# Inhibiting the JAK-STAT3 pathway in nucleus accumbens astrocytes alleviates cocaine-induced motor hyperactivity

Isabelle Arnoux, Anna Capano, Rachida Yakoubi, Claire Boulogne, Pascal Ezan, Carole Escartin, Nathalie Rouach

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Isabelle Arnoux, Anna Capano, Rachida Yakoubi, Claire Boulogne, Pascal Ezan, et al.. Inhibiting the JAK-STAT3 pathway in nucleus accumbens astrocytes alleviates cocaine-induced motor hyperactivity. Progress in Neurobiology, 2025, 255, pp.102852. 10.1016/j.pneurobio.2025.102852. hal-05425612

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# Inhibiting the JAK-STAT3 pathway in nucleus accumbens astrocytes alleviates cocaine-induced motor hyperactivity

Isabelle Arnoux<sup>a,1</sup>, Anna Capano<sup>a,1</sup>, Rachida Yakoubi<sup>a</sup>, Claire Boulogne<sup>b</sup>, Pascal Ezan<sup>a</sup>, Carole Escartin<sup>c,d</sup>, Nathalie Rouach<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Astrocytes

Cocaine

Reactivity

JAK-STAT signaling pathway

## ABSTRACT

Cocaine use disorder is a significant global health issue, and despite its widespread impact, effective treatments are lacking. While research has largely focused on the underlying neuronal mechanisms, the role of astrocytes, key regulators of synaptic transmission and plasticity, remains underexplored.

Using a multidisciplinary approach that combines immunohistochemistry, electron microscopy, 3D cell reconstruction, viral gene transfer, and behavioral assays, we investigated the early adaptive responses of astrocytes to repeated cocaine administration.

We report that cocaine administration induces astrocyte reactivity in the nucleus accumbens, characterized by structural remodeling, reduced synaptic coverage, and upregulation of reactivity-associated markers, including STAT3. Furthermore, we demonstrated that the JAK/STAT3 signaling pathway plays a critical role in the pathological structural astrocytic responses and in the cocaine-induced motor behavior.

Our findings highlight astrocytes as pivotal players in the initial neural adaptations underlying cocaine-induced behavior. These data may provide a basis for the development of novel therapeutic strategies targeting astrocytes to address the structural and functional disruptions associated with cocaine exposure.

## 1. Introduction

Cocaine is a highly rewarding and addictive drug, and its chronic consumption leads to compulsive drug-seeking and drug taking behavior with high rates of relapse despite negative consequences including medical, psychological and social issues (Koob and Volkow, 2010). Currently, cocaine is one of the most abused drugs, with over 20 million users worldwide reported in 2020 (United Nations Office on Drugs and Crime 2023 (UNODC, Global report on Cocaine 2023 – Local dynamics, global challenges (United Nations publications 2023))). Despite its broad impact, no effective pharmacological treatments are currently available.

Repeated administration of cocaine induces an enhanced behavioral response to subsequent drug exposure, a phenomenon known as behavioral sensitization (Robinson and Berridge, 1993). In animal models, this sensitization is typically reflected as progressively enhanced locomotor activity following repeated cocaine exposure

(Kalivas and Stewart, 1991; Robinson and Berridge, 2000). This behavioral response emerges from very early neuronal changes, including disrupted synaptic transmission due to dopamine transporter inhibition. Because of its robustness and temporal resolution, this experimental paradigm is widely used to investigate the initial cellular and circuit-level alterations that may precede and contribute to the development of compulsive drug-related behaviors. Early neuronal activity-dependent adaptations to cocaine administration occur in many brain regions, notably in the mesocorticolimbic reward circuits. The nucleus accumbens (NAc) is a central hub in the drug addiction circuit, which receives dopaminergic inputs from the ventral tegmental area and glutamatergic projections from the prefrontal cortex, hippocampus and amygdale (Scofield et al., 2016). Animals behaviorally sensitized to cocaine show an enhanced release of dopamine and glutamate in the NAc (Reid and Berger, 1996). However, therapeutic strategies targeting directly or indirectly dopamine and glutamate, like modafinil,

<footnote><sup>a</sup> Center for Interdisciplinary Research in Biology, Collège de France, CNRS, INSERM, PSL-Neuro, Université PSL, Paris, France</footnote>

<footnote><sup>b</sup> Institute for Integrative Biology of the Cell, CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France</footnote>

<footnote><sup>c</sup> Université Paris-Saclay, CEA, CNRS, MIRCen, Laboratoire des Maladies Neurodégénératives, Fontenay-aux-Roses, France</footnote>

<footnote><sup>d</sup> NeuroPSI, CNRS, Université Paris-Saclay, Saclay, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: nathalie.rouach@college-de-france.fr (N. Rouach).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
N-Acetylcysteine or topiramate, reported modest or no effect on this behavior (Dackis et al., 2012; LaRowe et al., 2013; Singh et al., 2016), indicating that the molecular and cellular mechanisms underlying the altered locomotor behavior are not fully elucidated.

Astrocytes are key partners of synapses, and actively regulate syn-
aptic transmission and plasticity through reciprocal interactions
(Araque et al., 1999; Haydon, 2001; Oliveira and Araque, 2022; Perea
et al., 2009), which influence animal behavior (Kofuji and Araque, 2021;
Lyon and Allen, 2022). A hallmark of astrocytes is their intricate
morphology, which plays a crucial role in maintaining extracellular
homeostasis and modulates synaptic transmission through their physical
coverage of synapses. Astrocytes indeed continuously monitor their
environment via their highly motile processes (Hirrlinger et al., 2004)
expressing various ion channels, transporters, and receptors. This allows
them to quickly detect local changes and adapt accordingly their
morphological, molecular, and functional phenotype, which can become
reactive in pathological conditions (Anderson et al., 2014; Escartin
et al., 2021). Studies on cocaine-induced plasticity have primarily
focused on deciphering neuronal mechanisms. However, recent data
indicate that cocaine exposure can alter the structural and functional
properties of astrocytes, including their morphology (Testen et al.,
2018), calcium signaling (Mongrédien et al., 2025; O'Donovan et al.,
2021) or regulation of glutamate homeostasis (Alberquilla et al., 2025;
Scofield et al., 2015). This suggests that astrocytic dysfunctions may
contribute to cocaine-induced behaviors. We thus here investigated the
early effects of repeated cocaine exposure on the phenotype of NAc as-
trocytes, and its role in cocaine-induced behavior by employing a 5-day
repeated cocaine administration paradigm. This experimental protocol
is a well-established model for investigating early neurobehavioral ad-
aptations associated with the initiation phase of drug exposure (Brown
et al., 2011; Kim et al., 2013; Zhou et al., 2016). By allowing precise
control over dose and timing, it provides a reproducible framework for
dissecting the cellular and circuit-level mechanisms underlying behav-
ioral sensitization and other initial responses to psychostimulants. We
found that cocaine induces astrocyte reactivity involving the JAK/
STAT3 signaling pathway, and that this pathway contributes to the
cocaine-induced pathological astrocytic structural remodeling and
mouse motor behavior. Our findings provide new insights into the role
of astrocytes in the early neural adaptations associated with repeated
drug exposure, and point to alternative therapeutic strategies targeting
astrocytes to modulate initial cocaine-induced behaviors.

