Activation Of Muscarinic Receptors Research Articles

Modulation of large rhythmic depolarizations in human large basket cells by norepinephrine and acetylcholine

Rhythmic brain activity is critical to many brain functions and is sensitive to neuromodulation, but so far very few studies have investigated this activity on the cellular level in vitro in human brain tissue samples. This study reveals and characterizes a novel rhythmic network activity in the human neocortex. Using intracellular patch-clamp recordings of human cortical neurons, we identify large rhythmic depolarizations (LRDs) driven by glutamate release but not by GABA. These LRDs are intricate events made up of multiple depolarizing phases, occurring at ~0.3 Hz, have large amplitudes and long decay times. Unlike human tissue, rat neocortex layers 2/3 exhibit no such activity under identical conditions. LRDs are mainly observed in a subset of L2/3 interneurons that receive substantial excitatory inputs and are likely large basket cells based on their morphology. LRDs are highly sensitive to norepinephrine (NE) and acetylcholine (ACh), two neuromodulators that affect network dynamics. NE increases LRD frequency through β-adrenergic receptor activity while ACh decreases it via M4 muscarinic receptor activation. Multi-electrode array recordings show that NE enhances and synchronizes oscillatory network activity, whereas ACh causes desynchronization. Thus, NE and ACh distinctly modulate LRDs, exerting specific control over human neocortical activity.

Pharmacologic blockade of nicotinic receptors in the suprachiasmatic nucleus increases ovarian atresia and inhibits follicular growth.

Reproduction in all mammalian species depends on the growth and maturation of ovarian follicles, that is, folliculogenesis. Follicular development can culminate with the rupture of mature follicles and the consequent expulsion of their oocytes (ovulation) or in atresia, characterized by the arrest of development and eventual degeneration. These processes are regulated by different neuroendocrine signals arising at different hypothalamic nuclei, including the suprachiasmatic nucleus (SCN). In the later, the activation of muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs) by acetylcholine is essential for the regulation of the pre-ovulatory signals that stimulate the rupture of mature follicles. To evaluate the participation of the nAChRs in the SCN throughout the oestrous cycle in the regulation of the hypothalamic-pituitary-ovarian axis. For this purpose, 90-day-old adult female rats in metoestrus, dioestrus, proestrus or oestrus were microinjected into the left- or right-SCN with 0.3 μL of saline solution as vehicle or with 0.225 μg of mecamylamine (Mec), a non-selective antagonist of the nicotinic receptors, diluted in 0.3 μL of vehicle. The animals were sacrificed when they presented vaginal cornification, indicative of oestrus stage, and the effects of the unilateral pharmacological blockade of the nAChRs in the SCN on follicular development, ovulation and secretion of oestradiol and follicle-stimulating hormone (FSH) were evaluated. The microinjection of Mec decreased the serum levels of FSH, which resulted in a lower number of growing and healthy follicles and an increase in atresia. The higher percentage of atresia in pre-ovulatory follicles was related to a decrease in the number of ova shed and abnormalities in oestradiol secretion. We also detected asymmetric responses between the left and right treatments that depended on the stage of the oestrous cycle. The present results allow us to suggest that during all the stages of the oestrous cycle, cholinergic signals that act on the nAChRs in the SCN are pivotal to modulate the secretion of gonadotropins and hence the physiology of the ovaries. Further research is needed to determine if such signals are generated by the cholinergic neurons in the SCN or by cholinergic afferents to the SCN.

Scopolamine infusion in the basolateral amygdala after saccharin intake induces conditioned taste avoidance in rats.

