Jérôme Wahis

Damien Kerspern

Ferdinand Althammer

Angel Baudon

Stéphanie Goyon

Daisuke Hagiwara

Arthur Lefèvre

Benjamin Boury-Jamot

Benjamin Bellanger

Marios Abatis

Miriam Silva Da Gouveia

Diego Benusiglio

Marina Eliava

Andrej Rozov

Ivan Weinsanto

Hanna Sophie Knobloch-Bollmann

Hong Wang

Marie Pertin

Perrine Inquimbert

Claudia Pitzer

Jan Siemens

Yannick Goumon

Benjamin Boutrel

Pascal Darbon

Christophe Maurice Lamy

Javier E Stern

Isabelle Décosterd

Jean-Yves Chatton

W Scott Young

Ron Stoop

Pierrick Poisbeau

Valery Grinevich

Dr Alexandre Charlet
email: acharlet@unistra.fr

Oxytocin Acts on Astrocytes in the Central Amygdala to Promote a Positive Emotional State

INTRODUCTION

The neuropeptide oxytocin (OT) modulates key neurophysiological functions [START_REF] Lee | Oxytocin: The great facilitator of life[END_REF] , including anxiety and pain, notably through its action on central amygdala (CeA) microcircuits [START_REF] Eliava | A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing[END_REF][START_REF] Huber | Vasopressin and oxytocin excite distinct neuronal populations in the central amygdala[END_REF][START_REF] Viviani | Oxytocin selectively gates fear responses through distinct outputs from the central amygdala[END_REF][START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF] .

Oxytocin receptors (OTR) are expressed in the lateral and capsular part of the CeA (CeL) [START_REF] Huber | Vasopressin and oxytocin excite distinct neuronal populations in the central amygdala[END_REF] . In this nucleus, OTR activation leads to increased firing of γ-aminobutyric acid (GABA) expressing interneurons. This directly inhibits projection neurons of the medial part of the CeA (CeM), the functional output of the CeA, thereby leading to clear behavioral outputs [START_REF] Huber | Vasopressin and oxytocin excite distinct neuronal populations in the central amygdala[END_REF][START_REF] Viviani | Oxytocin selectively gates fear responses through distinct outputs from the central amygdala[END_REF][START_REF] Hasan | A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System[END_REF] . Besides, the CeA is recognized as a major player in the pathophysiology of several neurological diseases, among which is neuropathic pain [START_REF] Neugebauer | The amygdala and persistent pain[END_REF] , for which recent studies also suggest a critical involvement of astrocytes [START_REF] Eto | The roles of cortical astrocytes in chronic pain and other brain pathologies[END_REF][START_REF] Hansen | Astrocytes -Multitaskers in chronic pain[END_REF][START_REF] Ji | Glia and pain: Is chronic pain a gliopathy?[END_REF] as well as the oxytocinergic system [START_REF] Poisbeau | Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels[END_REF][START_REF] Rash | Oxytocin and pain: a systematic review and synthesis of findings[END_REF] . However the possible involvement of astrocytes in the neuromodulatory effects of oxytocin has rarely been explored [START_REF] Kuo | An interaction of oxytocin receptors with metabotropic glutamate receptors in hypothalamic astrocytes[END_REF][START_REF] Parent | Oxytocin Facilitates Female Sexual Maturation through a Glia-to-Neuron Signaling Pathway[END_REF][START_REF] Scala-Guenot | Increase of intracellular calcium induced by oxytocin in hypothalamic cultures astrocytes[END_REF][START_REF] Wang | Oxytocin Rapidly Changes Astrocytic GFAP Plasticity by Differentially Modulating the Expressions of pERK 1/2 and Protein Kinase A[END_REF] . That, despite the numerous findings of astrocytes active involvement in the regulation of neural circuits in a brain area specific manner [START_REF] Adamsky | Astrocytic Activation Generates De Novo Neuronal Potentiation and Memory Enhancement[END_REF][START_REF] Allen | Astrocyte regulation of synaptic behavior[END_REF][START_REF] Khakh | The Emerging Nature of Astrocyte Diversity[END_REF][START_REF] Verkhratsky | Physiology of Astroglia[END_REF] , notably in the CeA [START_REF] Martin-Fernandez | Synapse-specific astrocyte gating of amygdala-related behavior[END_REF] , with yet many controversies remaining about the mechanisms involved [START_REF] Bazargani | Astrocyte calcium signaling: the third wave[END_REF][START_REF] Savtchouk | Gliotransmission: Beyond Black-and-White[END_REF] .

Herein, we employed patch-clamp techniques, calcium imaging, and behavioral assays to measure the consequences of in vivo and ex vivo direct manipulation of CeL astrocytes in both rats and mice. To this end, we used pharmacological approaches as well as genetic tools to ablate OTRs or expressed opsins specifically in CeL astrocytes.

We demonstrate that astrocytes are a necessary component in the oxytocinergic modulation of CeA neuronal circuits and propose a mechanism by which astrocytes relay OT effects in the CeA by gating neuronal N-methyl-D-aspartate receptors (NMDAR) activation through release of the co-agonist D-serine. This ultimately leads to promotion of the behavioral correlates of positive emotional states. With this study, we show for the first time that astrocyte-neuron communication is crucial for an efficient integration of positive emotions within the CeA, a structure involved in the processing of emotionallyrelevant cues.

RESULTS

CeL Astrocytes Express Functional Oxytocin Receptors

To specifically investigate whether CeA astrocytes express OTRs, we performed fluorescence in situ hybridizations (FISH) on rat CeA sections and found overlap between OTR mRNA and an astrocyte marker, glutamine synthetase (Figure 1a-c). We quantified cells positive for both markers and found that a randomly (Figure S1a) distributed subpopulation of about 13.6% of CeL astrocytes expresses the OTR mRNA (Figure 1c). We further corroborated this finding using the aldehyde dehydrogenase 1 family member L1 (ALDH1L1) as an astrocyte marker (Figure S1a), and confirmed it to be comparable in mice CeL astrocytes (Figures S1c-d; 2c).

To test whether endogenous activation of astrocytic OTR elicits physiological responses in CeL astrocytes, we performed calcium imaging in brain slices of rats using the calcium indicator Oregon Green® 488 BAPTA-1 (OGB1) and identified astrocytes through sulforhodamine 101 labelling (SR101) (Figure S2a). Indeed, measuring changes in intracellular calcium is the most commonly accepted readout of astrocytic activity [START_REF] Shigetomi | Probing the Complexities of Astrocyte Calcium Signaling[END_REF] . We first confirmed that SR101 positive cells of the CeL displayed typical electrophysiological properties of astrocytes (Figure S2b-c). We then expressed the opsin C1V1(t/t) under the oxytocin promoter through rAAVs (rAAV-OTp-C1V1(t/t)-mCherry) injected in the paraventricular, supraoptic, and accessory hypothalamic nuclei of rats, to enable lightevoked activation of oxytocin neurons and their distant axons localized within the CeL [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF][START_REF] Hasan | A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System[END_REF] (Figure 1d-e, S1e). We found that light-driven activation of C1V1(t/t) expressing oxytocinergic axons rapidly evoked long-lasting and oscillating calcium transients in astrocytes (Figure 1f-h). These calcium transients persisted during bath application of TTX, thereby excluding the possibility that they were evoked secondary to neuronal circuit activity.

OTR Activation Evokes Astrocytic Calcium Transients in CeL Astrocyte Syncitium

To confirm that the observed increase of intracellular calcium in CeL astrocytes is mediated by direct action on OTRs, we used the exogenous and OTR-selective agonist [Thr4Gly7]-oxytocin (TGOT). TGOT induced calcium transients in astrocytes, which were prevented by the incubation of the OTR antagonist [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (dOVT) and remained unaffected by the presence of TTX (Figure 2a).

Next, we sought to reinforce the conclusions drawn from our pharmacological experiments by testing whether the OTRs expressed in CeL astrocytes are responsible for the TGOT-induced increase of calcium transients in these cells. To specifically ablate OTRs in CeL astrocytes, an rAAV vector allowing expression of Cre recombinase under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter (rAAV-GFAPp-GFP-IRES-Cre) was injected into the CeL of an OTR conditional knockout (OTR-cKO) mouse line in which loxP sites flank the OTR coding sequence [START_REF] Lee | A conditional knockout mouse line of the oxytocin receptor[END_REF] (Figure 2c). The injection of a control vector (rAAV-GFAPp-GFP) in OTR-cKO mice did not alter the OTR expression pattern, as 15.9% of astrocytes remained positive for OTR mRNA (Figure 2c), a proportion similar as what was found in rats CeL (Figure 1a-c). In comparison, CeL injection of the rAAV-GFAPp-IRES-Cre vector significantly reduced the number of OTR mRNA-expressing astrocytes to 1.2% (Figure 2c, S2e) but did not alter the OTR mRNA signals in neurons (Figure S2f), thus resulting in the CeL astrocyte-specific genetic deletion of OTRs. We then performed calcium imaging in CeA slices of mice using the calcium indicator Rhod-2 and identified astrocytes through GFP fluorescence. In those animals, TGOT failed to evoke calcium transients in CeL astrocytes, which was in start contrast to what we observed in control mice (Figure 2d).

Altogether, these findings suggest that OTR-expressing CeL astrocytes are fully equipped to respond in an autonomous fashion to the activity of oxytocinergic axonal projections arising from the hypothalamus in both rats and mice.

One interesting discrepancy is that up to 50% of CeL astrocytes responded to evoked release of OT or bath application of TGOT (Figure 1f-h, 2a) while OTR mRNA was detected in only 13 to 15% of CeL astrocytes (Figure 1a-c, S1a, 2a-c). Astrocyte calcium responses are known to spread through the astrocytes network via gap-junctions or paracrine communication through ATP release [START_REF] Verkhratsky | Physiology of Astroglia[END_REF] . We first applied purinergic antagonists, PPADS, Suramin, and A438079 but failed to reveal any changes in the proportion or magnitude of CeL astrocytes responses to TGOT (Figure 2b,S2d). This suggests that purinergic transmission is not involved in the sequential activation of the CeL astrocytes network after OTR activation. However, carbenexolone (CBX), a widely-used blocker of gap junctions, significantly decreased the proportion of CeL astrocytes responding to OTR activation (16.5%) without impairing the calcium response dynamics in the remaining responsive CeL astrocytes (Figure 2b). These findings suggest that the initial OTR-induced activation of CeL astrocytes spreads across the CeL astrocytic network through gap junctions.

CeL Astrocytes are Sufficient to Recruit the CeL→CeM Neuronal Circuit.

To address the potential modulatory role of CeL astrocytes on the CeL→CeM neuronal circuit, we first asked whether CeL astrocyte activity was sufficient to solely modulate CeA neuronal circuits (Figure 3). Using rAAVs to express the light-gated opsin C1V1(t/t) under the control of the GFAP promoter (Figure 3a), we specifically targeted CeL astrocytes (Figure S3a) and tested the effect of light-evoked calcium transients elicited in CeL astrocytes on the CeL→CeM neuronal circuit (Figure 3b).

We hypothesized that a form of astrocytes-mediated neuromodulation could reveal itself through the presence of slow inward currents (SICs), a hallmark of neuronal NMDAR activation by astrocytes [START_REF] Perea | Properties of synaptically evoked astrocyte calcium signal reveal synaptic information processing by astrocytes[END_REF][START_REF] Shigetomi | Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons[END_REF] . SICs were indeed detected in CeL neurons (Figure 3c, S3b) and their frequency significantly increased after light-evoked calcium transients in CeL astrocytes (Figure 3c). To assess whether this increase in the SICs frequency is sufficient to increase excitability and ultimately firing in CeL neurons, we next performed ex vivo patch-clamp recordings of action potentials (APs) in CeL neurons upon CeL astrocytes stimulation (Figure 3b). We found that light-evoked calcium transients in CeL astrocytes led to an increase of APs frequencies in CeL neurons (Figure 3d). Crucially, this effect was prevented if we first loaded the Ca 2+ chelator BAPTA into the CeL astrocytes syncytium [START_REF] Jourdain | Glutamate exocytosis from astrocytes controls synaptic strength[END_REF] (Figure S3c).

