# Sex-dependent effects of stress on aIC-NAc circuit neuroplasticity: Role of the endocannabinoid system

Manon Gauthier, Léo-Paul Hebert, Emilie Dugast, Virginie Lardeux, Kevin Letort, Nathalie Thiriet, Laure Belnoue, Eric Balado, Marcello Solinas, Pauline Belujon

► To cite this version:

Manon Gauthier, Léo-Paul Hebert, Emilie Dugast, Virginie Lardeux, Kevin Letort, et al. Sex-dependent effects of stress on aIC-NAc circuit neuroplasticity: Role of the endocannabinoid system. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2025, 138, pp.111335. 10.1016/j.pnpbp.2025.111335. hal-05007846

HAL Id: hal-05007846

https://hal.science/hal-05007846v1

Submitted on 6 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Sex-dependent effects of stress on aIC-NAc circuit neuroplasticity: Role of the endocannabinoid system

Manon Gauthier<sup>a</sup>, Léo-Paul Hebert<sup>a</sup>, Emilie Dugast<sup>a,b</sup>, Virginie Lardeux<sup>a</sup>, Kevin Letort<sup>a,1</sup>, Nathalie Thiriet<sup>a</sup>, Laure Belnoue<sup>a,b</sup>, Eric Balado<sup>a</sup>, Marcello Solinas<sup>a</sup>, Pauline Belujon<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Stress

Synaptic plasticity

Insular cortex

Nucleus accumbens core

CB1 receptors

## ABSTRACT

Stress is a major risk factor for psychiatric disorders and affects neuroplasticity in brain areas like the nucleus accumbens core (NAcC) and the insular cortex (IC). This study examined neuroplasticity changes in the aIC-NAcC circuit after restraint stress in male and female rats, and explored the role of the endocannabinoid system.

Male and female rats underwent 2 h of acute restraint stress. Behavioral tests and *in vivo* electrophysiological recordings were performed immediately and 24 h after stress exposure. cFos was performed immediately after stress. Since stress effects were observed only in males, we evaluated the systemic and intra-NAc blockade of CB1 receptors in male rats.

We found increased c-Fos expression in the hypothalamus but not in the IC in both sexes after acute restraint stress, along with heightened anxiety and reduced exploratory behavior. Males and females exhibited different neuronal plasticity in the aIC-NAcC pathway. Under basal conditions, males showed equal proportions of long-term potentiation (LTP) and long-term depression (LTD), whereas females predominantly exhibited LTP. Stress disrupted synaptic plasticity in males by eliminating LTD in the aIC-NAcC pathway 24 h after exposure. This effect was reversed by systemic and local CB1 receptor blockade.

These findings suggest that integration of aIC information into NAcC differs by sex, with stress-induced neuroplasticity changes occurring only in males, dependent on the endocannabinoid system. This study provides insight into sex differences in stress reactivity, which may relate to stress-related psychiatric disorders.

## 1. Introduction

Stress intricately modulates the neurobiological substrates governing behavior and cognition (McEwen and Gianaros, 2011). Among its numerous effects, stress profoundly impacts neuronal plasticity, the fundamental property of neuronal networks, to adapt and reshape neuronal circuits in response to environmental stimuli. A brain region of interest in the investigation of stress-induced neuronal plasticity is the nucleus accumbens (NAc), a pivotal limbic-motor hub responsible for translating learned associations with motivational significance into goal-directed behavior (McEwen and Gianaros, 2011). Current research indicates that stress dynamically shapes synaptic strength and connectivity within the NAc, thereby influencing responses to stressors and reward-related cues (Campioni et al., 2009). Additionally, emerging

research highlight the critical role of the insular cortex (IC) in stress responses. The anterior IC (aIC) is involved in interoceptive awareness and emotional processing (Craig, 2009; Critchley et al., 2004; Gu et al., 2013) and serves as a key mediator of stress-related physiological and affective responses (Shi et al., 2023). Neuroimaging studies in humans have consistently identified the aIC as a central region affected in different stress-related psychiatric disorders, including depression and anxiety disorders. Indeed, increased activation of the aIC has been described in response to negative stimuli in depressed patients (Sliz and Hayley, 2012) and activation of the aIC is positively correlated to individual levels of anxiety (Terasawa et al., 2013). Similarly, preclinical studies in rodents have underscored the importance of the aIC circuit in modulating anxiety-related behaviors (Méndez-Ruette et al., 2019; Nicolas et al., 2023), and depression (Yokoyama et al., 2024).

<footnote><sup>a</sup> Université de Poitiers, INSERM, U-1084, Laboratoire de Neurosciences Expérimentales et Cliniques, Poitiers, France</footnote>

<footnote><sup>b</sup> CHU de Poitiers, Poitiers, France</footnote>

<footnote>* Corresponding author at: Laboratoire de Neurosciences Expérimentales et Cliniques, INSERM U1084, Université de Poitiers, Pôle Biologie Santé, Bâtiment B36, 1, rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9, France.</footnote>

<footnote>E-mail address: pauline.belujon@univ-poitiers.fr (P. Belujon).</footnote>

<footnote n="1">¹ Present address: Université de Bordeaux, CNRS, INCIA, UMR 5287, F-33000 Bordeaux, France.</footnote>
Considering the role of the aIC and the NAc in the stress response, and the IC's intricate connection with the NAc, in particular the NAc core (NAcC) (Reynolds and Zahm, 2005; Wright and Groenewegen, 1996), dysfunctions in the aIC-NAcC pathway might shape stress-induced maladaptive motivational behavior.

The endocannabinoid (eCB) system plays major roles in motivational (Solinas et al., 2008; Wenzel and Cheer, 2018) and stress processes (Hill et al., 2010). eCBs activate presynaptic cannabinoid type-1 receptors (CB1Rs) and modulate neurotransmitter release and synaptic plasticity across various brain regions, including the NAc (Covey and Yocky, 2021) and the insula (Zhao et al., 2024). Therefore, the eCB system may be involved in stress-induced changes in neuroplasticity in the aIC-NAcC pathway.

Here, we examined alterations in neuronal plasticity within the aIC-NAcC circuit following restraint stress in male and female rats. We first validated the stress protocol using behavioral assessments and c-Fos activation experiments, which revealed increased anxiety and c-Fos activation in the paraventricular nucleus (PVN) of the hypothalamus. Using *in vivo* electrophysiology and pharmacology, we show that 1) acute stress switches aIC-NAcC neuronal plasticity toward long-term potentiation 24 h after stress only in males, whereas it induces no changes in females and that 2) this switch is dependent on activation of CB1Rs.

## 2. Experimental procedures

### 2.1. Animals

Rats were used in this study because stress in rats can model many features of stress that are prevalent in human populations (Atrooz et al., 2021). One hundred and eighty-four adult (>9 weeks) male (n = 109) and female (n = 75) Sprague-Dawley rats (Janvier, France) arrived at least 5 days before experimentations and were handled consistently. Experiments were randomized, with equal habituation days for all groups. Rats were housed in pairs from arrival to the beginning of the stress protocol under a 12 h-light/-dark cycle (on at 7:00 am) with controlled temperature (22 °C) and humidity (47 %). Rats had *ad libitum* access to food and water in their homecage. All experiments were conducted during the light phase and in accordance with the European Union directives (2010/63/EU) for the care of laboratory animals. All experimental protocols were approved by the local ethics committee (COMETHEA, n°7147).

### 2.2. Stress protocol

Rats were subjected to a restraint stress which consisted of a 2 h- restraint session in a cylindric plexiglas size-adjusted tube (6 cm diameter for females and 7 cm for males, Phymep, Paris, France). For behavior and electrophysiology experiments, three groups were distinguished: a restraint stress group (RS) where experiments were performed immediately after restraint, restraint stress + 24 h group (RS24) where experiments were performed 24 h after restraint and a control group which remained in their homecage.

### 2.3. cFos experiments

The experimental protocol is illustrated in Fig. 1A. Rats were habituated to the experimental room in their homecage for 1 h. Rats were then restrained for 2 h and replaced 1 h in their homecage in the same experimental room before being isoflurane-anesthetized and euthanized with a lethal dose of pentobarbital (>150 mg/kg, i.p.). Rats were perfused intracardially with NaCl 0.9 % and paraformaldehyde 4 %. Control animals were exposed to the experimental room for 3 h without any manipulations before sacrifice. Brains were extracted and incubated 24 h in paraformaldehyde 4 % and cryoprotected 48 h in a 25 % sucrose solution. Once saturated, brains were coronally sliced (40 µm)

using a microtome (HM 450, Thermo Fischer Scientific, Illkirch-Graffenstaden, France). Brain slices were washed three times in phosphate buffered saline (PBS 1×).

