Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system

The study addresses how gut microbiota dysbiosis induced by chronic stress contributes to depressive-like behaviors and hippocampal dysfunction. Depression has heterogeneous pathophysiology involving HPA axis dysregulation, inflammation, reduced neuroplasticity, circuit dysfunctions, and neuromodulatory system perturbations, including endocannabinoid (eCB) signaling. Hippocampal alterations, such as reduced volume and impaired adult neurogenesis, are implicated in depression and can be modulated by antidepressant treatments. Emerging evidence links intestinal microbiota composition to brain function and mood disorders; dysbiosis has been observed in depressed patients and can influence anxiety, neuroinflammation, HPA axis activity, and neurogenesis. The authors hypothesize that stress-induced microbiota changes causally drive depressive-like behaviors via altered host lipid metabolism and impaired central eCB signaling, particularly in the hippocampus, and test whether restoring eCB tone or specific microbial taxa can rescue these phenotypes.

Prior clinical and preclinical studies indicate: (1) hippocampal volume loss and reduced adult neurogenesis are associated with depression; (2) gut microbiota modulates host immunity, metabolism, and CNS function, with dysbiosis reported in depressed patients; (3) microbiota from depressed patients can transfer behavioral alterations to rodents; (4) chronic stress models show microbiota changes, low-grade inflammation, abnormal HPA axis activity, and reduced neurogenesis; (5) probiotics, including Lactobacilli, can positively influence mood and stress responses in animal models and humans; (6) eCB system and CB1 receptor activity regulate mood and neurogenesis, with decreased eCB signaling reported under chronic stress and in depressed patients. These findings set the context for exploring a gut microbiota–lipid metabolism–eCB axis in depression.

- Animals: Adult male C57BL/6J mice under SPF conditions; germ-free C57BL/6J mice for colonization experiments. Ethical approvals obtained. - Depression model: Unpredictable chronic mild stress (UCMS) for 8 weeks with diverse stressors. - Fecal microbiota transplantation (FMT): Donor feces from control or UCMS mice transferred via oral gavage to germ-free or short-term antibiotic-treated SPF recipients; recipients housed in sterile isolators for 8 weeks before testing. - Behavioral assays: Novelty suppressed feeding, splash test, tail suspension test (TST), forced swim test (FST), and light/dark box (LDB) to assess depressive-like and anxiety-like behaviors. - Neurogenesis assays: Immunohistochemistry in dentate gyrus for Ki67 (proliferation) and DCX (immature neurons); EdU incorporation to assess survival of newborn neurons; analyses performed blinded. - Metabolomics: Serum untargeted metabolomics (GC/MS, LC/MS, LC/MS/MS) to profile lipids, including monoacylglycerols (MAG), diacylglycerols (DAG), and polyunsaturated fatty acids (PUFAs); targeted LC-MS/MS quantification of hippocampal 2-AG and AEA; total hippocampal PUFA profiling by SFC-MS. - eCB signaling readouts: Western blot for hippocampal mTOR pathway activation (p-mTOR S2448, p-p70S6K T389, p-rpS6 S235/236). - Pharmacology: MAGL inhibitor JZL184 (8 mg/kg i.p., every 2 days) to elevate 2-AG; CB1 antagonists rimonabant (central) and AM6545 (peripheral-restricted) to dissect receptor involvement. - Dietary and probiotic interventions: Oral arachidonic acid (AA; 8 mg/mouse/day) and Lactobacillus plantarum (LpWJL/LpWIL; 2×10^8 CFU, 5 days/week) supplementation in UCMS microbiota-recipient mice. - Microbiota profiling: 16S rDNA sequencing of fecal samples; OTU clustering and taxonomic assignment; analyses of alpha/beta diversity and differential abundance (e.g., Lactobacillaceae levels). - Additional measures: Gut permeability (FITC-dextran), immune cell profiling in gut, baseline corticosterone, and kynurenine levels. - Statistics: Mann–Whitney tests for two-group comparisons; one-way ANOVA with Tukey’s for multi-group; correlations (Pearson). Analyses blinded where applicable.