Given that the CeM is the main output of the CeA, we then conducted ex vivo patchclamp recordings of inhibitory post-synaptic currents (IPSCs) in CeM projection neurons (Figure 3b), whose frequency is known to increase after CeL interneurons firing [START_REF] Huber | Vasopressin and oxytocin excite distinct neuronal populations in the central amygdala[END_REF][START_REF] Viviani | Oxytocin selectively gates fear responses through distinct outputs from the central amygdala[END_REF][START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF] .Indeed, we found that light-evoked calcium transients in CeL astrocytes led to an increase of IPSCs frequencies in CeM neurons (Figure 3e) which was also prevented by BAPTA infusion in the CeL astrocytes syncytium (Figure S3d). Altogether, these results indicate that CeL astrocytes activity is sufficient to modulate the CeL→CeM neuronal circuit.

CeL Astrocytes are Necessary for the CeL→CeM Neuronal Circuit Modulation by

OTR Activation.

We then further examined whether CeL astrocytes are required to support the oxytocinergic modulation of the CeL→CeM neuronal circuit. We repeated the measurement of neuronal properties (Figure 3) and found that CeL neurons' SICs and APs frequencies, as well as CeM neurons' IPSCs frequency, were significantly increased after TGOT application in both rats and mice (Figure 4a1-a3, 4b1-b3; S4a).

Next, we loaded the Ca 2+ chelator BAPTA into the rat CeL astrocytes syncytium (Figure 4a). BAPTA loading of CeL astrocytes significantly reduced the OTR-induced increase of SICs and APs frequencies in CeL neurons as well as the increase in IPSCs frequencies in CeM neurons (Figure 4a1-3). These results indicate the necessity of functional CeL astrocytes for the OTR-mediated recruitment of the CeL→CeM neuronal circuit.

To further verify that the OTR-induced modulation of the CeL→CeM neuronal circuit depends on OTRs present on CeL astrocytes, we specifically ablated OTRs in CeL astrocytes of OTR-cKO mice (Figure 2c, S2e-f, 4b). In accordance with our previous results using pharmacological inactivation of astrocytes in rats, the specific deletion of OTR in mice CeL astrocytes significantly decreased the TGOT-induced increase of both SICs and APs frequencies in CeL neurons, as well as in IPSCs frequency in CeM neurons (Figure 4b1-3). These results highlight the necessity of OTR expression in CeL astrocytes for the OTR-mediated recruitment of the CeL→CeM neuronal circuit.

CeL Astrocytes Recruit the CeL→CeM Neuronal Circuit through NMDAR modulation

We then sought to confirm that the activation of neuronal NMDAR, which is underlying the presence of SICs [START_REF] Perea | Properties of synaptically evoked astrocyte calcium signal reveal synaptic information processing by astrocytes[END_REF][START_REF] Shigetomi | Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons[END_REF] , is indeed a key component of CeL astrocyte-to-CeL neuron communication (Figure 5a-b). While the blockade of NMDAR with (2R)-amino-5phosphonopentanoate (AP5, glutamate site) did not alter CeL astrocytic responses to TGOT (Figure S5a), AP5 prevented TGOT-induced modulation of SICs and APs frequencies in CeL neurons (Figure 5c-d). Moreover, OTR-induced CeL astrocytemediated SICs were linked to the activation of NR2B containing NMDARs, since ifenprodil prevented only the TGOT-induced SICs frequency increase (Figure 5c). We next tested whether repeated TGOT applications consistently modulate the CeL→CeM neuronal circuit via OTR-specific activation to assess the role of the NMDAR pathway in TGOT responsive CeM neurons (Figure S5b). We found that both AP5 and 5,7-dichlorokynurenic acid (DCKA, a blocker of the NMDAR glycine co-agonist site) significantly reduced the TGOT-evoked increase of IPSCs frequencies in CeM neurons (Figure S5c). On the contrary, the blockade of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had only a limited effect (Figure S5d). These results indicate that the oxytocin signal is directly conveyed from CeL astrocytes to CeL neurons via neuronal NMDAR activation.

We next sought to identify the molecular agent released by CeL astrocytes that gates the increase in CeL neuronal NMDAR activation. We used D-amino-acid oxidase (DAAO) to selectively catabolize the NMDAR co-agonist D-serine, a major astrocytic gliotransmitter [START_REF] Mothet | Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine[END_REF][START_REF] Oliet | Regulation of N-methyl-D-aspartate receptors by astrocytic D-serine[END_REF][START_REF] Robin | Astroglial CB1 Receptors Determine Synaptic D-Serine Availability to Enable Recognition Memory[END_REF] . TGOT had no effect after DAAO incubation, but subsequent incubation of the same cells in D-serine-containing ACSF partially restored the TGOTmediated increased of IPSCs frequencies in CeM neurons (Figure 5e). Altogether, these data suggest that OTR-mediated activation of CeL astrocytes leads to the release of Dserine, allowing an increased activation of NMDAR in CeL GABAergic interneurons which, by increasing these neurons firing rate, leads in an increase of IPSCs frequencies downstream of the circuit, in CeM projection neurons.

CeL Astrocytes are Sufficient to Modulate CeA Behavioral Correlates of Positive

Emotional State and are Necessary for their OTR-mediated Modulation

Finally, we aimed to determine whether the CeL astrocyte-mediated modulation of the CeL→CeM neuronal circuit by OTR activation contributes to known CeA behavioral correlates. Since the CeA is a key structure in the regulation of pain-associated disorders [START_REF] Neugebauer | Forebrain pain mechanisms[END_REF] and assigns emotionality to salient external stimuli [START_REF] Sah | The amygdaloid complex: anatomy and physiology[END_REF] , we first tested the effect of TGOT on increased pain sensitivity and anxiety exhibited by neuropathic rats 4 weeks after a spared nerve injury surgery procedure [START_REF] Alba-Delgado | Comorbid anxiety-like behavior and locus coeruleus impairment in diabetic peripheral neuropathy: A comparative study with the chronic constriction injury model[END_REF][START_REF] Yalcin | The sciatic nerve cuffing model of neuropathic pain in mice[END_REF] (SNI; Figure 6a). As neuropathy may cause synaptic changes in the amygdala [START_REF] Neugebauer | The amygdala and persistent pain[END_REF][START_REF] Han | Synaptic plasticity in the amygdala in a visceral pain model in rats[END_REF] , we conducted ex vivo patch-clamp recordings of IPSCs in CeM projection neurons 4 weeks after SNI or sham surgery. The TGOT-induced increase of IPSCs frequency was strictly similar in both conditions (Figure S6a4), indicating that the OTR-induced modulation of the CeL→CeM neuronal circuit is not altered after induction of SNI. Cannulae-guided micro-infusion of TGOT into the CeL slightly reduced mechanical hyperalgesia (Figure 6a1, S6a1) and clearly reduced anxiety levels in neuropathic animals (Figure 6a2, S6a2). We then performed similar experiments on SNI animals in which C1V1(t/t) was expressed under the control of a GFAP promoter in the CeL. Light-evoked stimulation of CeL astrocytes did not visibly affect mechanical pain thresholds of neuropathic animals, but was sufficient to reduce their anxiety (Figure 6a1-a2, S6a1-a2). Since the amygdala is critically involved in the regulation of emotions [START_REF] Phelps | Contributions of the Amygdala to Emotion Processing: From Animal Models to Human Behavior[END_REF] , we performed a conditioned place preference test (CPP) to assess if the OTR-induced modulation of the CeL→CeM pathway is linked to the emotional component of pain. Both cannulae-guided micro-infusion of TGOT in CeL and light-evoked activation of C1V1(t/t)expressing CeL astrocytes induced a strong place preference in neuropathic and shamoperated rats (Figure 6a3, S6a3).

To confirm that the OTR-induced modulation of the observed behaviors strictly depends of OTR present on CeL astrocytes, we specifically ablated OTRs in mice CeL astrocytes (Figure 2c, S2e-f, 6b) and tested the effect of TGOT on increased pain sensitivity and anxiety exhibited by neuropathic mice 4 weeks after a spared nerve injury surgery procedure [START_REF] Yalcin | The anxiodepressive comorbidity in chronic pain[END_REF] (SNI; Figure 6b). Cannula-guided micro-infusion of TGOT in CeL did not change mechanical hyperalgesia (Figure 6b1, S6b1) but reduced anxiety levels in control neuropathic animals (Figure 6b2, S6b2), an effect that was prevented by the CeL astrocyte specific deletion of OTRs (Figure 6b2). As previously shown in rats (Figure 6a3), we tested mice for CPP and found a strong place preference effect of micro-infusion of TGOT in CeL in both neuropathic and sham-operated control mice, which was absent after deletion of OTR in CeL astrocytes (Figure 6b3,S6b3).

Altogether, these findings point towards the OTR-mediated signaling in CeL astrocytes as a counterbalancing mechanism supporting the positive emotional valence under both chronic pain and healthy states. It indicates that the OTR-astrocytes mediated modulation of the CeL→CeM neuronal circuit is not only involved in the regulation of hyperalgesia or anxiety but also represents a more general cellular substrate underlying the promotion of a positive emotional state (Figure 7).

DISCUSSION

In addition to their classical functions related to structural and metabolic support, modulation of synaptic transmission and transmitter uptake and release, astrocytes have emerged as key players in intricate and diverse glia-neuronal network interactions [START_REF] Khakh | The Emerging Nature of Astrocyte Diversity[END_REF] .

However, the functional relevance of astro-neuronal interactions in the modulation of complex social and emotional behaviors remains elusive. Following the initial experiments on OTRs in cultured astrocytes [START_REF] Scala-Guenot | Increase of intracellular calcium induced by oxytocin in hypothalamic cultures astrocytes[END_REF] , we sought to investigate the role of these cells in OTR signaling in the CeA, a structure critically involved in the processing of emotionallyrelevant information. We showed that CeL astrocytes play a critical role in the oxytocininduced modulation of the CeL→CeM neuronal circuit in two rodent species, rats and mice. Here we report that a subpopulation of CeL astrocytes express functional OTR and convey the oxytocin signal among other (OTR-negative) astrocytes of the CeL through gap junctional coupling. This recruitment of the CeL astrocytes syncytium leads to an NMDAR-mediated tuning of CeA neuronal circuits. At the behavioral level, this oxytocinmediated modulation of the astro-neuronal network of the central amygdala might promote a positive emotional state.

The amygdala is a key region for emotional processing [START_REF] Phelps | Contributions of the Amygdala to Emotion Processing: From Animal Models to Human Behavior[END_REF] with a clear involvement in pain regulation and particularly persistent pain, which impacts ~8% of the global population [START_REF] Neugebauer | The amygdala and persistent pain[END_REF] . On the other hand, oxytocin is a neuropeptide well known to act on various behaviors throughout the cycle of life, with major involvement in a broad variety of physiological functions, all directed toward well-being, emotional balance and ultimately species survival [START_REF] Lee | Oxytocin: The great facilitator of life[END_REF][START_REF] Grinevich | Interplay between Oxytocin and Sensory Systems in the Orchestration of Socio-Emotional Behaviors[END_REF] . In this regard, it is interesting to observe that our results suggest that the oxytocin-induced modulation of CeA networks not only attenuates hypersensitivity symptoms related to nociception in itself, but more generally improves the positive emotional valence of the animals even under pain-free conditions. Pain management is a clinically difficult topic, and most of the newly developed analgesic drugs have failed or not met expectations and patients' needs. Therefore, it might be particularly relevant to consider new therapeutic strategies aiming at alleviating the pain spectrum globally, for example by targeting the oxytocin system, to improve the overall patient well-being.

