells, n = 424 GFP+ neurons from 3 animals) (Fig. 6C1). To allow for unbiased quantification of neuronal cFos expression, we employed a fully automated pipeline based on the Imaris software which analyzed cFos signals in more than 500,000 neurons across the seven different brain regions. Activating OTR+ inhibitory interneurons in the ILC resulted in an overall decrease of cFos expression in the ILC (main treatment effect, p = 0.0004), with a significant decrease in layer 2/3 (post-hoc layer 2/3, p < 0.0001, post-hoc layer 5/6 p = 0.0604) (Fig. 6C2), indicating extensive inhibition of OTR(GFP)-negative principal cells (Fig. 6B), particularly in layer 2/3 where the main population of OTR+ interneurons resides. As for the analyzed downstream regions, cFos expression was not significantly changed in the periaqueductal gray (PAG), lateral septum, supramammillary nuclei (Summ), or ventral tegmental area (VTA). However, it was significantly reduced in the basolateral amygdala (BLA) (15.4 ± 4.9% vs. 8.7 ± 2.0%; p = 0.0185) (Fig. 6C3). This suggests that experimental OT release in the ILC ultimately reduces the activity of BLA neurons. Notably, in the same paradigm, we observe a significant increase in cFos expression in the nucleus accumbens (NAc) (10.7 ± 2.3% vs. 22.2 ± 7.9%; p = 0.0084), hinting at differences between the possible functional dichotomy of BLA and NAc projections from the ILC. This may suggest that ILC output neurons projecting to the NAc are not directly (monosynaptically) inhibited by layer 2/3 OTR+ interneurons.

To test this hypothesis, we performed viral retrograde tracing of neurons projecting to the BLA or to the NAc by injection of a retrograde GFP-expressing rAAV (retro-rAAV-hSyn-GFP) into either BLA or NAc. The same animals were injected into the ILC with a Cre-dependent rAAV expressing mCherry in OTR+ interneurons (rAAV-DLX-DIO-mCherry) to visualize the overall topography of back-labeled projecting cells in the ILC in relation to the OTR+ cell population (Fig. 7A1, B1). Injection sites in the BLA or NAc were marked by co-injection of a small amount of soluble fluorescent beads into the tip of the injection pipette (Supplementary Fig. S7A). A substantial number of BLA-projecting neurons was found within the “inhibitory sleeve” formed by the vertical axonal cartridges of OTR+ Chandelier cells in layer 2/3 (18.1 ± 7.4 neurons per hemisphere in 50µm sections) as well as in the deeper layers of the ILC (31.8 ± 12.1 neurons per hemisphere in 50µm sections) (Fig. 7A2, and Supplementary Fig. S7C). In contrast, retrogradely labeled NAc-projecting neurons were found exclusively in the deeper layers of the ILC, but not within the “inhibitory sleeve” of layer 2/3 (Fig. 7B2). Furthermore, detailed morphological analysis of BLA-projecting neurons revealed that OTR+ interneurons form direct axo-axonic contacts with BLA-projecting pyramidal neurons in layer 2/3 (Fig. 7C, Supplementary Fig. S7B), but not with NAc-projecting pyramidal neurons in layer 5/6. Thus, we showed anatomical evidence of a direct and local OTR+-neuron-mediated inhibition of BLA-projecting neurons in layer 2/3 of the ILC, and focused on investigating this ILC-BLA downstream pathway.

Following this clear anatomical distinction, we next verified inhibitory action of ILC OTR+ interneurons on OTR- pyramidal neurons projecting to the BLA. In the first set of experiments, we injected a retrograde rAAV expressing GFP into the BLA (retro-rAAV-hSyn-GFP) and, in parallel, a Cre-dependent rAAV expressing ChR2 in interneurons (rAAV-DLX-DIO-ChR2-mCherry) into the ILC of OTR-Cre-rats. After a 3-week expression period, OTR+ interneurons were optogenetically activated by BL stimulation in an ex vivo experimental setup, while BLA-projecting OTR- pyramids were recorded in the whole cell mode (Fig. 7D1). Neurons were patched only if they were both GFP-positive (BLA-projecting) and mCherry-negative (OTR- neurons) (Fig. 7D2). Recorded neurons were identified as principal cells based on their typical discharge pattern and morphology after post-recording staining procedure (Supplementary Fig. S7D and Fig. 7D2). Notably, all recorded neurons (n = 11) responded with inhibition to BL induced OTR+ interneuron activation (Fig. 7D3 for responses of BLA-projecting OTR- pyramids, Supplementary Fig. S7E for representative trace verifying activation of OTR+ interneuron by BL).

Finally, we chemogenetically targeted the BLA-projecting population in the ILC, in order to verify its involvement in social behavior. We injected a rAAV expressing the excitatory DREADD Gq-mCherry in a Flp-dependent manner (rAAV-hSyn-FRT-Gq-mCherry) into the ILC of female rats, while simultaneously injecting a retro-rAAV expressing Flp (retro-rAAV-Ef1a-Flp) into the BLA (Fig. 7E1). With post-hoc verification of injection sites we could observe cell bodies in the ILC as well as dense fibers in the BLA (Fig. 7E2). Animals that received CNO injection prior to a free social interaction sessions spent significantly less time investigating the social partner (31.9 ± 18.1 s vs. 19.3 ± 15.0 s; p = 0.042) (Fig. 7E3, Supplementary Fig. S7F). Thus, we could show that activating BLA-projecting neurons had the same effect on social interaction as inhibiting OTR+ interneurons in the ILC (Fig. 5 B, right), supporting our hypothesis of OTR+ interneuron mediated inhibition of BLA-projecting neurons driving sociability in female rats.

Methods
Animals
Female Sprague-Dawley rats (3–8 weeks, Janvier Labs) and transgenic OTR-Cre rats bred in-house with a Sprague-Dawley background (Central Institute of Mental Health, Mannheim) were used. Adult virgin females (8–16 weeks) were included in anatomical, functional, and behavioral experiments. Age is provided as a range, as experiments always range over the course of several weeks, starting with stereotaxic injection, followed by 3 weeks of expression time, then behavioral experiments, and closed with sacrificing the animal and anatomy and/or ex vivo experiments. The age for injection was always 8–10 weeks, while the age for all social interaction and food vs. social behavior experiments were 11-16 weeks. For single-day behavioral tests (free social interaction, between-subject design), only animals in diestrus were included, as described previously by Tang, Benusiglio and colleagues4. Rats were group-housed under a 12-h light/dark cycle (light on 07:00, 22–24°C, 50 ± 5% humidity) with food and water ad libitum unless stated otherwise. All experiments were conducted under animal license G-193/20 and G-55/23 authorized by the German Animal Ethics Committee of the Baden Württemberg (Regierungspräsidium Karlsruhe, Germany) and in accordance with the German law, under license APAFIS #46161- 2024011918045241_v2 from the French Ministry, and under EU regulations.

Transgenic OTR-Cre Rats. Transgenic OTR-Cre rats32 express Cre recombinase in all OTR-expressing neurons, enabling cell type–specific expression of Cre-dependent (double-floxed inverted orientation, DIO) rAAVs. Animals were generated by breeding heterozygous Cre+ males with wild-type females, and offspring were genotyped using a PCR-based protocol to identify heterozygous Cre+ or homozygous Cre- animals.

Viral vectors
If not stated otherwise, plasmids for the production of rAAVs were generated as described previously5. Recombinant adeno-associated viruses (rAAVs) of serotype 1/2 were used following previously established methodologies5. HEK293T cells for viral production were obtained from Addgene (catalog number 240073). The OT-promoter controlled rAAV-OTp-ChR2-mCherry, rAAV-OTp-Venus, and rAAV-OTp-ChrimsonR-mScarlet, as well as the Cre-dependent rAAV-Ef1a-DIO-GFP, rAAV-Ef1a-DIO-mCherry, rAAV-Ef1a-DIO-GCaMP6s, and rAAV-Ef1a-DIO-ChR2-mCherry, were produced in-house (titers between 109 and 1010 genomic copies/μl). The plasmid for rAAV-hSyn-FLEX-FRT-Gq-mCherry (plasmid #161575) was obtained from Addgene and the viruses were produced in-house (titer > 109 genomic copies/μl). rAAVs for interneuron targeting (rAAV-DLX-DIO-GFP (number v277), rAAV-DLX-DIO-mCherry, rAAV-DLX-DIO-Gq-GFP (number v592), rAAV-DLX-DIO-Gi-GFP (number v703), rAAV-DLX-DIO-ChR2-mCherry (number v317)) were purchased from the ETH Zurich Viral Vector Facility (serotype DJ, titers 6.5 × 1012–8 × 1012 vg/ml). The serotype 9 rAAVs used for OTR knockdown (rAAV-hSyn-spCas9, rAAV-u6-gRNA(OTR)-hSyn-mCherry, rAAV-u6-gRNA(lacZ)-hSyn-mCherry) were obtained in collaboration with Larry Young and Arjen Boender (titers 3.0 × 1010 and 1.5 × 1010 genomic copies/μl for gRNA and Cas9 AAVs, respectively). The AAV-CRISPR/Cas9 plasmids to produce the viruses used in this study are available through Addgene: pAAV-hSyn-spCas9 (#231404), pAAV-u6-gRNA(OXTR.1)-CMV-eGFP, (#194015), pAAV-u6-gRNA(CTRL)-CMV-eGFP (#194017). Retrograde rAAVs (retro-rAAV-hSyn-GFP (plasmid #114213), retro-rAAV-Ef1a-Flp (plasmid #55637), titer ≥ 7 × 1012 vg/mL) were purchased from Addgene. The plasmid for GRAB-OT1.9 was obtained from the Yulong Li laboratory.

Stereotactic injections
Rats were anesthetized with either isoflurane (3-2% in 1 L/min O2) or ketamine/xylazine (75 mg/kg and 5 mg/kg, respectively). Viral solutions (300 nl per hemisphere) were delivered at 150 nl/min using glass micropipettes. Injection coordinates (mm from bregma) were: PVN (AP −1.8, ML ±0.3, DV −8.0), SON (AP −1.8, ML ±1.2, DV −9.25), ILC (AP +3.0, ML ±0.5, DV −4.6), BLA (AP −3.0, ML ±5.0, DV −8.4 to −9.0), and NAc (AP +1.2, ML ±1.0, DV −7.8 to −8.2). Following all experiments, injection sites and transgene expression were confirmed histologically.

Behavioral tests
Behavioral tests were performed in an odor-resistant square arena (60 × 60 × 60 cm) under dim light (<20 lux, SO 200K lux-meter, Sauter). Rats were habituated to the arena for 15 min the day before testing, and the arena was cleaned with 80% ethanol between trials. Experimental and stimulus rats were housed separately.

Free social interaction. Experimental rats were paired with novel, age- and weight-matched conspecifics for 5 min. Animals were placed in diagonally opposite corners at trial start. For optogenetic experiments, 470 nm light (10 mW/mm², 30 Hz, 10 ms pulses) was delivered during the first 2 min of the session. For chemogenetic activation, the DREADD agonist CNO (3 mg/kg) was injected 40 min prior to testing. Behaviors (anogenital/non-anogenital sniffing, following, approach, toy investigation) were manually scored using BORIS software (https://www.boris.unito.it) by two independent, blinded experimenters. Discrepancies were resolved by consensus. The order of animals tested was randomized. Additionally, the order of free social interaction or toy investigation was randomized. The assignment of interaction partners to the test animals was randomized, with the constraint that interaction partners had to be unknown.

Food vs. social preference test. The arena was equipped with triangular corner dividers containing mesh fronts, allowing indirect investigation of either an unfamiliar juvenile female rat or standard food pellets. Each 5-min test session followed a 15-min habituation period. The test was repeated after 24 h of food deprivation. Investigation time was analyzed using Ethovision XT v11.5 (Noldus) by measuring nose position within predefined zones. The following parameters were quantified: total investigation time, number of bouts, and mean bout duration (a bout was defined as each re-entry after leaving the zone). Control experiments used the same setup, but only food was presented in one corner, with the other left empty. The positions of the corners were assigned randomly, the order of animals tested was randomized.

Ex vivo electrophysiology and imaging
Slice Preparation. Brain slices were prepared 3–6 weeks after viral injections. Animals were anesthetized using isoflurane or injection of ketamine/xylazine, and decapitated. For experiments 1–4 (1: TGOT application in current clamp, 2: TGOT application in voltage clamp, 3: paired-patch recordings, 4: optogenetic stimulation of OTR⁺ neurons with recordings from OTR⁻ pyramidal cells), slice preparation and visualization followed established methods72,73. For experiment 5 (GRAB-OT1.9 sensor imaging combined with optogenetic stimulation of OT axons in the ILC), slice preparation followed Iwasaki, Lefevre32. For experiment 6 (optogenetic stimulation of OTR⁺ interneurons with recordings from BLA-projecting pyramidal neurons), rats were anesthetized with isoflurane, decapitated, and brains rapidly removed into chilled, carbogenated ACSF containing (in mM): 92 choline chloride, 2.1 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 Na-ascorbate, 2 thiourea, 3 Na-pyruvate, 5 N-acetyl-L-cysteine, 10 MgSO₄, and 0.5 CaCl₂, pH 7.3–7.4, 290–310 mOsm. Coronal vmPFC sections (400 µm) were cut using a Leica VT1200 vibratome, incubated for 10 min at 32 °C in the same solution, and then transferred to recovery ACSF containing (in mM): 92 NaCl, 2.1 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 10 glucose, 5 Na-ascorbate, 2 thiourea, 3 Na-pyruvate, 5 N-acetyl-L-cysteine, 1.3 MgSO₄, and 2.4 CaCl₂, pH 7.3–7.4, 290–310 mOsm, where they were kept for at least one h before recording. After recordings, selected slices were fixed overnight in 4% PFA at 4 °C and immunostained to examine recorded neurons. Sections were incubated with chicken anti-GFP (1:10,000), rabbit anti-RFP (1:2,000), and Streptavidin-iFluor750 (1:4000) in 0.5% Triton X-100/PBS for 72 h at 4 °C, washed in PBS, and then incubated with Alexa Fluor–conjugated secondary antibodies (1:2000) for 24 h. Sections were mounted with Mowiol and imaged on an Olympus VS200 slide scanner and Leica Stellaris 5 confocal microscope.

Patch-clamp recordings. Slices were transferred to a recording chamber on a Nikon Eclipse FN1 microscope and perfused at 2 ml/min with carbogenated ACSF (32 °C). For experiments 1–4 the ACSF contained (in mM): 125 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, and 25 glucose, pH 7.2, 290–300 mOsm. For experiment 5 the ACSF contained (in mM): 118 NaCl, 25 NaHCO₃, 3 KCl, 1.2 NaH₂PO₄, 2 CaCl₂, 1.3 MgSO₄, and 10 glucose, pH 7.4, 290–300 mOsm. Patch pipettes (5–7 MΩ) were filled with experiment-specific internal solutions. For experiments 1 and 4 the solution contained (in mM): 144 K-gluconate, 4 KCl, 4 Mg-ATP, 10 phosphocreatine, 0.3 GTP, and 10 HEPES, pH 7.3 with KOH, 293 mOsm. For experiment 2 it contained 144 Cs-gluconate, 4 CsCl, 4 Mg-ATP, 10 phosphocreatine, 0.3 GTP, and 10 HEPES, pH 7.3 with CsOH, 293 mOsm. For experiment 3 it contained 110 K-gluconate, 30 KCl, 8 NaCl, 4 Mg-ATP, 10 phosphocreatine, 0.3 GTP, and 10 HEPES, pH 7.3 with KOH, 293 mOsm. For experiment 6 it contained 145 K-gluconate, 2 MgCl₂, 4 Na₂ATP, 0.4 Na₃GTP, 5 EGTA, and 10 HEPES, pH 7.3 with KOH, 290–300 mOsm, and 0.05% biocytin for post-hoc neuron identification. Whole-cell configuration was achieved by gentle negative pressure. Signals were recorded with an EPC 10 USB amplifier using PATCHMASTER software (HEKA Elektronik), low-pass filtered at 2.9 kHz, digitized at 50 kHz.