- UCMS induces depressive-like but not anxiety-like behaviors in C57BL/6J mice: increased latency to feed (novelty suppressed feeding), increased grooming latency and decreased grooming (splash test), and increased immobility in TST and FST; no change in LDB anxiety measures. Reduced hippocampal neurogenesis (lower Ki67+ and DCX+ cell densities). - FMT transfers phenotype: Germ-free and antibiotic-treated recipient mice colonized with UCMS microbiota display depressive-like behaviors (NSF, splash test, TST, FST) without increased anxiety, and show reduced hippocampal neurogenesis (Ki67, DCX). - Lipid metabolism alterations: Serum MAG and DAG levels, including AA-containing species, are decreased in UCMS donors and UCMS microbiota recipients. n-6 PUFAs (linoleic acid 18:2n-6, intermediates, and AA 20:4n-6) are significantly reduced. Hippocampal PUFA levels trend lower in recipients. No changes in gut permeability or major gut immune populations; kynurenine and baseline corticosterone unchanged. - eCB deficits: Hippocampal 2-AG is significantly decreased in UCMS donors and recipients; AEA not significantly changed in recipients. Serum 1-AG inversely correlates with depressive severity (strong inverse correlation; r^2 ≈ 0.87). Hippocampal eCB downstream signaling is reduced, with decreased phosphorylation of mTOR, p70S6K, and rpS6. - Restoring eCB signaling rescues phenotype: JZL184 elevates hippocampal 2-AG, restores mTOR pathway phosphorylation, and reverses depressive-like behaviors in UCMS microbiota recipients; effects are blocked by central CB1 antagonist rimonabant but not by peripherally restricted CB1 antagonist AM6545, indicating central CB1 dependence. JZL184 also rescues proliferation, differentiation, and survival of newborn hippocampal neurons (Ki67, DCX, EdU). - Dietary precursor rescues: AA supplementation restores hippocampal 2-AG, improves depressive-like behaviors (TST, FST), and partially restores hippocampal neurogenesis; associated with increased hippocampal PUFAs and AEA. - Microbiota changes: UCMS alters fecal microbiota composition with decreased Lactobacillaceae and increases in Ruminococcaceae and Porphyromonadaceae; alpha diversity unchanged. These compositional differences are maintained after FMT. - Probiotic complementation rescues: L. plantarum supplementation in UCMS microbiota recipients restores hippocampal 2-AG, improves depressive-like behaviors (TST, FST), and partially rescues neurogenesis (Ki67, EdU); associated with increased hippocampal PUFAs and AEA.

The study demonstrates a causal role for gut microbiota in transmitting depressive-like behaviors and impaired hippocampal neurogenesis from chronically stressed mice to naïve hosts. Mechanistically, UCMS-associated dysbiosis reduces systemic availability of fatty acid precursors (n-6 PUFAs and AA-containing MAG/DAG), leading to decreased hippocampal 2-AG and diminished central CB1-mTOR signaling. Enhancing brain eCB tone via MAGL inhibition or replenishing AA restores eCB signaling, neurogenesis, and behavior, highlighting the eCB system as a key mediator linking microbiota-derived metabolic changes to mood-related brain function. The identification of decreased Lactobacillaceae as a hallmark of UCMS microbiota, and the ability of L. plantarum supplementation to normalize eCB levels, neurogenesis, and behavior, positions specific probiotics as potential modulators of host lipid metabolism and eCB bioavailability. These findings integrate gut microbiota composition, lipid metabolic pathways, and central eCB signaling into a coherent gut–brain axis contributing to depressive phenotypes, with therapeutic implications for dietary and probiotic strategies targeting eCB pathways.

Chronic stress-induced gut dysbiosis reduces systemic PUFAs and eCB precursors, lowering hippocampal 2-AG and CB1-mTOR signaling to impair adult neurogenesis and promote depressive-like behaviors. FMT establishes causality, and interventions that restore eCB tone—either by inhibiting 2-AG degradation (JZL184), supplementing the precursor AA, or adding L. plantarum—rescue neurogenesis and behavior. The work uncovers a microbiota–lipid metabolism–eCB pathway driving mood-related brain dysfunction and supports dietary/probiotic approaches as adjunctive therapies for stress-associated depression. Future research should delineate the precise bacterial taxa and metabolites responsible, define mechanisms of altered fatty acid absorption/synthesis, assess other brain regions and circuits affected, and translate biomarkers (e.g., serum MAG/DAG/PUFAs, 2-AG) and interventions to human studies.

- Mouse model and male-only C57BL/6J mice limit generalizability to females and humans. - FMT recipients were antibiotic-treated or germ-free mice, which may have inherent differences in physiology and behavior. - Focus on hippocampus; other brain regions involved in mood regulation were not assessed. - 16S rDNA profiling limits resolution to family/genus; specific causal bacterial strains beyond L. plantarum were not identified. - Mechanisms by which dysbiosis alters host fatty acid absorption/synthesis remain unresolved. - Probiotic strain and AA dosing regimens require optimization and safety evaluation for clinical translation. - Behavioral tests assess depressive-like phenotypes but do not capture the full spectrum of depression.