The oxytocinergic regulation of neural circuits and oxytocin's physiological functions is presently under intense scrutiny [START_REF] Hasan | A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System[END_REF][START_REF] Ferretti | Oxytocin Signaling in the Central Amygdala Modulates Emotion Discrimination in Mice[END_REF][START_REF] Hung | Gating of social reward by oxytocin in the ventral tegmental area[END_REF][START_REF] Marlin | Oxytocin enables maternal behaviour by balancing cortical inhibition[END_REF][START_REF] Menon | Oxytocin Signaling in the Lateral Septum Prevents Social Fear during Lactation[END_REF][START_REF] Nardou | Oxytocin-dependent reopening of a social reward learning critical period with MDMA[END_REF][START_REF] Oettl | Oxytocin Enhances Social Recognition by Modulating Cortical Control of Early Olfactory Processing[END_REF][START_REF] Xiao | Biased Oxytocinergic Modulation of Midbrain Dopamine Systems[END_REF] . Yet, to the best of our knowledge, these studies were solely based on the hypothesis that oxytocin signaling occurs predominantly in neurons, despite a sparse number of independent studies demonstrating expression of OTR in glia, notably astrocytes [START_REF] Kuo | An interaction of oxytocin receptors with metabotropic glutamate receptors in hypothalamic astrocytes[END_REF][START_REF] Scala-Guenot | Increase of intracellular calcium induced by oxytocin in hypothalamic cultures astrocytes[END_REF][START_REF] Wang | Oxytocin Rapidly Changes Astrocytic GFAP Plasticity by Differentially Modulating the Expressions of pERK 1/2 and Protein Kinase A[END_REF][START_REF] Mitre | A Distributed Network for Social Cognition Enriched for Oxytocin Receptors[END_REF][START_REF] Yoshida | Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice[END_REF] . Here, we propose a novel mechanism implicating astrocytes as key contributors of oxytocin action. The induction of long-lasting calcium oscillations in astrocytes following OTR activation leads to a sustained modulation of the CeL→CeM neuronal circuit, affecting known CeA functions such as emotional valence regulation. It may seem counter-intuitive that without a pristine astroglial function, CeA neurons appear unaffected by OTR activation, despite their clear transcription of OTR mRNA. We hypothesize that OTR-induced activation of CeL astrocytes gates CeL neurons responses to oxytocin, and probably other synaptic inputs, through sustained (co-) activation of NMDARs. Such mechanism of astrocytes to neuron communication through activation of extra-synaptic NMDARs has also been proposed by other studies to favor a synchronous increase in excitability across an ensemble of neurons [START_REF] Fellin | Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors[END_REF][START_REF] Angulo | Glutamate released from glial cells synchronizes neuronal activity in the hippocampus[END_REF] . In particular, activation of Gq or Gi/o G-protein coupled receptors (GPCRs) specifically in astrocytes enhances neuronal excitability and ultimately firing [START_REF] Durkee | G i/o protein-coupled receptors inhibit neurons but activate astrocytes and stimulate gliotransmission[END_REF] , a result which could parallel the activation of the same signaling pathways here by specific OTR activation, and GPCRs are known to be coupled to either of Gq or Gi/o proteins [START_REF] Busnelli | Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes[END_REF] . This proposed mechanism would allow for a synchronized and long-lasting switch in the gain of the CeA neuronal circuits, thereby amplifying the effect of oxytocin on CeA outputs in both the spatial and temporal domains. In light of the predominantly non-synaptic OT release from axons en passant, which could lead to CeL-restricted micro-volume transmission of the neuropeptide [START_REF] Chini | The Action Radius of Oxytocin Release in the Mammalian CNS: From Single Vesicles to Behavior[END_REF][START_REF] Grinevich | Assembling the Puzzle: Pathways of Oxytocin Signaling in the Brain[END_REF] , it seems plausible that astrocytes relay and amplify oxytocin signaling to neurons. This would provide control over the oxytocin effect on neuronal circuits, as has already been proposed for other neuromodulators and neurotransmitters [START_REF] Hirase | Volume transmission signalling via astrocytes[END_REF][START_REF] Kastanenka | A roadmap to integrate astrocytes into Systems Neuroscience[END_REF] . The different dynamics and connectivity of astro-neuronal networks provide new insights in the regulation of brain functions and ultimately animal behavior [START_REF] Oliveira | Do stars govern our actions? Astrocyte involvement in rodent behavior[END_REF] , with a complexity yet to be unraveled [START_REF] Verkhratsky | Physiology of Astroglia[END_REF][START_REF] Bazargani | Astrocyte calcium signaling: the third wave[END_REF][START_REF] Volterra | Astrocyte Ca(2+) signalling: an unexpected complexity[END_REF] .

From a larger perspective, our results further confirm that astrocytes are functionally diverse and can regulate central nervous system functions in a network-specific manner [START_REF] Khakh | The Emerging Nature of Astrocyte Diversity[END_REF][START_REF] Haim | Functional diversity of astrocytes in neural circuit regulation[END_REF][START_REF] Khakh | Diversity of astrocyte functions and phenotypes in neural circuits[END_REF] . Therefore, the study of astrocyte-neuron interactions is essential for a better comprehension of both physiological and pathological mechanisms affecting brain function [START_REF] Verkhratsky | Physiology of Astroglia[END_REF][START_REF] Khakh | Diversity of astrocyte functions and phenotypes in neural circuits[END_REF][START_REF] Filous | Targeting astrocytes in CNS injury and disease: A translational research approach[END_REF] .

Given that oxytocin modulates emotions and behaviors through a variety of actions on the peripheral and central nervous system 2,62-64 , our study adds a novel mechanism of oxytocin action: the modulation of astrocyte-neuron interactions. This mechanism underpins the oxytocinergic promotion of a positive emotional state, opening up new perspectives for studying the role of astroglial-neuronal networks in the modulation of emotional experiences [START_REF] Oliveira | Do stars govern our actions? Astrocyte involvement in rodent behavior[END_REF][START_REF] Bender | Astrocyte plasticity induced by emotional stress: A new partner in psychiatric physiopathology?[END_REF] , notably by neuropeptidergic systems [START_REF] Meyer-Lindenberg | Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine[END_REF] . On a broader scope, our present results demonstrate that the astroglia-mediated effects of oxytocin provide a new basis for the exploration of pathophysiologic mechanisms and enlighten a new vision of interplay between neuropeptides and glia that bears a general value for a better understanding of functional brain organization. This opens up new possibilities of research about the neuronal/glial substrates underlying positive emotional states, which is highlighted in our working model (Figure 7). The development of prospective treatments for somatic and mental diseases in humans may indeed be helped by targeting the oxytocin or other neuromodulators systems, and their joint action on multiple CNS cell types.

distribution of neurons in culture. Integr. Biol. 3, 1167 (2011). S1). S2). S3). S4). S5). S6). # P < 0.001, ## P < 0.01 Wilcoxon signed rank test; * P < 0.05, ** P < 0.01, *** P < 0.001; ANOVA or mixed-design ANOVA followed by posthoc Bonferroni test (Statistics in Table S6).

FIGURE TITLES AND LEGENDS

MATERIALS AND METHODS

KEY RESOURCES TABLE

All the reagents used are listed in the Supplementary Table 7 CONTACT FOR REAGENTS AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Alexandre Charlet (acharlet@unistra.fr). The requests for viral vectors should be addressed to Valery Grinevich (valery.grinevich@zi-mannheim.de).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

Animals were housed under standard conditions with food and water available ad libitum and maintained on a 12-hour light/dark cycle and all experiments were conducted in accordance with EU rules and approbation from French Ministry of Research (01597.05). For ex vivo and in vivo experiments, Wistar rats or C5BL/6 mice were used. Ex vivo experiments used animals between 18 and 25 days old, except in experiments where rAAVs were injected, in which case animals were between 2 and 6 months old at the time of sacrifice. In vivo experiments used 2-month-old animals at the time of the first surgery.

Cloning and Production of rAAV Vectors:

The generation of rAAVs allowing for the specific expression of the protein of interest in OT-cells is described in our previous work [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF] . Briefly, the conserved promoter region of 2.6 kb was chosen using the software BLAT from UCSC (http://genome.ucsc.edu/cgi-bin/hgBlat), was amplified from BAC clone RP24-388N9 (RPCI-24 Mouse, BACPAC Resources, CHORI, California, USA) and was subcloned into a rAAV2 backbone carrying an Ampicillin-resistance.

To construct the OTp-C1V1(t/t)-TS-mCherry AAV vector we used previously cloned OTp-DIO-GFP-WRE plasmid 2 equipped with the characterized 2.6 kb OT promoter [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF] . In this plasmid the DIO-GFP sequence was replaced by C1V1(t/t)-TS-mCherry from the rAAV CaMKIIa-C1V1(t/t)-TS-mCherry (Addgene, plasmid #35500).

To generate GfaP-C1V1(t/t)-TS-mCherry AAV vector, we replaced the CamKIIa promoter from the rAAV CaMKIIa-C1V1(t/t)-TS-mCherry by the Gfa promoter from the pZac2.1 gfaABC1D-tdTomato (Addgene, plasmid: 44332). The cell type specificity of the rAAV carrying the Gfa promoter was recently confirmed [START_REF] Shigetomi | Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses[END_REF] . In analogy, the generation of the GFAP-GFP-IRES-Cre vector was achieved using pZac2.1 gfaABC1D-tdTomato (Addgene, plasmid: 44332). First, the promoter was cloned into a rAAV2 backbone and sticky ends were blunted with EcoR1 and Basrg1. Next, pAAV-CamKIIa-C1V1(t/t)-TS-mCherry was blunted using BamHI and BsrGI. Finally, the pBS-ires cre construct was used and IRES-Cre was inserted into the GFAP-driven vector resulting in the GFAP-GFP-IRES-Cre construct.

Production of chimeric virions (recombinant Adeno-associated virus 1/2; rAAV 1/2) was described in [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF] . Briefly, human embryonic kidney cells 293 (HEK293; Agilent #240073) were calcium phosphate-transfected with the recombinant AAV2 plasmid and a 3-helper system. rAAV genomic titers were determined with QuickTiter AAV Quantitation Kit (Cell Biolabs, Inc., San Diego, California, USA) and are ~10 ^13 genomic copies per ml for all rAAV vectors used in this study.

METHOD DETAILS

Surgeries

Neuropathic Pain Model: Spared Nerve Injury (SNI) Procedure Animals were randomly separated in two groups to undergo either posterior left hindpaw SNI or sham procedure, with right hindpaw untouched. Animals were anaesthetized using isoflurane at 1.5-2.5%. Incision was made at mid-thigh level using the femur as a landmark and a section was made through the biceps femoris. The three peripheral branches (sural, common peroneal and tibial nerves) of the sciatic nerve were exposed. Both tibial and common peroneal nerves were ligated using a 5.0 silk suture and transected. The sural nerve was carefully preserved by avoiding any nerve stretch or nerve contact [START_REF] Decosterd | Spared nerve injury: an animal model of persistent peripheral neuropathic pain[END_REF] . For animals undergoing sham surgery, same procedure was performed but nerves remained untouched. Animals were routinely observed daily for 7 days after surgery and daily tested by the experimenter (Figure S4). Besides observing weight, social and individual behavior, the operated hindpaw was examined for signs of injury or autotomy. In case of autotomy or suffering, the animal was euthanized in respect of the ethical recommendations of the EU. No analgesia was provided after the surgery in order to avoid interference with chronic pain mechanisms and this is in accordance with our veterinary authorization. Suffering was minimized by careful handling and increased bedding.

