Differential Learned Helplessness Phenotypes in Male and Female Rats: Exploring the Therapeutic Impact of Cannabidiol Treatment

Differential Learned Helplessness Phenotypes in Male and Female Rats: Exploring the Therapeutic Impact of Cannabidiol Treatment | 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 Differential Learned Helplessness Phenotypes in Male and Female Rats: Exploring the Therapeutic Impact of Cannabidiol Treatment Irit Akirav, Uri Bright This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4270261/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Evidence suggests a bidirectional relationship between depressive symptoms and neuroinflammation. We studied the effects of chronic treatment with cannabidiol (CBD) in male and female rats exposed to an unpredictable chronic mild stress (UCMS) model of depression. We analyzed gene expression related to neuroinflammation, cannabinoids, and estrogen receptors, as well as specific microRNAs (miRs) in the ventromedial prefrontal cortex (vmPFC), CA1, and ventral subiculum (VS). We found sex- and brain region-dependent effects of UCMS and CBD. UCMS exerted sex-specific effects on immobility, increasing it in males while decreasing it in females; CBD reversed this effect in both sexes. Regarding neuroinflammation, CBD restored Tumor Necrosis Factor α (TNF-α) gene upregulation in the CA1 and VS in males. In both sexes, UCMS led to nuclear factor kappa B subunit 1 (NF-κB1) gene upregulation in the VS, unaffected by CBD. In males, UCMS-induced CB1 gene downregulation in the VS was restored by CBD. UCMS resulted in CB1 gene downregulation in the vmPFC in both sexes, with no CBD effect. In males, CBD restored UCMS-induced downregulation of VS ERα and ERβ genes. Finally, UCMS downregulated miR-146a-5p in the VS in females and upregulated it in the CA1 in males without CBD restoring effects. Our findings highlight sex-specific differences in learned helplessness and CBD’s modulation of neuroinflammatory, cannabinoid, and estrogen gene expression following exposure to chronic stress. Specifically, in males, hippocampal neuroinflammatory and estrogenic mechanisms play pivotal roles in mediating CBD's antidepressant effects. Biological sciences/Neuroscience/Molecular neuroscience Biological sciences/Drug discovery/Pharmacology/Pharmacogenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mounting evidence underscores the intricate interplay between depression and inflammation [ 1 ], with studies revealing elevated levels of inflammatory markers, such as Tumor Necrosis Factor α (TNF-α) and nuclear factor kappa B subunit 1 (NF-κB1), in individuals with depression [ 2 – 3 ]. Notably, attenuation of TNF-α and NF-κB1 expression has been associated with improvements in depressive symptoms. [ 4 – 5 ]. C annabidiol (CBD), renowned for its anti-inflammatory and antioxidative properties [ 6 ] has emerged as a promising candidate for treating depression [ 7 – 13 ]. Preclinical studies have demonstrated CBD's ability to reduce TNF-α expression and NF-κB1 activation [ 14 ]. CBD is an inverse agonist of both CB1 and CB2 receptors [ 15 ]; it inhibits the enzyme fatty acid amide hydrolase (FAAH), leading to increased levels of anandamide (AEA(. Additionally, CBD acts as an agonist of receptors such as transient receptor potential vanilloid 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARγ), and serotonin receptor 5-HT1a [ 16 ]. Our recent research has highlighted CBD's efficacy in alleviating depression-like behavior in male rats exposed to unpredictable chronic mild stress (UCMS), a widely used model of depression. These therapeutic effects have been linked to alterations in microRNAs (miR-16 and miR-135) within the ventromedial prefrontal cortex (vmPFC), mediated through the serotonergic 5-HT1a receptor [ 7 ]. Several miRNAs, including miR-146, miR-9, and miR-98, have been implicated in stress resilience and depression regulation, influencing inflammatory gene targets [ 17 – 31 ]. Elevated miR-146 levels have been associated with TNF-α treatment [ 23 ] and correlated with depressive symptoms [ 17 – 19 ]. MiR-9 mediated depressive-like symptoms and its downregulation or inhibition decreased depressive-like behavior in UCMS mice, improved regeneration of hippocampal cells [ 21 ], and improved learning and memory in a water maze test [ 32 ]. MiR-9 also targets NF-κB1 and inhibits its expression, as was found in studies with immune disease patients [ 25 – 26 , 28 ]. Mir-98 negatively correlated with depressive symptoms; its expression was lower in the PFC and hippocampus of mice that were subjected to chronic social stress while overexpressing miR-98 led to the alleviation of depressive-like symptoms [ 22 ]. MiR-98 also downregulated TNF-α expression [ 25 , 29 , 33 ], and its inhibition elevated TNF-α [ 31 ]. Sex differences in depression prevalence are well-documented, with women exhibiting nearly double the lifetime prevalence compared to men [ 34 ]. Preclinical studies suggest that male and female rats may respond differently to stress models and pharmacological treatments [ 35 – 37 ], possibly due to hormonal disparities, particularly estrogen; estrogen receptors are abundant both in the brains of males and females, though distributed differently [ 38 ]. The estrogen-α (ERα) and estrogen-β (ERβ) receptors play a pivotal role in mediating depressive-like symptoms [ 39 – 40 ], with ERβ implicated in the antidepressant effects of 17β-Estradiol in the forced swimming test (FST) in female rodents [ 41 – 42 ]; intra-hippocampal 17β-Estradiol had a similar antidepressant effect [ 40 ]. Our study aimed to elucidate whether the antidepressant properties of CBD are associated with alterations in genes encoding neuroinflammatory, estrogen, and cannabinoid receptors in key brain regions implicated in depression, namely the vmPFC, hippocampal CA1, and ventral subiculum (VS). Additionally, we investigated changes in specific miRNAs associated with neuroinflammation and depression, shedding light on potential mechanisms underlying CBD's therapeutic effects [ 19 , 22 , 28 ]. Materials and Methods For elaborated procedures, see supplementary information (SI). Subjects Male and female Sprague-Dawley rats aged 60 days were group-housed (No UCMS rats) or single-housed (UCMS rats) in a controlled environment at 22 ± 2°C under a 12-hour light/dark cycle. UCMS Protocol Rats were subjected to a four-week regimen of mild stressors, following a random sequence described previously in our lab [ 7 ] (see Table S1 ). Non-stressed rats were handled but not exposed to the stress protocol. Pharmacological agents During the final two weeks of the four-week UCMS model, both non-stressed and UCMS-exposed rats received daily intraperitoneal (i.p.) injections of either vehicle or CBD (10 mg/kg). Dosages were based on previous studies in our lab and elsewhere [ 7 – 8 ]. Depression-like behavioral tests Locomotor activity was measured in an Open Field Test (OFT) ; learned helplessness was assessed as immobility in The Forced Swim Test (FST) . Quantitative real-time PCR (qRT-PCR) Upon euthanasia, brain tissues from the vmPFC, CA1, and VS were collected for biochemical analysis. RNA extraction, cDNA preparation, and qRT-PCR were performed as previously described [ 7 , 43 – 44 ] to detect the expression of miRNAs (miR-9-5p, miR-98-5p, and miR-146a-5p) and mRNAs (cnr1, cnr2, tnf, nfkb1, esr1 and esr2; genes coding to CB1r, CB2r, TNF-α, NF-κB1, ERα and ERβ, respectively) (see Table S2 for mRNA primer sequences). Statistical Analyses Data were analyzed using SPSS 27 (IBM, Chicago, IL, USA). The results are expressed as means ± SEM. For statistical analysis, three-way ANOVA, two-way ANOVA, and Pearson bivariate correlation test were used. Post hoc comparisons were made using Tukey's range test. Significance was set at p ≤ 0.05. Results 1.1: The effects of chronic CBD administration during UCMS on depressive-like symptoms: We used a 2×2×2 design with main factors of Sex, Stress (No UCMS/UCMS) and Drug (Vehicle/CBD) (see Fig. 1 a for experimental design). In cases of a significant sex effect or three-way interaction, data from male and female rats were analyzed separately. See Table S3 for detailed analyses of three-way and two-way ANOVA. In the FST (Fig. 1 b), univariate ANOVA [sex×stress×drug (2×2×2)] revealed a significant effect of sex, drug, stress and the following interactions: stress×sex, sex×drug, and sex×stress×drug on immobility. No significant effect of stress×drug interaction was found. Two-way ANOVA [stress×drug (2×2)] revealed a significant effect of drug (males: F(1,39) = 19.412, p < 0.001; females: F(1,39) = 10.602, p < 0.01), stress (males: F(1,39) = 28.714, p < 0 < 001) and stressxdrug interaction (males: F(1,39) = 13.394, p < 0.01; females: F(1,39) = 7.199, p < 0,05). This suggests that in males, CBD restored the UCMS-induced increase in immobility, while in females, CBD restored the UCMS-induced decrease in immobility. A significant effect of stress, drug and stress×drug interaction on swimming was found in males, and of drug×stress in females. This suggests that in males, UCMS decreased swimming time and that CBD prevented this effect. No effects on climbing were found (data not shown, available in Table S3 ). In the OFT (Fig. 1 c), univariate ANOVA revealed a significant effect on locomotion of sex, stress and the following interactions: sex × stress and sex × stress × drug. Two-way ANOVA revealed a significant effect of stress (males: F(1,39) = 242.585, p < 0.001; females: F(1,39) = 46.286, p < 0.001) and stress×drug interaction (males: F(1,39) = 6.143, p < 0.05; females: F(1,39) = 6.376, p < 0.05), with no effect of drug (males: F(1,39) = 0.001, ns; females: F(1,39) = 0.943, ns), suggesting that UCMS led to increased locomotion in both sexes, regardless of CBD treatment. Also, we found no significant effect of stress nor CBD on the time spent in the center of the arena during the first 5 minutes of the test in males and females (data not shown, available in Table S3 ), suggesting that UCMS did not cause anxiety-like behavior. 1.2: The effects of chronic CBD administration during UCMS on cannabinoid receptors, inflammatory markers, and estrogen receptor gene expression : Raw ΔCt values for RT-PCR experiments are presented in Table S4 . 1.2.1: cnr1: In the vmPFC (Fig. 2 a), univariate ANOVA [sex×stress×drug (2×2×2)] revealed significant effects of stress but not of the drug or any of the interactions. Two-way ANOVA [stress×drug (2×2)] revealed a significant effect of stress (males: F(1,35) = 17.396, p < 0.001; females: F(1,34) = 17.912, p < 0.001) but not of drug (males: F(1,35) = 0.011, ns; females: F(1,34) = 0.098, ns) or stress×drug interaction (males: F(1,35) = 0.072, ns; females: F(1,34) = 0.019, ns), suggesting that UCMS led to downregulation of cnr1 regardless of CBD treatment. In the CA1 (Fig. 2 b), univariate ANOVA revealed significant effects of sex, stress and drug. Two-way ANOVA revealed a significant effect of drug (F(1,36) = 9.757, p < 0.01) and stress (F(1,36) = 13.617, p < 0.001) in males but not in females (drug: (F(1,37) = 0.493, ns; stress: F(1,37) = 0.124, ns). This suggests that CBD treatment to UCMS males resulted in the upregulation of cnr1. Stress×drug interaction was not significant in males (F(1,36) = 0.019, ns), or females (F(1,37) = 0.001, ns). In the VS (Fig. 2 c), univariate ANOVA revealed a significant effect of sex. Two-way ANOVA revealed a significant effect of drug (F(1,30) = 8.394, p < 0.01) and stress×drug interaction (F(1,30) = 6.915, p < 0.05) in males but not in females (drug: (F(1,28) = 0.001, ns; stress×drug: F(1,28) = 0.001, ns). This suggests that UCMS downregulated cnr1 in male rats and that CBD prevented this effect. Stress was not significant in males (F(1,30) = 2.708, ns) or females (F(1,28) = 0.066, ns), 1.2.2: cnr2: In the vmPFC (Fig. 2 d), univariate ANOVA (2×2×2) revealed a significant effect of sex and drug but not of stress or any of the interactions. Two-way ANOVA (2×2) revealed a significant effect of drug (males: F(1,34) = 6.927, p < 0.05; females: F(1,32) = 6.706, p < 0.05) but not of stress (males: F(1,34) = 0.614, ns; females: F(1,32) = 0.233, ns) or stress×drug interaction (males: F(1,34) = 0.081, ns; females: F(1,32) = 0.454, ns), suggesting that CBD downregulated cnr2 in both sexes irrespective of UCMS. In the CA1 (Fig. 2 e), univariate ANOVA revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA revealed a significant effect of drug (F(1,32) = 22.341, p < 0.001) and stress (F(1,32) = 0.5.439, p < 0.05) in males, but not in females: (drug: F(1,34) = 0.110, ns; stress: F(1,34) = 0.001, ns), suggesting that CBD treatment led to upregulation of cnr2 in males. Stress×drug interaction was not significant in males (F(1,32) = 1.302, ns) or females (F(1,34) = 0.004, ns). In the VS (Fig. 2 f), univariate ANOVA revealed a significant effect of sex but not of stress, drug or any of the interactions. Two-way ANOVA revealed no significant effect of drug (males: F(1,30) = 0.208, ns; females: F(1,29) = 0.024, ns), stress (males: F(1,30) = 2.174, ns; females: F(1,29) = 0.039, ns) or stress×drug interaction (males: F(1,30) = 0.290, ns; females: F(1,29) = 0.093, ns), suggesting that in both sexes, neither UCMS nor CBD affected cnr2 expression. 