Cannabinoid use generalizes stress responses by altering the astrocyte plasticity through extracellular matrix signaling in the nucleus accumbens core

Cannabinoid use generalizes stress responses by altering the astrocyte plasticity through extracellular matrix signaling in the nucleus accumbens core | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cannabinoid use generalizes stress responses by altering the astrocyte plasticity through extracellular matrix signaling in the nucleus accumbens core Ritchy Hodebourg, Lillian Duncan, Eric Dereschewitz, Peter Kalivas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7254957/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract The rising legal acceptance of cannabis and the high comorbidity between cannabis use disorder (CUD) and post-traumatic stress disorder (PTSD) highlight the importance of understanding how stress and cannabis influence the brain. We recently discovered that cannabis use promotes two PTSD-like symptoms: avoidance coping behaviors and the generalization of stress-coping responses to a neutral stimulus not previously linked to stress. To investigate the neuroadaptations behind these changes, we used in vivo zymography and confocal microscopy to examine how stress and cannabinoid use influence multipartite synaptic plasticity in the nucleus accumbens core (NAcore), including astroglial plasticity, Synapsin-I density, and matrix metalloproteinases (MMP-2,9) activity. For this purpose, rats were restraint stressed for 2h and simultaneously exposed to an odor that became the stress-conditioned stimulus (stress-CS). Three weeks later, rats self-administered cannabinoids (delta9-tetrahydrocannabinol + cannabidiol; THC + CBD) for 10 days, followed by 10 days of withdrawal. We then evaluated the effect of stress-CS or neutral odor (NS) on coping strategies in a defensive burying task. We demonstrated for the first time that THC + CBD generalized stress responses to the NS by causing astrocytes to retract from synapses and decreasing Synapsin-I density in the NAcore. Furthermore, cannabinoid use promoted avoidant coping behaviors in response to a stress-CS by triggering strong activation of MMP-2,9, driven largely by MMP-2, causing a re-association of astrocytes to synapses along with an increase of Synapsin-I density and astrocyte atrophy. However, these neuroadaptations only occurred in males. Overall, these findings highlight potential therapeutic targets like MMPs and astrocytes for treating co-occurring CUD/PTSD. Biological sciences/Neuroscience Biological sciences/Cell biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction For certain individuals, a single traumatic event can lead to trauma and stress-related disorders, including post-traumatic stress disorder (PTSD)[ 1 ]. Cannabis use disorder (CUD) is often comorbid with PTSD and is the most prevalent co-occurring condition among veterans diagnosed with CUD[ 2 ]. Patients with comorbid PTSD and CUD report higher PTSD symptom severity and poorer treatment outcomes relative to those without CUD[ 3 , 4 ]. Furthermore, recent longitudinal studies demonstrate that cannabis use is associated with higher PTSD symptom severity, including greater traumatic thought intrusions[ 5 , 6 ] and depressive symptoms[ 7 ]. Despite this evidence, PTSD is often listed as a qualified medical condition for the use of cannabis in the United States[ 8 , 9 ]. As of 2025, 31 states listed cannabis as a treatment for PTSD[ 10 ]. Consequently, the increasing use of cannabis for self-medication of PTSD and the comorbid PTSD and CUD underscores the need to identify the neurobiological mechanisms through which cannabis use alters and potentially exacerbates the symptoms associated with PTSD. Using an stress cue-conditioned (stress-CS) animal model of PTSD, we recently demonstrated that an acute stress potentiates cannabis consumption in male rats[ 11 ]. Additionally, we discovered that cannabis use promotes two significant PTSD-like symptoms: the generalization of stress-coping responses to a neutral stimulus that is not produced by stress exposure in the absence of cannabis use, and engaging in avoidance coping behaviors when faced with a conditioned stress odor[ 11 ]. Importantly, these findings differ from those observed after cocaine and heroin self-administration, where rats exposed to a stress-CS engage in active coping behaviors, without generalizing to an odor not associated with acute stress experience[ 12 – 14 ]. Finally, the cannabis-induced behavioral changes were accompanied by reduced spine density in the nucleus accumbens core (NAcore) and a further decrease in spine head diameter after exposure to the stress-CS[ 11 ], indicating that cannabis may be altering how environmental stimuli elicit synaptic plasticity. Nonneuronal components of the neuropil adjacent to synapses, including astroglia and extracellular matrix (ECM), form what is termed the multipartite synapse and function together with pre- and postsynapses to regulate synaptic plasticity[ 15 , 16 ]. For instance, astrocytes regulate the synaptic plasticity by insulating synapses, thereby providing them with metabolic support, gliotransmitters, and maintaining ion homeostasis[ 16 ]. In parallel, destabilizing the ECM via catalytic signaling induced by matrix metalloproteinases (MMPs) is associated with neuronal and astroglial plasticity. Both environmental stressors and addictive drugs alter ECM signaling and astroglial morphology and signaling, and contribute to the development of stress- and drug-related disorders[ 17 ]. However, the effects of cannabis use, either independently or in conjunction with stress, on the nonneuronal components of multipartite synapses remain unknown. The objective of this study was to assess whether the interaction between stress and cannabis use influences components of multipartite synapses, including astroglial plasticity, synapse density, and ECM catalytic signaling in the NAcore. For this purpose, following an acute restraint stress, astrocytes were labeled in rats that were trained to self-administer delta9-tetrahydrocannabinol + cannabidiol (THC + CBD)[ 18 ]. After assessing how cannabinoid use affected coping strategies through a defensive burying task (DBT), we employed confocal microscopy, in vivo zymography, and digital rendering techniques to quantify astrocyte morphology, the colocalization of astrocytes with the presynaptic marker Synapsin-I, and the gelatinolytic activity of MMP-2 and MMP-9 in the NAcore. Methods Animals Male and female Long-Evans rats (~ 250 g; Charles River Laboratories) were double-housed according to sex on a 12-h light/dark cycle with food and water available ad libitum. Experiments occurred in the light cycle. All procedures received approval from the Animal Care and Use Committee of the Medical University of South Carolina and were conducted following National Institutes of Health guidelines. Acute stress and odor pairing Rats were placed in a restraining device for 2 hours and paired with an odor (lemon or sandalwood; Sun essential, Phoenix, AZ, USA) that became the stress-CS. Control rats were exposed to the same odor in the home cage for 2 hours (Sham-CS). The odor that was not stress- or sham-paired was used during the DBT as a neutral odor control (stress-NS). Two weeks after the stress procedure, rats received indwelling catheters as previously described[ 18 ]. Viral labeling After catheter implantation, rats were implanted with bilateral guide cannulae into the NAcore (AP: 1.5mm, ML: ± 1.7mm, DV: -5.5mm). AAV5/GFAP-hM3dq-mCherry (University of Zurich) was injected into the left hemisphere (1.0 µl, 0.15 µl/min, 5 min diffusion) to label astrocytes. The microinjectors are 2mm longer than the guide cannulae to reach the target. THC + CBD self-administration Following surgery, rats underwent 5 days of vapor exposure to a THC + CBD mixture in a 10:1 ratio, as previously described[ 18 ]. Rats were then trained to self-administer THC + CBD on a fixed ratio 1 schedule, during 90 min sessions for 10 consecutive days. Active lever presses delivered 0.4 + 0.04 mg/kg THC + CBD and were paired with cues (light + tone) followed by a 20-s timeout. Another cohort of rats was trained to self-administer the vehicle (1% ethanol). Following self-administration, animals underwent 10 days of withdrawal to ensure the absence of THC metabolites during the DBT. Defensive burying task Coping strategies were quantified as previously described[ 11 ]. Briefly, bedding was placed in one half of the standard home cage, opposite the odor. Rats were placed facing away from the odor on the bedding side of the cage, and behavior was recorded for 15 min using Ethovision XT software (Leesburg, VA, USA). Active (burying) and avoidant (escape, immobility and grooming) coping mechanisms were quantified. In vivo zymography In vivo zymography was used to quantify MMP-2,9 activity as previously described in detail[ 19 ]. FITC-gelatin was microinjected into the right hemisphere (1.5µl, 0.5µl/min, 3 min diffusion) immediately before the DBT. Rats were then perfused with 4% PFA immediately after the DBT and brains were sliced at 100µm. Only slices containing the injection track and the anterior commissure on the same frame were analyzed (ImageJ; National Institute of Mental Health, Bethesda, MD). MMP-2 (444288, MilliporeSigma) and MMP-9 (444278, MilliporeSigma) inhibitors were dissolved in 1% DMSO and bilaterally microinjected before FITC-gelatin in a separate rat cohort as previously described[ 20 ]. Immunohistochemistry Left hemisphere slices, containing the GFAP-mCherry virus, were permeabilized for 15 min in PBS with 0.2% Triton X-100 (PBST), then blocked with 2% PBST containing 5% normal goat serum. Rabbit anti-synapsin-I antibody (1:1000, ab64581, Abcam) was incubated for 72 hours at 4ºC. Slices were washed with 0.2% PBST, then incubated with Alexa Fluor 405(1:1000, ab175652, Abcam) for 72 hours at 4ºC. Confocal microscopy Astrocyte images were acquired using a Leica SP5 with a 63X oil-immersion objective lens at 16-bit resolution with a 1-µm step size. Images were then imported into Imaris software (version 9.7.0). The mCherry signal was used for 3-dimensional reconstruction of astrocytes. The astrocyte-Synapsin-I proximity was quantified by using each rendered astrocyte as a region of interest (ROI) for colocalization analysis with Synapsin-I. The colocalization channel displays the results as the percentage of the ROI overlapping with Synapsin-I puncta. To determine the Synapsin-I density, we normalized the thresholded signal intensity to the volume of the frame from which it was acquired. Imaging and analysis were performed in a blinded manner. Statistics Data were analyzed with GraphPad Prism 10. Behavioral and in vivo zymography data were analyzed using two-way ANOVAs with Tukey’s post hoc comparison. For astroglial measures, a D’Agostino & Pearson test was initially used to assess the normality of each group. The Kruskal-Wallis test with Dunn’s post hoc was used for non-normally distributed treatment groups. Coping strategies and astroglial distribution were analyzed with a multiple Chi-square followed by a Bonferroni correction. Results Acute stress potentiated THC + CBD use in male rats Rats were subjected to acute restraint stress for 2 hours and simultaneously exposed to an odor. 3 weeks after the stress, rats were trained to self-administer THC + CBD or its vehicle for 10 days, followed by 10 days of withdrawal (Fig. 1A). In THC + CBD trained rats, stressed males pressed more on the active lever than sham rats (Fig. 1B). Stressed males consumed more THC + CBD than sham rats (Fig. 1C). Both stressed and sham rats showed equivalent discrimination between the active and inactive levers (Fig. 1D). No differences between stress and sham groups were observed in vehicle-trained rats (Fig. 1E-G). In female THC + CBD trained rats, no differences were measured between groups on the active lever presses and THC + CBD intake (Fig. S1 A-B). However, stressed rats had a higher discrimination index than sham rats (Fig. S1 C), indicating that the acute stress may increase the reinforcing value of the THC + CBD in females. No differences between stress and sham groups were observed in vehicle-trained rats (Fig. S1 D-F). THC + CBD promoted avoidant coping strategies in male rats Following the withdrawal period, we used DBT to evaluate the effect of THC + CBD on active (burying) and avoidant (escape, immobility and grooming) coping strategies. To this end, stressed rats were exposed to either the stress-CS odor previously associated with the stressful event or a neutral odor stimulus (NS) for 15 min. Non-stressed rats were exposed to the odor previously associated with the sham experience. Regarding active coping, in vehicle-trained male rats, the stress-CS caused an increase in burying compared to the sham group. Conversely, in THC + CBD-trained rats, the stress-NS group exhibited greater burying than the sham group. Furthermore, compared to vehicle rats, THC + CBD self-administration elevated burying in the stress-NS group while decreasing it in the stress-CS group (Fig. 2A). Regarding avoidant coping strategies, we found that THC + CBD self-administration induced a strong escape behavior under all stress conditions compared to vehicle rats (Fig. 2B). Conversely, the percent time spent immobile was reduced after THC + CBD (Fig. 2C). Moreover, stress-CS significantly increased grooming in vehicle-trained rats, while both stressed groups (CS and NS) exhibited a higher grooming frequency than the sham group in THC + CBD-trained rats (Fig. 2D). When comparing the distribution of coping strategies between groups, we found no difference among the stress-NS and sham-CS groups in vehicle-trained rats (Fig. 2E), while the stress-CS promoted an active coping strategy by increasing burying behavior. In summary, Fig. 2 shows that THC + CBD withdrawal promoted avoidant coping in the sham group, while a neutral odor stimulus facilitated both active and avoidant coping in stressed rats. Also, exposure to the stress-CS after THC + CBD further changed stress behaviors compared to the sham and stress-CS veh groups. Combined, these data indicate that THC + CBD use caused male rats exposed to an earlier stressor to generalize stress responses to neutral stimuli and worsen stress responses to conditioned stimuli. Surprisingly, in females, neither the stress nor the THC + CBD significantly affect coping strategies in the DBT (Fig. S2 ). Withdrawal from cannabinoids reduced the synaptic proximity and promoted astrocytic insulation of synapses after the stress-CS exposure in male rats To determine how stress and cannabinoids influence the plasticity of NAcore astrocytes, astroglia were labeled with a membrane-bound fluorescent reporter. Rats were perfused immediately after DBT, and NAcore slices labeled with the presynaptic marker Synapsin-I (Fig. 3A). To quantify proximity between astrocytes and synapses, each astrocyte was digitally rendered (Fig. 3B), and the co-labeling of the astrocyte surface with Synapsin-I was quantified (Fig. 3C). Consistent with our previous report[ 21 ], we found that acute stress (stress-NS) induced a constitutive reduction in astrocyte-Synapsin-I proximity, while exposure to the stress-CS resulted in a re-association between astrocytes and Synapsin-I in vehicle-trained male rats. Withdrawal from THC + CBD decreased astrocyte-Synapsin-I proximity in the sham group compared to vehicle rats. In stressed THC + CBD-trained rats, exposure to the NS further decreased the proximity between astrocytes and Synapsin-I relative to sham, while re-exposure to the stress-CS enhanced synaptic insulation by astroglia. Additionally, when compared to vehicle rats, the use of THC + CBD potentiated astrocytic insulation of synapses induced by the stress-CS (Fig. 3D, left). Because the cumulative frequency plots (Fig. 3D, middle) indicated different subpopulations of astrocytes, we divided the data into three subpopulations based on the values generating three equal populations in the sham-CS vehicle group. The multiple Chi 2 analysis revealed that exposure to the stress-CS did not fully restore the astrocytic insulation of synapses in vehicle-trained rats (Fig. 3D, right). Indeed, the astrocytes having less co-registration with Synapsin-I constituted a larger population in the stress-CS group than in the sham-CS group. Astrocyte-Synapsin-I proximity negatively correlated with grooming in vehicle-trained males (Fig. 3E, left). This correlation disappeared after THC + CBD use (Fig. 3E, right). Furthermore, while no correlation existed