Muscarinic receptor activity in the basolateral amygdala (BLA) is known to be involved in plasticity mechanisms that underlie emotional learning. The BLA is involved in the Attenuation of Neophobia, an incidental taste learning task in which a novel taste becomes familiar and recognized as safe. Here we assessed the role of muscarinic receptor activity in the BLA in incidental taste learning. Young adult male Wistar rats were bilaterally implanted with cannulas aimed at BLA. After recovery, rats were randomly assigned to either vehicle or muscarinic antagonist group, for each experiment. We tested the effect of specific and non-specific muscarinic antagonists administered either 1) 20min before novel taste presentation; 2) immediately after novel taste presentation; 3) immediately after retrieval (the second taste presentation on Day 5 -S2-) or immediately after the fifth taste presentation on Day 8 (S5). Non-specific muscarinic receptor antagonist scopolamine infused prior to novel taste, while not affecting novel taste preference, abolished AN, i.e., the increased preference observed in control animals on the second presentation. When administered after taste consumption, intra-BLA scopolamine not only prevented AN but caused a steep decrease in the taste preference on the second presentation. This scopolamine-induced taste avoidance was not dependent on taste novelty, nor did it generalize to another novel taste. Targeting putative postsynaptic muscarinic receptors with specific M1 or M3 antagonists appeared to produce a partial taste avoidance, while M2 antagonism had no effect. These data suggest that if a salient gustatory experience is followed by muscarinic receptors antagonism in the BLA, it will be strongly and persistently avoided in the future. The study also shows that scopolamine is not just an amnesic drug, and its cognitive effects may be highly dependent on the task and the structure involved.

The Relationship between Muscarinic and Cannabinoid Receptors in Neuronal Excitability and Epilepsy: A Review

<b><i>Background:</i></b> Of the seventy million people who suffer from epilepsy, 40 percent of them become resistant to more than one antiepileptic medication and have a higher chance of death. While the classical definition of epilepsy was due to the imbalance between excitatory glutamatergic and inhibitory γ-aminobutyric acid (GABA)-ergic signalling, substantial evidence implicates muscarinic receptors in the regulation of neural excitability. <b><i>Summary:</i></b> Cannabinoids have shown to reduce seizure activity and neuronal excitability in several epileptic models through the activation of muscarinic receptors with drugs which modulate their activity. Cannabinoids also have been effective in reducing antiepileptic activity in pharmaco-resistant individuals; however, the mechanism of its effects in temporal lobe epilepsy is not clear. <b><i>Key Messages:</i></b> This review seeks to elucidate the relationship between muscarinic and cannabinoid receptors in epilepsy and neural excitability.

Dysregulated Cholinergic Signaling Inhibits Oligodendrocyte Maturation Following Demyelination.

Dysregulation of oligodendrocyte progenitor cell (OPC) recruitment and oligodendrocyte differentiation contribute to failure of remyelination in human demyelinating diseases such as multiple sclerosis (MS). Deletion of muscarinic receptor enhances OPC differentiation and remyelination. However, the role of ligand-dependent signaling versus constitutive receptor activation is unknown. We hypothesized that dysregulated acetylcholine (ACh) release upon demyelination contributes to ligand-mediated activation hindering myelin repair. Following chronic cuprizone (CPZ)-induced demyelination (male and female mice), we observed a 2.5-fold increase in ACh concentration. This increase in ACh concentration could be attributed to increased ACh synthesis or decreased acetylcholinesterase-/butyrylcholinesterase (BChE)-mediated degradation. Using choline acetyltransferase (ChAT) reporter mice, we identified increased ChAT-GFP expression following both lysolecithin and CPZ demyelination. ChAT-GFP expression was upregulated in a subset of injured and uninjured axons following intraspinal lysolecithin-induced demyelination. In CPZ-demyelinated corpus callosum, ChAT-GFP was observed in Gfap+ astrocytes and axons indicating the potential for neuronal and astrocytic ACh release. BChE expression was significantly decreased in the corpus callosum following CPZ demyelination. This decrease was due to the loss of myelinating oligodendrocytes which were the primary source of BChE. To determine the role of ligand-mediated muscarinic signaling following lysolecithin injection, we administered neostigmine, a cholinesterase inhibitor, to artificially raise ACh. We identified a dose-dependent decrease in mature oligodendrocyte density with no effect on OPC recruitment. Together, these results support a functional role of ligand-mediated activation of muscarinic receptors following demyelination and suggest that dysregulation of ACh homeostasis directly contributes to failure of remyelination in MS.