Stereotaxic Surgery: Injections of rAAV Vectors

Stereotaxic surgery was performed under deep ketamine-xylazine anesthesia, using the KOPF (model 955) stereotaxic system. For specific control of rats CeA astroglial cells, 200 nl of rAAV serotype 1/2 (GFAPp-C1V1TSmCherry, Cloned from plasmids #35500 and 44332, Addgene) were injected bilaterally at the coordinates corresponding to CeL: rostro-caudal: -2.7mm, mediolateral: 4.2mm, dorso-ventral: -8.0mm (From Paxinos and Watson Atlas). For specific control of OT neurons, 200 nl of rAAV serotype 1/2 (OTp-C1V1TSmCherry or OTp-ChR2mCherry) were injected bilaterally at the coordinates corresponding to each hypothalamic OT nuclei. PVN: rostrocaudal: -1.8mm; medio-lateral: +/-0.4mm; dorso-ventral: -8.0mm; SON: rostro-caudal: -1.4mm; medio-lateral: +/-1.6mm; dorso-ventral: -9.0mm; AN: rostro-caudal: -2mm; medio-lateral: +/-1.2mm; dorso-ventral: -8.5mm (From Paxinos and Watson Atlas). For specific deletion of OTR in mice CeL astrocytes, 280 nl of rAAV serotype 1/2 (GFAPp-GFP-IRES-Cre) were injected bilaterally at the coordinates corresponding to CeL: rostro-caudal: -2.7mm, medio-lateral: 4.2mm, dorso-ventral: -8.0mm (From Paxinos and Watson Atlas) in OTR-cKO mice.

Stereotaxic Surgery: intra-CeL Cannulae

Cannulae Implantation. Animals were bilaterally implanted with guide cannulae for direct intra-CeL infusions. As guide cannulae we used C313G/Spc guide metallic cannulae (Plastics one, VA, USA) cut 5.8 mm below the pedestal. For this purpose, animals were deeply anesthetized with 4% isoflurane and their heads were fixed in a stereotaxic frame. The skull was exposed and two holes were drilled according to coordinates that were adapted from brain atlas (rat, 2.3 mm rostrocaudal; 4 mm lateral; 7.5 mm dorso-ventral relative to bregma; mice, 1.4 mm rostro-caudal; 2.6 mm lateral; 4.3 mm dorso-ventral relative to bregma) by comparing the typical bregma-lambda distance with the one measured in the experimental animal. Two screws were fixed to the caudal part of the skull in order to have an anchor point for the dental cement. Acrylic dental cement was finally used to fix the cannulae and the skin was sutured. In case of long lasting experiments (neuropathy-induced anxiety) with a cannula implantation at distance of the behavioral assay (> 4 weeks), cannulae were sometimes lost or cloaked, and concerned animals therefore excluded from testing.

Drugs Infusions. We used bilateral injections of 0.5 μl containing either vehicle (NaCl 0.9%) or oxytocin agonist TGOT (1 μM) dissolved in NaCl 0.9%. For this procedure two injectors (cut to fit 5.8 mm guide cannulae protruding 2 to 2.5 mm beyond the lower end of the cannula in older animals and 1.8 mm in 3-4 week old rats) were bilaterally lowered into the guide cannula, connected via polyten tubing to two Hamilton syringes that were placed in an infusion pump and 0.5 μl of liquid was injected in each hemisphere over a 2-minute-period. After the injection procedure, the injectors were kept in place for an additional minute in order to allow a complete diffusion of liquid throughout the tissue. Rats were subsequently left in the home cage for 15 minutes to recover from the stress of the injection and then handled for mechanical pain threshold or anxiety assessment. Animals that received TGOT injections for the first experiment (mechanical sensitivity assessment) were switched to the vehicle injected groups for the elevated plus maze experiment.

Stereotaxic Surgery: intra-CeL Optical Fiber

Optical Fiber Implantation. Sham and rAAVs injected animals both underwent a single surgical procedure in which after vector injection or no injection for sham, optical fibers designed to target the CeL were implanted and firmly maintained on the skull using dental cement. See "cannulae implantation" for the surgical procedure. Implantable optical fibers were homemade using optical fiber cut at appropriate length (FT200EMT, Thorlabs, NJ, USA) inserted and glued using epoxy based glue in ferrules (CFLC230-10, Thorlabs, NJ, USA).

Horizontal and Coronal Slices

Slices Preparations. In all cases, animals were anaesthetized using ketamine (Imalgene 90 mg/kg) and xylazine (Rompun, 10 mg / kg) administered intra-peritoneally. Transcardial perfusion was then performed using one of the following artificial cerebro-spinal fluids (ACSFs) dissection solutions. For animals between 18 and 25 days old, an ice-cold sucrose based dissection ACSF was used containing (in mM): Sucrose (170), KCl (2.5), NaH2PO4 (1.25), NaHCO3 (15), MgSO4 (10), CaCl2 (0.5), HEPES (20), D-Glucose (20), L-ascorbic acid (5), Thiourea (2), Sodium pyruvate (3), N-acetyl-L-cysteine (5), Kynurenic acid (2). For animals between 2 and 6 months old, an icecold NMDG based ACSF was used 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), Kynurenic acid (2). In both cases, pH was adjusted to 7.4 using either NaOH or HCl, this after bubbling in 95% O2-5% CO2 gas, bubbling which was maintained throughout the duration of use of the various ACSFs. Those ACSFs formulae were based on the work of [START_REF] Ikegaya | Large-scale imaging of cortical network activity with calcium indicators[END_REF] . Following decapitation, brain was swiftly removed in the same ice-cold dissection ACSFs as for transcardial perfusion, and 350 µm thick horizontal slices containing the CeA obtained using a Leica VT1000s vibratome. For experiments in Figure S1E, coronal slices of the same thickness containing the PVN were used. Upon slicing, brain slices were hemisected and placed, for 1 hour minimum before any experiments were conducted, in a room tempered holding chamber, containing normal ACSFs. For 2 to 6 month old animals, slices were first let for 10 minutes in 35°C NMDG ACSF before placing them in the holding chamber at room temperature. Normal ACSF, also used during all ex vivo experiments, is composed of (in mM): NaCl (124), KCl (2.5), NaH2PO4 (1.25), NaHCO3 (26), MgSO4 (2), CaCl2 (2), D-Glucose (15), adjusted for pH values of 7.4 with HCL or NaOH and continuously bubbled in 95% O2-5% CO2 gas. All ACSFs were checked for osmolality and kept for values between 305-310 mOsm/L. In electrophysiology or calcium imaging experiments, slices were transferred from the holding chamber to an immersion recording chamber and superfused at a rate of 2 ml/min with normal ACSFs unless indicated otherwise.

Drug Application. OTR agonists were bath applied through a 20s long pumping of agonist solution, corresponding to several times the volume of the recording chamber. Other drugs (antagonists, TTX, channel blockers etc.) were applied for at least 20 minutes in the bath before performing any experiments. BAPTA (or BAPTA-free solution for controls) loading of CeL astrocytes was realized following [START_REF] Serrano | GABAergic Network Activation of Glial Cells Underlies Hippocampal Heterosynaptic Depression[END_REF] protocol. Two distant CeL astrocytes per slice (label with SR101, 1 µM) were patched in whole cell configuration and voltage steps were applied (2 Hz, ∆40 mV) to help loading the BAPTA contained in the patch pipette (in mM): MgCl2 (1), NaCl (8), ATP (2) GTP (0.4) HEPES (10), BAPTA (40) and osmolality checked to be between 275-285 mOsm/l. The whole cell configuration was maintained during 45 min to allow BAPTA diffusion into the astrocytes syncytium.

Calcium Imaging and Identification of Astrocytes

To identify astrocytes, SR101 (1 μM) was added to ACSF in a culture well and slices were incubated for 20 minutes at 35°C. The specificity of SR101 labelling to astrocytes of the CeL was verified through patch-clamp experiments, the results of which can be found in Figure S2a-c. After SR101 loading, the synthetic calcium indicator OGB1 or Rhod-2 was bulk loaded following an adapted version of the method described previously [START_REF] Ikegaya | Large-scale imaging of cortical network activity with calcium indicators[END_REF] reaching final concentrations of either 0.0025 % (~20 μM) for OGB1 or 0.0025 % (~20 μM) for Rhod-2, 0.002% Cremophor EL, 0.01 % Pluronic F-127 and 0.5% DMSO in ACSF, and incubated for 45 to 60 minutes at 38°C. Upon incubation time, slices were washed in ACSF for at least an hour before any recording was performed. Astrocytes recorded for this study were then those co-labeled, in rats for SR101 and OGB1 and in mice for GFP and Rhod2. The spinning disk confocal microscope used to perform astrocyte calcium imaging was composed of a Zeiss Axio examiner microscope with a 40x water immersion objective (numerical aperture of 1.0), mounted with a X-Light Confocal unit -CRESTOPT spinning disk. Images were acquired at 2Hz with either a Rolera em-c² emCCD or an optiMOS sCMOS camera (Qimaging, BC, Canada). Cells within a confocal plane were illuminated for 100 to 150 ms for each wavelength (SR101 and Rhod-2: 575 nm, OGB1 and GFP: 475 nm) using a Spectra 7 LUMENCOR. The different hardware elements were synchronized through the MetaFluor software (Molecular Devices, LLC, Ca, USA) which was also used for online and offline quantitative fluorescence analysis. Astrocytic calcium levels were measured in hand drawn ROIs comprising the cell body plus, when visible, proximal processes, without distinction. In all recordings, the Fiji rolling ball algorithm was used to increase signal/noise ratio.. Absolute [Ca 2+ ]i variation were estimated as changes in fluorescence signals over the baseline (∆F/F). Baseline was established for each ROI as the average fluorescence over all pictures. To take into account micro-movements of the specimen on long duration recordings, the ∆F/F values were also calculated for SR101 / GFP and subsequently subtracted to the ones of OGB1 / Rhod2, except in the case of Figure 1f-h, where astrocytes were identified through SR101 fluorescence after the recordings, to avoid unwanted stimulation of the C1V1(t/t) opsin. On this last case, recordings in which movements / drifts were visible were discarded. Bleaching was further corrected by fitting a linear regression on the overall ∆F/F trace for each ROI, which values were then subtracted to the ∆F/F. Upon extraction of data, calculations and corrections of ∆F/F for each astrocyte, the area under the curve before and after OTR agonists' application (or light pulse for optogenetics experiments) were calculated and proportionally corrected relative to the different baseline and after stimulation recording durations. An astrocyte was considered as being responsive if the relative ratio of AUCs after drug/light application over baseline was higher or equal to a 20% increase. The relative AUCs ratios values of responding astrocytes were used for quantitative analysis and called "relative AUC increase", and in corresponding histograms the coordinate indicated as a 0 corresponds to a ratio of 1 (meaning neither increase nor decrease of the AUC). The maximal peak reached after drug/light application was also measured in responsive cells and used in quantitative analysis shown in various figures. Data were averaged across all responding astrocytes per slice, which were used as the statistical unit. For the response probability, calcium events were detected as an increases of fluorescence > 5% with a rising slope > 0,025 and a minimum inter-peak distance of 10 seconds. Then, the response probability was calculated as the number of astrocyte with a least 1 calcium event per time bin (30s) divide per the number of astrocyte in each recording. Image J software was also used on SR101 / OGB1 pictures to produce illustrative pictures. Finally, all calcium imaging experiments were conducted at controlled room temperature of 26°C.