between astrocyte-Synapsin-I proximity and burying in vehicle-trained rats (Fig. 3F, left), a negative correlation appeared after THC + CBD use (Fig. 3F, right). This illustrates that using cannabinoids alters the function of synaptic insulation in managing coping strategies. In females, exposure to the stress-CS increased the astrocyte-Synapsin-I proximity in both vehicle- and THC + CBD-trained rats (Fig. S3 A, left). Compared to vehicle rats, astrocytes near Synapsin-I were more abundant in the stress-NS group, while stress-CS exposure nearly eliminated astrocytes at mid-distance in THC + CBD females (Fig. S3 A right). Moreover, in THC + CBD-trained females, astrocyte-Synapsin-I proximity correlated positively with burying behavior and negatively with grooming (Fig. S3 B-C). Combination of acute stress and cannabinoid use reduced Synapsin-I density in NAcore Chronic stress is known to diminish the levels of synaptic markers, such as Synapsin-I[ 22 ]. We previously found that neither acute stress nor stress-CS exposure altered Synapsin-I density in the NAcore of male rats[ 21 ]. Nevertheless, the impact of acute stress followed by cannabinoid use on Synapsin-I remains unknown. Synapsin-I density was measured for each astrocyte digitally isolated. Similar to our prior report, Synapsin-I density remained unchanged in vehicle-trained males, regardless of the stress condition (Fig. 3G, left). Likewise, no difference was found between sham-CS groups. These findings suggest that the changes in astrocyte-Synapsin-I proximity observed in vehicle-trained rats and during withdrawal from THC + CBD result solely from astrocytic motility. However, the combination of acute stress and THC + CBD withdrawal lowered Synapsin-I density in stress-NS rats, while exposure to the stress-CS restored Synapsin-I density. The multiple Chi 2 analysis indicated that astrocytes with high Synapsin-I density formed a larger population in the stress-CS group compared to the sham-CS group (Fig. 3G, right). Furthermore, THC + CBD increased Synapsin-I density in the stress-CS group compared to the vehicle rats. This indicates that the potentiation of synaptic insulation induced by the drug following the stress-CS exposure observed in Fig. 3D could result largely from increased neuronal synapses associated with astroglia. Unlike males, the interaction between acute stress and cannabinoid use increased the Synapsin-I density in female NAcore (Fig. S3 D, left). In stressed vehicle females, astrocytes with low Synapsin-I density increased compared to the sham-CS group. These effects are reversed after THC + CBD use (Fig. S3 D, right). Stress-CS induced astrocyte atrophy after cannabinoid use in male rats Chronic stress causes astrocyte shrinkage in various brain areas[ 23 ]. Previously, we found that neither stress nor stress-CS influence astrocyte structure in the NAcore of males[ 21 ]. We replicated these negative data in vehicle males and demonstrated that after THC + CBD use the stress-CS reduced both the surface area and the volume of astrocytes (Fig. 4A-B left). Moreover, in Sham rats, THC + CBD use increased the subpopulation of astrocytes with smaller surface areas compared to Sham vehicle rats (Fig. 4A right). Additionally, in vehicle-trained rats, the stress-CS group exhibited a higher proportion of astrocytes with larger volumes than the sham group (Fig. 4B right). Cannabinoid use in males transformed a positive correlation between surface area and astrocyte-Synapsin-I proximity in vehicle-trained rats into a negative correlation (Fig. 4C). Additionally, in THC + CBD-trained rats, the volume showed a negative correlation with astrocyte-Synapsin-I proximity, unlike in vehicle rats (Fig. 4D). In summary, these data suggest that exposure to a stress-CS interacts with cannabinoid withdrawal to induce an increase in both astroglial coverage of synapses and astrocyte atrophy. In females, the acute stress induced a constitutive decrease in astrocyte surface area and volume in both vehicle- and THC + CBD-trained rats. Re-exposure to stress-CS restored astrocyte morphology in vehicle females, but THC + CBD prevented this effect (Fig. S4 A-B). Additionally, when comparing astrocyte morphology between sexes, THC + CBD withdrawal alone increased both the surface area and volume of astrocytes in females compared to males. (Fig. S4 C-D). MMP-2,9 activity is increased during withdrawal from cannabinoid use and is potentiated following the stress-CS exposure in male rats MMP- 2 and − 9 are key components of the ECM signaling and multipartite plasticity [ 16 , 24 ]. Both addictive drugs and stress exposure alter the activity of MMP-2 and MMP-9[ 17 ]. Using in vivo zymography we quantified the gelatinolytic activity of MMP-2,9 during the DBT[ 19 ]. Withdrawal from THC + CBD increased NAcore MMP-2,9 activity regardless of the stress condition in males (Fig. 5B). Moreover, stress-CS exposure increased the MMP-2,9 activity in both vehicle and THC + CBD males. However, THC + CBD use potentiated the MMP-2,9 activity compared to vehicle rats. Cannabinoid use reversed a positive correlation between MMP-2,9 activity and burying behavior in vehicle-trained rats into a negative correlation (Fig. 5C). Moreover, MMP-2,9 activity was positively correlated with the escape behavior in THC + CBD-trained, not vehicle rats (Fig. 5D). These data indicate that cannabinoid use modifies how ECM signaling regulates coping strategies, specifically by inducing a shift from active to avoidant coping. Additionally, after cannabinoid use, MMP-2,9 activity positively correlated with astrocyte-Synapsin I proximity, while negatively correlating with astrocyte surface area (Fig. 5E-F), suggesting that ECM signaling regulates the astroglial plasticity. Neither stress nor the THC + CBD alter the MMP-2,9 activity in females (Fig. S5 A). However, similar to males, MMP-2,9 activity showed a positive correlation with astrocyte-Synapsin-I proximity and a negative correlation with astrocyte volume after cannabinoid use (Fig. S5 B-C). Both MMP-2 and MMP-9 regulated the astroglial coverage of synapses, while MMP-2 influenced the astrocyte morphology To determine whether the changes in astroglial plasticity induced by the stress-CS in males are preceded by changes in the ECM catalytic signaling, we examined the effect of MMP-2 and MMP-9 inhibitors on stress-CS-induced astroglial plasticity. To this end, during THC + CBD withdrawal, another cohort of stressed males received bilateral microinjections of inhibitors (0.1 nmol) or their vehicle (1% DMSO) in NAcore, followed by a unilateral microinjection of the FITC-gelatin before stress-CS exposure (Fig. 6A). Compared to the DMSO microinjection, either MMP-2 or MMP-9 inhibitors altered the coping strategies. MMP-9 inhibitor notably reduced the burying behavior (Fig. 6B and S6). The coping strategies also significantly differ between the MMP-2 and MMP-9-treated groups (Fig. 6B), with the MMP-2 inhibitor significantly reducing grooming (Fig. 6B and S6). Interestingly, these alterations in stress-CS-induced behaviors were accompanied by only a nonsignificant reduction in gelatinolytic activity by either of the MMP inhibitors (Fig. 6C). Both MMP-2 and − 9 inhibitors reduced the astroglial coverage of synapses (Fig. 6D left). Furthermore, the MMP-2 inhibitor nearly eliminated astrocytes found near Synapsin-I (Fig. 6D right). A likely contributor to the reduction in synaptic coverage by astroglia was markedly reduced Synapsin-I density after treatment with the MMP-2, but not MMP-9 inhibitor (Fig. 6E). Finally, the MMP-2 inhibitor reversed astrocyte atrophy by increasing both surface area and volume (Fig. 6F-G). Discussion We recently demonstrated that cannabis use, after a single traumatic event, promotes two PTSD-like symptoms: generalization of stress responses to neutral stimuli and avoidant coping strategies following conditioned stress exposure[ 11 ]. To explore the neuroadaptations behind these changes, we used in vivo zymography and confocal microscopy to assess how stress and cannabinoid use affect multipartite synaptic plasticity in the NAcore, a brain region linked to stress and addiction cues responding[ 2 ]. We made four primary discoveries: 1) cannabinoid withdrawal, without any traumatic event, reduced the astroglial coverage of synapses and increased MMP-2,9 signaling in the ECM, 2) a prior traumatic event reduced Synapsin-I density during cannabinoid withdrawal, 3) exposure to a stress-CS after cannabinoid use led to avoidant coping strategies by activating MMP-2,9 in the NAcore, resulting in the re-association of astrocytes with synapses and astrocyte atrophy, and 4) these multipartite adaptations by stress and cannabinoids occurred only in males. THC + CBD disrupts coping strategies in males In line with our previous report, we found that THC + CBD use generalized stress responses to a neutral stimulus in stressed rats, by inducing both active (burying) and avoidant (escape and grooming) coping strategies. Importantly, a neutral stimulus did not trigger stress responses in vehicle-trained rats, suggesting that the effects seen in THC + CBD-trained rats result from the interaction between acute stress and cannabinoid withdrawal. Additionally, we found that cannabinoid use induced notable escape behavior in non-stressed rats. Both preclinical and clinical studies support a contention that extensive and prolonged cannabis use is associated with more severe withdrawal symptoms[ 25 – 27 ] and our data indicate that cannabinoid use, regardless of previous traumatic experiences, generalizes stress responses to unconditioned stimuli. Since stress generalization is a hallmark of PTSD[ 1 ], these preclinical data support longitudinal clinical studies[ 5 , 6 ] indicating that cannabis use worsens PTSD symptoms. Furthermore, we previously demonstrated that a stress-CS is sufficient to reinstate cocaine, heroin, and alcohol seeking, reflecting an active coping strategy[ 12 , 14 ]. Accordingly, we found that the stress-CS induced burying behavior in vehicle-trained rats. However, after cannabinoid use, re-exposure to the stress-CS reduced burying while promoting escape and grooming behaviors. Since avoidance behavior is a key criterion for defining PTSD, and avoidance coping strategies are associated with increased activation of the stress axis[ 28 ], our findings suggest that cannabis use heightened the salience of the stress-CS and thereby worsened PTSD symptoms. THC + CBD use alters how astroglial coverage regulates coping strategies in males The synaptic insulation provided by astrocytes plays a crucial role in synaptic plasticity by supplying metabolic support, gliotransmitters, and maintaining ion homeostasis[ 16 ]. Withdrawal from cocaine, heroin, and methamphetamine self-administration reduced the proximity between astrocytes and synapses in NAcore[ 29 – 32 ]. Similarly, we demonstrated that withdrawal from THC + CBD reduced the astroglial coverage of synapses in NAcore, suggesting that this effect is shared by many classes of addictive drugs. Akin to withdrawal from addictive drugs, an acute restraint stress also induced enduring decreases in astroglial coverage in NAcore[ 21 ]. Consistently, we found that acute stress reduced astrocyte-Synapsin-I proximity in both vehicle- and THC + CBD-trained rats. Finally, we demonstrated that re-exposure to the stress-CS increased the astroglial coverage of synapses in both vehicle- and THC + CBD-trained rats. However, while the stress-CS only partially restored the astrocytic insulation of synapses in vehicle-trained rats, cannabinoid exposure potentiated the re-association of astrocytes to synapses induced by the stress-CS. In examining astroglial insulation related to coping strategies, we found a negative correlation between astrocytes-Synapsin-I proximity and grooming behavior in vehicle-trained rats. This implies that astroglial coverage may partly countermand the expression of avoidant coping behaviors, although this correlation vanished following THC + CBD usage. Conversely, in THC + CBD-trained males, the proximity of astrocytes to Synapsin-I was negatively correlated with burying behavior. Thus, THC + CBD inhibits active coping strategies and potentially exacerbates the salience of the stress-CS by promoting synaptic insulation by astrocytes. Overall, this finding suggests that stress-CS exposure during cannabinoid withdrawal alters how astroglial coverage in the NAcore influences coping strategies. THC + CBD use following an acute stress reduces the Synapsin-I density in NAcore Severe stressors are known to decrease the levels of synaptic markers, including Synapsin-I and PSD95, in several brain regions. For example, chronic unpredictable mild stress (CUMS) decreases the expression of both Synapsin-I and PSD95 in the prefrontal cortex and hippocampus[ 22 , 33 ] and a single prolonged stress (SPS) reduces Synapsin-I and PSD95 in the hippocampus[ 34 ]. In NAcore, the combination of SPS and foot shock reduces PSD95[ 35 ]. In contrast, we found that neither 2 hours of acute restraint stress, stress-CS exposure, nor withdrawal from THC + CBD without stress pre-exposure affected the Synapsin-I density in the NAcore[ 7 ]. Thus, the reduction seen in astroglial coverage produced by stress pre-exposure (stress-NS) or THC + CBD without stress pre-exposure are largely due to astrocytic motility not an overall reduction in synapses. In contrast, we found that both Synapsin-I and astroglial synaptic proximity were reduced in the group receiving acute restraint stress and cannabinoid use (Stress-NS). Thus, the retraction of astrocytes induced by cannabinoid withdrawal, along with the loss of synapses resulting from the interplay between stress and cannabinoid use, may contribute to how THC + CBD generalizes stress responses to a neutral stimulus. THC + CBD use induces astrocyte atrophy during exposure to the stress-CS It is well-documented that stress alters astrocyte morphology[ 23 ]. For example, chronic restraint stress reduced the process length, branching, and volume of astrocytes in the prefrontal cortex[ 36 ], and astrocyte volume in the amygdala[ 37 ]. CUMS also induced astrocyte atrophy in the prefrontal cortex and hippocampus[ 38 , 39 ]. Moreover, the SPS protocol led to astrocyte atrophy in the hippocampus[ 40 ], where astrocyte atrophy is linked with anxiety and depressive-like behavior. For instance, the astrocyte morphology negatively correlates with anxiety, while treatments restoring the astrocyte morphology alleviate symptoms of anxiety and depression in animal models[ 39 – 41 ]. In NAcore, neither acute restraint stress nor the stress-CS affect astroglial morphology[ 21 ]. In contrast with stress, the effects of THC on astrocyte morphology are less understood. In organotypic hippocampal slices, prolonged incubation with THC reduces astrocytic processes[ 42 ]. Astrocytic processes are also reduced in the amygdala of rats treated with high doses of THC during adolescence[ 43 ]. However, the effects of voluntary cannabinoid consumption on astrocyte structure were unknown. Here, we demonstrated that THC + CBD self-administration, with or without a prior stressful experience, did not affect measures of astrocyte morphology, including surface area and volume. However, re-exposure to the stress-CS triggered astrocyte atrophy in THC + CBD, not vehicle-trained rats. Surprisingly, we observed a negative correlation between astrocyte surface area and volume and synapse coverage, indicating that cannabinoid use and stress-CS triggered both astrocyte atrophy and increased proximity of astroglia to synapses. THC + CBD use changes the function of MMP-2,9 in regulating coping strategies in males The gelatinases MMP-2 and MMP-9 play a necessary role in activity-dependent multipartite synaptic plasticity[ 16 ]. For example, MMP-9 controls dendritic spine enlargement induced by long-term potentiation[ 44 ]. Thus, over the last 20 years, research has focused on the role of MMP-2,9 in various psychiatric disorders, including drug and stress-related disorders[ 17 ]. Our laboratory demonstrated that drug seeking induced by Pavlovian cues requires transient activation of MMP-9 in the NAcore, which leads to both spine head expansion and the insertion of AMPA receptors on D1-medium spiny neurons[ 20 , 45 , 46 ]. In parallel, withdrawal from cocaine or heroin self-administration constitutively increases the MMP-2 activity around D2-MSNs [ 20 , 47 ]. Concerning stress-related disorders, both acute and chronic restraint stress increase MMP-9 activity in the hippocampus[ 48 , 49 ]. We also found that acute restraint stress increases