Cholinergic Mechanisms in Gastrointestinal Neoplasia.

Acetylcholine-activated receptors are divided broadly into two major structurally distinct classes: ligand-gated ion channel nicotinic and G-protein-coupled muscarinic receptors. Each class encompasses several structurally related receptor subtypes with distinct patterns of tissue expression and post-receptor signal transduction mechanisms. The activation of both nicotinic and muscarinic cholinergic receptors has been associated with the induction and progression of gastrointestinal neoplasia. Herein, after briefly reviewing the classification of acetylcholine-activated receptors and the role that nicotinic and muscarinic cholinergic signaling plays in normal digestive function, we consider the mechanics of acetylcholine synthesis and release by neuronal and non-neuronal cells in the gastrointestinal microenvironment, and current methodology and challenges in measuring serum and tissue acetylcholine levels accurately. Then, we critically evaluate the evidence that constitutive and ligand-induced activation of acetylcholine-activated receptors plays a role in promoting gastrointestinal neoplasia. We focus primarily on adenocarcinomas of the stomach, pancreas, and colon, because these cancers are particularly common worldwide and, when diagnosed at an advanced stage, are associated with very high rates of morbidity and mortality. Throughout this comprehensive review, we concentrate on identifying novel ways to leverage these observations for prognostic and therapeutic purposes.

Characterizing Muscarinic Receptor Subtype Roles in Inspiratory Burst Modulation of Hypoglossal Motoneurons in Mice

Loss of tongue muscle (innervated by hypoglossal (XII) motoneurons) tone is responsible for airway obstruction during sleep. Contributing factors include loss of noradrenergic drive and activation of muscarinic acetylcholine receptors (mAChRs). While noradrenergic effects are well studied, less is known about muscarinic effects. In mice, XII motoneurons express four mAChRs; M1, M3, and M5 are excitatory while M2 is inhibitory. We hypothesize that M2 is upregulated with postnatal maturation whereas M1, M3, and M5 are downregulated. We therefore expect that activation of M1, M3, and M5 receptors will potentiate inspiratory bursting early in postnatal development whereas activation of M2 receptors will inhibit inspiratory bursting later in postnatal development. To test this, we utilized rhythmic medullary slice preparations from mice at postnatal days 0-5 (P0-5) and P9-14, which contained preBötzinger Complex (site of inspiratory rhythm generation) and XII motoneurons. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarinic potentiation of inspiratory bursting in P0-5 mice at 100 μM (168±23% at 10 μM and 129±16% at 100 μM in muscarine [p<0.05] vs 183±34% muscarine (100 μM) control response, n=9). Activating M1 receptors locally with cevimeline (1 mM) for 30s or 60s did not mediate potentiation of inspiratory bursting as seen with muscarine (116±7% at 30s and 117±7% at 60s vs 181±40% control muscarine response [n=6, p<0.05]). Bath application of M2 antagonist methoctramine (MTH) in P0-5 mice at 1 μM had no effect on muscarine-potentiation of inspiratory bursting (207±109% at 1 μM MTH in muscarine vs 212±84% muscarine control response, n=9). Preliminary analysis of the P9-14 age group using 1 μM MTH suggests there may be an increase in muscarine-mediated potentiation of inspiratory bursting with M2 blockade (170±56% at 1 μM MTH in muscarine vs 152±56% muscarine control response, n=6, p>0.5). Bath application of M3 receptor antagonist 4-DAMP in P0-5 mice at 10 nM had no effect on muscarine-potentiated inspiratory bursting (172±12% vs 178±30% muscarine control), whereas application at 100 nM decreased muscarine-mediated inspiratory burst potentiation compared to control (133±12%, n=4, p<0.05). However, bath application with another M3 receptor antagonist, J Fumarate 104129, had no significant effect on muscarinic modulation of inspiratory bursting in P0-5 mice (188±46% at 0.6 nM [n=6], 185±32% at 6 nM [n=6], and 144±24% at 60 nM [n=4] vs 176±34% muscarine control [n=6]). Overall, our data partially supports a role of M1 receptors contributing to muscarinic potentiation of inspiratory bursting in XII motoneurons, with the contributions of M2 and M3 appearing insignificant early in postnatal life. Preliminary analysis suggests that M2 has a larger contribution later in maturation. Future research will evaluate the contribution of muscarinic receptor subtypes to inspiratory burst modulation later in postnatal life. NIH/NHLBI Funding: R15HL148870. This is the full abstract presented at the American Physiology Summit 2024 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.

