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

Depression is a leading cause of disability worldwide, with a complex and poorly understood pathophysiology. While several mechanisms have been implicated, including HPA axis dysregulation, inflammation, and alterations in neuromodulatory systems like the eCB system, recent research points to a potential role for the gut microbiota. Studies have revealed an association between mood disorders and dysbiosis, or an imbalance in the composition of the gut microbiota. Animal model studies demonstrate the microbiota's influence on anxiety and neurological diseases, and alterations in patient microbiota have been shown to alter behavior in recipient animals. However, the underlying molecular mechanisms connecting the gut microbiota and mood disorders remain largely elusive, prompting the need for further investigation using controlled experimental models. This study aims to elucidate a causal role of the gut microbiota in stress-induced depressive behaviors by using the well-established UCMS mouse model and FMT techniques.

The literature extensively documents the link between chronic stress and depression, often using the UCMS model to induce depressive-like behaviors in rodents. These models frequently exhibit hippocampal alterations, particularly reductions in adult hippocampal neurogenesis, a key feature associated with depression. Antidepressant treatments have been shown to reverse these neurogenic deficits. Separately, the influence of the gut microbiota on various host functions, including immunity, metabolism, and the central nervous system, has been increasingly acknowledged. Studies have noted dysbiosis in depressed patients and demonstrated that microbiota alterations can influence mood and behavior in animal models. However, the precise molecular mechanisms underlying the microbiota-depression connection remained unclear, providing the rationale for this study's focus on the eCB system as a potential mediator.

The study employed adult male C57BL/6J mice, including germ-free and SPF groups. Mice were subjected to an 8-week UCMS protocol to induce depressive-like states, characterized by various stressors. Control groups were similarly handled but without stress exposure. FMT was performed by transferring fecal microbiota from UCMS donors to naive germ-free or antibiotic-treated SPF recipients. Behavioral tests, including the novelty suppressed feeding test, light/dark box test, splash test, tail suspension test, and forced swim test, were conducted to assess depressive-like behaviors. Immunohistochemistry was used to evaluate adult hippocampal neurogenesis via Ki67 and DCX staining, and EdU labeling was employed for studying neuronal survival. Metabolomic profiling of serum and hippocampus was done to identify metabolic alterations. To manipulate eCB signaling, recipients were treated with the MAGL inhibitor JZL184, with or without CB1 antagonists rimonabant or AM6545. Dietary supplementation with arachidonic acid (AA), a precursor of eCBs, was also performed. Lastly, 16S rDNA sequencing analyzed the fecal microbiota composition to determine bacterial families, alpha diversity, and beta diversity. Statistical analyses used Mann-Whitney tests for comparing two groups and one-way ANOVA with Tukey's post-hoc test for multiple groups.

UCMS mice exhibited increased immobility in the tail suspension and forced swim tests, reflecting depressive-like behaviors. They also showed reduced hippocampal neurogenesis, indicated by decreased Ki67+ and DCX+ cell densities. FMT from UCMS mice successfully transferred these depressive behaviors and neurogenesis deficits to naive recipients. Metabolomic analysis revealed decreased levels of monoacylglycerols (MAGs), diacylglycerols (DAGs), and n-6 polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA) and its precursors, in both UCMS donors and recipients. Hippocampal 2-arachidonoylglycerol (2-AG) levels were also significantly reduced. This was accompanied by decreased phosphorylation of mTOR, p70S6K, and rpS6, suggesting impaired mTOR signaling. Treatment with JZL184, a MAGL inhibitor, significantly increased hippocampal 2-AG levels, reversed depressive-like behaviors, and restored hippocampal neurogenesis. This effect was CB1 receptor-dependent and primarily mediated by central CB1 signaling. Dietary AA supplementation or *Lactobacillus plantarum* WIL (LpWIL) complementation also reversed depressive-like behaviors and restored neurogenesis, with both increasing hippocampal 2-AG and AEA levels. UCMS mice showed a gut microbiota dysbiosis characterized by a decrease in *Lactobacillaceae*, which was maintained in recipient mice. Therefore, the decreased neurogenesis, depressive-like behaviors, and altered lipid metabolism were linked to the decreased eCB signaling mediated by the gut microbiota changes.

This research provides substantial evidence for a causal link between gut microbiota dysbiosis, altered lipid metabolism, eCB signaling disruption, and depressive-like behaviors. The study successfully demonstrates that the gut microbiota from stressed mice can transfer depressive phenotypes to naive recipients, highlighting the critical role of the gut-brain axis in mood regulation. The observed reduction in hippocampal 2-AG and the subsequent impairment of mTOR signaling directly implicate the eCB system in the pathogenesis of microbiota-induced depression. The successful reversal of these effects by enhancing central eCB signaling (using JZL184) or by supplementing with AA or LpWIL strongly supports this mechanistic scenario. These findings align with existing clinical data suggesting altered serum 2-AG levels in patients with depression and highlight the potential therapeutic value of targeting the eCB system or gut microbiota composition.

This study offers compelling evidence for a novel mechanistic pathway connecting gut microbiota dysbiosis, impaired eCB signaling, and stress-induced depressive-like behaviors. The transferability of depressive phenotypes via FMT underscores the significance of gut microbiota modulation in mood disorders. The successful restoration of normal behavior and neurogenesis through interventions targeting the eCB system or gut microbiota suggests promising therapeutic avenues for stress-associated depressive syndromes. Future research could explore the specific bacterial species and metabolites involved, and the precise mechanisms underlying the interaction between microbiota, lipid metabolism, and the eCB system.

The study primarily utilized a mouse model, which may not perfectly translate to human conditions. The study focused on male mice, potentially limiting the generalizability to female populations. While the study suggests a strong correlation between gut microbiota composition and depressive-like behaviors, the precise mechanisms linking these factors remain partially elucidated, requiring further investigation. While multiple behavioral tests were used, the inherent limitations of animal models of human depression must be acknowledged. The effects of AA and *L. plantarum* were not completely identical in all behavioral tests, requiring further research to fully understand the underlying mechanisms and potentially explore more detailed biomarkers of disease state and treatments efficacy.