MMP-9 in NAcore[ 19 ]. However, the effect of stress-CS on the activity of MMP-2,9 was unknown. Given that both stress-CS and drug cues promote spine head expansion[ 11 , 45 ], we hypothesized that stress-CS would increase MMP-2,9 activity. As anticipated, MMP-2,9 activity was increased after exposure to stress-CS in vehicle-trained rats. We also demonstrated that THC + CBD withdrawal constitutively increased MMP-2,9 activity. The combination of stress pre-exposure and cannabinoid withdrawal (stress-NS) did not alter the basal activity of MMP-2,9. However, re-exposure to the stress-CS during cannabinoid withdrawal increased the MMP-2,9 activity. Cannabinoid use not only elevated MMP-2,9 activity but also contributed to a shift from active to avoidant coping behaviors. Thus, while MMP-2,9 activity positively correlated with burying in vehicle-trained rats, cannabinoid use reversed the positive correlation to a negative correlation with burying and revealed a positive correlation with escape behavior. MMP-2 is a key regulator of the multipartite synaptic changes induced by the stress-CS following cannabinoid use. Considering that stress-CS re-exposure after cannabinoid use strongly activated MMP-2,9 and simultaneously increased astroglial coverage and astrocyte atrophy, we hypothesized that these events are interconnected. Supporting our hypothesis, MMP-2,9 activity positively correlated with astroglial coverage and negatively with astrocyte surface area. To establish a causal relationship between ECM catalytic signaling and astroglial plasticity, we administered inhibitors of both gelatinases before the DBT. We found that inhibiting either MMP-2 and MMP-9 reduced astroglial coverage of synapses in NAcore, while only the MMP-2 inhibitor reduced Synapsin-I density and prevented astrocyte atrophy. Additionally, inhibiting MMP-2 decreased grooming while increasing burying, indicating that MMP-2 activation by stress-CS contributes to the switch from active to avoidant behaviors in THC + CBD withdrawn rats. In summary, these data constitute the first in vivo evidence linking the catalytic activities of MMP-2 and MMP-9 to astroglial plasticity and stress-associated behaviors. Sexual dimorphism Women are twice as likely to develop PTSD and exhibit greater vulnerability for anxiety-related disorders [ 51 – 53 ]. However, historically, preclinical studies on stress-related disorders mostly used males and focused primarily on freezing behavior[ 50 ]. Including females and assessing other stress responses introduced new insights into coping strategies[ 51 , 52 ]. Indeed, research demonstrates that male rats freeze more than females, while females display higher escape-like behaviors[ 50 , 53 – 55 ]. Accordingly, we found that females escape more than males across all stress conditions. Surprisingly, neither stress nor THC + CBD had a significant impact on coping strategies in females. This may be due to high variability we observed in stress responses between females. Although the estrous cycle is not involved in escape-like behavior [ 42 ], it would be interesting to evaluate its influence on other coping responses. Despite not affecting behaviors, stress and cannabinoid use changed multipartite plasticity in females. Unlike males, the number of synapses (Synapsin-I puncta) is increased in stressed rats following THC + CBD use. Regarding astroglial plasticity, cannabinoid withdrawal without stress pre-exposure increased both the surface area and volume of NAcore astrocytes in females compared to males. This result differs from cocaine, where prolonged withdrawal elicits NAcore astrocyte atrophy in males without affecting female astrocytes[ 29 ]. Furthermore, while acute restraint stress did not affect astrocyte morphology in males, it induced astrocyte atrophy in females. The opposite effect is observed after chronic stress, where CUMS reduces astrocyte morphology in males without affecting females[ 56 ]. Our findings contribute to research on sexual dimorphism in astroglial plasticity. More studies are needed to understand how stress and cannabis differently affect female and male multipartite plasticity. Clinically relevant targets This study identified neurobiological mechanisms behind PTSD-like symptoms after cannabinoid use in male rats, highlighting potential therapeutic targets such as MMPs and astrocytes for comorbid CUD/PTSD. Recent studies demonstrate elevated plasma levels of MMP-2 and MMP-9 in PTSD patients[ 57 , 58 ]. Moreover, the nonselective MMP inhibitor doxycycline disrupts the consolidation of cued fear memory in humans[ 59 ]. Additionally, a single dose of minocycline, a more specific MMP-9 inhibitor, administered before a contextual fear memory paradigm, reduced the consolidation of fear memory[ 60 ]. These clinical data suggest that MMP inhibition represents a potentially effective strategy to treat the comorbidity between PTSD and CUD. Another interesting strategy would be to target astroglial plasticity. For example, the antidepressant fluoxetine alleviates depressive-like symptoms, partly by reversing astrocyte atrophy caused by CUMS[ 39 ]. Moreover, in humans, antidepressant treatment reverses astroglial atrophy in the hippocampus[ 61 ]. Thus, exploring the impact of antidepressant treatment on astrocyte atrophy resulting from stress-CS after cannabis use would be intriguing. Declarations Acknowledgements We thank Dr. Anna Kruyer and Dr. Anze Testen for their valuable advice on astroglial analysis. We thank Jordan Hopkins for technical support in conducting surgeries. Also, we thank the National Institute of Drug Abuse (NIDA) Drug Supply Program for providing (-)-trans-Delta9-tetrahydrocannabinol (THC) in ethyl alcohol (95%) and Cannabidiol (CBD) (ADL-19496-11) (Synthetic). This work was supported by the National Institute of Health NIH, K99DA058761 (R.H.) and U.S. Veterans Affairs VA BX004727 (P.K.). Author contributions R.H. designed the study, analyzed the data, and interpreted the results with input from P.W.K. R.H. performed surgeries. R.H., L.D., and E.D. conducted operant behavioral training. R.H. carried out confocal analyses. R.H. produced the figures and authored the manuscript with input and revisions from P.W.K. Competing interests The authors declare no competing interests. References Trauma and stressor-related discorders. Posttraumatic stress disorder. , in Diagnostic and statistical manual of mental disorders (5th Ed) , A.P. Association, Editor. 2013, American Psychiatric Association. Bonn-Miller, M.O., M.M. Bucossi, and J.A. Trafton, The underdiagnosis of cannabis use disorders and other Axis-I disorders among military veterans within VHA. Mil Med, 2012. 177 (7): p. 786-8. Bonn-Miller, M.O., et al., The impact of posttraumatic stress disorder on cannabis quit success. Am J Drug Alcohol Abuse, 2015. 41 (4): p. 339-44. Loflin, M., M. Earleywine, and M. Bonn-Miller, Medicinal versus recreational cannabis use: Patterns of cannabis use, alcohol use, and cued-arousal among veterans who screen positive for PTSD. Addict Behav, 2017. 68 : p. 18-23. Livingston, N.A., et al., Longitudinal course of mental health symptoms among veterans with and without cannabis use disorder. Psychol Addict Behav, 2022. 36 (2): p. 131-143. Metrik, J., et al., Cannabis use and posttraumatic stress disorder: prospective evidence from a longitudinal study of veterans. Psychol Med, 2022. 52 (3): p. 446-456. Murkar, A., et al., Increased cannabis intake during the COVID-19 pandemic is associated with worsening of depression symptoms in people with PTSD. BMC Psychiatry, 2022. 22 (1): p. 554. Kondev, V., N. Winters, and S. Patel, Cannabis use and posttraumatic stress disorder comorbidity: Epidemiology, biology and the potential for novel treatment approaches. Int Rev Neurobiol, 2021. 157 : p. 143-193. Hasin, D.S., US Epidemiology of Cannabis Use and Associated Problems. Neuropsychopharmacology, 2018. 43 (1): p. 195-212. PTSD and Medical Cannabis Programs . 2025 01/02/2025; Available from: https://www.mpp.org/issues/medical-marijuana/ptsd-medical-cannabis-programs/. Hodebourg, R., et al., Cannabinoid use is enhanced by stress and changes conditioned stress responses. Neuropsychopharmacology, 2022. 47 (5): p. 1037-1045. Carter, J.S., A.M. Kearns, and C.M. Reichel, Complex Interactions Between Sex and Stress on Heroin Seeking. Front Neurosci, 2021. 15 : p. 784365. Carter, J.S., et al., Long-term impact of acute restraint stress on heroin self-administration, reinstatement, and stress reactivity. Psychopharmacology (Berl), 2020. 237 : p. 1709-1721. Garcia-Keller, C., et al., N-Acetylcysteine treatment during acute stress prevents stress-induced augmentation of addictive drug use and relapse. Addict Biol, 2020. 25 (5): p. e12798. Dityatev, A. and D.A. Rusakov, Molecular signals of plasticity at the tetrapartite synapse. Current Opinion in Neurobiology, 2011. 21 (2): p. 353-359. Hodebourg, R., et al., Nonneuronal contributions to synaptic function. Neuron, 2025. Hodebourg, R., P.W. Kalivas, and A. Kruyer, Extrasynaptic therapeutic targets in substance use and stress disorders. Trends Pharmacol Sci, 2022. 43 (1): p. 56-68. Spencer, S., et al., A Model of Delta(9)-Tetrahydrocannabinol Self-administration and Reinstatement That Alters Synaptic Plasticity in Nucleus Accumbens. Biol Psychiatry, 2018. 84 (8): p. 601-610. Hodebourg, R., In Vivo Zymography: An Essential Activity Assay for Studying the Activity of Matrix Metalloproteinases in Specific Brain Cells. Methods Mol Biol, 2025. 2918 : p. 21-33. Chioma, V.C., et al., Heroin Seeking and Extinction From Seeking Activate Matrix Metalloproteinases at Synapses on Distinct Subpopulations of Accumbens Cells. Biol Psychiatry, 2020. Garcia-Keller, C., et al., Behavioral and accumbens synaptic plasticity induced by cues associated with restraint stress. Neuropsychopharmacology, 2021. Wang, X., et al., High-frequency repetitive transcranial magnetic stimulation improves depressive-like behaviors in CUMS-induced rats by modulating astrocyte GLT-1 to reduce glutamate toxicity. Journal of Affective Disorders, 2024. 348 : p. 265-274. Bansal, Y., S.A. Codeluppi, and M. Banasr, Astroglial Dysfunctions in Mood Disorders and Rodent Stress Models: Consequences on Behavior and Potential as Treatment Target. Int J Mol Sci, 2024. 25 (12). Beroun, A., et al., MMPs in learning and memory and neuropsychiatric disorders. Cell Mol Life Sci, 2019. 76 (16): p. 3207-3228. Bonnet, U. and U.W. Preuss, The cannabis withdrawal syndrome: current insights. Subst Abuse Rehabil, 2017. 8 : p. 9-37. Connor, J.P., et al., Clinical management of cannabis withdrawal. Addiction, 2022. 117 (7): p. 2075-2095. Trexler, K.R., et al., Novel behavioral assays of spontaneous and precipitated THC withdrawal in mice. Drug Alcohol Depend, 2018. 191 : p. 14-24. Koolhaas, J.M., et al., Coping styles in animals: current status in behavior and stress-physiology. Neuroscience & Biobehavioral Reviews, 1999. 23 (7): p. 925-935. Kim, R., et al., Abstinence-Dependent Effects of Long-Access Cocaine Self-Administration on Nucleus Accumbens Astrocytes Are Observed in Male, But Not Female, Rats. eNeuro, 2022. 9 (5). Kruyer, A., et al., Heroin Cue-Evoked Astrocytic Structural Plasticity at Nucleus Accumbens Synapses Inhibits Heroin Seeking. Biol Psychiatry, 2019. 86 (11): p. 811-819. Scofield, M.D., et al., Cocaine Self-Administration and Extinction Leads to Reduced Glial Fibrillary Acidic Protein Expression and Morphometric Features of Astrocytes in the Nucleus Accumbens Core. Biological Psychiatry, 2016. 80 (3): p. 207-215. Siemsen, B.M., et al., Effects of Methamphetamine Self-Administration and Extinction on Astrocyte Structure and Function in the Nucleus Accumbens Core. Neuroscience, 2019. 406 : p. 528-541. Ning, B., et al., The rapid antidepressant effect of acupuncture on two animal models of depression by inhibiting M1-Ach receptors regulates synaptic plasticity in the prefrontal cortex. Brain Res, 2024. 1822 : p. 148609. Peng, Y.-Y., et al., Deletion of p75NTR rescues behavioral and cognitive dysfunction in SPS-induced PTSD mice through hippocampal PI3K/Akt/mTOR pathway. International Journal of Biological Macromolecules, 2025. 308 : p. 142770. Gou, L., et al., (2R,6R)-hydroxynorketamine improves PTSD-associated behaviors and structural plasticity via modulating BDNF-mTOR signaling in the nucleus accumbens. Journal of Affective Disorders, 2023. 335 : p. 129-140. Tynan, R.J., et al., Chronic stress-induced disruption of the astrocyte network is driven by structural atrophy and not loss of astrocytes. Acta Neuropathol, 2013. 126 (1): p. 75-91. Naskar, S. and S. Chattarji, Stress Elicits Contrasting Effects on the Structure and Number of Astrocytes in the Amygdala versus Hippocampus. eneuro, 2019. 6 (1): p. ENEURO.0338-18.2019. Aten, S., et al., Chronic Stress Impairs the Structure and Function of Astrocyte Networks in an Animal Model of Depression. Neurochem Res, 2023. 48 (4): p. 1191-1210. Lin, S.S., et al., Electroacupuncture prevents astrocyte atrophy to alleviate depression. Cell Death Dis, 2023. 14 (5): p. 343. Xia, L., et al., FGF2 blocks PTSD symptoms via an astrocyte-based mechanism. Behavioural Brain Research, 2013. 256 : p. 472-480. Soto, J.S., et al., Astrocyte Gi-GPCR signaling corrects compulsive-like grooming and anxiety-related behaviors in Sapap3 knockout mice. Neuron, 2024. 112 (20): p. 3412-3423.e6. Landucci, E., et al., Neuronal and Astrocytic Morphological Alterations Driven by Prolonged Exposure with Δ9-Tetrahydrocannabinol but Not Cannabidiol. Toxics, 2022. 10 (2). Ferland, J.-M.N., et al., Dose mediates the protracted effects of adolescent THC exposure on reward and stress reactivity in males relevant to perturbation of the basolateral amygdala transcriptome. Molecular Psychiatry, 2022. Wang, X.-b., et al., Extracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine enlargement and long-term potentiation coordinately. Proceedings of the National Academy of Sciences, 2008. 105 (49): p. 19520-19525. Garcia-Keller, C., et al., Extracellular Matrix Signaling Through beta3 Integrin Mediates Cocaine Cue-Induced Transient Synaptic Plasticity and Relapse. Biol Psychiatry, 2019. 86 (5): p. 377-387. Garcia-Keller, C., et al., Relapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons. J Neurosci, 2020. 40 (44): p. 8463-8477. Smith, A.C., et al., Synaptic plasticity mediating cocaine relapse requires matrix metalloproteinases. Nat Neurosci, 2014. 17 (12): p. 1655-7. Aguayo, F.I., et al., Matrix Metalloproteinase 9 Displays a Particular Time Response to Acute Stress: Variation in Its Levels and Activity Distribution in Rat Hippocampus. ACS Chem Neurosci, 2018. 9 (5): p. 945-956. van der Kooij, M.A., et al., Role for MMP-9 in stress-induced downregulation of nectin-3 in hippocampal CA1 and associated behavioural alterations. Nat Commun, 2014. 5 : p. 4995. Mitchell, J.R., et al., Behavioral and neural correlates of diverse conditioned fear responses in male and female rats. Neurobiol Stress, 2024. 33 : p. 100675. Chu, A., et al., A fear conditioned cue orchestrates a suite of behaviors in rats. Elife, 2024. 13 . Dopfel, D., et al., Individual variability in behavior and functional networks predicts vulnerability using an animal model of PTSD. Nat Commun, 2019. 10 (1): p. 2372. Gruene, T.M., et al., Sexually divergent expression of active and passive conditioned fear responses in rats. Elife, 2015. 4 . Colon, L., et al., Sexual differentiation of contextual fear responses. Learn Mem, 2018. 25 (5): p. 230-240. Zambetti, P.R., B.P. Schuessler, and J.J. Kim, Sex Differences in Foraging Rats to Naturalistic Aerial Predator Stimuli. iScience, 2019. 16 : p. 442-452. Woodburn, S.C., J.L. Bollinger, and E.S. Wohleb, Synaptic and behavioral effects of chronic stress are linked to dynamic and sex-specific changes in microglia function and astrocyte dystrophy. Neurobiol Stress, 2021. 14 : p. 100312. Brahmajothi, M.V. and M.B. Abou-Donia, PTSD Susceptibility and Challenges: Pathophysiological Consequences of Behavioral Symptoms. Mil Med, 2020. 185 (Suppl 1): p. 279-285. Wu, L. and M. Jin, Clinical Significance of Serum MMP-9, S100-β and GFAP in Patients with Mental Disorders after Traumatic Brain Injury. Actas Esp Psiquiatr, 2025. 53 (1): p. 11-18. Bach, D.R., A. Tzovara, and J. Vunder, Blocking human fear memory with the matrix metalloproteinase inhibitor doxycycline. Mol Psychiatry, 2018. 23 (7): p. 1584-1589. Xia, Y., et al., Attenuating human fear memory retention with minocycline: a randomized placebo-controlled trial. Transl Psychiatry, 2024. 14 (1): p. 28. Cobb, J.A., et al., Density of GFAP-immunoreactive astrocytes is decreased in left hippocampi in major depressive disorder. Neuroscience, 2016. 