### 2.4. Immunochemistry

The protocol for cFos and CB1 immunochemistry was adapted from (Belnoue et al., 2011). For cFos, after three PBS washes, an incubation of 30 min in H₂O₂ 1.2 % + TrisHCl 0.01 M pH 7.6 was performed to inactivate peroxidases. Three washes in PBS 1× were done before a second incubation with bovine serum albumin (BSA) blocking buffer solution (PBS 1×-Triton 100 × 0.3 % + BSA 3 %) during 1 h30. Brain slices were incubated overnight at 4 °C in a solution of primary antibody mouse anti-cFos (1/10000 diluted in BSA buffer solution, C-10 sc271243, Santa Cruz Biotechnology, Dallas, TX, USA). After three PBS washes, secondary antibody incubation was done with a donkey anti-mouse biotinylated antibody (1/1000 diluted in BSA buffer solution, 715-065-150, Jackson, Cambridge, UK) under agitation for 1 h30 at room temperature. Sections were washed and incubated for 1 h with avidin-biotin complex (ABC kit, Vector, PK-6100, Vector laboratories, Newark, CA, USA). Following PBS washes, sections were incubated for 30 min in 3,3'-diaminobenzidine-4HCl (DAB substrate kit, SK-4100, Vector laboratories, Newark, CA, USA) containing H₂O₂ in Tris Buffer, and three washes in PBS. Brain slices were mounted on gelatinized slides and coverslipped with depex (Sigma-Aldrich, Saint-Quentin-Fallavier, France). Slides were imaged by a transmitted light microscope (Zeiss Imager.M2, Zeiss, Paris, France).

For CB1 receptors, after three PBS washes, brain slices were incubated with bovine serum albumin (BSA) blocking buffer solution (PBS 1×-Triton 100 × 0.3 % + BSA 3 %) for 1 h30. Then, brain slices were incubated overnight under agitation at 4 °C in a solution of primary antibody mouse anti-CB1 (1/500 diluted in BSA buffer solution, SYSY 258.011, Synaptic System, Göttingen, Germany) After three PBS washes, secondary antibody incubation was done with a donkey anti-mouse Alexa 647 (1/500 diluted in BSA buffer solution, A315571, Invitrogen, Carlsbad, California, USA)) for 1 h30 at room temperature. After three PBS washes and one with gelatined-water (0.2 %) brain slices were mounted on slides and coverslipped with depex (Sigma-Aldrich, Saint-Quentin-Fallavier, France). Slides were imaged by a fluorescent microscope (Zeiss Imager.M2, Zeiss, Paris, France).

### 2.5. Quantification of staining

Quantification of c-Fos positive nuclei was done by acquiring 3 slices (right and left hemispheres) per rat containing the paraventricular nucleus (PVN) of the hypothalamus and the aIC. The fluorescent intensity for CB1 staining was quantified by acquiring 6 slices (right and left hemispheres) containing the NAc. These analyses were performed with Qupath and the values obtained were averaged per rat.

### 2.6. Elevated plus maze (EPM)

The protocol for the EPM was adapted from (Mitra et al., 2005) and is illustrated in Fig. 1F. The EPM apparatus consisted of four elevated perpendicular Plexiglas arms: two opposite open arms and two opposite closed arms of 50 cm length and 11 cm width. All surfaces were black and the wall for the closed arms was 43 cm height. Open arms were around 25 lx illumination. Animals were habituated 30 min in the room where the elevated plus maze (EPM) apparatus was placed. Rats of control group were habituated to the room, then placed in the center of the EPM. Rats of RS group were habituated to the room, then stressed and placed in the center of the EPM. Rats of the RS24 group were stressed and replaced in their homecage for 22 h. The day after, they were habituated to 30 min in the EPM room, then placed in the center of the EPM (Fig. 1F). All animals were facing one open arm and then allowed to start exploring the maze freely during a 5-min test. Video
<figure><img src="image_4.png" /><figcaption>A</figcaption></figure>

Ctrl

Habituation (1h)

No Restraint stress (2h)

cFos expression (1h)

Perfusion

<figure><img src="image_5.png" /></figure>

RS

Habituation (1h)

Restraint stress (2h)

cFos expression (1h)

Perfusion

Paraventricular nucleus

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

Male

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

Female

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

Insular cortex

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

<figure><img src="image_10.png" /><figcaption>E</figcaption></figure>

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

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

Ctrl

Habituation (30 min)

EPM (5 min)

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

RS

Habituation (30 min)

Restraint stress (2h)

EPM (5 min)

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

RS24

Restraint stress (2h)

22h

Habituation (30 min)

EPM (5min)

<figure><img src="image_15.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_17.png" /></figure>

(caption on next page)
<figure><img src="image_18.png" /><figcaption>**Fig. 1.** Effect of restraint stress on cFos density in the paraventricular nucleus of the hypothalamus and insular cortex and avoidance and exploratory behavior. (A) Experimental groups and procedures for quantification of cFos expression in control (Ctrl) and restraint stressed (RS) rats. (B, D) Representative transmitted light images of coronal slices of the paraventricular nucleus of the hypothalamus (PVN) (B) and anterior the insular cortex (aIC) (D) showing cFos expression in a Ctrl and RS rat. (C, E) Impact of RS on mean ± SEM cFos density in the PVN (C) and in the aIC (E) in males and females. Male Ctrl, *n* = 8; Male RS, *n* = 5; Female Ctrl, *n* = 8; Female RS, *n* = 8; (F) Experimental groups and procedures for behavioral evaluation in elevated plus maze (EPM) in control (Ctrl) and restraint stressed rats immediately (RS) or 24 h after stress (RS24). Quantification of behaviors with percentage of time spent in open arms (G), distance in meter (H) and number of rearing (I) in males (purple) and females (green) (mean ± SEM). Male Ctrl, *n* = 8, Male RS, *n* = 8, Male RS24, *n* = 8; Female Ctrl, *n* = 10; Female RS, *n* = 9, Female RS24, *n* = 8; \*p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**Fig. 2.** Neuronal plasticity of the anterior insular cortex to nucleus accumbens core pathway in male and female rats. (A) Experimental group and procedure for *in vivo* electrophysiology in anesthetized control rats (Ctrl) (B) Schematic of the placements of the stimulation electrode in the anterior insular cortex (aIC) and the recording electrode in the core of the nucleus accumbens (NAcC). Grey lightning represents single pulse stimulation and yellow lightning represents high-frequency stimulation (HFS). (C) Representative aIC stimulation (black arrow) and NAcC recording sites (black triangle). (D) Left, representative electrophysiological trace showing aIC-evoked spike probability recorded from a NAcC neurons in a Ctrl male rat with an overview of 20 frames.; 12 spikes out of 20 stimulations were evoked. Right, spike latency distribution of an aIC-driven unit. Clearly visible is the peak latency at 19 ms in the NAcC to aIC stimulation and the spike jitter <5 ms. The lightning indicates the stimulation artifact (E) Representative electrophysiological trace showing aIC-evoked spike probability recorded from a NAcC neurons in a Ctrl male (left, purple) and female (right, green) rat with an overview of 10 frames, 10 min before HFS (baseline) and 15 min post-HFS. (F) Percentage change (± SEM) in aIC-evoked spike probability, normalized to the baseline, after HFS to the aIC in males (purple circles) and females (green circles) (F) Mean percent change post-HFS in aIC-evoked responses (± SEM) following HFS in male (purple) and female (green) rats. (G) Proportions of recorded neurons showing an LTP, no changes or LTD in males (purple) and female rats (green). Male *n* = 18; Female *n* = 17; \*p < 0.05; \#p < 0.05 in comparison to baseline. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
recordings were analyzed using video tracking software (Viewpoint, Lyon, France) to measure distance and time spent immobile. A manual analysis was performed by two experimenters who were blind to the experiment to measure the number of entries, the number of rearing and the time spent in open arms realized by the animal. An entry into an open arm was defined as occurring when all four paws of the animal were placed within the boundaries of the open arm (Pellow et al., 1985). Rearing was defined as a vertical movement where the rat lifted its forepaws off the ground and extended its body either against the walls of the testing apparatus (supported rearing) or without support from the walls (unsupported rearing). Each instance of rearing was counted when the rat's forepaws were raised completely off the floor for a minimum of 1 s.

## 2.7. *In-vivo single-unit extracellular recordings*

Electrophysiological recordings were adapted from previous studies (Belujon et al., 2010, 2012; Belujon and Grace, 2008). Rats were isoflurane-anesthetized (5 % induction, 2.5 % maintenance) and placed in a stereotaxic apparatus. Extracellular recording electrodes were pulled from glass micropipettes (capillaries 30–0057 GC150F-10, Harvard Apparatus, Holliston, MA, France; impedance, 8.5–14 MΩ) and filled with a 2 % Chicago Sky Blue dye (Sigma-Aldrich, Saint-Quentin-Fallavier, France) solution in 2 M NaCl. Evoked activity was recorded from the right NAcC (AP, +1.1–1.4 mm relative to bregma; L, 2–2.3 mm from the midline; D, −6.8–8 mm from the skull surface) according to the brain atlas (Paxinos and Watson, 2017). Signals were amplified 50 times, filtered (low pass: 300 Hz; high pass: 16 kHz; Multiclamp 700b, Axon Instruments, Union City, CA, USA) and digitized at 16 kHz (CED 1401, Cambridge Electronics Design, Cambridge, UK), acquired on a computer using Signal 6.05 software (Cambridge Electronics Design).