Distribution of muscarinic acetylcholine receptors in hypoglossal motoneurons across postnatal maturation

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. During REM sleep, hypoglossal motoneurons (XII MNs), which innervate genioglossus, receive less excitatory noradrenergic drive and may be inhibited by activation of muscarinic acetylcholine receptors. Data from intact adult rats during natural sleep implicates an inhibitory effect of muscarinic acetylcholine receptors on XII MNs. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at XII MNs. Our project examines the changes to distribution of muscarinic acetylcholine receptors (mAChRs) at XII MNs across postnatal maturation. We hypothesize there is an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) mAChRs at XII MNs with postnatal maturation. We performed double-labeled immunofluorescence experiments on perfused transverse brainstem slices (20μm) under identical conditions across six postnatal age groups: P0-P2, P3-P5, P6-P10, P11-P13, P14-P17, and adult against mAChR targets M1, M2, M3, M5. Slices were co-labeled with choline acetyltransferase (ChAT) and 4’,6-diamidino-2-phenylindole (DAPI). Images were collected using a confocal microscope. 10 individual XII MNs were identified in each slice using positive ChAT labeling as well as anatomical location. ImageJ was used to determine the average XII MN mAChR subtype intensity. Preliminary data (n = 3) indicate that M1 receptors showed a transitory increase in labeling intensity in XII MNs followed by a decrease across development (P1=89±11%, P4-5=100%, P9=76±18%, P12-13=67±8%, P17=67±4%, adult=56±7%, p = 0.13). M3 (n = 3) showed a trend for a transient increase in labeling intensity at P17 (P1=78±41%, P4-5=87±3%, P9=61±13%, P12-13=86±34%, P17=100%, adult=80±23, p = 0.60). M5 (n = 3) expression intensity decreased consistently over maturation (P1=100%, P4-5=92±34%, P9=75±23%, P12-13=70±10%, P17=66±14%, adult=39±12%, p = 0.0257). In contrast, M2 (n = 3) receptor expression intensity remained relatively consistent across maturation (P1 = 88±23%, P4-5 = 100%, P9=88±10%, P12-13 = 75±15%, P17 = 86±18%, adult = 75±10%). These data partly support our hypothesis of a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors across maturation. The decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift in muscarinic modulation from excitation to inhibition with postnatal maturation. Arizona Alzheimer's Consortium, NIH. This is the full abstract presented at the American Physiology Summit 2024 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.

