Stress induces oxytocin-Gαi-dependent remodeling of astrocytes to shape neuronal response in the amygdala

Domain: biology
Published: 2025-12-29
Editorial outcome: major_revision

Abstract
Anticipated reactions to stressful situations are vital for the survival and well-being of organisms, and abnormal reactions results in stress-related disorders. The neuropeptide oxytocin is a key modulator ensuring well-adapted stress responses. Oxytocin acts on both neurons and astrocytes, but the molecular and cellular mechanisms mediating stress response remain poorly understood. Here, we focus on the amygdala, a crucial hub that integrates and processes sensory information through oxytocin-dependent mechanisms. Using an acute stress paradigm in mice, genetic and pharmacological manipulations combined with proteomic, morphological, electrophysiological and behavioral approaches, we reveal that oxytocinergic modulation of the freezing response to stress is mediated by transient G\u03b1i-dependent retraction of astrocytic processes, followed by enhanced neuronal sensitivity to extracellular potassium in the amygdala. Our findings elucidate a pivotal role for astrocytes morphology-dependent modulation of brain circuits that is required for proper anticipated behavioral response to stressful situations.

Introduction
Anticipated reactions to potentially dangerous situations are essential for animal survival, but their dysregulation can lead to pathological conditions such as post-traumatic stress disorders and chronic anxiety1,2. Rodents display similar responses to stress as stereotyped freezing behavior3. Understanding the cellular mechanisms mediating stressor detection and the resulting behavioral and physiological adaptations is crucial to design effective treatments for anxiety and other stress-related disorders. Despite notable progress in this field, the cellular and molecular mechanisms mediating adequate physiological and behavioral responses to stress remain elusive. Importantly, stress response requires coordinated activity of various brain regions. Among these, the amygdala plays a central role, as lesions of this brain structure impair sensory information interpretation across mammalian species, including rodents, monkeys, and humans4. Projections from the amygdala basolateral complex (BLA) to the central nucleus (CeA) are particularly critical for stress-related behavior5. Sensory information is integrated in the BLA and transmitted to the CeA, which in turn modulates stress reactions as freezing behavior in rodents3,6. The activity of CeA neurons, especially in the laterocapsular part (CeL/C), is regulated by endogenous neuromodulators, notably oxytocin (OT)7,8.

OT is synthesized by neurons located in the hypothalamic accessory, supraoptic and paraventricular nuclei (PVN) that project to the pituitary and via axonal collaterals to various regions of the mammalian central nervous system9. OT modifies the balance between excitation and inhibition and regulates the salience of emotionally and socially relevant stimuli. In the context of the behavioral response to stress, this allows rapid switching between states of low and high arousal10\u201312. Within the CeL/C, OT modulates neuronal network activity and influences freezing. This highlights its role in regulating stress responses, and especially the acute stress-induced freezing in rodents6,7,11\u201316.

Remarkably, OT signaling involves not only neurons but also astrocytes as they express oxytocin receptors (OTR)17. Astrocytes are key regulators of neuronal function in the central nervous system18,19. They can detect and respond to neuronal activity by eliciting calcium transients; they release gliotransmitters, which in turn modulate synaptic transmission and they regulate neuronal excitability through neurotransmitter uptake and K+ buffering20. Consequently, astrocytes regulate the activity of neuronal circuits underlying behaviors in a brain-region specific manner17,20. Since OT is released in response to stressful conditions within the amygdala of rats21, this study aimed at determining whether and how astrocytes play a role in the oxytocinergic modulation of behavioral response to stress in vivo.

Here, we show that freezing response to acute stress is mediated by OTR- and Ca2+-dependent structural and functional changes in astrocytes, which in turn modulate neuronal activity in the CeL/C. Therefore, neuromodulatory signaling in astrocytes contributes to stress-induced neuronal hyperexcitability in the CeL/C.

Results
To investigate the role of astrocytes in oxytocinergic modulation of behavioral response to stress in vivo, we adapted a paradigm of acute stress-induced freezing in adult mice22. Animals were exposed to five 20-second-long sound stimuli followed by electrical foot shocks. These paired sound-shock sequences were randomly delivered over 10 minutes, and freezing was quantified during the 20 seconds following the electric foot shocks to assess the immediate stress response (Fig. 1a).

Astrocyte OTR in the CeL/C contributes to stress-induced freezing
We previously reported that roughly 20% of CeL/C astrocytes express OTR, and that those astrocytes are connected to neighbouring astrocytes through GAP junctions17. To directly assess whether astrocytic OT signaling influences the stress-induced freezing, we induced cell-specific loss of OTR by injecting an AAV encoding the Cre recombinase under the control of the glial fibrillary acidic protein (GFAP) promoter (rAAV-GFAP-Cre) to target astrocytes in the CeL/C of OTRfl/fl mice (GFAP OTR cKO, Fig. 1a and Supplementary Fig. 1a)17,23. This viral vector had a specificity between 95.8%-98,2% and an efficiency between 75,8% and 87,3% as indicated by astrocyte markers S100b, Aldh1l1 and GS (Supplementary Fig. 1a\u2013d). In addition, the vector causes a significant decrease in the number of astrocytes expressing OTR without altering OTR expression in other cell types (p<0.0001 and 0.7812, Supplementary Fig. 1e). We observed that GFAP OTR cKO mice failed to exhibit freezing behavior during the stress session as compared with WT control mice (p<0.0001, Fig. 1b and Supplementary Fig. 1f). Intriguingly, this effect was not observed in female mice (p=0.449, Supplementary Fig. 1g), indicating a putative sex-specific role of astrocytic OTR in the behavioral response to stress, which prompted us to focus on males. To test if the hormonal response to stress was similar between WT and GFAP OTR cKO mice, we quantified plasmatic corticosterone levels just after the stress protocol and the day before (to avoid any effect of the stress due to blood sampling). We observed a similar increase in plasmatic corticosterone levels in stressed versus na\u00efve animals in both WT and GFAP OTR cKO mice (p=0.677, Fig. 1c and Supplementary Fig. 1i), suggesting that the absence of freezing behavior was not due to an altered physiological response to stress. As observed by other studies24, females showed a higher corticosterone increase than males following the stress session (p=0.023, Supplementary Fig. 1j)

Importantly, we tested whether the absence of freezing of GFAP OTR cKO mice could stem from changes in their nociceptive sensitivity. We performed a comprehensive analysis of nociception and pain sensitivity in these animals and did not observe any significant differences in thermal cold (p=0.808), thermal heat (p=0.297) and mechanical (p=0.853) nociception, nor in spontaneous pain as tested with the conditioned place preference test compared to WT mice (p=0.577, Supplementary Fig. 1k, l). We next examined whether the absence of freezing could be explained by GFAP OTR cKO mice displaying depressive-like symptoms. This appears unlikely as GFAP OTR cKO and WT mice performed similarly in the sucrose preference test and in the direct social interaction test (Supplementary Fig. m-n).

Acute stress induces PVN OT neurons activation, CeA OT release, and astrocyte recruitment
Based on the behavioral characterization, astrocytes are involved in oxytocinergic modulation of stress-induced freezing in vivo. To decipher the underlying cellular mechanisms, we tested whether the acute stress paradigm activated PVN OT neurons and CeA astrocytes in vivo. To this end, we first recorded the calcium activity of OT neurons in the PVN in freely moving mice during the acute stress paradigm (Fig. 1d). To monitor individual neuronal calcium responses, we genetically targeted the calcium sensor GCaMP6f to OT neurons by breeding floxed GCaMP6f transgenic mice (line Ai148D) with OT-Cre mice25. Gradient-Index (GRIN) lenses were chronically implanted above the PVN for imaging of calcium activity using a micro-endoscope in freely-moving mice (Fig. 1d, Supplementary Fig. 2a\u2013c). Electric foot shocks induced calcium transients in 78% of the GCaMP6f-expressing OT neurons (n=67/86; Fig. 1e, f). Interestingly, PVN OT neurons also responded to stress induced by tail suspension (p=0.011) but not by cage shaking (p=0.750, Supplementary Fig. 2d, e). These findings established that the acute stress protocol reliably activated OT neurons in the hypothalamus.

Next, we investigated whether acute stress triggered OT release in the CeA, which is a well-know target region of PVN OT neurons7. To this end, we used CD1 mice, which have larger brains, and implanted microdialysis probes within the CeA region to measure the release of endogenous OT in freely moving mice (Fig. 1g). We found that stress induced by five randomly presented auditory tone-shock pairings induces an increase in CeA OT concentration (p=0.0162, Fig. 1h). This result showed that the foot shock-induced stress paradigm induces endogenous OT release in the CeA.

We then tested whether this acute stress paradigm induced a Ca2+ response in CeA astrocytes. To do so, we injected a rAAV carrying GCaMP6s under the control of the GFAP promoter (rAAV-GFAP-GCaMP6s) in the CeA and implanted optic fibers above the CeA to image Ca2+ activity in astrocyte population using fiber photometry in freely-moving mice (Fig. 1i). Similar to PVN OT neurons, electric foot shocks reliably increased Ca2+ transients in GCaMP6s-expressing astrocytes (p=0.010, Fig. 1j and Supplementary Fig. 2h). To test if this response can be observed in OTR-expressing astrocytes that represents ~25% of CeL/C astrocytes (Supplementary Fig. 1e)17, we aimed to express GCaMP in OTR-expressing astrocytes. To validate our targeting, we first injected a rAAV-GFAP-DIO-mScarlet in OTR-Cre mice (Supplementary Fig 2f, g). Following this verification, we injected OTR-Cre mice26 with a Cre-dependent AAV targeting astrocytes and expressing GCaMP6s. Following the electrical foot shock, the fluorescence significantly increased (p=0.0313, Fig. 1j and Supplementary Fig. 2h). Next, we aimed to test the involvement of OTR in this response. Given the non-ratiometric nature of the GCaMP6s, we opted for a longitudinal approach and achieved pharmacological inhibition of the OTR by injecting i.p. the antagonist L368 that cross the blood-brain barrier (Fig. 1k)27. We observed a significant reduction of the foot shock-induced Ca2+ response following L368, suggesting the involvement of the OTR in the stress-induced astrocyte Ca2+ signaling (Fig. 1l, Supplementary Fig. 2i, p=0.451, n=5, n=8). These results established that the acute stress paradigm activated OTR-expressing astrocytes and the general astrocyte population in the CeA.

Acute stress induces changes in the amygdala proteome
To gain insight into the molecular and cellular mechanisms induced by acute stress, we performed label-free quantitative mass spectrometry proteomics on amygdala samples snap frozen ~15 minutes following the acute stress protocol (Fig. 2a). Principal component analysis clearly separated na\u00efve from stressed mice (Fig. 2b), indicating an extensive stress-induced remodeling of the amygdala proteome. Two large clusters of proteins were either up- or down-regulated upon stress (Fig. 2c and Supplementary Fig. 3). Further analysis revealed that up- and down-regulated proteins are involved in axonal activity and morphological remodeling of astrocytes, indicating that stress modifies protein levels in CeA astrocytes. Indeed, profound expression changes were observed among 59 astrocyte proteins (Supplementary Fig. 3), showing, among others, an increase in GFAP cytoskeleton protein. Analysis of gene ontology (GO) terms and protein-protein interactions revealed multiple signaling components, pathways, transmitter receptors and ion channels, most of them related to synaptic activity (Fig. 2d). Gene set enrichment analysis (GSEA) indicated that differentially expressed proteins are involved in multiple metabolic and signaling processes, as well as in structural cellular components (Supplementary Fig. 3b-c). Among them, we found a significant change in intermediate filament proteins (Fig. 2e, f). Collectively, these data revealed that acute stress induces changes in astrocyte proteome, notably upregulation of intermediate filaments.

OTR signaling mediates acute stress-induced changes in astrocyte morphology and K+-dependent neuronal depolarization
We next investigated whether the acute stress-induced changes in proteins entail changes in astrocyte morphology. To achieve this, we injected a rAAV-GFAP-GFP in the CeL/C of na\u00efve and stressed mice. Using the IMARIS software, we performed three-dimensional reconstructions of GFP-expressing astrocytes from confocal images (Fig. 3a, b). We found that the acute stress protocol decreases the morphological complexity of astrocytes, which displayed reduced process ramification as measured by Sholl analysis (p<0.0001, Fig. 3b, c and Supplementary Fig. 4b). Notably, this morphological response was absent from GFAP OTR cKO mice (p=0.167, Fig. 3b, c and Supplementary Fig. 4b), suggesting an OTR-dependent mechanism. Stress-induced changes in morphology were accompanied by an OTR-dependent increase in GFAP expression in CeL/C astrocytes (p<0.001, Fig. 3d and Supplementary Fig. 4b\u2013d). Consistent with this result, the GFAP volume measured by immunofluorescence was inversely correlated to astrocyte process ramification (r\u00b2=0.569, p<0.0001, Fig. 3e and Supplementary Fig. 4b\u2013d). Together, these results revealed that acute stress concomitantly induces OTR-dependent upregulation of GFAP in astrocytes and a reduction of their morphologic complexity.

Given that astrocytic OTR are involved in acute stress-induced freezing (Fig. 1) and knowing that astrocytic regulation of neuronal excitability can depend on their morphology27,28, we first evaluated the spontaneous neuron activity by performing loose cell-attached recordings of CeL/C neurons (Fig. 3f). We observed an increased spontaneous firing frequency in neurons that come from stressed mice compared to their na\u00efve counterpart (p=0.028, Fig. 3g). To understand the origin of this increased activity of CeL/C neurons, we then characterized the excitability of CeL/C neurons in brain slices of naive and stressed mice using whole-cell patch-clamp recordings. We found that current injections induced similar voltage responses in neurons from WT na\u00efve and stressed mice (p=0.300, p=0.372, p=0.521, Fig. 3h and Supplementary Fig. 5a). Neuronal excitability also depends on extracellular K+ concentration, which is ~3 mM in resting conditions and could increase to 10-12 mM during neuronal activity and to even higher values in pathological conditions19. To mimic these conditions, we applied a K+ puff (30 mM) to induce depolarization of neuronal membrane potential (Fig. 3h, i and Supplementary Fig. 5b), and after verification that such application does not lead to cell death (Supplementary Fig. 5f), we observed higher depolarization in stressed mice compared to na\u00efve mice (p=0.0002). The kinetics of this depolarization was modified at the level of decay (p=0.086, p<0.0001, Fig. 3h and Supplementary Fig. 5b) and the K+ puff did not elicit higher spike frequency in neurons from stressed versus naive animals (p=0.094, Fig. 3h and Supplementary Fig. 5b). Importantly, we found that this stress-induced increase in K+-dependent neuronal depolarization was not significant in GFAP OTR cKO, as compared to naive mice (p=0.062, p=0.088, p=0.025, p=0,676, Fig. 3h, i and Supplementary Fig. 5b). To investigate whether acute stress modify extracellular K+ sensitivity in a defined subset of CeL/C neurons, we analyzed their electrophysiological features with a principal component analysis. However, we failed to identify any characteristics associated with the increased in extracellular K+ sensitivity observed after stress (Supplementary Fig. 5c, d). Furthermore, we demonstrated that under stress conditions, the various effects described above did not alter hyperpolarizing potassium currents following an action potential (IAHP) (Supplementary Fig. 5e). We further analyzed the timing of those changes and found that the stress-induced depolarization observed initially was no longer detectable after three days (Supplementary Fig. 5g). Taken together, the results revealed that OTR-dependent signaling in astrocytes mediates acute stress-induced morphologic changes in astrocytes and an increase of K+-dependent depolarization of CeL/C neurons.