Electrophysiology

Whole cell patch-clamp recordings of CeL neurons, CeL astrocytes and CeM neurons were visually guided by infrared oblique light visualization of neurons and completed by SR101 fluorescence observation for astrocytes. Patch-clamp recordings were obtained with an Axon MultiClamp 700B amplifier coupled to a Digidata 1440A Digitizer (Molecular Devices, CA, USA). Borosilicate glass electrodes (R = 3.5 -7 MΩ) with inner filament (OD 1.5 mm, ID 0.86 mm; Sutter Instrument, CA USA) were pulled using a horizontal flaming/brown micropipette puller (P97; Sutter Instrument, CA, USA). Recordings were filtered at 2 kHz, digitized at 40 kHz and stored with the pClamp 10 software suite (Molecular Devices; CA, USA). Analysis of patch-clamp data were performed using Clampfit 10.7 (Molecular Devices; CA, USA) and Mini analysis 6 software (Synaptosoft, NJ, USA) in a semi-automated fashion (automatic detection of events with chosen parameters followed by a visual validation).

Whole-Cell Recording of CeL Neurons and Astrocytes.

Recording pipettes were filled with an intracellular solution containing (in mM): KMeSO4 (125), CaCL2 (2), EGTA (1), HEPES (10), ATPNa2 (2), GTPNa (0.3). The pH was adjusted to 7.3 with KOH and osmolality checked to be between 290-295 mOsm/l, adjusted with sucrose if needed. All neurons were hold at a membrane potential of -65 mV, astrocytes -80 mV. Series capacitances and resistances were compensated electronically throughout the experiments using the main amplifier. For SICs measurements in CeL neurons (Figure 3, 4, S3, S4) , whole cell recordings were conducted in a Mg 2+ free ACSF, also containing biccuculin (10 µM) and TTX (1 µM) as in [START_REF] Shigetomi | Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons[END_REF] . Currents were categorized as SIC when their rise time (10% to 90%) and decay tau were strictly superior to 8 and 30 ms, respectively. Average SICs frequencies per cell was calculated over 10 minutes periods during basal condition and 10 minutes periods after the bath application of TGOT. CeL neurons were classified as TGOT-responsive when the average SIC frequency was increased by at least 30% after TGOT application when compared to its baseline.

Whole-cell Recording of CeM Neurons.

Pipettes were filled with an intracellular solution containing (in mM): KCl (150), HEPES (10), MgCl2 (4), CaCl2 (0.1), BAPTA (0.1), ATP Na salt (2), GTP Na salt (0.3). pH was adjusted to 7.3 with KOH and osmolality checked to be between 290-295 mOsm/L, adjusted with sucrose if needed. All cells were hold at a membrane potential of -70 mV. Series capacitances and resistances were compensated electronically throughout the experiments using the main amplifier. Average events frequencies per cell were calculated on 20s windows, chosen for TGOT or light stimulation during maximal effect, as determined by the visually identified maximal slope of the cumulative plot of the number of events. CeM neurons were classified as TGOT-responsive when the average IPSCs frequency was increased by at least 20% during at least 10s and up to 500s after TGOT application when compared to baseline average frequency. Baseline and recovery frequencies were measured respectively at the beginning and end of each recording. All patch-clamp experiments were conducted at room temperature.

Immunohistochemistry and in situ Hybridization

In situ Hybridization for OTR mRNA in Rat CeL. The probe for OTR mRNA was in vitro transcripted from a 902-bp fragment containing 133-1034 bases of the rat OTR cDNA (NCBI Reference Sequence: NM_012871.3) subcloned into pSP73 Vector (Promega). The digoxigenin (DIG)-labeled antisense and sense RNA probe from the linearized oxtr cDNA template was synthesized using DIG RNA Labeling Kit (SP6/T7) (Roche Diagnostics). Sections containing 2 consecutive sections of the CeL (corresponding to Bregma: 2.5) were processed for fluorescent in situ hybridization (FISH). Rats were transcardially perfused with PBS followed by 4% PFA. Brains were dissected out and post fixed overnight in 4% PFA at 4 °C with gentle agitation. 50 µm vibratome sections were cut, collected and fixed in 4% PFA at 4°C overnight. The free-floating sections were washed in RNase-free PBS, immersed in 0.75% glycine in PBS, treated with 0.5 μg/ml proteinase K for 30 min at 37 °C, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, and then hybridized with DIG-labeled RNA probe overnight at 65 °C. After RNase treatment and following intensive wash, the hybridized DIG-labeled probe was detected by incubation with Anti-Digoxigenin-POD (1:200; 11207733910; Roche Diagnostics) for 3 days at 4 °C. Signals were developed with tyramid signal amplification method. Rhodamine-conjugated tyramide was synthesized by coupling NHS-Rhodamine (Pierce Biotechnology, Thermo Fisher Scientific) to Tyramine-HCl (Sigma-Aldrich) in dimethylformamide with triethylamine. For the quantification of OTR mRNA-positive astrocytes, all confocal images were obtained using the same laser intensities and processed with the same brightness / contrast settings in Adobe Photoshop. Since the in situ signal for the OTR mRNA in astrocytes was weak, we first calculated the average intensity (signal intensity of all pixels divided by the total number of pixels) of the rhodamine-stained OTR mRNA signal for each individual section containing the CeL. Next, we calculated the standard deviation for each individual confocal image based on the intensity of all pixels comprising the image. We defined the threshold for OTR mRNA-positive astrocytes: If more than 1/4 of all pixels comprising an astrocyte soma displayed a signal intensity exceeding the average background intensity by more than 4-times the standard deviation, the astrocytes were considered as OTR mRNA-positive.

Astrocytes Markers. The aldehyde dehydrogenase 1 antibody is a commonly used marker for glial cells, including astrocytes. Therefore, we used the ALDH1L1 for immunohistochemistry in our initial experimental studies (Figure S1,S3). However, due to inconsistencies in staining quality as a result of batch-dependent antibody properties, especially in combination with the OTR mRNA FISH, we decided to employ Glutamine Synthetase (GluSyn, Figure 1a). GluSyn is a commonly used glial marker [START_REF] Anlauf | Glutamine synthetase as an astrocytic marker: its cell type and vesicle localization[END_REF] , which stains astrocyte cell bodies, faint processes and even astrocytes not expressing GFAP. Using GluSyn, we achieved consistent results in combination with our OTR mRNA FISH. S1 was performed on 25-μm cryostat-cut coronal sections prepared from fresh-frozen mouse brain (male C57BL/6J, P22). After extraction, brains were immediately frozen in Tissue-Tek O.C.T. compound and stored at -80 degrees Celsius. ISH was performed according to the manufacturer's instructions (Advanced Cell Diagnostics) for Fresh

Glutamine Synthase, ALDH1L1

Frozen RNAscope Multiplex Fluorescent Assay. Treatment of amygdala containing sections were adjusted with the 3-plex negative control and then coexpression of OTR and GFAP examined using ACD designed target probes as well as the nuclear stain DAPI. Single plan images were collected with an upright laser scanning microscope (LSM-710, Carl Zeiss) using a 40x-objective with keeping acquisition parameters constant between control and probe treated sections.

Specific deletion of OTRs in CeL astrocytes.

To specifically ablate OTRs in CeA astrocytes, transgenic cKO mice, in which loxP sites flank the OTR coding sequence (Lee et al., 2008), received bilateral injections (280 nl) of rAAV-GFAP-GFP-IRES-Cre. Following four weeks of expression of the viral proteins, mice were transcardially perfused with 1x PBS and 4% PFA. Brain sections were used for FISH (OTR mRNA) and IHC (GluSyn) to verify the validity of the approach.

Representative images and quantifications are provided in Figure 2c.

AAV-Gfa-C1V1TSmCherry Specificity. After 3 weeks of vector expression in the brain, rats were transcardially perfused with 4% paraformaldehyde solution. Tissue blocks, containing CeA were dissected from the fixed brain and Vibratome-cut into 50 µm thick free-floating sections. After several rinse steps sampled sections were blocked with 5% NGS in PBS and incubated for 48 h at 4°C with polyclonal rabbit anti-ALDH1L1 antibody (1:500, Abcam) in 1% Triton-PBS buffer, containing 0,1 % NGS. Appropriate secondary antibody (AlexaFluor488 conjugated goat antirabbit (1:1000, LifeTechnologies) was used for further antigene detection. Intrinsic mCherry fluorescence of vector-expressing cells was strong enough to detect them in the tissue without any additional antibody enhancement. The immunolabeled sections were mounted onto Superfrost slides, cover-slipped with Moviol, analyzed and documented using LEICA SP5 confocal microscope.

Biocytin Filling of Astrocytes. In the lateral part of the central amygdala slices visualized with infrared-differential contrast optics, astrocytes were identified by their morphological appearance and the absence of action potentials in response to depolarizing current injections. Cells were patched with pipettes filled with (in mM) 110 K-gluconate, 30 KCl, 4 ATP-Mg, 10 phosphocreatine, 0.3 GTP, and 10 HEPES (pH: 7.3; 310 mOsm). Concentration of biocytin was 2 mg/ml. After obtaining whole-cell configuration astrocytes were hold at -80 mV and typical filling time was 30 minutes. Then the pipettes were carefully retracted and slices were incubated for additional 20 minutes in the oxygenated ACSF before fixation. Only one cell was filled per slice. Slices with filled cells were immersion-fixed at 4°C for 5 days in 4% PFA-PBS solution. Next, the slices were flatembedded in 6% Agar-PBS, areas of interest were cut out of, re-embedded onto the Agar block and Vibratome-cut into 80 µm thick free-floating sections. The sections then were incubated with Avidin conjugated to Alexa Fluor488 (1:1000) (Thermo Fisher) in 1% Triton-PBS at 4°C, washed in PBS, mounted and cover-slipped. The tissue was analyzed and images taken at Leica TCS SP5 Confocal Microscope.

Optogenetics

Ex vivo. To elicit evoked activation of OT axons in combination with CeL or CeM neurons patchclamp, we used ChR2 expression in OT cells, as in [START_REF] Knobloch | Evoked axonal oxytocin release in the central amygdala attenuates fear response[END_REF] . Optical blue-light illumination of the CeL oxytocinergic axons expressing ChR2 was performed using light source XCite® 110LED from Excelitas Technologies through a GFP filter, controlled with Clampex driven TTL pulses for 20s at 30Hz with 10ms long pulses. To elicit light-evoked activation of OT axons in combination with astrocyte calcium imaging, or to elicit intracellular calcium increase in astrocytes, we used C1V1(t/t), a ChR1/VChR1 chimera with the combined mutations E122T/E162T (for more details, see [START_REF] Yizhar | Neocortical excitation/inhibition balance in information processing and social dysfunction[END_REF] . Optogenetic green light stimulation of C1V1(t/t) in ex vivo experiments was performed using either the Spectra 7 LUMENCOR (λ542 nm) or light source X-Cite® 110LED from Excelitas Technologies through a Cy3 filter, controlled via MetaFluor or Clampex driven TTL pulses, respectively.

In vivo. Animals were habituated to the fixation of an optical fiber on the ferrule without light stimulation for one week before the experiment. In all cases, optical fibers were attached to the ferrules using an adapter (ADAF2, Thorlabs, NJ, USA) and animals let free to move in a typical home cage for the duration of the stimulation. Implanted optical fibers were connected to two lasers (LRS-0532-GFM-00100-03 LaserGlow 532nm DPSS Laser System) and the output power adjusted to correspond to 20 to 30 mW measured at the tip of 200 µm diameter fibers similar to the one implanted. Stimulation of 500 ms duration at a frequency of 0.5Hz were given for 3 min.

