mAChR Activation Research Articles

Muscarinic modulation of the Xenopus laevis tadpole spinal mechanosensory pathway

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in spinal functions like locomotion. In Xenopus laevis tadpoles at two days old, a model vertebrate for motor control research, we investigated the role of mAChRs in the skin mechanosensory pathway. We found that mAChR activation by carbachol did not affect the sensory Rohon-Beard neuron properties. However, carbachol could hyperpolarise sensory interneurons and decrease their voltage responses to outward currents. Carbachol could increase the threshold for the mechanosensory pathway to start swimming, preventing the initiation of swimming at higher concentrations altogether. Recording from the sensory interneurons in carbachol showed that their spiking after skin stimulation was depressed. However, the general muscarinic antagonist atropine did not have a clear effect on the swimming threshold or the modulation of sensory interneuron membrane conductance. Our results suggest the skin mechanosensory pathway may be subject to muscarinic modulation in this simple vertebrate system.

M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

Heat shock protein (Hsp) regulation by muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus.

The cholinergic system plays a crucial role in modulating in the central nervous system physiological responses such as neurogenesis, neuronal differentiation, synaptic plasticity, and neuroprotection. In a recent study, we showed that Oxotremorine-M, a non-selective muscarinic acetylcholine receptor agonist, is able to transactivate the fibroblast growth factor receptor and to produce a significant increase in the hippocampal primary neurite outgrowth. In the present study we aimed to explore in the rat hippocampus the possible effect of acute or chronic treatment with Oxotremorine-M on some heat shock proteins (Hsp60, Hsp70, Hsp90) and on activation of related transcription factor heat shock factor 1 (HSF1). Following single injection of Oxotremorine-M (0.4 mg/kg) all Hsps examined were significantly increased in at least one of the time points studied (24, 48, and 72 hr). Treatment with Oxotremorine-M significantly increased the level of phosphorylated HSF1 in all time points studied, without change of protein levels. Similar pattern of Hsps changes was obtained following chronic Oxotremorine-M treatment (0.2 mg/kg) for 5 days. Surprisingly, following chronic treatment for 10 days no changes were observed in Hsps. The muscarinic acetylcholine receptor antagonist scopolamine (1 mg/kg) was able to completely block Oxotremorine-M effects on Hsps. In conclusion, considering the function of Hsps in protecting neuronal cells from deleterious proteotoxic stress, for example, protein mis-folding and aggregation, the results obtained indicate that muscarinic acetylcholine receptor activation may have implications in potential treatment of neurodegenerative disorders linked to protein aggregation, such as Alzheimer disease.

Inhibition of ZERO‐BK by PKC is involved in carbachol‐induced enhancement of rat colon smooth muscle motility

Muscarinic acetylcholine receptor (mAChR) activation is an important factor to enhance the motility of gastrointestinal (GI) smooth muscle. Large conductance Ca2+ -activated potassium (BK) channels are widely expressed in GI smooth muscle. Roles of BK in carbachol (a mAChR agonist) induced enhancement of GI motility and the molecular mechanisms remains unknown and were investigated in this study. Colonic smooth muscle (CSM) strip was perfused to record motility in vitro. The patch-clamp technique was used to record BK currents. RT-PCR was used to detect the expression of BK channels in rat CSM tissues. Two different types BK channels were constructed in HEK293 cells to investigate the regulation mechanism. Paired t tests were set with a P<.05 regarded as significant. Carbachol enhanced CSM contraction through M3 receptor (M3 R) were attenuated by IbTX, an inhibitor of BK. Carbachol inhibited BK currents in CSM cells and Go6983, an inhibitor of protein kinase C (PKC), reversed the effect. PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited BK currents. Two types of BK channels (ZERO-BK and STREX-BK) were detected in CSM. ZERO- but not STREX-BK channels expressed in HEK293 cells were inhibited by PMA. Our results provide strong evidence that inhibition of ZERO-BK but not STREX-BK channels via PKC pathway is involved in the enhancement of CSM motility by mAChR activation. Besides the activation of BK by an increase in intracellular calcium, inhibition of BK played an important role in GI motility regulation during mAChR activation.