Stress-induced changes in astroglial coverage of synapses and regulation of their efficacy within the amygdala CeL/C
The acute stress-induced changes in astrocyte morphology raised the question of whether this also affected the astrocytic coverage of synapses. This is of physiologic importance as such changes can influence circuit function29. To address this, we injected rAAV-GFAP-GFP in the CeL/C to label astrocytes. We then stained for vGluT1, vGluT2, and Homer1 to visualize pre- and postsynaptic elements of excitatory glutamatergic synapses (Fig. 4a, b and Supplementary Fig. 6a, b). Confocal microscopy revealed a significantly reduced number of synapses contacted by astrocytes from mice subjected to acute stress compared to those from na\u00efve mice (p=0.001, Fig. 4c). These results suggest that acute stress prompted the retraction of astrocytic processes away from synapses. Notably, this difference was not observed in GFAP OTR cKO mice, indicating that this change in synaptic coverage was mediated by astrocytic OTR (p=0.998, Fig. 4c). As a complementary method to visualize the coverage of synapses by astrocytic processes, we used transmission electron microscopy. In support of our observations by light microscopy, we observed a lower density of astrocytic processes surrounding synapses in CeL/C from stressed mice compared to na\u00efve ones (p<0.0001; Supplementary Fig. 6c). Together, these observations indicated that acute stress initiated an OTR-dependent retraction of astrocytic processes contacting synapses in the CeL/C.

We next tested whether the reduced coverage of synapses by astrocytic processes induced by acute stress affected their transmission properties. Previous studies suggested a major role of basolateral amygdala (BLA) to CeL/C projections in the adaptation to stress-induced freezing29,30. To optogenetically activate BLA neurons, we injected rAAV-CaMKII-ChR2-eYFP in the BLA (Fig. 4d). After visual verification of the BLA location of eYFP expression, we stimulated ChR2-expressing BLA neurons by light (5 ms pulse, 20 Hz during 5 s) and recorded the synaptic response of CeL/C neurons using patch-clamp recordings in acute brain slices of both na\u00efve and stressed animals (Fig. 4d and Supplementary Fig. 6d-g). Acute stress increased the overall amplitude of the evoked excitatory postsynaptic currents (eEPSCs) in CeL/C neurons as compared to na\u00efve mice (p=0.037, Fig. 4e, f), similarly to31. To test the involvement of OTR signaling in astrocytes, we performed the same recordings in na\u00efve and stressed GFAP OTR cKO mice. Under these conditions, the stress-induced increase in synaptic responses was abolished (p=0.948, Fig. 4e, f). Interestingly, the amplitude of EPSCs triggered by the two first photostimulations was not different between na\u00efve and stressed mice (Fig. 4e, Supplementary Fig. 6f, p=0.107). However, the BLA-evoked EPSCs amplitudes in the middle of the stimulation train was increased in stressed compared to na\u00efve mice (Fig. 4f, Supplementary Fig. 6f, p=0.031), indicating an incremental mechanism. This could arise from an accumulation of extracellular K+ with repeated stimulations. Crucially, we quantified synaptic parameters but did not observe any difference between na\u00efve and stressed animals of the same genotype (paired pulse ratio: p=0.3590, failure rate: p=0.4091, sEPSCs amplitude: p=0.7292, sEPSCs frequency: p=0.5835, eEPSCs rise time: p=0.3824, eEPSCs decay time: p=0.5280, Supplementary Fig. 6h). This indicates that direct synaptic plasticity is not responsible in the observed increased eEPSCs amplitude following stress exposure.

To test the implication of astrocytic K+ buffering in this phenomenon, we recorded K+ currents in CeL/C astrocytes following photostimulation of BLA neurons. We patch-clamped SR101-labelled astrocytes (Fig. 4g) and verified their astrocytic identity based on their electrophysiological response (IV curve, Supplementary Fig. 7a)32. To isolate the current due to K+ buffering, we subtracted from the current evoked by photostimulation of BLA neurons a second current recorded in the presence of Ba2+ (200 \u00b5M) to block K+ currents (Fig. 4g). This experiment revealed that the amplitudes of stimulation-induced K+ currents were significantly reduced in stressed mice compared with na\u00efve mice (p=0.030, Fig. 4h, i), indicating impaired K+ buffering in astrocytes. Since we cannot rule out a potential Ba2+ effect on neurons, we reproduced the experiment in GFAP OTR cKO mice. Crucially, the stimulation-induced K+ currents were not altered in GFAP OTR stressed mice compared with na\u00efve mice (p=0.159, Fig. 4h-i). A decrease in astrocyte K+ buffering capacity could arise from their process retraction but also from reduced expression of inwardly rectifying K 4.1 channels19,33. Thus, we extracted membrane proteins of CeA samples from stressed and na\u00efve mice and detected K 4.1 protein levels by western blot analysis (Fig. 4j and Supplementary Fig. 7b-c). We found that the acute stress paradigm reduced K 4.1 levels in CeA samples as compared with na\u00efve controls (Fig. 4j-l). These results suggested that acute stress induces a retraction of astrocytic processes from synapses and diminishes the K+ buffering capacity at the BLA-to-CeL/C synapses, thereby modifying the strength of this connection.

These findings indicate that acute stress initiates an OTR-dependent retraction of perisynaptic astrocytic processes, leading to a diminished K+ buffering capacity at the BLA-to-CeL/C synapses, enabling adequate behavioral stress response.

Identification of G proteins mediating OTR-dependent effects in astrocytes
We next aimed to identify the signaling pathways downstream of OTR underlying changes in Ca2+ transients in astrocytes after acute stress. Previous proteomic analysis suggested that acute stress modifies GPCR and cAMP signaling pathways (Fig. 2d, Supplementary Fig. 3c and 8a). Thus, we studied the subtype of G-proteins activated by OTR in astrocytes using bioluminescence resonance energy transfer (BRET)-based biosensors34 (Fig. 5a). We found that upon OT binding, OTR couple to both G\u03b1i and G\u03b1q in cultured astrocytes (Fig. 5b and Supplementary Fig. 8b). We next identified the G proteins coupling OTR to Ca2+ transients in CeL/C astrocytes by combining OGB1-based calcium imaging and pharmacological manipulations (Fig. 5c). We applied biased OTR agonists that favor either G\u03b1i (atosiban) or G\u03b1q (carbetocin) pathways or a full agonist ([Thr4,Gly7]-oxytocin, TGOT) that activates both pathways34,35. We then measured calcium transients in SR101-positive astrocytes in brain slices in the presence of TTX to block action potential-dependent neuronal activity (Fig. 5c). All three agonists increased the frequency and AUC of Ca2+ transients compared to baseline activity (frequency: p=0.024, p=0.001, p=0.003; AUC: p=0.002, p=0.0007, p=0.0008, Fig. 5d and Supplementary Fig. 8c). Similar proportions of cells responded to each agonists (~50%, Supplementary Fig. 8c). The G\u03b1q protein blocker YM254890 (500nM, 30min pre-incubation) prevented the carbetocin- and TGOT-induced calcium signaling (Carbetocin: Ca2+ transient frequency: p=0.296, TGOT: Ca2+ transient frequency: p=0.561, Supplementary Fig. 8d). The G\u03b1i protein blocker pertussistoxin (PeTX, 5 \u00b5g/mL, 5h pre-incubation) abolished calcium activity induced by atosiban (Ca2+ transient frequency: p=0.316, Supplementary Fig. 8e), while the response to TGOT remained unaffected (Ca2+ transient frequency: p=0.006, Supplementary Fig. 8e). In order to characterize the Ca2+ transients induced by OTR-G\u03b1i and OTR-G\u03b1q pathways, we compared the parameters of calcium event triggered by different G protein activation. We found that the rise constant and amplitude of Ca2+ transients induced by atosiban were lower as compared to those induced by carbetocin (p=0.016, p=0.013, Fig. 5e, f). Taken together, these pharmacological data indicated that activation of G\u03b1i and G\u03b1q proteins triggers distinct Ca2+ transient signatures in astrocytes.

OTR-G\u03b1i signaling mediates CeL/C astrocytes morphological reorganization and controls neurons sensitivity to extracellular K+
Given the involvement of OTR-G\u03b1i in CeL/C astrocytes Ca2+ activity, we sought to elucidate the potential role of OTR-G\u03b1i-mediated Ca2+ transients in CeL/C astrocytes. To do so, we incubated brain slices with different OTR agonists for 1h and assessed astrocyte GFAP levels by histology (Fig. 6a). While the full (TGOT) and G\u03b1i-biased (atosiban) agonists increased CeL/C astrocytes GFAP volume (p=0.007, p<0.001), the G\u03b1q-biased agonist (carbetocin) did not (p=0.404) (Fig. 6b). This suggested an involvement of G\u03b1i, but not G\u03b1q, in the modification of the GFAP cytoskeleton. Similarly, TGOT increased the K+-induced depolarization of CeL/C neurons (p=0.003), an effect prevented by the co-incubation of TGOT with the OTR antagonist dOVT (p>0.999) or by genetic deletion of OTR (GFAP OTR cKO, p>0.999) (Fig. 6c-d and Supplementary Fig. 9a\u2013c). These results indicate that astrocytic OTR-G\u03b1i mediate the OT-induced hypersensitivity of CeL/C neurons to extracellular K+. Similarly, atosiban but not carbetocin increased the K+-evoked depolarization of CeL/C neurons (p=0.022, p>0.999; Fig. 6d and Supplementary Fig. 9b, c).

Collectively, these findings suggest that the OTR-G\u03b1i pathway in CeL/C astrocytes recapitulate features of acute stress-induced morphological alterations in astrocytes and adjacent neurons sensitivity to extracellular K+ (Fig. 6e).

Methods
Animals
All experiments were conducted in accordance with European Union rules and approbation from the French Ministry of Research (01597.05). For ex vivo and in vivo experiments, male and female C57BL/6j mice were used except CD-1 mice (Charles Rivers, 022) for microdialysis. For ex vivo experiments animals were between 1 and 3 months old. To target GCaMP6f to OT neurons in the PVN, Ai148 mice (Jackson Laboratory, 030328) were crossbred with OXT-Cre (Jackson Laboratory, 024234). Animals were housed under standard conditions with food and water available ad libitum and maintained on a 12h light/dark cycle.

Viral transduction following stereotaxic injections
Animals were deeply anesthetized with 4% isoflurane, and their heads were fixed in a KOPF (model 955) stereotaxic frame. Metacam (Meloxicam, 20 mg/kg) was injected subcutaneously to limit inflammation and local analgesics were injected locally at the incision site (Bupivaca\u00efne, 5 mg/kg, and Luroca\u00efne, 2 mg/kg). The skull was then exposed, and two holes were drilled according to coordinates adapted from Paxinos and Watson brain Atlas. For injections in the CeL/C, we injected 200 nL of the appropriate rAAV at the following coordinates: rostrocaudal= -1.25 mm, mediolateral= \u00b13.1 mm, dorsoventral= -5.0 mm. To stimulate BLA neurons, we injected 100 nL of AAV9-CaMKIIa-hChR2(H134)-EYFP at the following coordinates: rostrocaudal= -1.8 mm, mediolateral= \u00b13.4 mm, dorsoventral= -5.0 mm. The wound was then sutured and the weight of the animals was followed for five days to verify the post-operatory recuperation. After ~3 weeks, mice were used for experiments.

Behavior analysis
Stress protocol. The day before the stress protocol, animals were introduced into the stress box with a smooth white plastic floor and walls for ten minutes for habituation and internal control. Mice were exposed five times to a 20 s-long CS tone (2 kHz, 60 dB) with a variable inter-tone interval (20 to 180 seconds). The next day, stripe patterns were placed on the walls of the box and the smooth white plastic floor was removed, revealing the grid floor of the box. Animals were introduced into the stress box for 10 min and exposed to five 20-second-long sound stimuli with different inter-tone time intervals. The last second of these tones coincided with a 1 s-long foot shock of 0.6 mA. Freezing behavior, speed and distance traveled was quantified using the ANY-maze software. Freezing was defined as periods longer than 2 seconds during which mice did not move in a time window of 20 s following the foot shock. For subsequent ex vivo experiments, mice were injected with anesthetics directly after the end of the stress protocol and slices were performed in the following 10 to 20 minutes.

Sucrose preference test. Cages were equipped with two feeding bottles, one with sucrose 1% and the other drinking water. At 24 h after the beginning of the experiment, we reversed the position of the bottles to avoid a potential place preference. Bottles were weighted before the experiment, 24 h and 48 h after. We then calculated the sucrose preference according to the following equation:

preference = (sucrose intake / total intake) * 100 (1)

Conditioned place preference. In this test, animals develop a preference to a clonidine-paired chamber due to both pain relief in this environment and avoidance for the saline-paired chamber associated with ongoing pain. The apparatus (42553 Conditioned Place Preference for Mice, Ugo Basile, Gemonio, Italy) consists of 2 Plexiglas chambers separated by manually operated doors. Two chambers (size 16x25x25 cm) distinguished by the texture of the floor and by the wall patterns. On the first, second and third days (pre-conditioning), animals were free to explore the apparatus during 30 min, and the time spent in each chamber was recorded using ANY-maze\u2122 software. Animals spending more than 75% or less than 25% of the total time in one chamber were excluded from the study. On the 4th day (conditioning) animals were intrathecally injected with either saline solution (10 \u00b5L, morning) or clonidine solution (10 \u00b5g in 10 \u00b5L in the afternoon) and restricted in one chamber for 15 min, switching chamber between the morning and the afternoon. On the 5th day the animals were free to explore the 3 chambers and the time spent in each chamber was recorded for 30.