Behavior

Mechanical Sensitivity Assessment.

In experiments with rats, we used a calibrated forceps (Bioseb, Chaville, France) previously developed in our laboratory to test the animal mechanical sensitivity [START_REF] Luis-Delgado | Calibrated forceps: A sensitive and reliable tool for pain and analgesia studies[END_REF] . Briefly, the habituated rat was loosely restrained with a towel masking the eyes in order to limit stress by environmental stimulations. The tips of the forceps were placed at each side of the paw and a graduate force applied. The pressure producing a withdrawal of the paw, or in some rare cases vocalization, was considered as the nociceptive threshold value. This manipulation was performed three times for each hind paw and the values were averaged as being the final nociceptive threshold value. In experiments with mice, we used von Frey filaments tests. Mechanical allodynia (a symptom of neuropathic pain) was tested using von Frey hairs and results were expressed in grams. Tests were performed during the morning starting at least 2 h after lights on. Mice were placed in clear Plexiglas boxes (7 cm x 9 cm x 7 cm) on an elevated mesh floor. Calibrated von Frey filaments (Bioseb) were applied to the plantar surface of each hindpaw until they just bent in a series of ascending forces up to the mechanical threshold. Filaments were tested five times per paw and the paw withdrawal threshold (PWT) was defined as the lower of two consecutive filaments for which three or more withdrawals out of the five trials were observed.

Elevated Plus Maze. Following protocol from [START_REF] Walf | The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents[END_REF] , the arena is composed of four arms, two open (without walls) and two closed (with walls; rats 30 cm high; mice 15 cm high). Arms are 10 cm wide, 50 cm long and elevated 50 cm off the ground for rats and 5 cm wide, 30 cm long and elevetad 40 cm of the ground for mice. Two lamps with intensity adjustable up to 50 watts were positioned on the top of the maze, uniformly illuminating it. Animals were video tracked using a video-tracking systems (Ethovision Pro 3.16 Noldus, Wageningen, Netherlands and Anymaze, Stoelting Europe, Ireland). After each trial, the maze was cleaned with 70% ethanol and dry with paper towel. Twenty minutes after intracerebral injections or directly after optical stimulation, the animal was let free at the center of the plus maze, facing the open arm opposite to where the experimenter is, and was able to freely explore the entire apparatus for six minutes. Total time and time spend in closed and open arms were recorded in seconds and the percentage of time spent in closed arms was calculated as a measure of anxiety. As internal control, the total distance traveled during the test period was quantified and compared between all different groups (Figure S6a2-b2). Animals falling from the apparatus during the test, freezing more than 50% of the total time, or with cannulae/optic fiber issues, were removed from the analysis.

Conditioned Place Preference.

The device is composed of two opaque conditioning boxes (rats: 30x32 cm; mice: 22x22 cm) and one clear neutral box (30x20 cm) Animals were video tracked using a video-tracking system (Anymaze, Stoelting Europe, Irland). After each trial, the device was cleaned with a disinfectant (Surfa'Safe, Anios laboratory). Based on [START_REF] King | Unmasking the tonic-aversive state in neuropathic pain[END_REF] , all rats underwent a 3 days habituation period during which they were able to freely explore the entire apparatus for 30 min. On the day 3, behavior was record for 15min to verify the absence of pre-conditioning chamber preference. The time spend in the different compartment were measured and paired compartment was chosen as the compartment in which rat spent the less time during the 3rd day of habituation. On day 4, animals were placed the morning in one compartment for 15 min with no stimulation (unpaired box). Four hours after, the animal were placed 15min in the opposite box (paired box) and CeL astrocyte expressing C1V1 vector were optogenetically stimulated (3 min -500ms light pulse at 0.5 Hz -λ542nm) or TGOT micro-infused through intracerebral cannulae. On day 5, the animals were place in the CPP box and allowed to freely explore the entire apparatus during 15min. As internal control, the total distance traveled during the test period was quantified and compared between all different groups (Figure S6). Rats falling spending more than 80% of the total time in a single chamber before the conditioning, or with cannulae/optic fiber issues, were removed from the analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS Statistical Analysis

All parametrical statistical tests presented in figure captions were performed following correct verification of the assumptions on the distribution of data, and if not non-parametric tests were used. Tests were performed using either SPSS 23 (IBM) or statview 5 (SAS institute Inc.). All values, group compositions and statistical tests for each experiment and figure panel are detailed in Supplementary Tables 123456.

Figure 1 .

 1 Figure 1. CeL Astrocytes Express Functional Oxytocin Receptors. (a) Overview of CeA fluorescent in situ hybridization of OTR mRNA (red) and glutamine synthetase immunostaining (GluSyn, green). (b) High magnification images of cells positive for OTR mRNA and/or GluSyn (double arrows); green arrows point GluSyn positive cells; red arrows point OTR mRNA-positive cells. Scale bars 100 (a) and 10µm (b). (c) Proportion of CeL astrocytes (GluSyn positive cells, left) and neurons (NeuN positive cells, right) positive for OTR mRNA (red, hatched) compared to the total number of OTR mRNA positive cells (n astrocytes = 1185, n neurons = 1254, n rats = 3). (d) Experimental rat model for acute brain slice calcium imaging. (e) Experimental scheme of the horizontal CeA slice preparation used, showing C1V1(t/t) expressing OT axons (yellow) arising from PVN and projecting to the CeL. (f-h) Effect of the activation of OT axons in the CeL through λ542 nm light pulses (10 ms width, 30 Hz, duration 20 s) on calcium transients of CeL astrocytes. (f) (Left) Typical ΔF/F0 traces; (right) raster plot displaying calcium events per responding astrocyte. (g) (Left) Time course of the calcium events probability per bin of 20s; (right) Δ calcium events probability before and after light-driven activation of CeL OT axons. (h) (Top) Pie chart of the proportion of responsive astrocytes; (bottom) Relative increase of ΔF/F0 AUC over baseline after light-driven activation of CeL OT axons and maximal peak values reached for responsive astrocytes. Data are expressed as means across slices plus SEM (n slices (ns) = 6, n astrocytes (na) = 37). White circles indicate averages across responding astrocytes per slices. (Statistics in TableS1).

Figure 2 .

 2 Figure 2. OTR Activation Evokes Astrocytic Calcium Transients in the CeL

Figure 3 .

 3 Figure 3. CeL Astrocytes Increased Activity is Sufficient to Recruit the CeL→CeM Neuronal Circuit. (a) Experimental rat model. (b) Scheme of the experimental setup,

Figure 4 .

 4 Figure 4. CeL Astrocytes are Necessary for the CeL→CeM Neuronal Circuit Modulation by OTR Activation. (a) Experimental rat model and experiments. (Bottom

Figure 5 .

 5 Figure 5. CeL Astrocytes Recruit the CeL→CeM Neuronal Circuit through NMDAR modulation. (a) Experimental rat model. (b) Scheme of the experimental setup. Letters

Figure 6 .

 6 Figure 6. CeL Astrocytes are Sufficient to Modulate CeA Behavioral Correlates of

Figure 7 .

 7 Figure 7. Oxytocin-dependent Interactions in the CeA. Based on our findings we

  Figure 1

  Figure S1

Colocalization with OTR mRNA in Rat CeL.

  A27042, ThermoFischer Scientific) for 2 hours at RT. Following intensive washing with PBS, sections were mounted using Mowiol.

	Double
	After
	development and washing steps the sections were stained with antibodies against glutamine
	synthase (mouse monoclonal, 1:500, ref: MAB302, MerckMilipore), ALDH1L1 (rabbit polyclonal,

1:500, ref: ab87117, abcam), in PBS and kept at 4°C on a shaker in a dark room overnight. After intensive washing with PBS, sections were stained with the respective secondary antibodies AlexaFluor488 (goat anti-mouse, 1:1000, ref: A11001, life technologies) and AlexaFluor680 (goat anti-mouse, 1:1000, ref:

in situ Hybridizations for OTR mRNA and GFAP mRNA in Mice CeL

. Fluorescent in situ hybridization (FISH) in Figure

TABLE S1 : Numerical values and statistical analysis of data presented in Fig. 1 and S1.

 S1

		Figure	Statistical unit	Groups tested	Paired or		N	Unit Mean SEM	Test used	p-value
					unpaired
	Figure		Statistical unit	TGOT TGOT+TTX Groups tested	4 22 Paired or	55,74 40,00 N Unit Mean SEM 4,76 7,07	Independent Sample t-test Test used	0,191 p-value
	3c	TGOT TGOT+TTX Baseline astrocytes averaged per slices AUC ratio before/after treatment of responding 20s λ542nm light pulse SICs frequencies across neurons recorded before and after treatment	4 unpaired 22		Hz	2,25 1,89 0,8457 0,2363 0,14 0,13 1,517 0,3781
				TGOT Baseline			4			0,29 0,25 0,1256 0,02
	3d	2a APs frequencies across neurons recorded before and after treatment	TGOT+TTX TGOT Wash 20s λ542nm light pulse			22			0,26 0,3 40,00 10,00 0,06 0,1287 0,745 0,3044
				TGOT+dOVT TGOT TGOT+dOVT TGOT NeuN rAAV-GFAPp-C1V1-mCherry + ALDH1L1 rAAV-GFAPp-C1V1-mCherry + Baseline 20s λ542nm light pulse				2,78 1,25 0,88 0,25 0,000 0,000 2,78 0,09 0,01 323 % 98,452 0,802 318 % 0,24 1,955 0,358 3,368 0,635	0,0038
		rAAV-GFAPp-C1V1-mCherry efficiency on 9 sections	TGOT+dOVT ALDH1L1 + rAAV-GFAPp-C1V1-mCherry			0,29 517 % 62,300 3,500 0,17
	S3c	APs frequencies across neurons recorded before and after treatment	TGOT+TTX Baseline+BAPTA 20s λ542nm light pulse+BAPTA	Paired	22	12 12	Hz	55,74 0,5292 0,1274 4,76 0,6833 0,3054	Dunn's multiple comparisons test	0,0395
	S3d	IPSCs frequencies across neurons recorded before and after treatment	TGOT+TTX+PPADS TGOT+TTX Baseline+BAPTA 20s λ542nm light pulse+BAPTA	Paired	6 22	9 9	Hz	57,66 2,25 0,994 0,222 3,83 0,14 0,992 0,197	Holm-Sidak's multiple comparisons test	0,072
				TGOT+TTX+PPADS			6			2,11	0,23
				TGOT+TTX			22			0,29	0,02
				TGOT+TTX+PPADS			6			0,29	0,02
				TGOT+TTX			22			55,74	4,76
				TGOT+TTX+CBX			8			16,52	5,78
				TGOT+TTX			22			2,25	0,14
				TGOT+TTX+CBX			5			2,17	0,51
				TGOT+TTX			22			0,29	0,02
				TGOT+TTX+CBX			5			0,30	0,04
				OTR cKO + rAAV-GFAP-GFP	Unpaired	897		%	15,90	1,40	Independent Sample t-test	<0,0001
				OTR cKO + rAAV-GFAP-Cre-GFP	Unpaired	940		%	1,20	0,30	Independent Sample t-test	<0,0001
				TGOT in control mice			13			0,49	0,05
				TGOT in OTR cKO + rAAV-GFAPp-Cre-GFP			10			0,09	0,02
				TGOT in control mice			13			1,24	0,18
				TGOT in OTR cKO + rAAV-GFAPp-Cre-GFP			10			1,84	0,17
				TGOT in control mice			13			0,05	0,02
				TGOT in OTR cKO + rAAV-GFAPp-Cre-GFP			10			0,11	0,05
				Astrocytes			82			108,00 9,92
				Neurons			20			47,43	5,72
				Astrocytes			82			22,25	2,39
				Neurons			20			210,80 43,22
				Astrocytes			82			10,17	0,79
				Neurons			13			8,96	1,94
				Astrocytes			20			-85,32 1,13
				Neurons			12			-60,25 1,42

TABLE S3 : Numerical values and statistical analysis of data presented in Fig. 3 and S3.