## 2. Materials and method

2.1. *Animals*

All experiments involving animals were conducted in accordance
with international, national, and/or institutional guidelines for the care
and use of animals. Wildtype (WT) mice were purchased from Janvier
laboratories (France) or bred in the CIRB animal facility. The GFAP-
eGFP (GFEC) mice, in which the enhanced green fluorescent protein
(eGFP) is expressed under the GFAP promoter, was generated by the
team of Dr. H. Kettenmann (Max-Delbrück Center for Molecular Medi-
cine, Berlin, Germany) (Nolte et al., 2001), then bred in house. All these
mouse lines were generated on a C57BL / 6 J background, and all mice
used in these experiments were adult males (between 2- and
6-month-old).

Mice were housed in a 12 h light/12 h dark circle, with ad libitum
access to food and water, according to the European Community
(Directive 2010-63/EEC) and French regulations (Code Rural R214/
87-130). The experimental procedures were approved by the local
ethics committee and registered with the French Research Ministry
(CEEA59, Paris 6). These experiments were conducted in accordance to
European Commission Council guidelines from January, 1st 2013
(2010/63/EU) and ethic committee (certificate A751901, Ministère de
l'Agriculture et de la Pêche), under the project number

2017121113344876, and all efforts were put in place to minimize the
number of animals used and their suffering.

## 2.2. AAV production and stereotaxic injection

For AAV *in vivo* gene transfer, the transgenes composed of GFP or SOCS3 cDNA were placed under the control of a GFA<sub>ABC1D</sub> promoter in an AAV shuttle plasmid containing the inverted terminal repeats (ITR) of AAV2. Pseudotyped serotype 9 AAV particles were produced by transient co-transfection of HEK-293T cells, as previously described (Berger et al., 2015). Viral titers were determined by quantitative PCR amplification of the ITR on DNase-resistant particles and expressed as vector genome per ml (vg/ml).

Mice were anesthetized with a mixture of ketamine (95 mg / kg, Merial) and xylazine (10 mg / kg, Bayer) in a solution of 0.9 % NaCl, and placed on a Stoelting's stereotaxic frame to perform AAV injection. Body temperature was monitored and buprenorphine analgesics (0.1 mg / kg; Centravet) were then sub-cutaneously administered to the mice. The AAV viral vectors were diluted in PBS 0.1 M, with 0.0001 % pluronic acid to get a concentration of 10¹³ vg / ml. The skull was exposed and 1 µl of pAAV9 GFA-ABC1D-GFP, to visualize the transduced cells, with or without pAAV2/9 GFA-ABC1D-SOCS3, were stereotaxically injected bilaterally in the nucleus accumbens (Ben Haim et al., 2015; Abjean et al., 2023) at the following stereotaxic coordinates: antero-posterior: + 1.7 mm from the bregma; medio-lateral: ± 1.2 mm from the medial suture line; dorso-ventral: - 4.6 mm from the skull bone.

Viral injections were performed at a rate of 0.2 µL / min using a 29-
gauge blunt-tip needle linked to a 2 µl Hamilton syringe (Phymep). The
cannula was then left in place for 5 min before being slowly removed.
The skin was sutured and mice were allowed to recover for at least 3
weeks before experiments were performed.

2.3. Cocaine injection

Cocaine (cocaine hydrochloride, Sigma Aldrich, C5776, 1 g) was diluted in saline solution (NaCl 0.9 % in H₂O) and injected intraperitoneally (I.P.) at 10 mg/kg for 5 consecutive days. The use of this narcotic is submitted to an authorization of the French drug agency (Agence Nationale de la Sécurité du Médicament et des produits de la santé, ANSM), number A-2016-9-2429-S.

2.4. Immunohistochemistry

Mice were deeply anesthetized with Euthasol (sodium pentobarbital (140 mg/kg)) and perfused intracardially with paraformaldehyde (PFA 2%). The brains were carefully dissected and postfixed overnight with PFA 2% at 4°C. The next day, PFA was removed and the brains were cryoprotected overnight in PBS containing 30% sucrose. The brains were then cut into 40 µm sections with a freezing microtome (Leica, Germany). Slices were stored in PBS at 4°C until use. For immunohistochemistry, slices were blocked with PBS-Gelatin-Triton (PBS with 0.002% of gelatin and 0.25% of Triton X-100), for 1 h at room temperature, incubated overnight at 4°C with primary antibodies in the blocking solution. Then, slices were washed 1 h at room temperature with PBS-Gelatin-Triton, and incubated 2 h with secondary antibodies and DAPI (1:2000, Sigma-Aldrich, D9542) in blocking solution and washed three times with PBS before mounting with Fluoromount (Invitrogen). For STAT3 staining, an antigen retrieval protocol was performed in methanol at -20°C for 20 min. Brain sections were blocked with 5% normal goat serum (Sigma) and 0.3% Triton 1 h at room temperature. The anti-STAT3 antibody was diluted in Signal Boost antibody diluent (Cell Signaling Technology) for 48 h at 4°C. Secondary antibody was incubated in 1% BSA with 0.3% Triton.

Low-magnification pictures were taken with an Axiozoom micro-
scope (Zeiss) to verify AAV injection site. Z-stack images were acquired
with a spinning disk confocal microscope with a 60X magnification
(CSUX1-A1 Yokogawa) then reconstructed using ImageJ software (NIH). Acquisition parameters were strictly conserved across all mice.

filament parameters and Sholl analysis were exported to Excel files and used for data analysis. The N used for statistical test is the number of astrocytes per condition.

