Intrinsic Cholinergic Innervation Research Articles

Regional differences of extrinsic and intrinsic cholinergic innervation and alterations by chronic opioid-induced constipation in the porcine colonic enteric nerve system

The colon is richly innervated by extrinsic (ext) and intrinsic (int) cholinergic nerves from central and enteric nervous system (CNS and ENS) modulating colonic motility, secretion and inflammation. However, a challenge lies in our inability to anatomically distinguish such innervation in the colon. The porcine colon is regarded as a useful translational research model due to many similarities with human. The present study developed an approach to differentiate ext- and int-cholinergic innervation of the porcine colon using double immunolabeling with a novel mouse antibody against human peripheral choline acetyltransferase (hpChAT) combined with a rabbit antibody anti-common ChAT (cChAT) respectively. The spatial configuration of ext- and int- cholinergic innervation in the ENS was assessed on 3D images generated from CLARITY-cleared porcine colonic samples. Their densities were computationally quantitated using Imaris 9 within the inner and outer submucosal and myenteric plexuses (ISP, OSP, MP) along the proximal, transverse and distal colonic regions (pC, tC, dC) collected from 18 adult Yucatan minipigs including 6 naïve (3 of each sex), 4 vehicle controls (2 of each sex), 8 treated with loperamide, a μ opioid receptor agonist (LOP, 0.4 or 3 mg/kg/day, po, 4 weeks, 4/group, 2 of each sex). The specificities of cChAT and hpChAT antibodies were confirmed with porcine cervical vagal trunk (CVT), sacral spinal cord (SSC, S2-S4) and colonic ENS. The double labeling showed the strong cChAT immunoreactive (+) fibers in the CVT and neuronal somata and fibers in the SSC, but no hpChAT+ labeling was visualized in these structures. In the colonic MP, the dense hpChAT+ neurons and fibers and varicose cChAT+ fibers were simultaneously visualized. 3D images demonstrated that int- cholinergic neurons (hpChAT+) were closely surrounded by ext- cholinergic varicose fibers and dot like structures (cChAT+), presumably nerve terminals. The density of cChAT+ but not hpChAT+ fibers+somata showed significant differences among three segments (dC>tC>pC) within three enteric plexuses (MP>OSP>ISP) in naïve pigs. Compared to control groups, the high dose LOP induced a 1.3-fold decrease of cChAT+ fiber density in males (p<0.05) and 1.5-fold in females (p<0.05), and 2.2-fold decrease of hpChAT+ fibers+somata density in males (p<0.05) and 3.6-fold in female (p<0.01) with a 1.5-fold greater decrease in females than males (p<0.05) in the MP of the dC. This study demonstrates for the first time the ext- and int- cholinergic innervation, the spatial configuration in 3D structure, regional variations in the porcine colonic ENS and the reduction by chronic opiate use with greater in females than males. These findings provide neuroanatomical evidence of CNS-gut cross-talk via cholinergic innervation of the colon and point to the neuromodulation of this pathway to alleviate functional colonic disorders like chronic opioid-induced constipation. Supported by NIH/SPARC OT2OD024899. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cover Image

The cover image is based on the Original Article 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 by Pu-Qing Yuan et al., https://doi.org/10.1111/nmo.14030.

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.

We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus. We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5. In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer. The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Centrality of Striatal Cholinergic Transmission in Basal Ganglia Function

Work over the past two decades revealed a previously unexpected role for striatal cholinergic interneurons in the context of basal ganglia function. The recognition that these interneurons are essential in synaptic plasticity and motor learning represents a significant step ahead in deciphering how the striatum processes cortical inputs, and why pathological circumstances cause motor dysfunction. Loss of the reciprocal modulation between dopaminergic inputs and the intrinsic cholinergic innervation within the striatum appears to be the trigger for pathophysiological changes occurring in basal ganglia disorders. Accordingly, there is now compelling evidence showing profound changes in cholinergic markers in these disorders, in particular Parkinson's disease and dystonia. Based on converging experimental and clinical evidence, we provide an overview of the role of striatal cholinergic transmission in physiological and pathological conditions, in the context of the pathogenesis of movement disorders.