Experimental protocols and analysis. For experiment 3 presynaptic neurons were stimulated with a 10 Hz train of three suprathreshold current pulses every 7 s, and postsynaptic responses were averaged over 50–100 sweeps. The success rate of synaptic coupling was calculated as the inverse (in %) of the number of neurons tested before a connection was found. In experiment 2 spontaneous EPSCs and IPSCs were recorded at −90 mV (EGABA) and 0 mV (EAMPA), respectively, and detected using a Wiener deconvolution algorithm in MATLAB (deconvwnr function) with τ_onset/τ_offset set to 4/15 ms for IPSCs and 2.5/10 ms for EPSCs. Peaks were identified in a 50 ms sliding window using dynamic and absolute thresholds, and PSC features were extracted from traces around each peak. In experiment 6 only layer 2/3 ILC pyramidal neurons projecting to the BLA were analyzed. Cells were voltage-clamped at −60 mV and stimulated with ten 470 nm light pulses (10 ms, ~10 mW) at 30 s intervals, which were later averaged. Data were analyzed with IGOR PRO (Wavemetrics), Signal (CED), and MATLAB. Electrophysiological results are reported as mean ± SD. Cumulative distributions were compared with the Kolmogorov–Smirnov test.

GRABOT1.9 oxytocin sensor imaging. Animals were euthanized with an overdose of ketamine (100 mg/kg) and xylazine (20 mg/kg), and decapitated, followed by brain extraction. Coronal brain slices containing the ILC were allowed to recover for at least one h in oxygenated ACSF at room temperature after preparation. Expression of rAAV-OTp-ChrimsonR-mScarlet in the PVN and SON was confirmed using an X-Cite 110 LED illumination system (XT-640-W). GRAB-OT1.9 imaging was performed on a Zeiss Axio Examiner microscope equipped with a CREST X-Light spinning disk confocal unit and a 20× water-immersion objective (NA 1.0). Images were acquired at 2 Hz using an optiMOS sCMOS camera (QImaging) controlled by MetaFluor software (v7.8.8.0). The OT1.9 sensor was excited at 475 nm (30 ms illumination), and ILC-projecting OT fibers were stimulated with 635 nm light at 30 Hz, 10 ms pulse width for 30 s after a 5 min baseline. Slices were then incubated in oxygenated ACSF with 1 µM Atosiban for ≥1 h before the second recording. Fluorescence intensity was quantified using Fiji (ImageJ) and processed with a custom Python script. Signals were corrected for photobleaching by fitting a mono-exponential or polynomial decay model, and light-evoked OT release was quantified as the area under the curve (AUC) in a 50–350 s window post-stimulation, as previously described32.

In Vivo Fiber-Optic Experiments
Optic Fiber Implantation. For in vivo optogenetic experiments, rats were anesthetized with either isoflurane (3-2% in 1 L/min O2) or ketamine/xylazine (75 mg/kg and 5 mg/kg, respectively), and bilaterally injected with rAAV-OTp-Venus or rAAV-OTp-ChR2-mCherry into the hypothalamic nuclei (SON, PVN) or with rAAV-Ef1a-DIO-ChR2-mCherry into the ILC, followed by bilateral optic fiber implantation above the ILC. Fibers (Ø 200 μm, NA 0.39, Thorlabs CFMC12L10) were cut to 5.5 mm length. For in vivo fiber photometry, rats received bilateral injections of rAAV-Ef1a-DIO-GCaMP6s into the ILC and bilateral optic fiber implants (Ø 400 μm, NA 0.50, 5.5 mm, Doric Lenses) above the ILC. Implantation coordinates for the ILC were AP +3.1 mm, ML ±1.52 mm, DV −4.75 mm, with a 10° angle to allow connection of patch cables.

Optogenetic Stimulation of OT Fibers. Two 473 nm blue lasers (DreamLasers, Shanghai) were coupled to patch cables (Thorlabs RJPFF2-FC/PC) and controlled by a Master-8 pulse generator. Light was delivered at 10 mW/mm², 30 Hz, 10 ms pulses for 120 s, as described previously5.

Fiber photometry of ILC OTR neurons. Rats were recorded for 10 min in a square open field arena. After 5 min habituation, an unfamiliar female rat or toy was introduced. Each rat underwent multiple sessions (44 total recordings across four animals). Data were acquired using a Doric Lenses photometry system and Doric Neuroscience Studio v6.1 software with 470 nm (Ca²⁺ signal) and 405 nm (isosbestic) excitation at 20 Hz and 0.4 mW output power. Videos were recorded in parallel for behavioral annotation. Raw signals were exported and preprocessed using the pMat toolbox74. High-frequency noise was removed by smoothing, and ΔF/F was calculated as (calcium signal – scaled isosbestic signal)/scaled isosbestic signal. The ΔF/F traces were then Z-scored relative to the mean and standard deviation of the baseline. For each behavioral event (anogenital sniffing, non-anogenital sniffing, toy sniffing), we compared the normalized signal averaged over 1 s before event onset to the signal averaged over the first 1 s after onset. Statistical analysis was performed in R (Version 4.0.4; R Core Team, 2021) using RStudio (Version 1.4.1106, PBC; Team R, 2019). Linear mixed-effects models were fitted using the lme475 and lmerTest76 packages, with type III ANOVA performed using Satterthwaite’s method. Post hoc Tukey’s multiple comparisons were conducted with the emmeans package77, and the Kenward–Roger approximation for denominator degrees of freedom. Model assumptions were verified by inspection of residual plots. The models compared Z-scored ΔF/F values before vs. during each behavioral event in a paired manner, with trial included as a random effect nested within session and sessions nested within animal. When recordings were performed bilaterally, hemisphere was added as a cross-nested random effect. Fixed factors included time in relation to trial onset (before vs. during the behavior), and in the case of comparison of active and received behaviors also behavior type. The model for the behaviors towards the toy was fitted separately, as these were recorded from separate sessions.

Histology
Rats were deeply anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed overnight in 4% PFA at 4 °C, sectioned coronally at 50 µm on a vibratome (Leica VT1000S), and collected into four series. Free-floating sections were processed using established protocols. Sections were incubated with primary antibodies in PBS with 0.5% Triton X-100 for 48–72 h at 4 °C, rinsed, and incubated with secondary antibodies for 24 h. Sections were mounted in Mowiol for imaging. Whole-section scans were acquired on an Olympus SLIDEVIEW VS200 slide scanner for overview images and verification of viral injection sites. Images of individual neurons, co-labeled markers, and OT fiber proximity were captured on a Leica Stellaris 5 confocal microscope in Z-stack mode. Primary antibodies included anti-OT (mouse, 1:2000, gift of H. Gainer, clone: PS38), anti-RFP (Rockland 600-401-379, rabbit, 1:2000, polyclonal), anti-GFP (Abcam ab13970, chicken, 1:1000, polyclonal), anti-cFos (Cell Signaling 2250, rabbit, 1:500, clone: 9F6), anti-NeuN (Merck MAB377, mouse, 1:1000, clone: A60), anti-NeuN (Abcam ab177487, rabbit, 1:2000, clone: EPR12763), anti-Ankyrin (Millipore MABN466, mouse, 1:1000, clone: N106/36), anti-Parvalbumin (Abcam ab11427, rabbit, 1:5000, polyclonal), anti-Somatostatin (BMAT-4103, rabbit, 1:3000, polyclonal), anti-Calbindin (SWANT 300, mouse, 1:3000, clone: CB300), and anti-Calretinin (SWANT 6B3, mouse, 1:5000 clone: 6B3). Secondary F(ab’)₂ fragment donkey antibodies (Jackson ImmunoResearch, 1:1000, polyclonal) included anti-mouse Alexa 488-conjugated (715-546-150), anti-chicken Alexa 488-conjugated (703-546-155), anti-mouse Cy3-conjugated (715-166-150), anti-rabbit Cy3-conjugated (711-166-152), anti-mouse Alexa 647-conjugated (715-606-150), anti-rabbit Alexa 647-conjugated (711-606-152).

Fiber quantification analysis. Densities of OT-positive fibers were quantified in serial sections collected between Bregma +3.72 and +2.52, corresponding to the rostral and caudal borders of the ILC (n = 11 sections from 5 animals). Anatomical borders between mPFC subdivisions were defined based on the Paxinos Rat Brain Atlas and previous cytoarchitectonic studies78. Whole-brain section scans containing Cg1, PrL, and ILC were captured with an Olympus VS200 slide scanner at 20× magnification and stitched into VSI image files. Fiber densities were quantified by counting crossing points of OT fibers in digitized images overlaid with a non-destructive grid of lines79 in ImageJ. Crossing points were averaged per animal, normalized to region area, and presented as fibers/mm².

Neuron quantification analysis. For each animal, one of four series of 50 µm sections through the ILC was analyzed to quantify OTR-expressing neurons. Two analyses were performed: (1) the proportion of inhibitory vs. excitatory OTR-expressing neurons across the ILC, and (2) co-expression of cytochemical markers. Neurons were manually counted in ImageJ using touch-count mode, averaged, and presented as percent of total OTR-expressing neurons.

Automated image analysis. Proximity of OT fibers to OTR neurons was quantified in Imaris (Bitplane, Oxford Instruments). Green (OTR⁺ interneurons) and red (OTR⁺ neurons) channels were surface-reconstructed using a grain size of 0.6 µm, sphere diameter 0.5 µm, with threshold and volume parameters adjusted per image (volumes > 1.7–5.25 µm³ included for green, > 10 µm³ for red). Blue-channel OT fibers were reconstructed using a grain size of 0.44 µm and included if > 3.85–10 µm³. Fiber segments were manually identified and assigned ID numbers for proximity mapping. GFP-, cFos-, and NeuN-positive cells were quantified in Imaris (Spots function, 10 µm diameter, quality threshold 1.21–1.35). The ILC was segmented manually into layers 1, 2/3, and 5/6 based on anatomical landmarks, and neurons were assigned accordingly. OT fiber distribution across ILC layers was calculated from surface reconstructions with a surface detail of 0.89 µm, absolute intensity threshold 2.2–6, and minimum volume 70 µm³.

Western blot
Protein was extracted from OTR-Cre and WT rat brains using RIPA lysis buffer, quantified with a BCA kit, separated on 4–12% NuPage Bis-Tris gels (Invitrogen), and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk/TBS-T, incubated overnight at 4 °C with rabbit anti-oxytocin receptor (Thermo Fisher, 1:1000) and mouse anti-GAPDH (Abcam, 1:5000), followed by HRP-conjugated secondary antibodies. Bands were visualized by chemiluminescence and quantified with ImageJ.

Multiplex fluorescent in situ hybridization
Expression profiles of OTR neurons in the ILC were characterized using RNAscope™ HiPlex (Advanced Cell Diagnostics, Hayward, CA, USA) on fresh-frozen 16 µm sections as previously described80. The following probes were used: vGlut1 (Rn-Slc17a7-T1, cat. no. 317001-T1), Cre (CRE-T2, cat. no. 312281-T2), vGAT1 (Rn-Slc32a1-T3, cat. no. 424541-T3), vGlut2 (Rn-Slc17a6-T5, cat. no. 31701-T5), and OTR (Rn-Oxtr-T6, cat. no. 483671-T6). Imaging was performed on a Zeiss Axio Imager M2 equipped with an automated z-stage and Axiocam 503 mono camera. Images were processed with Zen (3.1 blue edition and 3.0 SR black edition, Zeiss), CorelDraw 2020 (Corel), ImageJ81, and HiPlex Image Registration Software v1.0 (ACD). OTR mRNA-positive cells were identified by DAPI-stained nuclei or cell-like mRNA distribution and manually counted in ImageJ (Cell Counter), following Iwasaki, Lefevre32. Total neuronal populations were quantified in QuPath82 with the following parameters: detection[Channel 2]-vGlut1 75, detection[Channel 6]-vGAT 20, doSmoothing TRUE, splitByIntensity TRUE, splitByShape TRUE, spotSizeMicrons 2, minSpotSizeMicrons 0.15, maxSpotSizeMicrons 6, includeClusters TRUE.

Software
Statistical analysis was performed using SigmaPlot 11 (Systat, USA), GraphPad Prism 7.05 (GraphPad Software, San Diego, California, USA), and custom scripts written in MATLAB R2015a (MathWorks, USA). Experimental schemes and figures were created in Procreate (5.4) and Adobe Illustrator (v27.9).

