Choline Acetyltransferase (ChAT) Immunoreactivity in Paraffin Sections of Normal and Diseased Intestines

Abstract

SUMMARYThere is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 g/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.

Neonatal maternal separation of rat pups results in abnormal cholinergic regulation of epithelial permeability

“…ChAT was used to indicate the presence of acetylcholine, since it is essential in the synthesis of the neurotransmitter and is a specific marker of cholinergic nerves and acetylcholine-containing cells. ChAT has been identified by immunohistochemisty in the cell bodies and nerve terminals of both submucosal and myenteric plexus neurons (27); in addition, the cell membrane, endosomes, cytoskeleton, nucleus, and mitochondria of epithelial cells have also been shown to be immunoreactive (16). In our study, densitometry analysis of Western blots revealed a significant increase in the expression of ChAT relative to the expression of PGP9.5, a neuronal marker, suggesting that increased enzyme levels were present in the enteric nerves of MS pups.…”

Cholinergic inhibition of electrogenic sodium absorption in the guinea pig distal colon

“…The possibility cannot be excluded, however, that a small amount of ACh was constitutively released without action potential from the nerve terminals. Alternatively, epithelial and nonepithelial cells exhibiting immunoreactivity to choline acetyltransferase might have been the source of tissue ACh (30,38,39). A role of tissue ACh that was independent of the cholinergic nerve activity has been previously suggested from the finding that TTX inhibited the luminal propionate-induced Cl Ϫ secretion in the colon by 40%, whereas atropine inhibited it by 90% (55).…”

“…It has previously been shown that stimulation of the submucosal neurons activated anion secretion by activating both the cholinergic and noncholinergic pathways in the colon (12,18,22,23,32,33,51). Approximately half of the submucosal plexus neurons in the colon have been reported to contain ACh from choline acetyltransferase immunoreactivity and other morphological studies (13,14,30,32,33,38,39). Further evidence for the presence of cholinergic submucosal neurons has been the [ 3 H]ACh release induced by nerve stimulation (19,29,54,58).…”

“…Further evidence for the presence of cholinergic submucosal neurons has been the [ 3 H]ACh release induced by nerve stimulation (19,29,54,58). However, a morphological investigation of choline acetyltransferase immunoreactivity has failed to clearly demonstrate cholinergic nerve fibers in the lamina propria of colonic mucosa (38,39), although the concentration of choline acetyltransferase in the mucosal nerve fiber may not be high enough to be detected. Vasoactive intestinal peptide and substance P have been suggested to be released and to be responsible for the noncholinergic component of Cl Ϫ secretion (17,32,40,51).…”

The Cholinergic System Can Be of Unexpected Importance in Osteoarthritis

“…It is likely that the ACh produced in non-neuronal cells is released directly after synthesis, in contrast to the nerve-related ACh which is released via exocytosis. Cell types for which ACh production has been shown are cells in the airways , the keratinocytes of the skin (Grando et al, 1993(Grando et al, , 2006, cells of the intestinal epithelium (Klapproth et al, 1997;Ratcliffe et al, 1998;Jönsson et al, 2007), cells of the urinary bladder wall (Lips et al, 2007;Yoshida et al, 2008), cells in blood vessel walls Lips et al, 2003) and certain cancer cells (Song et al, 2003(Song et al, , 2008Paleari et al, 2008). A further celltype for which there is evidence of ACh production is the tenocyte of human patellar (Danielson et al, 2006(Danielson et al, , 2007 and Achilles (Bjur et al, 2008) tenocytes.…”