316 : p. 209-220. Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files FigS1.jpg Supplementary Figure S1 FigS2.jpg Supplementary Figure S2 FigS3V1.jpg Supplementary Figure S3 FigS4V1.jpg Supplementary Figure S4 FigS5V1.jpg Supplementary Figure S5 FigS6.jpg Supplementary Figure S6 TableS12025.docx Supplementary table S1 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 05 Nov, 2025 Review # 4 received at journal 31 Oct, 2025 Reviewer # 5 agreed at journal 26 Oct, 2025 Review # 3 received at journal 23 Oct, 2025 Review # 2 received at journal 21 Oct, 2025 Review # 1 received at journal 16 Oct, 2025 Reviewer # 4 agreed at journal 10 Oct, 2025 Reviewer # 3 agreed at journal 09 Oct, 2025 Reviewer # 2 agreed at journal 06 Oct, 2025 Reviewer # 1 agreed at journal 05 Oct, 2025 Reviewers invited by journal 05 Oct, 2025 Editor assigned by journal 01 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 31 Jul, 2025 Unknown event 31 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7254957","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":494397680,"identity":"74821538-2b83-4215-b261-e7f2fd4b0735","order_by":0,"name":"Ritchy Hodebourg","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACAyBiBjH4oQKMDURrkWwgWYvBAWK1mLM3b3xcUHE43/jaGeMPPxhsZDccIKDFsudYsfGMM4ctt93OMZPsYUgzJqjF4EaOmTRv22EDM6AWoAsPJxKjxfw3SIvx7BzjzwwM/4nSYsYM0mIgnWMgzcBwgAgtZ44VS884k24gcTutTLLHINl4JkEtx5s3fi6osDbgn528+cOPCjvZPkJa0E0gTfkoGAWjYBSMAhwAAEajQt5YSrZ5AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3998-6116","institution":"Medical University of South Carolina","correspondingAuthor":true,"prefix":"","firstName":"Ritchy","middleName":"","lastName":"Hodebourg","suffix":""},{"id":494397681,"identity":"5153685b-931b-45d8-94ca-d4dff191d40e","order_by":1,"name":"Lillian Duncan","email":"","orcid":"","institution":"Medical University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Lillian","middleName":"","lastName":"Duncan","suffix":""},{"id":494397682,"identity":"6bc1b043-1f00-455b-9f03-68d9f10d15f0","order_by":2,"name":"Eric Dereschewitz","email":"","orcid":"","institution":"Medical University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Dereschewitz","suffix":""},{"id":494397683,"identity":"a7e6457b-346f-4d8f-b83f-75a696ce42c7","order_by":3,"name":"Peter Kalivas","email":"","orcid":"https://orcid.org/0000-0001-9487-0119","institution":"Medical University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Kalivas","suffix":""}],"badges":[],"createdAt":"2025-07-30 16:26:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7254957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7254957/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89538103,"identity":"811b5b90-d302-4198-a370-1d21478c23d1","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":438421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcute restraint stress potentiated THC+CBD use in males. A) \u003c/strong\u003eExperimental timeline outlining THC+CBD self-administration, withdrawal and the defensive burying task. \u003cstrong\u003eB)\u003c/strong\u003e Acute restraint stress heightened active lever pressing during THC+CBD self-administration (stress: F\u003csub\u003e1,21\u003c/sub\u003e=4.54, p=0.045; days: F\u003csub\u003e9,189\u003c/sub\u003e, p=0.002). \u003cstrong\u003eC)\u003c/strong\u003e Stressed rats consumed more THC+CBD compared to sham animals (stress: F\u003csub\u003e1,21\u003c/sub\u003e=5.404, p=0.03). \u003cstrong\u003eD) \u003c/strong\u003eThe sham and stress groups showed no differences in their ability to distinguish between the active (THC+CBD infusion) and inactive (no consequence) levers during the final 5 days of self-administration. The dotted line represents a 2:1 ratio of active to inactive. \u0026nbsp;\u003cstrong\u003eE)\u003c/strong\u003e Acute stress did not affect the active lever presses during vehicle self-administration. \u003cstrong\u003eF)\u003c/strong\u003e Sham and stress groups consumed the same amount of vehicle. \u003cstrong\u003eG)\u003c/strong\u003e No difference was observed between sham and stress rats on the discrimination index during vehicle self-administration. See table S1 for complete analysis. Data are shown as mean ± SEM. Number of animals is expressed in brackets. *p\u0026lt; 0.05, comparing stress to sham.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/7d714ea687280921dfdeb104.jpg"},{"id":89538104,"identity":"e695ca2d-c4b5-4bde-8324-3f6361706858","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":491047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHC+CBD generalized stress responses in males. A) \u003c/strong\u003eStress-CS increased the time spent burying in vehicle-trained rats, while the Stress-NS led to an increase in the time spent burying in THC+CBD-trained rats (interaction: F\u003csub\u003e2,39\u003c/sub\u003e=14.77, p\u0026lt;0.0001). \u003cstrong\u003eB)\u003c/strong\u003e THC+CBD use generalized the escape behavior across all stress conditions (drug: F\u003csub\u003e1,39\u003c/sub\u003e=41.35, p\u0026lt;0.0001). \u003cstrong\u003eC)\u003c/strong\u003e THC+CBD reduced immobility in all three conditions (drug: F\u003csub\u003e1,39\u003c/sub\u003e=4.842, p=0.0338). \u003cstrong\u003eD) \u003c/strong\u003eStress-CS enhanced grooming frequency in vehicle-trained rats, whereas both Stress-NS and Stress-CS increased grooming in THC+CBD-trained rats (stress: F\u003csub\u003e2,39\u003c/sub\u003e=9.835, p=0.0003). \u0026nbsp;Data are shown as mean ± SEM. Each dot in bar represents one rat. \u003cstrong\u003eE)\u003c/strong\u003e Donut charts showing the distribution of the different coping strategies within each treatment. Stress-CS changed coping strategies in vehicle- (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3)\u003c/sub\u003e =34.06, p\u0026lt;0.0001 versus Sham-CS veh) and THC+CBD-trained rats (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3)\u003c/sub\u003e =34.06, p\u0026lt;0.0001 versus Sham-CS THC+CBD). However, THC+CBD promoted avoidant coping strategies during stress-CS exposure (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3)\u003c/sub\u003e =106.5, p\u0026lt;0.0001 versus Stress-CS veh). THC+CBD altered coping distribution in non-stressed rats (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3)\u003c/sub\u003e =974.4, p\u0026lt;0.0001 versus Sham-CS veh). THC+CBD modified stress responses during exposure to the neutral stimulus (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3)\u003c/sub\u003e =70.45, p\u0026lt;0.0001 versus stress-NS veh; X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3)\u003c/sub\u003e =113.2, p\u0026lt;0.0001 versus Sham-CS THC+CBD).\u0026nbsp; * p\u0026lt; 0.05, **p\u0026lt;0.01 compared to Sham-CS within each drug group using a Tukey or Chi-square post hoc; # p\u0026lt; 0.05, ## p\u0026lt;0.01 comparing THC+CBD to vehicle within each stress/sham group.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/0fd87261fbe8ceb43aec97d6.jpg"},{"id":89538672,"identity":"20f0e8c0-7684-4416-9108-dd43c6e96b33","added_by":"auto","created_at":"2025-08-21 05:55:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1022284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHC+CBD use reduced the astroglial coverage of synapses in NAcore but potentiated the synaptic insulation by astrocytes after the stress-CS exposure in males. A) \u003c/strong\u003eRepresentative confocal image of an astrocyte (red) surrounded by the presynaptic marker Synapsin-I (blue). \u003cstrong\u003eB)\u003c/strong\u003e Digital rendering of the astrocyte in A. \u003cstrong\u003eC)\u003c/strong\u003e Puncta showing co-labeling between astrocyte and Synapsin-I. \u003cstrong\u003eD) \u003c/strong\u003eLeft panel: Withdrawal from THC+CBD constitutively reduced astrocyte-Synapsin-I proximity, while re-exposure to the stress-CS strongly increased the colocalization between astrocytes and Synapsin-I (Kruskal-Wallis=95.97, p\u0026lt;0.0001). Middle panel: Cumulative frequency distribution of the astrocyte-Synapsin-I colocalization. Right panel: Stress-CS exposure did not fully restore the synaptic insulation by astroglia in vehicle-trained rats (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =16, p=0.0003 versus Sham-CS). See table S1 for complete analysis.\u0026nbsp; \u003cstrong\u003eE)\u003c/strong\u003e The negative correlation between the astrocyte-Synapsin-I colocalization and grooming frequency in vehicle-trained rats (Spearman r=-0.476, p=0.0435) disappeared following THC+CBD use. \u003cstrong\u003eF)\u003c/strong\u003e Negative correlation between the astrocyte-Synapsin-I colocalization and time spent burying in THC+CBD-trained rats (Pearson r=-0.444, p=0.0369), not in vehicle-trained rats. \u003cstrong\u003eG) \u003c/strong\u003eLeft panel: Acute stress and THC+CBD withdrawal reduced, while re-exposure to the stress-CS elevated Synapsin-I density (Kruskal-Wallis=62.39, p\u0026lt;0.0001). Middle panel: Cumulative frequency distribution of the Synapsin-I density. Right panel: Astrocytes surrounded by higher Synapsin-I density represented a larger population in the Stress-CS THC+CBD (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =17.5, p=0.0002 versus Sham-CS THC+CBD). Data are shown as mean ± SEM. Each dot in bar represents an astrocyte. * p\u0026lt; 0.05 compared to Sham-CS within each drug group using a Dunn or Chi-square post hoc; # p\u0026lt; 0.05 comparing THC+CBD to vehicle within each stress/sham group.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/9be2c90d6a34ebceae9c4658.jpg"},{"id":89538112,"identity":"1085fb8a-e1b8-4ef4-9dfe-246b60839ef5","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":763942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHC+CBD use induced astrocyte atrophy after the stress-CS exposure in males. A) \u003c/strong\u003eLeft panel: Stress-CS decreased the surface area in THC+CBD-trained rats (Kruskal-Wallis=47.67, p\u0026lt;0.0001). Middle panel: Cumulative frequency distribution of the surface area. Right panel: THC+CBD use increased the subpopulation of astrocytes with small surface area in Sham rats (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =14.33, p=0.0008 versus Sham-CS Veh), which was further augmented by stress-CS exposure (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =15.19, p=0.0005 versus Sham-CS THC+CBD).\u0026nbsp; \u003cstrong\u003eB)\u003c/strong\u003e Stress-CS decreased the volume in THC+CBD-trained rats (Kruskal-Wallis=51.84, p\u0026lt;0.0001). Middle panel: Cumulative frequency distribution of the volume. Right panel: Stress-CS increased the subpopulation of astrocytes with large volume in vehicle-trained rats (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =13.33, p=0.001 versus Sham-CS Veh), and the effect of stress-CS was reversed following THC+CBD use (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =64.4, p\u0026lt;0.0001 versus Stress-CS Veh). \u003cstrong\u003eC)\u003c/strong\u003e THC+CBD changed a positive correlation between astrocyte-Synapsin-I colocalization and surface area (left; Pearson r=0.486, p=0.039) into a negative one (right; Pearson r=-0.512, p=0.0177). \u003cstrong\u003eD)\u003c/strong\u003e Astrocyte-Synapsin-I colocalization negatively correlated with volume following THC+CBD use (Pearson r=-0.612, p=0.0039). Data are shown as mean ± SEM. Each dot in bar represents an analyzed astrocyte. * p\u0026lt; 0.05 compared to Sham-CS within each drug group using a Dunn or Chi-square post hoc; # p\u0026lt; 0.05 comparing THC+CBD to vehicle within each stress/sham group.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/9c8c84b8819847126ee1cc69.jpg"},{"id":89538124,"identity":"b2ead38d-87f5-48c4-a62c-1915b8c70fa3","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":639010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHC+CBD use potentiated the MMP-2,9 activity induced by the stress-CS in males. A) \u003c/strong\u003eRepresentative micrograph of in vivo zymography assay from each treatment group. \u003cstrong\u003eB)\u003c/strong\u003e THC+CBD withdrawal increased MMP-2,9 activity across all stress conditions (drug: F\u003csub\u003e1,31\u003c/sub\u003e=54.84, p\u0026lt;0.0001). Stress-CS increased the MMP-2,9 activity in NAcore of both vehicle- and THC+CBD-trained rats (stress: F\u003csub\u003e2,31\u003c/sub\u003e=33.03, p\u0026lt;0.0001), but THC+CBD use exacerbated the MMP-2,9 activity (interaction: F\u003csub\u003e2,31\u003c/sub\u003e=7.686, p=0.0019). \u003cstrong\u003eC)\u003c/strong\u003e THC+CBD shifted a positive correlation between burying and MMP-2,9 activity (left; Pearson r=0.485, p=0.03) into a negative correlation (right; Pearson r=-0.456, p=0.03). \u003cstrong\u003eD)\u003c/strong\u003e MMP-2,9 activity positively correlated with escape behavior in THC+CBD-trained rats (Spearman r=0.54, p=0.027). \u003cstrong\u003eE)\u003c/strong\u003e MMP-2,9 activity positively correlated with astrocyte-Synapsin-I colocalization (Pearson r=0.75, p=0.0049), and \u003cstrong\u003eF) \u003c/strong\u003enegatively correlated with astroglia surface area following THC+CBD use (Pearson r=-0.547, p=0.033). Data are shown as mean ± SEM. Each dot in bar represents an astrocyte. * p\u0026lt; 0.05 compared to Sham-CS within each drug group using a Dunn post hoc; # p\u0026lt; 0.05 comparing THC+CBD to vehicle within each stress/sham group.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/55068b5f312ad04dd38fe87e.jpg"},{"id":89538676,"identity":"41770608-5830-44c4-8eaa-4d8dabf39871","added_by":"auto","created_at":"2025-08-21 05:55:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":677902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMMP-2 regulated the multipartite synaptic plasticity following THC+CBD use. A) \u003c/strong\u003eExperimental timeline outlining THC+CBD self-administration, withdrawal and the defensive burying task. \u003cstrong\u003eB)\u003c/strong\u003e Both MMP-2i (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =10.6, p=0.005 versus DMSO) and MMP-9i (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =10.32, p=0.0058 versus DMSO) changed coping responses during exposure to the stress-CS.\u0026nbsp; Moreover, MMP-2i also changed the coping distribution compared to MMP-9i (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =96.79, p\u0026lt;0.0001). \u003cstrong\u003eC)\u003c/strong\u003e MMP-2i and MMP-9i did not alter the MMP-2,9 activity. \u003cstrong\u003eD)\u003c/strong\u003e Left panel: Both MMP-2i and MMP-9i reduced the astrocyte-Synapsin-I colocalization. Middle panel: Cumulative frequency distribution of the astrocyte-Synapsin-I colocalization. Right panel: MMP-9i increased the subpopulation of astrocytes located farther from Synapsin-I (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =18.13, p=0.0001 versus DMSO), while MMP-2i nearly eliminated astrocytes located near Synapsin-I (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(2)\u003c/sub\u003e =10.51, p=0.0052 versus MMP9i). \u003cstrong\u003eE)\u003c/strong\u003e MMP-2i reduced the Synapsin-I density (Kruskal-Wallis=12.32, p=0.0021). \u003cstrong\u003eF)\u003c/strong\u003e MMP-2i increased astroglia surface area (Kruskal-Wallis=9.37, p=0.0092). \u003cstrong\u003eG)\u003c/strong\u003e MMP-2i increased astroglia volume (Kruskal-Wallis=11.28, p=0.0035). Data are shown as mean ± SEM. Each dot in bar represents an astrocyte. * p\u0026lt; 0.05 compared to DMSO using a Dunn or Chi-square post hoc; # p\u0026lt; 0.05 comparing 9i to 2i.