## 2.8. *Stimulation protocol*

Concentric bipolar stimulating electrodes (FHC, USA) were lowered in the right aIC (AP, 10° angle, +3.4 mm from bregma; L, 4.0 mm from the midline; D, 5.0 mm from the brain surface). Stimulation of the aIC was delivered every 2 s (0.5 Hz, 300 µs width pulse), while recording electrode was lowered in the NAcC (Fig. 2B, C). Only NAcC neurons that responded monosynaptically (latency 20 ms (Belujon et al., 2013; Mulder et al., 1998), synaptic jitter <5 ms; (Kobayashi and Shinomoto, 2024)) were used for analysis (Fig. 2D). aIC-evoked-spike probability in the NAcC was calculated by dividing the number of action potentials by the number of stimulations. The current intensity was adjusted to obtain ~50 % of evoked response. Baseline aIC-evoked activity was recorded from NAcC neurons to have at least 10 min of stable recording. Spike probability was calculated at 5-min intervals. After a stable baseline (<15 % variability in spike probability over 10–15 min; (Floresco et al., 2001), high frequency stimulation known to induce synaptic plasticity in the cortex-accumbens pathway (HFS, 50 Hz, 2 s, 300 µs pulse width, Goto and Grace, 2005a, 2005b) was applied to the aIC with a current adjusted to obtain ~100 % of evoked-spike probability. Evoked-spike probability after HFS was recorded for 40 min with the same current used during baseline. Only recordings of 20 min or more post-HFS were analyzed. Changes of ±20 % in spike probability for more than 20 min were considered changes of synaptic plasticity (Floresco et al., 2001). Percentage of baseline (normalization) was used to control for unwanted source of variation.

Baseline current intensity, baseline spike probability and mean latency for the neurons recorded in the NAcC of male and female rats are shown in Table 1.

## 2.9. *Pharmacological injections*

For systemic injections, AM251 (2 mg/kg; NIDA) or its vehicle (DMSO 1 %, Tween 80 4 % NaCl 95 %) were injected intraperitoneally

<caption>**Table 1**</caption>

Latency to spike discharge and aIC stimulus intensity and baseline spike probability of neurons recording in the NAcC during the baseline period of animals.

<table><thead><tr><th rowspan="2"></th><th colspan="3">Males</th><th colspan="3">Females</th></tr><tr><th>Ctrl</th><th>RS</th><th>RS24</th><th>Ctrl</th><th>RS</th><th>RS24</th></tr></thead><tbody><tr><td>Number of neurons</td><td>18</td><td>14</td><td>9</td><td>15</td><td>14</td><td>13</td></tr><tr><td>Latency to spike discharge (ms)</td><td>19.12 ± 0.90</td><td>21.56 ± 1.17</td><td>22.19 ± 1.26</td><td>20.68 ± 1.34</td><td>22.74 ± 1.02</td><td>19.70 ± 0.84</td></tr><tr><td>Current intensity (mA)</td><td>0.95 ± 0.06</td><td>0.99 ± 0.08</td><td>0.98 ± 0.1</td><td>0.96 ± 0.06</td><td>0.86 ± 0.08</td><td>0.86 ± 0.09</td></tr><tr><td>Baseline spike probability</td><td>0.60 ± 0.04</td><td>0.53 ± 0.07</td><td>0.54 ± 0.06</td><td>0.48 ± 0.04</td><td>0.52 ± 0.03</td><td>0.50 ± 0.05</td></tr></tbody></table>

30 min before electrophysiological recordings. For local injections, AM251 (50 nM, 0.2 µl) or its vehicle (DMSO 1 %, Tween 80 4 % NaCl 95 %) were injected locally in the NAcC 30 min before electrophysiological recordings. The dose of AM251 was chosen based on pharmacological principles, ensuring it exceeded the dissociation constant (Kd = 7.5 nM) to achieve effective CB1 receptor blockade.

## 2.10. *Histology*

Location of the recording electrode was verified via Chicago Sky Blue dye (Sigma-Aldrich) electrophoretic ejection (−19 µA constant current, 20 min). Rats were then euthanized with a lethal dose of pentobarbital (>150 mg/kg, i.p.) and brains were collected. The tissue was fixed for 48 h in 8 % paraformaldehyde and then cryoprotected in a 25 % sucrose solution. Once saturated, brains were frozen with isopentane, and coronally sliced (30 µm) using a cryostat (CryoStar NX70, Thermo Fisher Scientific). Slices were mounted onto gelatin-coated slides and stained with a solution of neutral red and cresyl violet. Stimulating electrode sites were verified and only neurons recorded in the NAcC and stimulating electrode in the aIC were included in the data analysis (Supplementary Fig. 1).

Estrus cycle of females was determined in order to study whether the female estrus phase influences the experimental results. However, these data are purely descriptive. Cells of vaginal canal were collected using a cotton-tip soaked of distilled water, spread on slides, and stained with crystal violet solution (McLean et al., 2012; Marcondes et al., 2002) (Supplementary Fig. 2).

## 2.11. *Statistical analyses*

All the results are expressed as the mean ± SEM. A Grubb's test was used to identify statistical outlier animals. No outliers were found in this study. Statistical significance was set at *P* < 0.05. Data were analyzed with the statistical program GraphPad Prism (Version 9; GraphPad Pris San Diego, California, USA, RRID:SCR_002798).

For cFos and behavior analysis, a two-way ANOVA was performed with stress as the within-subject factor and sex as the between-subject factor, followed by Sidak's post hoc. When data did not pass the normality test, a mixed-effects model was used.

For CB1 receptors expression analyses, we used an unpaired *t*-test.

Electrophysiological data were analyzed using either a two-way ANOVA with time (minutes) and stress conditions (RS and RS24), sex (male and female) or drug (vehicle and AM251) as factors followed by Sidak's post hoc or a one-way ANOVA followed by Sidak's post hoc (mean percentage change after stress). Mean percentage change between control males and females and between local AM251 and vehicle in stressed males were analyzed using an unpaired *t*-test, since the data passed the normality test.

A chi-square test was conducted to compare the proportions of
neurons which displayed post-HFS LTP, LTD or no change across two groups (females and males) and across three experimental conditions per sex (control, acute stress, stress +24 h).

# 3. Results

## 3.1. Acute restraint stress increases cFos expression in the paraventricular nucleus of the hypothalamus and induces anxiety in male and female rats

Acute restraint stress in rodents has been shown to increase avoidance of the open arms and decrease exploratory behavior using the elevated plus maze (Padovan et al., 2000; Padovan and Guimarães, 2000; Reis et al., 2011) and increase cFos protein expression density in the paraventricular nucleus of the hypothalamus (PVN), which is part of the hypothalamo-pituitary-adrenal axis (HPA) (Zavala et al., 2011). The majority of studies have been done in males, and limited information is available in the literature for females. To validate the restraint model used in our study, we measured cFos expression in male and female rats in the PVN after exposure to acute stress. Additionally, we assessed cFos expression in the insular cortex to evaluate the impact of acute stress on this structure.

A two-way ANOVA was conducted to examine the effects of sex (male vs. female) and stress (control vs. restraint stress) on cFos levels in the PVN (Fig. 1B, C) and the aIC (Fig. 1D, E). There was a significant main effect of stress ($F_{(1, 25)} = 86.14, p < 0.0001$) in the PVN, indicating that acute stress significantly increased cFos levels in the PVN compared to the control group. However, the main effect of sex was not significant ($F_{(1, 25)} = 0.4762, p = 0.4762$). There was a significant interaction between sex and stress ($F_{(1, 25)} = 7.829, p = 0.0098$), suggesting that the effect of stress differed between sexes. Post-hoc comparisons using Sidak's multicomparison test revealed that cFos levels were significantly higher in the PVN after acute stress compared to the control group in males ($p < 0.0001$) and females ($p = 0.0003$) (Fig. 4C). For the aIC, there was a significant main effect of sex (two-way ANOVA, $F_{(1, 25)} = 40.62, p < 0.0001$) suggesting that cFos levels were lower in females in comparison to males, but the main effect of stress or the interaction between sex and stress were not significant (two-way ANOVA, $F_{(1, 25)} = 0.5016, p = 0.4853$ and $F_{(1, 25)} = 2.313, p = 0.1409$, respectively).

