Immunohistochemical study of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rat and guinea pig

Nakajima, Kyoji

doi:10.1016/s0168-0102(00)81191-8

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“…Here, we report the details of hpChAT antibody characterization and its use to visualize neuronal structures positive for the antiserum in human colon. The colon was chosen because of important cholinergic innervation demonstrated by functional studies in a wide variety of animal species (see Furness, 2006) and also because of the dense cholinergic innervation shown by IHC using rpChAT antiserum in rats and mice (Nakajima et al, 2000; Tooyama and Kimura, 2000; Yuan et al, 2005; Wang et al, 2007). Many potential applications exist for this new antiserum, in particular, to study cholinergic structures in the PNS, including the enteric nervous system, of normal and diseased human tissues.…”

Section: Introductionmentioning

confidence: 99%

A Novel Antiserum Against a Predicted Human Peripheral Choline Acetyltransferase (hpChAT) for Labeling Neuronal Structures in Human Colon

et al. 2019

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Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.

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