Muscarinic Receptor Function Research Articles

Therapeutic Potential of 1-(2-Chlorophenyl)-6,7-dimethoxy-3-methyl-3,4-dihydroisoquinoline.

The synthesized compound 1-(2-chlorophenyl) 6-7-dimethoxy-3-methyl-3,4-dihydroisoquinoline (DIQ) was investigated as a biological agent. Its potential to affect muscle contractility was predicted through in silico PASS analysis. Based on the in silico analysis, its capabilities were experimentally investigated. The study aimed to investigate the effects of DIQ on the ex vivo spontaneous contractile activity (CA) of smooth muscle (SM) tissue. DIQ was observed to reduce the strength of Ca2+-dependent contractions in SM preparations (SMP), possibly by increasing cytosolic Ca2+ levels through the activation of a voltage-gated L-type Ca2+ channel. DIQ potently affected calcium currents by modulating the function of muscarinic acetylcholine receptors (mAChRs) and 5-hydroxytryptamine (5-HT) receptors at a concentration of 50 μM. Immunohistochemical tests showed a 47% reduction in 5-HT2A and 5-HT2B receptor activity in SM cells and neurons in the myenteric plexus (MP), further confirming the effects of DIQ. Furthermore, a significant inhibition of neuronal activity was observed when the compound was co-administered with 5-HT to SM tissues. The conducted experiments confirm the ability of the isoquinoline analog to act as a physiologically active molecule to control muscle contractility and related physiological processes.

Muscarinic Cholinoreceptors in Skeletal Muscle: Localization and Functional Role.

The review focuses on the modern concepts of the functions of muscarinic cholinoreceptors in skeletal muscles, particularly, in neuromuscular contacts, and that of the signaling pathways associated with the activation of various subtypes of muscarinic receptors in the skeletal muscles of cold-blooded and warm-blooded animals. Despite the long history of research into the involvement of muscarinic receptors in the modulation of neuromuscular transmission, many aspects of such regulation and the associated intracellular mechanisms remain unclear. Now it is obvious that the functions of muscarinic receptors in skeletal muscle are not limited to the autoregulation of neurosecretion from motor nerve endings but also extend to the development and morphological rearrangements of the synaptic apparatus, coordinating them with the degree of activity. The review discusses various approaches to the study of the functions of muscarinic receptors in motor synapses, as well as the problems arising when interpreting experimental data. The final part of the review is devoted to an analysis of some of the intracellular mechanisms and signaling pathways that mediate the effects of muscarinic agents on neuromuscular transmission.

Systemic injection of nicotinic acetylcholine receptor antagonist mecamylamine affects licking, eyelid size, and locomotor and autonomic activities but not temporal prediction in male mice

Nicotinic acetylcholine receptors are thought to be associated with a wide range of phenomena, such as movement, learning, memory, attention, and addiction. However, the causal relationship between nicotinic receptor activity and behavior remains unclear. Contrary to the studies that examined the functions of muscarinic acetylcholine receptors, the role of the nicotinic acetylcholine receptors on behavior has not been examined as extensively. Here, we examined the effects of intraperitoneal injection of mecamylamine, a nicotinic acetylcholine receptor antagonist, on the performance of male mice in a head-fixed temporal conditioning task and a free-moving open-field task. The head-fixed experimental setup allowed us to record and precisely quantify the licking response while the mice performed the behavioral task with no external cues. In addition, by combining the utility of the head-fixed experimental design with computer vision analysis based on deep learning algorithms, we succeeded in quantifying the eyelid size of awake mice. In the temporal conditioning task, we delivered a 10% sucrose solution every 10 s using a blunt-tipped needle placed within the licking distance of the mice. After the training, the mice showed increased anticipatory licking toward the timing of sucrose delivery, suggesting that the mice could predict the timing of the reward. Systemic injection of mecamylamine decreased licking behavior and caused eye closure but had no effect on learned conditioned predictive behavior in the head-fixed temporal conditioning task. In addition, the injection of mecamylamine decreased spontaneous locomotor activity in a dose-dependent manner in the free-moving open-field task. The results in the open-field experiments further revealed that the effect of mecamylamine on fecal output and urination, suggesting the effects on autonomic activities. Our achievement of successful eyelid size recording has potential as a useful approach in initial screening for drug discovery. Our study paves a way forward to understanding the role of nicotinic acetylcholine receptors on learning and behavior.

