Butyrate Triggers Neuronal Signaling via Release of Colonic Serotonin in a Microfluidic Model of Gut-Brain Axis

Host-microbiota interactions play a central role in regulating gastrointestinal and cardiometabolic physiology, yet the mechanistic pathways linking microbial metabolites to sensory neuronal activation remain poorly defined. In particular, the rapid signaling events through which short-chain fatty acids such as butyrate influence epithelial and neuronal function in the colon have not been fully resolved due to limitations of existing models. Here, we developed and validated a compartmentalized microfluidic tri-channel model that spatially organizes primary colonic epithelial cells, sensory neurons, and microbial inputs to enable real-time, directional analysis of microbial-epithelial-neuronal communication to mimic the in vivo environment of the gastrointestinal tract. Using computational modeling and real-time imaging, we demonstrate a timeline in which butyrate elicits dose-dependent calcium (Ca 2+ ) influx in the sensory neurons, mediated through a multi-step signaling cascade: butyrate stimulation of colonic epithelial cells, via epithelial GPR41 and GPR43 receptors, triggering a rapid serotonin (5-HT) release that subsequently engages 5-HT3a receptors on sensory neurons to induce Ca 2+ influx, indicative of neuronal activation. Pharmacological inhibition of epithelial GPR41 and GPR43 receptors, or neuronal 5-HT3a receptors abolished these responses, demonstrating the requirement for both (epithelial and neuronal) components in the signaling pathway. Epithelial cells were essential intermediaries, as neurons cultured without them failed to respond to butyrate. Moreover, we establish the feasibility of a live microbiota–epithelial–neuronal tri‑culture model that can be used for future mechanistic dissection of microbiota–gut–brain signaling.