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

Major depressive disorder (MDD) affects over 300 million people and is poorly controlled by current treatments. Adult hippocampal neurogenesis (AHN) in the dentate gyrus (DG) supports learning, memory, and emotional regulation and is disrupted by chronic stress, contributing to depression pathophysiology. Antidepressant efficacy depends partly on AHN and is associated with increased neurogenesis. The endocannabinoid system (ECS), including cannabinoid type 2 receptors (CB2Rs), and neurotrophic factors such as brain-derived neurotrophic factor (BDNF), both regulate AHN and stress responses. Physical exercise (PE) enhances BDNF and modulates ECS activity, improving cognition and mood while reducing neuroinflammation. Human and preclinical data suggest synergistic interactions between ECS signaling and BDNF during exercise. The authors hypothesised that modulating CB2R activity in combination with PE would restore stress-induced behavioural and neurogenic impairments. They tested whether CB2R inhibition or activation, combined with PE, could reverse emotional and cognitive deficits and AHN disturbances after chronic stress, and whether these effects relate to neuroinflammation and BDNF levels.

Prior work identifies the ECS as a key regulator of AHN and stress adaptation, with cannabinoids producing anxiolytic and antidepressant-like effects via hippocampal neural stem cell regulation. CB2Rs, lacking psychotropic effects, influence hippocampal plasticity and stress responses; activation can be antidepressant, and CB2R overexpression confers resilience, though chronic CB2R blockade has also shown anxiolytic-like effects. MDD patients show reduced circulating endocannabinoids, implying ECS hypoactivity. Physical exercise improves cognition, reduces anxiety-like behaviour, enhances AHN, and elevates neurotrophic factors, particularly BDNF, which regulates survival, dendritic growth, and maturation of SGZ-born neurons. Exercise also suppresses neuroinflammation, a hallmark of MDD. Human studies reveal that single sessions of moderate exercise increase endocannabinoids and BDNF, improving memory consolidation, suggesting synergy between these systems; correlations between ECS activation and mood improvements after exercise have been observed in MDD. Preclinical work shows CB1R dependence for voluntary exercise-induced progenitor proliferation and links cannabidiol’s anxiolytic/antidepressant effects to ECS and BDNF increases. CB2Rs modulate BDNF-mediated neurogenesis postnatally, and CB2R inhibition can dampen BDNF signaling in stressed animals, indicating a complex CB2R–BDNF interaction. However, how CB2Rs direct exercise’s benefits under chronic stress had not been clarified prior to this study.

Animals: Male C57BL/6J mice (14 weeks; 26–30 g; 9–10 per group) were housed under standard conditions. All procedures were approved by institutional and national authorities. Design: After habituation and handling, mice underwent an 8-week unpredictable chronic mild stress (uCMS) protocol, followed by a 2-week milder stress period during which CB2R ligands and/or physical exercise (PE) were applied. A prior dose–response (0.5 vs 5 mg/kg) for CB2R ligands was conducted; the lowest dose (0.5 mg/kg) was used in uCMS experiments. CB2R agonist: HU308; CB2R inverse agonist/antagonist: AM630; drugs administered for 2 weeks. PE protocol lasted 2 weeks (moderate treadmill exercise as previously described). Four weeks before sacrifice, animals received BrdU (50 mg/kg, i.p., twice daily for 5 consecutive days) to label newborn cell survival. Behaviour: After treatment, the following were assessed: open field (OF), elevated plus maze (EPM), novel object recognition (NOR), forced swim test (FST), and sucrose splash test (SST) using standard procedures. Histology and stereology: After perfusion/fixation, 40 μm coronal sections were immunostained to quantify: proliferation (Ki67+), proliferating neuroblasts (Ki67+DCX+), immature neurons (DCX+), and survival of adult-born neurons (BrdU+NeuN+), with analyses across dorsal (dHip) and ventral (vHip) DG, suprapyramidal/infrapyramidal blades, and SGZ/GCL subregions. Confocal imaging and stereological quantification were performed (8–10 sections/animal; 5 animals/group). Biochemistry: Serum corticosterone (CORT) and hippocampal BDNF were quantified by ELISA (CORT n=8–10/group; BDNF n=7–8/group). Molecular: DG gene expression (n=6/group) assessed by qPCR for inflammatory markers (e.g., TNFα, IL-1β, IL-10) and enzymes (arginase 1, iNOS), normalised to β-actin using ΔΔCt. Statistics: Normality (Shapiro–Wilk). Two-tailed t-tests, one-/two-way ANOVA with Dunnett’s correction when appropriate. Pearson correlations for normally distributed variables. PCA conducted on behavioural, cellular, and molecular readouts. Significance p<0.05. Data shown as mean±SEM.