Muscarinic receptor M3 activation promotes fibrocytes contraction.

Fibrocytes are monocyte-derived cells able to differentiate into myofibroblasts-like cells. We have previously shown that they are increased in the bronchi of Chronic Obstructive Pulmonary Disease (COPD) patients and associated to worse lung function. COPD is characterized by irreversible airflow obstruction, partly due to an increased cholinergic environment. Our goal was to investigate muscarinic signalling in COPD fibrocytes. Fibrocytes were isolated from 16 patients with COPD’s blood and presence of muscarinic M3 receptor was assessed at the transcriptional and protein levels. Calcium signalling and collagen gels contraction experiments were performed in presence of carbachol (cholinergic agonist) ± tiotropium bromide (antimuscarinic). Expression of M3 receptor was confirmed by Western blot and flow cytometry in differentiated fibrocytes. Immunocytochemistry showed the presence of cytoplasmic and membrane-associated pools of M3. Stimulation with carbachol elicited an intracellular calcium response in 35.7% of fibrocytes. This response was significantly blunted by the presence of tiotropium bromide: 14.6% of responding cells (p < 0.0001). Carbachol induced a significant contraction of fibrocytes embedded in collagen gels (13.6 ± 0.3% versus 2.5 ± 4.1%; p < 0.0001), which was prevented by prior tiotropium bromide addition (4.1 ± 2.7% of gel contraction; p < 0.0001). Finally, M3-expressing fibrocytes were also identified in situ in the peri-bronchial area of COPD patients’ lungs, and there was a tendency to an increased density compared to healthy patient’s lungs. In conclusion, around 1/3 of COPD patients’ fibrocytes express a functional muscarinic M3 receptor. Cholinergic-induced fibrocyte contraction might participate in airway diameter reduction and subsequent increase of airflow resistance in patients with COPD. The inhibition of these processes could participate to the beneficial effects of muscarinic antagonists for COPD treatment.

Chronic Loss of Muscarinic M5 Receptor Function Manifests Disparate Impairments in Exploratory Behavior in Male and Female Mice despite Common Dopamine Regulation.

There are five cloned muscarinic acetylcholine receptors (M1-M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1-M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here, we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the muscarinic agonist oxotremorine, we revealed a unique concentration-response curve and a sensitivity to repeated forced swim stress or restraint stress exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia assayed using the sucrose preference test or on stress-coping strategy assayed using the forced swim test. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in response to environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety.SIGNIFICANCE STATEMENT The basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here, we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without affecting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.

The M3 Muscarinic Acetylcholine Receptor Promotes Epidermal Differentiation

The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3-/- neonatal mice. We found that Chmr3-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

Investigation of Cytotoxicity and Oxidative Stress Induced by the Pyrethroid Bioallethrin in Human Glioblastoma Cells: The Protective Effect of Vitamin E (VE) and Its Underlying Mechanism.

Bioallethrin belongs to the family of pyrethroid insecticides. Previous studies have shown that bioallethrin affected the function of muscarinic receptor and subsequently induced neurotoxicity in different brain models. Reactive oxygen species (ROS) are generated in the metabolic course of the human body, which can cause human damage when overactivated. However, whether bioallethrin evokes cytotoxicity through ROS signaling and whether the antioxidant Vitamin E (VE) protects these cytotoxic responses in human glial cell model are still elusive. This study investigated the effect of bioallethrin on cytotoxicity through ROS signaling and evaluated the protective effect of the antioxidant VE in DBTRG-05MG human glioblastoma cells. The cell counting kit-8 (CCK-8) was used to measure cell viability. Intracellular ROS and glutathione (GSH) levels were measured by a cellular assay kit. The levels of apoptosis- and antioxidant-related protein were analyzed by Western blotting. In DBTRG-05MG cells, bioallethrin (25-75 μM) concentration-dependently induced cytotoxicity by increasing ROS productions, decreasing GSH contents, and regulating protein expressions related to apoptosis or antioxidation. Furthermore, these cytotoxic effects were partially reversed by VE (20 μM) pretreatment. Together, VE partially lessened bioallethrin-induced apoptosis through oxidative stress in DBTRG-05MG cells. The data assist us in identifying the toxicological mechanism of bioallethrin and offer future development of the antioxidant VE to reduce brain damage caused by bioallethrin.