 S3

	Figure Figure	Statistical unit Statistical unit	Groups tested Groups tested	Paired or Paired or	N Unit Mean N Unit Mean	SEM SEM	Test used Test used	p-value p-value
						unpaired unpaired
			Baseline		Baseline		7 7		1,10 1,100	0,5088 0,509
			TGOT Baseline+Bapta TGOT		7 8 7		2,50 0,90 2,500	0,6437 0,2478 0,644
			Bapta +TGOT TGOT Bapta +TGOT Baseline TGOT Baseline+BAPTA Ifenprodil Ifenprodil + TGOT Baseline AP5 Baseline		Paired 10 8 Paired 7 10 8 9 7 9 10 9 9	0,88 0,425 19 2,50 0,367 Hz 10 0,88 0,43 1,100 0,92 0,105 0,20 0,433	0,2698 0,084 0,6437 0,136 0,2698 0,509 0,1277 0,027 0,1732 0,0898 0,128	Dunn's multuple comparisons Wilcoxon signed rank test	0,0003 0,0064
	3e	IPSCs frequencies across neurons recorded before and after treatment Bapta +TGOT TGOT Bapta +TGOT	TGOT Baseline AP5 TGOT +AP5		Paired 9 9 9 7 9 7	0,12 0,917 0,92 0,464 9 Hz 0,12 0,407	0,173 0,0595 0,1732 0,108 0,0595 0,150	Wilcoxon signed rank test
					TGOT		9		0,917	0,173
	S3a	rAAV-GFAPp-C1V1-mCherry specificity on 9 sections TGOT Bapta +TGOT	AP5 + TGOT TGOT+DAAO TGOT+D-Serine		7 17 17		0,407 5,05 0,611 2,711	0,150 0,691 0,182 0,634
									1,31	0,351
			baseline in control mice		8		1,10	0,3397
	Figure	TGOT in control mice Statistical unit Groups tested	Paired or	8 N Unit Mean 1,71	0,3669 SEM	Test used	p-value
			Baseline in OTR cKO mice + rAAV-Cre-GFP TGOT+TTX TGOT in OTR cKO mice + rAAV-Cre-GFP TGOT+TTX+AP5	unpaired	5 22 5 4		0,76 55,739 4,764 0,1939 0,75 0,1921 82,292 10,259
			TGOT in control mice TGOT+TTX		8 22		1,71 2,253	0,3669 0,142
			TGOT in OTR cKO mice + rAAV-Cre-GFP TGOT+TTX+AP5		5 4		0,75 2,163	0,1921 0,138
			baseline in control mice TGOT+TTX		7 22		0,26 0,293	0,1071 0,021
			TGOT in control mice Baseline in OTR cKO mice + TGOT+TTX+AP5		7 4		0,82 0,290	0,1495 0,018
			rAAV-Cre-GFP TGOT1		11		0,35 8,900	0,0691 1,500
			TGOT in OTR cKO mice + rAAV-Cre-GFP TGOT2		11		0,40 6,007	0,1263 1,062
			TGOT in control mice TGOT		7		0,82 9,217	0,1495 3,028
			TGOT in OTR cKO mice + rAAV-TGOT+dOVT				1,383	0,328
			Cre-GFP Baseline in control mice TGOT		11 27		0,40 1,52 12,371 2,558 0,1263 0,2621
			TGOT in control mice TGOT+AP5		27		2,80 6,121	0,3876 1,018
			Baseline in OTR cKO mice + rAAV-Cre-GFP TGOT		16		1,72 11,300 1,336 0,3677
			TGOT in OTR cKO mice + rAAV-TGOT+DCKA				5,338	1,133
			Cre-GFP TGOT in control mice TGOT		16 27		1,93 1,52 7,316	0,4365 0,2621 1,811
			TGOT in OTR cKO mice + rAAV-TGOT+DNQX				5,268	1,395
			Cre-GFP				16		1,93	0,4365
	Figure	Statistical unit		Groups tested	Paired or	N Unit Mean	SEM	Test used	p-value
						unpaired
		10 to 90% rise time	Baseline						31,68	7,158
		of SICs across cells before and after				Paired	6	ms	Paired t-test	0,922
		treatment	TGOT						32,28	3,482
	S3c	decay Tau of SICs across cells before and after treatment	Baseline TGOT				6	ms	113,78 25,468 217,26 68,062	Wilcoxon signed rank test	0,116
		amplitude of SICs	Baseline						-27,79	7,134
		across cells before				Paired	6	pA	Paired t-test	0,141
		and after treatment	TGOT						-17,22	1,304

TABLE S5 : Numerical values and statistical analysis of data presented in Fig. 5 and S5.

 S5

		Figure	Statistical unit	Groups tested		Time	Paw Unit	N	Mean	SEM	Test used	p-value
						CTRL		12	79,194	1,649	Time	<0,05
				Paired SNI : Vehicle		0.3h 3.5h		Wilcoxon signed 12 79,278 12 79,417 rank test	0,0156 1,876 2,315	Time*Group Time*Paw	<0,05 0,023
	5c	SICs frequencies across CeL neuron before and after treatment	Paired		n/min 24h CTRL		Wilcoxon signed 9 80,296 rank test 10 75,167	0,5703 2,956 2,616	Time*Group*Paw	<0,05
				Unpaired SNI : TGOT		3.5h 0.3h		Independent Mann-10 99,167 Whitney U test 10 122,633	3,662 0,0409 4,530
				Paired		Hz 24h		Wilcoxon signed rank test 9 76,963	0,0039 2,443
	5d	APs frequencies across CeL neuron before and after treatment	Paired Sham : Vehicle		Hz CTRL 0.3h		12 Wilcoxon signed 209,722 12 205,556 rank test	1,915 0,5313 1,614
				Unpaired		Hz 3.5h		Mann Whitney test 12 206,028	0,0122 2,017
	5e	IPSC frequencies across neurons recorded before and after	Paired	9	Hz 24h CTRL		Wilcoxon signed rank test 10 206,233 11 208,467	0,0076 3,004 2,189
				Sham : TGOT		0.3h		11	215,100	1,531
						3.5h		11	211,000	1,705
						24h		10	212,741	4,004
						CTRL		6	244,250	14,910	Time	0,001
		proportion of responding astrocytes averaged per slices	Unpaired Sham : C1V1		% 15 min 0 min		Independent Sample t-test 6 220,833 6 234,333	14,790 0,037 17,736	Time*Paw Time*Group	0,054 0,007
	S5a	AUC ratio before/after treatment of responding astrocytes averaged per slices	Unpaired		ratio 45 min CTRL		Independent Sample t-test 6 208,000 10 79,233	0,795 17,092 6,104	Time*Group*Paw	0,862
		MAX value reached after treatment of responding astrocytes averaged per slices IPSCs frequencies under TGOT	Unpaired SNI : C1V1		45 min 15 min % 0 min		10 t-test 10 10 Independent Sample 80,000 79,733 82,267	2,774 0,941 2,708 3,183
		across neurons recorded before	Paired	7	Hz CTRL		Paired t-test 9 73,815	3,517
	S5b	and after treatment across neurons recorded before IPSC frequencies under TGOT	SNI : Ctrl C1V1	3	Hz 15 min 0 min		9 9	83,148 79,667	0,0529 1,441 3,821
		and after treatment				45 min		9	80,222	2,180
	S5c	IPSC frequencies under TGOT across neurons recorded before rat threshold for pain per group S6a1 at different time points before and after treatment IPSC frequencies under TGOT and after drug(s) injection	SNI : Vehicle Paired	7	Hz CTRL 0.3h 3.5h	Right g	Paired t-test 12 216,139 12 213,139 12 212,361	0,0308 1,774 2,015 2,283
		across neurons recorded before	Paired	12 Hz		Paired t-test	<0.001
		and after treatment
	S5d	IPSC frequencies under TGOT across neurons recorded before and after treatment	Paired	8	Hz		Dunn's multiple comparisons	>0.05

TABLE S6 : Numerical values and statistical analysis of data presented in Fig. 6 and S6.