Von Frey test. The mechanical threshold of hind paw withdrawal was evaluated using von Frey hairs (Aniphy Vivo-Tech, Salon-de-Provence, France). Mice were placed in clear Plexiglas\u00ae boxes on an elevated mesh screen. After a habituation time of around 10 min, the filaments were pressed on the plantar surface of each hind paw in a series of ascending forces (0.6 to 8 grams). Each filament was applied five times per paw, until it just bent, and the threshold was defined as three or more withdrawals observed out of the 5 trials. The mean of sensitivity of the two hind paws was calculated.

Hargreaves test. The latency for hind paw withdrawal in response to thermal stimulation was determined using the Hargreaves method. Mice were placed in clear Plexiglas\u00ae boxes and testing began after exploration and grooming behaviors ended (15 min). The infrared beam of the radiant heat source (37570 Plantar Test, Ugo Basile, Gemonio, Italy) was applied to the plantar surface of each hind paw. The experimental cut-off to prevent damage to the skin was set at 15 s. Three measures of the paw withdrawal latency were obtained per animal and averaged for each hind paw.

Dry ice test. Hind paw withdrawal latency in response to cold thermal stimulation was determined using the dry ice method. Mice were placed in clear Plexiglas\u00ae boxes. After a 15 min habituation period, a cut 2 mL syringe filled with dry ice powder was placed against the glass surface under the animal\u2019s paws. This source of cold under the animal\u2019s plantar surface caused the animal\u2019s paw to retract. Latency times were recorded manually using a stopwatch. Three measurements of paw withdrawal latency (spaced at least 15 min apart) are then taken and averaged for each hind paw.

Social interaction test. Social interactions were assessed in the cage of the same size of the home cage, with a bit of dust at the bottom. For this test, interactions between a test subject and a stimulus mouse were systematically scored and videotaped for 5 min. Light intensity was 150 lux. All cages were bring in the test room for acclimatization of 30 min. Then, the stimulus and test mice were introduced in the same time in a clean cage for 5 min. The stimulus mice were from the same background C57BL6, same sex, same age and used maximum twice a day. Their weight was on average 7% less compared to test mice. Order of testing was counterbalanced according to genotype and stimulus mice. The times the test subject spent performing social behaviors (i.e. facial sniffing (oral-to oral contact), anogenital sniffing (oral-to-base sniffing), direct touching with a paw and pursuit of the stimulus mouse) were determined in real time with an ethological keyboard (ANY-maze software). After testing, each mouse was returned to its home cage and a new clean cage was used for the next mice. The percent of time spent engaged in social behavior compared to total time (5 min) was calculated for the measure of social interactions:

% total interaction = 100 * (facial sniffing time + anogenital sniffing time + direct touching time + pursuit time) / 300 s (2)

Corticosterone measurements in blood
Sampling. Blood sample (40 \u00b5l) were collected from tail tips following a scissor incision using a calibrated sodium-heparinized capillary (40 \u00b5l, Hirschmann\u2122 Disposable Capillary Tubes, Fisher Scientific, Illkirch, France). The blood was then supplemented with 10 \u00b5l of heparin (843 \u00b5M; Sigma-Aldrich, St. Quentin Fallavier, France). Subsequently, 10 \u00b5l of D4-CORT (5 \u00b5M, Sigma-Aldrich) in 99.1% H2O/0.1% formic acid (v/v; Fisher Scientific) was added to the mixture. Proteins were precipitated acetonitrile (ACN 100%, Fisher Scientific) and samples were centrifuged at 20,000 g (4\u00b0C, 30 min). The supernatant was collected and dried under vacuum (SpeedVac, Thermo Fisher). The dried samples were then resuspended in 20 \u00b5l of 20% ACN/0.1% formic acid (v/v). The supernatants was collected and stored at -80\u00b0C.

Dosage. Corticosterone measurements were conducted using a Dionex Ultimate 3000 HPLC system (Thermo Electron, Villebon-sur-Yvette, France) coupled to a triple quadrupole Endura mass spectrometer (Thermo Electron). The samples were loaded onto a heated Hypersil aQ column (100x1 mm, 1.9 \u00b5m, with a flow rate of 90 \u00b5l/min; Thermo Electron) at 40\u00b0C. Qualification and quantification were performed using multiple reaction monitoring (MRM) based on the isotopic dilution method. Compound identification was achieved by comparing the precursor ions, selective fragment ions, and retention times with those of the isotopically labeled standards (IS). The selection of monitored transitions, along with the optimization of collision energy and RF lens parameters, was automated (Supplementary Table \u201cHPLC and MSMS details\u201d). System control was driven by Xcalibur v4.0 software (Thermo Electron).

Microdialysis
Male CD1 mice (25-30 g; 7-8 weeks old; Charles River, Germany) were used due to their larger size. Animals were stereotaxically implanted with a U-shaped microdialysis probe (molecular cut-off: 18 kDa; length of membrane 1.5 mm) under isoflurane anaesthesia (Isoflurane Baxter, Baxter Deutschland GmbH, Germany) targeting the right CeA (AP: -1.0; lateral +2.4; DV: -5.0 mm from bregma; Paxinos and Watson, 1998). After a 48 h post-surgery recovery, the microdialysis probe was first perfused without sampling for 90 min (3.3 \u00b5l/min, Ringer\u2019s solution, adjusted to pH 7.4), before 3 consecutive 30 min microdialysates from the CeA were collected (1) under basal conditions, (2) during exposure to 5 random foot-shocks (0.6 mA, 1 s) over a 10 min period in a conditioning chamber (45x22x40 cm, transparent Perspex box with a steel grid floor), and (3) again in the home cage. Samples were kept at -20\u00b0C until radioimmunological analysis of OXT (detection limit: 0.5 pg/sample, intra-assay variability of <8% cross-reactivity with other neuropeptides including arginine vasopressin <0.7%). Brains were frozen and the perfusion sites were histologically verified on 40 \u00b5m cryo-cut stained brain slices.

In vivo imaging
In vivo micro-endoscope calcium imaging. To visualize calcium level in PVN OT neurons, Ai148:OXT-Cre mice were stereotactically implanted with a chronic GRIN lens (0.5 mm in diameter; 7.4 mm in length, Inscopix) \u00b1200 \u00b5m above the PVN at the following coordinates: rostrocaudal= -0.85 mm, mediolateral= +1.1 mm, dorsoventral= -4.9 mm with a 10-degree angle (right hemisphere). The lens was securely fixed to the skull with a three-component dental cement (C&B-Metabond, Parkell Inc.). After a recovery period of at least two weeks, animals were implanted with a baseplate to attach the micro-endoscope (nVista3, Inscopix). A tethered dummy micro-endoscope was connected to the baseplate for habituation to the recording procedures. Following an additional week for recovery and habituation, the real miniature microscope was attached to the baseplate one day before the experiment. Imaging data were collected at 10 Hz, with excitation light power 0.8-1.2 mW/mm\u00b2 (40-60% of maximal power, nVista3, Inscopix). Stacks of calcium images were first preprocessed to correct time-invariant pixels or reconstruct frames lost during the acquisition, and were then adjusted for motion artifacts in the x-y plans using Inscopix Data Processing software 1.9.1 (Inscopix). Subsequently, image stacks were exported to ImageJ, where the recording background was subtracted. Regions of interest (ROI) corresponding to individual neurons were manually defined over z-stacked images, and the raw average fluorescence from each ROI was quantified and exported using Fiji software. Raw traces are further processed and normalize using custom-made scripts (Matlab, MathWorks). Due to the potential bleaching of the GCaMP6f fluorescence within the session, an exponential curve was fitted and subtracted from the raw signal of each cell during each session to ensuring consistency in the fluorescence measurements. Subsequently, F0 was determined using Gaussian mixture modeling, where the observed intensity values from each cell in each session were approximated as a mixture of Gaussian distributions via an expectation-maximization procedure. The mean of the distribution containing the lowest values was identified as the F0 for each neuron and session. \u0394F/F0 are calculated base on bleaching-corrected calcium traces and the estimated F0, and then are normalized using envelope normalization. For control experiments, each mouse was subjected to a randomized protocol that included multiple instances (2\u20136 each) of tail suspension (lifting by the experimenter) and cage shaking, with intervals of 10\u201360 seconds. Baseline and post-event quantification windows were manually defined for each animal.

Fiber photometry. 3-4 weeks after viral injection, fiber-optic cannula (fibercore, 400 \u00b5m; 0.37 NA; 5 or 5.5 mm length; Doric Lenses, Quebec City, Canada) was implanted approximately 0.1 mm above the injection site and fixed to the skull with dental cement and Super-Bond C&B. After 5-7 days recovery, mice were handled by the experimenter for 3-5 days and acclimated to the fiber-optic patch cord connection for 5-7 additional days. Fluorescence intensity was measured with a dual-color fiber photometry setup equipped with two LED light sources, 465 and 560 nm (Doric Lenses). The light intensity at the end of fiber-optic patch cord was adjusted to between 50 and 80 \u00b5W. Data were acquired by lock-in mode in Doric Neuroscience Studio. Acquired data were processed by custom-made python codes. After data denoising with a Savitzky-Golay filter, photobleaching was corrected for by a computed control channel generated by fitting with an exponential curve with the corresponding signal. Normalized \u0394F (\u0394F %) was calculated by the equation:

\u0394F/F = (Fraw(t) - Fcontrol(t)) / Fcontrol(t) (3)

where Fraw is the Ca2+-dependent fluorescence changes and Fcontrol is the value in control channel computed for minimizing the photobleaching effect. For experiments using OTR antagonist, L368-899 (10 mg/kg) was injected 30 minutes before the stress session. Animals with inconsistent basal fluorescence between days were removed from the analysis.

Proteomic analysis
Tissue sample preparation. Mice brain were sliced as described for ex vivo electrophysiology. Using a 1 mm-diameter punch, the CeA was dissected bilaterally on two 400 \u00b5m slices per hemisphere (4 punches per mouse), snap-frozen and stored at -80\u00b0C. Anesthesia, slicing and punch sampling procedure took 10 to 20 minutes following the stress protocol. Isolated amygdala tissues from na\u00efve and stressed mice (n=5 per group) were disrupted using a lysis solution containing 1% Sodium-dodecyl sulfate, 50 mM Tris (pH 8.0), and a protease inhibitor cocktail (Merck) without EDTA. The tissues were subjected to sonication in AFA-tube TPX strips (Covaris) using a Covaris LE220R-Plus instrument (300 s, average power of 162.5, peak power of 325, 50% duty factor, 200 cycles per burst). Tolerance of the dithering settings were set at \u00b1 5 mm in the x and z axis, and 4.5 mm in the y axis, while operating at a velocity of 20 mm/s. Protein concentrations of the sonicated lysates were measured using a BCA assay (Pierce). For mass spectrometer analysis, protein digestion and peptide generation were performed using 1 \u00b5g of protein per condition. Due to the limited amount of initial material, we utilized the automated version of the SP3 protocol for protein digestion and peptide purification. The Bravo system is designed to handle 96 samples at once, performing all necessary steps such as protein reduction and alkylation, magnetic bead aliquoting, protein clean-up using SP3, protein digestion, and peptide recovery.

LC-MS/MS. The quantification of tryptic peptides from the whole proteome was performed using an EASY-nLC 1200 system (Thermo Fischer Scientific) connected to a Fusion mass spectrometer (Thermo Fischer Scientific). Peptides were separated by reverse-phase liquid chromatography employing 0.1% formic acid (solvent A) and 80% acetonitrile (solvent B) as the mobile phases. The separation of peptides was performed using an Acclaim PepMap trap column (Thermo Fisher Scientific, C18, 20 mm \u00d7 100 \u00b5m, 5 \u00b5m C18 particles, 100 \u00c5 pore size) and a nanoEase M/Z peptide BEH C18 analytical column (Waters, 250 mm \u00d7 75 \u00b5m 1/PK, 130 \u00c5, 1.7 \u00b5m). The gradient started with a 3% concentration of solvent B for the first 4 min, thereafter increasing to 8% and further rising to 10% after 6 min. The concentration of solvent B increased to 32% after 68 min and to 50% after 86 min. Between the 87th and 94th minute of the gradient, the concentration of solvent B reached 100%. The system was re-equilibrated after 95 min using 3% of solvent B for 10 min. The eluted peptides were subjected to ionization and introduced into the mass spectrometer using the Nanospray flex ion source (Thermo Fischer Scientific) and a Sharp Singularity nESI emitter (ID=20 \u00b5m, OD=365 \u00b5m, L=7 cm, \u03b1=7.5\u00b0) (FOSSILIONTECH), which was connected to a SIMPLELINK UNO-32 (FOSSILIONTECH). A constant spray voltage of 2.5 kV was applied to the emitter, and the capillary temperature of the ion transfer tube was set to 275\u00b0C. The Fusion mass spectrometer was used in data-independent (DIA) mode with a scan range of 400-1000 m/z, an Orbitrap resolution of 60000 FWHM, an AGC target of 3e6, and a maximum injection duration of 20 ms. The acquisition of data-independent MS/MS spectra was performed at a resolution of 30000 FWHM. The maximum injection duration was set to 50 ms and the AGC target was set to 1e6. The acquisition strategy included employing 26 isolation windows, each having a width of 23.3 m/z and overlapping by 1 Th. The collision energy used for fragmenting the precursor ions was set to 27, and a fixed initial mass of 200 m/z was used to get the MSMS spectra.

Data analysis. Raw data obtained from DIA measurements were analyzed using DIA-NN version 1.8.0. A spectral library was developed based on the predictions made by the fasta file. In addition, a fasta file containing commonly seen protein contaminant was included for the purpose of predicting the spectral library. The spectral library prediction used the default values. The default parameters of DIA-NN were used for the processing of the raw files. The precursor matrix output tables were filtered to include only values with a false discovery rate <0.01. Additionally, values were filtered based on a channel q-value <0.01 and a translated q-value <0.01. MBR feature was activated to enhance data integrity. Output tables generated by DIA-NN were then analyzed with R-Studio, using the dplyr package for data wrangling, and ggplot2 for visualisation. Differential expression and statistical analyses were carried out using the limma package. Gene Set Enrichment Analysis (GSEA) was conducted with the fgsea package, heat maps were created using the pheatmap package and Perseus software as explained thereafter.

Statistical analysis. GSEA was performed using the R package fgsea, which implements a fast preranked GSEA algorithm. Proteins were ranked according to the (log2 fold change) from the differential abundance analysis. For each protein set, fgsea computes an enrichment score using a Kolmogorov-Smirnov-like running-sum statistic on the ranked list. To assess significance, fgsea generates a null distribution of enrichment scores by repeated permutation of the protein-level statistics and recalculation of the enrichment score for each protein set. The nominal p-value is defined empirically as the proportion of null enrichment scores that are at least as extreme as the observed enrichment score for that protein set. Multiple testing correction across all tested protein sets was performed using the Benjamini-Hochberg procedure. The resulting FDR-adjusted p-values are reported in the Fig. 2e, f and Source Data Table 2.

