The gut microbiome (GM) is increasingly recognized for its impact on brain function and behavior, particularly in the context of mood disorders like depression. The hippocampus (HPC), crucial for learning, memory, and stress response, shows altered plasticity in depression. The vagus nerve (VN), a bidirectional communication pathway between the gut and the brain, is a likely mediator of GM-brain interactions. Impairments in HPC neurogenesis are associated with depressive states, while neurogenic stimuli counteract these states. Chronic stress, a significant risk factor for depression, disrupts the GM and impairs HPC function. Prior research established a causal link between stress-related GM disturbances and depressive behaviors, mediated by tryptophan metabolism and serotonin bioavailability. The VN innervates brainstem nuclei vital for serotonin bioavailability, raising the question of whether the GM utilizes the VN to regulate neurotransmission pathways, influence HPC plasticity, and affect behavior. This study used the unpredictable chronic mild stress (UCMS) model to investigate whether stress-induced GM changes require an intact VN to impact HPC neurogenesis and depressive-like behavior.

Existing literature demonstrates the gut microbiome's influence on hippocampal plasticity and affective behaviors, although the precise mechanisms remain unclear. The vagus nerve's role in conveying neural messages related to peripheral changes to the brain is well-established. Studies show that certain gut bacterial strains' ability to induce anxiety-like behaviors and alter BDNF and GABA receptor expression depends on vagal afferents. Similarly, gut vagal sensory signaling influences HPC plasticity and neurogenesis. Impairments in HPC neurogenesis are linked to depression, while neurogenic stimuli like fluoxetine or exercise counteract depression. Chronic stress induces GM changes and HPC impairments in both mice and humans, partly due to deficits in adult HPC neurogenesis. Prior work showed that chronic stress-related GM disturbances cause depressive states and HPC neurogenesis deficits by hijacking tryptophan metabolism and serotonin bioavailability. Vagus nerve afferents innervate crucial brainstem nuclei regulating serotonin bioavailability, impacting hippocampal neuroplasticity. VN stimulation increases serotonergic input to HPC neurons and influences affective behaviors. These findings prompted the study's investigation of the VN's role in GM's regulation of brain neurotransmission, HPC neuroplasticity, and behavior.

Adult male C57BL/6j mice were used. The unpredictable chronic mild stress (UCMS) model was employed to induce depressive-like behaviors. Mice underwent UCMS for 8 weeks, followed by fecal sample collection. Healthy recipient mice received fecal microbiota transplants (FMT) from UCMS or control (CT) mice after antibiotic treatment. A group of recipient mice underwent subdiaphragmatic vagotomy (Vx) two weeks prior to FMT. Behavioral assessments included open field, elevated plus maze, light-dark box, sucrose preference, novelty suppressed feeding, tail suspension, and forced swim tests. Histological analyses assessed adult HPC neurogenesis (using immunofluorescence for DCX and Ki67), and molecular analyses measured gene and protein expression (RT-qPCR and Western blotting) of neurotransmitters (serotonin, dopamine, GABA, glutamate), neurogenic factors (BDNF, CREB, FOXO), and inflammatory markers (TNFα, IL-1β, IL-6, COX2, Cx3cr1, TGFβ). 16S rRNA sequencing profiled the GM composition. Statistical analyses included Mann-Whitney tests and two-way ANOVA with Bonferroni post-hoc tests. Sample sizes were determined using G*Power software.

FMT with UCMS-derived microbiota activated the VN (increased c-Fos immunoreactivity in the nucleus tractus solitarius), rapidly altering neurotransmitter pathways in the brainstem and HPC. This included decreased expression of tryptophan hydroxylase (Tph2) and decreased dopamine-related enzymes, alongside alterations in glutamate receptors. In the HPC, UCMS-FMT increased c-Fos, decreased DCX+ neurons (indicating reduced neurogenesis), and decreased neurogenic factors (BDNF, CREB, FOXO). UCMS-FMT recipient mice exhibited depressive-like behaviors in various behavioral tests (reduced sucrose preference, increased latency to eat in the novelty suppressed feeding test, increased immobility in tail suspension and forced swim tests). These depressive-like behaviors were associated with lasting reduction in adult HPC neurogenesis. Vagotomy (Vx) abolished the effects of UCMS-FMT on behavior and hippocampal neurogenesis. Vx also prevented the UCMS-FMT induced neuroinflammation in the HPC (changes in TNFα, IL-1β, IL-6, COX2, Cx3cr1, and TGFβ). 16S rRNA sequencing confirmed that the UCMS treatment altered the gut microbiome composition and that the FMT procedure successfully transferred these changes to recipient animals.

This study demonstrates that stress-induced GM changes activate the VN, leading to neurotransmitter imbalances, decreased hippocampal neurogenesis, and neuroinflammation, ultimately resulting in depressive-like behaviors. Critically, the VN's integrity is essential for these effects. The rapid activation of the VN by UCMS-FMT, accompanied by early alterations in serotonin and dopamine neurotransmission, suggests a direct role for the VN in communicating GM-derived signals to the brain. The findings support the idea that the VN relays gut-derived signals to impact HPC function and neurogenesis through established pathways. The observed neuroinflammation further strengthens the link between GM dysbiosis, VN signaling, and the pathophysiology of depression. The protective effect of Vx underscores the VN's crucial role in mediating the effects of GM alterations on the brain and behavior. These findings align with clinical studies showing the efficacy of vagus nerve stimulation (VNS) in treating treatment-resistant depression.

This study provides strong evidence for the VN's role as a crucial mediator of GM's influence on hippocampal plasticity and depressive-like behaviors. The results demonstrate that stress-induced alterations in the GM require an intact VN to elicit their effects on the brain. These findings highlight the GM-VN-brain axis as a potential therapeutic target for mood disorders. Future research should focus on identifying specific bacterial strains and molecular mediators involved in this pathway, further refining VNS therapies and investigating other potential therapeutic interventions that target the GM-VN-brain axis.

The study utilized subdiaphragmatic vagotomy, which is not a selective method for abolishing VN activity. More precise methods, such as virus-based techniques, could improve future investigations. The study focused on male mice, limiting the generalizability of the findings to females. While the study identified associations between GM changes, VN activity, and behavior, it does not fully elucidate the exact molecular mechanisms involved. Further studies with more detailed mechanistic exploration are warranted.