Endogenous Acetylcholine Contributes to Flow-Mediated Dilation in Humans

Background & Objective. Flow-mediated dilation (FMD) is a crucial function of the vascular endothelium wherein intraluminal shear stress stimulates endothelium-dependent vasodilation mediated primarily by nitric oxide. Brachial artery FMD (baFMD) is widely evaluated in clinical research as an index of endothelial function, and impaired baFMD is an independent predictor of cardiovascular risk. Despite the physiological and clinical relevance of FMD, the mechanisms by which shear stress is transduced to endothelium-dependent vasodilation (i.e., upstream of nitric oxide production) remain unclear. baFMD corresponds closely with sensitivity to the endothelium-dependent vasodilator acetylcholine (ACh), and preclinical models suggest ACh is released from endothelial cells in response to shear stress. Therefore, the present study tested the hypothesis that activation of muscarinic ACh receptors facilitates baFMD in humans. Methods. baFMD was evaluated using Doppler ultrasound in healthy participants (n=11; 5F/6M; 23±2 y; means ± SE) under control conditions and following local inhibition of muscarinic ACh receptors via intra-arterial atropine infused proximal to the measurement site. baFMD was assessed in response to transient and sustained shear stimuli in separate trials: 1) reactive hyperemia following 5 min of arterial cuff occlusion (RH-FMD) and 2) steady-state rhythmic handgrip exercise at graded intensities (10, 15, 20, & 25% maximal voluntary contraction, MVC) to produce step increases in shear rate (EX-FMD). Blockade effcacy was confirmed via a dose-response to intra-arterial ACh infusion pre and post atropine. Results. Baseline brachial artery diameter and forearm blood flow were similar in all trials (all P>0.05). Atropine reduced peak brachial artery dilation following cuff release (RH-FMD, Δ diameter, atropine: 4.6±0.8 vs. control: 6.5±0.8%, P=0.04) and increased the shear stimulus (shear rate area under the curve, SRAUC: atropine, 29±3 vs. control: 21±3 s−1×103, P=0.01) such that the percent change in brachial diameter was diminished by 35±16% when normalized to SRAUC (baFMD:SRAUC ratio, atropine: 1.7±0.3 vs. control: 3.9±1.0, P=0.02). Similarly, in the EX-FMD trial, atropine reduced brachial artery diameter at all exercise intensities (25% MVC, percent change in diameter: atropine, 10.8±1.4 vs. control, 15.5±1.7%, P=0.04) despite a greater shear stimulus (shear rate: atropine, 10.9±1.1 vs. control, 9.3±0.9 s−1×102, P=0.01). Thus, atropine reduced the slope describing the relationship between brachial artery vasodilation (percent change) and the change in shear rate by 38±8% across all exercise intensities (slope, atropine: 1.3±0.1 vs. control: 2.4±0.4% Δ×s×10−2, P=0.007). As expected, atropine reduced brachial artery vasodilation in response to upstream infusion of ACh (1.5 μg ACh/dl forearm volume/min, Δ diameter, atropine: 2±1 vs. control: 23±4%, P=0.001). Conclusions. The results suggest endogenous ACh facilitates baFMD in response to both transient and sustained shear stimuli, which highlights a novel physiological role of endogenous ACh in vascular regulation in humans and provides insight regarding the basic mechanisms of flow-mediated vasodilation. NIH R01 HL119337 and F31 HL142240. This is the full abstract presented at the American Physiology Summit 2024 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.

Efficacy and Safety of Xanomeline-Trospium Chloride in Schizophrenia

A significant need exists for new antipsychotic medications with different mechanisms of action, greater efficacy, and better tolerability than existing agents. Xanomeline is a dual M1/M4 preferring muscarinic receptor agonist with no direct D2 dopamine receptor blocking activity. KarXT combines xanomeline with the peripheral muscarinic receptor antagonist trospium chloride with the goal of reducing adverse events due to xanomeline-related peripheral muscarinic receptor activation. In prior trials, xanomeline-trospium chloride was effective in reducing symptoms of psychosis and generally well tolerated in people with schizophrenia. To evaluate the efficacy and safety of xanomeline-trospium vs placebo in adults with schizophrenia. EMERGENT-3 (NCT04738123) was a phase 3, multicenter, randomized, double-blind, placebo-controlled, 5-week trial of xanomeline-trospium in people with schizophrenia experiencing acute psychosis, conducted between April 1, 2021, and December 7, 2022, at 30 inpatient sites in the US and Ukraine. Data were analyzed from February to June 2023. Participants were randomized 1:1 to receive xanomeline-trospium chloride (maximum dose xanomeline 125 mg/trospium 30 mg) or placebo for 5 weeks. The prespecified primary end point was change from baseline to week 5 in Positive and Negative Syndrome Scale (PANSS) total score. Secondary outcome measures were change from baseline to week 5 in PANSS positive subscale score, PANSS negative subscale score, PANSS Marder negative factor score, Clinical Global Impression-Severity score, and proportion of participants with at least a 30% reduction in PANSS total score. Safety and tolerability were also evaluated. A total of 256 participants (mean [SD] age, 43.1 [11.8] years; 191 men [74.6%]; 156 of 256 participants [60.9%] were Black or African American, 98 [38.3%] were White, and 1 [0.4%] was Asian) were randomized (125 in xanomeline-trospium group and 131 in placebo group). At week 5, xanomeline-trospium significantly reduced PANSS total score compared with placebo (xanomeline-trospium , -20.6; placebo, -12.2; least squares mean difference, -8.4; 95% CI, -12.4 to -4.3; P < .001; Cohen d effect size, 0.60). Discontinuation rates due to treatment-emergent adverse events (TEAEs) were similar between the xanomeline-trospium (8 participants [6.4%]) and placebo (7 participants [5.5%]) groups. The most common TEAEs in the xanomeline-trospium vs placebo group were nausea (24 participants [19.2%] vs 2 participants [1.6%]), dyspepsia (20 participants [16.0%] vs 2 participants [1.6%]), vomiting (20 participants [16.0%] vs 1 participant [0.8%]), and constipation (16 participants [12.8%] vs 5 participants [3.9%]). Measures of extrapyramidal symptoms, weight gain, and somnolence were similar between treatment groups. Xanomeline-trospium was efficacious and well tolerated in people with schizophrenia experiencing acute psychosis. These findings, together with the previously reported and consistent results from the EMERGENT-1 and EMERGENT-2 trials, support the potential of xanomeline-trospium to be the first in a putative new class of antipsychotic medications without D2 dopamine receptor blocking activity. ClinicalTrials.gov Identifier: NCT04738123.