Immunofluorecence staining and image analysis
Tissue fixation. Mice were anesthetized using ketamine (Ketamine 1000, 400 mg/kg) and xylazine (Paxman, 80 mg/kg) administered intraperitoneally. 10 to 20 minutes following stress protocol, mice were then perfused transcardially with PBS, followed by 4% PFA. Brains were dissected and post-fixed overnight in 4% PFA at 4\u00b0C.

Astrocyte morphology assessment. 50 \u00b5m vibratome sections were cut, collected, and stored in PBS 1X overnight at 4\u00b0C. Slices were then incubated in a PBS 1X-Gelatin 0.02%-Triton 0.25% solution for permeabilization and saturation of non-specific sites during 1 h at RT. Incubation of the first antibodies took place overnight at 4\u00b0C and slices were then washed and incubated with secondary antibodies overnight at 4\u00b0C. Finally, slices were mounted in an Abberior antifade medium and left for one day at 4\u00b0C in the dark for polymerization. All antibodies used and their concentrations are given in the reagent table (Supplementary Table \u201cList of reagents\u201d). Z-stack images of the CeL were captured (6-8 images per animal) with a depth of 50 \u00b5m and 1 \u00b5m intervals, employing a 40x magnification on the Nikon AX confocal system (Nikon Imaging Center, Heidelberg). All images used for analysis were taken with the same confocal settings (pinhole, laser intensity, digital gain, and digital offset). Imaris software (version 10.0, Oxford Instruments) was used for reconstruction and morphological analysis of astrocytes. CeL/C segmentation was performed by superimposing the putative CeL/C delimitation onto the corresponding CeL/C-containing brain images (Paxinos Mouse Brain Atlas) to achieve the best fitting overlay. Raw confocal files were imported into Imaris and subjected to surface reconstruction, involving background subtraction and setting surface detail to 0.5 \u00b5m with the largest diameter at 0.5 \u00b5m. Immunofluorescent signals from GFAP staining or virally-expressed eGFP were utilized as templates for surface and filament reconstructions. Astrocytes falling below a volume of <250 \u00b5m3 or exceeding >5500 \u00b5m3 were excluded from data analysis. A separate masked channel was generated for filament analysis, employing automated threshold detection with maximum seed point placements. Manual intervention ensured each astrocyte possessed precisely one starting point at the soma center. The efficiency of viruses were calculated as the number of cells co-expressing virus-dependent of GFP (or mScarlet) and astrocyte markers over the number of cells expressing astrocyte markers. The specificity were calculated as the number of cells co-expressing virus-dependent of GFP (or mScarlet) and astrocyte markers over the number of cells expressing GFP (or mScarlet). Morphological analysis involved calculating average values for each animal (surface area, volume, etc.). Sholl analysis was conducted using Imaris in the filament reconstruction mode. Each data point on the respective Sholl plots corresponds to the mean Sholl intersections, accounting for the average of the respective cohort with error bars indicating \u00b1 SEM, calculated across all astrocytes per animal. Identification of synapses in contact with astrocytes was performed after immunohistochemical amplification of virally expressed GFP in astrocytes, and staining of pre- and postsynaptic markers vGlut1/2 and Homer1. High magnification confocal images were acquired, which were then processed into our Imaris-assisted pipeline. The background was subtracted (surface detail at 0.2 \u00b5m, largest diameter at 0.2 \u00b5m), and debris measuring <0.1 \u00b5m3 was disregarded as nonspecific and filtered out in a subsequent step. vGlut1/2 and Homer1 puncta were reconstructed from high magnification confocal images. These putative synapses were kept for analysis if they followed the following criteria: 1) close apposition of pre- and postsynaptic components based on vGlut1/2 and Homer1 colocalisation, 2) volume of putative synapses must be at least 0.1 \u00b5m3, and 3) must be in a 50 nm-range of the astrocyte surface.

Ex vivo recordings
Slice preparations. Mice were anesthetized using ketamine (Ketamine 1000, 400 mg/kg) and xylazine (Paxman, 80 mg/kg) administered intraperitoneally. Intracardiac perfusion was then performed using an ice-cold NMDG-based aCSF containing (in mM): NMDG (93), KCl (2.5), NaH2PO4 (1.25), NaHCO3 (30), MgSO4 (10), CaCl2 (0.5), HEPES (20), D-glucose (25), L-ascorbic acid (5), thiourea (2), sodium pyruvate (3), N-acetyl-L-cysteine (10) and kynurenic acid (2). This solution was bubbled in 95% O2/5%CO2 gas all the time of the experiment and its pH was adjusted to 7.3-7.4 using HCl. After decapitation, the brain was swiftly removed in the same ice-cold dissection aCSFs and hemisectionned. 350 \u00b5m-thick horizontal slices containing the CeA were obtained using a Leica VT1000S vibratome. Slices were then stored in 37\u00b0C NMDG aCSF for 10 min before placing them in the holding chamber at room temperature containing normal aCSFs for 1 h minimum before any experiments were conducted. Normal aCSF used for all ex vivo experiments was composed of (in mM): NaCl (124), KCl (2.5), NaHPO4 (1.25), NaHCO3 (26), MgSO4 (2), CaCl2 (2), D-glucose (15), adjusted for pH values of 7.3-7.4 with HCl or NaOH and continuously bubbled in 95% O2/5%CO2 gas. Osmolality was set to 300\u00b110 mOsm/L. For pharmacologic experiments, slices were incubated in aCSF supplemented with 500 nM of agonist and/or antagonist for 1 h. Slices were then transferred to an immersion-recording chamber superfused at 2 ml/min with aCSF. The anesthesia and slicing procedure took 10 to 20 minutes.

Electrophysiology. Whole-cell patch-clamp recordings of CeL/C neurons were done using infrared oblique light visualization. For astrocyte recordings, slices were incubated in 1 \u00b5M SR101 at 37\u00b0C for 20 min and washed for 1 h. Subsequent patch-clamp recordings from astrocytes were guided by SR101 fluorescence. Borosilicate glass electrodes (OD 1.5 mm, ID 0.86 mm; Sutter Instrument) were pulled using a horizontal flaming/brown micropipette puller (P97; Sutter Instrument) to obtain 3.5-7 M\u03a9 recording pipettes. These pipettes were filled with an intracellular solution containing (in mM): K-Glu (125), HEPES (20), NaCl (10), and ATPNa2 (3). The pH was adjusted to 7.3-7.4 with KOH, and osmolality was adjusted at 300\u00b110 mOsm/L with water. Recordings were acquired with an Axon MultiClamp 700B Amplifier coupled to a Digidata 1440A Digitizer (Molecular Devices) at 20 kHz using either the pClamp 10 software suite for puff experiments or using WinWCP 4.2.2 freeware (John Dempster, SIPBS, University of Strathclyde, UK) for photostimulation experiments. Series resistance was monitored and manually compensated (~50% typically). Spontaneous action potential firing was measured over 10-minute-long recordings. At the beginning of each recording, current pulses ranging from -10 pA to 100 pA with 10 pA steps were applied for 300 ms per sweep to neurons or voltage pulses ranging from -100 mV to 100 mV with 20 mV steps lasting 100 ms for astrocytes. For neurons, the membrane potential drift was estimated with the Savitsky-Golay polynomial local filter from the scipy library and subtracted from the membrane potential. Spikes were then detected with the find_peaks function of the Scipy library for each sweep as peaks with a height and a prominence higher than 10 mV. The latency of spikes was quantified as the time between the beginning of the first step that triggers a spike and the first spike. The number of spikes was measured in the second step, which triggers action potentials. Spike hyperpolarizations were measured on the first step that evoked an action potential. This protocol was repeated three times, and the median results were used for further analysis. For astrocytes, voltage-current curves were constructed by measuring the current needed to reach the command voltage during the last 25 milliseconds of the voltage step. For K+ puff application, the injected current was adapted to hold the neuronal membrane at -60 mV. Basal activity of neurons was recorded for 20 s, and then a 20 s-long puff of a 30 mM KCl-containing aCSF was done at ~100 \u00b5m of the recorded neuron. The membrane potential drift was estimated after a stringent Gaussian filter from the pyABF library, thereby avoiding over-estimation of this value due to action potentials. This drift was then subtracted from the original traces, and spikes were detected with the find_peaks function of the Scipy library as peaks with a height and a prominence higher than 20 mV. Spikes and membrane potential were measured in the 20 s baseline, the 20 s K+ puff application, and the 20 s washout. The sum of spikes and the median of membrane potential were computed over these three periods. For depolarization kinetics, linear functions were fitted to the first five seconds of the rising/decay phase of the K+-induced depolarization on the Gaussian-filtered trace. This protocol was repeated three times, and the median values of measures were used for statistical analysis. For IAHP recordings, CeL/C neurons membrane potential were clamped at -20 mV, depolarized at +20 mV for 100 milliseconds, and clamped again at -20 mV for 50 seconds. We reproduced this protocol 5 times for each neuron. The AUC of each IAHP were measured as the integral from its maximum peak after the end of stimulation until its membrane potential return to baseline. The decay was measured as the time necessary for the current to decrease by 63%. For optostimulation of BLA neurons, the neuronal membrane potential was held in voltage clamp at -60 mV. Basal activity was recorded for 20 s of basal activity before stimulating glutamatergic BLA neurons with 5 ms blue light pulses at 20 Hz for 10 s. For each recording, EPSC were detected with the find_peaks function of the Scipy library as peaks higher than three times the standard deviation estimated during the 20 s baseline. Frequency and amplitude of these EPSC were measured in 5 seconds-long quantification windows taken just before the photostimulation, during the five first seconds of the photostimulation, and at the end of the recording. After each light pulse, we estimated a failure as the absence of evoked EPSC, or we estimated the rise and decay of the evoked EPSC. Rise and decay constants were measured on exponential and linear fits as the time needed to go from 10 to 90% or from 90 to 10% of the EPSC amplitude, respectively. This recording was repeated three times and the medial value of the three recordings was used for statistical analysis. The paired pulse ratio were calculated as the ratio between the second over the first EPSC evoked by the photostimulation of BLA axon terminals. For K+ currents recordings, astrocytes were patch-clamped at -80 mV and basal membrane current was recorded during 20 s before stimulating glutamatergic BLA neurons with 5 ms blue light pulses at 20 Hz for 10 s. Subsequently, recordings were performed in the presence of Ba2+ to block K currents and BLA neurons were stimulated again after 15 min. Evoked currents were estimated as the minimum value during the photostimulation subtracted from the minimum value during baseline. The K+ current was calculated by subtraction of currents evoked in the presence of Ba2+.

Quantification of cell mortality. A 20 s-long puff of aCSF or a 30 mM KCl-containing aCSF was done at ~100 \u00b5m of the recorded neuron in brain slices from naive and stress mice. Slices were then washed 5 min with PBS then incubated for 10 min with blue trypan (4%), washed again in PBS for 5 min 3 times, subjected to chemical fixation overnight in 4% PFA at 4\u00b0C and observed on by confocal microscopy.

Ex vivo calcium imaging. After SR101 incubation, the synthetic calcium indicator OGB1-AM was bulk loaded as previously described55, reaching final concentrations of 0.0025% (20 \u00b5M) for OGB1-AM, 0.002% for Cremophor EL, 0.01% for Pluronic F-127 and 0.5% for DMSO in aCSF and incubated for 1 h at 37\u00b0C Slices were then washed in aCSF for at least 1 h before imaging. Only astrocytes co-labeled for SR101 and OGB1 were used. The spinning disk confocal microscope for calcium imaging was composed of a Zeiss Axio examiner microscope with a \u00d740 water immersion objective (numerical aperture of 1.0), mounted with an X-Light Confocal Unit \u2013 CREST OPT spinning disk. Images were acquired at 2 Hz with an optiMOS sCMOS camera (Qimaging). Cells within a confocal plane were illuminated for 20 ms at 575 nm for SR101 and 80 ms at 475 nm for OGB1 using a Spectra 7 LUMENCOR. The different hardware elements were synchronized through the MetaFluor 7.8.8.0 software (Molecular Devices). Because astrocytes are mechanosensitive, OTR agonists (500 nM) were bath applied for 20 s and not puff-applied to avoid mechanical stimulation. All calcium imaging experiments were conducted at room temperature and cells with an unstable baseline were discarded.

All analyses were conducted as described55. Astrocytic calcium levels were measured in manually outlined regions of interest (ROI) comprising the cell body using ImageJ software. Subsequent offline data analysis was performed using a custom-written Python-based script. To take into account slice micro-movements, the SR101 fluorescence values were subtracted from the OGB1 ones. Then, a linear regression was applied to each trace to correct for photobleaching. Calcium transients were detected using the find_peaks function of the SciPy library as fluorescence variation exceeding 8 times the standard deviation and a prominence exceeding it 5 times (for more details, see55). The number of peaks and the area under the curve was quantified before and after the drug application. All data were normalized according to the duration of the recording, and astrocytes were labeled as \u2018responsive\u2019 when their AUC or their calcium transient frequency at least doubled after drug application. Because the time after stimulation is longer than the baseline (10 min vs. 5 min), the probability of observing a spontaneous calcium peak is stronger after stimulation. To avoid this bias, astrocytes with only one calcium peak during the whole recording were not considered responsive. Each calcium transients were then isolated, their area were estimated with the trapezoid method and their duration were measured as their half-maximum full width (HMFW). The rise and decay phases of Ca2+ transients were fitted with linear functions and the coefficient of these slopes was used as rise and decay constants. To compare effects of different agonists (Figure S6), evoked Ca2+ activity was normalized to the baseline values as ratio (baseline/drug effect).

BRET experiments in astrocytes
Purifed astrocytes were isolated from P2 C57BL6 mice whole brains by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) with anti-GLAST (ACSA1; (Jungblut et al., 2012)) MicroBeads according to the manufacturer\u2019s instructions. Cells were plated on poly-L-lysine-coated (Sigma Aldrich, St. Louis, MO, USA) T75 flasks in complete medium, composed by minimal essential medium (MEM, Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco, Life Technologies, Carlsbad, CA, USA) and glucose (5.5 g/L, Sigma Aldrich, St. Louis, MO, USA). Cells were grown in a humidified incubator at 37\u00b0C and 5% CO2. Two days before the experiment, astrocytes were harvested with trypsin 0,25% (Gibco, Life Technologies, Carlsbad, CA, USA) and plated in 12-well plates in complete medium at a density to ensure ~70% confluent cultures at 24 h after seeding. Typically, 80,000 cells/cm2 surface area of the culture dish were seeded in cell-specific medium. Astrocytes were transfected using Lipofectamine 3000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer\u2019s protocol with a DNA to Lipofectamine ratio of 1:3 w/v. A transfection enhancer, the 3000 enhancer reagent (1:2, DNA:Reagent, w/v), was used along with the Lipofectamine 3000 transfection reagent. 1.25 \u00b5g of plasmids DNA were transferred to each well of the 12-well plates. The cDNA amounts used per well were as following: G\u03b1x Rluc8 (300 ng), GFP10-G\u03b3 (250 ng), G\u03b2 (300 ng) and OTR (400 ng).