1.2.3: tnf: In the vmPFC (Fig. 3 a), univariate ANOVA (2×2×2) revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA (2×2) revealed no significant effect of drug (males: F(1,36) = 1.060, ns; females: F(1,33) = 0.576, ns), stress (males: F(1,36) = 1.211, ns; females: F(1,33) = 0.243, ns) or stress×drug interaction (males: F(1,36) = 0.122, ns; females: F(1,33) = 1.894, ns). In the CA1 (Fig. 3 b), univariate ANOVA revealed a significant effect of sex and stress but not of drug, or any of the interactions. Two-way ANOVA revealed a significant effect of drug (F(1,36) = 6.488, p < 0.05), stress (F(1,36) = 5.668 p < 0.05) and stress×drug interaction (F(1,36) = 4.085, p < 0.05) in males, but not in females (drug: F(1,36) = 0.086, ns; stress: F(1,36) = 1.097, ns; stress×drug interaction: F(1,36) = 0.003, ns), suggesting that CBD prevented UCMS-induced upregulation of tnf in males. In the VS (Fig. 3 c), univariate ANOVA revealed a significant effect of sex and sex×stress ×drug interaction, but not of stress, drug, or other interactions. Two-way ANOVA revealed a significant effect of drug (F(1,29) = 4.592, p < 0.05) and stress×drug interaction (F(1,29) = 6.718, p < 0.05) in males but not in females (drug: F(1,28) = 0.388, ns; sex×drug: F(1,28) = 0.462, ns), suggesting that CBD prevented UCMS-induced upregulation of tnf in males. Stress was not significant in males (F(1,29) = 3.704, ns) or females (F(1,28) = 0.290, ns). 1.2.4: nfkb1: In the vmPFC (Fig. 3 d), univariate ANOVA (2×2×2) revealed a significant effect of sex and sex×drug interaction, but not of stress, drug, or other interactions. Two-way ANOVA (2×2) revealed a significant effect of drug in females (F(1,34) = 14.913, p < 0.001) but not in males (F(1,32) = 0.138, ns), with no effect of stress (males: F(1,32) = 1.565, ns; females: F(1,34) = 0.195, ns) or stress×drug interaction (males: F(1,32) = 1.264, ns; females: F(1,34) = 0.157, ns), suggesting that in females, CBD resulted in the downregulation of nfkb1 irrespective of UCMS. In the CA1 (Fig. 3 e), univariate ANOVA revealed a significant effect of sex, stress, drug and stress×drug interaction. Two-way ANOVA revealed a significant effect of stress (males: F(1,36) = 4.865, p < 0.05; females: F(1,35) = 5.283, p < 0.05). Drug was significant in males (F(1,36) = 4.735, p < 0.05) but not in females (F(1,35) = 0.966, ns). Stress×drug interaction was not significant in males (F(1,36) = 3.419, ns) or females (F(1,35) = 3.953, ns). In the VS (Fig. 3 f), univariate ANOVA revealed a significant effect of sex, stress and sex×stress interaction, but not of drug or other interactions. Two-way ANOVA revealed a significant effect of stress (males: F(1,31) = 44.963, p < 0.001; females: F(1,27) = 15.462, p < 0.001) but not of drug (males: F(1,31) = 0.430, ns; females: F(1,27) = 0.214, ns) and stress×drug interaction (males: F(1,31) = 1.547, ns; females: F(1,27) = 0.001, ns), suggesting that in both sexes, UCMS led to the upregulation of nfkb1 regardless of CBD treatment. 1.2.5: esr1: In the vmPFC (Fig. 4 a), univariate ANOVA (2×2×2) revealed no significant effect of sex, stress, drug, or any of the interactions. Two-way ANOVA (2×2) revealed no significant effect of drug (males: F(1,35) = 0.031, ns; females: F(1,34) = 0.561, ns) stress (males: F(1,35) = 0.503, ns; females: F(1,34) = 0.762, ns) or stress×drug interaction (male: F(1,35) = 2.5601, ns; females: F(1,34) = 0.001, ns). In the CA1 (Fig. 4 b), univariate ANOVA revealed a significant effect of sex and stress but not of drug or any of the interactions. Two-way ANOVA revealed a significant effect of stress (males: F(1,35) = 5.650, p < 0.05; females: F(1,36) = 4.462, p < 0.05) but not drug (males: F(1,35) = 0.903, ns; females: F(1,36) = 0.270, ns) or stress×drug interaction (males: F(1,35) = 2.064, ns; females: F(1,36) = 1.744, ns), suggesting that in both sexes UCMS led to upregulation of est1 regardless of CBD treatment. In the VS (Fig. 4 c), univariate ANOVA revealed a significant effect of the following interactions: sex×stress, sex×drug, stress×drug and sex×stress×drug, but not of sex, stress and drug. Two-way ANOVA revealed a significant effect of drug in males (F(1,31) = 12.044, p < 0.01) and females (F(1,31) = 10.732, p < 0.01). Stress (F(1,31) = 6.010, p < 0.05) and stress×drug interaction (F(1,31) = 13.030, p < 0.01) were significant in males but not in females (stress: F(1,31) = 0.962, ns; stress×drug: F(1,31) = 0.275, ns), suggesting that in males CBD prevented the UCMS-induced downregulation of esr1. 1.2.6: esr2: In the vmPFC (Fig. 4 d), univariate ANOVA (2×2×2) revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA (2×2) revealed a significant effect of stress×drug interaction (F(1,35) = 4.821, p < 0.05) in males but not in females (F(1,35) = 0.088, ns). No significant effect was found of drug (males: F(1,35) = 0.004, ns; females: F(1,35) = 0.755, ns) or stress (males: F(1,35) = 2.008, ns; females: F(1,35) = 0.280, ns). In the CA1 (Fig. 4 e), univariate ANOVA revealed a significant effect of sex, stress and drug, but not any of the interactions. Two-way ANOVA revealed a significant effect of stress (males: F(1,35) = 6.485, p < 0.05; females: F(1,36) = 17.306, p < 0.001). Drug was significant in females (F(1,36) = 6.483, p < 0.05) but not in males (F(1,35) = 1.351, ns). Stress×drug interaction was not significant in males (F(1,35) = 0.023, ns) or females (F(1,36) = 0.611, ns), suggesting that UCMS led to upregulation of esr2 in both sexes. In the VS (Fig. 4 f), univariate ANOVA revealed a significant effect of sex, drug and the following interactions: sex×stress, sex×drug and stress×drug. Two-way ANOVA revealed a significant effect of drug (F(1,31) = 17.549, p < 0.001), stress (F(1,31) = 4.250, p < 0.05) and stress×drug interaction (F(1,31) = 12.1954, p < 0.01) in males, but not in females (drug F(1,31) = 0.115, ns; stress: F(1,31) = 3.955, ns; stress×drug interaction: F(1,31) = 1.687, ns), suggesting that in males CBD prevented the UCMS-induced downregulation of esr2. The distribution of estrus phases in each group of female rats was observed on the first day of behavioral tests. A similar distribution of rats across the diestrus, proestrus, and estrus phases was noted within each group (see Table S5 for estrus phase distribution and Table S6 for correlations between estrus phase and behavioral phenotype). To explore the association between depressive-like behavior and gene expression, Pearson bivariate correlation tests were conducted between the behavioral measurements and mRNA expression in the vmPFC, CA1, and VS in males (supplemental material, Table S7 ) and females ( Table S8 ). 1.4: The effects of chronic CBD administration during UCMS on miRNA expression in male and female rats : 1.4.1: miR-9-5p: In the vmPFC (Fig. 5 a), univariate ANOVA (2×2×2) revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA (2×2) revealed no significant effect of drug (males: F(1,31) = 0.068, ns; females: F(1,33) = 0.335, ns), stress (males: F(1,31) = 0.388, ns; females: F(1,33) = 1.564, ns) or stress×drug interaction (males: F(1,31) = 0.136, ns; females: F(1,33) = 0.071, ns). In the CA1 (Fig. 5 b), univariate ANOVA revealed a significant effect of sex and the following interactions: stress×drug and sex×stress×drug, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress×drug interaction (males: F(1,34) = 5.938, p < 0.05; females: F(1,34) = 17.087, p < 0.001). A significant effect of drug was found in males (F(1,34) = 5.753, p < 0.05) but not in females (F(1,34) = 1.026, ns). Stress was not significant in males (F(1,34) = 3.535, ns) or females (F(1,34) = 0.052, ns), suggesting that CBD treatment downregulated miR-9-5p in UCMS rats. In the VS (Fig. 5 c), univariate ANOVA revealed a significant effect of sex, stress and sex×stress interaction, but not of drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,34) = 24.911, p < 0.001) and stress×drug interaction (F(1,34) = 5.598, p < 0.05) in females, but not in males (stress: F(1,28) = 0.016, ns; stress ×drug: F(1,28) = 0.091, ns), suggesting that in females UCMS downregulated miR-9-5p. Drug was not significant in males (F(1,28) = 1.137, ns) or females (F(1,34) = 1.045, ns). 1.4.2: miR-98-5p: In the vmPFC (Fig. 5 d), univariate ANOVA (2×2×2) revealed a significant effect of sex, stress and the following interactions: stress×drug and sex×stress×drug but not of drug and any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,33) = 6.154, p < 0.05) and stress×drug interaction (F(1,33) = 24.91, p < 0.001) in females but not in males (stress: F(1,31) = 0.480, ns; stress×drug interaction: F(1,31) = 0.194, ns), suggesting CBD downregulated miR-98-5p in non-stressed females. Drug was not significant in both males (F(1,31) = 0.122, ns) and females (F(1,33) = 1.305, ns). In the CA1 (Fig. 5 e), univariate ANOVA revealed a significant effect of sex×stress×drug interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress×drug interaction in females (F(1,34) = 6.102, p < 0.05) but not in males (F(1,33) = 0.096, ns). Stress and drug were not significant in males (stress: F(1,33) = 0.281, ns; drug: F(1,33) = 0.064, ns) or females (stress: F(1,34) = 1.203, ns; drug: F(1,34) = 0.344, ns), In the VS (Fig. 5 f), univariate ANOVA revealed a significant effect of sex, stress and the following interactions: sex×drug and sex×stress×drug, but not of drug and the other interactions. Two-way ANOVA revealed a significant effect of drug in males (F(1,28) = 5.955, p < 0.05) but not in females (F(1,34) = 2.378, ns), and a significant effect of stress in females (F(1,34) = 17.302, p < 0.001) but not in males (F(1,28) = 0.587, ns), suggesting that CBD led to upregulation of miR-98-5p in UCMS males, while in females UCMS led to downregulation of miR-98-5p. Stress×drug interaction was not significant in males (F(1,28) = 3.296 ns) or females (F(1,34) = 1.097, ns). 1.4.3: miR-146a-5p: In the vmPFC (Fig. 5 g), univariate ANOVA (2×2×2) revealed a significant effect of sex and sex×stress×drug interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA (2×2) revealed a significant effect of stress×drug interaction in females (F(1,33) = 7.823, p < 0.01) but not in males (F(1,31) = 0.064, ns). Stress and drug were not significant in males (stress: F(1,31) = 0.160, ns; drug: F(1,31) = 1.246, ns) or females (stress: F(1,33) = 0.005, ns; drug: F(1,33) = 2.812, ns). In the CA1 (Fig. 5 h), univariate ANOVA revealed a significant effect of sex and sex×stress interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,34) = 13.529, p < 0.001) in males but not in females (F(1,34) = 0.465, ns), suggesting that in males UCMS led to upregulation of miR-146a-5p. Drug and stress×drug interaction were not significant in males (drug: F(1,34) = 0.286 ns; stress x drug: F(1,34) = 0.017, ns) or females (drug: F(1,34) = 0.259, ns; stress×drug: F(1,34) = 0.028, ns(. In the VS (Fig. 5 i), univariate ANOVA revealed a significant effect of sex and sex×stress interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,31) = 9.293, p < 0.01) in females but not in males (F(1,28) = 0.888, ns), suggesting that in females UCMS led to downregulation of miR-146a-5p. Drug and stress×drug interaction was not significant in males (drug: F(1,28) = 0.001, ns; stress×drug: F(1,28) = 0.527, ns) or females (drug: F(1,31) = 0.048, ns; stress×drug: F(1,31) = 2.262, ns). To explore the association between depressive-like behavior and microRNA expression, Pearson bivariate correlation tests were conducted between the behavioral measurements and microRNA expression in the vmPFC, CA1, and VS in males (supplemental material, Table S9 ) and females ( Table S10 ). Discussion In this study, we investigated the sex-specific effects of chronic CBD treatment on depressive-like behaviors in male and female rats exposed to UCMS. We explored the potential involvement of neuroinflammatory genes, ECS-related genes, estrogen-associated genes, and miRNAs known to modulate depression and neuroinflammation. Our findings shed light on the differential impact of CBD treatment on depressive phenotypes across sexes and elucidate the underlying molecular mechanisms implicated in these effects. Specifically, we demonstrate that CBD prevents the impact of UCMS on cnr1, tnf, esr1, and esr2 in the hippocampus of males exposed to UCMS. However, these genes do not appear to play a role in the effects of CBD in females. The effects of CBD on the behavioral phenotype in UCMS rats We found sex differences in the effects of UCMS on immobility in males and females, suggesting an opposite impact: UCMS increased passive