Choline acetyltransferase-containing neurons in the human parietal neocortex.

A number of immunocytochemical studies have indicated the presence of cholinergic neurons in the cerebral cortex of various species of mammals. Whether such cholinergic neurons in the human cerebral cortex are exclusively of subcortical origin is still debated. In this immunocytochemical study, the existence of cortical cholinergic neurons was investigated on surgical samples of human parietal association neocortex using a highly specific monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine biosynthesising enzyme. ChAT immunoreactivity was detected in a subpopulation of neurons located in layers II and III. These were small or medium-sized pyramidal neurons which showed cytoplasmic immunoreactivity in the perikarya and processes, often in close association to blood microvessels. This study, providing demonstration of ChAT neurons in the human parietal neocortex, strongly supports the existence of intrinsic cholinergic innervation of the human neocortex. It is likely that these neurons contribute to the cholinergic innervation of the intracortical microvessels.

Origins of spinal cholinergic pathways in amphibians demonstrated by retrograde transport and choline acetyltransferase immunohistochemistry

The existence of propriospinal cholinergic pathways and the origin of supraspinal cholinergic descending projections have been investigated in anuran and urodele amphibians. Retrograde tract tracing techniques with dextran amines injected in the spinal cord at different levels were combined with immunohistochemistry for choline acetyltransferase (ChAT). The analysis of the brachial, thoracic and lumbar spinal cord demonstrated that doubly labeled cells were present only close to the injection site. Thus, the participation of the spinal cholinergic cells in distant intersegmental connections is not present, or is very limited, in amphibians. In anurans, tracer applications to the brachial cord revealed cholinergic cells of origin of spinal projections located in four distinct brain nuclei. The most rostrally located cells were found bilaterally in the preoptic area, among the magnocellular cells. In the ipsilateral isthmic region, the laterodorsal tegmental nucleus also showed doubly labeled cells. Throughout the brainstem, abundant codistribution was observed but actual coexistence of the tracer and ChAT was only found in the nucleus of the solitary tract and the inferior reticular nucleus. In the case of the urodele, abundant codistribution between retrogradely labeled cells and ChAT-positive neurons in zones like the suprachiasmatic nucleus, the isthmic region and the rhombencephalic reticular formation was observed, but the only doubly labeled cells were the Mauthner neurons. The present results in amphibians contrast with previous data in mammals in which is striking the presence of a widespread intrinsic cholinergic innervation of the spinal cord and the virtual absence of cholinergic projections descending from the brainstem.

Reduction in CHT1-mediated choline uptake in primary neurons from presenilin-1 M146V mutant knock-in mice

The memory loss in Alzheimer's disease (AD) has been linked to cholinergic hypoactivity. Mutations in presenilin-1 (PS-1) may regulate cholinergic signaling, although their precise roles in cholinergic neurotransmission in AD are unsettled. Neuronal uptake of choline via the high affinity choline transporter (CHT1) is essential for cholinergic neurotransmission. CHT1 is a Na+-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter. Although cholinergic neurons in the nucleus basalis of Meynert are a major source of cholinergic projections for the cerebral cortex, it is unclear whether cortical neurons exhibit intrinsic CHT1 activity that is altered in AD. We now report that primary cortical neurons express intrinsic and biologically active CHT1, and that, in these neurons, CHT1-mediated choline uptake activity is significantly reduced in PS-1 M146V mutant knock-in mice. Further kinetic studies using HC-3 binding and cell surface biotinylation assays showed that the PS-1 mutation inhibits CHT1 mediated choline uptake by reducing the ligand binding affinity of CHT1 without significantly altering levels of CHT1 expression in the plasma membrane. Since human neocortex has recently been shown to possess intrinsic cholinergic innervation, our results indicate that alterations in CHT1-mediated high affinity choline uptake in cortical neurons may contribute to Alzheimer's dementia.

Involvement of intrinsic cholinergic and GABAergic innervation in the effect of NMDA on striatal dopamine efflux and metabolism as assessed by microdialysis studies in freely moving rats.

