While morphology and development of the zebrafish enteric nervous system (ENS) are known, neurochemical coding of enteric neurons is still incomplete. Using immunofluorescence, we aimed to unravel the neurochemical coding of zebrafish enteric neurons. Tyrosine hydroxylase (TH), VIP and PACAP were observed in nerve fibres, while other markers were detected in neuronal cell bodies. In embryos, all markers, except for choline acetyltransferase (ChAT) and TH, were present from 72 hpf on. Nitrergic neurons, which were evenly distributed and remained constant in time, constituted a major subpopulation. The neuronal proportions of the other markers increased during development. These results support previous data that the ENS is well‐developed before the onset of feeding. In the adult, all markers were expressed in the ENS. A major percentage of neurons displayed calbindin and calretinin, while serotonin was the only marker showing significant distribution differences in the three intestinal regions. Serotonin was not coexpressed with any of the other markers. At least five neuronal subpopulations were determined. A functional classification of the subpopulations has been suggested.
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