Characterizing the diversity of enteric neurons using Dopamine Transporter (DAT)-Cre reporter mice

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic, although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture given previous work has showed that some DA neurons contain little if any TH and its levels tend to be decreased in response to cellular stress. The study herein provides a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, validated by <em>in situ</em>hybridization. We observed distinct subtypes of DAT neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, and a smaller population likely releasing nitric oxide; while others were positive for a slew of canonical DA markers. Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which were ChAT/DAT-positive neurons and characterised by the expression of <em>Th</em>, <em>Grp</em>,<em>Calcb</em> and<em> Sst</em>. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and discover any inherent vulnerabilities in disease.