The PVN activation suggests that the procedure used in the present study activates the hypothalamic-pituitary-adrenal axis in both sexes and validate our model.

In the elevated plus maze (EPM) test (Fig. 1F), the percentage of time spent in open arms decreased after stress in males and females (Fig. 1G). There was a significant main effect of stress (two-way ANOVA, $F_{(2, 45)} = 10.97, p = 0.0001$), but no main effect of sex ($F_{(1, 45)} = 0.02063, p = 0.8864$) and no interaction between stress and sex ($F_{(2, 45)} = 0.4497, p = 0.6406$). Sidak's post-hoc comparisons revealed that the percentage of time spent in open arms decreased in males between Ctrl vs RS ($p = 0.0055$) and between Ctrl vs RS24 ($p = 0.0096$) but not between RS and RS24 ($p = 0.9963$). In females, it decreased between Ctrl and RS24 ($p = 0.0284$) but not between Ctrl and RS ($p = 0.0947$), or between RS and RS24 ($p = 0.9261$).

Exploratory behavior was investigated by measuring the distance travelled in the EPM (Fig. 1H) and the number of rearing (Fig. 1I). The distance was decreased after stress in males and females. There was a main effect of stress (two-way ANOVA; $F_{(2, 45)} = 16.83, p < 0.0001$) but no main effect of sex ($F_{(1, 45)} = 0.5920, p = 0.4457$) or interaction between sex and stress ($F_{(2, 45)} = 0.2284, p = 0.7968$). Sidak's post hoc analysis showed a difference between Ctrl and RS rats ($p = 0.0007$) and between Ctrl and RS24 rats ($p = 0.0143$) in males but not between RS and RS24 ($p = 0.6589$) and between Ctrl and RS in females ($p = 0.0006$) but neither between Ctrl and RS24 ($p = 0.1029$), nor between RS and RS24 ($p = 0.2460$). Moreover, the number of rearing was decreased after stress in males and females. There was a main effect of stress (two-way ANOVA; $F_{(2, 45)} = 38.87, p < 0.0001$), but no main effect of sex ($F_{(1, 45)} = 0.4946, p = 0.3493$) and no interaction between sex and stress ($F_{(2, 45)} = 0.4673, p = 0.6297$).

Sidak's post-hoc analysis revealed a difference between Ctrl vs RS ($p < 0.0001$, RS vs RS24 ($p < 0.0001$) but not between Ctrl vs RS24 ($p = 0.1492$) in males and a difference between Ctrl vs RS ($p < 0.0001$), between RS vs RS24 ($p < 0.0001$) but not between Ctrl vs RS24 ($p = 0.1298$).

These results suggest that acute stress induces anxiety in both male and female rats.

## 3.2. Insula-accumbens neuronal plasticity is different between naive males and females

To examine neuronal plasticity in the aIC-NAcC pathway in females and males, aIC-evoked activity was assessed in the NAcC after HFS to the aIC (Fig. 2A–D).

HFS applied to the aIC had a different effect on aIC-induced spike probability in the NAcC of control female and male rats (Fig. 2E–H). Indeed, a mixed effect analysis indicates an interaction between time and sex ($F_{(9, 258)} = 2.233, p = 0.0204$), although no effect of time ($F_{(9, 258)} = 2.365, p = 0.1240$) and no effect of sex ($F_{(1, 31)} = 3.221, p = 0.0825$) was observed. Post hoc analysis showed a significant increase of spike probability 15 min post-HFS in comparison to baseline in females ($p = 0.0461$), but no changes in males ($p = 0.9935$) (Fig. 2F). When comparing the mean percentage change in aIC-evoked NAcC spike probability across the 40 min post HFS, we found a trend toward a difference between male and female rats (Fig. 2G; Unpaired t-test, $t = 1.88, p = 0.0691$). A strong inter-individual variability was observed in the control groups for males and females (Fig. 2G). We thus clustered the different response types in relation to the mean percentage change in spike probability after HFS (Fig. 2H). In males, aIC HFS induced LTP (mean percentage change of spike probability >20 %) in 33 % of recorded neurons, LTD (mean percentage change of spike probability <−20 %) in 33 % and no changes (mean percentage change of spike probability between +20 % and −20 %) in 33 %. In females, 60 % of recorded neurons showed an LTP post-HFS, 13.3 % an LTD, and 26.7 % no changes. The proportion of responses was different between males and females (Fig. 2H; Chi square, $p = 0.0002$). It should be noted that this high variability did not depend on the location of recording and/or stimulating electrode (Supplementary Fig. 1).

This suggests that under control conditions, high frequency stimulation of the aIC is more efficient in males than in females to induce long-term depression and is more efficient in females than in males to induce long-term potentiation.

## 3.3. Restraint stress induces lasting changes in synaptic plasticity in the aIC-NAcC pathway in male, but not female, rats

To examine potential changes of neuronal transmission in the aIC-NAcC pathway in stressed females and males, aIC-evoked activity was assessed in the NAcC after HFS to the aIC in Ctrl, RS and RS24 rats (Fig. 3A–G).

In males, immediately after restraint stress, aIC HFS induced LTP in 43 % of recorded neurons and no changes in 57 %. Twenty-four hours after RS, aIC HFS induced LTP in 78 % of recorded neurons, and 11 % of LTD or no changes (Fig. 3B). The proportion of responses was significantly different between control and RS males (Chi square, $p < 0.0001$), control and RS24 males (chi square, $p < 0.0001$) and RS and RS24 males (chi square, $p < 0.0001$) (Fig. 3B). Analysis of the percentage change in aIC evoked NAcC spike probability (Fig. 3C) showed a significant main effect of time (mixed effect analysis, $F_{(18, 316)} = 5.380, p = 0.0105$), a significant interaction between time and stress condition ($F_{(18, 316)} = 1.742, p = 0.0317$) and a trend toward a significant main effect of stress condition ($F_{(2, 38)} = 3.186, p = 0.0526$). Analysis of the mean percentage change across the 40 min period post-HFS (Fig. 3D) revealed a significant effect of stress in males (Kruskal-Wallis test, $p = 0.0418$). Dunn's multiple comparison test showed that immediately after stress, there was no significant difference in comparison to ctrl ($p = 0.1438$) but a
A

<figure><img src="image_20.png" /></figure>

Ctrl

Anesthesia

In vivo electrophysiology

RS

Restraint stress (2h)

Anesthesia

In vivo electrophysiology

RS24

Restraint stress (2h)

22h

Anesthesia

In vivo electrophysiology

Male

B

Naive

<figure><img src="image_21.png" /></figure>

Restraint Stress

<figure><img src="image_22.png" /></figure>

Restraint Stress + 24h

<figure><img src="image_23.png" /></figure>

C

<figure><img src="image_24.png" /></figure>

D

<figure><img src="image_25.png" /></figure>

E

<figure><img src="image_26.png" /></figure>

Female

F

Naive

<figure><img src="image_27.png" /></figure>

Restraint Stress

<figure><img src="image_28.png" /></figure>

Restraint Stress + 24h

<figure><img src="image_29.png" /></figure>

G

<figure><img src="image_30.png" /></figure>

H

<figure><img src="image_31.png" /></figure>

I

<figure><img src="image_32.png" /></figure>

(caption on next page)
**Fig. 3.** Effect of restraint stress on neuronal plasticity of the anterior insular cortex to nucleus accumbens core pathway in male and female rats. (A) Experimental groups and procedures for in vivo electrophysiology in anesthetized control rats (Ctrl) and restraint stressed rats anesthetized immediately (RS) or 24 h after stress (RS24). (B, F) Representative electrophysiological trace showing aIC-evoked spike probability recorded from a NAcc neurons in a Ctrl (left), after RS (middle) and 24 h after RS (right) in male (B) and female (F) rats with an overview of 10 frames, 10 min before HFS (baseline) and 15 min post-HFS. (C, G) Proportions of recorded neurons showing an LTP, no changes or LTD in Ctrl, after RS and 24 h after RS (RS24) males (purple, C) and female rats (green, G). (D, H) Mean percentage change (± SEM) in aIC-evoked spike probability, normalized to the baseline, after HFS to the aIC in Ctrl (circle), RS (square) and RS24 (triangle) in male (purple, D) and female (green, H) rats. (E, I) Mean percentage change (± SEM) in aIC-evoked spike probability following HFS to the aIC in ctrl (circle), immediately after stress (RS) and 24 h after stress (RS24) in male (purple, E) and female (green, I) rats. Male Ctrl, n = 18; Male RS, n = 14, Male RS24, n = 9; Female Ctrl, n = 17, Female RS, n = 15, Female RS24, n = 14. \*p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

difference between ctrl and RS24 ($p = 0.0410$).