BRET experiments were performed 72 h after transfection. Astrocytes were detached with trypsin 0,25% and resuspended in Krebs-Ringer\u2019s-HEPES (KRH) buffer containing (in mM) NaCl (125), KCl (5), MgSO4 (1.2), KH2PO4 (1.2), CaCl2 (2), glucose (6) and HEPES-NaOH (25), pH 7.4.

For specific validation experiments, BRET biosensors were expressed in HEK293 cells. To this end, cells were co-transfected using plasmids encoding G\u03b1q-97-Rluc8 or G\u03b1i2-91-Rluc8 and GFP10-G\u03b32, G\u03b21, and the OTR. Two days after transfection, cells were washed twice, detached, and resuspended with PBS with 0.5 mM MgCl2 at room temperature. Cells were distributed on 96-well microplates (100 \u00b5g of proteins/well) (Optiplate, PerkinElmer Life Sciences) and incubated at room temperature for 5 min in the presence or absence of YM (60 nM) and two minutes with PBS or OT (10 \u00b5M).

BRET measurements were performed as previously described using the Infinite F500 multimode microplate reader (Tecan)34. The signals emitted by the Rluc8 donor that allows the sequential integration of light signals was detected with two filter settings (Rluc8 filter, 370-450 nm; GFP10 filter, 510-540 nm). The data were recorded by Infinite F500 after the addition of 5 \u00b5M DeepBlueC coelenterazine and the BRET signal was calculated as the ratio between GFP10 emission and the light emitted by Rluc8 after 2 min stimulations with maximal dose of OT (1 \u00b5M) or vehicle (PBS). All measurements were performed at 37\u00b0C. The BRET signal was calculated as the ratio between GFP10 emission and the light emitted by Rluc8. The changes in BRET induced by OT were expressed on graphs as \u201cOT-induced changes in BRET ratio\u201d using the following formula:

BRET ratio = (emission GFP10 OT / emission Rluc8 OT) - (emission GFP10 PBS / emission Rluc8 PBS) (4)

Tissue preparation, protein extraction and western blotting
Ten to twenty minutes following the end of the acute stress protocol, brains from stressed or na\u00efve (home cage controls) mice were sliced as described for ex vivo electrophysiology. Using a 1 mm-diameter punch, the CeA was dissected bilaterally on two 400 \u00b5m slices per hemisphere (4 punches per mouse), snap-frozen and stored at -80\u00b0C until processing. To enrich membrane protein fraction protein extraction was performed using the Mem-PERTM Plus Membrane Protein Extraction Kit (Thermo scientific) according to the manufacturer\u2019s instructions. Briefly, after rinsing, CeA punches were transferred into grinders (Wheaton; Dounce tissue grinder 1 mL, #357538) with 500 \u00b5L of permeabilization buffer containing protease (1:100, Protease inhibitor cocktail, Sigma-Aldrich, #P8340) and phosphatase (1:100, Phosphatase inhibitor cocktail 2, Sigma-Aldrich, #P5726) inhibitors and grinded until even suspension. Homogenates were transferred in 2 mL tubes, incubated for 10 min at 4\u00b0C under constant rotation followed by centrifugation at 16,000 g for 15 min at 4\u00b0C. After removal of the supernatant containing cytosolic proteins, the pellet was resuspended in 500 \u00b5L of solubilization buffer with protease and phosphatase inhibitors. Samples were incubated for 30 min at 4\u00b0C under constant rotation, centrifuged at 16,000 g for 15 min at 4\u00b0C and the supernatant containing membrane proteins was collected and stored at -80\u00b0C. Because of very low amounts of input tissue, we used a protein concentration method. Membrane protein samples were incubated with 77% trichloroacetic acid on ice for 30 min and centrifuged at 16,000 g for 15 min at 4\u00b0C. After supernatant removal, pellets were rinsed with ice cold acetone and centrifuged at 16,000 g for 5 min at 4\u00b0C. After supernatant removal, pellets were resuspended in loading buffer (NuPAGE\u00ae LDS sample buffer and sample reducing agent, Invitrogen) and denaturated 10 min at 70\u00b0C. Samples were loaded on 4-20% Criterion\u2122 TGX Stain-Free Precast Gels (Bio-rad) and migration was performed 1 h at 170 V in Tris-glycine buffer (Bio-rad) followed by Stain-Free gel activation using a ChemiDoc MP Imaging System (Bio-rad). Proteins were transferred on a nitrocellulose membrane with the Trans-Blot Turbo\u2122 Transfer System (Bio-Rad). Total protein load per well was evaluated after imaging the membrane under UV light. After 3x10 min rinses in 9 mM NaCl Tris-HCl Buffer + 0.1% Tween 20 (TBST), membranes were blocked in TBST + 5% milk for 1 h at room temperature (RT) and incubated overnight at 4\u00b0C sequentially with the following primary antibodies: anti-K 4.1 (1:1000, Rabbit; Alomone, #APC-035), anti-actin or anti-Tom20 in TBST + 5% milk. After 3x10 min washed in TBST, membranes were incubated for 1 h at RT with Horse Radish Peroxidase (HRP)-conjugated secondary antibodies (1:5000, Vector laboratories) diluted in TBST + 5% milk. Membranes were incubated with Clarity Western ECL substrate (Bio-rad) and the signal was detected with ChemiDoc MP Imaging System. Band intensity was quantified with Image Lab Version 5.2.1 and normalized to total loaded protein load. In-between signal detections, peroxidase activity was inactivated using a 20 min incubation of membranes in 1% Na-Azide in TBST at RT followed by 3 x 10 min wash in TBST. When necessary, antibodies were stripped using a 20 min incubation in stripping solution (ReBlot Plus Mild Antibody Stripping Solution, Millipore). Western blotting was performed with Biorad Criterion Stain Free gels, which allow to detect all proteins on the membrane via a reaction of the trial compound with tryptophan residues and thus do not require to normalize the protein concentration per well prior to protein migration.

CeL/C ultrastructural analysis
Mice were anesthetized with intraperitoneal injection of 100 mg/kg ketamine chlorhydrate and 5 mg/kg xylazine and transcardially perfused with glutaraldehyde (2.5% in 0.1 M PBS at pH 7.4). 1 mm amygdala punches were immersed in the same fixative overnight, rinsed several times in phosphate buffer, and postfixed in 1% osmium for 1 hour. Finally, samples were dehydrated in graded ethanol series and embedded in Embed812 (EMS). Ultrathin sections were cut with an ultramicrotome (Leica), stained with uranyl acetate (1% (w/v) in 50% ethanol), and examined by transmission electron microscopy (TEM; Hitachi H7500 equipped wuthan AMT Hamamatsu digital camera). The areas for image acquisition by TEM were chosen randomly (50 images per side per animal) and the density of synapses was identical in both na\u00efve and acute stress conditions (3.47\u00b10.08 vs 3.62\u00b10.08, respectively).