coping in males but decreased it in females compared to a non-stressed control group. However, in both sexes, the administration of CBD restored this phenotype. Previous studies that examined the effects of UCMS on immobility in females showed contradictory results, some showing increased or decreased immobility, others showing no effect, depending on the stress protocol, the rodent type, etc. [ 45 – 47 ]. In our study we saw lower baseline immobility measures in females compared to males, which were similar to those that were found in another study that showed lower immobility in female rats due to chronic stress [ 47 ]. This suggests that females are less prone to immobility behavior to begin with, implying that the FST might not be ideal to detect depressive phenotype in female rodents. Interestingly, it has been suggested that UCMS should be considered as the first stress session and FST as the second stress session and that females previously subjected to chronic mild stress cope better by exhibiting increased active behavior in the second FST in comparison with males [ 48 ]. This suggests that the behavioral paradigms assessing the stress response (for example, the combination of stressful procedures) may affect this sex-dependent outcome and should be considered in studying the pathophysiology of stress-related depression. In the OFT, exposure to UCMS similarly affected locomotor behavior in males and females, and UCMS-induced increase in locomotion was not restored with CBD treatment. We and others have previously shown increased locomotion following chronic stress [ 7 , 49 – 50 ], and an earlier study showed a similar effect of UCMS in female mice [ 46 ] [but see 46, 51]. The lack of effect of CBD on UCMS-induced hyperlocomotion aligns with studies showing similar results in different behavioral and genetic models [ 7 , 52 ], suggesting that CBD does not lead to changes in locomotor behavior. In addition, locomotion in No UCMS rats was significantly higher in females compared to males, indicating that female rats exhibit higher locomotion behavior than males in a novel environment. The effects of CBD on neuroinflammation in UCMS rats Sex-dependent differences in neuroinflammatory markers were also observed following exposure to UCMS and treatment with CBD. In males, CBD prevented upregulation of the TNF-α gene in the CA1 and VS, suggesting a role of neuroinflammatory genes in the therapeutic-like effects of CBD in males exposed to UCMS. Positive correlations were observed between immobility and the neuroinflammatory genes, suggesting that their upregulation is highly associated with increased passive coping. These findings corroborate with previous studies that showed a positive correlation between depressive symptoms and TNF-α and NF-κB1 expression, both in humans [ 2 – 4 ] and animals [ 5 , 53 – 57 ], and specifically in the hippocampus [ 5 , 53 – 57 ]. CBD’s anti-inflammatory effect (i.e., decreasing the expression of TNF-α and NF-κB1) is also in line with studies suggesting that its antidepressant properties may be mediated by changes in these inflammatory markers in the hippocampus [ 14 , 58 – 71 ]. In both males and females, UCMS upregulated the NF-κB1 gene in the VS, with no effect of CBD. This effect was positively correlated with hyperactivity in the OFT, suggesting that hippocampal NF-κB1 may be involved in activity, in line with previous findings of lower locomotion in the OFT in nfkb1-knockout mice [ 72 – 73 ]. The effects of CBD on CB1 and CB2 genes in UCMS rats In males, but not in females, UCMS induced downregulation of the CB1 gene in the VS that was prevented d by CBD. The effect of UCMS corroborates with a previous study that demonstrated the downregulation of CB1 expression in the ventral hippocampus of male rats – but not females – exposed to the chronic mild stress (CMS) model of depression [ 74 ]. Notably, following CMS, these rats demonstrated anhedonic behavior. An earlier study showed similar results regarding the downregulation of hippocampal CB1 following UCMS [ 75 ]. UCMS males and females demonstrated downregulation of the CB1 gene in the vmPFC, with no effect of CBD. This corroborates with findings from our previous study in UCMS males, showing downregulation of cnr1 in the vmPFC, with no effect of CBD [ 7 ]. Also, there was a negative correlation between this effect and the distance travelled in the OFT, suggesting that the downregulation is associated with increased locomotion. This is in line with previous findings regarding the involvement of CB1 in locomotion behavior [ 76 – 78 ]. Interestingly, in both sexes, CBD led to the downregulation of cnr2 in the vmPFC, regardless of stress. These findings were not correlated with behavioral measures, providing further evidence of the wide spectrum of the effects of CBD on ECS mechanisms, including those not directly interconnected with symptoms of depression. The effects of CBD on estrogen genes in UCMS rats Sex-dependent differences were also observed in the effects of CBD on genes coding for estrogenic receptors following UCMS. In males, CBD prevented UCMS-induced downregulation of VS ERα and ERβ genes. Negative correlations were observed between immobility in the FST and the estrogen genes, suggesting that their downregulation is highly associated with increased passive coping. It has been suggested that ERβ is involved in social and mood-related behaviors in males [ 79 ]. In fact, it was previously shown that male mice lacking the ERβ gene spent more time immobile and a reduced time swimming and climbing in the FST [ 79 ], further establishing a possible role of hippocampal ERβ in learned helplessness in males. In UMCS-exposed rats of both sexes, an upregulation of CA1 esr1 and esr2 was observed, with the latter positively correlated with the total distance travelled in the OFT. This suggests that increased CA1 ERβ levels are associated with increased locomotion. It has been shown that male ERβ knockout (BERKO) mice demonstrated deficits in motor behavior compared to control mice, thereby establishing a connection to ERβ [ 80 ]. Previous findings in female rodents showed that ERα and ERβ receptors are important mediators of the antidepressant effects of 17β-Estradiol and other ligands [ 39 – 42 ]. In our study, UCMS did not lead to the downregulation of hippocampal esr1 in females. Possibly, the involvement of ERβ in mood-related behaviors in females is through signaling in the central amygdala, as ERβ blocking in this region improved sucrose intake (i.e., an antidepressant effect) in female rats exposed to chronic stress [ 80 ]. Moreover, the central amygdala ERβ is involved in other behaviors, such as sociosexual behaviors and anxiety [ 82 – 83 ]. The effects of CBD on miRNAs in UCMS rats In females, UCMS + CBD decreased CA1 miR-9-5p to control levels compared to the UCMS and CBD groups. This aligns with findings suggesting that MiR-9 is upregulated in the hippocampus of depressed mice and that silencing miR-9 in the hippocampus can improve depressive-like symptoms [ 21 ]. Yet, a different effect was observed in the VS in which UCMS exposure decreased the expression of VS miR-9-5p compared to the No UCMS groups. In UCMS-exposed females, CBD downregulated miR-98-5p in the VS compared to the control non-stressed groups, and this effect was negatively correlated with locomotion in the OFT, suggesting that miR-98-5p downregulation is associated with increased activity in females. In UCMS-exposed males, CBD upregulated miR-98-5p in the VS compared to the UCMS group; this aligns with a recent study suggesting that depressive symptoms are associated with lower levels of miR-98 in the hippocampus [ 22 ]. In UCMS-exposed females, CBD upregulated vmPFC miR-98-5p compared to the UCMS and CBD groups. This aligns with earlier studies showing male-female differences in miR-98 expression in different models [ 84 – 85 ]. In females, UCMS led to the downregulation of miR-146a-5p in the VS. In the vmPFC, CBD upregulated miR-146 in UCMS-exposed females compared to the UCMS group. These results are in line with several studies that showed that in both humans and animals, elevation of miR-146 is associated with worsening of depressive symptoms, and vice versa [ 17 – 20 ]. In males, UCMS upregulated miR-146a-5p in the CA1 compared to the control groups, with no restoring effect of CBD, and this upregulation was positively correlated with increased immobility in the FST. Overall, our results suggest that CBD modulates the expression of specific microRNAs in a region- and sex-specific manner in response to chronic stress, potentially contributing to its antidepressant effect. Conclusions This study set out to investigate molecular alterations in the brain which are associated with the therapeutic efficacy of CBD in male and female rats subjected to UCMS. Our findings reveal notable distinctions in behavioral responses between male and female rats under UCMS conditions, particularly in the FST. While CBD effectively mitigated UCMS-induced despair-like behavior in both sexes, it did not influence locomotor activity. Sex-specific variations emerged in the expression of neuroinflammatory markers and estrogen receptor genes. In males, CBD administration reversed UCMS-induced alterations in hippocampal CB1 expression, as well as inflammatory and estrogenic markers, suggesting the involvement of hippocampal cannabinoid, neuroinflammatory and estrogenic mechanisms in CBD's antidepressant-like effects. Conversely, CBD failed to reverse UCMS-induced changes in any markers or estrogenic receptors in females, indicating the potential engagement of alternative pathways. It is essential to recognize that male and female brains can respond in different manners to the same experimental manipulations. Notably, there are indications of male-female differences in endocannabinoid, serotonergic, inflammatory, and estrogenic markers and activity, all of which may influence depressive symptoms [ 86 – 99 ]. The observed sex differences in stress response likely stem from a complex interplay of factors, including variations in serotonergic function, fluctuations in the estrous cycle, and activity within the hypothalamic-pituitary-adrenal (HPA) axis. These findings underscore the necessity of considering sex-specific neurobiological mechanisms in the development of therapeutic interventions targeting stress-related disorders. Further exploration of the intricate interactions between CBD, sex hormones, and stress-responsive pathways is essential for advancing our understanding and refining treatment strategies tailored to individual needs. Declarations Acknowledgments We extend our gratitude to Sharon Zorin for her invaluable assistance in conducting the behavioral paradigms. Author Contribution Conceptualization, design and interpretation of data: U.B. and I.A.; formal analysis, investigation, visualization, writing—original draft preparation: U.B.; writing—review and editing, resources, supervision, funding acquisition, I.A. . All authors have read and agreed to the final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Funding Statement This work was supported by the Israel Science Foundation (ISF), grant number 993/20 to IA. Conflict of Interest The authors have nothing to disclose. References Beurel E, Toups M, Nemeroff CB. The bidirectional relationship of depression and inflammation: double trouble. Neuron 2020;107:234–56. Himmerich H, Fulda S, Linseisen J, Seiler H, Wolfram G, Himmerich S et al . Depression, comorbidities and the TNF-α system. 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Gender-specific decrease in NUDR and 5-HT1A receptor proteins in the prefrontal cortex of subjects with major depressive disorder. International Journal of Neuropsychopharmacology 2009;12:155–68. Elgellaie A, Thomas SJ, Kaelle J, Bartschi J, Larkin T. Pro-inflammatory cytokines IL‐1α, IL‐6 and TNF‐α in major depressive disorder: Sex‐specific associations with psychological symptoms. European Journal of Neuroscience 2023. Birur B, Amrock EM, Shelton RC, Li L. Sex differences in the peripheral immune system in patients with depression. Frontiers in psychiatry 2017;8:108. Kühnemann S, Brown TJ, Hochberg RB, MacLusky NJ. Sex differences in the development of estrogen receptors in the rat brain. Hormones and behavior 1994;28:483–91. Derry HM, Padin AC, Kuo JL, Hughes S, Kiecolt-Glaser JK. Sex differences in depression: does inflammation play a role?. Current psychiatry reports 2015;17:1–0. Wilson ME, Westberry JM, Trout AL. Estrogen receptor-alpha gene expression in the cortex: sex differences during development and in adulthood. Hormones and behavior 2011;59:353–7. Kurian JR, Olesen KM, Auger AP. Sex differences in epigenetic regulation of the estrogen receptor-α promoter within the developing preoptic area. Endocrinology 2010;151:2297–305. Zuloaga DG, Zuloaga KL, Hinds LR, Carbone DL, Handa RJ. Estrogen receptor β expression in the mouse forebrain: age and sex differences. Journal of Comparative Neurology 2014;522:358–71. Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplementaryMaterialMethodsandResults15.4.docx SupplementaryTable3.pdf SupplementaryTable4.xlsx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4270261","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":292626761,"identity":"9cc868f6-f535-444a-b3b0-04b29e71e4b9","order_by":0,"name":"Irit Akirav","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie2RMUvEMBiGA4HUIWfW1+X6F3IU9Ib+mN6SLh4UXByOI7ec27n2Z3iT3JYSqMuBq6Mi6FqXTnKYWnVr6yiYJ0O+hDx870cI8Xj+IMwt4/Zxc6CEQLT3cjqoRN/KiW4V9PZpmOlWIV8K6VaOg3JmLxc2vRV7SbPFFELTp4pk3QrjyhT70s53+bmkeQnAsAh9wRhJdaGZmt88cEk5w9KNdto7CxOvTjmoVN67YPwAhCao+xW4YKt1nEjjgo3WgDR8oAtekmK1iSe7XGV2tAEmll8g6VHCaxW96RrhmbDbZ14vMb672lbVe9yp/CDJkTSfVfM7ybDQKMHjb955PB7PP+QDuk1KTLLyg6YAAAAASUVORK5CYII=","orcid":"","institution":"University of Haifa, Israel","correspondingAuthor":true,"prefix":"","firstName":"Irit","middleName":"","lastName":"Akirav","suffix":""},{"id":292626762,"identity":"e74bb9fc-d6a9-4488-a4ed-f2b6d40e866f","order_by":1,"name":"Uri Bright","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Uri","middleName":"","lastName":"Bright","suffix":""}],"badges":[],"createdAt":"2024-04-15 14:02:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4270261/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4270261/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55169301,"identity":"74612626-64b9-4952-aeed-2dabf86c802e","added_by":"auto","created_at":"2024-04-23 14:54:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84432,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Experimental design: Male and female rats were exposed to the UCMS model for four weeks or not. No-UCMS and UCMS-exposed rats received daily injections (i.p.) of vehicle or CBD (10 mg/kg) during the last two weeks of the 4-week UCMS model. Behavioral tests, including FST and OFT, followed this. On day 35, rats were harvested and examined for the expression of the cnr1, cnr2, tnf, nfkb1, esr1, and esr2 genes (that encode for CB1, CB2, TNF-α, NF-κB1, ER-α, and ER-β, respectively) and miRNAs (miR-9-5p, miR-98-5p, miR-146a-5p) in the vmPFC, CA1 and VS. All analyses were conducted using two-way ANOVA [stress × drug (2 × 2); n = 8-10of each sex in each group]. (b) Males: UCMS rats treated with a vehicle spent more time immobile than all groups in the FST; Females: UCMS rats treated with a vehicle spent less time immobile than all groups. (c)\u003cstrong\u003e:\u003c/strong\u003e Males and Females: UCMS rats travelled more distance than no UCMS rats in the OFT.\u003c/p\u003e\n\u003cp\u003eCBD: cannabidiol; FST: forced swim test; miRNAs, miRs: micro RNAs; OFT: open field test; UCMS: unpredictable chronic mild stress; vmPFC: ventromedial prefrontal cortex; VS: ventral subiculum *, p\u0026lt;0.05, ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Binder41.png","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/6308d04acb4fcac60b4149c1.png"},{"id":55169302,"identity":"9ac046d5-f140-41ac-92b7-6547d53098cf","added_by":"auto","created_at":"2024-04-23 14:54:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57817,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of CBD treatment on CB1r and CB2r mRNA (cnr1 and cnr2, respectively) expression in the vmPFC, CA1, and VS in rats exposed to UCMS. (a) Males and Females: UCMS downregulated cnr1 levels in the vmPFC. (b) Males: UCMS rats treated with CBD demonstrated cnr1 upregulation in the CA1 compared to No UCMS rats treated with a vehicle. Females: there were no differences between the groups. (c) Males: UCMS rats treated with a vehicle demonstrated downregulated cnr1 levels in the VS compared to all groups. Females: there were no differences between the groups. \u003cstrong\u003ecnr2\u003c/strong\u003e: (d) Males and females: CBD downregulated cnr2 in the vmPFC. (e) Males: No UCMS rats treated with CBD demonstrated upregulated cnr1 levels in the CA1 compared to No UCMS and UCMS rats treated with vehicle. Females: there were no differences between the groups. (f) Males and Females: there were no differences between the groups in cnr2 in the VS.\u003c/p\u003e\n\u003cp\u003eCBD: cannabidiol; UCMS: unpredictable chronic mild stress; vmPFC: ventromedial prefrontal cortex; VS: ventral subiculum. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001 indicate statistically significant effects followed by post hoc comparisons; #, p \u0026lt; 0.05 indicate statistical significance in main effects\u003c/p\u003e","description":"","filename":"Binder42.png","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/6b097cafeb6812db8655c280.png"},{"id":55169306,"identity":"8781c16b-de2e-44aa-a006-f652c0c37b26","added_by":"auto","created_at":"2024-04-23 14:54:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56663,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of CBD treatment on TNF-α and NF-κB1 mRNA (tnf and nfkb1, respectively) expression in the vmPFC, CA1, and VS in rats exposed to UCMS. (a) Males and Females: there were no differences between the groups in tnf in the vmPFC. (b) Males: UCMS rats treated with vehicle demonstrated tnf upregulation in the CA1 compared to all groups. Females: there were no differences between the groups. (c) Males: UCMS rats treated with vehicle demonstrated tnf upregulation in the VS compared to all groups. Females: there were no differences between the groups. (d): Males: there were no differences between the groups in nfkb1 in the vmPFC. Females: UCMS rats treated with vehicle showed higher nfkb1 expression compared to No UCMS and UCMS rats treated with CBD. (e) Males: UCMS rats treated with CBD demonstrated upregulated nfkb1 levels in the CA1 compared to all groups. Females: UCMS rats treated with CBD showed higher nfkb1 expression than No UCMS rats treated with CBD. (f) Males: UCMS rats demonstrated nfkb1 upregulation in the VS compared to no UCMS rats. Females: UCMS rats treated with vehicle showed higher nkfb1 expression in the VS than the No UCMS groups. UCMS rats treated with CBD showed higher nfkb1 expression than No UCMS rats treated with CBD.\u003c/p\u003e\n\u003cp\u003eCBD: cannabidiol; UCMS: unpredictable chronic mild stress; vmPFC: ventromedial prefrontal cortex; VS: ventral subiculum. *, p\u0026lt;0.05, **, p\u0026lt;0.01, ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Binder43.png","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/4a39c143ca2d58e93acc5a6e.png"},{"id":55169303,"identity":"edab1682-2527-401a-9d78-38e39e4cd04a","added_by":"auto","created_at":"2024-04-23 14:54:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57785,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of CBD treatment on ERα and ERβ mRNA (esr1 and esr2, respectively) expression in the vmPFC, CA1, and VS in rats exposed to UCMS. (a) Males and Females: there were no differences between the groups in esr1 in the vmPFC. (b) Males and females: UCMS rats demonstrated esr1 upregulation in the CA1. (c) Males: UCMS rats treated with vehicle demonstrated esr1 downregulation in the VS compared to all groups. Females: UCMS rats treated with vehicle demonstrated higher esr1 expression than No UCMS-CBD rats. (d) Males and females: there were no differences between the groups in esr2 in the vmPFC. (e) Males: UCMS upregulated esr2 in the CA1. Females: UCMS rats demonstrated esr2 upregulation compared to no UCMS rats treated with a vehicle. (f) Males: UCMS rats treated with vehicle demonstrated esr2 downregulation in the VS compared to all groups. Females: there were no differences between the groups.\u003c/p\u003e\n\u003cp\u003eCBD: cannabidiol; UCMS: unpredictable chronic mild stress; vmPFC: ventromedial prefrontal cortex; VS: ventral subiculum. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001 indicate statistically significant effects followed by post hoc comparisons; #, p \u0026lt; 0.05 indicate statistical significance in main effects.\u003c/p\u003e","description":"","filename":"Binder44.png","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/1f14f373eaf06c8fc188bb31.png"},{"id":55169307,"identity":"b2d69746-299b-46b4-86a6-3773b975cd8e","added_by":"auto","created_at":"2024-04-23 14:54:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86533,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of CBD treatment on miR-9-5p, miR-98-5p, and miR-146a-5p expression in the vmPFC, CA1, and VS in rats exposed to UCMS. (a) Males and females:there were no differences between the groups in miR-9-5p\u003cstrong\u003ein the vmPFC\u003c/strong\u003e. (b) Males: there were no differences between the groups in the CA1. Females: UCMS rats treated with CBD demonstrated lower miR-9-5p expression than UCMS rats treated with vehicle and No UCMS rats treated with CBD. (c) Males: there were no differences between the groups in the VS. Females: UCMS rats treated with a vehicle showed lower miR-9-5p expression than No UCMS rats. UCMS rats treated with CBD showed lower miR-9-5p expression than No UCMS rats treated with vehicle. (d)Males: there were no differences in miR-98-5p in the vmPFC between the groups. Females: No UCMS rats treated with CBD showed lower miR-98-5p expression than No UCMS-Veh rats and UCMS-CBD rats. UCMS rats treated with a vehicle showed lower miR-98-5p expression than UCMS-CBD rats. (e) Males and females: there were no differences between the groups in miR-98-5p in the CA1. (f) Males: UCMS rats treated with CBD demonstrated upregulation of miR-98-5p in the VS, while UCMS rats treated with vehicle demonstrated downregulation of miR-98-5p. Females: UCMS rats treated with CBD demonstrated lower miR-98-5p expression than No UCMS rats. (g) Males and females: there were no differences between the groups in miR-146a-5p in the vmPFC. (h) Males: UCMS rats treated with a vehicle showed upregulation of miR-146a-5p in the CA1 and were not significantly different from UCMS rats treated with CBD. Females: there were no differences between the groups. (i) Males: there were no differences between the groups miR-146a-5p in the VS. Females: UCMS rats treated with a vehicle demonstrated lower miR-146a-5p expression than No UCMS-Veh rats.\u003c/p\u003e\n\u003cp\u003eCBD: cannabidiol; miR; micro RNA; UCMS: unpredictable chronic mild stress; vmPFC: ventromedial prefrontal cortex; VS: ventral subiculum. *, p\u0026lt;0.05, **, p\u0026lt;0.01, ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Binder45.png","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/ad2ed7f18ced2690b64c4b7c.png"},{"id":60152348,"identity":"b636125a-a759-42e5-868f-5a3c80a063aa","added_by":"auto","created_at":"2024-07-12 11:13:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":966647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/00e7b840-1737-46c1-88ac-c51094897bc9.pdf"},{"id":55169894,"identity":"52b28f6d-ce5b-4282-b4b3-f89c706e305e","added_by":"auto","created_at":"2024-04-23 15:02:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":241934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryMaterialMethodsandResults15.4.docx","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/03239320ece03cf7c141edc9.docx"},{"id":55169304,"identity":"068c7291-42f8-4600-b494-e805d8bdc694","added_by":"auto","created_at":"2024-04-23 14:54:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":345491,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/d58d295639a5759faf029e47.pdf"},{"id":55169308,"identity":"b461924b-8bfd-4ee3-9857-c7d19020d7a5","added_by":"auto","created_at":"2024-04-23 14:54:12","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":75863,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4270261/v1/3b0eae826e53039d8db16943.xlsx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Differential Learned Helplessness Phenotypes in Male and Female Rats: Exploring the Therapeutic Impact of Cannabidiol Treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMounting evidence underscores the intricate interplay between depression and inflammation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], with studies revealing elevated levels of inflammatory markers, such as Tumor Necrosis Factor α (TNF-α) and nuclear factor kappa B subunit 1 (NF-κB1), in individuals with depression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, attenuation of TNF-α and NF-κB1 expression has been associated with improvements in depressive symptoms. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eC\u003c/em\u003eannabidiol (CBD), renowned for its anti-inflammatory and antioxidative properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] has emerged as a promising candidate for treating depression [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Preclinical studies have demonstrated CBD's ability to reduce TNF-α expression and NF-κB1 activation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CBD is an inverse agonist of both CB1 and CB2 receptors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; it inhibits the enzyme fatty acid amide hydrolase (FAAH), leading to increased levels of anandamide (AEA(. Additionally, CBD acts as an agonist of receptors such as transient receptor potential vanilloid 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARγ), and serotonin receptor 5-HT1a [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur recent research has highlighted CBD's efficacy in alleviating depression-like behavior in male rats exposed to unpredictable chronic mild stress (UCMS), a widely used model of depression. These therapeutic effects have been linked to alterations in microRNAs (miR-16 and miR-135) within the ventromedial prefrontal cortex (vmPFC), mediated through the serotonergic 5-HT1a receptor [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral miRNAs, including miR-146, miR-9, and miR-98, have been implicated in stress resilience and depression regulation, influencing inflammatory gene targets [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Elevated miR-146 levels have been associated with TNF-α treatment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and correlated with depressive symptoms [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MiR-9 mediated depressive-like symptoms and its downregulation or inhibition decreased depressive-like behavior in UCMS mice, improved regeneration of hippocampal cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and improved learning and memory in a water maze test [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. MiR-9 also targets NF-κB1 and inhibits its expression, as was found in studies with immune disease patients [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Mir-98 negatively correlated with depressive symptoms; its expression was lower in the PFC and hippocampus of mice that were subjected to chronic social stress while overexpressing miR-98 led to the alleviation of depressive-like symptoms [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. MiR-98 also downregulated TNF-α expression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and its inhibition elevated TNF-α [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSex differences in depression prevalence are well-documented, with women exhibiting nearly double the lifetime prevalence compared to men [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Preclinical studies suggest that male and female rats may respond differently to stress models and pharmacological treatments [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], possibly due to hormonal disparities, particularly estrogen; estrogen receptors are abundant both in the brains of males and females, though distributed differently [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The estrogen-α (ERα) and estrogen-β (ERβ) receptors play a pivotal role in mediating depressive-like symptoms [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], with ERβ implicated in the antidepressant effects of 17β-Estradiol in the forced swimming test (FST) in female rodents [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]; intra-hippocampal 17β-Estradiol had a similar antidepressant effect [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study aimed to elucidate whether the antidepressant properties of CBD are associated with alterations in genes encoding neuroinflammatory, estrogen, and cannabinoid receptors in key brain regions implicated in depression, namely the vmPFC, hippocampal CA1, and ventral subiculum (VS). Additionally, we investigated changes in specific miRNAs associated with neuroinflammation and depression, shedding light on potential mechanisms underlying CBD's therapeutic effects [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eFor elaborated procedures, see supplementary information (SI).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSubjects\u003c/strong\u003e \u003cp\u003eMale and female Sprague-Dawley rats aged 60 days were group-housed (No UCMS rats) or single-housed (UCMS rats) in a controlled environment at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 12-hour light/dark cycle.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eUCMS Protocol\u003c/strong\u003e \u003cp\u003eRats were subjected to a four-week regimen of mild stressors, following a random sequence described previously in our lab [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] (see \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Non-stressed rats were handled but not exposed to the stress protocol.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePharmacological agents\u003c/strong\u003e \u003cp\u003eDuring the final two weeks of the four-week UCMS model, both non-stressed and UCMS-exposed rats received daily intraperitoneal (i.p.) injections of either vehicle or CBD (10 mg/kg). Dosages were based on previous studies in our lab and elsewhere [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDepression-like behavioral tests\u003c/strong\u003e \u003cp\u003eLocomotor activity was measured in an \u003cb\u003eOpen Field Test (OFT)\u003c/b\u003e; learned helplessness was assessed as immobility in \u003cb\u003eThe Forced Swim Test (FST)\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eQuantitative real-time PCR (qRT-PCR)\u003c/strong\u003e \u003cp\u003eUpon euthanasia, brain tissues from the vmPFC, CA1, and VS were collected for biochemical analysis. RNA extraction, cDNA preparation, and qRT-PCR were performed as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] to detect the expression of miRNAs (miR-9-5p, miR-98-5p, and miR-146a-5p) and mRNAs (cnr1, cnr2, tnf, nfkb1, esr1 and esr2; genes coding to CB1r, CB2r, TNF-α, NF-κB1, ERα and ERβ, respectively) (see \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e for mRNA primer sequences).\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS 27 (IBM, Chicago, IL, USA). The results are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. For statistical analysis, three-way ANOVA, two-way ANOVA, and Pearson bivariate correlation test were used. Post hoc comparisons were made using Tukey's range test. Significance was set at p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.1: The effects of chronic CBD administration during UCMS on depressive-like symptoms:\u003c/h2\u003e \u003cp\u003eWe used a 2\u0026times;2\u0026times;2 design with main factors of Sex, Stress (No UCMS/UCMS) and Drug (Vehicle/CBD) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea for experimental design). In cases of a significant sex effect or three-way interaction, data from male and female rats were analyzed separately. See \u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e for detailed analyses of three-way and two-way ANOVA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the FST (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), univariate ANOVA [sex\u0026times;stress\u0026times;drug (2\u0026times;2\u0026times;2)] revealed a significant effect of sex, drug, stress and the following interactions: stress\u0026times;sex, sex\u0026times;drug, and sex\u0026times;stress\u0026times;drug on immobility. No significant effect of stress\u0026times;drug interaction was found. Two-way ANOVA [stress\u0026times;drug (2\u0026times;2)] revealed a significant effect of drug (males: F(1,39)\u0026thinsp;=\u0026thinsp;19.412, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; females: F(1,39)\u0026thinsp;=\u0026thinsp;10.602, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), stress (males: F(1,39)\u0026thinsp;=\u0026thinsp;28.714, p\u0026thinsp;\u0026lt;\u0026thinsp;0\u0026thinsp;\u0026lt;\u0026thinsp;001) and stressxdrug interaction (males: F(1,39)\u0026thinsp;=\u0026thinsp;13.394, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; females: F(1,39)\u0026thinsp;=\u0026thinsp;7.199, p\u0026thinsp;\u0026lt;\u0026thinsp;0,05). This suggests that in males, CBD restored the UCMS-induced increase in immobility, while in females, CBD restored the UCMS-induced decrease in immobility.\u003c/p\u003e \u003cp\u003eA significant effect of stress, drug and stress\u0026times;drug interaction on swimming was found in males, and of drug\u0026times;stress in females. This suggests that in males, UCMS decreased swimming time and that CBD prevented this effect. No effects on climbing were found (data not shown, available in \u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), univariate ANOVA revealed a significant effect on locomotion of sex, stress and the following interactions: sex \u0026times; stress and sex \u0026times; stress \u0026times; drug. Two-way ANOVA revealed a significant effect of stress (males: F(1,39)\u0026thinsp;=\u0026thinsp;242.585, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; females: F(1,39)\u0026thinsp;=\u0026thinsp;46.286, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and stress\u0026times;drug interaction (males: F(1,39)\u0026thinsp;=\u0026thinsp;6.143, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; females: F(1,39)\u0026thinsp;=\u0026thinsp;6.376, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no effect of drug (males: F(1,39)\u0026thinsp;=\u0026thinsp;0.001, ns; females: F(1,39)\u0026thinsp;=\u0026thinsp;0.943, ns), suggesting that UCMS led to increased locomotion in both sexes, regardless of CBD treatment. Also, we found no significant effect of stress nor CBD on the time spent in the center of the arena during the first 5 minutes of the test in males and females (data not shown, available in \u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e), suggesting that UCMS did not cause anxiety-like behavior.\u003c/p\u003e \u003cp\u003e \u003cem\u003e1.2: The effects of chronic CBD administration during UCMS on cannabinoid receptors, inflammatory markers, and estrogen receptor gene expression\u003c/em\u003e:\u003c/p\u003e \u003cp\u003eRaw ΔCt values for RT-PCR experiments are presented in \u003cb\u003eTable S4\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1.2.1: cnr1:\u003c/h3\u003e\n\u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), univariate ANOVA [sex\u0026times;stress\u0026times;drug (2\u0026times;2\u0026times;2)] revealed significant effects of stress but not of the drug or any of the interactions. Two-way ANOVA [stress\u0026times;drug (2\u0026times;2)] revealed a significant effect of stress (males: F(1,35)\u0026thinsp;=\u0026thinsp;17.396, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; females: F(1,34)\u0026thinsp;=\u0026thinsp;17.912, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) but not of drug (males: F(1,35)\u0026thinsp;=\u0026thinsp;0.011, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.098, ns) or stress\u0026times;drug interaction (males: F(1,35)\u0026thinsp;=\u0026thinsp;0.072, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.019, ns), suggesting that UCMS led to downregulation of cnr1 regardless of CBD treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), univariate ANOVA revealed significant effects of sex, stress and drug. Two-way ANOVA revealed a significant effect of drug (F(1,36)\u0026thinsp;=\u0026thinsp;9.757, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01) and stress (F(1,36)\u0026thinsp;=\u0026thinsp;13.617, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in males but not in females (drug: (F(1,37)\u0026thinsp;=\u0026thinsp;0.493, ns; stress: F(1,37)\u0026thinsp;=\u0026thinsp;0.124, ns). This suggests that CBD treatment to UCMS males resulted in the upregulation of cnr1. Stress\u0026times;drug interaction was not significant in males (F(1,36)\u0026thinsp;=\u0026thinsp;0.019, ns), or females (F(1,37)\u0026thinsp;=\u0026thinsp;0.001, ns).\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), univariate ANOVA revealed a significant effect of sex. Two-way ANOVA revealed a significant effect of drug (F(1,30)\u0026thinsp;=\u0026thinsp;8.394, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and stress\u0026times;drug interaction (F(1,30)\u0026thinsp;=\u0026thinsp;6.915, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in males but not in females (drug: (F(1,28)\u0026thinsp;=\u0026thinsp;0.001, ns; stress\u0026times;drug: F(1,28)\u0026thinsp;=\u0026thinsp;0.001, ns). This suggests that UCMS downregulated cnr1 in male rats and that CBD prevented this effect. Stress was not significant in males (F(1,30)\u0026thinsp;=\u0026thinsp;2.708, ns) or females (F(1,28)\u0026thinsp;=\u0026thinsp;0.066, ns),\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e1.2.2: cnr2:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex and drug but not of stress or any of the interactions. Two-way ANOVA (2\u0026times;2) revealed a significant effect of drug (males: F(1,34)\u0026thinsp;=\u0026thinsp;6.927, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05; females: F(1,32)\u0026thinsp;=\u0026thinsp;6.706, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) but not of stress (males: F(1,34)\u0026thinsp;=\u0026thinsp;0.614, ns; females: F(1,32)\u0026thinsp;=\u0026thinsp;0.233, ns) or stress\u0026times;drug interaction (males: F(1,34)\u0026thinsp;=\u0026thinsp;0.081, ns; females: F(1,32)\u0026thinsp;=\u0026thinsp;0.454, ns), suggesting that CBD downregulated cnr2 in both sexes irrespective of UCMS.