In female rats, immediately after restraint stress, aIC HFS induced LTP in 36 % of recorded neurons, no changes in 50 % and an LTD in 14 %. Twenty-four hours after RS, aIC HFS induced LTP in 61 % of recorded neurons, and LTD in 8 % and no changes in 31 % (Fig. 3E). The proportion of responses were not significantly different between groups but a trend toward a difference between RS and RS24 (Chi square, ctrl vs RS, $p = 0.1024$; ctrl vs RS24, $p = 0.0931$; RS vs RS24, $p = 0.0577$). Analysis of the percentage change in aIC evoked NAcc spike probability (Fig. 3F) showed a significant main effect of time (mixed effect analysis, $F(18, 354) = 11.60, p = 0.0005$), but no effect of stress ($F(2, 43) = 0.6036, p = 0.5514$) and no interaction between time and stress ($F(18, 354) = 0.8707, p = 0.6149$). Analysis of the mean percentage change across the 40 min period (Fig. 3G) showed no change in post-HFS spike probability (One-Way ANOVA, $F(2, 39) = 0.6221, p = 0.5421$).

Altogether these results suggest that in males, stress impairs synaptic mechanisms that allows long-term depression of excitability, which lasts up to 24 h. These results also suggest that in females, stress has no effect on aIC-NAc neuronal plasticity.

### 3.4. Blocking CB1 receptors restores neuronal plasticity 24 h after stress in males

Functional CB1 receptors are present at glutamatergic synapses in the NAcc (Deroche et al., 2020) and play a crucial role in regulating plasticity within the brain structures where they are expressed (Robbe et al., 2002). Previous studies have demonstrated that stress can shift CB1 receptor-dependent plasticity from LTD to LTP in the Bed Nucleus of the Stria Terminalis (Glangetas et al., 2013). Based on this, we hypothesis that in our model, stress may similarly alter plasticity in the NAcc in response to aIC stimulation in males and that blocking CB1 receptors could potentially restore it.

We first establish CB1 receptor involvement on baseline plasticity in naïve males, and 24 h after restraint stress by blocking CB1 receptors by systemic injection of AM251, a CB1 receptor antagonist (Fig. 4A–E).

In naïve males, AM251 had no effect on the HFS-induced plasticity, whereas 24 h after stress, aIC HFS blocked the stress-induced LTP in the NAcc (Fig. 4B, C). A mixed effect analysis revealed a main effect of time ($F(27, 137) = 4.466, p = 0.0344$), a main effect of stress ($F(1, 8) = 12.89, p = 0.0071$) and an interaction between time and drug ($F(9, 67) = 7.4, p \le 0.0001$). Posthoc analysis revealed a significant difference between vehicle and AM251 only in the stressed group (Fig. 4D). Analysis of the mean percentage change across the 40 min period post-HFS (Fig. 4E) showed a significant difference in spike probability between the RS24 + vehicle group and the RS24 + AM251 group (Fig. 4D). Thus, a mixed effect analysis revealed a main effect of the drug (vehicle or AM251) ($F(1, 16) = 6.505, p = 0.0214$), no effect of stress ($F(1, 16) = 0.00094, p = 0.09759$) and an interaction between stress and drug ($F(1, 16) = 6.22, p = 0.024$). Posthoc analysis revealed a significant difference between vehicle and AM251 only in the stressed group.

Given the data indicating increased aIC drive in the NAcc in stressed animals, along with the effects observed following systemic AM251 injection in these animals, we investigated whether CB1 receptors in the NAcc specifically mediate the stress-induced changes in synaptic plasticity (Fig. 4A, F–H). Consistent with our hypothesis, we found that AM251 infusion directly into the NAcc prevented the HFS-induced LTP

previously observed in the aIC-NAcC pathway (Fig. 4G–H). Thus, there is a significant difference between spike probability after HFS with pre-infusion of AM251 and with pre-infusion of vehicle in stressed males. Indeed, a mixed effect analysis revealed no effect of time ($F(9, 76) = 2.501, p = 0.138$), a main effect of the drug (vehicle or AM251) ($F(1, 9) = 6.41, p = 0.035$) and an interaction between time and drug ($F(9, 76) = 3.322, p = 0.0018$) (Fig. 4G). Analysis of the mean percentage change across the 40 min period post-HFS (Fig. 4G) showed a significant difference in spike probability between the RS24 + vehicle group and the RS24 + AM251 group (Unpaired t-test, $t = 2.482, p = 0.0349$) (Fig. 4H). These data suggest that the eCB system in the NAcc of stressed rats is responsible for the abnormal increase of aIC-NAcC drive.

### 4. Discussion

To our knowledge, this is the first study to examine neuronal plasticity in the anterior insular cortex-nucleus accumbens core pathway in both male and female rats and the impact of stress on aIC information processing in the NAcc. Our data reveal that acute stress switches the long-term impact of aIC stimulation on NAcc neurons into LTP only in males, with no effect in females. In addition, we found that the eCB system, through the stimulation of CB1 receptors in the NAcc, plays a key role in this plasticity switch.

While most studies on stress have been conducted in males, less is known about the neurobiological effects of stress in females. In this study, we found an increase in cFos expression in the PVN after acute restraint stress, consistent with previous studies (Chowdhury et al., 2000; Kovács et al., 2018). In the aIC, we found no changes in cFos expression after stress, which is inconsistent with a study showing a decrease in cell activation after 1 h of restraint stress (Tomeo et al., 2022). The discrepancies could be due to the difference in the time spent in the restraint tube, or other experimental conditions. The absence of cFos changes in the aIC, despite alterations in aIC-NAcC synaptic plasticity suggests that stress-induced plasticity occurs without requiring strong immediate neuronal activation. Indeed, c-Fos expression primarily reflects acute neuronal activation (Dragunow and Faull, 1989), whereas synaptic plasticity changes involve longer-lasting modifications in synaptic strength (Citri and Malenka, 2008), that may persist beyond the window of c-Fos expression.

In the EPM, we found a decrease in the % time spent in open arms, as well as a decrease in exploratory behavior in males and females. Our data suggest that in both males and females, restraint stress reduces activity and decreases the time spent in anxiogenic environments, such as the open arms of the EPM, with effects lasting up to 24 h. This is consistent with previous studies showing a lasting effect of stress in males (Mitra et al., 2005). This enhanced avoidance, and decreased exploratory behavior can be described as hypervigilance in anticipation of a possible future threat (anxiety), triggered by acute stress in both males and females (Endler and Kocovski, 2001).

Our electrophysiological data indicate different information processing in the NAc from aIC inputs between naïve males and females. First, it is important to note that the high frequency stimulation employed in our protocol could potentially engage other interconnected brain structures (Gogolla, 2017; Hernández-Ortiz et al., 2023), which may contribute to or modulate the observed effects in the NAcc. In naïve rats, HFS of the aIC induced no overall changes in spike probability in
<figure><img src="image_33.png" /><figcaption>**Fig. 4.** Effect of CB1 antagonist, AM251, on stress-induced neuroplastic changes in the NAcC in male rats. (A) Experimental groups for in vivo electrophysiology in anesthetized rats or 24 h after stress (RS24). (B) Representative electrophysiological trace showing aIC-evoked spike probability recorded from a NAcC neurons 24 h after RS after systemic vehicle injection (left) and AM251 (right) in a male with an overview of 10 frames, 10 min before HFS (baseline) and 15 min post-HFS. (C) Stimulation, recording and systemic injection protocol. (D) Percent change in aIC-evoked responses (± SEM) following vehicle (full triangle or circle) or AM251 (white triangle or circle) systemic injection and aIC HFS in control males (ctrl, circle) and in males 24 h after restraint stress (RS24, triangle). (E) Post-HFS mean percentage change (± SEM) in aIC-evoked spike probability, normalized to the baseline, to the aIC in control + vehicle, control + AM251, RS24 + vehicle and RS24 + AM251 in male rats. (F) Stimulation, recording and local infusion protocol. (G) Percent change in aIC-evoked responses (± SEM) following vehicle (full triangle) or AM251 (white triangle) local injection and aIC HFS in males 24 h after restraint stress (RS24). (H) Post-HFS mean percentage change (± SEM) in aIC-evoked spike probability, normalized to the baseline, to the aIC in RS24 + vehicle and RS24 + AM251 in male rats. Male control + i.p. vehicle, n = 5; control + i.p. AM251, n = 5; RS24 + i.p. vehicle, n = 5; RS24 + i.p. AM251, n = 5; RS24 + local vehicle, n = 5; RS24 + local AM251, n = 6. \*p < 0.05.</figcaption></figure>
males but an overall long-term potentiation in females. This is due in part to a large proportion of neurons recorded in males that displayed an LTD (33 %), whereas only a small proportion of neurons recorded in females showed this type of plasticity. This sex difference indicates difference in synaptic plasticity in basal conditions. Neuromodulators such as dopamine or endocannabinoids play a key role in regulating synaptic plasticity (Citri and Malenka, 2008) and may potentially contribute to the observed variations between males and females. It has been shown that females have a higher proportion of spines with large heads near tyrosine-hydroxylase immunoreactive fibers in the NAc, suggesting potential sex differences in dopaminergic modulation of large spines synapses (Wissman et al., 2012). Since dopamine is a potent modulator of synaptic plasticity in the NAcC (Goto and Grace, 2005a, 2005b), different dopaminergic modulation in the NAcC between males and females could potentially lead to different synaptic plasticity in the NAcC in basal conditions. Another possible explanation is the difference in the endocannabinoid (eCB) system between males and females. We did not find any differences in the expression of CB1 receptors in the NAcC between males and females (Supplemental Fig. 3), which is consistent with previous studies (Castelli et al., 2014). However, previous studies have shown that affinity of eCB (anandamide and 2-arachidonoylglycerol, 2-AG) for CB1 receptors are different in males and females in the striatum (de Fonseca et al., 1994). Males eCB receptor affinity in the striatum was higher compared to those of females at random stage of the estrous cycle (de Fonseca et al., 1994). Considering that eCB-dependent plasticity has been shown to be induced by tetanus stimulation in the NAc (Robbe et al., 2002), it is possible that differences in eCB synthesis, and/or release, or the signaling downstream of CB1R in the NAc of males and females explain differences in synaptic plasticity (Piette et al., 2020) in the aIC-NAcC pathway. Further investigation into the role of sex differences in endocannabinoid signaling or receptor modulation will be needed for understanding the underlying mechanisms.