Microdialysis perfusion was used to study the participation of striatal cholinergic and gamma-aminobutyric acid-ergic (GABAergic) neurotransmission in basal and N-methyl-D-aspartate (NMDA) receptor-modulated dopamine release and metabolism in the striatum of the freely moving rat. Reverse dialysis of atropine (1-50 microM) induced a concentration-related increase in dopamine efflux and decrease in 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) efflux. (+)-Bicuculline (10-100 microM) similarly increased dopamine efflux, but was without consistent effect on metabolite efflux. Reverse dialysis of NMDA (1 mM) evoked an approximately twofold increase in dopamine efflux and decreased DOPAC and HVA efflux to 30-40% of basal levels. The effect of NMDA on dopamine efflux was completely abolished by coadministration of tetrodotoxin (TTX; 1 microM) or atropine (10 microM), and markedly potentiated (approximately fourfold) by coadministration of (+)-bicuculline (50 microM). The NMDA-induced decrease in dopamine metabolite efflux was inhibited by coadministration of TTX or (+)-bicuculline, but was unaffected by atropine. Our data suggest that dopamine release in the striatum is subject to both cholinergic and GABAergic tonic inhibitory mechanisms mediated through muscarinic and GABAA receptors, respectively. Furthermore, NMDA-stimulated dopamine release also involves obligatory cholinergic facilitation and an inhibitory GABAergic component mediated through these respective receptors.

Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. I. Central nervous system

Antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter mark expression of this specifically cholinergic protein in perinuclear regions of the soma and on secretory vesicles concentrated within cholinergic nerve terminals. In the central nervous system, the vesicular acetylcholine transporter terminal fields of the major putative cholinergic pathways in cortex, hippocampus, thalamus, amygdala, olfactory cortex and interpeduncular nucleus were examined and characterized. The existence of an intrinsic cholinergic innervation of cerebral cortex was confirmed by both in situ hybridization histochemistry and immunohistochemistry for the rat vesicular acetylcholine transporter and choline acetyltransferase. Cholinergic interneurons of the olfactory tubercle and Islands of Calleja, and the major intrinsic cholinergic innervation of striatum were fully characterized at the light microscopic level with vesicular acetylcholine transporter immunohistochemistry. Cholinergic staining was much more extensive for the vesicular acetylcholine transporter than for choline acetyltransferase in all these regions, due to visualization of cholinergic nerve terminals not easily seen with immunohistochemistry for choline acetyltransferase in paraffin-embedded sections. Cholinergic innervation of the median eminence of the hypothalamus, previously observed with vesicular acetylcholine transporter immunohistochemistry, was confirmed by the presence of vesicular acetylcholine transporter immunoreactivity in extracts of median eminence by western blotting. Cholinergic projections to cerebellum, pineal gland, and to the substantia nigra were documented by vesicular acetylcholine transporter-positive punctate staining in these structures. Additional novel localizations of putative cholinergic terminals to the subependymal zone surrounding the lateral ventricles, and putative cholinergic cell bodies in the sensory mesencephalic trigeminal nucleus, a primary sensory afferent ganglion located in the brainstem, are documented here. The cholinergic phenotype of neurons of the sensory mesencephalic trigeminal nucleus was confirmed by choline acetyltransferase immunohistochemistry. A feature of cholinergic neurons of the central nervous system revealed clearly with vesicular acetylcholine transporter immunohistochemistry in paraffin-embedded sections is the termination of cholinergic neurons on cholinergic cell bodies. These are most prominent on motor neurons of the spinal cord, less prominent but present in some brainstem motor nuclei, and apparently absent from projection neurons of the telencephalon and brainstem, as well as from the preganglionic vesicular acetylcholine transporter-positive sympathetic and parasympathetic neurons visualized in the intermediolateral and intermediomedial columns of the spinal cord. In addition to the large puncta decorating motor neuronal perikarya and dendrites in the ventral horn, vesicular acetylcholine transporter-positive terminal fields are distributed in lamina X surrounding the central canal, where additional small vesicular acetylcholine transporter-positive cell bodies are located, and in the superficial layers of the dorsal horn. Components of the central cholinergic nervous system whose existence has been controversial have been confirmed, and the existence of new components documented, with immunohistochemistry for the vesicular acetylcholine transporter. Quantitative visualization of terminal fields of known cholinergic systems by staining for vesicular acetylcholine transporter will expand the possibilities for documenting changes in synaptic patency accompanying physiological and pathophysiological changes in these systems.