Statistical analysis
Except for the principal component analysis that was performed using the scikit python library, all statistical tests were performed using GraphPad Prism version 8.0.0 (GraphPad Software). Parametrical tests were performed if data showed normal distribution and equality of group variances. Otherwise, non-parametric tests were used. All values, group compositions, and statistical tests for each experiment are detailed in Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The paper by Baudon et al describes a study of stress-induced retraction of astrocytic processes from around glutamate synapses in the central amygdala that is mediated by oxytocin receptor and Gai activation and that leads to potentiation of excitatory synaptic transmission between the BLA and the central lateral amygdala. This astrocytic OTR activation in astrocytes is largely responsible for the freezing response to repeated foot shock presentations. They show that the synaptic potentiation following glial retraction is mediated by reduced K+ uptake and increased [K+] around excitatory synapses. This study takes a multi-pronged, multi-disciplinary approach that creates a compelling case for oxytocin’s role in astrocytic structural remodeling that contributes to synaptic potentiation during associative learning. While the study is impressive in its breadth and depth, I nevertheless have a few concerns with respect to the data and its interpretations.\nThis is a concern that has been raised repeatedly. The spread of calcium signal to nearly all the astrocytes from a 20% subset of OTR-expressing astrocytes via gap junction coupling is not very compelling, even with the additional evidence provided. Based on the gap junction figure provided from Wahis et al., 2021, the reductions in the responses following Cx knockout and carbenexolone blockade are partial and the residual responses occur in significantly more than the 20% of astrocytes that express OTR. The calcium response to OT agonist in the OTR-expressing astrocytes is very weak and it’s hard to imagine that it is sufficient to spread from the 20% of OTR+ astrocytes to the other 80% of OTR- astrocytes. If the hypothesis is true, then repeating the experiment performed in Fig. 1 to measure calcium changes in all CeL astrocytes in response to foot shock in the astrocyte Cx KO mouse should show a significant reduction, if not complete loss, of the calcium response.\nIf the auditory tones presented in Fig. 1e were also presented in the behavioral freezing experiment shown in Fig. 1a and b, this needs to be stipulated in the figure or text. I was surprised to see that a conditioning stimulus, and not just the foot shock stress stimulus, was included since testing associative memory formation was not a part of the plan. Perhaps a justification should be added to provide a rationale for applying an associative learning paradigm rather than just the foot shock stressor alone.\nIt should be stated in the text that the Microdialysis experiments were done in CD1 mice. Was there a reason for switching mouse strains? Related, how constrained is the spatial resolution of the Microdialysis probe? Can it distinguish between CeA and, for example, BLA?\nThe increase in GCaMP fluorescence in OTR-expressing astrocytes in response to foot shock was weak and did not reach statistical significance (p = 0.055). It appears nonetheless that the increase was consistent in each of the 4 mice tested, which suggests that this would reach statistical significance if powered more. I appreciate the technical difficulty of this experiment and that the authors have added an additional replicate since their original submission, but the small magnitude of the response, the low number of replicates, and the failure to reach statistical significance call for additional powering of the experiment.\nThe reduction in the Ca response after OTR antagonist application did not reach statistical significance (note p = 0.0519 and not 0.519), suggesting that this experiment was also underpowered. That said, the AUC data presented in the Ext Fig were significant and could perhaps be presented in the regular figure. The question arose, however, as to why the experiment wasn’t performed in OTR KO mice to confirm the OTR dependence of the astrocyte Ca response. This would provide an OTR dependence in the CeL astrocytes more directly than the IP injection of OTR antagonist.\nLines 201-202: “the K+ puff did not elicit higher spike frequency in neurons from stressed versus naive animals (pnumber of spikes=0.094).” Most of the neurons recorded showed no change in the spike frequency in hi K - were they silent at baseline and did not respond to K or were they spontaneously active and did not change their frequency in K? This is important because more depolarization may be necessary to drive silent neurons to threshold than to increase firing frequency in spontaneously firing neurons. If the non-responding neurons were not counted, or counted separately as failures, then there would likely be a statistically significant difference in the K-induced change in AP freq.\nAnother measure of excitability that is not accounted for in Fig. 3 is membrane resistance, which should be compared between groups. A stress-induced change in membrane resistance could explain why a bigger K-induced depolarization does not lead to a higher AP frequency in high K, despite the higher AP frequency at baseline in the stressed group. There are Rinput data presented in the Ext Figure, but they should be presented along with the other changes in intrinsic properties.\nThe AP response to Puff K (>50 Hz) in Ext Fig. 5 is not representative of the responses included in the analysis.\nHow were contacts between astrocytes and synapses measured? It is not clear from the photomicrograph in Fig. 4b and the Methods description how it was determined that astrocyte processes were in contact with synaptic profiles. Similarly, the electron micrograph in Ext Fig. 6b depicts total number of putative astrocyte processes within a region without distinguishing their proximity to synapses. How was it determined from this measure that there was a lower density of astrocytic processes surrounding synapses? In order to make this determination, juxtaposition of astrocytic processes with synaptic profiles should be assessed rather than density of astrocytic profiles.\nIt is not clear why changes in cAMP-associated proteins are presented in Ext Fig. 8a, since Gs is not one of the G proteins implicated in OTR signaling.\nGiven their variability, it is hard to see how the Ca transients presented in Fig. 5d can be analyzed for changes in waveform.\nSince the Ca response to OT in OTR-expressing astrocytes was very weak and because LTP-induced astrocytic restructuring is independent of Ca signaling (Henneberger et al., 2020), is it possible that the stress-induced morphological changes in astrocytes seen here are mediated by a Ca-independent mechanism?", "reviewer_2": "The authors have addressed most of my previous comments, and the dataset is generally strong. However, two critical concerns remain unaddressed. These unresolved points substantially limit the interpretability of the study.\nFirst, the issue of AAV-mediated targeting specificity. This is raised by three of four reviewers - was not addressed directly. Extended Data Fig. 1 is insufficient, as S100β can also label oligodendrocyte precursor cells. This is a central issue in the field: without proper validation, the core findings based on cKO manipulations remain unconvincing. The GFAP promoter is known to drive off-target expression in GFAP-positive stem/progenitor lineages. Better validated astrocyte-targeting strategies are available (PMID: 32042146; 37973910) and should be considered. As previously noted, functional astrocyte studies demand rigorous, case-by-case validation—particularly when major conclusions rely on cell-type specificity.\nSecond, the authors state that no suitable cAMP sensor is available. However, a highly sensitive sensor (PMID: 38514778) was publicly available as a preprint before their submission and is now peer-reviewed and published. It has the dynamic range required, has been validated in vivo in astrocytes, and would enable direct measurement of Gi/Gs signaling in ex vivo as well, especially solving a problem derived from the non-cell specific nature of pharmacology.", "reviewer_3": "The authors have conducted a number of additional experiments and undertaken substantial revisions that address at least several of the critiques raised by the reviewers. While this reviewer retains some reservations regarding the conceptual novelty and the causal underpinnings of the findings, multiple specific lines of investigation presented in the manuscript open promising avenues that could significantly enhance our understanding of brain machinery.\nIn this context, I suggest that the authors discuss two recently raised issues that bear directly on their arguments. First, whether relatively rapid fear extinction, which is as critical as fear acquisition (https://doi.org/10.1073/pnas.2501331122), might reverse the observed changes. Second, how their interpretation is influenced by evidence that seems to link mechanistically Kir4.1 expression levels to the regulation of local circuit excitability (https://doi.org/10.1016/j.celrep.2025.115299).", "reviewer_4": "While I appreciate the authors’ efforts to address the reviewers’ concerns, several key issues remain unresolved. In many cases, the responses are either insufficient or unconvincing, and numerous clarifications rely on circular reasoning that does not adequately support the conclusions drawn. Furthermore, although the number of animals used in specific experiments was increased from 3 to 4, this remains insufficient to support statistically robust conclusions. The statistical analyses also appear more opaque in the revised version rather than clarified.\nBelow, I provide specific comments:\n- Oxytocin signaling and astrocyte activation (Page 4): The authors report that ~20% of CeL/C astrocytes express OTR and highlight modulation of the oxytocin signaling pathway in their proteomic dataset after stress. However, this is difficult to reconcile with the data shown in Figure 1j, where approximately 80% of astrocytes show robust calcium activation following acute stress. The authors have not adequately explained what alternative signaling pathways might underlie this widespread activation in OTR-negative astrocytes. If relevant data are available in the proteomic dataset, they should be clearly analyzed and discussed.\n- Validation of viral targeting and functional consequences: Although the authors provide new data regarding the efficiency and specificity of rAAV-GFAP-Cre targeting, the validation remains insufficient. It is based on only two mice and 12 fields of view, which raises concerns about representativeness. Moreover, an independent analysis of the images suggests a targeting efficiency closer to ~40%, which could significantly affect the interpretation of the results. Crucially, the authors did not address a previously raised point: whether there is a linear correlation between the number of OTR-deleted astrocytes and the severity of the freezing behavior. This analysis is essential to support a causal relationship between the extent of astrocytic deletion and behavioral effects.\n- Astrocytic calcium activity – lack of single-cell resolution: The authors justify not performing single-cell calcium analyses in astrocytes due to technical limitations related to astrocyte morphology and the depth of the CeL/C. While these difficulties are acknowledged, the concern remains unaddressed. Since neuronal activity was analyzed at the single-cell level, failing to apply the same resolution to astrocytes limits interpretability and prevents meaningful comparisons. Sparse GCaMP expression, although more technically demanding, could be achieved using alternative viral serotypes, concentration or promoter strategies. No such attempts were made, and no alternative solutions were proposed.\n- Use of 30 mM extracellular K⁺ and lack of mechanistic insight into potassium signaling: The authors acknowledge that 30 mM K⁺ is a supraphysiological concentration and cite studies suggesting such levels may occur transiently in restricted microdomains. However, they provide no data to confirm that this concentration does not induce cellular stress or toxicity in their model. I previously suggested measuring K⁺-dependent currents (e.g., IAHP) to clarify the underlying mechanisms. Instead, the authors report a non-significant trend (p = 0.0824) in afterhyperpolarization and proceed to amplify the effect by applying exogenous K⁺. This approach does not clarify the physiological relevance of the observations and leaves core mechanistic questions unanswered.\n- Use of ChR2 and lack of complementary stimulation approaches: The authors argue that ChR2-mediated stimulation of the BLA→CeL/C pathway offers a more physiological alternative to exogenous K⁺ application by eliciting endogenous K⁺ release. While this rationale is understandable, ChR2 is known to produce non-physiological synchrony and transient surges in extracellular K⁺ (Octeau et al., 2019). I had suggested employing complementary approaches such as chemogenetics or electrical stimulation to validate the findings. The authors dismissed these options due to their limitations, but they did not attempt any such control experiments. As a result, their conclusions remain heavily reliant on a single technique, which weakens the robustness of the study.\n- Corticosterone levels and sex differences: I appreciate that the authors included corticosterone data from wild-type females and acknowledge that baseline levels were higher than in males. Their ethical justification for not extending this analysis to OTR KO females is understandable. However, given the reported sex differences in behavior, the potential contribution of sex-specific OTR expression or signaling remains an important and unresolved issue that warrants further discussion.\nDespite the revisions and clarifications provided, the manuscript still presents several conceptual and methodological limitations that undermine the strength of its conclusions. Key issues, including insufficient sample size, limited physiological relevance, lack of mechanistic depth, and incomplete methodological validation, remain unresolved."}, "author_response": "We thank the reviewers for dedicating their time and effort to evaluate our manuscript. We are grateful for their thoughtful and constructive feedback, which we address point by point below. We hope that our responses will resolve their concerns and support the publication of this study. For ease of reference, all responses and newly added data in the revised manuscript are typed in blue.\nReviewer #1:\nThe paper by Baudon et al describes a study of stress-induced retraction of astrocytic processes from around glutamate synapses in the central amygdala that is mediated by oxytocin receptor and Gai activation and that leads to potentiation of excitatory synaptic transmission between the BLA and the central lateral amygdala. This astrocytic OTR activation in astrocytes is largely responsible for the freezing response to repeated foot shock presentations. They show that the synaptic potentiation following glial retraction is mediated by reduced K+ uptake and increased [K+] around excitatory synapses. This study takes a multi-pronged, multi-disciplinary approach that creates a compelling case for oxytocin’s role in astrocytic structural remodeling that contributes to synaptic potentiation during associative learning. While the study is impressive in its breadth and depth, I nevertheless have a few concerns with respect to the data and its interpretations.\nWe appreciate the reviewer's positive assessment of our manuscript. As outlined below, we have responded to the different points. We have performed additional experiments, reanalysed data, and modified the manuscript. The changes have greatly improved our manuscript.\nThis is a concern that has been raised repeatedly. The spread of calcium signal to nearly all the astrocytes from a 20% subset of OTR-expressing astrocytes via gap junction coupling is not very compelling, even with the additional evidence provided. Based on the gap junction figure provided from Wahis et al., 2021, the reductions in the responses following Cx knockout and carbenexolone blockade are partial and the residual responses occur in significantly more than the 20% of astrocytes that express OTR. The calcium response to OT agonist in the OTR-expressing astrocytes is very weak and it’s hard to imagine that it is sufficient to spread from the 20% of OTR+ astrocytes to the other 80% of OTR- astrocytes. If the hypothesis is true, then repeating the experiment performed in Fig. 1 to measure calcium changes in all CeL astrocytes in response to foot shock in the astrocyte Cx KO mouse should show a significant reduction, if not complete loss, of the calcium response.\nWe agree with the reviewer that elucidating the mechanism of communication between astrocytes is an important point. In our previous publication, we showed that ~20% of astrocytes were positive for OTR mRNA (Wahis et al., 2021). In the revised extended data figure 1, we reproduced this quantification and observed OTR mRNA in ~25% of CeL/C astrocytes (Extended data Figure 1e).\n[...]\nConcerning our ex vivo Ca2+ imaging experiments, we observed that 54% of astrocytes respond to OT (Figure 5 and Extended data Figure 8b), equivalent to the percentage found in our previous manuscript (Wahis et al., 2021 Figure 2 and 3), indicating that a subpopulation but not all astrocytes respond to OT. For in vivo experiments however, it is important to note that we do not know the proportion of astrocytes showing an increased Ca2+ signaling during acute stress since we do not have a single-cell resolution.\nAs mentioned by the reviewer, our past study indicated that 40% of astrocytes still responded to OT in mice lacking Cx30 and Cx43, and 34% still responded in the presence of the gap junctions blocker carbenexolone. These observations suggest that connexin-dependent and -independent mechanism mediate OT-induced Ca2+ signaling in astrocytes (Wahis et al., 2021, Figure 3b-c). Therefore, replicating our calcium imaging experiments in Cx KO mice would mainly confirm our previous findings (Wahis et al., 2021). Even if a reduced signal were observed, this outcome would only reproduce published results without providing new mechanistic insight. Nonetheless, we take the reviewer’s comment seriously and agree that this question warrants deeper investigation in a future manuscript.\nIf the auditory tones presented in Fig. 1e were also presented in the behavioral freezing experiment shown in Fig. 1a and b, this needs to be stipulated in the figure or text. I was surprised to see that a conditioning stimulus, and not just the foot shock stress stimulus, was included since testing associative memory formation was not a part of the plan. Perhaps a justification should be added to provide a rationale for applying an associative learning paradigm rather than just the foot shock stressor alone.\nWe thank the reviewer for pointing at this topic and we have modified corresponding statements in the manuscript. Given the numerous reports on oxytocin effects during fear and anxiety behaviour, we originally intended to assess the involvement of oxytocin in long-term memory, and in fear extinction. Nevertheless, when we tried to train GFAP OTR cKO mice for fear learning, we observed a complete absence of freezing during the conditioning stage. For this reason, we decided to investigate the absence of the freezing immediate reaction - a.k.a acute stress. Based on reviewer’s comment, we now mention the sound stimuli in the Results, Discussion, and Material/Method (Stress protocol).\n[...]\nIt should be stated in the text that the Microdialysis experiments were done in CD1 mice. Was there a reason for switching mouse strains? Related, how constrained is the spatial resolution of the Microdialysis probe? Can it distinguish between CeA and, for example, BLA?\nThe reviewer is right. Therefore, we now explain better our use of the CD1 strain. CD1 mice are larger compared to C57BL/6 mice, which facilitates anatomical differentiation during histological processing. In particular, their larger brain volume permits to target adjacent brain regions more efficiently. Given the physical dimensions of the microdialysis probe used in our study (0.45 mm in width × 1.5 mm in length), we can better target the CeA in CD1 compared to C57BL/6 mice. Admittedly, the central (CeA) and basolateral (BLA) amygdala cannot be reliably distinguished. Nevertheless, since almost no oxytocinergic fibers have been found in the BLA of rats or mice (Knobloch et al., 2012, Son et al., 2022 – see panels thereafter), we expect that most of the OT detected by the microdialysis probe was released in the CeA.