Choline transporter: an additional marker of cholinergic nerves in the enteric nervous system

Purpose: The cholinergic circuitry of the enteric nervous system (ENS) is labelled using antibodies against molecules involved in acetylcholine (ACh) synthesis and release. A new component of ACh synthesis, the high affinity choline transporter (CHT), which is essential in the uptake of choline into cholinergic nerves, has been isolated and cloned. Recently, antiserum against CHT has been used in the central nervous system to label cholinergic nerves, but has not been located in the ENS. The aim of this study was to localise CHT immunoreactivity (IR) within rat and human intestine and determine if CHT colocalised with other cholinergic markers in the ENS. Methods: Segments of rat (n = 3) ileum and colon and human paediatric (n = 3) colon were fixed, prepared for sections and wholemounts and incubated with antisera against the full length human CHT sequence, followed by a fluorescent secondary antibody. Tissue was double labelled using antibodies to neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP) and vesicular acetylcholine transporter (VAChT). Colocalisation was quantitated in 3 confocal images from each segment. Results: In human and rat intestine, CHT-IR was present in nerve fibres in the circular muscle and myenteric ganglia and in myenteric nerve cell bodies. In human colon, CHT-IR was present in almost all VAChT-IR cholinergic nerve fibres in the circular muscle and myenteric ganglia. In contrast, in the rat only 56% of circular muscle VAChT-IR nerve fibres were CHT-IR, with this accounting for 79% of CHT-IR nerve fibres. In the myenteric ganglia, 48% of VAChT-IR nerve fibres were CHT-IR, accounting for 50% of CHT-IR nerve fibres. Surprisingly, no CHT-IR nerve cell bodies were cChAT-IR. Yet, 40% of CHT-IR nerve cell bodies were SP-IR (tachykinergic), accounting for 12% of SP-IR nerve cell bodies. In both species, there was little localisation of CHT-IR in nitrergic NOSIR inhibitory nerve fibres. Conclusions: In human paediatric colon, there was almost complete colocalisation of CHT-IR and VAChT-IR in nerve fibres in the circular muscle and myenteric ganglia. Therefore the CHT antisera would be useful for studying the enteric cholinergic circuitry in human intestine and complimentary to currently used labels. In rat, CHT-IR identified VAChT-IR positive, VAChT-IR negative nerve fibres and cChAT-IR negative cell bodies. This result suggests the antibody does not bind to all the cholinergic circuitry identified by cChAT-IR or VAChT-IR. Further studies are needed to determine if the CHT antibody is specifically labelling cholinergic nerves in the rat.Andrea M. Harrington, Margaret Lee, Sim-Yee Ong, Eric Yong, Pam Farmer, John M. Hutson and Bridget R. Southwel