\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), univariate ANOVA revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA revealed a significant effect of drug (F(1,32)\u0026thinsp;=\u0026thinsp;22.341, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and stress (F(1,32)\u0026thinsp;=\u0026thinsp;0.5.439, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in males, but not in females: (drug: F(1,34)\u0026thinsp;=\u0026thinsp;0.110, ns; stress: F(1,34)\u0026thinsp;=\u0026thinsp;0.001, ns), suggesting that CBD treatment led to upregulation of cnr2 in males. Stress\u0026times;drug interaction was not significant in males (F(1,32)\u0026thinsp;=\u0026thinsp;1.302, ns) or females (F(1,34)\u0026thinsp;=\u0026thinsp;0.004, ns).\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), univariate ANOVA revealed a significant effect of sex but not of stress, drug or any of the interactions. Two-way ANOVA revealed no significant effect of drug (males: F(1,30)\u0026thinsp;=\u0026thinsp;0.208, ns; females: F(1,29)\u0026thinsp;=\u0026thinsp;0.024, ns), stress (males: F(1,30)\u0026thinsp;=\u0026thinsp;2.174, ns; females: F(1,29)\u0026thinsp;=\u0026thinsp;0.039, ns) or stress\u0026times;drug interaction (males: F(1,30)\u0026thinsp;=\u0026thinsp;0.290, ns; females: F(1,29)\u0026thinsp;=\u0026thinsp;0.093, ns), suggesting that in both sexes, neither UCMS nor CBD affected cnr2 expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e1.2.3: tnf:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA (2\u0026times;2) revealed no significant effect of drug (males: F(1,36)\u0026thinsp;=\u0026thinsp;1.060, ns; females: F(1,33)\u0026thinsp;=\u0026thinsp;0.576, ns), stress (males: F(1,36)\u0026thinsp;=\u0026thinsp;1.211, ns; females: F(1,33)\u0026thinsp;=\u0026thinsp;0.243, ns) or stress\u0026times;drug interaction (males: F(1,36)\u0026thinsp;=\u0026thinsp;0.122, ns; females: F(1,33)\u0026thinsp;=\u0026thinsp;1.894, ns).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), univariate ANOVA revealed a significant effect of sex and stress but not of drug, or any of the interactions. Two-way ANOVA revealed a significant effect of drug (F(1,36)\u0026thinsp;=\u0026thinsp;6.488, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05), stress (F(1,36)\u0026thinsp;=\u0026thinsp;5.668 p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and stress\u0026times;drug interaction (F(1,36)\u0026thinsp;=\u0026thinsp;4.085, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in males, but not in females (drug: F(1,36)\u0026thinsp;=\u0026thinsp;0.086, ns; stress: F(1,36)\u0026thinsp;=\u0026thinsp;1.097, ns; stress\u0026times;drug interaction: F(1,36)\u0026thinsp;=\u0026thinsp;0.003, ns), suggesting that CBD prevented UCMS-induced upregulation of tnf in males.\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), univariate ANOVA revealed a significant effect of sex and sex\u0026times;stress \u0026times;drug interaction, but not of stress, drug, or other interactions. Two-way ANOVA revealed a significant effect of drug (F(1,29)\u0026thinsp;=\u0026thinsp;4.592, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and stress\u0026times;drug interaction (F(1,29)\u0026thinsp;=\u0026thinsp;6.718, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in males but not in females (drug: F(1,28)\u0026thinsp;=\u0026thinsp;0.388, ns; sex\u0026times;drug: F(1,28)\u0026thinsp;=\u0026thinsp;0.462, ns), suggesting that CBD prevented UCMS-induced upregulation of tnf in males. Stress was not significant in males (F(1,29)\u0026thinsp;=\u0026thinsp;3.704, ns) or females (F(1,28)\u0026thinsp;=\u0026thinsp;0.290, ns).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e1.2.4: nfkb1:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex and sex\u0026times;drug interaction, but not of stress, drug, or other interactions. Two-way ANOVA (2\u0026times;2) revealed a significant effect of drug in females (F(1,34)\u0026thinsp;=\u0026thinsp;14.913, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in males (F(1,32)\u0026thinsp;=\u0026thinsp;0.138, ns), with no effect of stress (males: F(1,32)\u0026thinsp;=\u0026thinsp;1.565, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.195, ns) or stress\u0026times;drug interaction (males: F(1,32)\u0026thinsp;=\u0026thinsp;1.264, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.157, ns), suggesting that in females, CBD resulted in the downregulation of nfkb1 irrespective of UCMS.\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), univariate ANOVA revealed a significant effect of sex, stress, drug and stress\u0026times;drug interaction. Two-way ANOVA revealed a significant effect of stress (males: F(1,36)\u0026thinsp;=\u0026thinsp;4.865, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; females: F(1,35)\u0026thinsp;=\u0026thinsp;5.283, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Drug was significant in males (F(1,36)\u0026thinsp;=\u0026thinsp;4.735, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not in females (F(1,35)\u0026thinsp;=\u0026thinsp;0.966, ns). Stress\u0026times;drug interaction was not significant in males (F(1,36)\u0026thinsp;=\u0026thinsp;3.419, ns) or females (F(1,35)\u0026thinsp;=\u0026thinsp;3.953, ns).\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), univariate ANOVA revealed a significant effect of sex, stress and sex\u0026times;stress interaction, but not of drug or other interactions. Two-way ANOVA revealed a significant effect of stress (males: F(1,31)\u0026thinsp;=\u0026thinsp;44.963, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; females: F(1,27)\u0026thinsp;=\u0026thinsp;15.462, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not of drug (males: F(1,31)\u0026thinsp;=\u0026thinsp;0.430, ns; females: F(1,27)\u0026thinsp;=\u0026thinsp;0.214, ns) and stress\u0026times;drug interaction (males: F(1,31)\u0026thinsp;=\u0026thinsp;1.547, ns; females: F(1,27)\u0026thinsp;=\u0026thinsp;0.001, ns), suggesting that in both sexes, UCMS led to the upregulation of nfkb1 regardless of CBD treatment.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e1.2.5: esr1:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), univariate ANOVA (2\u0026times;2\u0026times;2) revealed no significant effect of sex, stress, drug, or any of the interactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwo-way ANOVA (2\u0026times;2) revealed no significant effect of drug (males: F(1,35)\u0026thinsp;=\u0026thinsp;0.031, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.561, ns) stress (males: F(1,35)\u0026thinsp;=\u0026thinsp;0.503, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.762, ns) or stress\u0026times;drug interaction (male: F(1,35)\u0026thinsp;=\u0026thinsp;2.5601, ns; females: F(1,34)\u0026thinsp;=\u0026thinsp;0.001, ns).\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), univariate ANOVA revealed a significant effect of sex and stress but not of drug or any of the interactions. Two-way ANOVA revealed a significant effect of stress (males: F(1,35)\u0026thinsp;=\u0026thinsp;5.650, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; females: F(1,36)\u0026thinsp;=\u0026thinsp;4.462, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not drug (males: F(1,35)\u0026thinsp;=\u0026thinsp;0.903, ns; females: F(1,36)\u0026thinsp;=\u0026thinsp;0.270, ns) or stress\u0026times;drug interaction (males: F(1,35)\u0026thinsp;=\u0026thinsp;2.064, ns; females: F(1,36)\u0026thinsp;=\u0026thinsp;1.744, ns), suggesting that in both sexes UCMS led to upregulation of est1 regardless of CBD treatment.\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), univariate ANOVA revealed a significant effect of the following interactions: sex\u0026times;stress, sex\u0026times;drug, stress\u0026times;drug and sex\u0026times;stress\u0026times;drug, but not of sex, stress and drug. Two-way ANOVA revealed a significant effect of drug in males (F(1,31)\u0026thinsp;=\u0026thinsp;12.044, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and females (F(1,31)\u0026thinsp;=\u0026thinsp;10.732, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Stress (F(1,31)\u0026thinsp;=\u0026thinsp;6.010, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and stress\u0026times;drug interaction (F(1,31)\u0026thinsp;=\u0026thinsp;13.030, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were significant in males but not in females (stress: F(1,31)\u0026thinsp;=\u0026thinsp;0.962, ns; stress\u0026times;drug: F(1,31)\u0026thinsp;=\u0026thinsp;0.275, ns), suggesting that in males CBD prevented the UCMS-induced downregulation of esr1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1.2.6: esr2:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex but not of stress, drug, or any of the interactions. Two-way ANOVA (2\u0026times;2) revealed a significant effect of stress\u0026times;drug interaction (F(1,35)\u0026thinsp;=\u0026thinsp;4.821, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in males but not in females (F(1,35)\u0026thinsp;=\u0026thinsp;0.088, ns). No significant effect was found of drug (males: F(1,35)\u0026thinsp;=\u0026thinsp;0.004, ns; females: F(1,35)\u0026thinsp;=\u0026thinsp;0.755, ns) or stress (males: F(1,35)\u0026thinsp;=\u0026thinsp;2.008, ns; females: F(1,35)\u0026thinsp;=\u0026thinsp;0.280, ns).\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), univariate ANOVA revealed a significant effect of sex, stress and drug, but not any of the interactions. Two-way ANOVA revealed a significant effect of stress (males: F(1,35)\u0026thinsp;=\u0026thinsp;6.485, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05; females: F(1,36)\u0026thinsp;=\u0026thinsp;17.306, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Drug was significant in females (F(1,36)\u0026thinsp;=\u0026thinsp;6.483, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not in males (F(1,35)\u0026thinsp;=\u0026thinsp;1.351, ns). Stress\u0026times;drug interaction was not significant in males (F(1,35)\u0026thinsp;=\u0026thinsp;0.023, ns) or females (F(1,36)\u0026thinsp;=\u0026thinsp;0.611, ns), suggesting that UCMS led to upregulation of esr2 in both sexes.\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), univariate ANOVA revealed a significant effect of sex, drug and the following interactions: sex\u0026times;stress, sex\u0026times;drug and stress\u0026times;drug. Two-way ANOVA revealed a significant effect of drug (F(1,31)\u0026thinsp;=\u0026thinsp;17.549, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), stress (F(1,31)\u0026thinsp;=\u0026thinsp;4.250, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and stress\u0026times;drug interaction (F(1,31)\u0026thinsp;=\u0026thinsp;12.1954, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in males, but not in females (drug F(1,31)\u0026thinsp;=\u0026thinsp;0.115, ns; stress: F(1,31)\u0026thinsp;=\u0026thinsp;3.955, ns; stress\u0026times;drug interaction: F(1,31)\u0026thinsp;=\u0026thinsp;1.687, ns), suggesting that in males CBD prevented the UCMS-induced downregulation of esr2.\u003c/p\u003e \u003cp\u003eThe distribution of estrus phases in each group of female rats was observed on the first day of behavioral tests. A similar distribution of rats across the diestrus, proestrus, and estrus phases was noted within each group (see \u003cb\u003eTable S5\u003c/b\u003e for estrus phase distribution and \u003cb\u003eTable S6\u003c/b\u003e for correlations between estrus phase and behavioral phenotype).\u003c/p\u003e \u003cp\u003eTo explore the association between depressive-like behavior and gene expression, Pearson bivariate correlation tests were conducted between the behavioral measurements and mRNA expression in the vmPFC, CA1, and VS in males (supplemental material, \u003cb\u003eTable S7\u003c/b\u003e) and females (\u003cb\u003eTable S8\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003e1.4: The effects of chronic CBD administration during UCMS on miRNA expression in male and female rats\u003c/em\u003e:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e1.4.1: miR-9-5p:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex but not of stress, drug, or any of the interactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwo-way ANOVA (2\u0026times;2) revealed no significant effect of drug (males: F(1,31)\u0026thinsp;=\u0026thinsp;0.068, ns; females: F(1,33)\u0026thinsp;=\u0026thinsp;0.335, ns), stress (males: F(1,31)\u0026thinsp;=\u0026thinsp;0.388, ns; females: F(1,33)\u0026thinsp;=\u0026thinsp;1.564, ns) or stress\u0026times;drug interaction (males: F(1,31)\u0026thinsp;=\u0026thinsp;0.136, ns; females: F(1,33)\u0026thinsp;=\u0026thinsp;0.071, ns).