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/10d8cb2bf299a7209549fdbc.jpg"},{"id":89539781,"identity":"610a1738-8c3e-4c59-9cf2-0d89057f922d","added_by":"auto","created_at":"2025-08-21 06:11:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5458045,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/95f5d0bd-d67b-40f1-ae88-ea8e88d2ec40.pdf"},{"id":89538105,"identity":"9f2eec04-0f8a-4591-8234-3d81f88f7c54","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":350337,"visible":true,"origin":"","legend":"Supplementary Figure S1","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/1736ec098a1bd2a4ae462bdd.jpg"},{"id":89538108,"identity":"c149b7f3-460c-4120-854a-12ac71612488","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":245708,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure S2\u003c/p\u003e","description":"","filename":"FigS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/2f8bf97d1e836fa89563fb54.jpg"},{"id":89538111,"identity":"1ed2ef0c-3e93-4fa5-9b7b-be4e1f62d93c","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":715446,"visible":true,"origin":"","legend":"Supplementary Figure S3","description":"","filename":"FigS3V1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/577bc31e0335176da6fda14b.jpg"},{"id":89538115,"identity":"171188bc-3571-4c56-8668-28dcd126821e","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":710676,"visible":true,"origin":"","legend":"Supplementary Figure S4","description":"","filename":"FigS4V1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/8ed304cf13ae1bc66df9031b.jpg"},{"id":89538119,"identity":"c0872b8e-9249-476f-a53a-8f39d194df29","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":298578,"visible":true,"origin":"","legend":"Supplementary Figure S5","description":"","filename":"FigS5V1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/436a37c6db9a2547b05060ad.jpg"},{"id":89538117,"identity":"e94c2704-a24e-4da2-ad22-7ba6574a3a24","added_by":"auto","created_at":"2025-08-21 05:47:48","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":202157,"visible":true,"origin":"","legend":"Supplementary Figure S6","description":"","filename":"FigS6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/2c7fb1acbb23c3805d7f95eb.jpg"},{"id":89538922,"identity":"6d7d60d1-2c4c-46cb-9f6b-7f95159da0f7","added_by":"auto","created_at":"2025-08-21 06:03:48","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":28538,"visible":true,"origin":"","legend":"Supplementary table S1","description":"","filename":"TableS12025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7254957/v1/74901bf2bed2ffb5ac4d9ca1.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Cannabinoid use generalizes stress responses by altering the astrocyte plasticity through extracellular matrix signaling in the nucleus accumbens core","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor certain individuals, a single traumatic event can lead to trauma and stress-related disorders, including post-traumatic stress disorder (PTSD)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cannabis use disorder (CUD) is often comorbid with PTSD and is the most prevalent co-occurring condition among veterans diagnosed with CUD[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Patients with comorbid PTSD and CUD report higher PTSD symptom severity and poorer treatment outcomes relative to those without CUD[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, recent longitudinal studies demonstrate that cannabis use is associated with higher PTSD symptom severity, including greater traumatic thought intrusions[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and depressive symptoms[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite this evidence, PTSD is often listed as a qualified medical condition for the use of cannabis in the United States[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As of 2025, 31 states listed cannabis as a treatment for PTSD[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, the increasing use of cannabis for self-medication of PTSD and the comorbid PTSD and CUD underscores the need to identify the neurobiological mechanisms through which cannabis use alters and potentially exacerbates the symptoms associated with PTSD.\u003c/p\u003e\u003cp\u003eUsing an stress cue-conditioned (stress-CS) animal model of PTSD, we recently demonstrated that an acute stress potentiates cannabis consumption in male rats[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, we discovered that cannabis use promotes two significant PTSD-like symptoms: the generalization of stress-coping responses to a neutral stimulus that is not produced by stress exposure in the absence of cannabis use, and engaging in avoidance coping behaviors when faced with a conditioned stress odor[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Importantly, these findings differ from those observed after cocaine and heroin self-administration, where rats exposed to a stress-CS engage in active coping behaviors, without generalizing to an odor not associated with acute stress experience[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Finally, the cannabis-induced behavioral changes were accompanied by reduced spine density in the nucleus accumbens core (NAcore) and a further decrease in spine head diameter after exposure to the stress-CS[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], indicating that cannabis may be altering how environmental stimuli elicit synaptic plasticity.\u003c/p\u003e\u003cp\u003eNonneuronal components of the neuropil adjacent to synapses, including astroglia and extracellular matrix (ECM), form what is termed the multipartite synapse and function together with pre- and postsynapses to regulate synaptic plasticity[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For instance, astrocytes regulate the synaptic plasticity by insulating synapses, thereby providing them with metabolic support, gliotransmitters, and maintaining ion homeostasis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In parallel, destabilizing the ECM via catalytic signaling induced by matrix metalloproteinases (MMPs) is associated with neuronal and astroglial plasticity. Both environmental stressors and addictive drugs alter ECM signaling and astroglial morphology and signaling, and contribute to the development of stress- and drug-related disorders[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the effects of cannabis use, either independently or in conjunction with stress, on the nonneuronal components of multipartite synapses remain unknown. The objective of this study was to assess whether the interaction between stress and cannabis use influences components of multipartite synapses, including astroglial plasticity, synapse density, and ECM catalytic signaling in the NAcore. For this purpose, following an acute restraint stress, astrocytes were labeled in rats that were trained to self-administer delta9-tetrahydrocannabinol + cannabidiol (THC + CBD)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After assessing how cannabinoid use affected coping strategies through a defensive burying task (DBT), we employed confocal microscopy, in vivo zymography, and digital rendering techniques to quantify astrocyte morphology, the colocalization of astrocytes with the presynaptic marker Synapsin-I, and the gelatinolytic activity of MMP-2 and MMP-9 in the NAcore.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMale and female Long-Evans rats (~ 250 g; Charles River Laboratories) were double-housed according to sex on a 12-h light/dark cycle with food and water available ad libitum. Experiments occurred in the light cycle. All procedures received approval from the Animal Care and Use Committee of the Medical University of South Carolina and were conducted following National Institutes of Health guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAcute stress and odor pairing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRats were placed in a restraining device for 2 hours and paired with an odor (lemon or sandalwood; Sun essential, Phoenix, AZ, USA) that became the stress-CS. Control rats were exposed to the same odor in the home cage for 2 hours (Sham-CS). The odor that was not stress- or sham-paired was used during the DBT as a neutral odor control (stress-NS). Two weeks after the stress procedure, rats received indwelling catheters as previously described[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eViral labeling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter catheter implantation, rats were implanted with bilateral guide cannulae into the NAcore (AP: 1.5mm, ML: ± 1.7mm, DV: -5.5mm). AAV5/GFAP-hM3dq-mCherry (University of Zurich) was injected into the left hemisphere (1.0 µl, 0.15 µl/min, 5 min diffusion) to label astrocytes. The microinjectors are 2mm longer than the guide cannulae to reach the target.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC + CBD self-administration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing surgery, rats underwent 5 days of vapor exposure to a THC + CBD mixture in a 10:1 ratio, as previously described[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Rats were then trained to self-administer THC + CBD on a fixed ratio 1 schedule, during 90 min sessions for 10 consecutive days. Active lever presses delivered 0.4 + 0.04 mg/kg THC + CBD and were paired with cues (light + tone) followed by a 20-s timeout. Another cohort of rats was trained to self-administer the vehicle (1% ethanol). Following self-administration, animals underwent 10 days of withdrawal to ensure the absence of THC metabolites during the DBT.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDefensive burying task\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCoping strategies were quantified as previously described[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, bedding was placed in one half of the standard home cage, opposite the odor. Rats were placed facing away from the odor on the bedding side of the cage, and behavior was recorded for 15 min using Ethovision XT software (Leesburg, VA, USA). Active (burying) and avoidant (escape, immobility and grooming) coping mechanisms were quantified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo zymography\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn vivo zymography was used to quantify MMP-2,9 activity as previously described in detail[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. FITC-gelatin was microinjected into the right hemisphere (1.5µl, 0.5µl/min, 3 min diffusion) immediately before the DBT. Rats were then perfused with 4% PFA immediately after the DBT and brains were sliced at 100µm. Only slices containing the injection track and the anterior commissure on the same frame were analyzed (ImageJ; National Institute of Mental Health, Bethesda, MD). MMP-2 (444288, MilliporeSigma) and MMP-9 (444278, MilliporeSigma) inhibitors were dissolved in 1% DMSO and bilaterally microinjected before FITC-gelatin in a separate rat cohort as previously described[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLeft hemisphere slices, containing the GFAP-mCherry virus, were permeabilized for 15 min in PBS with 0.2% Triton X-100 (PBST), then blocked with 2% PBST containing 5% normal goat serum. Rabbit anti-synapsin-I antibody (1:1000, ab64581, Abcam) was incubated for 72 hours at 4ºC. Slices were washed with 0.2% PBST, then incubated with Alexa Fluor 405(1:1000, ab175652, Abcam) for 72 hours at 4ºC.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConfocal microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAstrocyte images were acquired using a Leica SP5 with a 63X oil-immersion objective lens at 16-bit resolution with a 1-µm step size. Images were then imported into Imaris software (version 9.7.0). The mCherry signal was used for 3-dimensional reconstruction of astrocytes. The astrocyte-Synapsin-I proximity was quantified by using each rendered astrocyte as a region of interest (ROI) for colocalization analysis with Synapsin-I. The colocalization channel displays the results as the percentage of the ROI overlapping with Synapsin-I puncta. To determine the Synapsin-I density, we normalized the thresholded signal intensity to the volume of the frame from which it was acquired. Imaging and analysis were performed in a blinded manner.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData were analyzed with GraphPad Prism 10. Behavioral and in vivo zymography data were analyzed using two-way ANOVAs with Tukey’s post hoc comparison. For astroglial measures, a D’Agostino \u0026amp; Pearson test was initially used to assess the normality of each group. The Kruskal-Wallis test with Dunn’s post hoc was used for non-normally distributed treatment groups. Coping strategies and astroglial distribution were analyzed with a multiple Chi-square followed by a Bonferroni correction.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAcute stress potentiated THC\u0026thinsp;+\u0026thinsp;CBD use in male rats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRats were subjected to acute restraint stress for 2 hours and simultaneously exposed to an odor. 3 weeks after the stress, rats were trained to self-administer THC\u0026thinsp;+\u0026thinsp;CBD or its vehicle for 10 days, followed by 10 days of withdrawal (Fig.\u0026nbsp;1A). In THC\u0026thinsp;+\u0026thinsp;CBD trained rats, stressed males pressed more on the active lever than sham rats (Fig.\u0026nbsp;1B). Stressed males consumed more THC\u0026thinsp;+\u0026thinsp;CBD than sham rats (Fig.\u0026nbsp;1C). Both stressed and sham rats showed equivalent discrimination between the active and inactive levers (Fig.\u0026nbsp;1D). No differences between stress and sham groups were observed in vehicle-trained rats (Fig.\u0026nbsp;1E-G).\u003c/p\u003e\u003cp\u003eIn female THC\u0026thinsp;+\u0026thinsp;CBD trained rats, no differences were measured between groups on the active lever presses and THC\u0026thinsp;+\u0026thinsp;CBD intake (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B). However, stressed rats had a higher discrimination index than sham rats (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC), indicating that the acute stress may increase the reinforcing value of the THC\u0026thinsp;+\u0026thinsp;CBD in females. No differences between stress and sham groups were observed in vehicle-trained rats (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC\u0026thinsp;+\u0026thinsp;CBD promoted avoidant coping strategies in male rats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing the withdrawal period, we used DBT to evaluate the effect of THC\u0026thinsp;+\u0026thinsp;CBD on active (burying) and avoidant (escape, immobility and grooming) coping strategies. To this end, stressed rats were exposed to either the stress-CS odor previously associated with the stressful event or a neutral odor stimulus (NS) for 15 min. Non-stressed rats were exposed to the odor previously associated with the sham experience. Regarding active coping, in vehicle-trained male rats, the stress-CS caused an increase in burying compared to the sham group. Conversely, in THC\u0026thinsp;+\u0026thinsp;CBD-trained rats, the stress-NS group exhibited greater burying than the sham group. Furthermore, compared to vehicle rats, THC\u0026thinsp;+\u0026thinsp;CBD self-administration elevated burying in the stress-NS group while decreasing it in the stress-CS group (Fig.\u0026nbsp;2A). Regarding avoidant coping strategies, we found that THC\u0026thinsp;+\u0026thinsp;CBD self-administration induced a strong escape behavior under all stress conditions compared to vehicle rats (Fig.\u0026nbsp;2B). Conversely, the percent time spent immobile was reduced after THC\u0026thinsp;+\u0026thinsp;CBD (Fig.\u0026nbsp;2C). Moreover, stress-CS significantly increased grooming in vehicle-trained rats, while both stressed groups (CS and NS) exhibited a higher grooming frequency than the sham group in THC\u0026thinsp;+\u0026thinsp;CBD-trained rats (Fig.\u0026nbsp;2D). When comparing the distribution of coping strategies between groups, we found no difference among the stress-NS and sham-CS groups in vehicle-trained rats (Fig.\u0026nbsp;2E), while the stress-CS promoted an active coping strategy by increasing burying behavior. In summary, Fig.\u0026nbsp;2 shows that THC\u0026thinsp;+\u0026thinsp;CBD withdrawal promoted avoidant coping in the sham group, while a neutral odor stimulus facilitated both active and avoidant coping in stressed rats. Also, exposure to the stress-CS after THC\u0026thinsp;+\u0026thinsp;CBD further changed stress behaviors compared to the sham and stress-CS veh groups. Combined, these data indicate that THC\u0026thinsp;+\u0026thinsp;CBD use caused male rats exposed to an earlier stressor to generalize stress responses to neutral stimuli and worsen stress responses to conditioned stimuli.\u003c/p\u003e\u003cp\u003eSurprisingly, in females, neither the stress nor the THC\u0026thinsp;+\u0026thinsp;CBD significantly affect coping strategies in the DBT (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWithdrawal from cannabinoids reduced the synaptic proximity and promoted astrocytic insulation of synapses after the stress-CS exposure in male rats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine how stress and cannabinoids influence the plasticity of NAcore astrocytes, astroglia were labeled with a membrane-bound fluorescent reporter. Rats were perfused immediately after DBT, and NAcore slices labeled with the presynaptic marker Synapsin-I (Fig.\u0026nbsp;3A). To quantify proximity between astrocytes and synapses, each astrocyte was digitally rendered (Fig.\u0026nbsp;3B), and the co-labeling of the astrocyte surface with Synapsin-I was quantified (Fig.