Cannabinoid type 2 receptor inhibition enhances the antidepressant and proneurogenic effects of physical exercise after chronic stress

Major depressive disorder (MDD), affecting over 300 million worldwide, is often triggered by chronic stress. Current treatments are frequently ineffective, necessitating new therapeutic avenues. Adult hippocampal neurogenesis (AHN), the generation of new neurons in the adult hippocampus, is a promising target. AHN is crucial for learning, memory, and emotional regulation, and is negatively impacted by stress, with reduced AHN linked to MDD pathophysiology. Human and preclinical studies show a correlation between AHN suppression, stress response, and depressive-like behavior, while AHN enhancement improves these phenotypes. Antidepressants' efficacy also depends on AHN, indicating a complex interplay. Furthermore, chronic stress and antidepressant treatment differentially affect endocannabinoid and neurotrophic factor expression, key regulators of AHN. The endocannabinoid system (ECS), particularly CB2 receptors (devoid of psychotropic effects), is implicated in stress response and AHN regulation. CB2R activation shows antidepressant effects, while CB2R deficiency exacerbates stress-induced neuroinflammation. Conversely, chronic CB2R inhibition displays anxiolytic effects. Physical exercise (PE) also improves cognitive function, exerts anxiolytic effects, regulates hippocampal function, and enhances AHN, partly via neurotrophic factors like brain-derived neurotrophic factor (BDNF). Emerging evidence suggests an interaction between PE and the ECS in regulating AHN, with cannabinoids and neurotrophic factors modulating cognitive plasticity and mood. This study investigated whether modulating CB2R activity, in combination with PE, could reverse the neurobiological and behavioral consequences of chronic stress.

The literature extensively documents the detrimental effects of chronic stress on adult hippocampal neurogenesis (AHN) and its association with depression. Studies consistently demonstrate that chronic stress reduces hippocampal volume, dendritic arborization, and the generation of new neurons. Conversely, resilience to stress is associated with larger hippocampal volumes and increased neurogenesis. Preclinical models have confirmed that AHN suppression contributes to vulnerability to stress and depressive-like behavior, whereas interventions that enhance AHN mitigate these negative effects. The endocannabinoid system (ECS), particularly CB2 receptors, has emerged as a significant modulator of AHN and stress response. Research indicates that CB2 receptor activation can exert antidepressant effects, while its inhibition shows anxiolytic-like effects. The role of physical exercise (PE) in enhancing AHN, improving cognitive function, and reducing depressive-like symptoms has been well-established. PE's effects are partly mediated by neurotrophic factors, particularly BDNF, which plays a crucial role in the survival, growth, and maturation of newly born neurons. The interaction between PE and the ECS in regulating AHN is a relatively new area of investigation, with emerging evidence suggesting a synergistic relationship between cannabinoids, neurotrophic factors, and the beneficial effects of exercise on mood and cognitive function.

Male C57BL/6J mice were randomly assigned to experimental groups after habituation and handling periods. A dose-response study was conducted using the CB2R agonist HU308 and the inverse agonist/antagonist AM630 (0.5 and 5 mg/kg) for two weeks. Unpredictable chronic mild stress (uCMS) was induced for eight weeks, followed by a two-week milder stress protocol during which CB2R ligands were administered with a PE protocol. BrdU (50 mg/kg) injections were given for five consecutive days four weeks before sacrifice to assess newborn neuron survival. Behavioral assessments included open field (OF), elevated plus maze (EPM), novel object recognition (NOR), forced swimming test (FST), and sucrose splash test (SST). After sacrifice, immunostaining was performed to evaluate cell proliferation (Ki67), neuronal differentiation (DCX), and survival of newborn neurons (BrdU/NeuN). Glial cell density (Iba1 for microglia, GFAP for astrocytes, MBP for myelin) was also assessed. Serum corticosterone and hippocampal BDNF levels were quantified using ELISA kits. Gene expression analysis of DG tissue was conducted using qRT-PCR to measure TNFa, IL-1β, IL-10, arginase, and iNOS. Statistical analyses included two-tailed unpaired and paired Student's t-tests, one-way and two-way ANOVA, and Pearson correlation.

CB2R agonist HU308 administration showed no significant effects on behavior or neurogenesis in physiological conditions. AM630 at lower doses induced anxiety- and depressive-like behaviors and impaired cognition, but did not significantly affect AHN. PE alone showed trends towards increased exploratory behavior in naïve mice. uCMS exposure negatively impacted weight gain, increased corticosterone levels, and induced anxiety-like, anhedonic-like behaviors, impaired coping, and cognitive deficits. The combination of AM630 (0.5 mg/kg) and PE significantly reversed uCMS-induced behavioral deficits, while HU308 + PE did not. uCMS reduced Ki67+ cells, Ki67+/DCX+ cells, and BrdU+/NeuN+ cells, effects counteracted by AM630 + PE, especially in the ventral hippocampus. AM630 + PE restored the subregional distribution of these cell populations disrupted by uCMS. uCMS increased Iba1 immunoreactivity (microgliosis) and decreased MBP immunoreactivity (myelination) in the DG granular layer, effects reversed by AM630 + PE. uCMS reduced GFAP immunoreactivity (astrocytes) in the DG hilus, also counteracted by AM630 + PE. While uCMS did not significantly affect pro-inflammatory cytokines or neuroinflammatory enzymes, AM630 + PE treatment reduced TNFa and neuroinflammatory enzymes. AM630 + PE increased hippocampal BDNF levels, correlating with improved behavior and neurogenesis. Principal component analysis showed clustering of AM630 + PE treated uCMS mice with the control group.

This study reveals a crucial role for CB2 receptors in mediating the beneficial effects of physical exercise in counteracting chronic stress. CB2R inhibition, but not activation, is essential for the antidepressant, anxiolytic, and pro-cognitive actions of PE. The contrasting effects of CB2R modulation in physiological versus pathological conditions suggest a dual role, possibly related to receptor constitutive activity and regional/cellular localization. The findings expand on previous research demonstrating the interaction between PE, the ECS, and neurotrophic factors in regulating AHN and mood. The study demonstrates that the combination of CB2R inhibition and PE is more effective than either intervention alone in reversing stress-induced behavioral and neurobiological deficits. The observed improvements in AHN, reduced neuroinflammation, and increased BDNF levels contribute to the behavioral recovery. The subregional analysis of AHN highlights the impact of chronic stress on distinct hippocampal areas and the ability of the combined treatment to restore the regional balance of neurogenic cell populations. The finding that AM630 may act as a protean ligand is discussed, suggesting it might regulate CB2 receptor constitutive activity and modulate BDNF signaling.

This study demonstrates that combining CB2R inhibition with physical exercise is a promising therapeutic strategy for mitigating the effects of chronic stress. This multimodal approach effectively reverses behavioral deficits, enhances adult hippocampal neurogenesis, and reduces neuroinflammation. The results highlight the importance of understanding the intricate interplay between the ECS, neurotrophic factors, and lifestyle interventions in treating stress-related disorders. Future research should explore the underlying mechanisms, investigate the effects in other brain regions, and examine the generalizability of the findings to other populations.

The study used only male mice, limiting the generalizability to females. The correlation between CB2R modulation, PE, and the observed effects does not establish direct causation. The study focuses primarily on the hippocampus, and further investigation is needed to explore the effects in other brain regions. The mechanism through which CB2R inhibition enhances PE's effects needs further investigation. Finally, the long-term effects of the combined treatment require additional study.