\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), univariate ANOVA revealed a significant effect of sex and the following interactions: stress\u0026times;drug and sex\u0026times;stress\u0026times;drug, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress\u0026times;drug interaction (males: F(1,34)\u0026thinsp;=\u0026thinsp;5.938, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; females: F(1,34)\u0026thinsp;=\u0026thinsp;17.087, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A significant effect of drug was found in males (F(1,34)\u0026thinsp;=\u0026thinsp;5.753, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not in females (F(1,34)\u0026thinsp;=\u0026thinsp;1.026, ns). Stress was not significant in males (F(1,34)\u0026thinsp;=\u0026thinsp;3.535, ns) or females (F(1,34)\u0026thinsp;=\u0026thinsp;0.052, ns), suggesting that CBD treatment downregulated miR-9-5p in UCMS rats.\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), univariate ANOVA revealed a significant effect of sex, stress and sex\u0026times;stress interaction, but not of drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,34)\u0026thinsp;=\u0026thinsp;24.911, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and stress\u0026times;drug interaction (F(1,34)\u0026thinsp;=\u0026thinsp;5.598, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in females, but not in males (stress: F(1,28)\u0026thinsp;=\u0026thinsp;0.016, ns; stress \u0026times;drug: F(1,28)\u0026thinsp;=\u0026thinsp;0.091, ns), suggesting that in females UCMS downregulated miR-9-5p. Drug was not significant in males (F(1,28)\u0026thinsp;=\u0026thinsp;1.137, ns) or females (F(1,34)\u0026thinsp;=\u0026thinsp;1.045, ns).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e1.4.2: miR-98-5p:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex, stress and the following interactions: stress\u0026times;drug and sex\u0026times;stress\u0026times;drug but not of drug and any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,33)\u0026thinsp;=\u0026thinsp;6.154, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and stress\u0026times;drug interaction (F(1,33)\u0026thinsp;=\u0026thinsp;24.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in females but not in males (stress: F(1,31)\u0026thinsp;=\u0026thinsp;0.480, ns; stress\u0026times;drug interaction: F(1,31)\u0026thinsp;=\u0026thinsp;0.194, ns), suggesting CBD downregulated miR-98-5p in non-stressed females. Drug was not significant in both males (F(1,31)\u0026thinsp;=\u0026thinsp;0.122, ns) and females (F(1,33)\u0026thinsp;=\u0026thinsp;1.305, ns).\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), univariate ANOVA revealed a significant effect of sex\u0026times;stress\u0026times;drug interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress\u0026times;drug interaction in females (F(1,34)\u0026thinsp;=\u0026thinsp;6.102, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) but not in males (F(1,33)\u0026thinsp;=\u0026thinsp;0.096, ns). Stress and drug were not significant in males (stress: F(1,33)\u0026thinsp;=\u0026thinsp;0.281, ns; drug: F(1,33)\u0026thinsp;=\u0026thinsp;0.064, ns) or females (stress: F(1,34)\u0026thinsp;=\u0026thinsp;1.203, ns; drug: F(1,34)\u0026thinsp;=\u0026thinsp;0.344, ns),\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), univariate ANOVA revealed a significant effect of sex, stress and the following interactions: sex\u0026times;drug and sex\u0026times;stress\u0026times;drug, but not of drug and the other interactions. Two-way ANOVA revealed a significant effect of drug in males (F(1,28)\u0026thinsp;=\u0026thinsp;5.955, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not in females (F(1,34)\u0026thinsp;=\u0026thinsp;2.378, ns), and a significant effect of stress in females (F(1,34)\u0026thinsp;=\u0026thinsp;17.302, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in males (F(1,28)\u0026thinsp;=\u0026thinsp;0.587, ns), suggesting that CBD led to upregulation of miR-98-5p in UCMS males, while in females UCMS led to downregulation of miR-98-5p. Stress\u0026times;drug interaction was not significant in males (F(1,28)\u0026thinsp;=\u0026thinsp;3.296 ns) or females (F(1,34)\u0026thinsp;=\u0026thinsp;1.097, ns).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e1.4.3: miR-146a-5p:\u003c/h2\u003e \u003cp\u003eIn the vmPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg), univariate ANOVA (2\u0026times;2\u0026times;2) revealed a significant effect of sex and sex\u0026times;stress\u0026times;drug interaction, but not of stress, drug, or any of the other interactions.\u003c/p\u003e \u003cp\u003eTwo-way ANOVA (2\u0026times;2) revealed a significant effect of stress\u0026times;drug interaction in females (F(1,33)\u0026thinsp;=\u0026thinsp;7.823, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but not in males (F(1,31)\u0026thinsp;=\u0026thinsp;0.064, ns). Stress and drug were not significant in males (stress: F(1,31)\u0026thinsp;=\u0026thinsp;0.160, ns; drug: F(1,31)\u0026thinsp;=\u0026thinsp;1.246, ns) or females (stress: F(1,33)\u0026thinsp;=\u0026thinsp;0.005, ns; drug: F(1,33)\u0026thinsp;=\u0026thinsp;2.812, ns).\u003c/p\u003e \u003cp\u003eIn the CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), univariate ANOVA revealed a significant effect of sex and sex\u0026times;stress interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,34)\u0026thinsp;=\u0026thinsp;13.529, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) in males but not in females (F(1,34)\u0026thinsp;=\u0026thinsp;0.465, ns), suggesting that in males UCMS led to upregulation of miR-146a-5p. Drug and stress\u0026times;drug interaction were not significant in males (drug: F(1,34)\u0026thinsp;=\u0026thinsp;0.286 ns; stress x drug: F(1,34)\u0026thinsp;=\u0026thinsp;0.017, ns) or females (drug: F(1,34)\u0026thinsp;=\u0026thinsp;0.259, ns; stress\u0026times;drug: F(1,34)\u0026thinsp;=\u0026thinsp;0.028, ns(.\u003c/p\u003e \u003cp\u003eIn the VS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei), univariate ANOVA revealed a significant effect of sex and sex\u0026times;stress interaction, but not of stress, drug, or any of the other interactions. Two-way ANOVA revealed a significant effect of stress (F(1,31)\u0026thinsp;=\u0026thinsp;9.293, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in females but not in males (F(1,28)\u0026thinsp;=\u0026thinsp;0.888, ns), suggesting that in females UCMS led to downregulation of miR-146a-5p. Drug and stress\u0026times;drug interaction was not significant in males (drug: F(1,28)\u0026thinsp;=\u0026thinsp;0.001, ns; stress\u0026times;drug: F(1,28)\u0026thinsp;=\u0026thinsp;0.527, ns) or females (drug: F(1,31)\u0026thinsp;=\u0026thinsp;0.048, ns; stress\u0026times;drug: F(1,31)\u0026thinsp;=\u0026thinsp;2.262, ns).\u003c/p\u003e \u003cp\u003eTo explore the association between depressive-like behavior and microRNA expression, Pearson bivariate correlation tests were conducted between the behavioral measurements and microRNA expression in the vmPFC, CA1, and VS in males (supplemental material, \u003cb\u003eTable S9\u003c/b\u003e) and females (\u003cb\u003eTable S10\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the sex-specific effects of chronic CBD treatment on depressive-like behaviors in male and female rats exposed to UCMS. We explored the potential involvement of neuroinflammatory genes, ECS-related genes, estrogen-associated genes, and miRNAs known to modulate depression and neuroinflammation. Our findings shed light on the differential impact of CBD treatment on depressive phenotypes across sexes and elucidate the underlying molecular mechanisms implicated in these effects. Specifically, we demonstrate that CBD prevents the impact of UCMS on cnr1, tnf, esr1, and esr2 in the hippocampus of males exposed to UCMS. However, these genes do not appear to play a role in the effects of CBD in females.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of CBD on the behavioral phenotype in UCMS rats\u003c/h2\u003e \u003cp\u003eWe found sex differences in the effects of UCMS on immobility in males and females, suggesting an opposite impact: UCMS increased passive coping in males but decreased it in females compared to a non-stressed control group. However, in both sexes, the administration of CBD restored this phenotype. Previous studies that examined the effects of UCMS on immobility in females showed contradictory results, some showing increased or decreased immobility, others showing no effect, depending on the stress protocol, the rodent type, etc. [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our study we saw lower baseline immobility measures in females compared to males, which were similar to those that were found in another study that showed lower immobility in female rats due to chronic stress [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This suggests that females are less prone to immobility behavior to begin with, implying that the FST might not be ideal to detect depressive phenotype in female rodents. Interestingly, it has been suggested that UCMS should be considered as the first stress session and FST as the second stress session and that females previously subjected to chronic mild stress cope better by exhibiting increased active behavior in the second FST in comparison with males [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This suggests that the behavioral paradigms assessing the stress response (for example, the combination of stressful procedures) may affect this sex-dependent outcome and should be considered in studying the pathophysiology of stress-related depression.\u003c/p\u003e \u003cp\u003eIn the OFT, exposure to UCMS similarly affected locomotor behavior in males and females, and UCMS-induced increase in locomotion was not restored with CBD treatment. We and others have previously shown increased locomotion following chronic stress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and an earlier study showed a similar effect of UCMS in female mice [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] [but see 46, 51]. The lack of effect of CBD on UCMS-induced hyperlocomotion aligns with studies showing similar results in different behavioral and genetic models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], suggesting that CBD does not lead to changes in locomotor behavior. In addition, locomotion in No UCMS rats was significantly higher in females compared to males, indicating that female rats exhibit higher locomotion behavior than males in a novel environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of CBD on neuroinflammation in UCMS rats\u003c/h2\u003e \u003cp\u003eSex-dependent differences in neuroinflammatory markers were also observed following exposure to UCMS and treatment with CBD. In males, CBD prevented upregulation of the TNF-α gene in the CA1 and VS, suggesting a role of neuroinflammatory genes in the therapeutic-like effects of CBD in males exposed to UCMS. Positive correlations were observed between immobility and the neuroinflammatory genes, suggesting that their upregulation is highly associated with increased passive coping. These findings corroborate with previous studies that showed a positive correlation between depressive symptoms and TNF-α and NF-κB1 expression, both in humans [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and animals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and specifically in the hippocampus [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. CBD\u0026rsquo;s anti-inflammatory effect (i.e., decreasing the expression of TNF-α and NF-κB1) is also in line with studies suggesting that its antidepressant properties may be mediated by changes in these inflammatory markers in the hippocampus [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR59 CR60 CR61 CR62 CR63 CR64 CR65 CR66 CR67 CR68 CR69 CR70\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn both males and females, UCMS upregulated the NF-κB1 gene in the VS, with no effect of CBD. This effect was positively correlated with hyperactivity in the OFT, suggesting that hippocampal NF-κB1 may be involved in activity, in line with previous findings of lower locomotion in the OFT in nfkb1-knockout mice [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of CBD on CB1 and CB2 genes in UCMS rats\u003c/h2\u003e \u003cp\u003eIn males, but not in females, UCMS induced downregulation of the CB1 gene in the VS that was prevented d by CBD. The effect of UCMS corroborates with a previous study that demonstrated the downregulation of CB1 expression in the ventral hippocampus of male rats \u0026ndash; but not females \u0026ndash; exposed to the chronic mild stress (CMS) model of depression [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Notably, following CMS, these rats demonstrated anhedonic behavior. An earlier study showed similar results regarding the downregulation of hippocampal CB1 following UCMS [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUCMS males and females demonstrated downregulation of the CB1 gene in the vmPFC, with no effect of CBD. This corroborates with findings from our previous study in UCMS males, showing downregulation of cnr1 in the vmPFC, with no effect of CBD [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Also, there was a negative correlation between this effect and the distance travelled in the OFT, suggesting that the downregulation is associated with increased locomotion. This is in line with previous findings regarding the involvement of CB1 in locomotion behavior [\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, in both sexes, CBD led to the downregulation of cnr2 in the vmPFC, regardless of stress. These findings were not correlated with behavioral measures, providing further evidence of the wide spectrum of the effects of CBD on ECS mechanisms, including those not directly interconnected with symptoms of depression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of CBD on estrogen genes in UCMS rats\u003c/h2\u003e \u003cp\u003eSex-dependent differences were also observed in the effects of CBD on genes coding for estrogenic receptors following UCMS. In males, CBD prevented UCMS-induced downregulation of VS ERα and ERβ genes. Negative correlations were observed between immobility in the FST and the estrogen genes, suggesting that their downregulation is highly associated with increased passive coping. It has been suggested that ERβ is involved in social and mood-related behaviors in males [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. In fact, it was previously shown that male mice lacking the ERβ gene spent more time immobile and a reduced time swimming and climbing in the FST [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], further establishing a possible role of hippocampal ERβ in learned helplessness in males.