\u0026nbsp;3C). Consistent with our previous report[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we found that acute stress (stress-NS) induced a constitutive reduction in astrocyte-Synapsin-I proximity, while exposure to the stress-CS resulted in a re-association between astrocytes and Synapsin-I in vehicle-trained male rats. Withdrawal from THC\u0026thinsp;+\u0026thinsp;CBD decreased astrocyte-Synapsin-I proximity in the sham group compared to vehicle rats. In stressed THC\u0026thinsp;+\u0026thinsp;CBD-trained rats, exposure to the NS further decreased the proximity between astrocytes and Synapsin-I relative to sham, while re-exposure to the stress-CS enhanced synaptic insulation by astroglia. Additionally, when compared to vehicle rats, the use of THC\u0026thinsp;+\u0026thinsp;CBD potentiated astrocytic insulation of synapses induced by the stress-CS (Fig.\u0026nbsp;3D, left). Because the cumulative frequency plots (Fig.\u0026nbsp;3D, middle) indicated different subpopulations of astrocytes, we divided the data into three subpopulations based on the values generating three equal populations in the sham-CS vehicle group. The multiple Chi\u003csup\u003e2\u003c/sup\u003e analysis revealed that exposure to the stress-CS did not fully restore the astrocytic insulation of synapses in vehicle-trained rats (Fig.\u0026nbsp;3D, right). Indeed, the astrocytes having less co-registration with Synapsin-I constituted a larger population in the stress-CS group than in the sham-CS group.\u003c/p\u003e\u003cp\u003eAstrocyte-Synapsin-I proximity negatively correlated with grooming in vehicle-trained males (Fig.\u0026nbsp;3E, left). This correlation disappeared after THC\u0026thinsp;+\u0026thinsp;CBD use (Fig.\u0026nbsp;3E, right). Furthermore, while no correlation existed between astrocyte-Synapsin-I proximity and burying in vehicle-trained rats (Fig.\u0026nbsp;3F, left), a negative correlation appeared after THC\u0026thinsp;+\u0026thinsp;CBD use (Fig.\u0026nbsp;3F, right). This illustrates that using cannabinoids alters the function of synaptic insulation in managing coping strategies.\u003c/p\u003e\u003cp\u003eIn females, exposure to the stress-CS increased the astrocyte-Synapsin-I proximity in both vehicle- and THC\u0026thinsp;+\u0026thinsp;CBD-trained rats (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA, left). Compared to vehicle rats, astrocytes near Synapsin-I were more abundant in the stress-NS group, while stress-CS exposure nearly eliminated astrocytes at mid-distance in THC\u0026thinsp;+\u0026thinsp;CBD females (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA right). Moreover, in THC\u0026thinsp;+\u0026thinsp;CBD-trained females, astrocyte-Synapsin-I proximity correlated positively with burying behavior and negatively with grooming (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB-C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCombination of acute stress and cannabinoid use reduced Synapsin-I density in NAcore\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChronic stress is known to diminish the levels of synaptic markers, such as Synapsin-I[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We previously found that neither acute stress nor stress-CS exposure altered Synapsin-I density in the NAcore of male rats[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nevertheless, the impact of acute stress followed by cannabinoid use on Synapsin-I remains unknown. Synapsin-I density was measured for each astrocyte digitally isolated. Similar to our prior report, Synapsin-I density remained unchanged in vehicle-trained males, regardless of the stress condition (Fig.\u0026nbsp;3G, left). Likewise, no difference was found between sham-CS groups. These findings suggest that the changes in astrocyte-Synapsin-I proximity observed in vehicle-trained rats and during withdrawal from THC\u0026thinsp;+\u0026thinsp;CBD result solely from astrocytic motility. However, the combination of acute stress and THC\u0026thinsp;+\u0026thinsp;CBD withdrawal lowered Synapsin-I density in stress-NS rats, while exposure to the stress-CS restored Synapsin-I density. The multiple Chi\u003csup\u003e2\u003c/sup\u003e analysis indicated that astrocytes with high Synapsin-I density formed a larger population in the stress-CS group compared to the sham-CS group (Fig.\u0026nbsp;3G, right). Furthermore, THC\u0026thinsp;+\u0026thinsp;CBD increased Synapsin-I density in the stress-CS group compared to the vehicle rats. This indicates that the potentiation of synaptic insulation induced by the drug following the stress-CS exposure observed in Fig.\u0026nbsp;3D could result largely from increased neuronal synapses associated with astroglia.\u003c/p\u003e\u003cp\u003eUnlike males, the interaction between acute stress and cannabinoid use increased the Synapsin-I density in female NAcore (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD, left). In stressed vehicle females, astrocytes with low Synapsin-I density increased compared to the sham-CS group. These effects are reversed after THC\u0026thinsp;+\u0026thinsp;CBD use (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD, right).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStress-CS induced astrocyte atrophy after cannabinoid use in male rats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChronic stress causes astrocyte shrinkage in various brain areas[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Previously, we found that neither stress nor stress-CS influence astrocyte structure in the NAcore of males[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We replicated these negative data in vehicle males and demonstrated that after THC\u0026thinsp;+\u0026thinsp;CBD use the stress-CS reduced both the surface area and the volume of astrocytes (Fig.\u0026nbsp;4A-B left). Moreover, in Sham rats, THC\u0026thinsp;+\u0026thinsp;CBD use increased the subpopulation of astrocytes with smaller surface areas compared to Sham vehicle rats (Fig.\u0026nbsp;4A right). Additionally, in vehicle-trained rats, the stress-CS group exhibited a higher proportion of astrocytes with larger volumes than the sham group (Fig.\u0026nbsp;4B right).\u003c/p\u003e\u003cp\u003eCannabinoid use in males transformed a positive correlation between surface area and astrocyte-Synapsin-I proximity in vehicle-trained rats into a negative correlation (Fig.\u0026nbsp;4C). Additionally, in THC\u0026thinsp;+\u0026thinsp;CBD-trained rats, the volume showed a negative correlation with astrocyte-Synapsin-I proximity, unlike in vehicle rats (Fig.\u0026nbsp;4D). In summary, these data suggest that exposure to a stress-CS interacts with cannabinoid withdrawal to induce an increase in both astroglial coverage of synapses and astrocyte atrophy.\u003c/p\u003e\u003cp\u003eIn females, the acute stress induced a constitutive decrease in astrocyte surface area and volume in both vehicle- and THC\u0026thinsp;+\u0026thinsp;CBD-trained rats. Re-exposure to stress-CS restored astrocyte morphology in vehicle females, but THC\u0026thinsp;+\u0026thinsp;CBD prevented this effect (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-B). Additionally, when comparing astrocyte morphology between sexes, THC\u0026thinsp;+\u0026thinsp;CBD withdrawal alone increased both the surface area and volume of astrocytes in females compared to males. (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC-D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMMP-2,9 activity is increased during withdrawal from cannabinoid use and is potentiated following the stress-CS exposure in male rats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMMP- 2 and \u0026minus;\u0026thinsp;9 are key components of the ECM signaling and multipartite plasticity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Both addictive drugs and stress exposure alter the activity of MMP-2 and MMP-9[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Using in vivo zymography we quantified the gelatinolytic activity of MMP-2,9 during the DBT[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Withdrawal from THC\u0026thinsp;+\u0026thinsp;CBD increased NAcore MMP-2,9 activity regardless of the stress condition in males (Fig.\u0026nbsp;5B). Moreover, stress-CS exposure increased the MMP-2,9 activity in both vehicle and THC\u0026thinsp;+\u0026thinsp;CBD males. However, THC\u0026thinsp;+\u0026thinsp;CBD use potentiated the MMP-2,9 activity compared to vehicle rats.\u003c/p\u003e\u003cp\u003eCannabinoid use reversed a positive correlation between MMP-2,9 activity and burying behavior in vehicle-trained rats into a negative correlation (Fig.\u0026nbsp;5C). Moreover, MMP-2,9 activity was positively correlated with the escape behavior in THC\u0026thinsp;+\u0026thinsp;CBD-trained, not vehicle rats (Fig.\u0026nbsp;5D). These data indicate that cannabinoid use modifies how ECM signaling regulates coping strategies, specifically by inducing a shift from active to avoidant coping. Additionally, after cannabinoid use, MMP-2,9 activity positively correlated with astrocyte-Synapsin I proximity, while negatively correlating with astrocyte surface area (Fig.\u0026nbsp;5E-F), suggesting that ECM signaling regulates the astroglial plasticity.\u003c/p\u003e\u003cp\u003eNeither stress nor the THC\u0026thinsp;+\u0026thinsp;CBD alter the MMP-2,9 activity in females (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA). However, similar to males, MMP-2,9 activity showed a positive correlation with astrocyte-Synapsin-I proximity and a negative correlation with astrocyte volume after cannabinoid use (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB-C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBoth MMP-2 and MMP-9 regulated the astroglial coverage of synapses, while MMP-2 influenced the astrocyte morphology\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine whether the changes in astroglial plasticity induced by the stress-CS in males are preceded by changes in the ECM catalytic signaling, we examined the effect of MMP-2 and MMP-9 inhibitors on stress-CS-induced astroglial plasticity. To this end, during THC\u0026thinsp;+\u0026thinsp;CBD withdrawal, another cohort of stressed males received bilateral microinjections of inhibitors (0.1 nmol) or their vehicle (1% DMSO) in NAcore, followed by a unilateral microinjection of the FITC-gelatin before stress-CS exposure (Fig.\u0026nbsp;6A). Compared to the DMSO microinjection, either MMP-2 or MMP-9 inhibitors altered the coping strategies. MMP-9 inhibitor notably reduced the burying behavior (Fig.\u0026nbsp;6B and S6). The coping strategies also significantly differ between the MMP-2 and MMP-9-treated groups (Fig.\u0026nbsp;6B), with the MMP-2 inhibitor significantly reducing grooming (Fig.\u0026nbsp;6B and S6). Interestingly, these alterations in stress-CS-induced behaviors were accompanied by only a nonsignificant reduction in gelatinolytic activity by either of the MMP inhibitors (Fig.\u0026nbsp;6C).\u003c/p\u003e\u003cp\u003eBoth MMP-2 and \u0026minus;\u0026thinsp;9 inhibitors reduced the astroglial coverage of synapses (Fig.\u0026nbsp;6D left). Furthermore, the MMP-2 inhibitor nearly eliminated astrocytes found near Synapsin-I (Fig.\u0026nbsp;6D right). A likely contributor to the reduction in synaptic coverage by astroglia was markedly reduced Synapsin-I density after treatment with the MMP-2, but not MMP-9 inhibitor (Fig.\u0026nbsp;6E). Finally, the MMP-2 inhibitor reversed astrocyte atrophy by increasing both surface area and volume (Fig.\u0026nbsp;6F-G).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe recently demonstrated that cannabis use, after a single traumatic event, promotes two PTSD-like symptoms: generalization of stress responses to neutral stimuli and avoidant coping strategies following conditioned stress exposure[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To explore the neuroadaptations behind these changes, we used in vivo zymography and confocal microscopy to assess how stress and cannabinoid use affect multipartite synaptic plasticity in the NAcore, a brain region linked to stress and addiction cues responding[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. We made four primary discoveries: 1) cannabinoid withdrawal, without any traumatic event, reduced the astroglial coverage of synapses and increased MMP-2,9 signaling in the ECM, 2) a prior traumatic event reduced Synapsin-I density during cannabinoid withdrawal, 3) exposure to a stress-CS after cannabinoid use led to avoidant coping strategies by activating MMP-2,9 in the NAcore, resulting in the re-association of astrocytes with synapses and astrocyte atrophy, and 4) these multipartite adaptations by stress and cannabinoids occurred only in males.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC\u0026thinsp;+\u0026thinsp;CBD disrupts coping strategies in males\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn line with our previous report, we found that THC\u0026thinsp;+\u0026thinsp;CBD use generalized stress responses to a neutral stimulus in stressed rats, by inducing both active (burying) and avoidant (escape and grooming) coping strategies. Importantly, a neutral stimulus did not trigger stress responses in vehicle-trained rats, suggesting that the effects seen in THC\u0026thinsp;+\u0026thinsp;CBD-trained rats result from the interaction between acute stress and cannabinoid withdrawal. Additionally, we found that cannabinoid use induced notable escape behavior in non-stressed rats. Both preclinical and clinical studies support a contention that extensive and prolonged cannabis use is associated with more severe withdrawal symptoms[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and our data indicate that cannabinoid use, regardless of previous traumatic experiences, generalizes stress responses to unconditioned stimuli. Since stress generalization is a hallmark of PTSD[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], these preclinical data support longitudinal clinical studies[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] indicating that cannabis use worsens PTSD symptoms. Furthermore, we previously demonstrated that a stress-CS is sufficient to reinstate cocaine, heroin, and alcohol seeking, reflecting an active coping strategy[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Accordingly, we found that the stress-CS induced burying behavior in vehicle-trained rats. However, after cannabinoid use, re-exposure to the stress-CS reduced burying while promoting escape and grooming behaviors. Since avoidance behavior is a key criterion for defining PTSD, and avoidance coping strategies are associated with increased activation of the stress axis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], our findings suggest that cannabis use heightened the salience of the stress-CS and thereby worsened PTSD symptoms.