Activation of M4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

Current pharmacotherapies for Tourette syndrome (TS) are often unsatisfactory and poorly tolerated, underscoring the need for novel treatments. Insufficient striatal acetylcholine has been suggested to contribute to tic ontogeny. Thus, we tested whether activating M1 and/or M4 receptors-the two most abundant muscarinic receptors in the striatum-reduced tic-related behaviours in mouse models of TS. Studies were conducted using CIN-d and D1CT-7 mice, two TS models characterized by early-life depletion of striatal cholinergic interneurons and cortical neuropotentiation, respectively. First, we tested the effects of systemic and intrastriatal xanomeline, a selective M1/M4 receptor agonist, on tic-like and other TS-related responses. Then, we examined whether xanomeline effects were reduced by either M1 or M4 antagonists or mimicked by the M1/M3 agonist cevimeline or the M4 positive allosteric modulator (PAM) VU0467154. Finally, we measured striatal levels of M1 and M4 receptors and assessed the impact of VU0461754 on the striatal expression of the neural marker activity c-Fos. Systemic and intrastriatal xanomeline reduced TS-related behaviours in CIN-d and D1CT-7 mice. Most effects were blocked by M4, but not M1, receptor antagonists. VU0467154, but not cevimeline, elicited xanomeline-like ameliorative effects in both models. M4, but not M1, receptors were down-regulated in the striatum of CIN-d mice. Additionally, VU0467154 reduced striatal c-Fos levels in these animals. Activation of striatal M4, but not M1, receptors reduced tic-like manifestations in mouse models, pointing to xanomeline and M4 PAMs as novel putative therapeutic strategies for TS.

Agonist-selective activation of individual G-proteins by muscarinic receptors

Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

Acetylcholine Engages Distinct Amygdala Microcircuits to Gate Internal Theta Rhythm.

Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in the basolateral amygdala (BLA) in both humans and rodents have been shown to underlie emotional memory, yet their mechanism remains unclear. Here, using brain slice electrophysiology in male and female mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potentials that depend upon M3 muscarinic receptor activation of cholecystokinin (CCK) interneurons (INs) without the need for external glutamate signaling. Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, providing a functional SOM→CCK IN circuit connection gating BLA theta. Parvalbumin (PV) INs, which can drive BLA oscillations in baseline states, are not involved in the generation of ACh-induced theta, highlighting that ACh induces a cellular switch in the control of BLA oscillatory activity and establishes an internally BLA-driven theta oscillation through CCK INs. Theta activity is more readily evoked in BLA over the cortex or hippocampus, suggesting preferential activation of the BLA during high ACh states. These data reveal a SOM→CCK IN circuit in the BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.