Auditory mismatch responses are differentially sensitive to changes in muscarinic acetylcholine versus dopamine receptor function.

The auditory mismatch negativity (MMN) has been proposed as a biomarker of NMDA receptor (NMDAR) dysfunction in schizophrenia. Such dysfunction may be caused by aberrant interactions of different neuromodulators with NMDARs, which could explain clinical heterogeneity among patients. In two studies (N = 81 each), we used a double-blind placebo-controlled between-subject design to systematically test whether auditory mismatch responses under varying levels of environmental stability are sensitive to diminishing and enhancing cholinergic vs. dopaminergic function. We found a significant drug × mismatch interaction: while the muscarinic acetylcholine receptor antagonist biperiden delayed and topographically shifted mismatch responses, particularly during high stability, this effect could not be detected for amisulpride, a dopamine D2/D3 receptor antagonist. Neither galantamine nor levodopa, which elevate acetylcholine and dopamine levels, respectively, exerted significant effects on MMN. This differential MMN sensitivity to muscarinic versus dopaminergic receptor function may prove useful for developing tests that predict individual treatment responses in schizophrenia.

Muscarinic receptor M3 activation promotes COPD fibrocyte contraction

Fibrocytes are circulating monocytes-derived cells, able to migrate to injured organs in order to differentiate into myofibroblasts-like cells. We have previously shown that their densities are increased in the lungs of Chronic Obstructive Pulmonary Disease (COPD) patients and associated to worse lung function (Eur Respir J 2019 54: 1802173). COPD is characterized by irreversible airflow obstruction, partly due to an increased cholinergic environment (increased muscarinic M3 signalling, targeted by short and long-acting muscarinic antagonists). We hypothesize that the contractile status of differentiated fibrocytes is under control of a cholinergic stimulation. Fibrocytes were isolated from COPD patients’ blood and cultured for 14 days to reach full differentiation. Presence of M3 receptor was quantified by qPCR, flow cytometry and immunocytochemistry. Calcium signalling as well as contraction experiments using collagen gels were performed in presence of carbachol (cholinergic agonist) ± tiotropium bromide (M2/M3 antagonist). qPCR showed that differentiated fibrocytes expressed all isoforms of muscarinic receptors (M1 to M5). Moreover, 59,7% (± 15) of fibrocytes were found to express the M3 receptor by flow cytometry. Immunocytochemistry showed the existence of cytoplasmic and membrane-associated pools of M3 ( Fig. 1 A). Stimulation with carbachol (10 −4 M) elicited an intracellular calcium response in 39% (± 20) of fibrocytes, with a mean peak amplitude of 0,86 (± 0,66). Such response was blunted in presence of tiotropium bromide (10 –6 M): 28% (± 4) of responding cells and mean peak amplitude of 0,2 ± 0,04. Moreover, carbachol induced a significant contraction of fibrocytes embedded in collagen gels (86,4 ± 0,3% of initial gel surface versus 97,5 ± 4,1%; p < 0,0001), which was prevented by prior tiotropium bromide addition (resulting in 95,9 ± 2,7% of initial surface; p < 0,0001, Fig. 1 B). Around 1/3 of COPD patient's fibrocytes express a functional muscarinic M3 receptor. Cholinergic-induced fibrocyte contraction could participate in airway diameter reduction and subsequent increase of airflow resistance in patients with COPD. The inhibition of these processes could participate to the beneficial effects of muscarinic antagonists for COPD treatment.

Differential Actions of Muscarinic Receptor Subtypes in Gastric, Pancreatic, and Colon Cancer.