 S6

	Figure Figure Figure Figure Figure Figure	Statistical unit Statistical unit Statistical unit Statistical unit Statistical unit Statistical unit	Groups tested Groups tested Groups tested Groups tested Groups tested Groups tested	Time Time Paired Time Time Time	Paw Unit Paw Unit N Unit Paw Unit Paw Unit Paw Unit	N N Mean N N N	Mean Mean SEM Mean Mean Mean	SEM SEM Test used SEM SEM SEM	Test used Test used p-value Test used Test used Test used	p-value p-value p-value p-value p-value
		IPSC frequencies under TGOT in sham and SNI animals	SNI : Vehicle + Ctrl C1V1 Sham TGOT SNI OTR cKO mice + Sham OTR cKO mice + Vehicule rAAV-Cre-GFP -> TGOT rAAV-Cre-GFP ->	24h CTRL Unpaired 9 CTRL	Hz	9 10 20 7,083 1,357 214,037 213,433 956,437 6 5,333333 4 -153 5 12,23	2,370 2,471 297,666 Independent Sample t-test 0,421637 178,1 0,3877	Mixed-design ANOVA with Bonferroni Mixed-design ANOVA 0,592	Time Time*Group Treatment 0,0378	<0,05 <0,05 >0.05
	S6a2 6c2 S4a S6b2	total distance per rat basal IPSC frequencies in sham and SNI animals Delta time spent in the paired chamber per mice IPSC frequencies before and after TGOT in SNI animals total distance per mice	SNI : TGOT Sham : Vehicle SNI : TGOT Sham : Vehicle Sham : TGOT Sham : C1V1 SNI : C1V1 SNI TGOT Sham Sham control mice -> TGOT Sham control mice -> Vehicule SNI OTR cKO mice + rAAV-Cre-GFP -> TGOT SNI Baseline TGOT SNI OTR cKO mice + SNI control mice -> Vehicule Sham control mice -> TGOT SNI control mice -> TGOT Sham OTR cKO mice +	0.3h 3.5h 24h CTRL 0.3h 3.5h Unpaired 0 min Paired 15 min 45 min	9 16 Left 16 9	Hz m g s Hz m	10 10 9 12 12 12 9 10 8 9 7 6,639 2,183 213,267 213,433 212,296 207,222 207,278 207,417 1076,448 1154,124 1233,782 1088,825 1174,686 4,322 1,461 6 5,333333 5 -19,36 7 12,68 2,866 0,595 2,167 0,677 6,639 2,183 6 5 6 5 7 12,13 8 338,1 8 13,78	1,695 2,231 2,846 1,543 2,055 1,970 94,766 268,971 156,472 338,957 566,612 Independent Sample t-test 0,421637 31,57 0,8074 Paired t-test 0,4472136 0,4472136 0,3323 95,74 1,144	Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons adjustment for multiple comparisons 0,484 Kruskal-Wallis test with Dunn's multiple 0,0039 comparisons Kruskal-Wallis test with Dunn's multiple	Time*Paw Time*Group*Paw Treatment * Surgery 0,0063	0,023 <0,05 >0.05
	6a1 6a2 6c2 S6a1 S6a3 6a3 Figure 6b1 S6c2 6b2 S6b1	rat threshold for pain per group at different time points before and after drug(s) injection Time spent in closed arm per rat Delta time spent in the paired chamber per mice injection rat threshold for pain per group at different time points before and after drug(s) injection total distance per rat Delta time spent in the paired chamber per rat Statistical unit mice threshold for pain per group at different time points before and after drug(s) IPSC frequencies before and after TGOT in sham animals total distance per mice Time spent in closed arm per mice mice threshold for pain per group at different time points before and after drug(s) injection	SNI : Vehicle + Ctrl C1V1 SNI : TGOT Sham : Vehicle + Ctrl C1V1 Sham : TGOT Sham OTR cKO mice + rAAV-Cre-GFP -> TGOT Vehicule SNI OTR cKO mice + rAAV-Cre-GFP -> TGOT SNI control mice -> Vehicule TGOT SNI control mice -> TGOT Sham control mice -> Sham control mice -> TGOT Sham control mice -> Vehicule rAAV-Cre-GFP -> TGOT Sham OTR cKO mice + rAAV-Cre-GFP -> Vehicule SNI OTR cKO mice + rAAV-Cre-GFP -> SNI : Ctrl C1V1 Sham : TGOT SNI : C1V1 Sham : C1V1 SNI : Vehicle + Ctrl C1V1 SNI : TGOT Sham : Vehicle + Ctrl C1V1 Sham : TGOT Sham : C1V1 SNI : C1V1 SNI : Vehicle + Ctrl C1V1 SNI : TGOT Sham : Vehicle Sham : TGOT Sham : C1V1 SNI : C1V1 Baseline rAAV-Cre-GFP -> rAAV-Cre-GFP -> TGOT Groups tested Vehicule Vehicule SNI OTR cKO mice + rAAV-Cre-GFP -> TGOT SNI control mice -> Vehicule SNI OTR cKO mice + Sham OTR cKO mice + rAAV-Cre-GFP -> TGOT rAAV-Cre-GFP -> TGOT Sham control mice -> Vehicule SNI OTR cKO mice + rAAV-Cre-GFP -> TGOT SNI control mice -> TGOT SNI control mice -> TGOT Sham OTR cKO mice + rAAV-Cre-GFP -> Vehicule Sham OTR cKO mice + rAAV-Cre-GFP -> TGOT Sham control mice -> Vehicule Sham control mice -> TGOT Sham OTR cKO mice + rAAV-Cre-GFP -> TGOT Sham OTR cKO mice + TGOT SNI control mice -> Sham control mice -> TGOT SNI OTR cKO mice + rAAV-Cre-GFP -> Vehicule SNI control mice -> TGOT SNI OTR cKO mice + rAAV-Cre-GFP -> TGOT SNI control mice -> Vehicule	24h CTRL 0.3h 3.5h 24h CTRL 0 min 15 min 45 min CTRL 0 min 15 min 45 min CTRL 0 min 15 min 45 min 45 min CTRL 0 min 15 min 45 min CTRL 0 min 15 min 45 min CTRL Time CTRL 0 min 15 min 45 min CTRL 0 min 15 min Paired 0 min 15 min 0 min 15 min 45 min CTRL 0 min 15 min 45 min CTRL	Left Left Right Right Paw Unit g s s g m s g 9 Hz Right Left Left Right m s Right g	10 10 10 10 9 6 6 6 6 10 10 10 10 9 9 9 9 20 9 10 8 5 8 8 8 6 5 5 6 8 6 6 6 5 4 4 8 5 10 8 13 6 5 8 10 8 13 6 5 8 3,339 0,694 211,033 209,967 212,033 207,900 209,889 247,167 254,278 232,667 222,278 182,767 216,200 186,400 185,700 186,407 209,130 198,074 200,370 72,656 59,635 57,967 56,711 1,32 4,5 4,5 3,8 -31,75 -103,5 282,8 4,666667 29,2 5,333333 5,333333 5 34,82 31,13 -108,3 4,25 27,61 -95,47 267,438 -35,646 435,667 476,7 259,125 15,16 27,67 19,56 15,37 15,94 13,84 5 60,06 13 4,307693 5 13,32 7,083 1,357 N Mean 13 1,923077 13 2,138462 13 1,646154 0,09910845 1,975 1,996 1,572 3,374 2,673 13,531 10,786 10,491 4,576 10,286 11,015 11,411 11,335 10,872 9,747 5,709 2,720 2,181 1,575 2,458 0,3273268 0,3273268 0,646971 25,34 43,41 95,21 0,421637 2,579 0,421637 0,4472136 5,108 0,421637 5,921 0,08 126,3 0,4531635 2,189 9,139 56,206 74,858 71,396 112,894 69,796 110,36 1,591 11,08 2,565 3,165 3,037 2,052 1,1 0,3820427 0,8245 SEM 0,1915369 0,3024554 13 2,646154 0,4852458 5 1,08 0,08 5 1,32 0,08 5 1,08 0,08 Paired t-test 7 59,51 0,5815 7 61,2 1,343 13 4,153846 0,1538462 13 3,538461 0,2432521 7 10,71 0,6737 7 11,57 0,2496 8 47,58 1,173 5 60,56 1,078 7 59,01 1,535 7 49,27 6 46,97 5,195 8 4 0,3779645 8 4,5 0,5 5 30,68 3,288 2,344 13 4,923077 0,2878198 6 12,34 1,525 5 4,4 0,4 4 32,38 7,6 5 4 0 5 3,6 0,4 5 4,4 0,4 8 4,5 0,3273268 6 24,34 1,381 5 22,6 3,947	Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons Kruskal-Wallis test with Dunn's multiple comparisons comparisons multiple test with Dunn's Kruskal-Wallis Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons Mixed-design ANOVA Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons comparisons 0,0007 Test used Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons Kruskal-Wallis test with Dunn's multiple comparisons Mixed-design ANOVA with Bonferroni adjustment for multiple comparisons Kruskal-Wallis test with Dunn's multiple comparisons	Time Time*Group Time*Paw Time*Group*Paw Treatment Treatment * Surgery 0,0061 0,5496 Treatment Surgery Treatment * Surgery Treatment Treatment * Surgery p=0.5416 0,001 0,007 0,054 0,862 <0,0001 0,0408 <0.0001 0,0316 0,8856 p=0.6272 < 0,0001 Interaction 0,1894 Treatment P < 0,0001 Treatment * Surgery P < 0,0001 p-value Neuropathy 0,3355 Group 0,0246 0,3644
			Sham : C1V1 Sham OTR cKO mice +				9	56,893	7,415
			rAAV-Cre-GFP ->				5	-42,64	96,75
			SNI : C1V1 Vehicule	45 min			7 8	51,067 4,675	6,594 0,5842425

ACKNOWLEDGEMENTS

This work was supported by the IASP Early Career Research grant 2012, FP7 Career Integration grant 334455, Initiative of Excellence (IDEX) Attractiveness grant 2013, IDEX Interdisciplinary grant 2015, University of Strasbourg Institute for Advanced Study (USIAS) fellowship 2014-15, Foundation Fyssen research grant 2015, NARSAD Young Investigator Grant 24821, ANR JCJC grant (to AC); ANR-DFG grant GR 3619/701 (to AC and VG); the Schaller Research Foundation, DFG grant GR 3619/13-1, DFG within the Collaborative research Center SFB 1134, (to VG) and SFB 1158 (to CP and VG); SNSF-DFG grant GR 3619/8-1 (to RS and VG), Fritz Thyssen foundation (to VG); Alexander von Humboldt Foundation (to DH); Fyssen foundation and PROCOP grant and SFB1158 seed grant for young scientists (to AL); Research Foundation -Flanders, fellowship (12V7519N) (to JW); Russian science foundation RSF (17-75-10061) and the Program of Competitive Growth of Kazan University (to AR); the intramural research program of the NIMH (ZIAMH002498) (to WSY); National Institutes of Health grants R01NS094640 and R01HL090948 (to JES). The authors thank Vincent Lelièvre for in situ hybridization advices; Romain Goutagny for in vivo optogenetics assistance; Fulvio Magara for anxiety behavior advices; Barbara Kurpiers and the Interdisciplinary Neurobehavioral Core Facility of Heidelberg University for experiments performed there; Sophie Reibel, Dominique Ciocca and the Chronobiotron UMS 3415 for all animal care; Thomas Splettstoesser (www.scistyle.com) for its initial help with the preparation of figures.

AUTHOR CONTRIBUTIONS

Conceptualization, AC; Methodology, AC, BBo, CML, CP, DK, FA, ID, JW, JYC, PD, PP, RS, VG, WSY, YG; Analysis, AC, BBe, BBJ, CML, DK, FA, HSKB, JW, SG; In situ hybridization, DH, FA, HSKB, HW, JS, ME; Immunohistochemistry, AL, DH, FA, JW, ME, MdSG; Ex vivo patch-clamp electrophysiology, AB, AC, JW, SG, DK, IW, BBe, MA; Ex vivo calcium imaging, AB, CML, DK, JW; Astrocytes characterization, AR, DK, IW, ME, SG; Behavior, AC, BBJ, DK, JW; Mice line validation, WSY; Viral vectors validation, MdSG, ME, VG; Spared nerve injuries, PI, MP; Writing, AC, BBo, JES, JYC, JW, RS, VG; Funding acquisition AC, VG; Supervision, AC; Project administration, AC.

DECLARATION OF INTERESTS

The authors declare no competing interest Spatial Distribution Analysis of Astrocytes within the CeL.

For the analysis of spatial distribution of astrocytes within the CeL, confocal images were analyzed and all OTR+ astrocytes were manually marked. A separate image with the location of identified astrocytes in the CeL was exported for spatial distribution analysis. The coordinate's points of OTR+ astrocytes (events) were extracted with custom-written MATLAB (MathWorks) script and the centroid of each event was calculated with the function regionprops. The univariate first-order nearest neighbor (NN) test (Clark-Evans statistic) was derived from the distance to the closest event for each data point in the image. The test estimates the departure of the observed NN distances from those expected under a null model of complete spatial randomness (CSR; a homogeneous Poisson process). We calculated the mean NN distance for all events in each image as:

∑ 𝑟 𝑁 where 𝑟 is the distance from a given event to its nearest neighbor, and N is the number of events in a given population. The ratio 𝑅 = 𝑟̅ 𝐴 𝑟̅ 𝐸 is the observed mean NN distance by mean distance which would be expected if this population was distributed randomly. In a random distribution 𝑅 = 1. The values of 𝑅 can vary between 0 (maximum aggregation or clustering) and 2.1491 (maximum spacing or uniformity). For details see [START_REF] Clark | Distance to Nearest Neighbor as a Measure of Spatial Relationships in Populations[END_REF][START_REF] Millet | Pattern analysis and spatial[END_REF] . Thus, 𝑅 values are indicative of uniform, random, and clustered patterns of point distribution. To evaluate the significance of 𝑅, it is possible to calculate the standard variate of the normal curve, namely the c-score:

where 𝜎 𝑟̅ 𝐸 is the standard error of the mean distance to NN in a randomly distributed population with the same number of events as the observed population. The c value 1.96 represents the 5% level of significance for a two-tailed test. We calculated the mean NN distance for 12 randomly selected images, 𝑅, and the c-score for image of the CeL (Figure S1b): Only in two images the cscore was equal to or greater than 1.96, therefore only in two of the images it was possible to reject the null hypothesis of randomly distributed population (Statistics in Table S1). S1). S2). S3). S4). S5). S6).

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