## 2.5. Antibodies

<table><thead><tr><td>Antibody</td><td>Target</td><td>Species</td><td>Type</td><td>Clonality</td><td>Concentration</td><td>Company</td><td>Reference</td></tr></thead><tbody><tr><td>GFP</td><td>GFP</td><td>Chicken</td><td>Primary</td><td>Poly</td><td>1:500</td><td>Aves</td><td>1020</td></tr><tr><td>STAT3</td><td>STAT3</td><td>Rabbit</td><td>Primary</td><td>Mono</td><td>1:200</td><td>Cell Signaling</td><td>8768S</td></tr><tr><td>S100 beta</td><td>Astrocytes</td><td>Rabbit</td><td>Primary</td><td>Poly</td><td>1:500</td><td>Proteintech</td><td>15146-1-AP</td></tr><tr><td>Sox9</td><td>Astrocytes</td><td>Goat</td><td>Primary</td><td>Poly</td><td>1:500</td><td>R&amp;D Systems</td><td>AF3075</td></tr><tr><td>Vimentin</td><td>Astrocytes</td><td>Goat</td><td>Primary</td><td>Poly</td><td>1:500</td><td>Millipore</td><td>AB1620</td></tr><tr><td>GLT-1</td><td>Astrocytes</td><td>Rabbit</td><td>Primary</td><td>Poly</td><td>1:500</td><td>Frontier Institute</td><td>Af670</td></tr><tr><td>Glutamine Synthetase</td><td>Astrocytes</td><td>Rabbit</td><td>Primary</td><td>Poly</td><td>1:500</td><td>Sigma</td><td>G2781</td></tr><tr><td>Anti rabbit Alexa 555</td><td>Rabbit IgG</td><td>Goat</td><td>Secondary</td><td>Poly</td><td>1:1000</td><td>Life technologies</td><td>A27039</td></tr><tr><td>Anti chicken Alexa 488</td><td>Chicken IgG</td><td>Goat</td><td>Secondary</td><td>Poly</td><td>1:1000</td><td>Life technologies</td><td>A11039</td></tr><tr><td>Anti-chicken Alexa 488</td><td>Chicken IgG</td><td>Donkey</td><td>Secondary</td><td>Poly</td><td>1:1000</td><td>Life technologies</td><td>A78948</td></tr><tr><td>Anti-rabbit Alexa 555</td><td>Goat IgG</td><td>Donkey</td><td>Secondary</td><td>Poly</td><td>1:1000</td><td>Life technologies</td><td>A31572</td></tr><tr><td>Anti-goat Alexa 555</td><td>Goat IgG</td><td>Donkey</td><td>Secondary</td><td>Poly</td><td>1:1000</td><td>Life technologies</td><td>A21447</td></tr></tbody></table>

## 2.6. Quantification of Vimentin, GFAP, GLT-1, S100β and GS integrated density

Fluorescence intensity measurements for Vimentin, GFAP, GLT-1, S100β and GS were performed using an inverted confocal laser-scanning microscope (Leica SP5). Images were acquired with a 63x objective as sequential stacks of consecutive confocal images at 600 - 1000 nm intervals, using two excitation lasers (argon 488 nm and helium-neon 543 nm or 647 nm). Z-projections were reconstructed using ImageJ software, and fluorescence intensity was quantified by measuring the integrated density for each marker. The N used for statistical test is the number of fields of view per condition.

## 2.7. Quantification of STAT3 and Sox9 cell density

All images were acquired with identical settings at the spinning disk confocal microscope. 30 optical sections of 0.2 µm thickness were imported into ImageJ and processed for quantitative analysis. The number of STAT3 and Sox9 positive cells were obtained by counting the number of cells in three dimensions in z-stacks by using Cell Counter, an ImageJ plug-in. The N used for statistical test is the number of fields of view per condition.

## 2.8. Three-dimensional analysis of astrocyte morphology

Z-stack images were acquired at a spinning disk confocal microscope with a 60X magnification (CSUX1-A1 Yokogawa). 50 optical sections of 0.2 µm thickness were imported for quantitative analysis. Isolated astrocytes were selected as regions of interest based on their GFP staining in GFAP-eGFP mice and GFP virus-injected mice. We used Imaris software (Oxford Instruments) to reconstruct astrocytes by employing the module filament tracer. All astrocytes with incomplete soma (cut in the x, y or z plane) were manually removed and not included in the analysis. To differentiate signal from background, an automated thresholding approach was applied during the filament reconstruction process. The following custom settings were used: largest diameter 7.00 µm, seed points 0.300 µm, remove seed points around starting points: diameter of sphere regions: 10.8 µm. To ensure consistency, seed point creation thresholds were normalized across images by setting them as a fixed proportion of the image's background-corrected mean intensity. Seed points were manually corrected (either placed in or removed from the center of the soma) if the IMARIS algorithm placed them incorrectly. All

## 2.9. Electron microscopy

For cryofixation, mice were decapitated, the brain immediately removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) composed of (in mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 0.1 CaCl2, 3 MgCl2, 25 glucose, 3 myo-inositol, 2 Na-pyruvate, 0.4 ascorbic acid, pH 7.4, and bubbled with 95 % oxygen. Slices were made with a vibratome (Leica Microsystems VT1200S) at 150 µm thickness in the coronal or tangential plane. After cutting, each slice was transferred immediately to a Petri dish containing the same medium completed with 20 % BSA. 1.2 mm-diameter pieces of nucleus accumbens were punched under a stereomicroscope (Leica Microsystems M80) and placed in HPF carriers (Leica Microsystems, 16706897). To ensure that no air bubbles became trapped inside the sample holder with the tissue, a small drop of 20 % BSA was added. This was then high-pressure frozen using a Leica EMPACT2 (Leica Microsystems). This entire procedure was completed as rapidly as possible, in less than 1 min between vibratome sectioning and freezing. Frozen samples were then stored in liquid nitrogen until further processing.

Frozen tissue was stained, dehydrated, and embedded at low temperature using a low temperature embedding device (AFS2; Leica Microsystems). Frozen samples were transferred to this device in liquid nitrogen and were incubated in 2 % osmium tetroxide and 2 % water in acetone, for 36 h at -90°C. The temperature was then raised over 14 h to -20°C, then maintained at -20°C for 16 h. Finally, temperature was raised to 4°C over 4 h. Finally, samples were mixed with increasing concentrations of resin (Agar Scientific, R1165) over 24 h whilst the temperature rose to 20°C. They were then added to 100 % resin for 24 h and then placed in silicon moulds for 24 h at 60°C for the resin to harden.