A cholinergic propriospinal innervation of the rat spinal cord

Previous work has suggested the presence of a widespread, intrinsic cholinergic innervation of the spinal cord. A combination of retrograde axonal tract tracing and choline acetyltransferase immunocytochemistry was used to show that there is a propriospinal cholinergic innervation of the rat spinal cord that arises from short-range projections (spanning up to six spinal segments) of cholinergic neurons known as ‘central canal’ and ‘partition’ cells.

Development of cholinergic nerve transmission in the chick oesophagus

1. The onset and development of cholinergic mechanisms in the smooth muscle of the chick oesophagus were studied by estimating the changes in mechanical response and biochemical parameters between 9 days of incubation and 7 days after hatching. 2. Transmural and vagal nerve stimulation first evoked contraction in the oesophagus at 10 days and 11 days of incubation, respectively. These contractions were inhibited by atropine (1-2 microM) and potentiated by physostigmine (0.2 microM). On the other hand, hexamethonium (200 microM) had an inhibitory effect on vagal nerve stimulation but not on transmural nerve stimulation. 3. The relative amplitude of contraction induced by both vagal nerve and transmural stimulations compared to high K+ (80 mM)-induced contractions, progressively increased with age in embryos up to 19 days of incubation. 4. The activity of choline acetyltransferase (ChAT), an enzyme synthesizing acetylcholine (ACh), also gradually increased in the oesophagus during the period from 9 days to 19 days of incubation, which was similar to the change in the nerve-mediated contraction. On the other hand, the cholinesterase activity reached a maximum at 13 days of incubation and decreased until 7 days after hatching. 5. The contractile response to ACh and binding sites of [3H]-quinuclidinyl benzilate ([3H]-QNB) were observed in the oesophagus at 9 days of incubation. The maximum response produced by ACh (300 microM) tended to be greater in early stages (9-13 days of incubation) than in later stages. The sensitivity estimated from pD2 values increased up to 15 days of incubation. The maximum response produced by ACh (300 microM) tended to be greater in early stages (9-13 days of incubation) than in later stages. The sensitivity estimated from pD2 values increased up to 15 days of incubation. During the embryonic period, the number of muscarinic receptors estimated from the binding of [3H]-QNB changed very little. 6. These results suggest that in the chick oesophagus, extrinsic and intrinsic cholinergic innervation start to function at 10 days and 11 days of incubation, respectively and continue to develop progressively up to the time of hatching. It seems likely that the functional and biochemical maturation of receptive mechanisms on the smooth muscle precede those of cholinergic innervation.

A pharmacological comparison of organophosphorus and carbamate anti-cholinesterase agents on guinea pig ileum

The pharmacological effects of organophosphorus (4-methylthiophenyl dipropyl phosphate:propaphos, diisopropylfluorophosphate:DEP) and carbamate (physostigmine, neostigmine) anti-cholinesterase (anti-ChE) on guinea pig isolated ileum were examined. Physostigmine (0.7 or 7.2 μ m) and neostigmine (0.1, 1, or 10 μ m) caused a phasic and tonic contraction of the ileum, while a sustained application of propaphos (0.3, 1, 10 μ m) and DFP (0.1 or 1 μ m) produced intermittent contractions of slow onset. The organophosphate-induced contractions were markedly reduced by morphine (0.1 μ m), tetrodotoxin (31 n m), hexamethonium (1 μ m), and atropine (14 n m), which suggested a possible involvement of intrinsic cholinergic innervation in the development of its spasmogenicity. On the other hand, the carbamate-induced contraction was not significantly affected by these antagonists except atropine which inhibited it by 52.5 ± 9.8% ( n = 7). The transmural stimulation- and picric acid (1 μ m)-induced contractions of the ileum were significantly enhanced by lower concentrations of physostigmine (72 n m) and neostigmine (10 n m) but not by the organophosphates. Similarly, the Ca 2+-induced contraction of the K +-depolarized ileum was effectively enhanced only by the carbamates. Physostigmine (7.2 μ m), neostigmine (10 μ m), and DFP (100 μ m) did not affect the specific [ 3H]quinuclidinyl benzilate (QNB) binding to ileal longitudinal muscles, suggesting no direct effect of muscarinic cholinergic receptors. The acetylcholine (ACh)-induced inhibition of the [ 3H]QNB binding was significantly enhanced only by carbamates. Thus we conclude that organophosphates may contract guinea pig isolated ileum by an action on the intrinsic cholinergic nerves and that the carbamate-induced contraction may relate to its anti-ChE effect in cholinergic synapses as well as a direct effect on the smooth muscles.