\n[...]\nThe increase in GCaMP fluorescence in OTR-expressing astrocytes in response to foot shock was weak and did not reach statistical significance (p = 0.055). It appears nonetheless that the increase was consistent in each of the 4 mice tested, which suggests that this would reach statistical significance if powered more. I appreciate the technical difficulty of this experiment and that the authors have added an additional replicate since their original submission, but the small magnitude of the response, the low number of replicates, and the failure to reach statistical significance call for additional powering of the experiment.\nWe thank the reviewer for understanding the complexity of this experiment. We have performed additional experiments to address the concerns. We fully agree to the importance of sufficiently powered samples. Therefore, we have increased the number of animals. The reinforced results show a constant and significant increase of calcium signaling in OTR-expressing astrocytes after an electric shock. The data are shown in figure 1j for the traces. The quantitative analyses are in the extended data figure 2h. As predicted by the referee, the changes do reach statistical significance at the 0.05 level:\n[...]\nThe reduction in the Ca response after OTR antagonist application did not reach statistical significance (note p = 0.0519 and not 0.519), suggesting that this experiment was also underpowered. That said, the AUC data presented in the Ext Fig were significant and could perhaps be presented in the regular figure. The question arose, however, as to why the experiment wasn’t performed in OTR KO mice to confirm the OTR dependence of the astrocyte Ca response. This would provide an OTR dependence in the CeL astrocytes more directly than the IP injection of OTR antagonist.\nWe thank the referee for the comments and fully agree to the importance of sufficient sample size. We have performed additional experiments to increase the number of recordings in antagonist condition (n=8). Clearly, injection of an OTR antagonist reduces significantly the astrocytic calcium response in the central lateral amygdala. The data are presented in Figure 1l and extended data figure 2i:\n[...]\nRegarding the referee's second comment concerning \"use of OTR cKO mice to confirm this result\", we agree that deletion of the OTR in astrocytes to observe a potential decrease in calcium response would be a nice complementary experiment. However, the non-ratiometric nature of the GCaMP indicator precludes stringent comparison between different animals. To obtain longitudinal data, we expressed the CreERT2 in astrocytes (using GFAP-CreERT2 viruses) of OTR fl/fl mice and measured stress-mediated calcium transients before and after tamoxifen-induced deletion of astrocytic OTRs. To do so, we administered 4-hydroxytamoxifen (4-OHT, 30 mg/kg) i.p. for two days following the first FC trial to induce OTR ablation. The second FC trial was conducted 10 days after the first.\nUnfortunately, the expression of Cre recombinase did not allow for efficient OTR deletion, and we were unable to obtain convincing data. The inability of CreERT2 to eliminate astrocytic OTRs may be due to the fact that 1) CreERT2 is transiently and not constitutively active, and 2) both construct (GFAP-GCaMP and GFAP-CreERT2) are under the control of same promoter whose transcriptional activity may not be high enough to trigger strong expressing of both constructs.\nRegardless the failure of this experimental design, the highly complementary approaches including behavioral analysis of the OTR cKO model (Figure 1a-b), calcium imaging of OTR expressing astrocytes (Figure 1j), and pharmacological blockade of OTR activity (Figure 1k-l) clearly indicate that OTR expressing astrocytes actively participate in the behavioral response to an acute stressor.\nLines 201-202: “the K+ puff did not elicit higher spike frequency in neurons from stressed versus naive animals (pnumber of spikes=0.094).” Most of the neurons recorded showed no change in the spike frequency in hi K - were they silent at baseline and did not respond to K or were they spontaneously active and did not change their frequency in K? This is important because more depolarization may be necessary to drive silent neurons to threshold than to increase firing frequency in spontaneously firing neurons. If the non-responding neurons were not counted, or counted separately as failures, then there would likely be a statistically significant difference in the K-induced change in AP freq.\nWe appreciate the reviewer’s comment which prompted us to reconsider the relevant readouts of these experiments and to re-analyse our data. Thanks to referee's comment we have realized that the spike frequency or number is not the sole relevant readout. Almost all neurons we recorded were silent during the baseline. When we applied the K+ puff, approximately half of them showed action potentials, and among those, some neurons rapidly adapted and returned to a non-firing state (see example thereafter). Thus, we believe that the lack of difference in spike number between naïve and stressed animals is likely not due to insufficient depolarization, but rather reflects an adaptation or a ceiling effect that pushes the neuron beyond its physiological action range.\nUpon reviewer’s comment, we examined the proportion of neurons firing action potentials during K puff in each condition (see bar graph below). We observed an increase in the percentage of neurons exhibiting K -evoked action potentials, further suggesting that stress enhances the excitability of CeL/C neurons. We include this analysis in the Extended Data Figure 5b.\n[...]\nAnother measure of excitability that is not accounted for in Fig. 3 is membrane resistance, which should be compared between groups. A stress-induced change in membrane resistance could explain why a bigger K-induced depolarization does not lead to a higher AP frequency in high K, despite the higher AP frequency at baseline in the stressed group. There are Rinput data presented in the Ext Figure, but they should be presented along with the other changes in intrinsic properties.\nWe thank the reviewer for this comment and we followed the suggestion to include these data. Comparing membrane resistances of neurons from naïve and stressed mice revealed no difference (p = 0,4374). We have inserted this graph in the extended data figure 5d.\n[...]\nThe AP response to Puff K (>50 Hz) in Ext Fig. 5 is not representative of the responses included in the analysis.\nWe thank the reviewer for pointing this out. Accordingly, we have replaced the example trace.\n[...]\nHow were contacts between astrocytes and synapses measured? It is not clear from the photomicrograph in Fig. 4b and the Methods description how it was determined that astrocyte processes were in contact with synaptic profiles.\nThe referee's comment prompted us to add a corresponding explanation in the manuscript.\n[...]\nSimilarly, the electron micrograph in Ext Fig. 6b depicts total number of putative astrocyte processes within a region without distinguishing their proximity to synapses. How was it determined from this measure that there was a lower density of astrocytic processes surrounding synapses? In order to make this determination, juxtaposition of astrocytic processes with synaptic profiles should be assessed rather than density of astrocytic profiles.\nWe fully agree with the referee. Indeed, the number of astrocytes per se does not indicate a relation to synapses. Therefore, we have reanalyzed all micrographs (100 electron micrographs per animal, 2 animals per experimental condition) and counted the number of synapses per micrograph. We now show the ratio (number of astrocytic processes per number of synapses) in each image in addition to the previously shown number of astrocytes processes a proximity to synapses (extended data figure 6c).\n[...]\nIt is not clear why changes in cAMP-associated proteins are presented in Ext Fig. 8a, since Gs is not one of the G proteins implicated in OTR signaling.\nThe reviewer is right, no previous studies have shown coupling of OTR to Gs-dependent signaling. However, in addition to its well-described activation of Gq proteins, numerous studies showed that OTR also activates Gi proteins (Hoare et al., 1999, Zhou et al., 2007, Busnelli et al., 2012). Given the ability of these proteins to inhibit the adenylate cyclase, and to decrease the quantity of cytosolic cAMP (Sadana and Dessauer, 2008), we thought to investigate whether activation of the OTR could modify the expression of cAMP-related genes.\nGiven their variability, it is hard to see how the Ca transients presented in Fig. 5d can be analyzed for changes in waveform.\nWe agree with the reviewer that the Ca2+ transients observed after OT application vary in size, frequency and shape. These stochastic features of astrocytic Ca2+ signals have been observed by many scientists investigating Ca2+ transients in astroglial cells (Volterra et al., 2014, Gorzo and Gordon, 2022, Semyanov et al., 2020, Covelo et al., 2022). To extract meaningful signals, we thus recorded responses from a large number of cells to take into account this high variability. From our point of view, it is more meaningful to analyse population-level activity rather than a cell-specific response.\nSince the Ca response to OT in OTR-expressing astrocytes was very weak and because LTP-induced astrocytic restructuring is independent of Ca signaling (Henneberger et al., 2020), is it possible that the stress-induced morphological changes in astrocytes seen here are mediated by a Ca-independent mechanism?\nWe agree with the referee that Ca-independent mechanisms contribute to the stress-induced morphological changes in astrocytes. We now mention this possibility in the discussion:\n[...]\nReviewer #2:\nThe authors have addressed most of my previous comments, and the dataset is generally strong. However, two critical concerns remain unaddressed. These unresolved points substantially limit the interpretability of the study.\nFirst, the issue of AAV-mediated targeting specificity. This is raised by three of four reviewers - was not addressed directly. Extended Data Fig. 1 is insufficient, as S100β can also label oligodendrocyte precursor cells. This is a central issue in the field: without proper validation, the core findings based on cKO manipulations remain unconvincing. The GFAP promoter is known to drive off-target expression in GFAP-positive stem/progenitor lineages. Better validated astrocyte-targeting strategies are available (PMID: 32042146; 37973910) and should be considered. As previously noted, functional astrocyte studies demand rigorous, case-by-case validation—particularly when major conclusions rely on cell-type specificity.\nWe thank the reviewer for this insightful input. Indeed GFAP is expressed in non-astrocytic cells, especially in neurogenic niches. We injected the GFAP promoter-containing virus in adult mice central amygdala, where no neurogenesis have been reported to date. To address the referees' important comment, we have performed additional experiments to further validate the efficacy and specificity of our viral tools and genetic mouse models. We have:\n1) Validated viral GFP expression and correct astrocyte targeting with the widely accepted astrocyte markers glutamine synthetase and Aldh1l1\n2) Injected OTR-Cre mice with pGFAP-DIO-mScarlet virus and revealed Glutamine Synthetase to validate the astrocyte specificity of mScarlet expression,\n3) Validated our astrocyte-specific OTR ablation strategy via multiplexed RNAscope,\n4) Illustrated the injection sites and the viral spread.\n[...]\nSecond, the authors state that no suitable cAMP sensor is available. However, a highly sensitive sensor (PMID: 38514778) was publicly available as a preprint before their submission and is now peer-reviewed and published. It has the dynamic range required, has been validated in vivo in astrocytes, and would enable direct measurement of Gi/Gs signaling in ex vivo as well, especially solving a problem derived from the non-cell specific nature of pharmacology.\nWe agree with the reviewer that a direct measure of a Gi-mediated drop of cAMP levels would be neat. However, to date, no study - including those by researchers who developed fluorescent cAMP probes - has reported a decreasing signal using such probes. While a new sensor has indeed been published and will soon be available in our lab (agreed with Prof. Sakamoto Masayuki), implementing these experiments, establishing an appropriate analysis workflow, and generating convincing data would require a substantial investment of time and effort for an uncertain outcome.\nFrom a scientific perspective, our manuscript already presents multiple complementary approaches that corroborate our conclusions. We employ validated biased agonists that preferentially target the Gq or Gi pathways, as well as specific inhibitors of the intracellular cascades mediated by Gq (YM254890) or Gi (Pertussis toxin). Therefore, an additional experiment measuring decreased cAMP signaling following OTR-Gi recruitment would not provide significant new insight, but rather would merely confirm the well-established principle that Gi pathway activation reduces cAMP levels.\nReviewer #3:\nThe authors have conducted a number of additional experiments and undertaken substantial revisions that address at least several of the critiques raised by the reviewers. While this reviewer retains some reservations regarding the conceptual novelty and the causal underpinnings of the findings, multiple specific lines of investigation presented in the manuscript open promising avenues that could significantly enhance our understanding of brain machinery.\nIn this context, I suggest that the authors discuss two recently raised issues that bear directly on their arguments. First, whether relatively rapid fear extinction, which is as critical as fear acquisition (https://doi.org/10.1073/pnas.2501331122), might reverse the observed changes. Second, how their interpretation is influenced by evidence that seems to link mechanistically Kir4.1 expression levels to the regulation of local circuit excitability (https://doi.org/10.1016/j.celrep.2025.115299).\nWe thank the reviewer for raising these interesting points. As suggested, we now include those references in the discussion, as thereafter:\n[...]\nReviewer #4:\nWhile I appreciate the authors’ efforts to address the reviewers’ concerns, several key issues remain unresolved. In many cases, the responses are either insufficient or unconvincing, and numerous clarifications rely on circular reasoning that does not adequately support the conclusions drawn. Furthermore, although the number of animals used in specific experiments was increased from 3 to 4, this remains insufficient to support statistically robust conclusions. The statistical analyses also appear more opaque in the revised version rather than clarified.\nWe would like to thank the reviewer for these comments. They prompted us to perform again additional experiments and helped to further improve our manuscript.\nWe fully agree to the importance of sufficient sample size. Therefore, we increased the number of animals in figure 1j and extended data 2h. The new results corroborate a constant and significant increase of calcium signaling in OTR-expressing astrocytes after an electric shock. We also increased the sample size for experiments shown in figure 1l, and extended data figure 2i.\n[...]\n- Oxytocin signaling and astrocyte activation (Page 4): The authors report that ~20% of CeL/C astrocytes express OTR and highlight modulation of the oxytocin signaling pathway in their proteomic dataset after stress. However, this is difficult to reconcile with the data shown in Figure 1j, where approximately 80% of astrocytes show robust calcium activation following acute stress. The authors have not adequately explained what alternative signaling pathways might underlie this widespread activation in OTR-negative astrocytes. If relevant data are available in the proteomic dataset, they should be clearly analyzed and discussed.\nWe thank the reviewer for these important comments.\nWe acknowledge that, in addition to oxytocin signaling, other pathways such as glutamatergic, GABAergic, BDNF, and CRF signaling have been implicated in fear conditioning (Li et al., 2013; Penzo et al., 2015; Pomrenze et al., 2019; Hou et al., 2024). Astrocytes are known to express receptors for these signaling molecules, and thus, we agree that the observed astrocytic Ca² responses to foot shock are not solely attributable to oxytocin signaling. Nevertheless, through a combination of genetic and pharmacological approaches, we specifically demonstrated the involvement of OTR-expressing astrocytes in response to acute stress (Figure 1i-l and Extended Data Figure 2h-i), indicating their contribution of entire CeA astrocyte activities.\n[...]\n- Validation of viral targeting and functional consequences: Although the authors provide new data regarding the efficiency and specificity of rAAV-GFAP-Cre targeting, the validation remains insufficient. It is based on only two mice and 12 fields of view, which raises concerns about representativeness. Moreover, an independent analysis of the images suggests a targeting efficiency closer to ~40%, which could significantly affect the interpretation of the results. Crucially, the authors did not address a previously raised point: whether there is a linear correlation between the number of OTR-deleted astrocytes and the severity of the freezing behavior. This analysis is essential to support a causal relationship between the extent of astrocytic deletion and behavioral effects.\nBased on this reviewers's comment, we have performed additional experiments to further validate the efficacy and specificity of our viral tools and genetic mouse models. We have:\n1) Validated viral GFP expression and correct astrocyte targeting with the widely accepted astrocyte markers glutamine synthetase and Aldh1l1,\n2) Injected OTR-Cre mice with pGFAP-DIO-mScarlet virus and revealed Glutamine Synthetase to validate the astrocyte specificity of mScarlet expression,\n3) Validated our astrocyte-specific OTR ablation strategy via multiplexed RNAscope,\n4) Illustrated the injection sites and the viral spread.\n[...]\n- Astrocytic calcium activity – lack of single-cell resolution: The authors justify not performing single-cell calcium analyses in astrocytes due to technical limitations related to astrocyte morphology and the depth of the CeL/C. While these difficulties are acknowledged, the concern remains unaddressed. Since neuronal activity was analyzed at the single-cell level, failing to apply the same resolution to astrocytes limits interpretability and prevents meaningful comparisons. Sparse GCaMP expression, although more technically demanding, could be achieved using alternative viral serotypes, concentration or promoter strategies. No such attempts were made, and no alternative solutions were proposed.\nWe agree with the reviewer that single cell resolution in vivo experiments would nicely complement our fiber photometry data. Nevertheless, the Ca2+ transients observed in astrocytes are very variable in size, frequency and shape. These stochastic features of astrocytic Ca2+ signalling have been observed by many scientists investigating Ca2+ transients in astroglial cells and seem to be a characteristic shared among astrocytes (Volterra et al., 2014, Gorzo and Gordon, 2022, Semyanov et al., 2020, Covelo et al., 2022). Therefore, from our point of view, it is more physiologically meaningful to analyse astrocytes activity at the population-level than a single cell responses. Along these lines, we think that measuring the population-wide calcium signals using fiber photometry is more relevant than single cell resolution, as the latter does not bring provide more insight in system level mechanisms.\n- Use of 30 mM extracellular K and lack of mechanistic insight into potassium signaling: The authors acknowledge that 30 mM K is a supraphysiological concentration and cite studies suggesting such levels may occur transiently in restricted microdomains. However, they provide no data to confirm that this concentration does not induce cellular stress or toxicity in their model.\nWe have addressed the reviewer's concern regarding a potential toxicity of extracellular potassium concentration at 30 mM with new experiments. We applied ACSF or K+ puffs as previously (20 seconds/per minutes, 3 times), and quantified cell mortality using trypan blue. We observed no difference between the number of trypan blue-stained cells following ACSF and 30mM K+ puffs, indicating that transient application of the latter solution does not induce particular cell death.