Statistics and reproducibility
An Excel file containing all statistical tests and results is provided as Supplementary Data 1. For each dataset, normality was assessed before selecting the appropriate test. Parametric tests (paired or unpaired two-tailed t-tests, two- or three-way ANOVA) were used when assumptions were met; otherwise, non-parametric tests (Wilcoxon signed-rank, Mann–Whitney U) were applied. For repeated-measures or unbalanced designs, linear mixed-effects models were used. Unless stated otherwise, α was set at 0.05 and post hoc comparisons were corrected for multiple testing. In figures, asterisks (*) indicate significant main effects and hash symbols (#) indicate significant post hoc effects. Fiber-photometry data were analyzed using linear mixed-effects models as described in the section Fiber Photometry of ILC OTR Neurons.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Schimmer et al. present a connectivity diagram in female rats that details the neural circuits involved in social interaction, facilitated by oxytocin (OXT) release from PVN-OXT neurons to OTR-positive interneurons in layer 2/3 of the infralimbic cortex (ILC). The authors further elucidate that these OTR-positive interneurons are predominantly Chandelier cells. These Chandelier cells inhibit OTR-negative pyramidal neurons in layer2/3 that project to the basolateral amygdala (BLA). The study is particularly exciting because it is the first to demonstrate the involvement of OTR-positive Chandelier cells in social behaviors. A series of in vivo and in slice experiments provide compelling data to support the main claim of the paper. cFos based downstream circuit mapping is a plus. The use of a toy rat as a control is a great idea. Despite these strengths, the impact of this manuscript is diminished by several major concerns outlined below:\nMajor comments:\n- The ILC has been previously implicated in social behaviors (PMID: 32668253, PMID: 32327679, social odor: PMID: 31768051) as well as reward-based motivational behaviors (PMID: 32393535, PMID: 34624366). Additionally, OTR-positive inhibitory neurons in the ILC are already known to play a role in social behaviors (PMID: 25303526). Furthermore, there are existing reports linking ILC to BLA connectivity in social behaviors—ILC neurons projecting to the BLA are preferentially activated by social cues, and inhibition of this circuit impairs social behavior (PMID: 32668253). It is important to discuss these previous findings in detail and compare them with the observations in this manuscript to contextualize the significance of the current results.\n- Figure 2C3: The plot is unclear as it currently compares trials from the same mouse during the entire 5-minute lights OFF phase (grey dots) and trials where the light (BL) was ON for 2 minutes followed by an OFF phase. Instead of comparing the entire 5 minutes, it would be more effective to compare the light ON phase (first 2 minutes) with the light OFF phase (last 3 minutes) to allow for clearer comparisons from the same animals. Alternatively, if there was a light OFF phase followed by a light ON phase within the same animal, one could observe a significant increase in social interaction from the timepoint the BL is turned ON.\n- Fig 3A1-A6: The RNA in situ quantifications (Fig 3A5) were performed using data from 2 animals, with 2 sections per brain. This sample size seems underpowered for reliable quantification and statistics. I would recommend using at least 3 animals (preferably 4 or more) for more robust comparisons.\n- Fig 3C: This is a weak statement, what’s the definition of the close proximity here? Any other cell type to compare against? There should be some quantitative analysis to back the statement.\n- Figure 3B1-3: The data in Fig 3B1-3 do not agree with the RNA in situ data presented in Fig 3A5. According to the RNA in situ results, 93% of OTR neurons in layer 2/3 are inhibitory, while in layer 5/6, 85% are inhibitory. However, in the OTR-Cre rats, this ratio is not consistent after virus injections. In the OTR- Cre rats, layer 2/3 remains approximately 100% inhibitory, but the images depicted show nearly 100% excitatory OTR neurons in layer 5/6. The methods section mentions that a mixture of two viruses was injected. Please clarify the reason for this inconsistency.\n- Figure 4B: The activation of L2/3 ILC OTR interneurons leads to increased social interaction. Additionally, in Figure 5A2, chemo-genetic activation of ILC interneurons shows reduced c-Fos activation in both the ILC and BLA. This suggests that the activation of L2/3 OTR-positive interneurons in the ILC inhibits pyramidal neurons projecting to the BLA, resulting in increased social interaction. Does this imply that the overall inhibition of the ILC-BLA circuit is responsible for driving the increase in social interaction? If so, how does this finding align with the results reported in PMID: 32663438, where an overall increase in ILC activity (19% of neurons showed increased GCaMP activity, with the majority of cells being non-responsive) was observed?\n- Figure 4C, Line 321-328: By looking at the data, CNO stimulation after 24h or food deprivation remove (or nullify) preference toward food rather than shifting its preference toward the social. I would suggest to be more conservative in interpreting the result. Moreover, in C3, how is the bout defined and quantified? It’s not clearly stated in the method section.\n- Figure 5D: Chandelier cells in layer 2/3 are known for making axo-axonic inhibitory connections. However, the image in Figure 5D appears to show axo-dendritic connections (explained as axo-axonic in line 431-432). It would be valuable to quantify the colocalizations between OTR-positive interneurons and Chandelier cells in layer 2/3 to confirm majority of OTR interneurons are Chandelier cells.\n- Figure 5F: The LFP recordings using MEA appear more confusing than informative. How is the LFP result aligned with the rest of the results was not well described. An alternative approach to convincingly demonstrate Oxtr-Chandler to ILC excitatory to BLA neurons for social behavioral regulation would be to activate ILC-BLA projection neurons by injecting retro-Chr2 into the BLA, then activate these neurons and compare social interaction levels to a BL OFF control. Additionally, LFP oscillations between the ILC and BLA in the context of social behaviors have been described in PMID: 35580019. How does the activation of OTR-positive Chandelier cells compare with those previously recorded oscillations?\n- Figure 1C4, 2D2: Is the social interaction scoring done blindly by at least two different experts? Rigorous quantification methods should be included in the method section.\n- Fig5B and C needs quantification; “A substantial number of BLA-projecting neurons was found within the “inhibitory sleeve” in layer 2/3 (Figure 5 B2)”. Moreover, actual majority of BLA projecting ILC neurons are in L5/6 rather than L2/3 based on 5B2 and C2. Is it possible that L2/3 Ex neurons communicated with L5/6, then control BLA circuit?\nMinor comments:\n- Figure 3B2: The OT (IHC) image in blue is difficult to see, particularly due to the poor visibility of fibers and excessive background noise in layer 5/6. Enhancing the clarity of the image with different color and reducing background noise would improve its readability.\n- Figure 3C1: If the OT fibers shown are identified through immunohistochemistry (IHC), please indicate this by labeling them as OT (IHC), not mcherry, in the figure.\n- Extended data S4 A1: Extended Data S4 A1:Social memory tests performed with saline (control) injections did not show significant differences between novel and familiar rats, although a trend is present. The discussion regarding social memory appears to be based on this non-significant observation. It would be important to address this limitation and consider whether the trend warrants further investigation or if additional controls are needed.", "reviewer_2": "Schimmer et al is presenting a manuscript describing how oxytocin release in the infra-limbic area of the prefrontal cortex (ILC) facilitates social interactions between female rats. First, they characterize OT projections to the PFC and demonstrate that OT release promote sociability. Then, they leverage fiberphotometry of calcium signaling to show OTR+ neurons are activated during social interactions initiated by the test rat. They also show that chemogenetic activation of OTR+ neurons promote social interaction while CRISPR/Cas9-mediated knockout of OTR has the opposite effect. Then, they demonstrate the OTRS are predominantly expressed by GABAergic neurons in L2/3 of the ILC using in situ hybridization and viral injections. They also show that some L2/3 interneurons and L5 pyramidal neurons are functionally modulated by OTR agonists using patch-clamp recordings. The authors perform chemogenetic manipulation of OTR interneurons and repeat their previous effect on sociability while showing social novelty preference is not affected. Manipulation of OTR+ INs can however facilitate the preference for social interaction over food. Then, they perform paired recordings in slices and show PN receive direct inhibition from OTR+ INs. Finally, they use chemogenetic activation of OTR+ INs coupled with c-fos labelling to test which downstream area is modulated and found that BLA activity is decreased while NAc activity is increased. Viral expression of fluorescent protein, optogenetic and chemogenetic coupled to MEA recording achieve to demonstrate that OTR INs silence L5 PN projecting to the BLA.\nThe article is well-written, the experiments are thorough and use state-of-the-art techniques to probe the function and mechanism of OT release in the ILC (promoter-specific expression coupled to Cre-dependent viruses, fiberphometry, paired patch recordings, chemogenetic an optogenetic coupled with slice recording or behaviors). The authors blend these techniques in a very elegant fashion to successfully and convincingly demonstrate their claims. The results are novel and timely as mechanistic studies of the cortical neuromodulatory circuits are still lacking despite knowing for a long-time that neuropeptide and their receptor are widely expressed throughout the cortex.\nIn only have minor concerns:\n- Statistics are sometimes a bit light with the name or p values of the test missing (Fig. S4A2 for example). Systematically provide F values and degrees of freedom for ANOVAs. Perform nested t tests when multiple observations are done in the same mice. Consider putting all the statistic in a separate table rather that in the text. Try to be consistent with the number of significant digits when reporting p values (same degree of precision).\n- Line 159: “9.5% of neurons expressed OTRs with a higher occurrence in superficial layers 2/3 compared to the deeper layers 5/6 (12.7% and 8.9%, respectively)”. Provide statistical comparison.\n- Line 169: “p<0.0001 for anogenital and non-anogenital”. What test is used?\n- Fig. 2B4. What are the tests used. Give exact p values.\n- Line 252: “Similar to our findings with the RNAscope approach, we could identify an OTR+ interneuron population in layer 2/3 (yellow)and an OTR+ pyramidal population in layer 5/6 (red).” Give percentages so one can compare with the ISH results.\n- Line 254: “Notably, we could also observe OT fibers throughout the ILC, with denser innervation in layer 2/3”. Quantify the density of fibers per ILC layers.\n- Line 304 (social memory test). I would prefer that the authors avoid calling this test the social memory test but rather call it social novelty preference test. There’s no indication the PFC is needed to store or consolidate memory. Rather, the PFC is likely to utilize social cues from vCA1 to guide the expression of a social memory during recall. In the data presented in Fig. S4A2, comment on the fact that rats did not show a significant preference for the novel rat. Calculate the preference indexes as well and analyze both graphs according to the indications given in the next comment.\n- Fig. 4C and Fig. S4B. Chemogenetic activation during food vs. social preference. Please show the interaction times with both corners followed with the preference index (4C2). Data in 4C2 should be analyzed with a two-way ANOVA (injection x feeding status). Also, perform one-sample t tests to compare each group against 0 and determine whether there is a preference for social or feeding. Graphs C3 and C4 should be plotted with social and food next to each other (do the same for the social and food interaction time graph, as you did in Fig. S4A2). Given that interaction with one compartment is not independent from the interaction with the other compartment I would limit the analysis of this graph to comparing social and food within each group, not across group. Calculating the index allows across group comparison by reducing the number of variables to one variable per group.\n- Line 314: “where in a satiated state animals are expected to highly prefer social interaction, while food restriction for 24 hours leads to a shift of preference towards food (Extended Data Figure S4 B1-2).” Figure reference is incorrect, it should be Fig. 4C2\n- Given the clear effect of OT release on facilitating social interaction with another female, the effect on social vs. food preference appears to be a consequence of facilitating the social interaction rather than choice-specific. Please comment on this briefly in the discussion.\n- Fig. 5E1. It should be ChR2 instead of mCherry in the virus construct.\n- Fig. 4S2. Check the scale for the layer 2/3 FS OTR+ interneuron recording.\n- Fig. S1. Can you show the ratio of time spent in the surround divide by the time spent in the center?\n- Fig. S5E. Can you orient the coronal slices with dorsal axis upward? I find the current orientation confusing.\n- Line 401. Can you first discard any long-range projections from OTR+ interneurons in the ILC? This way performing the complex c-fos analysis following manipulation of the OTR+ interneurons would make more sense. In addition, explicit what kind of behavior perform the rats prior to perfusing them? A nested t test should be used for Fig. 4A2, give the p value.\n- Fig. 5: The treatment to the NAc projections differs quite a lot from the BLA projection. Can you investigate a bit further or comment in the discussion about the putative microcircuit?", "reviewer_3": "This manuscript by Schimmer et al aims to elucidate the circuit mechanism through which oxytocin modulates PFC circuits regulating social behavior. The authors do a very thorough job tracing the axons of hypothalamic OT neurons into the PFC, genetically labeling the neurons expressing the OT receptor (OTR), and detailing how activation of this receptor can alter the activity of PFC excitatory neuron populations that are thought to control pro-social behaviors. The experiments are conducted to a high standard, and the manuscript is clear and well-written. While the authors do refer to some relevant previous work, they do miss a few important citations that should be mentioned, some of which containing results that the authors’ findings should be reconciled with. Much work has been done on PFC circuits, and specifically on the reciprocal interactions between the PFC and BLA. Referring to these important studies can help the reader interpret the results of the current one. This is an important study that will be of great interest to the broader neuroscience audience as it provides a potential mechanism for how OT modulates the cortical aspect of social behavior regulation. I would be happy to hear the authors’ response to my specific comments below - all are relatively minor, but can improve the validity of the findings and their interpretation.\nIntroduction\n“With the exception of the auditory cortex in nursing mice, which has been extensively studied by Froemke and colleagues (Marlin, Mitre et al. 2015, Valtcheva and Froemke 2018, Schiavo, Valtcheva et al. 2020), the vast majority of studies demonstrate that the behavioral OT effects are mediated via subcortical brain regions, whereas OT signaling in the cortex is much less explored” - the authors mention specifically the important work of Robert Froemke while overlooking significant contribution by Nathaniel Heinz (2014, 2016), Oettl et al 2016 and Tan et al 2019, who have also studied OT-mediated cortical modulation of social behavior.\n“Despite the inspiring demonstrations that OT action and/or OTR-expressing neurons (OTR+ neurons) in the PFC modulate paternal (He, Young et al. 2019) and sexual behavior (Nakajima, Görlich and Heintz 2014) in voles and mice, respectively, it remains unknown how OT signaling in the PFC affects basic social behavior.” - the term “basic” social behavior is not clearly defined, which may confuse the reader about its distinction from “sociosexual” behavior (Nakajima et al 2014 & Li et al 2016). In this regard, the authors should also relate to Li et al (2016, Cell), which demonstrates how mPFC-projecting OT neurons differentially facilitate social behavior in females compared to males.\nThe authors might also want to refer to work by Josh Z. Huang detailing the molecular identity of chandelier interneurons in L2 of the PFC and how they innervate amygdala-projecting neurons in mice (see Lu et al., Nat Neurosci 2017, DOI: 10.1038/nn.4624). While the Lu paper failed to demonstrate a behavioral role for these neurons, the current study sheds important light on the topic.\nFigure 1:\nPanels B1-2 : The demonstration of PVN- and SON-originating OT neurons in the mPFC is compelling. What is the unique contribution of each nucleus to the density presented in B3? This could be achieved by performing the viral-based tracing, which is mentioned in the main text but not presented in Fig1, with two fluorophores within the same animal\nPanel B3: Fiber density should be normalized to the area/volume. Although the area/volume used for the current analysis is missing, the results of PrL+Cgt seem lower than those reported in Knobloch et al 2012 (ILC is not reported in that paper). How do you explain these differences?