Prevention of MK-801-induced amnestic effect with combined activation of 5-HT1A and muscarinic receptors in mice

BackgroundMuscarinic or 5-HT1A receptors are crucial in learning and memory processes, and their expression is evident in the brain areas involved in cognition. The administration of the activators of these receptors prevents the development of cognitive dysfunctions in animal models of schizophrenia induced by MK-801 (N-methyl-d-aspartate receptor antagonist) administration. GABAergic dysfunction is considered as one of the most important causes of MK-801-induced spatial learning deficits. MethodsNovel object recognition (NOR) and Morris water maze (MWM) tests were used to study the anti-amnestic effect of the biased 5-HT1A receptor agonist (F15599) alone or in combinations with VU0357017 (M1 receptor allosteric agonist), VU0152100 (M4 receptor positive allosteric modulator), and VU0238429 (M5 receptor positive allosteric modulator) on MK-801-induced dysfunctions. The compounds were administered for 5 consecutive days. Animals tested with the MWM underwent 5-day training. Western blotting was used to study the expressions of 5-HT1A receptors and the level of GAD65 in the frontal cortices (FCs) and hippocampi of the animals. ResultsF15599 prevented the amnestic effect induced by MK-801 in the MWM at a dose of 0.1 mg/kg. The co-administration of the compound with muscarinic receptors activators had no synergistic effect. The additive effect of the combinations was evident in the prevention of declarative memory dysfunctions investigated in NOR. The administration of MK-801 impaired 5-HT1A expression in the hippocampi and decreased GAD65 levels in both the FCs and hippocampi. The administration of muscarinic ligands prevented these MK-801-induced deficits only in the hippocampi of MWM-trained animals. No effects of the compounds were observed in untrained mice. ConclusionOur results indicate that F15599 prevents schizophrenia-related spatial learning deficits in the MWM; however, the activity of the compound is not intensified with muscarinic receptors activators. In contrast, the combined administration of the ligands is effective in the NOR model of declarative memory. The muscarinic receptors activators reversed MK-801-induced 5-HT1A and GAD65 dysfunctions in the hippocampi of MWM-trained mice, but not in untrained mice.

Cholinergic signaling via muscarinic M1 receptor confers resistance to docetaxel in prostate cancer

Docetaxel is the most commonly used chemotherapy for advanced prostate cancer (PC), including castration-resistant disease (CRPC), but the eventual development of docetaxel resistance constitutes a major clinical challenge. Here, we demonstrate activation of the cholinergic muscarinic M1 receptor (CHRM1) in CRPC cells upon acquiring resistance to docetaxel, which is manifested in tumor tissues from PC patients post- vs. pre-docetaxel. Genetic and pharmacological inactivation of CHRM1 restores the efficacy of docetaxel in resistant cells. Mechanistically, CHRM1, via its first and third extracellular loops, interacts with the SEMA domain of cMET and forms a heteroreceptor complex with cMET, stimulating a downstream mitogen-activated protein polykinase program to confer docetaxel resistance. Dicyclomine, a clinically available CHRM1-selective antagonist, reverts resistance and restricts the growth of multiple docetaxel-resistant CRPC cell lines and patient-derived xenografts. Our study reveals a CHRM1-dictated mechanism for docetaxel resistance and identifies a CHRM1-targeted combinatorial strategy for overcoming docetaxel resistance in PC.