Series of ultrathin sections (70 nm thickness; comprising 32-45 sections) were collected on slot grids, contrasted with 2 % uranyl acetate and Reynold's lead citrate, and examined at the EM level, to investigate the astroglial coverage, where the region of interest was photographed throughout consecutive sections in the entire series of ultrathin sections at 44 000 X using a Hitachi HT7700 TEM at 100 kV, equipped with a AMT41 B 4 K camera; Milexia France SAS. The same series above were used to estimate the astroglial coverage by measuring the shortest distance between astrocytic processes and the closest synaptic cleft using ImageJ (Schneider et al., 2012).

## 2.10. Quantitative volumetric analysis of the glial coverage

To quantify the astrocytic coverage of the synaptic boutons and their
target structures in wild type and cocaine treated mice, the following experimental approach was used. The first, the middle and the last images of a series were used for a further quantitative volumetric analysis using the interactive software ImageJ. In each section a grid (grid size 10 × 10 µm2) was placed over the EM image, and in each square, the abundance of fine astrocytic processes was documented throughout these images and averaged. Using the Cavalieri method [Unbiased Steeplechey: Three-Dimensional Measurement in Microscopy (Advanced Methods) Paperback—7 January 2005 by Vyvyan Howard Matthew Reed], the (absolute) volume contribution of astrocytic processes was

determined according to the Cavalieri estimator:

$$V=a(p) \times \Sigma P \times t$$

where a(p) is the size of one square (10 × 10 µm2), P is the number of squares counted, and t is the thickness of the slice (70 nm=0.07 µm).

## 2.11. Locomotor activity

We used 40*40*40 cm white opaque PVC openfield (Noldus, The Netherlands). Each mouse was removed from its home cage and put into

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Cocaine induces structural remodeling of accumbal astrocytes. (A) Experimental design and timeline showing intraperitoneal injection of saline or cocaine (10 mg/kg) daily for five consecutive days. The brains were collected 24 h after the last injection. (B) Representative travel paths of WT mice injected with a solution of saline (black) or cocaine (10 mg/kg, red). Only the last 10 min of the recordings of day 5 are shown. (C) The distance traveled by cocaine-injected mice (red) increases compared to saline-injected mice (black). Two-way repeated measures ANOVA Saline vs Cocaine ***p < 0.0001, F(1,37) = 184.2, Post hoc analysis using Sidak's multiple comparisons test revealed p < 0.001 between saline and cocaine groups for each day. No sensitization was detected in the saline group (Day1 (D1) vs. Day5 (D5), p = 0.4773, df= 14, Dunnett's multiple comparisons test), whereas significant sensitization was observed between D1 and D5 in the cocaine group (p < 0.001, df = 21, Dunnett's multiple comparisons test), saline n = 15 mice, cocaine n = 24 mice. (D) The time spent in the center is increased in cocaine-injected mice (red) compared to saline-injected mice (black) on day 1 and day 5. Day 1: *p = 0.0492, t = 2.2727, df = 9, Day 5: *p = 0.0242, t = 2.864, df = 7, Unpaired t-test, saline n = 5 mice on day 1 and day 5, cocaine n = 6 at day 1 and n = 4 mice at day 5. (E) The number of entries in the center of the open-field arena is increased in cocaine-injected mice (red) compared to saline-injected mice (black) at day 1 and day 5. Day 1: *p = 0.0217, t = 2.771, df = 9, Day 5: **p = 0.0093, t = 3.555, df = 7, Unpaired t-test, saline n = 5 mice on day 1 and day 5, cocaine n = 6 at day 1 and n = 4 mice at day 5. (F) Three-dimensional reconstruction of accumbal astrocytes in saline (white) and cocaine- (red) injected GFAP-eGFP mice using IMARIS software. Scale bar: 10 µm. (G) The domain volume of astrocytes is reduced in cocaine- (red) compared to saline-injected mice (black). **p = 0.0072, t = 2.835, df = 39, Unpaired t-test, saline n = 17 astrocytes from 3 mice, cocaine n = 25 astrocytes from 3 mice. (H) The sum of the lengths of astrocytic processes was diminished in cocaine-injected mice (red) compared to saline-injected mice (black). ***p < 0.0001, t = 5.617, df = 40 Unpaired t-test, saline n = 17 astrocytes from 3 mice, cocaine n = 25 astrocytes from 3 mice. (I) Sholl analysis showing the number of intersections per radius in saline- (black) and cocaine- (red) injected mice. The concentric circles were spaced by 5 µm and centered on the soma of the astrocyte. p = 0.0001, F(11,371) = 3.429, Two-way ANOVA. Post-hoc analysis using Sidak's multiple comparisons test revealed p = 0.0063 at 5 µm, p < 0.0001 at 10 µm, p < 0.0001 at 15 µm, p = 0.0001 at 20 µm, p = 0.0046 at 25 µm. (J) Electron micrographs depicting fine astrocytic processes in nucleus accumbens of saline-injected mice (left) and cocaine-injected mice (right). de: dendrite, sp: spine, sb: synaptic bouton. Scale bar: 1 µm. (K) The volume of astrocyte processes measured by electron microscopy is significantly decreased in cocaine-injected (red) mice compared to saline-injected (black) mice. p = 0.0004 Mann-Whitney test, saline: n = 9 astrocytes from 3 mice, cocaine: n = 9 astrocytes from 3 mice. (L) Electron micrographs showing fine astrocytic perisynaptic processes in saline-injected (left) and cocaine-injected (right) mice. de: dendrite, b: synaptic bouton. Scale bar: 200 nm. (M) The distance between an astrocytic process to the nearest synapse is increased in cocaine-injected mice (red) compared to saline-injected mice (black). p < 0.0001, Mann-Whitney test, saline: n = 69 distances from 3 mice, cocaine: n = 69 distances from 3 mice.</figcaption></figure>
a holding box next to the testing box. Subsequently, the mouse was put into the testing box facing the rear wall. Path tracking has been recorded with Ethovision XT 15 (Noldus, The Netherlands). Locomotor activity was measured for 30 min before injections of cocaine or saline solution (habituation) and then 30 min directly after injection (test). Time in the center, which was determined as 30 cm away from each wall of the box, was measured automatically, when the center-point of the mouse moved into it. All results shown in this paper are the measurements obtained during the test phase.

## 2.12. Statistical analysis

Statistical significance was tested in GraphPad Prism (GraphPad Software Inc., La Jolla, CA. *p* < 0.05, **p** < 0.01, ***p* < 0.001). For all data, we first tested normal distribution using the one-sample Shapiro test, and we used nonparametric tests in case of non-normal distribution. Graphs show mean ± s.e.m (standard error of the mean).