\nNumber of sections, animals and statistics mentioned in legends are different than what is reported in the Methods section.\nComparing different areas within the same animal can be achieved by averaging each area per rat and use paired t-test (and not unpaired) to compare between areas or by using a linear model with random effect to control for pseudoreplication.\nPanels C\nGeneral comments: To support the main claim of this figure, the authors should provide supplemental schematics or images of the optic fiber targeting the ILC for all animals.\nSome explanation is needed for the choice of blue light stimulation protocol (30 Hz for 120 s) for ChR2 activation, given the potential opsin desensitization (Nagel 2003; Lin 2011), the dynamics of large dense core vesicle release and potential depletion from the terminals under such strong stimulation. Have the authors tried recording OT release under this protocol?\nC4 - for the comparison of total interaction time the statistics should be unpaired t-test, rather than a two-way ANOVA.\nFigure 2:\nPanels B: The fiber photometry result, while consistent with later results in the paper, might not be specific to OTR+ neurons. I assume that other populations in the PFC would show the same pattern of activity as these neurons. Is this activity pattern consistent with the activity of PVN OT neurons? Does it change when OT release is suppressed in the PFC or more broadly? This should be discussed in the Discussion section if not experimentally addressed.\nPanels C1-2: The rationale for using a stimulation protocol in OTR+ neurons in the ILC, is not fully clear until Fig3 D3. Consider moving this experiment to Fig 3 or reorganize these data to better support the rationale for this experiment.\nPanel D: The authors use an interesting approach to study the specific role of OTR by functionally disrupting OTR signalling in the ILC using gene editing. It’s important to note that while most of the mutations induced to the OTR by this method result in complete loss of its ligand binding, some residual functionality can not be excluded (e.g. receptor dimerization; Boender et al 2023), as seen in the TGOT experiment (Extended Data Fig. 2). Therefore, this approach does not fall under the definition of “knockout”, and this term should be avoided to prevent misinterpretation.\nFigure 3:\nGeneral comment: Given the small sample size in this figure (n=2), the conclusions drawn in this part should be taken with a grain of salt. Especially, regarding the distribution and expression of OTR which is considered sex-, age- and state-dependent. Specifically, the identity of OTR+ neurons in the mPFC and their characteristics appear inconsistent with previous literature (Nakajima et al., 2014; Li et al., 2016; Tan et al. 2019). This inconsistency should be addressed at least in the Discussion section, if not with further experiments.\nIn this regard, the authors don’t mention the age of the animals, virginity status in all experiments and the esterous cycle for their non-behavioral experiments, which might be relevant for OTR expression (Bale et al 1995).\nB2 - The OT channel (blue) seems much noisier in layer 5/6 compared to layer 2/3, which maked it hard to appreciate the heavier OT innervation in layer 2/3. This result should be quantified by comparing OT fiber density in layer 2/3 and layer 5/6.\n“Histological quantification after viral labelling of OTR+ cell types qualitatively confirmed…” - It was either quantified or qualitatively confirmed, but not both.\nFigure 4:\nThe authors show that OTR+ neurons target L2/3 PYR cells over L5 PYR cells. This is almost expected from the anatomical configuration and the projection patterns of L2/3 OTR+ neurons. Lu et al (Nat Neurosci 2017) demonstrated that within L2, chandelier interneurons preferentially target BLA-projecting over CC-projecting PYR cells. It would be interesting to see whether similar specificity exists for OTR+ interneurons.\nC1-4: If I understand correctly, this experiment equally suggests that activation of ILC OTR interneurons in layer 2/3 promotes satiety, which corresponds with the anorexogenic effect of OT. It would be beneficial to include a control experiment to verify that such activation does not alter feeding behavior, supporting a specific effect on sociability. Furthermore, inclusion of a control group in this experiment seems to be important, given the known retroconversion of CNO to clozapine, particularly in rats. There is not much information on how hunger states interact with clozapine or CNO responses, and it would be good to make sure that the effects are indeed specific to the OT manipulation.\nThe term \"cornered social interaction\" is not a commonly-used one. Please rephrase.\nFigure 5:\nGeneral comment: While the suppression of the ILC-BLA pathway by activation of ILC OTR+ interneurons is well demonstrated in-vitro and in-vivo, the lack of its effect on anxiety-related behavior in females is puzzling (Extended Data Figure S1). This finding may require further discussion, especially considering the results from Li et al. (2016), which highlighted sex-specific effects on anxiety-related behavior following activation of OTR+ interneurons in the PFC.\nF: Since the authors claim a specific involvement of the ILC-BLA pathway in social interactions, it would be crucial to include an analysis that specifically compares changes in local field potentials during social versus non-social interactions (i.e. interaction with food), rather than presenting the change during the entire “encounter” period. Also, were these changes correlated with the social preference index described in Fig. 4 C2?\nGeneral note: when providing the construct names for Cre-dependent viral vectors (e.g. \"rAAV-DLX-hm3Dq-GFP or rAAV-DLX-hm4Di-GFPC\" as mentioned in Fig. 4) the Cre/lox cassette should be included.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "ANSWERS TO REVIEWERS’ COMMENTS\nWe thank the reviewers for dedicating their time and effort to evaluate our manuscript. We are\ngrateful for their thoughtful and constructive feedback, which we have addressed point by point\nbelow. We hope that our responses will resolve their concerns and support the publication of this\nstudy. For ease of reference, all responses and newly added data in the revised manuscript are\ntyped in blue.\nReviewer #1 (Remarks to the Author):\nSchimmer et al. present a connectivity diagram in female rats that details the neural circuits involved\nin social interaction, facilitated by oxytocin (OXT) release from PVN-OXT neurons to OTR-positive\ninterneurons in layer 2/3 of the infralimbic cortex (ILC). The authors further elucidate that these OTR-\npositive interneurons are predominantly Chandelier cells. These Chandelier cells inhibit OTR-\nnegative pyramidal neurons in layer2/3 that project to the basolateral amygdala (BLA). The study is\nparticularly exciting because it is the first to demonstrate the involvement of OTR-positive Chandelier\ncells in social behaviors. A series of in vivo and in slice experiments provide compelling data to\nsupport the main claim of the paper. cFos based downstream circuit mapping is a plus. The use of\na toy rat as a control is a great idea. Despite these strengths, the impact of this manuscript is\ndiminished by several major concerns outlined below:\nMajor comments:\n- The ILC has been previously implicated in social behaviors (PMID: 32668253, PMID: 32327679,\nsocial odor: PMID: 31768051) as well as reward-based motivational behaviors (PMID: 32393535,\nPMID: 34624366). Additionally, OTR-positive inhibitory neurons in the ILC are already known to play\na role in social behaviors (PMID: 25303526). Furthermore, there are existing reports linking ILC to\nBLA connectivity in social behaviors—ILC neurons projecting to the BLA are preferentially activated\nby social cues, and inhibition of this circuit impairs social behavior (PMID: 32668253). It is important\nto discuss these previous findings in detail and compare them with the observations in this\nmanuscript to contextualize the significance of the current results.\nWe thank the reviewer for this very detailed comment, and have now added new paragraphs about\nthe role of the ILC in social behaviors and reward-based motivational behaviors:\n“Population imaging in the mouse mPFC shows that ensembles preferentially encode social olfactory cues\nand refine with experience (Levy, Tamir et al. 2019). This is consistent with a role for ILC circuits in driving\nsocial actions rather than merely reflecting sensory processing.”\n“Independent of OT signaling, work in rats shows that inactivation of the ILC broadly impaired both active\nand inhibitory avoidance and suppressed inappropriate reward seeking (Capuzzo and Floresco 2020).\nConsistent with this, a comprehensive review concluded that ILC function is highly context-dependent,\nvarying with task demands and the specific neuronal ensembles recruited (Nett and LaLumiere 2021).”\nAlso, we added a paragraph about the specific ILC-BLA pathway in social behaviors into our\ndiscussion, where we reference these papers and discuss our findings in context:\n“Notably, a study by Lu, Tucciarone et al. (2017) demonstrated that a subset of layer 2 Chandelier cells in\nthe PrL cortex selectively innervates BLA-projecting principal cells ( PCs) over contralateral-cortex-\nBLA\nprojecting principal cells ( PCs), while receiving preferential input from PCs, thereby establishing a\nCC CC\ndirectional inhibitory module that gates BLA-related output. Interestingly, they also found PCs in the\nBLA\nsame laminar layers as Chandelier cells, while PCs were located in deeper layers, resembling our finding\nCC\nof BLA- vs. NAc-projecting neurons. This spatial coincidence suggests that OTR⁺ interneurons are well-\npositioned to modulate PCs ensembles. While their study did not indicate a behavioral role of this\nBLA\nneuronal subset, our work suggests its prosocial role.”\n“Importantly, several studies indicate the ILC as a key node for social approach via amygdala-directed\noutputs. In mice, BLA-projecting ILC neurons are preferentially recruited by social cues, and normal\nsociability requires intact ILC-BLA signaling (Huang, Zucca et al. 2020). In hamsters, chemogenetic\n1\nactivation of the ILC-BLA connection during social defeat confers resilience and reduces defeat-associated\nbehavior while lowering BLA c-Fos (Dulka, Bagatelas et al. 2020). Our finding that activating OTR+\ninterneurons in the rat ILC increases sociability, coupled with reduced BLA cFos expression, aligns with a\nmodel in which ILC microcircuits gate amygdala-directed output to favor social approach under challenge.\nThis is further strengthened by the antisocial effects of stimulation of ILC-BLA-projecting pyramidal neurons\nwe observed.”\n- Figure 2C3: The plot is unclear as it currently compares trials from the same mouse during the\nentire 5-minute lights OFF phase (grey dots) and trials where the light (BL) was ON for 2 minutes\nfollowed by an OFF phase. Instead of comparing the entire 5 minutes, it would be more effective to\ncompare the light ON phase (first 2 minutes) with the light OFF phase (last 3 minutes) to allow for\nclearer comparisons from the same animals. Alternatively, if there was a light OFF phase followed\nby a light ON phase within the same animal, one could observe a significant increase in social\ninteraction from the timepoint the BL is turned ON.\nWe thank the reviewer for this valid point and agree that the initial\nrepresentation of the data may have been unclear. To improve clarity\nand consistency with other parts of our study, we have reanalyzed the\ndata and now present both the overall 5-minute session and the\nseparated sub-behaviors, analogous to our other behavioral\nexperiments (Fig. 2 C3, see on the right), including the optogenetic\nstimulation of OT fibers shown in Figure 1, which used the same\nstimulation protocol (Fig. 1 E4, Fig. 2 D2).\nFig. 2 C3 Animals spend\nWe also agree that comparing light ON vs. light OFF phases within the significantly more time socially\nsame animal might seem intuitive; however, this comparison would be interacting if the BL is ON in the\nfirst two minutes of the session,\nconfounded by the well-documented time-dependent decline in social\ncompared to when it is OFF (n=5\ninterest, as observed in our control conditions (gray line in the original animals). This effect is\ngraph, see the graph on the right showing the significant decrease in significantly driven by anogenital\nand non-anogenital sniffing.\ninteraction time over a 5min session, this data is from control groups of\nseveral optogenetic experiments). This would likely produce an\nartificial difference unrelated to the stimulation effect. The design\nwe’ve chosen (within subject light ON and light OFF sessions) might\nseem suboptimal and we can see its weaknesses, however we made\nsure to control for habituation effects (part of the rats started with light\nON, the rest with light OFF sessions). Moreover, we performed and\npresented several follow up chemogenetic experiments which yielded\ncongruent results, serving as additional controls.\nThe idea of reversing the stimulation order (i.e., light OFF followed by\nlight ON) is indeed interesting. However, implementing such a design\nwould not directly address the central question of our study and would\nrequire repeating an entire cohort of animals. Given that this additional\nexperimental setup is unlikely to provide critical new insights, and in light of the 3R principles\n(Replacement, Reduction, and Refinement), we consider such an approach not justified at this stage.\n- Fig 3A1-A6: The RNA in situ quantifications (Fig 3A5) were performed using data from 2 animals,\nwith 2 sections per brain. This sample size seems underpowered for reliable quantification and\nstatistics. I would recommend using at least 3 animals (preferably 4 or more) for more robust\ncomparisons.\nWe fully agree with the reviewer and have extended this experiment with another 3 animals (so 5\nanimals in total). While the overall picture does not change by this added data, we can now\nconfidently perform reliable quantifications and comparisons. For results see Fig. 3 A4-6 below.\nthe ILC are GABAergic interneurons, with a small fraction of glutamatergic neurons, which are predominantly found in the layers 5/6. A6\nIn-situ-hybridization shows that 1.35% of all neurons in the ILC are OTR+ neurons, with 6.82% of all GABAergic neurons being OTR+.\n- Fig 3C: This is a weak statement, what’s the definition of the close proximity here? Any other cell\ntype to compare against? There should be some quantitative analysis to back the statement.\nWe thank the reviewer for this concern, and have accordingly performed a new experiment utilizing\nsemi-automated, high-throughput analysis in IMARIS to quantify the proximity of OT fibers to OTR+\nneurons in layer 2/3 and layer 5/6. The results are now depicted in Fig S3 D, and described in the\ntext:\nFig. S3 D Automated, high-\n“Notably, OT fibers were found to be significantly closer throughput analysis of the\nto OTR+ neurons in layer 2/3 compared to layer 5/6 distance of OT fibers to OTR\nneurons in the ILC. Fibers are\n(0.04±0.07µm vs. 0.91±0.55µm, p<0.0001) (Extended\ncloser to neurons in layer 2/3\nData Figure S3 D).” compared to layer 5/6 (n=24\nneurons and n=20 neurons,\nsections obtained from 4\nanimals).\n- Figure 3B1-3: The data in Fig 3B1-3 do not agree with the RNA in situ\ndata presented in Fig 3A5. According to the RNA in situ results, 93% of OTR neurons in layer 2/3\nare inhibitory, while in layer 5/6, 85% are inhibitory. However, in the OTR-Cre rats, this ratio is not\nconsistent after virus injections. In the OTR- Cre rats, layer 2/3 remains approximately 100%\ninhibitory, but the images depicted show nearly 100% excitatory OTR neurons in layer 5/6. The\nmethods section mentions that a mixture of two viruses was injected. Please clarify the reason for\nthis inconsistency.\nWe thank the reviewer for pointing out this\nimportant inconsistency. To address this\nconcern, we performed a new, semi-\nautomated high-throughput Imaris-based\nquantitative analysis of rAAV-labeled\ninterneurons and pyramidal neurons in the\ndifferent layers of the ILC, now presented in\nFig. 3 C4, please see below. This updated\nquantification yields results that more\nclosely match the RNA in situ data:\nFig. 3 C4 Semi-automated, high-throughput\napproximately 93% of OTR+ neurons in analysis of the number of OTR+ neurons\nlayer 2/3 and 63% in layer 5/6 are identified as interneurons (yellow) or\nclassified as interneurons. We also agree\npyramidal neurons (red) in the ILC (n=4\nwith the reviewer that the microscope bregma levels).\nanimals, each with 3 sections at three different\nimage originally shown in Fig. 3B2\nFig. 3 B2 Representative\nwas not representative in light of the confocal image depicting\nOTR+ interneurons\nRNA in situ findings. We have\ndominating layers 2/3\ntherefore replaced it with a more (yellow), and individual OTR+\nrepresentative image (see below).\npyramidal neurons localized\nin layers 5/6 (red), with OT\nAdditional images throughout the\nfibers (blue, white arrows) in\nmanuscript (Fig. 3C, Fig. 4A2, Fig.\nboth subdivisions of the ILC.\nS4C3) further demonstrate the presence of interneurons in layer 5/6.\nScalebar 100µm.\nwhich labels all OTR+ neurons in our OTR-Cre line red, and rAAV-DLX-DIO-GFP, which specifically\nlabels OTR+ interneurons green. Since all GFP+ cells also express mCherry, these interneurons\nappear yellow in merged images. A likely explanation for the underrepresentation of interneurons in\nlayer 5/6 in the viral labeling data is the reliance on GFP expression to identify interneurons. This\napproach assumes near-complete infection and expression in all interneurons, which may not be the\ncase. Weak expression or incomplete infection, particularly among certain subtypes (Yazdan-\nShahmorad et al., 2024), can lead to underestimation of interneuron numbers and overestimation of\nexcitatory neurons in layer 5/6. Moreover, our earlier RNAscope analysis revealed that vGAT mRNA\npuncta are more abundant in layer 2/3 (average 9 dots) compared to layer 5/6 (average 4 dots).