- Dose selection: In naive mice, HU308 (0.5/5 mg/kg) did not alter behaviour but increased DCX+ and BrdU+DCX+ cells; AM630 (0.5/5 mg/kg) induced anxiety-/depressive-like behaviours and cognitive deficits without altering AHN counts. The 0.5 mg/kg dose was used for uCMS studies. - Behaviour under uCMS: uCMS reduced weight gain and increased nadir CORT. It induced anxiety-like behaviour (reduced EPM open-arm time), anhedonia-like behaviour (increased SST latency), impaired coping (increased FST immobility), and memory deficits (reduced NOR novel object preference). AM630 combined with PE reversed anxiety-like, anhedonia-like, and coping deficits to control-like levels and improved OF centre time; HU308 did not confer behavioural recovery under uCMS. NOR deficits were prevented by PE alone and by PE+AM630, but not by HU308±PE. - AHN: uCMS significantly reduced proliferation (Ki67+), proliferating neuroblasts (Ki67+DCX+), immature neurons (DCX+), and survival (BrdU+NeuN+)—particularly in vHip. AM630+PE restored all stages of AHN to control-like levels across total, dorsal, and ventral DG. PE alone partially rescued DCX+ pool and BrdU+NeuN+ survival but not proliferation. AM630 alone did not fully normalize AHN. - Subregional DG distributions: uCMS disrupted the normal supra/infrapyramidal and SGZ/GCL distributions of Ki67+, Ki67+DCX+, and BrdU+NeuN+ populations, including a shift from SGZ to GCL and from suprapyramidal to infrapyramidal blades, with a progenitor:immature DCX+ ratio shift (~25:75 in controls to ~34:66). AM630+PE normalized these distributions to control-like patterns; PE alone partially corrected several disturbances, especially in dHip SGZ. - Neuroinflammation and glial/myelin markers: uCMS increased Iba1 immunoreactivity (microgliosis) and decreased MBP in the DG granular layer; hilar GFAP (astrocytes) decreased, while granular GFAP was unchanged. AM630+PE reversed granular Iba1 increase, restored MBP reduction to control-like levels, and prevented hilar GFAP loss. HU308+PE reduced Iba1 and restored MBP and hilar GFAP toward control but overall HU308 regimens showed tendencies toward increased IL-1β/IL-10/iNOS expression. At the transcript level, AM630+PE reduced TNFα (further than PE alone), arginase 1, and iNOS vs uCMS. - BDNF and CORT: uCMS increased CORT and showed a negative correlation between CORT and hippocampal BDNF (Spearman p=0.0192). AM630+PE significantly increased hippocampal BDNF relative to uCMS (p≈0.0494), suggesting enhanced pro-survival signaling. - Multivariate integration: PCA of all readouts (features 1+2 explained 83.83% variance) showed AM630+PE-treated uCMS animals clustering with controls, unlike other groups. Correlation matrices indicated positive behaviour–neurogenesis associations in controls and in AM630+PE-treated uCMS mice, but negative or disrupted associations in uCMS, uCMS+PE, and uCMS+AM630 alone. Overall, CB2R inhibition combined with PE, but not CB2R activation, robustly prevents/reverses uCMS-induced emotional and cognitive deficits, restores AHN across stages and subregions, reduces neuroinflammatory signatures, and elevates hippocampal BDNF.

The study demonstrates that CB2R inhibition enables and amplifies the beneficial effects of physical exercise on behaviour and hippocampal plasticity under chronic stress. uCMS-induced anxiety-/depressive-like behaviours and cognitive deficits were accompanied by reductions and subregional misallocation of AHN populations, increased microgliosis, hypomyelination in the DG granular layer, and reduced hilar astrocytic expression. Combining AM630 (CB2R inverse agonist) with PE reversed these changes, restoring AHN proliferation, differentiation, and survival to control-like levels, normalising DG subregional distributions, reducing microglial activation and inflammatory gene expression, and restoring MBP and GFAP signatures. Increased hippocampal BDNF and its inverse relation to elevated CORT support a mechanism wherein CB2R inhibition plus exercise facilitates pro-survival and proneurogenic pathways that foster adaptive, resilient behaviour. The data suggest a state-dependent, dual role of CB2Rs: in physiological conditions, CB2R activation can be proneurogenic, whereas under pathological stress, reducing CB2R constitutive activity (via AM630) synergises with exercise to normalise neurogenesis and behaviour. Potential mechanisms include protean ligand behaviour of AM630 adjusting CB2R constitutive activity, ECS–BDNF crosstalk, and exercise-driven modulation of neuroimmune status. The multivariate clustering and restored positive behaviour–plasticity correlations further indicate system-level recovery with the combined treatment.

CB2R inhibition combined with physical exercise counteracts chronic stress-induced behavioural deficits and restores hippocampal plasticity. This multimodal approach normalises AHN across stages and subregions, reduces neuroinflammatory load, rescues myelin and astrocytic alterations, and elevates hippocampal BDNF, leading to behavioural recovery. The findings highlight CB2Rs as crucial mediators of exercise benefits in the stressed brain and support combining lifestyle interventions with targeted endocannabinoid modulation as a potential therapeutic strategy for stress-related disorders. Future work should: (1) test efficacy in females and across ages to enhance translational relevance; (2) examine other brain regions and circuit-level effects; (3) delineate cell type-specific and intracellular mechanisms underlying CB2R–exercise interactions (e.g., BDNF signaling pathways); and (4) optimise dosing/timing and evaluate long-term outcomes and safety.

- Sex limitation: Experiments were conducted only in male mice; applicability to females remains to be determined. - Mechanistic inference: Associations between behavioural recovery, AHN, neuroinflammation, and BDNF increases are correlative; causal pathways and cell type-specific mechanisms were not directly manipulated. - Pharmacology: Potential complexities of CB2R pharmacology (e.g., ligand selectivity, protean/inverse agonism, receptor constitutive activity) and dose/chronicity effects may influence generalisability to other contexts or compounds. - Scope: Focus was on the hippocampal DG; effects in other brain regions or systemic endpoints were not assessed. - Stress/exercise parameters: Specific uCMS and PE regimens may limit extrapolation to different stress intensities or exercise modalities.