A gut-derived metabolite alters brain activity and anxiety behaviour in mice.

Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.

Three-dimensional imaging for the quantification of spatial patterns in microbiota of the intestinal mucosa.

Improving our understanding of host­microbe relationships in the gut requires the ability to both visualize and quantify the spatial organization of microbial communities in their native orientation with the host tissue. We developed a systematic procedure to quantify the three-dimensional (3D) spatial structure of the native mucosal microbiota in any part of the intestines with taxonomic and high spatial resolution. We performed a 3D biogeographical analysis of the microbiota of mouse cecal crypts at different stages of antibiotic exposure. By tracking eubacteria and four dominant bacterial taxa, we found that the colonization of crypts by native bacteria is a dynamic and spatially organized process. Ciprofloxacin treatment drastically reduced bacterial loads and eliminated Muribaculaceae (or all Bacteroidetes entirely) even 10 d after recovery when overall bacterial loads returned to preantibiotic levels. Our 3D quantitative imaging approach revealed that the bacterial colonization of crypts is organized in a spatial pattern that consists of clusters of adjacent colonized crypts that are surrounded by unoccupied crypts, and that this spatial pattern is resistant to the elimination of Muribaculaceae or of all Bacteroidetes by ciprofloxacin. Our approach also revealed that the composition of cecal crypt communities is diverse and that Lactobacilli were found closer to the lumen than Bacteroidetes, Ruminococcaceae, and Lachnospiraceae, regardless of antibiotic exposure. Finally, we found that crypts communities with similar taxonomic composition were physically closer to each other than communities that were taxonomically different.

Peripheral neuronal activation shapes the microbiome and alters gut physiology.

The gastrointestinal (GI) tract is innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Here, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic contractility and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally activated neurons differentially regulate the gut microbiome and GI physiology in mice without involvement of signals from the brain.

Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems.

Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.

Emerging evidence linking the gut microbiome to neurologic disorders.

The gut microbiome contributes to the development and function of the immune, metabolic, and nervous systems. Furthermore, commensal bacteria modulate symptoms and pathology in mouse models of neuropsychiatric and neurodevelopmental diseases. Uncovering mechanisms that are utilized by the microbiome to mediate gut-brain connections may provide novel opportunities to target therapies to the gut in order to treat neurologic disorders.