Gut microbiota regulates diurnal oscillations in stress responsiveness

Many neuropsychiatric disorders are intricately linked to stress, and patients with these conditions often display alterations in their gut microbiota profile and in circadian regulation of hormonal and behavioral timekeeping processes. In this work we explore how the gut microbiota modulate diurnal oscillations in neuroendocrinal systems resulting in different stress responsiveness across the day, to shed light into how these gut microbes could be playing a role in neuropsychiatric disorders. Glucocorticoid hormones are key to regulating the organism’s response to the environment and display a robust rhythm with a distinct peak around the sleep-wake transition, which is one of the entraining cues from the brain to peripheral tissues. Stress-induced glucocorticoid secretion will act on the same tissues leading to changes in state to cope with any stressors. Although the relationship between gut microbiota and stress is well established, it is still largely unknown how the circadian component of glucocorticoid secretion plays a role in such modulation. In this study we aimed to explore the influence of the gut microbiota in the rhythmicity of the brain transcriptome and metabolome, the HPA-axis, and the downstream effects on the stress response and behavior. This study used germ-free and microbial depleted animals as models of microbial disruption. We first used RNA-sequencing of the master clock in the brain – the suprachiasmatic nucleus – as well as hippocampus, and amygdala to dissect the influence of the gut microbiota in the brain’s diurnal gene expression oscillations. Rhythmicity analysis was performed using an in-house developed R package. Our analysis revealed that gut microbes are key to maintain normal gene expression across the day in these regions and that the disrupted genes are associated with pathways related to circadian rhythm and the stress response. Next, we used a multi-omics approach, combining RNA-sequencing and metabolomics of the amygdala and hippocampus for a join pathway analysis, which indicated that glutamate metabolism is disrupted in animals with differential microbial status. Glutamate is the most abundant excitatory neurotransmitter and plays a key role in maintaining appropriate stress responsiveness. Then, to interrogate how the gut microbiota could be modulating these pathways related to the stress response in the brain, we assessed the daily oscillations of glucocorticoids in the microbially-disrupted animals and showed that corticosterone rhythmicity is disrupted by microbial status (Rhythmicity analysis, One-way ANOVA with Kruskal-Wallis post hoc test). Gene expression profiling throughout the day was performed by quantitative RT-PCR on the paraventricular nucleus of the hypothalamus, pituitary and adrenal glands and revealed alterations in clock gene expression and in genes that compose pathways important to appropriate HPA-axis function (Rhythmicity analysis, Unpaired t-test). Lastly, we demonstrated that these baseline alterations in microbially-depleted animals led to a disruption of the stress response and stress sensitive behavior in a time-of-day dependent manner (Two-way ANOVA with Kruskal-Wallis post hoc test). In summary these findings reveal that gut microbiota may be a crucial factor in maintaining appropriate stress responsiveness across the day, helping to elucidate mechanisms of microbial modulation of brain function and behavior.