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

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.

Intrinsic cholinergic innervation in the human sigmoid colon revealed using CLARITY, three‐dimensional (3D) imaging, and a novel anti‐human peripheral choline acetyltransferase (hpChAT) antiserum

“…The antibody protein gene product 9.5 (PGP9.5), a pan‐neuronal marker, 22 was used as a counterstain to display all neuronal components. To further characterize the specificity of the novel hpChAT antiserum in labeling of human colonic enteric cholinergic neurons, 20 double labeling was performed with hpChAT antiserum and other two classic ChAT antibodies: a goat anti‐human ChAT antibody from Chemicon (AB144p) or a rabbit anti‐porcine ChAT antibody from Dr. Michael Schemann (P3YEB). These two antibodies represented the most popular ChAT antibodies commonly used to label human enteric cholinergic neurons 23,24 .…”

“…20 This antiserum labels peripheral cholinergic nerve cell bodies and fibers in the human large intestine without cross-reacting with the common ChAT expressed in the central nervous system (CNS). 20 Secondly, we reconstructed 3D images from Z-stacks of confocal images and traced digitally the local process projections and connectivity of hpChAT immunoreactive (ir) neurons in the SMP and MP using Imaris 9.2 and 9.5 for neuroscientist. Then, the density and intensity of hpChAT-ir fibers innervating the circular and longitudinal muscle layers were computationally quantitated and compared.…”

“…The protocol of passive CLARITY for brain 7 the specificity of the novel hpChAT antiserum in labeling of human colonic enteric cholinergic neurons, 20 double labeling was performed with hpChAT antiserum and other two classic ChAT antibodies: a goat anti-human ChAT antibody from Chemicon (AB144p) or a rabbit anti-porcine ChAT antibody from Dr. Michael Schemann (P3YEB).…”

“…The main objective of this study was to fill up the gap in anatomical mapping of the intrinsic cholinergic innervation of the human sigmoid colon, particularly in 3D structures. To this end, firstly, we developed a modified CLARITY protocol adapted to tissue properties of human sigmoid colon and compatible with immunofluorescence using a recently characterized mouse anti‐human peripheral ChAT (hpChAT) protein antiserum 20 . This antiserum labels peripheral cholinergic nerve cell bodies and fibers in the human large intestine without cross‐reacting with the common ChAT expressed in the central nervous system (CNS) 20 .…”

“…To this end, firstly, we developed a modified CLARITY protocol adapted to tissue properties of human sigmoid colon and compatible with immunofluorescence using a recently characterized mouse anti‐human peripheral ChAT (hpChAT) protein antiserum 20 . This antiserum labels peripheral cholinergic nerve cell bodies and fibers in the human large intestine without cross‐reacting with the common ChAT expressed in the central nervous system (CNS) 20 . Secondly, we reconstructed 3D images from Z‐stacks of confocal images and traced digitally the local process projections and connectivity of hpChAT immunoreactive (ir) neurons in the SMP and MP using Imaris 9.2 and 9.5 for neuroscientist.…”