Cancers arising from gastrointestinal epithelial cells are common, aggressive, and difficult to treat. Progress in this area resulted from recognizing that the biological behavior of these cancers is highly dependent on bioactive molecules released by neurocrine, paracrine, and autocrine mechanisms within the tumor microenvironment. For many decades after its discovery as a neurotransmitter, acetylcholine was thought to be synthesized and released uniquely from neurons and considered the sole physiological ligand for muscarinic receptor subtypes, which were believed to have similar or redundant actions. In the intervening years, we learned this former dogma is not tenable. (1) Acetylcholine is not produced and released only by neurons. The cellular machinery required to synthesize and release acetylcholine is present in immune, cancer, and other cells, as well as in lower organisms (e.g., bacteria) that inhabit the gut. (2) Acetylcholine is not the sole physiological activator of muscarinic receptors. For example, selected bile acids can modulate muscarinic receptor function. (3) Muscarinic receptor subtypes anticipated to have overlapping functions based on similar G protein coupling and downstream signaling may have unexpectedly diverse actions. Here, we review the relevant research findings supporting these conclusions and discuss how the complexity of muscarinic receptor biology impacts health and disease, focusing on their role in the initiation and progression of gastric, pancreatic, and colon cancers.

Pioglitazone prevents obesity-related airway hyperreactivity and neuronal M2 receptor dysfunction.

Obesity-related asthma often presents with more severe symptoms than non-obesity-related asthma and responds poorly to current treatments. Both insulin resistance and hyperinsulinemia are common in obesity. We have shown that increased insulin mediates airway hyperreactivity in diet-induced obese rats by causing neuronal M2 muscarinic receptor dysfunction, which normally inhibits acetylcholine release from parasympathetic nerves. Decreasing insulin with streptozotocin prevented airway hyperreactivity and M2 receptor dysfunction. The objective of the present study was to investigate whether pioglitazone, a hypoglycemic drug, prevents airway hyperreactivity and M2 receptor dysfunction in obese rats. Male rats fed a low- or high-fat diet were treated with pioglitazone or PBS by daily gavage. Body weight, body fat, fasting insulin, and bronchoconstriction and bradycardia in response to electrical stimulation of vagus nerves and to aerosolized methacholine were recorded. Pilocarpine, a muscarinic receptor agonist, was used to measure M2 receptor function. Rats on a high-fat diet had potentiated airway responsiveness to vagal stimulation and dysfunctional neuronal M2 receptors, whereas airway responsiveness to methacholine was unaffected. Pioglitazone reduced fasting insulin and prevented airway hyperresponsiveness and M2 receptor dysfunction but did not change inflammatory cytokine mRNA expression in alveolar macrophages. High-fat diet, with and without pioglitazone, had tissue-specific effects on insulin receptor mRNA expression. In conclusion, pioglitazone prevents vagally mediated airway hyperreactivity and protects neuronal M2 muscarinic receptor function in obese rats.

Model-based prediction of muscarinic receptor function from auditory mismatch negativity responses

Drugs affecting neuromodulation, for example by dopamine or acetylcholine, take centre stage among therapeutic strategies in psychiatry. These neuromodulators can change both neuronal gain and synaptic plasticity and therefore affect electrophysiological measures. An important goal for clinical diagnostics is to exploit this effect in the reverse direction, i.e., to infer the status of specific neuromodulatory systems from electrophysiological measures.In this study, we provide proof-of-concept that the functional status of cholinergic (specifically muscarinic) receptors can be inferred from electrophysiological data using generative (dynamic causal) models. To this end, we used epidural EEG recordings over two auditory cortical regions during a mismatch negativity (MMN) paradigm in rats. All animals were treated, across sessions, with muscarinic receptor agonists and antagonists at different doses. Together with a placebo condition, this resulted in five levels of muscarinic receptor status. Using a dynamic causal model - embodying a small network of coupled cortical microcircuits - we estimated synaptic parameters and their change across pharmacological conditions. The ensuing parameter estimates associated with (the neuromodulation of) synaptic efficacy showed both graded muscarinic effects and predictive validity between agonistic and antagonistic pharmacological conditions.This finding illustrates the potential utility of generative models of electrophysiological data as computational assays of muscarinic function. In application to EEG data of patients from heterogeneous spectrum diseases, e.g. schizophrenia, such models might help identify subgroups of patients that respond differentially to cholinergic treatments. Significance StatementIn psychiatry, the vast majority of pharmacological treatments affect actions of neuromodulatory transmitters, e.g. dopamine or acetylcholine. As treatment is largely trial-and-error based, one of the goals for computational psychiatry is to construct mathematical models that can serve as “computational assays” and infer the status of specific neuromodulatory systems in individual patients. This translational neuromodeling strategy has great promise for electrophysiological data in particular but requires careful validation. The present study demonstrates that the functional status of cholinergic (muscarinic) receptors can be inferred from electrophysiological data using dynamic causal models of neural circuits. While accuracy needs to be enhanced and our results must be replicated in larger samples, our current results provide proof-of-concept for computational assays of muscarinic function using EEG.