# 3. Results

## 3.1. Cocaine induces astrocyte structural remodeling in the nucleus accumbens

Astrocytes are highly dynamic and adaptive cells, capable of rapidly remodeling their structure in response to environmental changes. To investigate the impact of cocaine on astrocytes morphology, we used a model of cocaine sensitization in wild-type (WT) mice. Animals were injected intraperitoneally with either saline (NaCl 0.9%) or cocaine (10 mg/kg) solution on five consecutive days and behavioral activities were recorded in an open-field box for 30 min after each injection (Fig. 1A). This 5-day repeated cocaine model is a powerful tool to study processes relevant to early neurobehavioral consequences in the initiation phase of drug use in controlled conditions for dose and timing. The locomotor activity was increased in cocaine-sensitized mice and they traveled in the entire open-field area, whereas saline-injected mice spent more time at the border of the arena, avoiding the central area (Fig. 1B). We found that the distance traveled in cocaine-injected mice was significantly higher compared to saline-injected mice and the distance traveled increased in response to repeated cocaine injection, which is characteristic of behavioral sensitization (Kalivas and Duffy, 1993) (Fig. 1C). The time spent in the center and the number of entries into the center of the arena were increased at days 1 and 5 in cocaine-injected mice (Fig. 1D and E), indicating an anxiolytic effect of cocaine sensitization.

To examine the impact of cocaine administration on astrocyte morphology in the NAc, we used GFAP-eGFP mice, in which astrocytes express GFP in their cytoplasm, allowing optimal visualization of the soma and processes (Nolte et al., 2001). Astrocytes of the NAc were analyzed from brains collected 24 h after the last injection of saline or cocaine solution, corresponding to a short-term withdraw (Fig. 1A). Using three-dimensional reconstructions, we found that accumbal astrocytes in cocaine-sensitized mice were smaller and less ramified compared to saline-injected mice (Fig. 1F). The volume of the domain and the sum of their process length were reduced in astrocytes from cocaine-sensitized mice (Fig. 1G and H). In addition, we found a reduction in astrocyte arborization in cocaine-sensitized mice, as revealed by the decrease in astrocyte morphology complexity using Sholl analysis (Fig. 1I). These results indicate that astrocytes undergo a robust structural remodeling in the NAc in response to cocaine sensitization.

To gain further insights into the morphological changes of astrocytes at the ultrastructural level and to investigate their impact on synapses coverage, we examined series of ultrathin sections (comprising 32–45 sections) using high-end fine-scale transmission electron microscopy. We found an overall decrease of astroglial coverage of synapses, as confirmed by the fine analysis of astrocyte-synapse complexes, which

indicates an important increase by ~ 2.5 fold of the shortest distance between astrocytic processes and the closest synapse in cocaine-sensitized mice (Fig. 1L and M). Furthermore, quantitative analysis on 3D volume reconstructions of astrocytes in the NAc confirmed a decrease in the volume occupied by astrocytes in cocaine-sensitized mice (Figs. 1J and 1K). These results indicate that cocaine sensitization alters the volume and distribution of astrocyte processes that retracted from synapses in the NAc.

## 3.2. Cocaine induces astrocyte reactivity in the nucleus accumbens

The reduction in territory size is an adaptive response associated with reactive astrocytes (Escartin et al., 2021). Using electron microscopy, we observed prominent intermediate filament bundles within enlarged processes in NAc astrocytes in response to cocaine exposure (Fig. 2A). This is another hallmark of reactive astrocytes, highlighting the structural remodeling of intermediate filaments. To further assess the phenotype of astrocytes upon cocaine sensitization, we analyzed the expression of several astroglial proteins known to vary upon reactivity. Using immunohistochemistry, we found that cocaine increased the expression of vimentin and GFAP, and decreased the expression of glutamine synthetase (GS) in accumbal astrocytes (Fig. 2B-C). In addition, we observed a decrease in the expression of GLT-1 and S100β, and an increase in the number of Sox9 positive cells (Figure S1). Consistent with the increase of intermediate filament bundles observed in astrocytes upon cocaine sensitization, these changes correspond to a typical reactive response, which includes alterations in astrocyte structure and homeostatic functions, as well as transcriptional reprogramming (Hol and Pekny, 2015; Ohtaki et al., 2007; Sun et al., 2017; Yamada et al., 1992). Multiple pathways are involved in astrocyte reactivity, including the JAK-STAT3 pathway, which is active in several models of acute injury or neurodegenerative disorders (Abjean et al., 2023; Ben Haim et al., 2015; Justicia et al., 2000; O'Callaghan et al., 2014).

We thus investigated whether the JAK-STAT3 signaling pathway was activated in astrocytes upon cocaine sensitization. To this end, we measured STAT3 accumulation in the nucleus of astrocytes labelled by Sox9 using immunohistochemistry, as an index of pathway activation (Fig. 2D). We found that cocaine-sensitized mice display a significant increase in the percentage of STAT3-positive astrocytes (+25%, Fig. 2E), indicating an activation of the JAK-STAT3 signaling pathway. Altogether, these data show that cocaine triggers astrocyte reactivity in the NAc.

