Expression analysis of colonic neurons stimulated with AhR agonist (3-methylcholanthrene)

Neural control of visceral organ function is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence and is often dysregulated in gastrointestinal (GI) disorders. Luminal factors, such as diet and microbiota regulate neurogenic programs of gut motility, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor Aryl hydrocarbon Receptor (AhR) functions as a biosensor in intestinal neural circuits linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons representing distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles controlled by the combined effects of host genetic programmes and microbial colonisation. Microbiota-induced expression of AhR in neurons of the distal gastrointestinal tract enables them to respond to the luminal environment and induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in enteric neurons of antibiotic-treated mice partially restores intestinal motility. Taken together, our experiments identify AhR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits towards gut homeostasis and health. The enteric nervous system (ENS) encompasses the intrinsic neural networks of the gastrointestinal (GI) tract, which regulate most aspects of intestinal physiology, including peristalsis. In addition to host-specific genetic programmes, microbiota and diet have emerged as critical regulators of gut tissue physiology and changes in the microbial composition of the lumen often accompany GI disorders. We found that gut environmental sensor Aryl hydrocarbon receptor (AhR) is induced in colonic neurons in response to microbiota colonisation and regulates intestinal peristalsis in an AhR ligand-dependent manner. In this experiment, we used RNA sequencing to identify genes regulated in mouse colonic neurons by AhR activation. We developed an adeno-associated virus (AAV)-based strategy for isolation and RNA sequencing of enteric neuron nuclei (nRNAseq) representing different intestinal segments and microbiota states. For isolation of neuronal nuclei from the myenteric plexus, mice were injected intravenously with AAV9-CaMKII-EGFPKASH. 5 weeks after intravenous injection of AAV, the longitudinal smooth muscle layer and associated myenteric plexus were peeled off the wall of the smal intestine (SI) and colon and subjected to Dounce homogenization. After filtering the homogenate to remove large debris, samples were centrifuged at 1000 x G for 10 min at 4°C to obtain a pellet containing muscularis externa nuclei. For flow cytometric analysis, doublet discrimination gating was applied to exclude aggregated nuclei, and intact nuclei were determined by subsequent gating on the area and height of DAPI intensity. Both EGFP+ (neuronal) and EGFP- (non-neuronal) nuclear populations were collected directly into 1.5mL tube containing Trizol LS reagent.