\u003c/p\u003e \u003cp\u003eIn UMCS-exposed rats of both sexes, an upregulation of CA1 esr1 and esr2 was observed, with the latter positively correlated with the total distance travelled in the OFT. This suggests that increased CA1 ERβ levels are associated with increased locomotion. It has been shown that male ERβ knockout (BERKO) mice demonstrated deficits in motor behavior compared to control mice, thereby establishing a connection to ERβ [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious findings in female rodents showed that ERα and ERβ receptors are important mediators of the antidepressant effects of 17β-Estradiol and other ligands [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In our study, UCMS did not lead to the downregulation of hippocampal esr1 in females. Possibly, the involvement of ERβ in mood-related behaviors in females is through signaling in the central amygdala, as ERβ blocking in this region improved sucrose intake (i.e., an antidepressant effect) in female rats exposed to chronic stress [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Moreover, the central amygdala ERβ is involved in other behaviors, such as sociosexual behaviors and anxiety [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of CBD on miRNAs in UCMS rats\u003c/h2\u003e \u003cp\u003eIn females, UCMS\u0026thinsp;+\u0026thinsp;CBD decreased CA1 miR-9-5p to control levels compared to the UCMS and CBD groups. This aligns with findings suggesting that MiR-9 is upregulated in the hippocampus of depressed mice and that silencing miR-9 in the hippocampus can improve depressive-like symptoms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Yet, a different effect was observed in the VS in which UCMS exposure decreased the expression of VS miR-9-5p compared to the No UCMS groups.\u003c/p\u003e \u003cp\u003eIn UCMS-exposed females, CBD downregulated miR-98-5p in the VS compared to the control non-stressed groups, and this effect was negatively correlated with locomotion in the OFT, suggesting that miR-98-5p downregulation is associated with increased activity in females. In UCMS-exposed males, CBD upregulated miR-98-5p in the VS compared to the UCMS group; this aligns with a recent study suggesting that depressive symptoms are associated with lower levels of miR-98 in the hippocampus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn UCMS-exposed females, CBD upregulated vmPFC miR-98-5p compared to the UCMS and CBD groups. This aligns with earlier studies showing male-female differences in miR-98 expression in different models [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn females, UCMS led to the downregulation of miR-146a-5p in the VS. In the vmPFC, CBD upregulated miR-146 in UCMS-exposed females compared to the UCMS group. These results are in line with several studies that showed that in both humans and animals, elevation of miR-146 is associated with worsening of depressive symptoms, and vice versa [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn males, UCMS upregulated miR-146a-5p in the CA1 compared to the control groups, with no restoring effect of CBD, and this upregulation was positively correlated with increased immobility in the FST.\u003c/p\u003e \u003cp\u003eOverall, our results suggest that CBD modulates the expression of specific microRNAs in a region- and sex-specific manner in response to chronic stress, potentially contributing to its antidepressant effect.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study set out to investigate molecular alterations in the brain which are associated with the therapeutic efficacy of CBD in male and female rats subjected to UCMS. Our findings reveal notable distinctions in behavioral responses between male and female rats under UCMS conditions, particularly in the FST. While CBD effectively mitigated UCMS-induced despair-like behavior in both sexes, it did not influence locomotor activity.\u003c/p\u003e \u003cp\u003eSex-specific variations emerged in the expression of neuroinflammatory markers and estrogen receptor genes. In males, CBD administration reversed UCMS-induced alterations in hippocampal CB1 expression, as well as inflammatory and estrogenic markers, suggesting the involvement of hippocampal cannabinoid, neuroinflammatory and estrogenic mechanisms in CBD's antidepressant-like effects. Conversely, CBD failed to reverse UCMS-induced changes in any markers or estrogenic receptors in females, indicating the potential engagement of alternative pathways.\u003c/p\u003e \u003cp\u003eIt is essential to recognize that male and female brains can respond in different manners to the same experimental manipulations. Notably, there are indications of male-female differences in endocannabinoid, serotonergic, inflammatory, and estrogenic markers and activity, all of which may influence depressive symptoms [\u003cspan additionalcitationids=\"CR87 CR88 CR89 CR90 CR91 CR92 CR93 CR94 CR95 CR96 CR97 CR98\" citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. The observed sex differences in stress response likely stem from a complex interplay of factors, including variations in serotonergic function, fluctuations in the estrous cycle, and activity within the hypothalamic-pituitary-adrenal (HPA) axis.\u003c/p\u003e \u003cp\u003eThese findings underscore the necessity of considering sex-specific neurobiological mechanisms in the development of therapeutic interventions targeting stress-related disorders. Further exploration of the intricate interactions between CBD, sex hormones, and stress-responsive pathways is essential for advancing our understanding and refining treatment strategies tailored to individual needs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to Sharon Zorin for her invaluable assistance in conducting the behavioral paradigms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, design and interpretation of data: U.B. and I.A.; formal analysis, investigation, visualization, writing—original draft preparation: U.B.; writing—review and editing, resources, supervision, funding acquisition, I.A. .\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Israel Science Foundation (ISF), grant number 993/20 to IA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have nothing to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eBeurel E, Toups M, Nemeroff CB. 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Neuroscience 2005;135:703\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFarooq RK, Isingrini E, Tanti A, Le Guisquet AM, Arlicot N, Minier F \u003cem\u003eet al\u003c/em\u003e. Is unpredictable chronic mild stress (UCMS) a reliable model to study depression-induced neuroinflammation?. Behavioural brain research 2012;231:130\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhu S, Wang J, Zhang Y, Li V, Kong J, He J \u003cem\u003eet al\u003c/em\u003e. Unpredictable chronic mild stress induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase in mice. Brain Research 2014;1576:81\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhao X, Cao F, Liu Q, Li X, Xu G, Liu G \u003cem\u003eet al\u003c/em\u003e. Behavioral, inflammatory and neurochemical disturbances in LPS and UCMS-induced mouse models of depression. 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Pro-inflammatory cytokines IL‐1\u0026alpha;, IL‐6 and TNF‐\u0026alpha; in major depressive disorder: Sex‐specific associations with psychological symptoms. European Journal of Neuroscience 2023.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBirur B, Amrock EM, Shelton RC, Li L. Sex differences in the peripheral immune system in patients with depression. Frontiers in psychiatry 2017;8:108.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eK\u0026uuml;hnemann S, Brown TJ, Hochberg RB, MacLusky NJ. Sex differences in the development of estrogen receptors in the rat brain. Hormones and behavior 1994;28:483\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDerry HM, Padin AC, Kuo JL, Hughes S, Kiecolt-Glaser JK. Sex differences in depression: does inflammation play a role?. Current psychiatry reports 2015;17:1\u0026ndash;0.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWilson ME, Westberry JM, Trout AL. 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Journal of Comparative Neurology 2014;522:358\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4270261/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4270261/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEvidence suggests a bidirectional relationship between depressive symptoms and neuroinflammation. We studied the effects of chronic treatment with cannabidiol (CBD) in male and female rats exposed to an unpredictable chronic mild stress (UCMS) model of depression. We analyzed gene expression related to neuroinflammation, cannabinoids, and estrogen receptors, as well as specific microRNAs (miRs) in the ventromedial prefrontal cortex (vmPFC), CA1, and ventral subiculum (VS).\u003c/p\u003e \u003cp\u003eWe found sex- and brain region-dependent effects of UCMS and CBD. UCMS exerted sex-specific effects on immobility, increasing it in males while decreasing it in females; CBD reversed this effect in both sexes. Regarding neuroinflammation, CBD restored Tumor Necrosis Factor α (TNF-α) gene upregulation in the CA1 and VS in males. In both sexes, UCMS led to nuclear factor kappa B subunit 1 (NF-κB1) gene upregulation in the VS, unaffected by CBD. In males, UCMS-induced CB1 gene downregulation in the VS was restored by CBD. UCMS resulted in CB1 gene downregulation in the vmPFC in both sexes, with no CBD effect. In males, CBD restored UCMS-induced downregulation of VS ERα and ERβ genes. Finally, UCMS downregulated miR-146a-5p in the VS in females and upregulated it in the CA1 in males without CBD restoring effects.\u003c/p\u003e \u003cp\u003eOur findings highlight sex-specific differences in learned helplessness and CBD\u0026rsquo;s modulation of neuroinflammatory, cannabinoid, and estrogen gene expression following exposure to chronic stress. Specifically, in males, hippocampal neuroinflammatory and estrogenic mechanisms play pivotal roles in mediating CBD's antidepressant effects.\u003c/p\u003e","manuscriptTitle":"Differential Learned Helplessness Phenotypes in Male and Female Rats: Exploring the Therapeutic Impact of Cannabidiol Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-23 14:54:06","doi":"10.21203/rs.3.rs-4270261/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67d045d3-5b67-4bed-b481-add90ea257a5","owner":[],"postedDate":"April 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30830625,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":30830626,"name":"Biological sciences/Drug discovery/Pharmacology/Pharmacogenetics"}],"tags":[],"updatedAt":"2024-07-12T11:05:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-23 14:54:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4270261","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4270261","identity":"rs-4270261","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}