## 3.3. Activation of the JAK-STAT3 pathway in accumbal astrocytes controls cocaine-induced structural remodeling

To investigate whether activation of the JAK-STAT3 pathway in astrocytes mediates the cocaine-induced structural remodeling of astrocytes, we selectively expressed the pathway inhibitor suppressor of cytokine signaling 3 (SOCS3), by viral gene transfer in accumbal astrocytes (Ben Haim et al., 2015; Kershaw et al., 2013). To visualize transduced cells in the NAc, we co-injected two adeno-associated vector (AAV) encoding either SOCS3 or GFP under an astrocyte-specific GFA<sub>ABCD</sub> promoter. The control group was injected only with the AAV vector encoding GFP (Fig. 3A). Cocaine sensitization was performed 4 weeks later and brains were collected 24 h after the last injection (Fig. 3B). The transgenes were strongly expressed in the NAc following this procedure, as shown by GFP expression (Figures S2C and S2D), and were selectively expressed by astrocytes, as GFP expression was exclusively found in cells positive for S100β, a marker of astrocytes (Figure S2E) and Sox9 (Figures S2F-G). To analyze the level of JAK-STAT3 activation in cocaine-sensitized mice expressing SOCS3, we performed immunolabeling for STAT3 (Figure S2F). SOCS3 expression in astrocytes inhibited the cocaine sensitization-induced increase in STAT3, restoring expression to the levels observed in saline-injected mice (Figure S2H). In addition, SOCS3 expression significantly
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Cocaine administration induces astrocyte reactivity. (A) Electron micrographs showing the presence of prominent bundle of intermediate filaments in enlarged astrocytic processes (pink and asterisks) in cocaine-injected (right) compared to saline-injected (left) mice. Scale bar left: 500 nm, scale bar right: 1 µm. (B) Confocal images of immunostaining for vimentin (cyan), GFAP (orange) and glutamine synthetase (GS, green) in the nucleus accumbens. Scale bar: 10 µm. (C) The labeling intensity of vimentin and GFAP increases, while the intensity of GS decreases in cocaine-sensitized mice (red) compared to saline-injected mice (black). Vimentin: p = 0.0068, t = 2.987, df = 22, Unpaired t test saline = 8 fields from 3 mice, cocaine = 16 fields from 3 mice. GFAP: p = 0.0472 Mann-Whitney test, saline n = 7 fields from 3 mice, cocaine = 20 fields from 3 mice. GS: p = 0.0017 Mann-Whitney test, saline n = 8 fields from 3 mice, cocaine n = 17 fields from 3 mice. (D) Confocal images of STAT3 (top panel) and Sox9- (middle panel) immunostainings. STAT3-positive nucleus is highlighted with dashed lines for clarity. The bottom panel presents a merged image, illustrating the spatial overlap between STAT3 and Sox9 expression in astrocytes. Scale bar: 10 µm. (E) The density of STAT3 positive astrocytes increases in cocaine-sensitized mice (red) compared to control saline-injected mice (black). \*p = 0.0376, t = 2.488, df = 8, Unpaired t-test, saline: n = 29 fields from 5 mice, cocaine: n = 25 fields from 5 mice.</figcaption></figure>

reduced the cocaine-mediated rise in Sox9-positive astrocytes, thereby validating our approach as Sox9 is a downstream transcriptional target of JAK-STAT3 (Hall et al., 2017) (Figure S2I). These results indicate that SOCS3 expression is sufficient to inhibit the JAK-STAT3 signaling pathway in astrocytes upon cocaine sensitization.

To test whether the inhibition of the JAK-STAT3 signaling pathway impacts astrocyte remodeling upon cocaine sensitization, we analyzed the effect of SOCS3 on astrocyte morphology in three-dimensional reconstructions (Fig. 3C). We found no significant difference in the structure of SOCS3-expressing astrocytes in saline- and cocaine-injected mice (Fig. 3C-F). Cocaine sensitization indeed failed to reduce the domain volume or the total process length of astrocytes expressing SOCS3 (Fig. 3D and E), indicating that inhibition of the JAK-STAT3 pathway prevented astrocyte shrinkage. In addition, the complexity of astrocyte arborization was also unchanged in cocaine-sensitized mice when the JAK-STAT3 signaling pathway was inhibited, as assessed by Sholl analysis (Fig. 3F). Altogether, these data show that JAK-STAT3

pathway inhibition prevents the structural remodeling of astrocytes induced by cocaine.

### 3.4. Activation of the JAK-STAT3 pathway in accumbal astrocytes contributes to cocaine-induced hyperlocomotion

We then investigated the effects of JAK-STAT3 inhibition in NAc astrocytes on the behavioral alterations triggered by cocaine administration, i.e., increased locomotor activity (Fig. 1C-D) and anxiolytic effect (Fig. 1E-F). We found that the cocaine-induced hyperlocomotion was significantly reduced in mice expressing SOCS3 selectively in accumbal astrocytes compared to GFP control mice (Fig. 4A and B), suggesting that JAK-STAT3 signaling contributes to the early neuro-adaptive processes underlying cocaine-evoked hyperlocomotion. The effect of SOCS3 on locomotion was specific to the cocaine administration, as SOCS3 had no effect on basal locomotion in saline-injected WT mice (Figure S3). Furthermore, the SOCS3 effect was specific to the
A

gfaABC,D — SOCS3

gfaABC,D — GFP

<figure><img src="image_6.png" /></figure>

B

Virus
injection

4 weeks p.i.

D1 D2 D3 D4 D5

Injection
Behavior
Tissue
collection

P50-70

C

SOCS3 GFP
Saline

<figure><img src="image_7.png" /></figure>

SOCS3 GFP
Cocaine

<figure><img src="image_8.png" /></figure>

GFP
Cocaine

<figure><img src="image_9.png" /></figure>

D

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E

<figure><img src="image_11.png" /></figure>

F

<figure><img src="image_12.png" /></figure>

(caption on next page)
**Fig. 3.** Inhibition of the JAK-STAT3 pathway in accumbal astrocytes rescues astrocyte morphology in cocaine-treated mice. (A) Experimental design showing bilateral co-injection of AAV-GFAP<sub>ABCD1</sub>-SOCS3 and AAV-GFAP<sub>ABCD1</sub>GFP in the NAc of WT mice. Control mice were only injected with AAV-GFAP<sub>ABCD1</sub>-GFP. (B) Timeline of the experimental design. AAV injections were performed at P50-70, followed by saline/cocaine injection and behavioral recordings 4 weeks post-injection (p.i.). The brains were collected 24 h after the last injection. (C) Three-dimensional reconstruction of accumbal astrocytes in SOCS3-GFP mice after saline injections (grey) and in SOCS3-GFP (pink) or GFP (purple) mice after cocaine sensitization using IMARIS software. Scale bar: 10 µm. (D) After cocaine sensitization, the total length of astrocytic processes was unchanged in SOCS3-GFP mice (pink) compared to GFP mice (purple) and was similar to SOCS3 saline-injected mice (grey). \*p = 0.0006, F(2,34) = 0.9584, One-way ANOVA followed by post hoc analysis using Tukey's multiple comparisons test \*p = 0.0259 and \*\*\*p = 0.0005, SOCS3-GFP saline n = 8 cells from 3 mice, SOCS3-GFP cocaine n = 14 cells from 6 mice, GFP cocaine n = 15 cells from 6 mice. (E) After cocaine sensitization, the domain volume of astrocytes was unchanged in SOCS3-GFP mice (pink) compared to GFP mice (purple), and was similar to SOCS3 saline-injected mice (grey). \*\*p = 0.0016, F(2.35) = 0.6743, One-way ANOVA followed by post hoc analysis using Tukey's multiple comparisons test \*p = 0.0283 and \*\*p = 0.018, SOCS3 GFP saline n = 8 cells from 3 mice, SOCS3-GFP cocaine n = 15 cells from 6 mice, GFP cocaine n = 15 cells from 7 mice. (F) Sholl analysis showing the number of intersections per radius in SOCS3 saline-injected mice (grey) and SOCS3-GFP mice (pink) or GFP mice (purple) mice after cocaine sensitization. The concentric circles were spaced by 5 µm and centered on the soma of astrocytes. SOCS3 expression in astrocyte from cocaine-sensitized mice rescues the ramification complexity. p = 0.0005, Two-way ANOVA followed by post hoc analysis using Tukey's multiple comparisons test \*\*\*p = 0.0027 and \*\*\*\* p < 0.0001 two-way ANOVA, SOCS3-GFP saline n = 8 cells from 3 mice SOCS3-GFP cocaine n = 15 cells from 6 mice, GFP cocaine n = 15 cells from 7 mice.