\nWhile both would be counted equally in RNAscope, these differences in mRNA levels may be\nreflected in lower rAAV-DLX-DIO-GFP expression in layer 5/6, again biasing classification.\nIn summary, while technical limitations in viral labeling likely account for some discrepancies, our\nnew quantitative analysis using Imaris provides a more accurate and consistent picture of OTR\nneuron identity across layers, and aligns well with our RNA in situ data.\n- Figure 4B: The activation of L2/3 ILC OTR interneurons leads to increased social interaction.\nAdditionally, in Figure 5A2, chemo-genetic activation of ILC interneurons shows reduced c-Fos\nactivation in both the ILC and BLA. This suggests that the activation of L2/3 OTR-positive\ninterneurons in the ILC inhibits pyramidal neurons projecting to the BLA, resulting in increased social\ninteraction. Does this imply that the overall inhibition of the ILC-BLA circuit is responsible for driving\nthe increase in social interaction? If so, how does this finding align with the results reported in PMID:\n32663438, where an overall increase in ILC activity (19% of neurons showed increased GCaMP\nactivity, with the majority of cells being non-responsive) was observed?\nWe agree with the conclusion of the reviewer that the overall inhibition of the ILC-BLA circuit\n(mediated by OTR+ interneurons) is responsible for driving the increase in social interaction. The\npaper they refer to however has several differences to our study, and does not contradict our\nfindings: Kingsbury and colleagues found that in mice (not rats), who are interacting with both\nfemales and males (not just females), 22% of neurons in the dmPFC (not vmPFC/ILC) respond to\nsocial interaction. This response however is both an increase as well as a decrease in activity, with\nslightly more neurons decreasing in their activity. So overall, while this study focuses on a different\nsubdivision of the PFC and looks at the entire neuron population (not specific to OTR+ neurons),\nthey also find a heterogenous population of neurons modulated by social interaction.\nAdditionally, we performed chemogenetic stimulation of ILC–BLA projecting pyramidal neurons (Fig.\n5F, see below), which led to a decrease in social interaction time. This provides additional evidence\nsupporting our claim that inhibition of the ILC–BLA pathway likely promotes social interaction in\nfemale rats.\nFig. 5 F Social interaction after chemogenetic activation of BLA-projecting neurons originating in the ILC. F1 Injection scheme depicting\nthe injection of a retrogradely Flp-expressing rAAV into the BLA with simultaneous injection of a Flp-dependent (Frt) Gq-mCherry-\nexpressing rAAV into the ILC to chemogenetically activate BLA-projecting ILC neurons after i.p. application of CNO. F2 Representative\ninjection site showing expression of the rAAV-hSyn-FRT-Gq-mCherry in cell bodies and fibers in the ILC and projecting fibers in the BLA.\nScalebar 250µm. F3 Chemogenetic manipulation of BLA-projecting neurons via CNO injection before a 5 min social interaction (SI)\nsession with an unknown conspecific in the open field. Activation of BLA-projecting neurons decreases social interaction time (n=11\nanimals).\n4\n- Figure 4C, Line 321-328: By looking at the data, CNO stimulation after 24h or food deprivation\nremove (or nullify) preference toward food rather than shifting its preference toward the social.\nI would suggest to be more conservative in interpreting the result. Moreover, in C3, how is the bout\ndefined and quantified? It’s not clearly stated in the method section.\nWe thank the reviewer for this comment. Based on another reviewers’ comment we have now also\nrepeated the food vs social experiment with more appropriate control groups (to control for CNO off-\ntargets), and are now depicting the results as ‘time investigating food/social corner’ (Fig. 4 C1, please\nsee below). This direct readout (instead of using the calculated SPI) allows for more intuitive\ninterpretation. Moreover, we performed a dedicated experiment to verify the influence of ILC OTR\ninterneurons chemogenetic activation solely on food interest (Fig S4 D1, see below). In this improved\ndesign, both the food vs social experiment as well as food interest experiment clearly show no effect\nof ILC OTR interneurons activation on interaction with food corner, with clear effects on social corner.\nTaken together, these results strengthen our interpretation, while we also acknowledge and point\nout the crucial limitations of the design.\nFig. 4 C1 Chemogenetic activation of OTR+ interneurons Fig. S4 D1 The time spent investigating the food was only\nincreases time spent investigating the social corner (n=8 animals). influenced by hunger, but not chemogenetic activation of OTR+\ninterneurons.\nWe have also added a definition of the number of bouts and general quantification in the method\nsection.\n„Corner zones were determined uniformly prior to the experiment, and the position of the nose of the rat\nwithin this zone was counted. The parameters quantified were a) the total duration of the nose being within\nthe zone, b) the number of bouts and c) the average length of bouts, with one bout being defined as every\nnew re-entry into the zone after previously exiting it.“\n- Figure 5D: Chandelier cells in layer 2/3 are known for making axo-axonic inhibitory connections.\nHowever, the image in Figure 5D appears to show axo-dendritic connections (explained as axo-\naxonic in line 431-432). It would be valuable to quantify the colocalizations between OTR-positive\ninterneurons and Chandelier cells in layer 2/3 to confirm majority of OTR interneurons are Chandelier\ncells.\nWe thank the reviewer for this valid concern, and have addressed it with an additional experiment\nthat has now been added to the manuscript: we have performed biocytin fillings of OTR+\ninterneurons in layer 2/3 with subsequent immunohistochemical staining for ankyrin (an axon-initial\nsegment marker) and therefore could quantify that 86% of OTR+ interneurons showed the\nmorphological features typical for Chandelier cells (Fig. 4 E2, see below).\nexchanged the image in Fig. 5D (see below) for one which should not raise further concerns,\ndepicting pyramids projecting to the BLA with axons of OTR+ Chandelier cells.\nFig. 5 D Representative\naxo-axonic contacts\n(white arrows) of OTR+\ninterneurons (red) on\nBLA-projecting\npyramidal neurons\n(green). The axon-initial\nsegment is stained\nimmunohistochemically\nfor ankyrin (blue).\nScalebar 50µm\noverview, 25µm zoom.\nFig. 4 E2 Biocytin filling (blue) of OTR+ neurons (green) and\nstaining for ankyrin (red) verifies typical chandelier\nmorphology in 12 of 14 OTR+ interneurons. Scalebar 50µm\noverview, 25µm zoom.\n- Figure 5F: The LFP recordings using MEA appear more confusing than informative. How is the\nLFP result aligned with the rest of the results was not well described. An alternative approach to\nconvincingly demonstrate Oxtr-Chandler to ILC excitatory to BLA neurons for social behavioral\nregulation would be to activate ILC-BLA projection neurons by injecting retro-Chr2 into the BLA, then\nactivate these neurons and compare social interaction levels to a BL OFF control. Additionally, LFP\noscillations between the ILC and BLA in the context of social behaviors have been described in\nPMID: 35580019. How does the activation of OTR-positive Chandelier cells compare with those\npreviously recorded oscillations?\nWe sincerely appreciate this comment by the reviewer, and have decided to exclude this MEA\nexperiment from the manuscript, as it does in fact lead to more confusion than information at its\ncurrent state. Thorough and prolonged studies will be required to complete this subset of\nexperiments. We feel like it was not crucial for the conclusion and elucidation of the other findings,\nand will be working on it for a future publication.\nHowever, we agree with the idea of the reviewer that another behavioral experiment can be utilized\nto show the behavioral effect of manipulating ILC-BLA projecting neurons, and, as mentioned above,\nhave performed it similarly to what the reviewer suggested: we chemogenetically activated BLA-\nprojecting ILC neurons (utilizing a Flp-FRT rAAV system), and could show that this in fact decreased\nsocial interaction (Fig 5F, please see below).\nFig. 5 F Social interaction after chemogenetic activation of BLA-projecting neurons originating in the ILC. F1 Injection scheme depicting\nthe injection of a retrogradely Flp-expressing rAAV into the BLA with simultaneous injection of a Flp-dependent (Frt) Gq-mCherry-\nexpressing rAAV into the ILC to chemogenetically activate BLA-projecting ILC neurons after i.p. application of CNO. F2 Representative\ninjection site showing expression of the rAAV-hSyn-FRT-Gq-mCherry in cell bodies and fibers in the ILC and projecting fibers in the BLA.\nScalebar 250µm. F3 Chemogenetic manipulation of BLA-projecting neurons via CNO injection before a 5 min social interaction (SI)\nsession with an unknown conspecific in the open field. Activation of BLA-projecting neurons decreases social interaction time (n=11\nanimals).\n6\n- Figure 1C4, 2D2: Is the social interaction scoring done blindly by at least two different experts?\nRigorous quantification methods should be included in the method section.\nThe different behavioral experiments were scored by ARa, SS, and AL. All videos were blinded\nbefore analysis. We have added the appropriate information in the method section as:\n“Behavioral videos were manually scored by two independent experimenters blinded to experimental\nconditions. Scorers initially annotated videos separately; discrepancies were reviewed jointly and resolved\nby consensus to produce a final dataset”\n- Fig5B and C needs quantification; “A substantial number of BLA-projecting neurons was found\nwithin the “inhibitory sleeve” in layer 2/3 (Figure 5 B2)”. Moreover, actual majority of BLA projecting\nILC neurons are in L5/6 rather than L2/3 based on 5B2 and C2. Is it possible that L2/3 Ex neurons\ncommunicated with L5/6, then control BLA circuit?\nWe fully agree with the reviewer and have conducted this quantification now (see Fig S5 C) as below:\nFig. S5 C Quantification of BLA-projecting\nneurons in layer 2/3 and layer 5/6 of the ILC.\nC1 Overview of the ILC with BLA-projecting\nneurons in red (rAAV-FRT-Gq-mCherry in\nILC, retro-rAAV-Flp in BLA) and NeuN\nstaining (blue). Scalebar 250µm. C2 BLA-\nprojecting neurons in layer 2/3 (white\narrow) and layer 5/6 (yellow arrows).\nScalebar 100µm. C3 Number of BLA-\nprojecting neurons in layer 2/3 and layer 5/6\nof the ILC.\nWe could show that 36% of BLA projecting neurons are located in layer 2/3, with the remaining 64%\nlocated in layer 5/6. Our main point was the general existence of BLA-projecting neurons in layer\n2/3, because the same is not true for NAc-projecting neurons (Fig5 C2). We are convinced that the\n(smaller) population of BLA-projecting neurons in layer 2/3 would be sufficient to control the ILC-BLA\ncircuit, but it has also been reported previously that Chandelier cells in layer 2/3 innervate pyramidal\nneurons in cortical layers 2, 3, and 5 (Jiang et al., 2013; Lee et al., 2015) and that while they have\nmostly vertically oriented axonal segments in layer 2/3, individual long descending axons reached\nand arborized in layer 6 (Woodruff et al., 2010). These new references have now been included in\nthe discussion.\n“It has been reported previously that Chandelier cells in layer 2/3 innervate pyramidal neurons in cortical\nlayers 2, 3, and 5 (Jiang, Wang et al. 2013, Lee, Wang et al. 2015), and that while they have mostly vertically\noriented axonal segments in layer 2/3, individual long descending axons reached and arborized in layer 6\n(Woodruff, Anderson and Yuste 2010). However, in our experiments, chemogenetic activation of ILC OTR+\ninterneurons did not alter cFos expression in deep ILC layers, suggesting that their effects are largely\nconfined to layer 2/3.”\nAdditionally, the reviewer raised an interesting and valid\nFig. 5 A2 Local\nquestion about L2/3 and L5/6 communication. As we cannot reduction of cFos\nfully exclude such interactions in our case (as they have been expression in the ILC\nafter chemogenetic\nreported: Collins et al., 2019; PMID: 29628187) we reanalyzed\nactivation of OTR+\nour cFos experiment (Fig. 5 A1-3) and quantified the cFos interneurons in the\nILC, specific to layer\nexpression separately for L2/3 and L5/6 to be able to infer any\n2/3.\nsignificant activity changes in deep layers upon OTR\ninterneurons chemogenetic activation. As depicted the\nnumber of cFos positive nuclei was significantly altered only in\nL2/3 suggesting lack of robust activity changes in L5/6 and\ntherefore lack of putative layer interactions in this case.\n- Figure 3B2: The OT (IHC) image in blue is difficult to see, particularly due to the poor visibility of\nfibers and excessive background noise in layer 5/6. Enhancing the clarity of the image with different\ncolor and reducing background noise would improve its readability.\nWe thank the reviewer for this comment and have exchanged the image for one of better quality\n(Fig3 B2), where OT fibers are clearly visible with minimal background (please see below).\nFig. 3 B2 Representative\nconfocal image depicting OTR+\ninterneurons dominating layers\n2/3 (yellow), and individual OTR+\npyramidal neurons localized in\nlayers 5/6 (red), with OT fibers\n(blue, white arrows) in both\nsubdivisions of the ILC.\nScalebar 100µm.\n- Figure 3C1: If the OT fibers shown are identified through immunohistochemistry (IHC), please\nindicate this by labeling them as OT (IHC), not mCherry, in the figure.\nWe apologize for not stating this clearer, but while (old) Fig 3 B2 does show OT fibers that were\nidentified through immunohistochemistry, the fibers in (old) Fig 3 C1 (now Fig S3 C) were in fact\nlabelled after viral injection with rAAV-OTp-mCherry in the PVN and SON and therefore stained for\nmCherry (IHC).\nIn line, we have adapted the figure legend accordingly as:\n“OT fibers in close proximity to OTR+ neurons. OT fibers labelled by viral injection of rAAV-OTp-mCherry\nin the PVN and SON are found…”\n- Extended data S4 A1: Extended Data S4 A1: Social memory tests performed with saline (control)\ninjections did not show significant differences between novel and familiar rats, although a trend is\npresent. The discussion regarding social memory appears to be based on this non-significant\nobservation. It would be important to address this limitation and consider whether the trend warrants\nfurther investigation or if additional controls are needed.\nWe thank the reviewer for this valid comment, and have adapted the text accordingly to tone down\nthe interpretation of the novel preference test as:\n“Moreover, we conducted a social novelty preference test and observed no difference induced by\nchemogenetic activation of ILC OTR+ interneurons (corner: p=0.0100, treatment: p=0.1474, corner x\ntreatment: p=0.1937, post-hoc tests all ns) (Extended Data Figure S4C). Taken together, these experiments\nshowed that recruitment of ILC OTR+ interneurons is sufficient to promote social behavior without\nalterations of social novelty preference or anxiety.”\nReviewer #2 (Remarks to the Author):\nSchimmer et al is presenting a manuscript describing how oxytocin release in the infra-limbic area\nof the prefrontal cortex (ILC) facilitates social interactions between female rats. First, they\ncharacterize OT projections to the PFC and demonstrate that OT release promote sociability. Then,\nthey leverage fiberphotometry of calcium signaling to show OTR+ neurons are activated during\nsocial interactions initiated by the test rat. They also show that chemogenetic activation of OTR+\nneurons promote social interaction while CRISPR/Cas9-mediated knockout of OTR has the opposite\neffect. Then, they demonstrate the OTRS are predominantly expressed by GABAergic neurons in\nL2/3 of the ILC using in situ hybridization and viral injections. They also show that some L2/3\ninterneurons and L5 pyramidal neurons are functionally modulated by OTR agonists using patch-\nclamp recordings. The authors perform chemogenetic manipulation of OTR interneurons and repeat\ntheir previous effect on sociability while showing social novelty preference is not affected.\nManipulation of OTR+ INs can however facilitate the preference for social interaction over food.\nThen, they perform paired recordings in slices and show PN receive direct inhibition from OTR+ INs.\nFinally, they use chemogenetic activation of OTR+ INs coupled with c-fos labelling to test which\n8\ndownstream area is modulated and found that BLA activity is decreased while NAc activity is\nincreased. Viral expression of fluorescent protein, optogenetic and chemogenetic coupled to MEA\nrecording achieve to demonstrate that OTR INs silence L5 PN projecting to the BLA.\nThe article is well-written, the experiments are thorough and use state-of-the-art techniques to probe\nthe function and mechanism of OT release in the ILC (promoter-specific expression coupled to Cre-\ndependent viruses, fiberphotometry, paired patch recordings, chemogenetic an optogenetic coupled\nwith slice recording or behaviors). The authors blend these techniques in a very elegant fashion to\nsuccessfully and convincingly demonstrate their claims. The results are novel and timely as\nmechanistic studies of the cortical neuromodulatory circuits are still lacking despite knowing for a\nlong-time that neuropeptide and their receptor are widely expressed throughout the cortex.\nWe thank the reviewer for their positive view of our study. We appreciate particularly the comments\naimed at improving the quantitative analysis of our anatomical data, which we thoroughly followed\nand provided requested analyses (details below).