Whereas stress impacted the aIC-NAcC pathway in males for at least 24 h, it had no effect in females. Indeed, in males, we demonstrated a total loss of LTD after stress that is maximal after 24 h. Previous studies have shown sex-specific role for corticotropin-releasing factor (CRF), released by PVN neurons in response to a stressor or a stressed conspecific, as a modulator of aIC synaptic activity (Rieger et al., 2022). Indeed, CRF injected in the aIC induced a depolarization of pyramidal neurons in both males and females but a reduction in presynaptic inhibitory tone in males only. Therefore, in our study, CRF release in response to stress could affect aIC synaptic activity only in males, and therefore the aIC-NAcC pathway in a different manner in males and females. Further, stress induces sex-specific variations in neurotrophin expression in the brain (Bath et al., 2013). For example, brain-derived neurotrophic factor (BDNF) expression in the hippocampus increases in female rats (Lin et al., 2009) but not in male rats after acute stress (Lakshminarasimhan and Chattarji, 2012; Lin et al., 2009). In prefrontal regions, BDNF mRNA levels are higher in female controls compared to males. Additionally, acute stress does not alter these levels in females; however, in males, BDNF mRNA levels increase in response to stressors (Bland et al., 2005). Although, data on BDNF levels in the NAcC or the aIC are lacking in females following stress, it is possible that changes in BDNF levels occur differently between males and females in the NAcC or the aIC following stress. Our data are consistent with preclinical studies using acute stress paradigm showing sexual dimorphism in the effect of stress but it is not yet known how these factors may directly influence neuronal physiology in the NAcC or the aIC in a sex-specific manner. This will need to be further investigated.

The finding that both males and females are vulnerable to behavioral changes after acute stress but only males exhibit alterations in aIC-NAc plasticity suggests that the stress circuitry may be sex-dependent. Indeed, females may utilize alternative pathways or mechanism for stress adaptation. For example, the aIC-basolateral amygdala (BLA) pathway has been implicated in processing emotional valence (Nicolas

et al., 2023), and stress-induced changes in synaptic plasticity in the BLA-NAc pathway has been described consistently (Gill and Grace, 2013; Madur et al., 2023; Segev and Akirav, 2016). In males, changes in aIC-NAc plasticity may directly reflect the neural impact of acute stress, whereas in females, regions such as the BLA may play a more prominent role. Further studies focusing on females will be needed to identify alternative stress circuitry.

Our data support the idea that stress blocks LTD in the aIC-NAc pathway only in males via the eCB system. eCBs are key mediators of synaptic plasticity (Heifets and Castillo, 2009). They play a role in short term plasticity, and they can influence the induction of eCB-independent plasticity forms such as LTP (Chevaleyre et al., 2006). Many studies have shown that acute stress increases 2-AG and decreases anandamide signaling in the majority of structures in the brain (Morena et al., 2016). 2-AG is more abundant than anandamide in most brain regions, is a full agonist for CB1 receptors and is the primary eCB involved in CB1-mediated inhibition of presynaptic release (Ohno-Shosaku and Kano, 2014). Changes in eCB signaling following acute stress can result in deficits in eCB-mediated synaptic plasticity and in homeostatic adaptations at presynaptic CB1 receptors (Melis et al., 2014). CB1 receptors are located at glutamatergic afferents to the NAcC (Deroche et al., 2020) but also at fast-spiking interneurons (Winters et al., 2012). In our model, blocking CB1 receptors, whether systemically or locally in the NAc, restored the stress-elicited shift in plasticity. However, it not clear if this effect occurs via CB1Rs located on glutamatergic terminals in the NAcC or via CB1Rs population located on NAcC GABA interneurons. This will need to be further investigated.

## 5. Conclusion

Taken together, our study provides novel insights into the impact of stress on neuronal plasticity in the aIC-NAcC pathway and reveals significant sex differences in this neural circuitry. Our findings suggest that while both males and females exhibit behavioral changes following acute stress, only males experience alterations in aIC-NAcC plasticity, suggesting alternative pathways for stress adaptation in females. This sex-specific switch is mediated by the eCB system, highlighting its crucial role in stress-induced adaptations.

These findings contribute to a deeper understanding of the neurobiological basis of stress-related disorders, particularly given the higher prevalence of such conditions in women. Future research should aim to elucidate the specific pathways and molecular mechanisms involved in female stress adaptation, providing a stronger foundation for developing targeted therapeutic interventions.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pnpbp.2025.111335.

## CRediT authorship contribution statement

**Manon Gauthier:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Léo-Paul Hebert:** Data curation, Formal analysis, Methodology, Writing – review & editing. **Emilie Dugast:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Virginie Lardeux:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Kevin Letort:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Nathalie Thiriet:** Writing – review & editing, Conceptualization. **Laure Belnoue:** Writing – review & editing, Methodology, Formal analysis. **Eric Balado:** Writing – review & editing, Software, Methodology, Formal analysis. **Marcello Solinas:** Writing – review & editing, Methodology, Conceptualization. **Pauline Belujon:** Writing – review & editing, Writing – original draft, Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
## Ethical statement

All experiments were conducted in accordance with the European Union directives (2010/63/EU) for the care of laboratory animals. All experimental protocols were approved by the local ethics committee (COMETHEA, n°7147).

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT in order to improve readability, and grammar. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

## Funding

This work was supported by the Institute National de la Santé et de la Recherche Médicale, the Centre National pour la Recherche Scientifique, the University of Poitiers, CHU of Poitiers, and the Région Nouvelle-Aquitaine.

## Declaration of competing interest

The authors declare no potential conflict of interest.

## Acknowledgements

This work has benefited from the facilities and expertise of PREBIOS platform (University of Poitiers). This work was supported by the Institute National de la Santé et de la Recherche Médicale, the Centre National pour la Recherche Scientifique, the University of Poitiers, CHU of Poitiers, and the Région Nouvelle-Aquitaine. The authors thank Catherine Le Goff for technical support and Luigi Bellochio for helpful discussions.

## Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## References

* Atrooz, F., Alkadhi, K.A., Salim, S., 2021. Understanding stress: insights from rodent models. Current Research in Neurobiology 2, 100013. https://doi.org/10.1016/j.cnrneur.2021.100013.

* Bath, K.G., Schilit, A., Lee, F.S., 2013. Stress effects on BDNF expression: effects of age, sex, and form of stress. Neuroscience 239, 149–156. https://doi.org/10.1016/j.neuroscience.2013.01.074.

* Belnoue, L., Grosjean, N., Abrous, D.N., Koehl, M., 2011. A critical time window for the recruitment of bulbar newborn neurons by olfactory discrimination learning. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 31 (3), 1010–1016. https://doi.org/10.1523/JNEUROSCI.3941-10.2011.

* Belujon, P., Grace, A.A., 2008. Critical role of the prefrontal cortex in the regulation of hippocampus-accumbens information flow. J. Neurosci. 28 (39), 9797–9805. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18815264.

* Belujon, P., Lodge, D.J., Grace, A.A., 2010. Aberrant striatal plasticity is specifically associated with dyskinesia following levodopa treatment. Mov. Disord. 25 (11), 1568–1576. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20623773.

* Belujon, P., Dollish, H.K., Grace, A.A., 2012. Ketamine restores activity of the dopamine system selectively in rats exhibiting learned helplessness in an animal model of depression. In: Neuroscience Meeting Planner. Program No: 774.07.