Muscarinic receptor activation promotes destabilization and updating of object location memories in mice

The storage of long-term memories is a dynamic process. Reminder cues can destabilize previously consolidated memories, rendering them labile and modifiable. However, memories that are strongly encoded or relatively remote at the time of reactivation can resist destabilization only being rendered labile under conditions that favour memory updating. Using the object location recognition task, here we show in male C57BL/6 mice that novelty-induced destabilization of strongly-encoded memories requires muscarinic acetylcholine receptor (mAChR) activation. Furthermore, we use the objects-in-updated locations task to show that updating of object location memories is mAChR-dependent. Thus, mAChR stimulation appears to be critical for spatial memory destabilization and related memory updating. Enhancing our understanding of the role of ACh in memory updating should inform future research into the underlying causes of behavioural disorders that are characterized by persistent maladaptive memories, such as age-related cognitive inflexibility and post-traumatic stress disorder.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Activation of M1 muscarinic acetylcholine receptors by proline-rich oligopeptide 7a (

The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability.

Increased GIRK channel activity prevents arrhythmia in mice with heart failure by enhancing ventricular repolarization

Ventricular arrhythmia causing sudden cardiac death is the leading mode of death in patients with heart failure. Yet, the mechanisms that prevent ventricular arrhythmias in heart failure are not well characterized. Using a mouse model of heart failure created by transverse aorta constriction, we show that GIRK channel, an important regulator of cardiac action potentials, is constitutively active in failing ventricles in contrast to normal cells. Evidence is presented indicating that the tonic activation of M2 muscarinic acetylcholine receptors by endogenously released acetylcholine contributes to the constitutive GIRK activity. This constitutive GIRK activity prevents the action potential prolongation in heart failure ventricles. Consistently, GIRK channel blockade with tertiapin-Q induces QT interval prolongation and increases the incidence of arrhythmia in heart failure, but not in control mice. These results suggest that constitutive GIRK channels comprise a key mechanism to protect against arrhythmia by providing repolarizing currents in heart failure ventricles.

Acetylcholine Receptor Agonists Exerted Neuroprotection Against Doxorubicin‐Induced Chemobrain

AbstractBackgroundAlthough doxorubicin has been broadly used in treating a wide range of cancers, it frequently causes a serious neurological consequences referred to as chemobrain in cancer patients. Chemobrain shares several key pathological features with Alzheimer’s disease (AD) including neuroinflammation and Tau pathology. In addition to the inhibition of acetylcholinesterase, the activation of a7nicotinic and muscarinic acetylcholine receptors (a7nAChR and mAChR) using their agonists has been considered an effective therapeutic target against dementia. However, the benefit of intervention with nAChR and mAChR agonists in DOX‐induced chemobrain has never been investigated.MethodTwenty‐five male Wistar rats were assigned to received either (0.9% NSS) or doxorubicin (3 mg/kg; 6 doses) via intraperitoneal injection. Then, doxorubicin‐treated rats were divided into 4 intervention groups to treat with either vehicle, an a7nAChR agonist (PNU‐282987; 3 mg/kg), a mAChR agonist (bethanechol; 12 mg/kg), or a combined treatment of PNU‐282987 and bethanechol via intraperitoneal injection as a concomitant treatment with doxorubicin for a period of 30 days. After completion of the treatment paradigm, cognitive function was evaluated using the novel object location task (NOLT). The hippocampal tissues were dissected to enable further investigation.ResultSystemic administration of doxorubicin impaired long‐term cognitive function in rats as shown by a decreased preference index in the NOLT (Figure A). Furthermore, doxorubicin‐treated rats exhibited neuroinflammation in the hippocampus as demonstrated by an increase in TNF‐a protein expression (Figure B) and an alteration in microglial morphology to a pro‐inflammatory amoeboid‐liked phenotype (Figure C). Notably, injection of doxorubicin markedly reduced the phosphorylation of GSK3b at Ser9 (Figure D) and upregulated the phosphorylation of Tau at Thr181 (Figure E). Also, a decline in the dendritic spine density in the CA1 region was observed in doxorubicin‐treated rats compared with the controls (Figure F). Interestingly, all AChR agonists restored cognitive function in rats in a similar manner by attenuating neuroinflammation and Tau phosphorylation while preserving dendritic spine density (Figure A‐F).ConclusionTogether, activation of AChRs by their agonists protected against the neuropathology of doxorubicin‐induced chemobrain, indicating their potential role as a novel strategy to prevent chemobrain in patients receiving chemotherapy.