\nI only have minor concerns:\n- Statistics are sometimes a bit light with the name or p values of the test missing (Fig. S4A2 for\nexample). Systematically provide F values and degrees of freedom for ANOVAs. Perform nested t\ntests when multiple observations are done in the same mice. Consider putting all the statistic in a\nseparate table rather that in the text. Try to be consistent with the number of significant digits when\nreporting p values (same degree of precision).\nWe want to thank the reviewer for this important comment, and have now created a Supplemental\nDocument with a table including all statistical tests and corresponding values as suggested. In the\ntext we have also corrected all p-values to have a uniform number of significant digits.\n- Line 159: “9.5% of neurons expressed OTRs with a higher occurrence in superficial layers 2/3\ncompared to the deeper layers 5/6 (12.7% and 8.9%, respectively)”. Provide statistical comparison.\nThe absolute values of this analysis changed as we performed\nan additional semi-automated, high-throughput tracking of Fig. S2 A More OTR+\nneurons are found in layer\nneurons and increased the n substantially as suggested by\n2/3 of the ILC, compared\nreviewer #1, but we have also now performed the suggested to layer 5/6.\nstatistical comparison and included it into the manuscript and\nthe data table (Fig. S2 A, see on the right).\n- Line 169: “p<0.0001 for anogenital and non-anogenital”. What test is used?\nThe fiber photometry data was tested using a linear mixed effect model, all additional details (nesting,\nfixed factors) can be found in the methods section as:\n“Statistical analysis was performed in R (Version 4.0.4; R Core Team, 2021) using RStudio (Version\n1.4.1106, PBC; Team R, 2019). Linear mixed-effects models were fitted using the lme4 (Douglas Bates,\nBolker and Walker 2015) and lmerTest (Kuznetsova, Brockhoff and Christensen 2017) packages, with type\nIII ANOVA performed using Satterthwaite’s method. Post hoc Tukey’s multiple comparisons were\nconducted with the emmeans package (Lenith 2023), and the Kenward–Roger approximation for\ndenominator degrees of freedom. Model assumptions were verified by inspection of residual plots. The\nmodels compared Z-scored ΔF/F values before vs. during each behavioral event in a paired manner, with\ntrial included as a random effect nested within session and sessions nested within animal. When recordings\nwere performed bilaterally, hemisphere was added as a cross-nested random effect. Fixed factors included\ntime in relation to trial onset (before vs. during the behavior), and in the case of comparison of active and\nreceived behaviors also behavior type. The model for the behavior towards the toy was fitted separately,\nas these were recorded from separate sessions.”\nWe have also now added test details in the Supplemental table.\n- Fig. 2B4. What are the tests used. Give exact p values.\n- Line 252: “Similar to our findings with the RNAscope approach, we could\nidentify an OTR+ interneuron population in layer 2/3 (yellow)and an OTR+\npyramidal population in layer 5/6 (red).” Give percentages so one can compare\nwith the ISH results.\nWe thank the reviewer for this important comment! The previously mentioned\npopulations were identified only qualitatively, we have now performed a semi-\nautomated, high-throughput analysis of OTR+ neurons in the ILC via Imaris. In\nthe manuscript we have plotted the data further subdivided along the rostro-\ncaudal axis (Fig3 C4), and are mentioning the percentages in the text. On the\nright we have plotted the same data in analogy to the ISH results for direct and\neasy comparison. The RNAscope and viral results are comparable, for reasons\nthat might explain the small discrepancies please refer to our answer to\nReviewer #1 regarding the same concern (Reviewer #1, Comment 4).\n- Line 254: “Notably, we could also observe OT fibers throughout the ILC, with\ndenser innervation in layer 2/3”. Quantify the density of fibers per ILC layers.\nWe appreciate this important comment by the reviewer, and have now\nperformed additional viral injections aimed at addressing this question\nand others (Reviewer #3, first comment to Figure 1) and injected\nrAAV-OTp-Venus into the PVN and SON of separate animals to study\nthe anatomical features of the OT input to the ILC. We observed that\ncombined OT innervation of the superficial cortical layers (L1 + 2/3)\nis actually comparable with the deeper L5/6 (Fig. 1 C3, please see on\nthe right). In our original experiment. we visualized OT fibers with\nimmunohistochemical staining for OT and observed the fibers mostly\nin the superficial layers. One of the contributing factors might have\nbeen the high background staining we observed in the deep cortical\nlayers, and which was pointed out by Reviewer #3. Moreover, layer\n5/6 of the ILC has very blurry boundaries with the forceps of corpus\nFig. 1 C3 Localized analysis of\ncallosum. All OT fibers ipsi- and contralaterally projecting to the fiber innervation originating from\ninsula, AON, aPir etc. travel along the white matter and branch in the the PVN or SON depending on\nthe layer in the ILC (layer 1,\ndeep ILC layers. All those thin OT branches became visible in virus-\nlayer 2/3 or layer 5/6) reveals\ninjected brain tissue, though they are not detectable in antibody- most fibers in layer 5/6 (n=6\nstained tissue. Notably, we later describe the population of OTR+ sections obtained from 3\nanimals per group).\nchandelier cells in layer 2/3 driving our observed behavior and\ndownstream circuit, and their apical dendrites are located in layer 1,\nwhere we now accurately analyzed this layer and found substantial\ninnervation.\n- Line 304 (social memory test). I would prefer that the authors avoid calling this test the social\nmemory test but rather call it preference test. There’s no indication the PFC is needed to store or\nconsolidate memory. Rather, the PFC is likely to utilize social cues from vCA1 to guide the\nexpression of a social memory during recall. In the data presented in Fig. S4A2, comment on the\nfact that rats did not show a significant preference for the novel rat. Calculate\nthe preference indexes as well and analyze both graphs according to the\nindications given in the next comment.\nWe appreciate this comment and have rephrased the ‘social memory test’ to\n‘social novelty preference test’ throughout the manuscript. We apologize for\nthe confusing description and depiction of statistical tests for this paradigm,\nas the ANOVA does in fact produce a significant effect on the corner term\n(novel vs familiar, p=0.0100), however post-hoc tests for the saline and CNO\ncondition only show a trend (and this was reported misleadingly as non-\n10\nsignificant). Most importantly, our main finding of this experiment, which is the lack of effect after\nchemogenetic manipulation (treatment: p=0.1474, corner x treatment: p=0.1937) persist. We now\npresent the statistical results clearly in the results section. We also have performed SPI (social\npreference index) analysis (see on the right). The SPI test also shows no significant difference\nbetween the saline and CNO group, our hypothesis that ILC OTR-neuron activation does not\nmodulate social novelty preference remains unaltered.\n- Fig. 4C and Fig. S4B. Chemogenetic activation during food vs. social preference. Please show the\ninteraction times with both corners followed with the preference index (4C2). Data in 4C2 should be\nanalyzed with a two-way ANOVA (injection x feeding status). Also, perform one-sample t tests to\ncompare each group against 0 and determine whether there is a preference for social or feeding.\nGraphs C3 and C4 should be plotted with social and food next to each other (do the same for the\nsocial and food interaction time graph, as you did in Fig. S4A2). Given that interaction with one\ncompartment is not independent from the interaction with the other compartment I would limit the\nanalysis of this graph to comparing social and food within each group, not across group. Calculating\nthe index allows across group comparison by reducing the number of variables to one variable per\ngroup.\nWe thank the reviewer for this detailed and constructive comment. This experiment has\nbeen scrutinized by all reviewers, who commented on the potential modulation of food interest\n(reviewer 1 comment to figure 4C), lack of essential controls for food interest and CNO effects\n(reviewer 3 comment to figure 4C) as well as the detailed analysis pipeline (this comment).\nTo comprehensively address these points as well as our own concerns, we repeated the experiment\nwith a between-subject design. Instead of administering saline and CNO in the same rats as before,\nwe used two separate animal groups, one injected with the rAAV-DLX-DIO-Gq, and the new group\nwith a control virus rAAV-DLX-DIO-GFP, and administered CNO to both groups. This allowed us to\ncorrectly control for any unspecific CNO effects, and investigate properly the potential influence\nof ILC OTR interneuron modulation on food interest, clearly showing a lack of anorexigenic\neffects (see figure S4 D1-4 below).\nFig. S4 D Food interest. Animals were placed in an OF with food pellets behind a mesh in one corner. D1 The time spent investigating\nthe food was only influenced by hunger, but not chemogenetic activation of OTR+ interneurons. D2 Number of bouts while investigating\nfood in the OF. D3 Average speed in the OF as well as D4 distance travelled in the OF (n=8 animals per group).\nAdditionally, the cohort of rats used in this experiment showed much stronger preference towards\nthe social corner, even when hungry, compared to the cohort used in the original experiment.\nThis rendered the SPI to be a less effective parameter to visualize the observed effects of ILC OTR\ninterneuron modulation on the social vs food preference.\nTherefore, for clarity and transparency we decided to remove the original experiment from the\nmanuscript, replacing it with the new between subject design. Moreover, we swapped the SPI for an\nunprocessed data approach: analyzing and depicting raw interaction time with the corners. Because\ntime in the two corners is complementary within a fixed session, corner-wise raw times plus a model\nwith a ‘corner’ factor provides more information than a single index. The original SPI panels are\ntherefore removed from the main text to avoid redundancy. Rather than a two-way ANOVA on an\nindex, we analyzed raw times using a linear mixed-effects model (fixed effects: food\nrestriction [satiated vs 24 h], virus [GFP vs Gq], corner [social vs food]). This approach (i) respects\nthe repeated-measures structure across corners, (ii) avoids distributional distortions that can arise\nwith ratios/indices, and (iii) allows the specific virus × corner contrast that directly tests whether\n11\nhM3Dq activation selectively increases social vs food interaction time. All results and detailed data\ncan be found in the Supplemental Table. In summary the test revealed that:\n corner: p < 0.0001 (****) animals spend more time at the social than food corner overall;\n food restriction × corner: p = 0.0004 (***) deprivation changes allocation between corners\n(expected modulation of preference);\n virus × corner: p = 0.0431 (*) hM3Dq activation increases social relative to food time (selective\neffect on social engagement). This result is of special importance, as it shows that CNO\nincreases social interest regardless of hunger. The post hoc test specifically points to a\nsignificant effect only in the Gq-hungry group.\nMain effects of food restriction (p = 0.7023) and virus (p = 0.0654) were not significant, consistent\nwith a selective, corner-specific modulation rather than a global change in exploration.\nRegarding the suggestion to plot social and food corner directly next to each other: in the new\nexperiment the social interest visibly and significantly dominates the food interest regardless of\nhunger. Therefore, the most interesting and crucial comparison is the effect of CNO on social\ninteraction. Hence, we decided to leave the social bar next to each other to emphasize the\ndifference.\n- Line 314: “where in a satiated state animals are expected to highly prefer social interaction, while\nfood restriction for 24 hours leads to a shift of preference towards food (Extended Data Figure S4\nB1-2).” Figure reference is incorrect; it should be Fig. 4C2\nWe apologize for the confusing placement of this Figure reference; we have moved it to another\npoint in the manuscript.\n- Given the clear effect of OT release on facilitating social interaction with another female, the effect\non social vs. food preference appears to be a consequence of facilitating the social interaction rather\nthan choice-specific. Please comment on this briefly in the discussion.\nWe thank the reviewer for this valid comment and have added this into the discussion as:\n“In our social vs. food choice paradigm, activation of OTR⁺ ILC interneurons selectively increased social\ninvestigation without generalized arousal, consistent with the proposed role of the ILC in resolving\ncompeting motivations by suppressing non-social drives when social cues are behaviorally salient.\nIntriguingly, the prosocial effect of ILC OTR+ interneuron modulation emerges stronger under hunger. This\nsuggests that OT signaling in the ILC acts not necessarily as a tonic enhancer of sociability, but as a\ncontext-dependent biasing mechanism that promotes social engagement specifically when motivational\nconflict is strong.”\n- Fig. 5E1. It should be ChR2 instead of mCherry in the virus construct.\nWe thank the reviewer for pointing out this error, which we have corrected.\n- Fig. 4S2. Check the scale for the layer 2/3 FS OTR+ interneuron recording.\nWe want to thank the reviewer for spotting this error in Fig. 4F2. We checked the original recording\ndata and have indeed adjusted the scale to correct the mistake that was previously made during\nformatting of the figure.\n- Fig. S1. Can you show the ratio of time spent in the surround Fig. S1 C6 The\ndivide by the time spent in the center?\npercentage of time\nspent in the center\nof the arena\nWe have added this calculation and added it in Fig. S1 C6 (see remains\non the right). unchanged\n12\n- Fig. S5E. Can you orient the coronal slices with dorsal axis upward? I find the current orientation\nconfusing.\nWe have omitted this experiment from the publication (for reasons refer to answers point 9 of\nreviewer #1), therefore the image has been removed.\n- Line 401. Can you first discard any long-range projections from OTR+ interneurons in the ILC?\nThis way performing the complex c-fos analysis following manipulation of the OTR+ interneurons\nwould make more sense. In addition, explicit what kind of behavior perform the rats prior to\nperfusing them? A nested t test should be used for Fig. 4A2, give the p value.\nWe have never observed long-range projections of OTR+ neurons originating from the ILC (rAAV-\nDLX-DIO-GFP) in any of the investigated regions (BLA, NAc, VTA, PAG, Sep, Summ), therefore we\ncan confidently assume that any cFos changes in these regions are caused by indirect inhibition of\nprojecting non-OTR neurons. Moreover, we cannot exclude multisynaptic effects.\nFor the cFos analysis, rats were not subjected to specific behavioral tests, they received i.p.\ninjections of CNO or saline and were placed back into the home-cage into their familiar social\nenvironment.\nFor Fig 5A2, we have further split the analysis in the ILC into the layers and separately analyzed the\ncFos expression in projecting regions. Therefore, we have performed t-tests and corrected for\nmultiple testing (Sidak). The p values can be found in the Supplemental Table.\n- Fig. 5: The treatment to the NAc projections differs quite a lot from the BLA projection. Can you\ninvestigate a bit further or comment in the discussion about the putative microcircuit?\nWe thank the reviewer for pointing out this lack in the manuscript, and have now added more\nexplanation about the projections to the NAc and why we did not investigate them further (see below).\nTo quickly summarize: anatomically, we did not find NAc-projecting neurons in the ILC layer 2/3 (as\nopposed to the BLA-projecting neurons, we now even quantified this population in Fig S5 C), and\ninitial preliminary patch-clamp experiments (not shown) did show a clear picture of inhibition of BLA-\nprojecting neurons in layer 2/3, which we later verified with more precise optogenetic patch\nexperiments. Therefore, we chose to focus on the BLA-projecting neurons, as we anticipated that\nthey would exert a more direct modulation of social behavior.\n“Chemogenetic activation of OTR+ ILC neurons in our experiment led to downregulation of cFos expression\nin the BLA and increased cFos expression in the NAc. We propose that the observed reduction in the\nnumber of cFos-positive cells in the BLA is caused by the ILC OTR+ interneurons-mediated inhibition of\nILC principal cells providing excitatory input to the BLA. Among the mPFC subdivisions, the ILC in particular\nis known to target a dense population of GABAergic interneurons in the BLA (Davis, Zaki et al. 2017) and\nthe BLA in turn densely innervates the NAc (Correia, McGrath et al. 2016). We hypothesize that the\nobserved cFos upregulation in the NAc might be caused by the suppression of BLA interneuron activity,\nthereby increasing the excitatory signaling from the BLA to the NAc. As mapping circuits via cFos activity\npatterns comes with the caveat of spatial and temporal ambiguity, we further investigated the OTR-ILC-\nBLA/NAc circuit both anatomically and electrophysiologically. Retrograde tracing following injections in the\nBLA and NAc revealed BLA-projecting principal cells in the ILC that were embedded within a dense\n“inhibitory sleeve” and NAc-projecting principal cells only in deeper cortical layers. Additionally, we showed\nOTR+ interneurons forming axonal cartridges at the initial axonal segment of neurons projecting to the BLA\nwhich was not observed in the case of NAc-projecting principal cells. This was further confirmed by ex vivo\nstudies which showed direct monosynaptic coupling between OTR+ interneurons and BLA-projecting\nprincipal cells.”