* Belujon, P., Patton, M.H., Grace, A.A., 2013. Disruption of prefrontal cortical- hippocampal balance in a developmental model of schizophrenia: reversal by sulpiride. Int. J. Neuropsychopharmacol. 16 (3), 507–512. https://doi.org/10.1017/S146114571200106X.

* Bland, S.T., Schmid, M.J., Der-Avakian, A., Watkins, L.R., Spencer, R.L., Maier, S.F., 2005. Expression of c-fos and BDNF mRNA in subregions of the prefrontal cortex of male and female rats after acute uncontrollable stress. Brain Res. 1051 (1–2), 90–99. https://doi.org/10.1016/j.brainres.2005.05.065.

* Campioni, M.R., Xu, M., McGehee, D.S., 2009. Stress-induced changes in nucleus accumbens glutamate synaptic plasticity. J. Neurophysiol. 101 (6), 3192–3198. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19357347.

* Castelli, M., Fadda, P., Casu, A., Spano, M., Casti, A., Fratta, W., Fattore, L., 2014. Male and female rats differ in brain cannabinoid CB1 receptor density and function and in behavioural traits predisposing to drug addiction: effect of ovarian hormones. Curr. Pharm. Des. 20 (13), 2100–2113. https://doi.org/10.2174/13816128113199990430.

* Chevaleyre, V., Takahashi, K.A., Castillo, P.E., 2006. Endocannabinoid-mediated synaptic plasticity in the CNS. Annu. Rev. Neurosci. 29, 37–76. https://doi.org/10.1146/annurev.neuro.29.050165.112834.

* Chowdhury, G.M., Fujioka, T., Nakamura, S., 2000. Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress. Brain Res. Bull. 52 (3), 171–182. http://www.ncbi.nlm.nih.gov/pubmed/10822158.

* Citri, A., Malenka, R.C., 2008. Synaptic plasticity: multiple forms, functions, and mechanisms. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology 33 (1), 18–41. https://doi.org/10.1038/sj.npp.1301559.

* Covey, D.P., Yocky, A.G., 2021. Endocannabinoid modulation of nucleus accumbens microcircuitry and terminal dopamine release. Frontiers in Synaptic Neuroscience 13. https://doi.org/10.3389/fnsyn.2021.734975.

* Craig, A.D., 2009. How do you feel—now? The anterior insula and human awareness. Nat. Rev. Neurosci. 10 (1), 59–70. https://doi.org/10.1038/nrn2555.

* Critchley, H.D., Wiens, S., Rotstein, P., Ohman, A., Dolan, R.J., 2004. Neural systems supporting interoceptive awareness. Nat. Neurosci. 7 (2), 189–195. https://doi.org/10.1038/nn1176.

* de Fonseca, F.R., Cebeira, M., Ramos, J.A., Martín, F., Fernández-Ruiz, J.J., 1994. Cannabinoid receptors in rat brain areas: sexual differences, fluctuations during estrous cycle and changes after gonadectomy and sex steroid replacement. Life Sci. 54 (3), 159–170. https://doi.org/10.1016/0024-3205(94)00585-0.

* Deroche, M.A., Lassalle, O., Castell, L., Valjent, E., Manzoni, O.J., 2020. Cell-type- and endocannabinoid-specific synapse connectivity in the adult nucleus accumbens core. J. Neurosci. 40 (5), 1028–1041. https://doi.org/10.1523/JNEUROSCI.1100-19.2019.

* Dragunow, M., Faull, R., 1989. The use of c-fos as a metabolic marker in neuronal pathway tracing. J. Neurosci. Methods 29 (3), 261–265. http://www.ncbi.nlm.nih.gov/pubmed/2507830.

* Endler, N.S., Kocovski, N.L., 2001. State and trait anxiety revisited. J. Anxiety Disord. 15 (3), 231–245. https://doi.org/10.1016/s0887-6185(01)00060-3.

* Floresco, S.B., Blaha, C.D., Yang, C.R., Phillips, A.G., 2001. Modulation of hippocampal and amygdala-evoked activity of nucleus accumbens neurons by dopamine: cellular mechanisms of input selection. J. Neurosci. 21 (8), 2851–2860. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11306637.

* Gill, K.M., Grace, A.A., 2013. Differential effects of acute and repeated stress on hippocampus and amygdala inputs to the nucleus accumbens shell. Int. J. Neuropsychopharmacol. 1–13. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=23745764.

* Glangetas, C., Girard, D., Groc, L., Marsicano, G., Chaouloff, F., Georges, F., 2013. Stress switches cannabinoid type-1 (CB1) receptor-dependent plasticity from LTD to LTP in the bed nucleus of the stria terminalis. J. Neurosci. Off. J. Soc. Neurosci. 33 (50), 19657–19663. https://doi.org/10.1523/JNEUROSCI.3175-13.2013.

* Gogolla, N., 2017. The insular cortex. Curr. Biol. 27 (12), R580–R586. https://doi.org/10.1016/j.cub.2017.05.010.

* Goto, Y., Grace, A.A., 2005a. Dopamine-dependent interactions between limbic and prefrontal cortical plasticity in the nucleus accumbens: disruption by cocaine sensitization. Neuron 47 (2), 255–266. https://doi.org/10.1016/j.neuron.2005.06.017.

* Goto, Y., Grace, A.A., 2005b. Dopaminergic modulation of limbic and cortical drive of nucleus accumbens in goal-directed behavior. Nat. Neurosci. 8 (6), 805–812. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15908948.

* Gu, X., Hof, P.R., Friston, K.J., Fan, J., 2013. Anterior insular cortex and emotional awareness. J. Comp. Neurol. 521 (15), 3371–3388. https://doi.org/10.1002/cne.23368.

* Heifets, B.D., Castillo, P.E., 2009. Endocannabinoid signaling and long-term synaptic plasticity. Annual Review of Physiology 71, 283–306. https://doi.org/10.1146/annurev.physiol.010908.163149. Volume 71, 2009.

* Hernández-Ortiz, E., Luis-Islas, J., Tecuapetla, F., Gutierrez, R., Bermúdez-Rattoni, F., 2023. Top-down circuitry from the anterior insular cortex to VTA dopamine neurons modulates reward-related memory. Cell Rep. 42 (11), 113365. https://doi.org/10.1016/j.celrep.2023.113365.

* Hill, M.N., McLaughlin, R.J., Bingham, B., Shrestha, L., Lee, T.T.Y., Gray, J.M., Hillard, C. J., Gorzalka, B.B., Viau, V., 2010. Endogenous cannabinoid signaling is essential for stress adaptation. Proc. Natl. Acad. Sci. U. S. A. 107 (20), 9406–9411. https://doi.org/10.1073/pnas.0914661107.

* Kobayashi, R., Shinomoto, S., 2024. Inference of monosynaptic connections from parallel spike trains: a review. Neurosci. Res. https://doi.org/10.1016/j.neures.2024.07.006.

* Kovács, L.A., Schiessl, J.A., Nafz, A.E., Csernus, V., Gaszner, B., 2018. Both basal and acute restraint stress-induced c-Fos expression is influenced by age in the extended amygdala and brainstem stress centers in male rats. Front. Aging Neurosci. 10, 248. https://doi.org/10.3389/fnagi.2018.00248.

* Lakshminarasimhan, H., Chattarji, S., 2012. Stress leads to contrasting effects on the levels of brain derived neurotrophic factor in the hippocampus and amygdala. PLoS One 7 (1), e30481. https://doi.org/10.1371/journal.pone.0030481.
Lin, Y., Ter Horst, G.J., Wichmann, R., Bakker, P., Liu, A., Li, X., Westenbroek, C., 2009. Sex differences in the effects of acute and chronic stress and recovery after long-term stress on stress-related brain regions of rats. Cerebral Cortex (New York, N.Y.: 1991) 19 (9), 1978-1989. https://doi.org/10.1093/cercor/bhn225.

Madur, L., Ineichen, C., Bergamini, G., Greter, A., Poggi, G., Cuomo-Haymour, N., Sigrist, H., Sych, Y., Paterna, J.-C., Bornemann, K.D., Viollet, C., Fernandez-Albert, F., Alanis-Lobato, G., Hengerer, B., Pryce, C.R., 2023. Stress deficits in reward behaviour are associated with and replicated by dysregulated amygdala-nucleus accumbens pathway function in mice. Communications Biology 6 (1), 1-20. https://doi.org/10.1038/s42003-023-04811-4.

Marcondes, F.K., Bianchi, F.J., Tanno, A.P., 2002. Determination of the estrous cycle phases of rats: Some helpful considerations. Brazilian Journal of Biology = Revista Brasileira De Biologia 62 (4A), 609-614. https://doi.org/10.1590/s1519-69842002000400008.

McEwen, B.S., Gianaros, P.J., 2011. Stress- and allostasis-induced brain plasticity. Annu. Rev. Med. 62, 431-445. https://doi.org/10.1146/annurev-med-052209-100430.