\n[...]\n- Use of ChR2 and lack of complementary stimulation approaches: The authors argue that ChR2-mediated stimulation of the BLA→CeL/C pathway offers a more physiological alternative to exogenous K application by eliciting endogenous K release. While this rationale is understandable, ChR2 is known to produce non-physiological synchrony and transient surges in extracellular K (Octeau et al., 2019). I had suggested employing complementary approaches such as chemogenetics or electrical stimulation to validate the findings. The authors dismissed these options due to their limitations, but they did not attempt any such control experiments. As a result, their conclusions remain heavily reliant on a single technique, which weakens the robustness of the study.\nWe agree with the reviewer that ““ChR2 is known to produce non-physiological synchrony and transient surges in extracellular K ”: it is for this exact reason that we used an optogenetic approach to mimic synchronous activity-induce increase in extracellular K+, that can be observed in several patho-physiological conditions (Hashimoto et al., 1979, Jensen and Yaari, 1997, Richter and Lehmenkühler, 2008).\nWe understand the reviewer’s suggestion of using a chemogenetic approach to repeat our optogenetic experiment. Nevertheless, we think that this approach raises a number of critical drawbacks: first, chemogenetic do not allows a precise time control of the stimulation, neither in onset nor offset; second, there are increasing number of publication pointing toward the DREADD ligands as having a number of undesirable side effects. Therefore, we cannot use this approach to address the astrocytes involvements in the BLA-CeA synapse.\n[...]\n- Corticosterone levels and sex differences: I appreciate that the authors included corticosterone data from wild-type females and acknowledge that baseline levels were higher than in males. Their ethical justification for not extending this analysis to OTR KO females is understandable. However, given the reported sex differences in behavior, the potential contribution of sex-specific OTR expression or signaling remains an important and unresolved issue that warrants further discussion.\nWe appreciate the reviewer's comment. We fully agree that sex-dependent differences are an important issue that needs more investigation, and we started to explore this in a separate study.\n[...]\nANSWERS TO REVIEWERS’ COMMENTS\nWe thank again the reviewers for their efforts to help improving the manuscript.\nReviewer #1:\nThe authors have addressed my concerns to my satisfaction. I have just a few minor suggestions:\nPresumably, the foot shocks were presented along with the auditory tone at the conclusion of the tone (i.e., the two were co-presented), but this is not clear from the description (i.e., presentation “after” the sound). If so, were they presented at the same time for each of the sound stimuli? The way it is stated makes it sound like the foot shocks were presented randomly, but I assume that you mean that the paired sound-shock sequence was presented randomly over the 10 min.\nWe thank the reviewer for pointing this mistake. Indeed, the shock was always presented at the end of the sound, and the paired sound-shock sequence was presented randomly over the 10 min. This is now more clearly stated in the updated manuscript.\n[...]\nChange the description of the 2nd microdialysis group to “5 randomly presented auditory tone-shock pairings.”\nThe sentence is modified in the updated manuscript.\n[...]\nThe reasoning for not using the GFAP OTR KO for the in vivo imaging experiment is reasonable (i.e., not ratiometric nature of GCaMP and necessity for unpaired comparisons) and I appreciate the attempt, albeit failed, to use the tamoxifen-inducible KO model. I would recommend (but do not require) that this statement be used to introduce and provide a rationale for the OTR antagonist experiment.\nWe thank the reviewer for her/his understanding and support. Because we are not sure why the tamoxifen-inducible KO model fail, we do prefer avoiding its mention in the main manuscript. However, we modified the text in order to highlight the importance of a longitudinal approach:\n[...]\nIn the identification of astrocyte-synapse association, the debris exclusion criterion of < 0.5 µm3 is not consistent with criterion #2 - volume of synapses > 0.1 µm3, unless I am not understanding correctly.\nWe thank the reviewer for pointing out this apparent contradiction and we sincerely apologize for this oversight. In fact, the original criterion should have stated <0.05 µm3 not <0.5 µm3. However, after the initial round of reviewer comments and in line with other similar studies, we have refined our criterion for a more strict, conservative debris filtering threshold at 0.1 µm3. We have now adjusted the Methods Section accordingly to correctly highlight our synapse debris filtering threshold at 0.1 µm3.\n[...]\nIn the EM analysis, was there a change in the number of synaptic profiles in the stress vs. naive micrographs? Were ROI's selected randomly or was the density of synapses used to select an ROI? A decrease in the ratio of astrocytes to synapses could be caused by an increase in synapse numbers rather than decrease in astrocyte numbers, which might imply a change in astrocytic coverage but could also have other, equally impactful implications.\nWe agree with the referee that this point needs to be clarified. The areas for image acquisition by TEM were chosen randomly (50 images per lateral and 50 contralateral side per animal). As shown in the attached figure, the densities of synapses were very similar in control and fear-conditioned animals, whereas the densities of astrocytic processes and thus their ratio were strongly decreased by fear conditioning. In the figure, boxplots show synapse and process counts per image (n=100 images per animal) and ratios (astro process/synapse) per image. Individual points represent individual images. This is now mentioned in the method:\n[...]\nIn addition at p. 13, lines 418-420, change the word “complimentary” to “complementary”.\nDone.\nReviewer #2:\nOne of my previous points has been well addressed. However, the limitation of the pharmacological approach in Fig. 6 should be discussed, as it cannot ensure astrocyte-specific effects. Future studies using cell-specific monitoring are needed to validate these findings.\nWe thank the reviewer for pointing out the limitation of the pharmacological approach. In the figure 6d, we performed an astrocyte-specific deletion of the OTR, identifying OTR astrocytes to be responsible for TGOT-induced increased in K-evoked neuronal depolarization. Upon reviewer comment, we double checked this figure and figure out that we ticked the red bar graph “OTR cKO” instead of “GFAP OTR cKO”, and we believe that this can be the source of this misunderstanding. We now fixed this mistake and hope that the reviewer will be satisfied.\n[...]\nReviewer #3:\nWhile the authors have addressed my remaining comments, I find the claim that “astrocyte process retraction shapes behaviour” to be highly exaggerated. Virtually any change in the brain can be correlated with or even causally linked to some of the hundreds of potentially measurable animal behaviors. Establishing a comprehensive combinatorial map of such relationships seems neither feasible nor realistic.\nWe thank the reviewer for this caution. Accordingly, we modified the manuscript title: “Stress induces an oxytocin Gαi signaling mediated modulation of amygdala astrocytes-neurons network”.\nReviewer #4:\nThe revised manuscript is substantially improved. The additional animals in key experiments, the clearer presentation of statistical analyses, and especially the expanded validation of the viral strategy (including astrocyte specificity and OTR deletion) all strengthen the study. I appreciate the clarification regarding the proportion of OTR-positive astrocytes, the discussion of alternative signaling pathways that may recruit OTR-negative astrocytes, and the inclusion of sex-related observations.\nWe thank the reviewer for his/her support and appreciation of our new experiments.\nMy main remaining reservation concerns the physiological relevance of the 30 mM K manipulation. While I agree that the new experiments indicate that brief K application is not overtly toxic and that IAHP measurements do not reveal obvious differences between naïve and stressed animals, I still think the link between these ex vivo manipulations and in vivo K dynamics during stress is somewhat indirect.\nWith that caveat stated in the Discussion, I am satisfied with the revisions and I am supportive of publication.\nWe agree with the reviewer and therefore have included a discussion sentence about the putative indirect link between ex vivo manipulations and in vivo K+ dynamics.\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns to my satisfaction. I have just a few minor suggestions:\nPresumably, the foot shocks were presented along with the auditory tone at the conclusion of the tone (i.e., the two were co-presented), but this is not clear from the description (i.e., presentation “after” the sound). If so, were they presented at the same time for each of the sound stimuli? The way it is stated makes it sound like the foot shocks were presented randomly, but I assume that you mean that the paired sound-shock sequence was presented randomly over the 10 min.\nChange the description of the 2nd microdialysis group to “5 randomly presented auditory tone-shock pairings.”\nThe reasoning for not using the GFAP OTR KO for the in vivo imaging experiment is reasonable (i.e., not ratiometric nature of GCaMP and necessity for unpaired comparisons) and I appreciate the attempt, albeit failed, to use the tamoxifen-inducible KO model. I would recommend (but do not require) that this statement be used to introduce and provide a rationale for the OTR antagonist experiment.\nIn the identification of astrocyte-synapse association, the debris exclusion criterion of < 0.5 um3 is not consistent with criterion #2 - volume of synapses > 0.1 um3, unless I am not understanding correctly.\nIn the EM analysis, was there a change in the number of synaptic profiles in the stress vs. naive micrographs? Were ROI's selected randomly or was the density of synapses used to select an ROI? A decrease in the ratio of astrocytes to synapses could be caused by an increase in synapse numbers rather than decrease in astrocyte numbers, which might imply a change in astrocytic coverage but could also have other, equally impactful implications.\nIn addition at p. 13, lines 418-420, change the word “complimentary” to “complementary”.", "reviewer_2": "One of my previous points has been well addressed. However, the limitation of the pharmacological approach in Fig. 6 should be discussed, as it cannot ensure astrocyte-specific effects. Future studies using cell-specific monitoring are needed to validate these findings.", "reviewer_3": "While the authors have addressed my remaining comments, I find the claim that “astrocyte process retraction shapes behaviour” to be highly exaggerated. Virtually any change in the brain can be correlated with or even causally linked to some of the hundreds of potentially measurable animal behaviors. Establishing a comprehensive combinatorial map of such relationships seems neither feasible nor realistic.", "reviewer_4": "The revised manuscript is substantially improved. The additional animals in key experiments, the clearer presentation of statistical analyses, and especially the expanded validation of the viral strategy (including astrocyte specificity and OTR deletion) all strengthen the study. I appreciate the clarification regarding the proportion of OTR-positive astrocytes, the discussion of alternative signaling pathways that may recruit OTR-negative astrocytes, and the inclusion of sex-related observations.\nMy main remaining reservation concerns the physiological relevance of the 30 mM K⁺ manipulation. While I agree that the new experiments indicate that brief K⁺ application is not overtly toxic and that IAHP measurements do not reveal obvious differences between naïve and stressed animals, I still think the link between these ex vivo manipulations and in vivo K⁺ dynamics during stress is somewhat indirect.\nWith that caveat stated in the Discussion, I am satisfied with the revisions and I am supportive of publication."}, "author_response": "We thank again the reviewers for their efforts to help improving the manuscript.\nReviewer #1:\nThe authors have addressed my concerns to my satisfaction. I have just a few minor suggestions:\nPresumably, the foot shocks were presented along with the auditory tone at the conclusion of the tone (i.e., the two were co-presented), but this is not clear from the description (i.e., presentation “after” the sound). If so, were they presented at the same time for each of the sound stimuli? The way it is stated makes it sound like the foot shocks were presented randomly, but I assume that you mean that the paired sound-shock sequence was presented randomly over the 10 min.\nWe thank the reviewer for pointing this mistake. Indeed, the shock was always presented at the end of the sound, and the paired sound-shock sequence was presented randomly over the 10 min. This is now more clearly stated in the updated manuscript.\nPage 4 Line 92-94: Animals were exposed to five 20-second-long sound stimuli followed by electrical foot shocks. These paired sound-shock sequences were randomly delivered over 10 minutes, and freezing was quantified during the 20 seconds following the electric foot shocks to assess the immediate stress response (Fig. 1a).\nChange the description of the 2nd microdialysis group to “5 randomly presented auditory tone-shock pairings.”\nThe sentence is modified in the updated manuscript.\nPage 5 Line 142-143: We found that stress induced by five randomly presented auditory tone-shock pairings induces an increase in CeA OT concentration.\nThe reasoning for not using the GFAP OTR KO for the in vivo imaging experiment is reasonable (i.e., not ratiometric nature of GCaMP and necessity for unpaired comparisons) and I appreciate the attempt, albeit failed, to use the tamoxifen-inducible KO model. I would recommend (but do not require) that this statement be used to introduce and provide a rationale for the OTR antagonist experiment.\nWe thank the reviewer for her/his understanding and support. Because we are not sure why the tamoxifen-inducible KO model fail, we do prefer avoiding its mention in the main manuscript. However, we modified the text in order to highlight the importance of a longitudinal approach:\nPage 6 Line 155-160: Next, we aimed to test the involvement of OTR in this response. Given the non-ratiometric nature of the GCaMP6s, we opted for a longitudinal approach and achieved pharmacological inhibition of the OTR by injecting i.p. the antagonist L368 that cross the blood-brain barrier (Fig. 1k)27. We observed a significant reduction of the foot shock-induced Ca2+ response following L368, suggesting the involvement of the OTR in the stress-induced astrocyte Ca2+ signaling\nIn the identification of astrocyte-synapse association, the debris exclusion criterion of < 0.5 µm3 is not consistent with criterion #2 - volume of synapses > 0.1 µm3, unless I am not understanding correctly.\nWe thank the reviewer for pointing out this apparent contradiction and we sincerely apologize for this oversight. In fact, the original criterion should have stated <0.05 µm3 not <0.5 µm3. However, after the initial round of reviewer comments and in line with other similar studies, we have refined our criterion for a more strict, conservative debris filtering threshold at 0.1 µm3. We have now adjusted the Methods Section accordingly to correctly highlight our synapse debris filtering threshold at 0.1 µm3.\nPage 20 Line 687-688: […] debris measuring <0.1µm³ was disregarded as nonspecific and filtered out in a subsequent step.\nIn the EM analysis, was there a change in the number of synaptic profiles in the stress vs. naive micrographs? Were ROI's selected randomly or was the density of synapses used to select an ROI? A decrease in the ratio of astrocytes to synapses could be caused by an increase in synapse numbers rather than decrease in astrocyte numbers, which might imply a change in astrocytic coverage but could also have other, equally impactful implications.\nWe agree with the referee that this point needs to be clarified. The areas for image acquisition by TEM were chosen randomly (50 images per side per animal) and the density of synapses was identical in both naïve and acute stress conditions (3.47±0.08 vs 3.62±0.08, respectively). As shown in the attached figure, the densities of synapses were very similar in control and fear-conditioned animals, whereas the densities of astrocytic processes and thus their ratio were strongly decreased by fear conditioning. In the figure, boxplots show synapse and process counts per image (n=100 images per animal) and ratios (astro process/synapse) per image. Individual points represent individual images. This is now mentioned in the method:\nPage 24, lines 883-885: The areas for image acquisition by TEM were chosen randomly (50 images per side per animal) and the density of synapses was identical in both naïve and acute stress conditions (3.47±0.08 vs 3.62±0.08, respectively).\nIn addition at p. 13, lines 418-420, change the word “complimentary” to “complementary”.\nDone.\nReviewer #2:\nOne of my previous points has been well addressed. However, the limitation of the pharmacological approach in Fig. 6 should be discussed, as it cannot ensure astrocyte-specific effects. Future studies using cell-specific monitoring are needed to validate these findings.\nWe thank the reviewer for pointing out the limitation of the pharmacological approach. In the figure 6d, we performed an astrocyte-specific deletion of the OTR, identifying OTR astrocytes to be responsible for TGOT-induced increased in K-evoked neuronal depolarization. Upon reviewer comment, we double checked this figure and figure out that we ticked the red bar graph “OTR cKO” instead of “GFAP OTR cKO”, and we believe that this can be the source of this misunderstanding. We now fixed this mistake and hope that the reviewer will be satisfied.\nReviewer #3:\nWhile the authors have addressed my remaining comments, I find the claim that “astrocyte process retraction shapes behaviour” to be highly exaggerated. Virtually any change in the brain can be correlated with or even causally linked to some of the hundreds of potentially measurable animal behaviors. Establishing a comprehensive combinatorial map of such relationships seems neither feasible nor realistic.\nWe thank the reviewer for this caution. Accordingly, we modified the manuscript title: “Stress induces an oxytocin Gαi signaling mediated modulation of amygdala astrocytes-neurons network”.\nReviewer #4:\nThe revised manuscript is substantially improved. The additional animals in key experiments, the clearer presentation of statistical analyses, and especially the expanded validation of the viral strategy (including astrocyte specificity and OTR deletion) all strengthen the study. I appreciate the clarification regarding the proportion of OTR-positive astrocytes, the discussion of alternative signaling pathways that may recruit OTR-negative astrocytes, and the inclusion of sex-related observations.\nWe thank the reviewer for his/her support and appreciation of our new experiments.\nMy main remaining reservation concerns the physiological relevance of the 30 mM K manipulation. While I agree that the new experiments indicate that brief K application is not overtly toxic and that IAHP measurements do not reveal obvious differences between naïve and stressed animals, I still think the link between these ex vivo manipulations and in vivo K dynamics during stress is somewhat indirect.\nWith that caveat stated in the Discussion, I am satisfied with the revisions and I am supportive of publication.\nWe agree with the reviewer and therefore have included a discussion sentence about the putative indirect link between ex vivo manipulations and in vivo K+ dynamics.\nPage 12 Line 393-394: While our results are based on ex vivo experiments and may not fully reflect in vivo conditions, the data indicate that the variation of neuronal excitability is due to a defect in K+ clearance."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns and have conducted a very rigorous and thorough study and a nice paper."}, "author_response": ""}]

Decision letter