\nReviewer #3 (Remarks to the Author):\nThis manuscript by Schimmer et al aims to elucidate the circuit mechanism through which oxytocin\nmodulates PFC circuits regulating social behavior. The authors do a very thorough job tracing the\naxons of hypothalamic OT neurons into the PFC, genetically labeling the neurons expressing the OT\nreceptor (OTR), and detailing how activation of this receptor can alter the activity of PFC excitatory\n13\nneuron populations that are thought to control pro-social behaviors. The experiments are conducted\nto a high standard, and the manuscript is clear and well-written. While the authors do refer to some\nrelevant previous work, they do miss a few important citations that should be mentioned, some of\nwhich containing results that the authors’ findings should be reconciled with. Much work has been\ndone on PFC circuits, and specifically on the reciprocal interactions between the PFC and BLA.\nReferring to these important studies can help the reader interpret the results of the current one. This\nis an important study that will be of great interest to the broader neuroscience audience as it provides\na potential mechanism for how OT modulates the cortical aspect of social behavior regulation. I\nwould be happy to hear the authors’ response to my specific comments below - all are relatively\nminor, but can improve the validity of the findings and their interpretation.\nIntroduction\n“With the exception of the auditory cortex in nursing mice, which has been extensively studied by\nFroemke and colleagues (Marlin, Mitre et al. 2015, Valtcheva and Froemke 2018, Schiavo, Valtcheva\net al. 2020), the vast majority of studies demonstrate that the behavioral OT effects are mediated via\nsubcortical brain regions, whereas OT signaling in the cortex is much less explored” - the authors\nmention specifically the important work of Robert Froemke while overlooking significant contribution\nby Nathaniel Heinz (2014, 2016), Oettl et al 2016 and Tan et al 2019, who have also studied OT-\nmediated cortical modulation of social behavior.\nWe thank the reviewer for pointing out these important studies in the field of cortical OT research.\nWhile we did not mention these works at this point in the introduction, we referenced all mentioned\nstudies (except for Oettl et al. 2016) either towards the end of the introduction referring to specific\nPFC OT action, or in the discussion. We have now added a short paragraph in the introduction to\nfurther acknowledge these studies:\n“While most studies show that OT’s behavioral effects arise from subcortical circuits, emerging work has\nstarted to uncover its role in cortical signaling. OT signaling in the murine auditory cortex and its role in\nnursing, has been extensively studied by Froemke and colleagues (Marlin, Mitre et al. 2015, Valtcheva and\nFroemke 2018, Schiavo, Valtcheva et al. 2020). Moreover, OT has been shown to drive top-down\nmodulation of early sensory processing in the olfactory bulb to enhance social recognition (Oettl, Ravi et\nal. 2016). Additionally, several studies have directly implicated cortical OT signaling in the regulation of\nsocio-sexual behaviors: in the medial prefrontal cortex (mPFC), Heintz and colleagues identified OTR-\nexpressing (OTR+) interneurons that gate female sociosexual behavior and revealed a sex-dimorphic\nOTR–CRH interaction shaping social and anxiety-related responses (Nakajima, Görlich and Heintz 2014,\nLi, Nakajima et al. 2016). Moreover, OTR-expressing glutamatergic neurons in the mPFC have been shown\nto selectively regulate social recognition memory (Tan, Singhal et al. 2019).”\nDespite the inspiring demonstrations that OT action and/or OTR-expressing neurons (OTR+\nneurons) in the PFC modulate paternal (He, Young et al. 2019) and sexual behavior (Nakajima,\nGörlich and Heintz 2014) in voles and mice, respectively, it remains unknown how OT signaling in\nthe PFC affects basic social behavior.” - the term “basic” social behavior is not clearly defined, which\nmay confuse the reader about its distinction from “sociosexual” behavior (Nakajima et al 2014 &\nLi et al 2016). In this regard, the authors should also relate to Li et al (2016, Cell), which demonstrates\nhow mPFC-projecting OT neurons differentially facilitate social behavior in females compared to\nmales.\nWe thank the reviewer for pointing out the suboptimal terminology and have changed the wording in\nthe manuscript to affiliative (non-sexual) social behavior / same-sex social interactions / conspecific\nsociability. We also included the work by Li et al., 2016 in the introduction (see previous answer),\nand refer to it in more detail in the discussion, as:\n“Notably, we did not observe any effects on anxiety behaviors when activating ILC OTR+ interneurons. This\nis in line with work from Li, Nakajima et al. (2016), who showed that activating OTR-expressing interneurons\nin the mPFC reduces anxiety-like behavior in males but not females (with OTR loss producing the opposite,\nmale-specific anxiogenic effect). In contrast, the same manipulation enhances social behavior in females\nbut not males, revealing complementary sex-specific routing of OT’s influence on anxiety vs. sociability, a\nsex difference yet unexplored in rats.”\nchandelier interneurons in L2 of the PFC and how they innervate amygdala-projecting neurons in\nmice (see Lu et al., Nat Neurosci 2017, DOI: 10.1038/nn.4624). While the Lu paper failed to\ndemonstrate a behavioral role for these neurons, the current study sheds important light on the topic.\nWe thank the reviewer for mentioning this study and included it in the manuscript. Considering we\nonly point to the role Chandelier cells play in the ILC over the course of the result section, mentioning\nthis study in the introduction might be confusing for potential readers, but we are dedicating a new\nparagraph in our discussion to this study as:\n“Notably, a study by Lu, Tucciarone et al. (2017) demonstrated that a subset of layer 2 Chandelier cells in\nthe PrL cortex selectively innervates BLA-projecting principal cells ( PCs) over contralateral-cortex-\nBLA\nprojecting principal cells ( PCs), while receiving preferential input from PCs, thereby establishing a\nCC CC\ndirectional inhibitory module that gates BLA-related output. Interestingly, they also found PCs in the\nBLA\nsame laminar layers as Chandelier cells, while PCs were located in deeper layers, resembling our finding\nCC\nof BLA- vs. NAc-projecting neurons. This spatial coincidence suggests that OTR⁺ interneurons are well-\npositioned to modulate PCs ensembles. While their study did not indicate a behavioral role of this\nBLA\nneuronal subset, our work suggests its prosocial role.”\nFigure 1:\nPanels B1-2: The demonstration of PVN- and SON-originating OT neurons in the mPFC is\ncompelling. What is the unique contribution of each nucleus to the density presented in B3? This\ncould be achieved by performing the viral-based tracing, which is mentioned in the main text but not\npresented in Fig1, with two fluorophores within the same animal.\nWe thank the reviewer for this excellent comment, and have indeed performed an additional\nexperiment followed by semi-automated quantification of OT fibers originating from the PVN and\nSON. While we initially performed this experiment exactly as suggested by the reviewer (within\nsubject comparison, rAAV-OTp-Venus in the PVN, rAAV-OTp-mCherry in the SON, and vice versa),\nwe found that expression and detection levels of markers in the two viruses were significantly\ndifferent. Therefore, we switched to an approach with between subject comparison, where animals\nreceived rAAV-OTp-Venus injections either in the PVN or SON. For results see Fig 1C below.\nFig. 1 C Innervation from the PVN and SON. C1 Injection scheme depicting the injection of a rAAV expressing Venus under the control\nof the OT promoter (rAAV-OTp-Venus) into either the PVN or SON in separate animals, with respective injection sites. Scalebar 250µm.\nC2 Automated analysis of fibers in the ILC quantifying their density by the sum of their volume originating from the PVN or SON reveals\nstronger innervation from the SON. C3 Localized analysis of fiber innervation originating from the PVN or SON depending on the layer in\nthe ILC (layer 1, layer 2/3 or layer 5/6) reveals most fibers in layer 5/6 (n=6 sections obtained from 3 animals per group).\nPanel B3: Fiber density should be normalized to the area/volume. Although the area/volume used\nfor the current analysis is missing, the results of PrL+Cgt seem lower than those reported in Knobloch\net al 2012 (ILC is not reported in that paper). How do you explain these differences?\nIn line with the reviewer´s suggestion, we have now normalized the fiber density to ‘fibers per mm2’,\nexactly how we did it in Knobloch et al., 2013. While the data in Knobloch et al., 2013 is conducted\nin lactating female rats (where we reported that a 3-fold increase in fluorescence intensity is\nobserved in late pregnancy/lactating rats compared to virgin rats), here we used virgin female rats\nand therefore lower expression and potentially detection rates would be possible. Additionally,\n15\nmethodological differences might explain slight discrepancies, as in Knobloch’s study 15 years ago\nwe used DAB staining of thick brain slices and manual analysis with a camera lucida, which is clearly\nless advanced than the automated Imaris analysis we used now. Nevertheless, we found on average\n50 fibers per mm2 in the ILC, and while the ILC was not specifically analyzed in the Knobloch´s\npaper, other cortical forebrain structures reported mostly ‘up to 10’ or ‘up to 50’ fibers per mm2,\nproviding comparable results.\nNumber of sections, animals and statistics mentioned in legends are different than what is reported\nin the Methods section.\nWe thank the reviewer for realizing this error, we have now corrected it. Additionally, we’ve added a\nsupplementary spreadsheet summarizing all performed statistical comparisons to improve clarity.\nComparing different areas within the same animal can be achieved by averaging each area per rat\nand use paired t-test (and not unpaired) to compare between areas or by using a linear model with\nrandom effect to control for pseudoreplication.\nWe agree with the reviewer and have now performed the statistics on averaged counting per animal,\nusing a paired t-test (please also see the new Supplemental Table).\nPanels C\nGeneral comments: To support the main claim of this figure, the authors should provide\nsupplemental schematics or images of the optic fiber targeting the ILC for all animals.\nWe routinely perform fiber implantations in rats over years (Knobloch et al., Neuron (2012), Hasan\net al., Neuron (2019), Tang et al., Nature Neuroscience (2020), Tang et al., STAR Protocols (2021),\nIwasaki et al., Nature Communications (2023) and others) and always verify implantation site post\nhoc by inspecting brain slices at the light microscope, excluding any animals that were mistargeted.\nThis was also the case for this experiment, and based on the experimental protocols all the included\nanimals were correctly implanted. To be fully transparent, this experiment was performed more than\na decade ago, and we failed to retrieve requested specimen as it has inevitably degraded over the\nyears.\nTo strengthen the claim of this figure we decided to perform a new experiment – utilizing a novel\nGRAB OT sensor to visualize ex vivo OT release in the ILC upon similar optogenetic stimulation of\nOT fibers, which was additionally pharmacologically verified by administration of atosiban – a potent\nOTR (and therefore GRAB OT sensor) antagonist (Fig. 1 D, see below).\nFig. 1 D Axonal OT release in the ILC ex vivo. D1 Injection scheme depicting the injection of a rAAV expressing the excitatory ChrimsonR\nunder the control of the OT promoter (rAAV-OTp-ChrimsonR-mScarlet) into the hypothalamic nuclei of female rats and injection of a rAAV\nexpressing an OT-sensor (rAAV-OT1.9-GRAB-sensor) in the ILC. Stimulation of the OT fibers in the ILC (625nm light, 30 Hz, 10 ms light\non, for 30s). D2 Peri-stimulus GRAB-OT1.9 fluorescence changes in the ILC in ACSF-only solution (blue) or in the presence of Atosiban\n(OTR-antagonist, gray). Orange square marks the stimulation period. Bracket indicates the timeframe for area under the curve (AUC)\nanalysis in panel D3. Data presented as mean±sem. D3 Recordings in ACSF condition showed higher AUC, calculated between 50 and\n350 seconds after light stimulation, compared to recordings in the Atosiban condition (n=10 sections).\nWe hope that, although we have failed to provide requested data, we have satisfactorily addressed\nthe reviewers’ concerns with this new, sophisticated experiment.\nChR2 activation, given the potential opsin desensitization (Nagel 2003; Lin 2011), the dynamics of\nlarge dense core vesicle release and potential depletion from the terminals under such strong\nstimulation. Have the authors tried recording OT release under this protocol?\nWe thank the reviewer for raising this important point. Our intention with the 30 Hz, 10 ms, 120 s\ntrain was to robustly engage dense-core vesicle exocytosis from long-range oxytocin (OT) axons. In\nour study Knobloch et al., 2012 we showed that 30 Hz as well as continuous stimulation with blue\nlight reliably induce action potentials in PVN and SON neurons with our opsin constructs. Consistent\nwith this rationale, Qian et al., 2022 (our co-authored study) used an OT sensor in PFC together with\noptogenetic activation of PVN OT neurons (20 Hz, 10 ms pulses; up to 10 s) and detected reliable\nOT release (Fig. 5f,g). Although that protocol is milder than ours, it demonstrates that sustained high-\nfrequency optical stimulation of PVN OT neurons evokes measurable cortical OT release under\nclosely related conditions.\nMoreover, as mentioned in our response to the previous comment, we addressed this issue in a new\nexperiment, proving that this stimulation leads to OT release in ILC, detectable with the GRAB OT\nsensor.\nTo additionally address potential opsin rundown\nand the ability of OT neurons to follow high-rate\ntrains, we performed new whole-cell recordings\nfrom identified PVN OT neurons expressing\nChR2-EYFP or ChrimsonR-mScarlet. 30Hz,\n10ms light trains drove robust, time-locked\nresponses in current clamp and sustained\nphotocurrents in voltage clamp at room\ntemperature (Fig S1 A; representative traces Fig. S1 A Functional verification of OTp-ChR2 and OTp-\nattached on the right). These data indicate that our ChrimsonR through patch-clamp recordings.\nopsins provide stable drive to OT neurons at the\nfrequencies used.\nWhen it comes to the concern of potential overstimulation, it is important to note that we use a very\nstrong protocol as established in previous studies (Knobloch et al., 2012) to circumvent potential\nreduction of stimulation due to tissue light absorption. While causing the depletion of OT vesicles is\na valid concern of the reviewer, we want to point out that this is not only unproblematic for this\nexperiment (as we only stimulate the system once), but rather wanted (as we want to release all\navailable OT). Importantly, OT action lasts for several minutes, so following our stimulation, the\nILC is plausibly flooded with OT throughout the entire length of the behavioral experiment (5 mins).\nC4 - for the comparison of total interaction time the statistics should be unpaired t-test, rather than a\ntwo-way ANOVA.\nWe fully agree with the reviewer and have performed separate statistics for the total interaction time\n(t-test) and sub-behaviors (2-way-ANOVA). For details, please refer to the new Supplemental Table.\nFigure 2:\nPanels B: The fiber photometry result, while consistent with later results in the paper, might not be\nspecific to OTR+ neurons. I assume that other populations in the PFC would show the same pattern\nof activity as these neurons. Is this activity pattern consistent with the activity of PVN OT neurons?\nDoes it change when OT release is suppressed in the PFC or more broadly? This should be\ndiscussed in the Discussion section if not experimentally addressed.\nTo address the reviewer’s concern that “other PFC populations might show a similar pattern,” we\nemphasize that our claim is restricted to the genetically defined OTR⁺ population; while other\npopulations could show correlated dynamics at the network level, the signal we measure arises from\nOTR-Cre-positive neurons only. Unfortunately, at this point we don’t have reliably established tools\n17\n(e.g. dual color photometry) to properly address this interesting issue, which we believe is beyond\nthe scope of the current study. Regarding consistency with PVN OT neuron activity, our prior work\nshows that PVN OT neurons increase activity during social touch/interaction (e.g., Tang et al., 2020,\nNat Neurosci), and that optogenetic activation of PVN OT neurons produces measurable OT release\nin the PFC (Qian et al., 2022, Fig. 5f, g). Thus, the direction and timescale of our OTR⁺ PFC\npopulation signal are consistent with known PVN OT neuron dynamics and with evoked OT release\nin the cortex.\nRegarding dependence on OT signaling: while we did not manipulate OT signaling during our\nphotometry experiments, we performed a viral OTR knockdown in the ILC (Fig. 2D) and found that\nit reversed the behavioral effect in our social-interaction assay. Thus, ILC OTR signaling is necessary\nfor the behavioral phenotype measured in the same paradigm, supporting the physiological\nrelevance of the OTR⁺ population activity we report.