High affinity choline transporter immunoreactivity in rat ileum myenteric nerves

Abstract

Recently, an antibody against the choline transporter (CHT), an essential molecule involved in ACh uptake, was used to label cholinergic nerves in the central nervous system; however, the enteric nervous system (ENS) was not examined. The present study localised CHT immunoreactivity (CHT-IR) within the rat ileum ENS and determined whether it colocalised with immunoreactivity for markers of cholinergic, tachykinergic and nitrergic circuitry. Segments of rat ileum were fixed, prepared for sectioning or whole-mounts and incubated with anti-CHT antisera followed by a fluorescent secondary antibody. Samples were double-labelled with antibodies to nitric oxide synthase, substance P (SP), common choline acetyltransferase (cChAT) and vesicular acetylcholine transporter (VAChT). CHT-IR was present in varicosities of nerve fibres in the myenteric plexus and muscle layers of rat ileum. In the myenteric ganglia, CHT-IR was found in nerve fibres and the cytoplasm of some nerve cell bodies. In the myenteric ganglia, no CHT/cChAT-immunoreactive neurons were present. A small number of CHT/SP-immunoreactive neurons and CHT/SP-immunoreactive nerve fibres clustered around unlabelled neurons. CHT-IR colocalised with VAChT-IR in the myenteric plexus but only half of the CHT-immunoreactive myenteric nerve fibres were VAChT-immunoreactive and half of VAChT-immunoreactive fibres were CHT-immunoreactive. In the circular muscle, 75% of CHT-immunoreactive fibres were VAChT-immunoreactive. Thus, the anti-CHT antiserum labels neurons and nerve fibres in the rat ENS. It does not label cholinergic cChAT-immunoreactive neurons, although it does immunostain cholinergic VAChT-immunoreactive nerve fibres and a population of nerves that are not VAChT-immunoreactive.