Abstract
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.
Highlights
Acetylcholine (ACh) was the first molecule identified to act as a neurotransmitter (Loewi and Navratil, 1926)
In the blotted membrane pre-fixed with PFA, human pChAT (hpChAT)-ir staining was seen at a band of approximately 47 kDa in the colon, while no labeling was detectable in the cervical segment of the ventral spinal cord
We cannot rule out the possibility that hpChAT antiserum may recognize 72 kDa common ChAT (cChAT) in the cord under a non-PFA fixed condition
Summary
Acetylcholine (ACh) was the first molecule identified to act as a neurotransmitter (Loewi and Navratil, 1926). ChAT-IHC shows a high morphological resolution that has enabled the detailed mapping of cholinergic pathways in the CNS of many species including rat and human (Mesulam, 1990; Woolf, 1991) This contrast with the peripheral nervous system (PNS), where ChAT-IHC often gives vague and faint staining of cholinergic cells and fibers. In the human PNS, the detailed organization of cholinergic structures remains unclear as mentioned in studies reporting failures in staining of notable components such as cholinergic nerves in intestinal mucosae (Porter et al, 1996; Ratcliffe et al, 1998)
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