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

**Fig. 4.** Inhibition of the JAK/STAT3 pathway in accumbal astrocytes reduces cocaine-induced motor behavior. (A) Representative travel paths in SOCS3 saline-injected mice (grey) and in SOCS3-GFP (pink) and GFP (purple) mice after cocaine sensitization. Only the last 10 min of the recordings of day 5 are shown here. (B) The distance traveled by SOCS3-GFP cocaine-injected mice (pink) is reduced compared to GFP cocaine-injected mice (purple), but remains higher compared to SOCS3 saline-injected mice (grey). p = 0.0053, F(8112) = 2.922, Two-way ANOVA followed by post hoc analysis using Tukey's multiple comparisons test \*\*\*p < 0.001, SOCS3-GFP saline n = 9 mice, SOCS3-GFP cocaine n = 9, GFP cocaine n = 13 mice. A significant sensitization was observed between D1 and D5 in GFP cocaine-injected mice (p = 0.05, df=12, Dunnett's multiple comparisons test) whereas no sensitization was detected in the SOCS3-GFP cocaine-injected mice (p = 0.2132, df=8, Dunnett's multiple comparisons test) (C) The time spent in the center is unchanged in SOCS3-GFP mice (pink) and GFP mice (purple) after cocaine sensitization at D1 and D5, and remains high compared to SOCS3-GFP saline-treated mice. At day 1: \*p = 0.0215 Kruskal-Wallis test followed by post-hoc analysis Tukey's multiple comparisons test \*p = 0.028403, SOCS3-GFP saline n = 9 mice, SOCS3-GFP cocaine n = 7 mice and GFP cocaine n = 6 mice. On day 5: \*\*p = 0.0080, F(2,15) = 3.369, One-way ANOVA test followed by post-hoc analysis Tukey's multiple comparisons test \*p = 0.0344 and \*\*p = 0.009, SOCS3-GFP saline n = 6 mice, SOCS3-GFP cocaine n = 5 mice and GFP cocaine n = 7 mice. (D) The number of entries in the center of the open field arena is similar in SOCS3-GFP mice (pink) and GFP mice (purple) after cocaine sensitization at D1 and D5. At day 1: \*\*\*p = 0.0005, F(2,19) = 2.797, One-way ANOVA test followed by post-hoc analysis Tukey's multiple comparisons test, \*\*p = 0.0031 and p = 0.0011 SOCS3-GFP saline n = 9 mice, SOCS3-GFP cocaine n = 7 mice and GFP cocaine n = 6 mice. On day 5: \*\*\*p = 0.0022, F(2,15) = 6.807, One-way ANOVA test followed by post-hoc analysis Tukey's multiple comparisons test \*\*\*p = 0.0045 and p = 0.0056 SOCS3-GFP saline n = 6 mice, SOCS3-GFP cocaine n = 5 mice and GFP cocaine n = 7 mice.
locomotion behavior, as the anxiolytic effect of cocaine sensitization was unaltered. The time spent in the center of the arena and the number of entries were indeed unchanged in cocaine-sensitized mice expressing SOCS3 in astrocytes compared to the GFP control mice (Fig. 4C and D). These findings indicate that inhibition of the JAK-STAT3 pathway in NAc astrocytes decreases cocaine-induced hyperlocomotion, but not its anxiolytic effect.

# 4. Discussion

We here show that cocaine leads in the NAc to astrocyte reactivity through the JAK-STAT3 signaling pathway, which plays a key role in the cocaine-induced pathological structural remodeling of astrocytes and mouse hyperlocomotion.

## 4.1. Cocaine-induced astrocyte reactivity

Our results reveal that repeated cocaine administration induces a significant structural plasticity in astrocytes. They undergo morphological changes characteristic of reactive astrocytes, including a reduction in size and process complexity. This structural remodeling leads to a retraction of astrocytic processes, which decreases synaptic coverage. With fewer perisynaptic contacts and a decrease expression of the GLT1 glutamate transporter, the neurotransmitters and ions buffering capacity of astrocytes is expected to be reduced, resulting functionally in impaired glutamate clearance and ionic dysregulation. This impairment of the astroglial homeostatic control likely results in extracellular glutamate accumulation, which could induce neuronal hyperexcitability mediating the hyperlocomotor response to cocaine (Pierce et al., 1996). While these findings are consistent with previous studies reporting cocaine-induced alterations in astrocyte morphology during prolonged withdrawal period after cocaine administration (Bowers and Kalivas, 2003) or following cocaine self-administration (Scofield et al., 2016), our results extend this knowledge by uncovering a signaling pathway within astrocytes that controls this structural remodeling. In addition to these morphological changes of astrocytes, we found that cocaine exposure changes the expression of important astrocyte proteins (namely GLT1, GS, S100β, Sox9, GFAP and vimentin) often associated with reactive astrocytes (Escartin et al., 2021). Astrocyte reactivity can, depending on the context, have beneficial or detrimental effects. Our findings indicate that in the present model, STAT3-dependent reactive astrocytes contribute to cocaine-induced hyperlocomotion. While earlier studies have reported that cocaine can induce structural changes in astrocytes (Harder et al., 2024; Wang et al., 2022), which is reflected by the altered expression of the GFAP cytoskeleton protein (Jarvis et al., 2020; Scofield et al., 2016), our study provides the first conceptual evidence that inhibiting astrocyte reactivity, which prevents morphological rearrangements, can mitigate motor symptoms associated with cocaine exposure. This astrocytic mechanism provides insights into the brain's plasticity mechanisms underlying drug-induced behavior and complements well-established neuronal adaptations triggered by repeated cocaine exposure. Briefly, it was shown that cocaine primarily acts by inhibiting dopamine transporters, resulting in a buildup of dopamine in the synaptic cleft. This disruption of dopamine reuptake intensifies dopaminergic signaling, which drives an immediate hyperlocomotion (Giros et al., 1996). Prolonged exposure to cocaine induces effects by altering the expression of genes involved in neurotransmission (Nestler, 2001), and long-term effects by triggering a structural remodeling of dendrites in NAc neurons (Robinson and Kolb, 2004). Altogether, these neuronal adaptations underlie the progressive behavioral changes observed with repeated drug exposure. Our findings extend this framework by demonstrating that astrocytes are also engaged during the initial phase of sensitization, suggesting that glial plasticity contributes to the early adaptations to cocaine.