\nPanels C1-2: The rationale for using a stimulation protocol in OTR+ neurons in the ILC, is not fully\nclear until Fig3 D3. Consider moving this experiment to Fig 3 or reorganize these data to better\nsupport the rationale for this experiment.\nWe thank the reviewer for this comment. The rationale of performing this experiment at this point is\nthat in the previous experiment we showed how social interaction activates OTR+ neurons (fiber\nphotometry), and we now want to artificially mimic the observed effect of endogenous OT action by\noptogenetic activation of OTR+ neurons, in order to show the direct effect on behavior.\nPanel D: The authors use an interesting approach to study the specific role of OTR by functionally\ndisrupting OTR signaling in the ILC using gene editing. It’s important to note that while most of the\nmutations induced to the OTR by this method result in complete loss of its ligand binding, some\nresidual functionality cannot be excluded (e.g. receptor dimerization; Boender et al 2023), as seen\nin the TGOT experiment (Extended Data Fig. 2). Therefore, this approach does not fall under the\ndefinition of “knockout”, and this term should be avoided to prevent misinterpretation.\nWe fully agree with the reviewer and now refer to this manipulation as a CRISPR/Cas9-mediated\nOTR depletion / functional knockdown, not a knockout. As the reviewer notes, CRISPR editing in\nvivo can yield mosaic indels and incomplete transduction, so residual OTR signaling cannot be\nexcluded. Moreover, ligand-independent/heteromeric effects of OTR (e.g., potential dimerization\nreported in Boender et al., 2023) could preserve some downstream function even when ligand\nbinding is reduced, consistent with the residual TGOT response we observe (Fig. S2 F). We added\na paragraph to the Discussion to state these caveats, and emphasize that our conclusions concern\nnecessity of OTR signaling in ILC rather than complete receptor ablation, as:\n“Our in vivo CRISPR/Cas9-mediated OTR targeting should be interpreted as a partial loss-of-function rather\nthan a complete receptor knockout. First, viral transduction and CRISPR editing are inherently mosaic, so\na fraction of OTR-expressing neurons likely retain intact receptors. Second, indel heterogeneity may\ngenerate alleles with partial activity. Third, residual receptor functionality unrelated to high-affinity ligand\nbinding (for example heteromerization/dimerization) may persist and maintain some downstream signaling\n(Boender, Boon et al. 2023). These features likely explain the residual responsiveness to TGOT we\ndetected (n=1 neuron, Extended Data Figure S2 F) and indicate that our behavioral effects probably\nunderestimate the full impact of abolishing OTR signaling in the ILC.”\nFigure 3:\nGeneral comment: Given the small sample size in this figure (n=2), the conclusions drawn in this\npart should be taken with a grain of salt. Especially, regarding the distribution and expression of OTR\nwhich is considered sex-, age- and state-dependent. Specifically, the identity of OTR+ neurons in\nthe mPFC and their characteristics appear inconsistent with previous literature (Nakajima et al.,\n2014; Li et al., 2016; Tan et al. 2019). This inconsistency should be addressed at least in the\nDiscussion section, if not with further experiments.\ntotal n=5). We are happy to report no major changes in the overall results, but can draw much more\nreliable conclusions from this data (please see below):\nFig. 3 A4 Colocalization of OTR and Cre mRNA shows high specificity of the transgenic Cre rat line. A5 The majority of OTR+ neurons in\nthe ILC are GABAergic interneurons, with a small fraction of glutamatergic neurons, which are predominantly found in the layers 5/6. A6\nIn-situ-hybridization shows that 1.35% of all neurons in the ILC are OTR+ neurons, with 6.82% of all GABAergic neurons being OTR+.\nThe previous literature is already quite broad: early work by Nakajima et al., 2014 and Li et al., 2016\nreports that OTR⁺ cells in mPFC are predominantly Sst⁺ interneurons with strong outputs onto L2/3\nand L5 pyramidal neurons; Li also shows CRHBP expression and sex-dependent effects, while Tan\n(2019) demonstrated a glutamatergic OTR⁺ subset with distinct circuit/behavioral consequences.\nThese studies also vary in various details from our study:\n Species & line differences: mice in Nakajima 2014; Li 2016, still the overall conclusion of\npredominantly interneurons stays consistent. Also mice in Tan 2019, it’s plausible that rat ILC\nhas fewer glutamatergic OTR⁺ neurons than mouse PL/mPFC, or that driver-line and reporter\nsensitivity differ (BAC transgenics/knock-ins can label partly non-overlapping sets)\n Different anatomical compartment (ILC vs “mPFC” as a whole): several mouse papers pooled\nPL + IL + cingulate or emphasized PL, whereas we targeted the ILC specifically. Laminar and\nareal composition of OTR cells varies across PFC subfields; a PL-skewed sample could\noverrepresent a glutamatergic OTR subset that is sparse in ILC.\n Sex & hormonal state: many mouse studies were males or mixed without cycle control. We\nuse specifically females in the diestrus cycle.\nWe have added an additional section to the discussion to address these differences, as:\n“This aligns with mouse mPFC reports emphasizing a predominantly interneuronal OTR population\n(Nakajima, Görlich and Heintz 2014, Li, Nakajima et al. 2016). By contrast, besides the interneuron\npopulation, Tan, Singhal et al. (2019) identified a substantial VGluT1⁺ OTR+ population in the mouse\nmPFC. We suspect these differences reflect a combination of species (rat vs. mouse), subregion (ILC vs.\nbroader PFC/PL), sex (female rats), and methodological factors (driver lines, reporter/probe sensitivity, and\npositivity thresholds). Notably, our OTR-Cre specificity was validated by RNAscope (Cre↔OTR mRNA\noverlap ~83-93%) and pharmacology (all OTR+ neurons marked with GFP responded to bath application\nof TGOT in our patch clamp experiments).”\nIn this regard, the authors don’t mention the age of the animals, virginity status in all experiments\nand the estrous cycle for their non-behavioral experiments, which might be relevant for OTR\nexpression (Bale et al 1995).\nWe thank the reviewer for noticing that we only mentioned age, virginity status and estrous cycle for\nbehavioral animals. We did in fact use the same age (8-12 weeks) and virginity status (virgin).\nWe have added the respective sentence in the method section.\nhard to appreciate the heavier OT innervation in layer 2/3. This result should be quantified by\ncomparing OT fiber density in layer 2/3 and layer 5/6.\nWe thank the reviewer for this comment and have exchanged the\nimage for one of better quality (Fig3 B2), where OT fibers are clearly\nvisible with minimal background as below.\nWe also have now performed the suggested quantification of fiber\ndensity in layer 2/3 and layer 5/6. It can be found in Fig1 C3 (on the\nright). Please refer to our previous answer to Reviewer #2 (Comment\n6) concerning further explanations.\nFig. 3 B2 Representative\nconfocal image depicting\nOTR+ interneurons\ndominating layers 2/3\nFig. 1 C3 Localized analysis of fiber\n(yellow), and individual OTR+\ninnervation originating from the\npyramidal neurons localized\nPVN or SON depending on the\nin layers 5/6 (red), with OT\nlayer in the ILC (layer 1, layer 2/3 or\nfibers (blue, white arrows) in\nlayer 5/6) reveals most fibers in\nboth subdivisions of the ILC.\nlayer 5/6 (n=6 sections obtained\nScalebar 100µm.\nfrom 3 animals per group).\n“Histological quantification after viral labelling of OTR+ cell types qualitatively confirmed…” - It was\neither quantified or qualitatively confirmed, but not both.\nWe thank the reviewer for this point and have corrected the sentence in the manuscript as:\n“Histological quantification after viral labelling of OTR+ cell types confirmed that the majority of OTR+\nneurons were GAD-expressing interneurons immunopositive for parvalbumin and/or calbindin (Extended\nData Figure S3 B).”\nFigure 4:\nThe authors show that OTR+ neurons target L2/3 PYR cells over L5 PYR cells. This is almost\nexpected from the anatomical configuration and the projection patterns of L2/3 OTR+ neurons. Lu\net al (Nat Neurosci 2017) demonstrated that within L2, chandelier interneurons preferentially target\nBLA-projecting over CC-projecting PYR cells. It would be interesting to see whether similar specificity\nexists for OTR+ interneurons.\nWe thank the reviewer for the insightful suggestion! Lu et al. (2017) showed that in the prelimbic\ncortex, layer-2 Chandelier cells (ChCs) preferentially innervate BLA-projecting pyramidal neurons\n( PCs) over contralateral cortex-projecting neurons ( PCs) within L2, thereby exerting directional\nBLA CC\ninhibitory control over PC ensembles.\nBLA\nOur study in the ILC was not designed to resolve the projection identity of our L2/3 recordings, as\nwe did not combine OTR⁺-cell activation with dual retrograde labeling. Consequently, we cannot\ndirectly test whether OTR⁺ interneurons display the same BLAPC-over-CCPC specificity within L2/3.\nWe agree that this is clearly an interesting question, but does not lie within the scope of our study.\nNevertheless, we have added the work of Lu and colleagues into our discussion:\n“Notably, a study by Lu, Tucciarone et al. (2017) demonstrated that a subset of layer 2 Chandelier cells in\nthe PrL cortex selectively innervates BLA-projecting principal cells ( PCs) over contralateral-cortex-\nBLA\nprojecting principal cells ( PCs), while receiving preferential input from PCs, thereby establishing a\nCC CC\ndirectional inhibitory module that gates BLA-related output. Interestingly, they also found PCs in the\nBLA\nsame laminar layers as Chandelier cells, while PCs were located in deeper layers, resembling our finding\nCC\nof BLA- vs. NAc-projecting neurons. This spatial coincidence suggests that OTR⁺ interneurons are well-\npositioned to modulate PCs ensembles. While their study did not indicate a behavioral role of this\nBLA\nneuronal subset, our work suggests its prosocial role.”\ninterneurons in layer 2/3 promotes satiety, which corresponds with the anorexogenic effect of OT. It\nwould be beneficial to include a control experiment to verify that such activation does not alter feeding\nbehavior, supporting a specific effect on sociability. Furthermore, inclusion of a control group in this\nexperiment seems to be important, given the known retroconversion of CNO to clozapine, particularly\nin rats. There is not much information on how hunger states interact with clozapine or CNO\nresponses, and it would be good to make sure that the effects are indeed specific to the OT\nmanipulation.\nWe fully agree with these comments and have decided to repeat this experiment employing a\nbetween subject design. Instead of administering saline and CNO in the same rats as before, we\nused two separate animal groups, one injected with the rAAV-DLX-DIO-Gq, and the new group\nwith a control virus rAAV-DLX-DIO-GFP, and administered CNO to both groups. This allowed us to\ncorrectly control for any unspecific CNO effects, and investigate properly the potential influence\nof ILC OTR interneuron modulation on food interest, clearly showing a lack of anorexigenic\neffects (see figure S4 D1-4). For detailed discussion on statistical tests used please refer to our\nanswer to reviewer #2 and the Supplemental Table containing all statistics.\nFig. S4 D Food interest. Animals were placed in an OF with food pellets behind a mesh in one corner. D1 The time spent investigating\nthe food was only influenced by hunger, but not chemogenetic activation of OTR+ interneurons. D2 Number of bouts while investigating\nfood in the OF. D3 Average speed in the OF as well as D4 distance travelled in the OF (n=8 animals per group).\nThe term \"cornered social interaction\" is not a commonly-used one. Please rephrase.\nWe thank the reviewer for this comment and refrain from using the term in the manuscript.\nFigure 5:\nGeneral comment: While the suppression of the ILC-BLA pathway by activation of ILC OTR+\ninterneurons is well demonstrated in-vitro and in-vivo, the lack of its effect on anxiety-related\nbehavior in females is puzzling (Extended Data Figure S1). This finding may require further\ndiscussion, especially considering the results from Li et al. (2016), which highlighted sex-specific\neffects on anxiety-related behavior following activation of OTR+ interneurons in the PFC.\nWe thank the reviewer for this interesting point. Li et al. (2016) actually found a very interesting\nsexual dimorphism: when activating OxtrINs in the mPFC (prelimbic) it reduced anxiety-like behavior\n(↑ open-arm time in EPM; ↑ center time in OF) without changing sociability in males; in females it\nactually increased sociability without affecting anxiety. Even though the species and subdivision of\nthe PFC differs from our study, we have included this very interesting finding in our discussion:\n“Notably, we did not observe any effects on anxiety behaviors when activating ILC OTR+ interneurons. This\nis in line with work from Li, Nakajima et al. (2016), who showed that activating OTR-expressing interneurons\nin the mPFC reduces anxiety-like behavior in males but not females (with OTR loss producing the opposite,\nmale-specific anxiogenic effect). In contrast, the same manipulation enhances social behavior in females\nbut not males, revealing complementary sex-specific routing of OT’s influence on anxiety vs. sociability, a\nsex difference yet unexplored in rats.”\nactivation in the ILC, and performed additional chemogenetic experiments (activation of OTR+\ninterneurons) in the open filed and elevated zero maze. In line with our previous observations, we\ndid not find any significant effects on center time in the open field or time or entries to the open arm\nin the EZM. For the full results see Fig. S4 A and B (as below).\nFig. S4 A Exploratory behavior in the open field (OF) after chemogenetic activation of OTR+ interneurons in the ILC. A1 Distance travelled\nin the OF, A2 Time in the center of the OF, A3 number of entries into the OF, and A4 average speed in the OF (n=8 animals).\nB Exploratory behavior in the elevated zero maze (EZM). B1 Distance travelled in the EZM, B2 Time spent in the open parts of the EZM,\nB3 number of entries into the open parts of the EZM, and B4 average speed in the EZM (n=8 animals).\nF: Since the authors claim a specific involvement of the ILC-BLA pathway in social interactions, it\nwould be crucial to include an analysis that specifically compares changes in local field potentials\nduring social versus non-social interactions (i.e. interaction with food), rather than presenting the\nchange during the entire “encounter” period. Also, were these changes correlated with the social\npreference index described in Fig. 4 C2?\nWe sincerely appreciate this comment by the reviewer, and have decided (also based on comments\nby the other reviewers) to exclude this MEA experiment from the manuscript, as it does lead to more\nconfusion than information at its current state. Thorough and prolonged studies will be required to\ncomplete this subset of experiments. After thorough deliberation we concluded it was not crucial for\nthe conclusion and elucidation of the other findings, and will be working on it for a future publication.\nInstead, we have performed a new experiment specifically manipulating the BLA-projecting neurons\nin a social interaction experiment (Fig. 5 F, please see below), which shows the suppressing effect\non social interactions after activating these neurons.\nFig. 5 F Social interaction after chemogenetic activation of BLA-projecting neurons originating in the ILC. F1 Injection scheme depicting\nthe injection of a retrogradely Flp-expressing rAAV into the BLA with simultaneous injection of a Flp-dependent (Frt) Gq-mCherry-\nexpressing rAAV into the ILC to chemogenetically activate BLA-projecting ILC neurons after i.p. application of CNO. F2 Representative\ninjection site showing expression of the rAAV-hSyn-FRT-Gq-mCherry in cell bodies and fibers in the ILC and projecting fibers in the BLA.\nScalebar 250µm. F3 Chemogenetic manipulation of BLA-projecting neurons via CNO injection before a 5 min social interaction (SI)\nsession with an unknown conspecific in the open field. Activation of BLA-projecting neurons decreases social interaction time (n=11\nanimals).\nGeneral note: when providing the construct names for Cre-dependent viral vectors (e.g. \"rAAV-DLX-\nhm3Dq-GFP or rAAV-DLX-hm4Di-GFPC\" as mentioned in Fig. 4) the Cre/lox cassette should be\nincluded.\nWe thank the reviewer for pointing out this error, and have corrected it in the manuscript.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for their work on our manuscript.\nWe thank the reviewers for dedicating their time and effort to evaluate the revision of our manuscript.\nFor ease of reference, all responses are typed in blue.\nReviewer #1 (Remarks to the Author):\nThank you for throughly responding all comments. I'm very pleased with the response and updated\nmanuscript. No further comments. I applaud all the effort by authors and congrat on this\nmanuscript with high quality work.\nWe thank the reviewer for this very supportive response, and want to thank them again for their\nfeedback that helped us improve the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have thoroughly and adequately answered to all my concerns. I fully support the\npublication of this manuscript.\nWe thank the reviewer for their support, and want to thank them again for their feedback that\nhelped us improve the manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all of my comments with additional experiments, data analysis and\nrefinement of statistical tests. I have no further comments on the revised manuscript.\nCongratulations to the authors on this very important study.\nWe thank the reviewer for their supportive response, and want to thank them again for their\nfeedback that helped us improve the manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer for their work on our manuscript.\n1"}]

Decision letter