Characterizing enteric neurons in Dopamine Transporter (DAT)-Cre reporter mice reveals dopaminergic subtypes with dual-transmitter content

Abstract

AbstractThe 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. This study herein provides a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein (tdTomato) 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, as well as validated usingin situhybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DA markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which were ChAT/DAT-tdTomato-immunoreactive neurons and were characterised by the expression ofGrp,CalcbandSst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and discover any inherent vulnerabilities in disease.Abstract FigureUsing a reporter mouse line, expressing a fluorescent protein under control of the dopamine transporter (DAT) promoter, discrete subtypes of dopaminergic neurons were unveiled across the ganglionated plexuses of the gut. A novel subpopulations of enteric DA neurons, expressing genes previously reported involved in dopamine signaling in the brain, exhibit a cholinergic phenotype.