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC\u0026thinsp;+\u0026thinsp;CBD use alters how astroglial coverage regulates coping strategies in males\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe synaptic insulation provided by astrocytes plays a crucial role in synaptic plasticity by supplying metabolic support, gliotransmitters, and maintaining ion homeostasis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Withdrawal from cocaine, heroin, and methamphetamine self-administration reduced the proximity between astrocytes and synapses in NAcore[\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, we demonstrated that withdrawal from THC\u0026thinsp;+\u0026thinsp;CBD reduced the astroglial coverage of synapses in NAcore, suggesting that this effect is shared by many classes of addictive drugs. Akin to withdrawal from addictive drugs, an acute restraint stress also induced enduring decreases in astroglial coverage in NAcore[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Consistently, we found that acute stress reduced astrocyte-Synapsin-I proximity in both vehicle- and THC\u0026thinsp;+\u0026thinsp;CBD-trained rats. Finally, we demonstrated that re-exposure to the stress-CS increased the astroglial coverage of synapses in both vehicle- and THC\u0026thinsp;+\u0026thinsp;CBD-trained rats. However, while the stress-CS only partially restored the astrocytic insulation of synapses in vehicle-trained rats, cannabinoid exposure potentiated the re-association of astrocytes to synapses induced by the stress-CS. In examining astroglial insulation related to coping strategies, we found a negative correlation between astrocytes-Synapsin-I proximity and grooming behavior in vehicle-trained rats. This implies that astroglial coverage may partly countermand the expression of avoidant coping behaviors, although this correlation vanished following THC\u0026thinsp;+\u0026thinsp;CBD usage. Conversely, in THC\u0026thinsp;+\u0026thinsp;CBD-trained males, the proximity of astrocytes to Synapsin-I was negatively correlated with burying behavior. Thus, THC\u0026thinsp;+\u0026thinsp;CBD inhibits active coping strategies and potentially exacerbates the salience of the stress-CS by promoting synaptic insulation by astrocytes. Overall, this finding suggests that stress-CS exposure during cannabinoid withdrawal alters how astroglial coverage in the NAcore influences coping strategies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC\u0026thinsp;+\u0026thinsp;CBD use following an acute stress reduces the Synapsin-I density in NAcore\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSevere stressors are known to decrease the levels of synaptic markers, including Synapsin-I and PSD95, in several brain regions. For example, chronic unpredictable mild stress (CUMS) decreases the expression of both Synapsin-I and PSD95 in the prefrontal cortex and hippocampus[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and a single prolonged stress (SPS) reduces Synapsin-I and PSD95 in the hippocampus[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In NAcore, the combination of SPS and foot shock reduces PSD95[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast, we found that neither 2 hours of acute restraint stress, stress-CS exposure, nor withdrawal from THC\u0026thinsp;+\u0026thinsp;CBD without stress pre-exposure affected the Synapsin-I density in the NAcore[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, the reduction seen in astroglial coverage produced by stress pre-exposure (stress-NS) or THC\u0026thinsp;+\u0026thinsp;CBD without stress pre-exposure are largely due to astrocytic motility not an overall reduction in synapses. In contrast, we found that both Synapsin-I and astroglial synaptic proximity were reduced in the group receiving acute restraint stress and cannabinoid use (Stress-NS). Thus, the retraction of astrocytes induced by cannabinoid withdrawal, along with the loss of synapses resulting from the interplay between stress and cannabinoid use, may contribute to how THC\u0026thinsp;+\u0026thinsp;CBD generalizes stress responses to a neutral stimulus.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC\u0026thinsp;+\u0026thinsp;CBD use induces astrocyte atrophy during exposure to the stress-CS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIt is well-documented that stress alters astrocyte morphology[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For example, chronic restraint stress reduced the process length, branching, and volume of astrocytes in the prefrontal cortex[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and astrocyte volume in the amygdala[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. CUMS also induced astrocyte atrophy in the prefrontal cortex and hippocampus[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, the SPS protocol led to astrocyte atrophy in the hippocampus[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], where astrocyte atrophy is linked with anxiety and depressive-like behavior. For instance, the astrocyte morphology negatively correlates with anxiety, while treatments restoring the astrocyte morphology alleviate symptoms of anxiety and depression in animal models[\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In NAcore, neither acute restraint stress nor the stress-CS affect astroglial morphology[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast with stress, the effects of THC on astrocyte morphology are less understood. In organotypic hippocampal slices, prolonged incubation with THC reduces astrocytic processes[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Astrocytic processes are also reduced in the amygdala of rats treated with high doses of THC during adolescence[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, the effects of voluntary cannabinoid consumption on astrocyte structure were unknown. Here, we demonstrated that THC\u0026thinsp;+\u0026thinsp;CBD self-administration, with or without a prior stressful experience, did not affect measures of astrocyte morphology, including surface area and volume. However, re-exposure to the stress-CS triggered astrocyte atrophy in THC\u0026thinsp;+\u0026thinsp;CBD, not vehicle-trained rats. Surprisingly, we observed a negative correlation between astrocyte surface area and volume and synapse coverage, indicating that cannabinoid use and stress-CS triggered both astrocyte atrophy and increased proximity of astroglia to synapses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTHC\u0026thinsp;+\u0026thinsp;CBD use changes the function of MMP-2,9 in regulating coping strategies in males\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe gelatinases MMP-2 and MMP-9 play a necessary role in activity-dependent multipartite synaptic plasticity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For example, MMP-9 controls dendritic spine enlargement induced by long-term potentiation[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, over the last 20 years, research has focused on the role of MMP-2,9 in various psychiatric disorders, including drug and stress-related disorders[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our laboratory demonstrated that drug seeking induced by Pavlovian cues requires transient activation of MMP-9 in the NAcore, which leads to both spine head expansion and the insertion of AMPA receptors on D1-medium spiny neurons[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In parallel, withdrawal from cocaine or heroin self-administration constitutively increases the MMP-2 activity around D2-MSNs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Concerning stress-related disorders, both acute and chronic restraint stress increase MMP-9 activity in the hippocampus[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. We also found that acute restraint stress increases MMP-9 in NAcore[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the effect of stress-CS on the activity of MMP-2,9 was unknown. Given that both stress-CS and drug cues promote spine head expansion[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], we hypothesized that stress-CS would increase MMP-2,9 activity. As anticipated, MMP-2,9 activity was increased after exposure to stress-CS in vehicle-trained rats. We also demonstrated that THC\u0026thinsp;+\u0026thinsp;CBD withdrawal constitutively increased MMP-2,9 activity. The combination of stress pre-exposure and cannabinoid withdrawal (stress-NS) did not alter the basal activity of MMP-2,9. However, re-exposure to the stress-CS during cannabinoid withdrawal increased the MMP-2,9 activity. Cannabinoid use not only elevated MMP-2,9 activity but also contributed to a shift from active to avoidant coping behaviors. Thus, while MMP-2,9 activity positively correlated with burying in vehicle-trained rats, cannabinoid use reversed the positive correlation to a negative correlation with burying and revealed a positive correlation with escape behavior.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMMP-2 is a key regulator of the multipartite synaptic changes induced by the stress-CS following cannabinoid use.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsidering that stress-CS re-exposure after cannabinoid use strongly activated MMP-2,9 and simultaneously increased astroglial coverage and astrocyte atrophy, we hypothesized that these events are interconnected. Supporting our hypothesis, MMP-2,9 activity positively correlated with astroglial coverage and negatively with astrocyte surface area. To establish a causal relationship between ECM catalytic signaling and astroglial plasticity, we administered inhibitors of both gelatinases before the DBT. We found that inhibiting either MMP-2 and MMP-9 reduced astroglial coverage of synapses in NAcore, while only the MMP-2 inhibitor reduced Synapsin-I density and prevented astrocyte atrophy. Additionally, inhibiting MMP-2 decreased grooming while increasing burying, indicating that MMP-2 activation by stress-CS contributes to the switch from active to avoidant behaviors in THC\u0026thinsp;+\u0026thinsp;CBD withdrawn rats. In summary, these data constitute the first in vivo evidence linking the catalytic activities of MMP-2 and MMP-9 to astroglial plasticity and stress-associated behaviors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSexual dimorphism\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWomen are twice as likely to develop PTSD and exhibit greater vulnerability for anxiety-related disorders [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, historically, preclinical studies on stress-related disorders mostly used males and focused primarily on freezing behavior[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Including females and assessing other stress responses introduced new insights into coping strategies[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Indeed, research demonstrates that male rats freeze more than females, while females display higher escape-like behaviors[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Accordingly, we found that females escape more than males across all stress conditions. Surprisingly, neither stress nor THC\u0026thinsp;+\u0026thinsp;CBD had a significant impact on coping strategies in females. This may be due to high variability we observed in stress responses between females. Although the estrous cycle is not involved in escape-like behavior [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], it would be interesting to evaluate its influence on other coping responses. Despite not affecting behaviors, stress and cannabinoid use changed multipartite plasticity in females. Unlike males, the number of synapses (Synapsin-I puncta) is increased in stressed rats following THC\u0026thinsp;+\u0026thinsp;CBD use. Regarding astroglial plasticity, cannabinoid withdrawal without stress pre-exposure increased both the surface area and volume of NAcore astrocytes in females compared to males. This result differs from cocaine, where prolonged withdrawal elicits NAcore astrocyte atrophy in males without affecting female astrocytes[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, while acute restraint stress did not affect astrocyte morphology in males, it induced astrocyte atrophy in females. The opposite effect is observed after chronic stress, where CUMS reduces astrocyte morphology in males without affecting females[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Our findings contribute to research on sexual dimorphism in astroglial plasticity. More studies are needed to understand how stress and cannabis differently affect female and male multipartite plasticity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinically relevant targets\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study identified neurobiological mechanisms behind PTSD-like symptoms after cannabinoid use in male rats, highlighting potential therapeutic targets such as MMPs and astrocytes for comorbid CUD/PTSD. Recent studies demonstrate elevated plasma levels of MMP-2 and MMP-9 in PTSD patients[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Moreover, the nonselective MMP inhibitor doxycycline disrupts the consolidation of cued fear memory in humans[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Additionally, a single dose of minocycline, a more specific MMP-9 inhibitor, administered before a contextual fear memory paradigm, reduced the consolidation of fear memory[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. These clinical data suggest that MMP inhibition represents a potentially effective strategy to treat the comorbidity between PTSD and CUD. Another interesting strategy would be to target astroglial plasticity. For example, the antidepressant fluoxetine alleviates depressive-like symptoms, partly by reversing astrocyte atrophy caused by CUMS[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, in humans, antidepressant treatment reverses astroglial atrophy in the hippocampus[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Thus, exploring the impact of antidepressant treatment on astrocyte atrophy resulting from stress-CS after cannabis use would be intriguing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Anna Kruyer and Dr. Anze Testen for their valuable advice on astroglial analysis. We thank Jordan Hopkins for technical support in conducting surgeries. Also, we thank the National Institute of Drug Abuse (NIDA) Drug Supply Program for providing (-)-trans-Delta9-tetrahydrocannabinol (THC) in ethyl alcohol (95%) and Cannabidiol (CBD) (ADL-19496-11) (Synthetic). This work was supported by the National Institute of Health NIH, K99DA058761 (R.H.) and U.S. Veterans Affairs VA BX004727 (P.K.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.H. designed the study, analyzed the data, and interpreted the results with input from P.W.K. R.H. performed surgeries. R.H., L.D., and E.D. conducted operant behavioral training. R.H. carried out confocal analyses. R.H. produced the figures and authored the manuscript with input and revisions from P.W.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cem\u003eTrauma and stressor-related discorders. Posttraumatic stress disorder.\u003c/em\u003e, in \u003cem\u003eDiagnostic and statistical manual of mental disorders (5th Ed)\u003c/em\u003e, A.P. Association, Editor. 2013, American Psychiatric Association.\u003c/li\u003e\n\u003cli\u003eBonn-Miller, M.O., M.M. Bucossi, and J.A. Trafton, \u003cem\u003eThe underdiagnosis of cannabis use disorders and other Axis-I disorders among military veterans within VHA.\u003c/em\u003e Mil Med, 2012. \u003cstrong\u003e177\u003c/strong\u003e(7): p. 786-8.\u003c/li\u003e\n\u003cli\u003eBonn-Miller, M.O., et al., \u003cem\u003eThe impact of posttraumatic stress disorder on cannabis quit success.\u003c/em\u003e Am J Drug Alcohol Abuse, 2015. \u003cstrong\u003e41\u003c/strong\u003e(4): p. 339-44.\u003c/li\u003e\n\u003cli\u003eLoflin, M., M. Earleywine, and M. Bonn-Miller, \u003cem\u003eMedicinal versus recreational cannabis use: Patterns of cannabis use, alcohol use, and cued-arousal among veterans who screen positive for PTSD.\u003c/em\u003e Addict Behav, 2017. \u003cstrong\u003e68\u003c/strong\u003e: p. 18-23.\u003c/li\u003e\n\u003cli\u003eLivingston, N.A., et al., \u003cem\u003eLongitudinal course of mental health symptoms among veterans with and without cannabis use disorder.\u003c/em\u003e Psychol Addict Behav, 2022. \u003cstrong\u003e36\u003c/strong\u003e(2): p. 131-143.\u003c/li\u003e\n\u003cli\u003eMetrik, J., et al., \u003cem\u003eCannabis use and posttraumatic stress disorder: prospective evidence from a longitudinal study of veterans.\u003c/em\u003e Psychol Med, 2022. \u003cstrong\u003e52\u003c/strong\u003e(3): p. 446-456.\u003c/li\u003e\n\u003cli\u003eMurkar, A., et al., \u003cem\u003eIncreased cannabis intake during the COVID-19 pandemic is associated with worsening of depression symptoms in people with PTSD.\u003c/em\u003e BMC Psychiatry, 2022. \u003cstrong\u003e22\u003c/strong\u003e(1): p. 554.\u003c/li\u003e\n\u003cli\u003eKondev, V., N. Winters, and S. Patel, \u003cem\u003eCannabis use and posttraumatic stress disorder comorbidity: Epidemiology, biology and the potential for novel treatment approaches.\u003c/em\u003e Int Rev Neurobiol, 2021. \u003cstrong\u003e157\u003c/strong\u003e: p. 143-193.\u003c/li\u003e\n\u003cli\u003eHasin, D.S., \u003cem\u003eUS Epidemiology of Cannabis Use and Associated Problems.\u003c/em\u003e Neuropsychopharmacology, 2018. \u003cstrong\u003e43\u003c/strong\u003e(1): p. 195-212.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003ePTSD and Medical Cannabis Programs\u003c/em\u003e. 2025 01/02/2025; Available from: https://www.mpp.org/issues/medical-marijuana/ptsd-medical-cannabis-programs/.\u003c/li\u003e\n\u003cli\u003eHodebourg, R., et al., \u003cem\u003eCannabinoid use is enhanced by stress and changes conditioned stress responses.\u003c/em\u003e Neuropsychopharmacology, 2022. \u003cstrong\u003e47\u003c/strong\u003e(5): p. 1037-1045.\u003c/li\u003e\n\u003cli\u003eCarter, J.S., A.M. Kearns, and C.M. Reichel, \u003cem\u003eComplex Interactions Between Sex and Stress on Heroin Seeking.\u003c/em\u003e Front Neurosci, 2021. \u003cstrong\u003e15\u003c/strong\u003e: p. 784365.\u003c/li\u003e\n\u003cli\u003eCarter, J.S., et al., \u003cem\u003eLong-term impact of acute restraint stress on heroin self-administration, reinstatement, and stress reactivity.