Long-term effects of early postnatal nicotine exposure on cholinergic function in the mouse hippocampal CA1 region

In rodent models of smoking during pregnancy, early postnatal nicotine exposure results in impaired hippocampus-dependent memory, but the underlying mechanism remains elusive. Given that hippocampal cholinergic systems modulate memory and rapid development of hippocampal cholinergic systems occurs during nicotine exposure, here we investigated its impacts on cholinergic function. Both nicotinic and muscarinic activation produce transient or long-lasting depression of excitatory synaptic transmission in the hippocampal CA1 region. We found that postnatal nicotine exposure impairs both the induction and nicotinic modulation of NMDAR-dependent long-term depression (LTD). Activation of muscarinic receptors decreases excitatory synaptic transmission and CA1 network activity in both wild-type and α2 knockout mice. These muscarinic effects are still observed in nicotine-exposed mice. M1 muscarinic receptor activity is required for mGluR-dependent LTD. Early postnatal nicotine exposure has no effect on mGluR-dependent LTD induction, suggesting that it has no effect on the function of m1 muscarinic receptors involved in this form of LTD. Our results demonstrate that early postnatal nicotine exposure has more pronounced effects on nicotinic function than muscarinic function in the hippocampal CA1 region. Thus, impaired hippocampus-dependent memory may arise from the developmental disruption of nicotinic cholinergic systems in the hippocampal CA1 region.

Castanea sativa Mill. bark extract cardiovascular effects in a rat model of high-fat diet.

Ellagitannins may have a beneficial impact in cardiovascular diseases. The aim of the study was to evaluate the effect of high-fat diet (HFD) and the efficacy of Castanea sativa Mill. bark extract (ENC) on cardiac and vascular parameters. Rats were fed with regular diet, (RD, n = 15), HFD (n = 15), RD + ENC (20 mg/kg/day by gavage, n = 15), and HFD + ENC (same dose, n = 15) and the effects on body weight, biochemical serum parameters, and inflammatory cytokines determined. Cardiac functional parameters and aorta contractility were also assessed on isolated atria and aorta. Results showed that ENC reduced weight gain and serum lipids induced by HFD. In in vitro assays, HFD decreased the contraction force of left atrium, increased right atrium chronotropy, and decreased aorta K+ -induced contraction; ENC induced transient positive inotropic and negative chronotropic effects on isolated atria from RD and HFD rats and a spasmolytic effect on aorta. In ex vivo experiments, ENC reverted inotropic and chronotropic changes induced by HFD and enhanced Nifedipine effect more on aorta than on heart. In conclusion, ENC restores metabolic dysfunction and cardiac cholinergic muscarinic receptor function, and exerts spasmolytic effect on aorta in HFD rats, highlighting its potential as nutraceutical tool in obesity.

M1 muscarinic acetylcholine receptor dysfunction in moderate Alzheimer's disease pathology.