## 4.2. Involvement of the JAK-STAT3 pathway in cocaine-induced astrocyte reactivity

The JAK-STAT3 signaling pathway plays a pivotal role in modulating astrocyte reactivity (Ceyzériat et al., 2016). Although we did not directly dissect the upstream triggers in this study, it is noteworthy that cocaine is known to increase cytokines such as IL-6 and CNTF, as well as inflammatory and neurotransmitter-related signaling within the NAc (Berhow et al., 1996; Cheng et al., 2023), which could all activate STAT3 in astrocytes (Ben Haim et al., 2015; Bonni et al., 1997; Heinrich et al., 2003). Here, we demonstrated that inhibition of the JAK-STAT3 pathway in astrocytes prevents the structural alterations of astrocytes and the motor hyperactivity triggered by cocaine. Mechanistically, cocaine exposure activates JAK-STAT3 signaling, which in turn drives astrocytic hypertrophy and retraction of processes, leading to a loss of perisynaptic coverage. This structural remodeling combined to the decreased expression of the GLT1 glutamate transporter should reduce the capacity of astrocytes to buffer extracellular glutamate and ions, and result in glutamate spillover and neuronal hyperexcitability, which is known to translate behaviorally in hyperlocomotion (Pierce et al., 1996). Dampening astrocyte reactivity through SOCS3-mediated inhibition of the pathway prevented both the morphological remodeling and the associated behavioral phenotype, while leaving the anxiolytic-like effect of cocaine unaffected. These effects are likely cell-autonomous due to the viral strategy designed to selectively manipulate JAK-STAT3 signaling within astrocytes, and the observed reduction in the number of STAT3 + astrocytes together with the prevention of morphological remodeling. However, these effects may also secondarily involve feedback interactions with neurons or other glial cells. It is worth noting that SOCS3, while widely recognized as a negative regulator of JAK-STAT3 signaling, especially in astrocytes (Abjean et al., 2023; Ben Haim et al., 2015; Ceyzériat et al., 2018; Guillemaud et al., 2020), through direct binding of receptor-JAK complexes (Kershaw et al., 2013), may also have potential effects on other signaling cascades such as NF-κB (Chhabra et al., 2014), notably by a reduction in cytokine production by SOCS3-expressing cells. In our model, SOCS3 over-expression did not induce structural or behavioral alterations in the absence of cocaine (Figs. 3 and 4, Supplementary Figures 2 and 3), suggesting that such non-specific effects are limited under our conditions. This differential effect of astrocytes on two distinct behavioral outcomes (motor vs anxiolytic effect) highlights the selective influence of accumbal astrocytes on specific neuronal circuits. Additional studies are required to clarify whether, and through which mechanisms, astrocyte reactivity alters the motivational and reward processes induced by cocaine. Taken together, these results underscore the complexity of astrocytic responses to cocaine, and their context-dependent role in influencing neuronal circuits and modulating behavioral outcomes.

## 4.3. Therapeutic potential of the JAK-STAT3 pathway for substance use disorders

The identification of the JAK-STAT3 pathway as a key regulator of astrocyte reactivity underscores its therapeutic potential to modulate early responses induced by cocaine exposure. Interestingly, previous studies have shown that activation of this signaling pathway exerts neuroprotective effects, and reduces the intensity of convulsions under conditions of high cocaine exposure (60 mg/kg) (Mai et al., 2019), suggesting a dose-dependent effect. By targeting the JAK-STAT3 pathway, therapeutic interventions can modulate glial activity to counteract the maladaptive changes induced by cocaine. This approach is particularly promising as it focuses on the critical, but underexplored, role of astrocytes in cocaine-mediated behavior, complementing existing neuron-centric treatments (Liddelow and Barres, 2017). Glial-centered strategies could provide a novel avenue to alleviate the structural and functional disruptions associated with substance use
disorders, offering a broader therapeutic landscape. Future studies should explore the implications of astrocytic signaling pathways in cocaine-induced behaviors and their potential for clinical intervention.

## Funding

This work was funded by grants from ANR (ANR-22-CE16-0009-03, ANR-22-CE16-0027) to N.R., IRESP and Alliance Aviesan program (IRESP-19-ADDICTIONS-06) to N.R. and addiction prevention fund (CAD-V124-030) as part of the 2024 call for research projects entitled "Addictive Behaviors and Drugs (CAD): Prevention, Mechanisms, Identification, and Support," jointly sponsored by IRESP and INCa to N.R. and C.E., from ANR to C.E. (ANR-16-TERC-0016-01), and from Sorbonne University doctoral school ED3C and Labex Memolife to A.C. This work has also received support under the Major Research Program of PSL Research University "PSL-Neuro" launched by PSL Research University and implemented by ANR (ANR-10-IDEX-0001).

## CRediT authorship contribution statement

**Isabelle Arnoux:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Anna Capano:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Yakoubi Rachida:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Claire Boulogne:** Writing – review & editing, Project administration, Methodology, Investigation, Formal analysis. **Pascal Ezan:** Writing – review & editing, Methodology, Investigation. **Carole Escartin:** Writing – review & editing, Resources. **Nathalie Rouach:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We gratefully acknowledge the Orion technological core (IMACHEM-IBiSA) of the Center for Interdisciplinary Research in Biology for their support, and especially Julien Dumont and Estelle Anceaume for assisting us with slide scanner acquisition, and Philippe Mailly for his expertise and guidance in the 3D reconstruction of astrocytes. We sincerely thank Dominique Langui and Asha Baskaran from the ICM Quant imaging platform for assisting us with EM image acquisition, Astrid Rollenhagen for her advises on EM analysis, and all the members of the Neuroglial Interactions in Cerebral Physiology and Pathologies laboratory for constructive discussions.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.pneurobio.2025.102852.

## Data availability

All published reagents will be shared on an unrestricted basis after completion of a material transfer agreement; reagent requests should be directed to the lead contact.

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