McLean, A.C., Valenzuela, N., Fai, S., Bennett, S.A.L., 2012. Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. Journal of Visualized Experiments: JoVE 67, 4389. https://doi.org/10.3791/4389.

Melis, M., Greco, B., Tonini, R., 2014. Interplay between synaptic endocannabinoid signaling and metaplasticity in neuronal circuit function and dysfunction. Eur. J. Neurosci. 39 (7), 1189-1201. https://doi.org/10.1111/ejn.12501.

Méndez-Ruette, M., Linsambarth, S., Moraga-Amaro, R., Quintana-Donoso, D., Méndez, L., Tamburini, G., Cornejo, F., Torres, R.F., Stehberg, J., 2019. The role of the rodent insula in anxiety. Front. Physiol. 10. https://doi.org/10.3389/fphys.2019.00330.

Mitra, R., Jadhav, S., McEwen, B.S., Vyas, A., Chattarji, S., 2005. Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala. Proc. Natl. Acad. Sci. U. S. A. 102 (26), 9371-9376. https://doi.org/10.1073/pnas.0504011102.

Morena, M., Patel, S., Bains, J.S., Hill, M.N., 2016. Neurobiological interactions between stress and the endocannabinoid system. Neuropsychopharmacology 41 (1), 80-102. https://doi.org/10.1038/npp.2015.166.

Mulder, A.B., Hodenpijl, M.G., Lopes da Silva, F.H., 1998. Electrophysiology of the hippocampal and amygdaloid projections to the nucleus accumbens of the rat: convergence, segregation, and interaction of inputs. J. Neurosci. 18 (13), 5095-5102. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9634575.

Nicolas, C., Ju, A., Wu, Y., Eildirdi, H., Delcasso, S., Couderc, Y., Fornari, C., Mitra, A., Supiot, L., Vérité, A., Masson, M., Rodriguez-Rozada, S., Jacky, D., Wiegert, J.S., Beyeler, A., 2023. Linking emotional valence and anxiety in a mouse insula-amygdala circuit. Nature Communications 14 (1), 1. https://doi.org/10.1038/s41467-023-40517-1.

Ohno-Shosaku, T., Kano, M., 2014. Endocannabinoid-mediated retrograde modulation of synaptic transmission. Curr. Opin. Neurobiol. 29, 1-8. https://doi.org/10.1016/j.conb.2014.03.017.

Padovan, C.M., Del Bel, Guimarães, F.S., 2000. Behavioral effects in the elevated plus maze of an NMDA antagonist injected into the dorsal hippocampus: Influence of restraint stress. Pharmacology, Biochemistry and Behavior 67, 325-330. https://doi.org/10.1016/S0091-3057(00)00361-0.

Padovan, C.M., Guimarães, F.S., 2000. Restriction-induced hypoactivity in an elevated plus-maze. Brazilian Journal of Medical and Biological Research 33, 79-83. https://doi.org/10.1590/S0100-879X200000100011.

Paxinos, G., Watson, C., 2017. The Rat Brain in Stereotatic Coordinates. Academic Press, San Diego.

Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14 (3), 149-167. https://doi.org/10.1016/0165-0270(85)90031-7.

Piette, C., Cui, Y., Gervasi, N., Venance, L., 2020. Lights on endocannabinoid-mediated synaptic potentiation. Front. Mol. Neurosci. 13. https://doi.org/10.3389/fmol.2020.00132.

Reis, D.G., Scopinho, A.A., Guimarães, F.S., Corrêa, F.M.A., Resstel, L.B.M., 2011. Behavioral and Autonomic Responses to Acute Restraint Stress Are Segregated within the Lateral Septal Area of Rats. PLoS ONE 6 (8), e23171. https://doi.org/10.1371/journal.pone.0023171.

Reynolds, S.M., Zahm, D.S., 2005. Specificity in the projections of prefrontal and insular cortex to ventral striatopallidum and the extended amygdala. J. Neurosci. 25 (50), 11757-11767. https://doi.org/10.1523/JNEUROSCI.3432-05.2005.

Rieger, N.S., Varela, J.A., Ng, A.J., Granata, L., Djerdjaj, A., Brenhouse, H.C., Christianson, J.P., 2022. Insular cortex corticotropin-releasing factor integrates stress signaling with social affective behavior. Neuropsychopharmacology 47 (6), 1156-1168. https://doi.org/10.1038/s41386-022-01292-7.

Robbe, D., Kopf, M., Remaury, A., Bockaert, J., Manzoni, O.J., 2002. Endogenous cannabinoids mediate long-term synaptic depression in the nucleus accumbens. Proc. Natl. Acad. Sci. U. S. A. 99 (12), 8384-8388. https://doi.org/10.1073/pnas.122149199.

Segev, A., Akirav, I., 2016. Cannabinoids and glucocorticoids in the basolateral amygdala modulate hippocampal-accumbens plasticity after stress. Neuropsychopharmacology 41 (4), 1066-1079. https://doi.org/10.1038/npp.2015.238.

Shi, T., Feng, S., Zhou, Z., Li, F., Fu, Y., Zhou, W., 2023. Stress-altering anterior insular cortex activity affects risk decision-making behavior in mice of different sexes. Front. Cell. Neurosci. 17, 1094808. https://doi.org/10.3389/fncel.2023.1094808.

Sliz, D., Hayley, S., 2012. Major depressive disorder and alterations in insular cortical activity: a review of current functional magnetic imaging research. Front. Hum. Neurosci. 6, 323. https://doi.org/10.3389/fnhum.2012.00323.

Solinas, M., Goldberg, S.R., Piomelli, D., 2008. The endocannabinoid system in brain reward processes. Br. J. Pharmacol. 154 (2), 369-383. https://doi.org/10.1038/bjp.2008.130.

Terasawa, Y., Shibata, M., Moriguchi, Y., Umeda, S., 2013. Anterior insular cortex mediates bodily sensibility and social anxiety. Soc. Cogn. Affect. Neurosci. 8 (3), 259-266. https://doi.org/10.1093/scan/nss108.

Tomeo, R.A., Gomes-de-Souza, L., Benini, R., Reis-Silva, L.L., Crestani, C.C., 2022. Site-specific regulation of stress responses along the rostrocaudal axis of the insular cortex in rats. Front. Neurosci. 16. https://doi.org/10.3389/fnins.2022.878927.

Wenzel, J.M., Cheer, J.F., 2018. Endocannabinoid regulation of reward and reinforcement through interaction with dopamine and endogenous opioid signaling. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology 43 (1), 103-115. https://doi.org/10.1038/npp.2017.126.

Winters, B.D., Krüger, J.M., Huang, X., Gallaher, Z.R., Ishikawa, M., Czaja, K., Krueger, J. M., Huang, Y.H., Schlüter, O.M., Dong, Y., 2012. Cannabinoid receptor 1-expressing neurons in the nucleus accumbens. Proc. Natl. Acad. Sci. U. S. A. 109 (40), E2717-E2725. https://doi.org/10.1073/pnas.1206303109.

Wissman, A.M., May, R.M., Woolley, C.S., 2012. Ultrastructural analysis of sex differences in nucleus accumbens synaptic connectivity. Brain Struct. Funct. 217 (2), 181-190. https://doi.org/10.1007/s00429-011-0353-6.

Wright, C.I., Groenewegen, H.J., 1996. Patterns of overlap and segregation between insular cortical, intermediiodorsal thalamic and basal amygdaloid afferents in the nucleus accumbens of the rat. Neuroscience 73 (2), 359-373. https://doi.org/10.1016/0306-4522(95)00592-7.

Yokoyama, R., Ago, Y., Igarashi, H., Higuchi, M., Tanuma, M., Shimazaki, Y., Kawai, T., Seiriki, K., Hayashida, M., Yamaguchi, S., Tanaka, H., Nakazawa, T., Okamura, Y., Hashimoto, K., Kasai, A., Hashimoto, H., 2024. (R)-ketamine restores anterior insular cortex activity and cognitive deficits in social isolation-reared mice. Mol. Psychiatry 29 (5), 1406-1416. https://doi.org/10.1038/s41380-024-02419-6.

Zavala, J.K., Fernandez, A.A., Gosselink, K.L., 2011. Female responses to acute and repeated restraint stress differ from those in males. Physiology & Behavior 104 (2), 215-221. https://doi.org/10.1016/j.physbeh.2011.03.022.

Zhao, Z., Cavelo, A., Couderc, Y., Mitra, A., Varilh, M., Wu, Y., Jacky, D., Fayad, R., Cannich, A., Bellocchio, L., Marsicano, G., Beyeler, A., 2024. Cannabinoids regulate an insula circuit controlling water intake. Curr. Biol. 34 (9), 1918-1929.e5. https://doi.org/10.1016/j.cub.2024.03.053.