\u003c/em\u003e Psychopharmacology (Berl), 2020. \u003cstrong\u003e237\u003c/strong\u003e: p. 1709-1721.\u003c/li\u003e\n\u003cli\u003eGarcia-Keller, C., et al., \u003cem\u003eN-Acetylcysteine treatment during acute stress prevents stress-induced augmentation of addictive drug use and relapse.\u003c/em\u003e Addict Biol, 2020. \u003cstrong\u003e25\u003c/strong\u003e(5): p. e12798.\u003c/li\u003e\n\u003cli\u003eDityatev, A. and D.A. Rusakov, \u003cem\u003eMolecular signals of plasticity at the tetrapartite synapse.\u003c/em\u003e Current Opinion in Neurobiology, 2011. \u003cstrong\u003e21\u003c/strong\u003e(2): p. 353-359.\u003c/li\u003e\n\u003cli\u003eHodebourg, R., et al., \u003cem\u003eNonneuronal contributions to synaptic function.\u003c/em\u003e Neuron, 2025.\u003c/li\u003e\n\u003cli\u003eHodebourg, R., P.W. Kalivas, and A. Kruyer, \u003cem\u003eExtrasynaptic therapeutic targets in substance use and stress disorders.\u003c/em\u003e Trends Pharmacol Sci, 2022. \u003cstrong\u003e43\u003c/strong\u003e(1): p. 56-68.\u003c/li\u003e\n\u003cli\u003eSpencer, S., et al., \u003cem\u003eA Model of Delta(9)-Tetrahydrocannabinol Self-administration and Reinstatement That Alters Synaptic Plasticity in Nucleus Accumbens.\u003c/em\u003e Biol Psychiatry, 2018. \u003cstrong\u003e84\u003c/strong\u003e(8): p. 601-610.\u003c/li\u003e\n\u003cli\u003eHodebourg, R., \u003cem\u003eIn Vivo Zymography: An Essential Activity Assay for Studying the Activity of Matrix Metalloproteinases in Specific Brain Cells.\u003c/em\u003e Methods Mol Biol, 2025. \u003cstrong\u003e2918\u003c/strong\u003e: p. 21-33.\u003c/li\u003e\n\u003cli\u003eChioma, V.C., et al., \u003cem\u003eHeroin Seeking and Extinction From Seeking Activate Matrix Metalloproteinases at Synapses on Distinct Subpopulations of Accumbens Cells.\u003c/em\u003e Biol Psychiatry, 2020.\u003c/li\u003e\n\u003cli\u003eGarcia-Keller, C., et al., \u003cem\u003eBehavioral and accumbens synaptic plasticity induced by cues associated with restraint stress.\u003c/em\u003e Neuropsychopharmacology, 2021.\u003c/li\u003e\n\u003cli\u003eWang, X., et al., \u003cem\u003eHigh-frequency repetitive transcranial magnetic stimulation improves depressive-like behaviors in CUMS-induced rats by modulating astrocyte GLT-1 to reduce glutamate toxicity.\u003c/em\u003e Journal of Affective Disorders, 2024. \u003cstrong\u003e348\u003c/strong\u003e: p. 265-274.\u003c/li\u003e\n\u003cli\u003eBansal, Y., S.A. Codeluppi, and M. Banasr, \u003cem\u003eAstroglial Dysfunctions in Mood Disorders and Rodent Stress Models: Consequences on Behavior and Potential as Treatment Target.\u003c/em\u003e Int J Mol Sci, 2024. \u003cstrong\u003e25\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eBeroun, A., et al., \u003cem\u003eMMPs in learning and memory and neuropsychiatric disorders.\u003c/em\u003e Cell Mol Life Sci, 2019. \u003cstrong\u003e76\u003c/strong\u003e(16): p. 3207-3228.\u003c/li\u003e\n\u003cli\u003eBonnet, U. and U.W. Preuss, \u003cem\u003eThe cannabis withdrawal syndrome: current insights.\u003c/em\u003e Subst Abuse Rehabil, 2017. \u003cstrong\u003e8\u003c/strong\u003e: p. 9-37.\u003c/li\u003e\n\u003cli\u003eConnor, J.P., et al., \u003cem\u003eClinical management of cannabis withdrawal.\u003c/em\u003e Addiction, 2022. \u003cstrong\u003e117\u003c/strong\u003e(7): p. 2075-2095.\u003c/li\u003e\n\u003cli\u003eTrexler, K.R., et al., \u003cem\u003eNovel behavioral assays of spontaneous and precipitated THC withdrawal in mice.\u003c/em\u003e Drug Alcohol Depend, 2018. \u003cstrong\u003e191\u003c/strong\u003e: p. 14-24.\u003c/li\u003e\n\u003cli\u003eKoolhaas, J.M., et al., \u003cem\u003eCoping styles in animals: current status in behavior and stress-physiology.\u003c/em\u003e Neuroscience \u0026amp; Biobehavioral Reviews, 1999. \u003cstrong\u003e23\u003c/strong\u003e(7): p. 925-935.\u003c/li\u003e\n\u003cli\u003eKim, R., et al., \u003cem\u003eAbstinence-Dependent Effects of Long-Access Cocaine Self-Administration on Nucleus Accumbens Astrocytes Are Observed in Male, But Not Female, Rats.\u003c/em\u003e eNeuro, 2022. \u003cstrong\u003e9\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eKruyer, A., et al., \u003cem\u003eHeroin Cue-Evoked Astrocytic Structural Plasticity at Nucleus Accumbens Synapses Inhibits Heroin Seeking.\u003c/em\u003e Biol Psychiatry, 2019. \u003cstrong\u003e86\u003c/strong\u003e(11): p. 811-819.\u003c/li\u003e\n\u003cli\u003eScofield, M.D., et al., \u003cem\u003eCocaine Self-Administration and Extinction Leads to Reduced Glial Fibrillary Acidic Protein Expression and Morphometric Features of Astrocytes in the Nucleus Accumbens Core.\u003c/em\u003e Biological Psychiatry, 2016. \u003cstrong\u003e80\u003c/strong\u003e(3): p. 207-215.\u003c/li\u003e\n\u003cli\u003eSiemsen, B.M., et al., \u003cem\u003eEffects of Methamphetamine Self-Administration and Extinction on Astrocyte Structure and Function in the Nucleus Accumbens Core.\u003c/em\u003e Neuroscience, 2019. \u003cstrong\u003e406\u003c/strong\u003e: p. 528-541.\u003c/li\u003e\n\u003cli\u003eNing, B., et al., \u003cem\u003eThe rapid antidepressant effect of acupuncture on two animal models of depression by inhibiting M1-Ach receptors regulates synaptic plasticity in the prefrontal cortex.\u003c/em\u003e Brain Res, 2024. \u003cstrong\u003e1822\u003c/strong\u003e: p. 148609.\u003c/li\u003e\n\u003cli\u003ePeng, Y.-Y., et al., \u003cem\u003eDeletion of p75NTR rescues behavioral and cognitive dysfunction in SPS-induced PTSD mice through hippocampal PI3K/Akt/mTOR pathway.\u003c/em\u003e International Journal of Biological Macromolecules, 2025. \u003cstrong\u003e308\u003c/strong\u003e: p. 142770.\u003c/li\u003e\n\u003cli\u003eGou, L., et al., \u003cem\u003e(2R,6R)-hydroxynorketamine improves PTSD-associated behaviors and structural plasticity via modulating BDNF-mTOR signaling in the nucleus accumbens.\u003c/em\u003e Journal of Affective Disorders, 2023. \u003cstrong\u003e335\u003c/strong\u003e: p. 129-140.\u003c/li\u003e\n\u003cli\u003eTynan, R.J., et al., \u003cem\u003eChronic stress-induced disruption of the astrocyte network is driven by structural atrophy and not loss of astrocytes.\u003c/em\u003e Acta Neuropathol, 2013. \u003cstrong\u003e126\u003c/strong\u003e(1): p. 75-91.\u003c/li\u003e\n\u003cli\u003eNaskar, S. and S. Chattarji, \u003cem\u003eStress Elicits Contrasting Effects on the Structure and Number of Astrocytes in the Amygdala versus Hippocampus.\u003c/em\u003e eneuro, 2019. \u003cstrong\u003e6\u003c/strong\u003e(1): p. ENEURO.0338-18.2019.\u003c/li\u003e\n\u003cli\u003eAten, S., et al., \u003cem\u003eChronic Stress Impairs the Structure and Function of Astrocyte Networks in an Animal Model of Depression.\u003c/em\u003e Neurochem Res, 2023. \u003cstrong\u003e48\u003c/strong\u003e(4): p. 1191-1210.\u003c/li\u003e\n\u003cli\u003eLin, S.S., et al., \u003cem\u003eElectroacupuncture prevents astrocyte atrophy to alleviate depression.\u003c/em\u003e Cell Death Dis, 2023. \u003cstrong\u003e14\u003c/strong\u003e(5): p. 343.\u003c/li\u003e\n\u003cli\u003eXia, L., et al., \u003cem\u003eFGF2 blocks PTSD symptoms via an astrocyte-based mechanism.\u003c/em\u003e Behavioural Brain Research, 2013. \u003cstrong\u003e256\u003c/strong\u003e: p. 472-480.\u003c/li\u003e\n\u003cli\u003eSoto, J.S., et al., \u003cem\u003eAstrocyte Gi-GPCR signaling corrects compulsive-like grooming and anxiety-related behaviors in Sapap3 knockout mice.\u003c/em\u003e Neuron, 2024. \u003cstrong\u003e112\u003c/strong\u003e(20): p. 3412-3423.e6.\u003c/li\u003e\n\u003cli\u003eLanducci, E., et al., \u003cem\u003eNeuronal and Astrocytic Morphological Alterations Driven by Prolonged Exposure with \u0026Delta;9-Tetrahydrocannabinol but Not Cannabidiol.\u003c/em\u003e Toxics, 2022. \u003cstrong\u003e10\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eFerland, J.-M.N., et al., \u003cem\u003eDose mediates the protracted effects of adolescent THC exposure on reward and stress reactivity in males relevant to perturbation of the basolateral amygdala transcriptome.\u003c/em\u003e Molecular Psychiatry, 2022.\u003c/li\u003e\n\u003cli\u003eWang, X.-b., et al., \u003cem\u003eExtracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine enlargement and long-term potentiation coordinately.\u003c/em\u003e Proceedings of the National Academy of Sciences, 2008. \u003cstrong\u003e105\u003c/strong\u003e(49): p. 19520-19525.\u003c/li\u003e\n\u003cli\u003eGarcia-Keller, C., et al., \u003cem\u003eExtracellular Matrix Signaling Through beta3 Integrin Mediates Cocaine Cue-Induced Transient Synaptic Plasticity and Relapse.\u003c/em\u003e Biol Psychiatry, 2019. \u003cstrong\u003e86\u003c/strong\u003e(5): p. 377-387.\u003c/li\u003e\n\u003cli\u003eGarcia-Keller, C., et al., \u003cem\u003eRelapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons.\u003c/em\u003e J Neurosci, 2020. \u003cstrong\u003e40\u003c/strong\u003e(44): p. 8463-8477.\u003c/li\u003e\n\u003cli\u003eSmith, A.C., et al., \u003cem\u003eSynaptic plasticity mediating cocaine relapse requires matrix metalloproteinases.\u003c/em\u003e Nat Neurosci, 2014. \u003cstrong\u003e17\u003c/strong\u003e(12): p. 1655-7.\u003c/li\u003e\n\u003cli\u003eAguayo, F.I., et al., \u003cem\u003eMatrix Metalloproteinase 9 Displays a Particular Time Response to Acute Stress: Variation in Its Levels and Activity Distribution in Rat Hippocampus.\u003c/em\u003e ACS Chem Neurosci, 2018. \u003cstrong\u003e9\u003c/strong\u003e(5): p. 945-956.\u003c/li\u003e\n\u003cli\u003evan der Kooij, M.A., et al., \u003cem\u003eRole for MMP-9 in stress-induced downregulation of nectin-3 in hippocampal CA1 and associated behavioural alterations.\u003c/em\u003e Nat Commun, 2014. \u003cstrong\u003e5\u003c/strong\u003e: p. 4995.\u003c/li\u003e\n\u003cli\u003eMitchell, J.R., et al., \u003cem\u003eBehavioral and neural correlates of diverse conditioned fear responses in male and female rats.\u003c/em\u003e Neurobiol Stress, 2024. \u003cstrong\u003e33\u003c/strong\u003e: p. 100675.\u003c/li\u003e\n\u003cli\u003eChu, A., et al., \u003cem\u003eA fear conditioned cue orchestrates a suite of behaviors in rats.\u003c/em\u003e Elife, 2024. \u003cstrong\u003e13\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eDopfel, D., et al., \u003cem\u003eIndividual variability in behavior and functional networks predicts vulnerability using an animal model of PTSD.\u003c/em\u003e Nat Commun, 2019. \u003cstrong\u003e10\u003c/strong\u003e(1): p. 2372.\u003c/li\u003e\n\u003cli\u003eGruene, T.M., et al., \u003cem\u003eSexually divergent expression of active and passive conditioned fear responses in rats.\u003c/em\u003e Elife, 2015. \u003cstrong\u003e4\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eColon, L., et al., \u003cem\u003eSexual differentiation of contextual fear responses.\u003c/em\u003e Learn Mem, 2018. \u003cstrong\u003e25\u003c/strong\u003e(5): p. 230-240.\u003c/li\u003e\n\u003cli\u003eZambetti, P.R., B.P. Schuessler, and J.J. Kim, \u003cem\u003eSex Differences in Foraging Rats to Naturalistic Aerial Predator Stimuli.\u003c/em\u003e iScience, 2019. \u003cstrong\u003e16\u003c/strong\u003e: p. 442-452.\u003c/li\u003e\n\u003cli\u003eWoodburn, S.C., J.L. Bollinger, and E.S. Wohleb, \u003cem\u003eSynaptic and behavioral effects of chronic stress are linked to dynamic and sex-specific changes in microglia function and astrocyte dystrophy.\u003c/em\u003e Neurobiol Stress, 2021. \u003cstrong\u003e14\u003c/strong\u003e: p. 100312.\u003c/li\u003e\n\u003cli\u003eBrahmajothi, M.V. and M.B. Abou-Donia, \u003cem\u003ePTSD Susceptibility and Challenges: Pathophysiological Consequences of Behavioral Symptoms.\u003c/em\u003e Mil Med, 2020. \u003cstrong\u003e185\u003c/strong\u003e(Suppl 1): p. 279-285.\u003c/li\u003e\n\u003cli\u003eWu, L. and M. Jin, \u003cem\u003eClinical Significance of Serum MMP-9, S100-\u0026beta; and GFAP in Patients with Mental Disorders after Traumatic Brain Injury.\u003c/em\u003e Actas Esp Psiquiatr, 2025. \u003cstrong\u003e53\u003c/strong\u003e(1): p. 11-18.\u003c/li\u003e\n\u003cli\u003eBach, D.R., A. Tzovara, and J. Vunder, \u003cem\u003eBlocking human fear memory with the matrix metalloproteinase inhibitor doxycycline.\u003c/em\u003e Mol Psychiatry, 2018. \u003cstrong\u003e23\u003c/strong\u003e(7): p. 1584-1589.\u003c/li\u003e\n\u003cli\u003eXia, Y., et al., \u003cem\u003eAttenuating human fear memory retention with minocycline: a randomized placebo-controlled trial.\u003c/em\u003e Transl Psychiatry, 2024. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 28.\u003c/li\u003e\n\u003cli\u003eCobb, J.A., et al., \u003cem\u003eDensity of GFAP-immunoreactive astrocytes is decreased in left hippocampi in major depressive disorder.\u003c/em\u003e Neuroscience, 2016. \u003cstrong\u003e316\u003c/strong\u003e: p. 209-220.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7254957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7254957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rising legal acceptance of cannabis and the high comorbidity between cannabis use disorder (CUD) and post-traumatic stress disorder (PTSD) highlight the importance of understanding how stress and cannabis influence the brain. We recently discovered that cannabis use promotes two PTSD-like symptoms: avoidance coping behaviors and the generalization of stress-coping responses to a neutral stimulus not previously linked to stress. To investigate the neuroadaptations behind these changes, we used in vivo zymography and confocal microscopy to examine how stress and cannabinoid use influence multipartite synaptic plasticity in the nucleus accumbens core (NAcore), including astroglial plasticity, Synapsin-I density, and matrix metalloproteinases (MMP-2,9) activity. For this purpose, rats were restraint stressed for 2h and simultaneously exposed to an odor that became the stress-conditioned stimulus (stress-CS). Three weeks later, rats self-administered cannabinoids (delta9-tetrahydrocannabinol\u0026thinsp;+\u0026thinsp;cannabidiol; THC\u0026thinsp;+\u0026thinsp;CBD) for 10 days, followed by 10 days of withdrawal. We then evaluated the effect of stress-CS or neutral odor (NS) on coping strategies in a defensive burying task. We demonstrated for the first time that THC\u0026thinsp;+\u0026thinsp;CBD generalized stress responses to the NS by causing astrocytes to retract from synapses and decreasing Synapsin-I density in the NAcore. Furthermore, cannabinoid use promoted avoidant coping behaviors in response to a stress-CS by triggering strong activation of MMP-2,9, driven largely by MMP-2, causing a re-association of astrocytes to synapses along with an increase of Synapsin-I density and astrocyte atrophy. However, these neuroadaptations only occurred in males. Overall, these findings highlight potential therapeutic targets like MMPs and astrocytes for treating co-occurring CUD/PTSD.\u003c/p\u003e","manuscriptTitle":"Cannabinoid use generalizes stress responses by altering the astrocyte plasticity through extracellular matrix signaling in the nucleus accumbens core","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 05:47:43","doi":"10.21203/rs.3.rs-7254957/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-11-05T14:17:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-01T02:12:16+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-26T07:16:12+00:00","index":5,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-23T13:54:16+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-21T20:41:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-16T19:13:39+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-10T19:18:35+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-09T14:05:56+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-06T15:52:11+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-06T01:18:53+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-05T20:18:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T15:20:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T09:43:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2025-07-31T14:32:58+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-07-31T11:32:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a80f6975-878a-431d-bba2-beb573033211","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52511587,"name":"Biological sciences/Neuroscience"},{"id":52511588,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2026-04-27T16:04:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 05:47:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7254957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7254957","identity":"rs-7254957","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}