Neural regulation and pharmacology of the gut of Chione stutchburyi, a bivalve mollusc

1. The extrinsic nerve supply to the gut of Chione stutchburyi, a New Zealand venerid bivalve mollusc, consists of exitatory serotonergic and inhibitory dopaminergic nerves. 2. There is excitatory cholinergic innervation of the hindgut, but not the cardiac gut. We postulate the presence of cholinergic ganglion cells in the wall of the hindgut and that this intrinsic cholinergic innervation intiates spontaneous activity of the gut. 3. High levels of monomines are found in gut tissues. Concentrations of catecholamines fall to very low levels in winter.

The anatomy and innervation of the sphincter of Oddi in the dog and cat.

AbstractThe fine morphology of the choledocho‐duodenal junctions of dogs and cats was studied by means of microdissection, classical histological staining techniques and by demonstrating in serial sections non‐specific cholinesterases, which stain the smooth muscle components excellently. The inbuilt cholinergic nervous apparatus was studied in serial sections by demonstrating acetylcholinesterase. By combining these methods, the three‐dimensional structural arrangement of the intrinsic cholinergic innervation system, with reference to the various substructures to be innervated, could be studied and mapped in detail.The most important features in the morphology, with respect to the function, were the tapering of the terminal ductus choledochus into a nozzle, the existence of concentric retrograde saccules around the ampulla and the intramural part of the bile duct, and the complex construction of the sphincters. Normally such a structure makes possible an active suction‐pressure pumping for evacuation of bile and pancreatic juice, being at the same time effective in preventing regurgitation of the duodenal fluid. On the contrary, if the orificium of the ampulla is obstructed, this structural arrangement probably facilitates regurgitation, especially into the pancreatic duct.The intrinsic cholinergic nervous apparatus of the choledocho‐duodenal junction was extremely rich, especially if compared with the cholinergic innervation of the muscular layer of the gut wall. This can probably be considered as an indication of complex motor (and possibly associated sensory) functions of the sphincters, in contrast to only slow gross contractions of the gut.To summarize: both morphological and neuroanatomical bases seem to be provided for rhythmic suction‐pressure pumping as well as for sensitive protective reflex functions in the choledocho‐duodenal junctions of the dog and cat. The finely graded complex interactions of the various muscle components are regulated by connecting and integrating neural elements between the individual smooth muscle units.

Relation between Gut Motility and Innervation in Man

Various factors combine to produce regional differences in the motility of the human gastrointestinal tract. Important contributions are made by the intrinsic nerves, and there is now evidence for four types of fibres, which include cholinergic and nor-adrenergic nerves, inhibitory fibres which are non-adrenergic, and excitatory fibres which are non-cholinergic. Studies using drugs or electric impulses to stimulate nerves demonstrate a relatively weak intrinsic cholinergic innervation of the stomach, an organ which relies substantially on extrinsic vagal fibres. There is strong excitatory innervation in the small intestine, but predominantly inhibitory innervation in the colon. These patterns of nerve distribution correlate with regional differences in behaviour and function of the gut.

The intrinsic adrenergic and cholinergic innervation of the genitalia of Galago and Tupaia. The penis.

The intrinsic innervation of the penises of Galago and Tupaia were studied using modern histochemical techniques. The adrenergic and cholinergic innervation of the penile vessels and intrinsic musculature in these two species were similar. However, the adrenergic nerves of Tupaia were more intensely fluorescent than in Galago. The possibility of an efferent component of the dorsal nerve of the penis is discussed.