Aggregation of amyloid beta and loss of cholinergic innervation in the brain are predominant components of Alzheimer’s disease pathology and likely underlie cognitive impairment. Acetylcholinesterase inhibitors are one of the few treatment options for Alzheimer’s disease, where levels of available acetylcholine are enhanced to counteract the cholinergic loss. However, these inhibitors show limited clinical efficacy. One potential explanation for this is a concomitant dysregulation of cholinergic receptors themselves as a consequence of the amyloid beta pathology. We tested this hypothesis by examining levels of M1 muscarinic acetylcholine receptors in the temporal cortex from seven Alzheimer’s disease and seven non-disease age-matched control brain tissue samples (control: 85 ± 2.63 years old, moderate Alzheimer’s disease: 84 ± 2.32 years old, P-value = 0.721; eight female and six male patients). The samples were categorized into two groups: ‘control’ (Consortium to Establish a Registry for Alzheimer’s Disease diagnosis of ‘No Alzheimer’s disease’, and Braak staging pathology of I–II) and ‘moderate Alzheimer’s disease’ (Consortium to Establish a Registry for Alzheimer’s Disease diagnosis of ‘possible/probable Alzheimer’s disease’, and Braak staging pathology of IV). We find that in comparison to age-matched controls, there is a loss of M1 muscarinic acetylcholine receptors in moderate Alzheimer’s disease tissue (control: 2.17 ± 0.27 arbitrary units, n = 7, Mod-AD: 0.83 ± 0.16 arbitrary units, n = 7, two-tailed t-test, t = 4.248, P = 0.00113). Using a functional rat cortical brain slice model, we find that postsynaptic muscarinic acetylcholine receptor function is dysregulated by aberrant amyloid beta-mediated activation of metabotropic glutamate receptor 5. Crucially, blocking metabotropic glutamate receptor 5 restores muscarinic acetylcholine receptor function and object recognition memory in 5XFAD transgenic mice. This indicates that the amyloid beta-mediated activation of metabotropic glutamate receptor 5 negatively regulates muscarinic acetylcholine receptor and illustrates the importance of muscarinic acetylcholine receptors as a potential disease-modifying target in the moderate pathological stages of Alzheimer’s disease.

The function of intracellular muscarinic acetylcholine receptor: influence of physiological stress

The function of intracellular muscarinic acetylcholine receptor: influence of physiological stress

Endocytic sorting and downregulation of the M2 acetylcholine receptor is regulated by ubiquitin and the ESCRT complex

Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.

Inhibitory muscarinic acetylcholine receptors enhance aversive olfactory learning in adult Drosophila.

Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. Here, we show that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. Our results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons.

M2 receptors activation modulates cell growth, migration and differentiation of rat Schwann-like adipose-derived stem cells

Schwann cells (SCs) play a central role in peripheral nervous system physiology and in the response to axon injury. The ability of SCs to proliferate, secrete growth factors, modulate immune response, migrate and re-myelinate regenerating axons has been largely documented. However, there are several restrictions hindering their clinical application, such as the difficulty in collection and a slow in vitro expansion. Adipose-derived stem cells (ASCs) present good properties for peripheral nerve regenerative medicine. When exposed to specific growth factors in vitro, they can acquire a SC-like phenotype (dASCs) expressing key SCs markers and assuming spindle-shaped morphology. Nevertheless, the differentiated phenotype is unstable and several strategies, including pharmacological stimulation, are being studied to improve differentiation outcomes. Cholinergic receptors are potential pharmacological targets expressed in glial cells. Our previous work demonstrated that muscarinic cholinergic receptors, in particular M2 subtype, are present in SCs and are able to modulate several physiological processes. In the present work, muscarinic receptors expression was characterised and the effects mediated by M2 muscarinic receptor were evaluated in rat dASCs. M2 receptor activation, by the preferred agonist arecaidine propargyl ester (APE), caused a reversible arrest of dASCs cell growth, supported by the downregulation of proteins involved in the maintenance of cell proliferation and upregulation of proteins involved in the differentiation (i.e., c-Jun and Egr-2), without affecting cell survival. Moreover, M2 receptor activation in dASCs enhances a pronounced spindle-shaped morphology, supported by Egr2 upregulation, and inhibits cell migration. Our data clearly demonstrate that rat dASCs express functional muscarinic receptors, in particular M2 subtype, which is able to modulate their physiological and morphological processes, as well as SCs differentiation. These novel findings could open new opportunities for the development of combined cell and pharmacological therapies for peripheral nerve regeneration, harnessing the potential of dASCs and M2 receptors.

Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility.

The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.