Muscarinic M1 Receptor Modulation of Synaptic Plasticity in Nucleus Accumbens of Wild-Type and Fragile X Mice.

We investigated how metabotropic acetylcholine receptors control excitatory synaptic plasticity in the mouse nucleus accumbens core. Pharmacological and genetic approaches revealed that M1 mAChRs (muscarinic acetylcholine receptors) trigger multiple and interacting forms of synaptic plasticity. As previously described in the dorsal striatum, moderate pharmacological activation of M1 mAChR potentiated postsynaptic NMDARs. The M1-potentiation of NMDAR masked a previously unknown coincident TRPV1-mediated long-term depression (LTD). In addition, strong pharmacological activation of M1 mAChR induced canonical retrograde LTD, mediated by presynaptic CB1R. In the fmr1-/y mouse model of Fragile X, we found that CB1R but not TRPV1 M1-LTD was impaired. Finally, pharmacological blockade of the degradation of anandamide and 2-arachidonylglycerol, the two principal endocannabinoids restored fmr1-/y LTD to wild-type levels. These findings shed new light on the complex influence of acetylcholine on excitatory synapses in the nucleus accumbens core and identify new substrates of the synaptic deficits of Fragile X.

Discovery and Development of Muscarinic Acetylcholine M4 Activators as Promising Therapeutic Agents for CNS Diseases.

Among the muscarinic acetylcholine receptor (mAChR) subtypes, the M4 receptor has been investigated as a promising drug target for the treatment of schizophrenia. These investigations have been based on findings from M4-deficient mice studies as well as on the results of a clinical trial that used xanomeline, an M1/M4 mAChRs-preferring agonist. Both orthosteric agonists and positive allosteric modulators of M4 mAChR have been reported as promising ligands that not only have antipsychotic effects, but can also improve cognitive impairment and motor dysfunction. However, challenges remain due to the high homology of the orthosteric binding site among all muscarinic receptors. In this review, we summarize our approach to the identification of M4 mAChR activators, orthosteric agonists, and positive allosteric modulators based on M4 mAChR structural information and structure-activity relationship studies. These findings indicate that selective M4 mAChR activators are promising potential therapeutic agents for several central nervous system conditions.

Tonic inhibition of brown adipose tissue sympathetic nerve activity via muscarinic acetylcholine receptors in the rostral raphe pallidus.

A tonically active, muscarinic cholinergic inhibition of rostral raphe pallidus (rRPa) neurons influences thermogenesis of brown adipose tissue (BAT) independent of ambient temperature conditions. The tonically active cholinergic input to rRPa originates caudal to the hypothalamus. Muscarinic acetylcholine receptor (mAChR) activation in rRPa contributes to the inhibition of BAT sympathetic nerve activity (SNA) evoked by activation of neurons in the rostral ventrolateral medulla (RVLM). The RVLM is not the sole source of the muscarinic cholinergic input to rRPa. Activation of GABA receptors in rRPa does not mediate the cholinergic inhibition of BAT SNA. We sought to determine if body temperature and energy expenditure are influenced by a cholinergic input to neurons in the rostral raphe pallidus (rRPa), the site of sympathetic premotor neurons controlling thermogenesis of brown adipose tissue (BAT). Nanoinjections of the muscarinic acetylcholine receptor (mAChR) agonist, oxotremorine, or the cholinesterase inhibitor, neostigmine (NEOS), in the rRPa of anaesthetized rats decreased cold-evoked BAT sympathetic nerve activity (SNA, nadirs: -72 and -95%), BAT temperature (Tbat, -0.5 and -0.6°C), expired CO2 (Exp. CO2 , -0.3 and -0.5%) and heart rate (HR, -22 and -41bpm). NEOS into rRPa reversed the increase in BAT SNA evoked by blockade of GABA receptors in rRPa. Nanoinjections of the mAChR antagonist, scopolamine (SCOP), in the rRPa of warm rats increased BAT SNA (peak: +1087%), Tbat (+1.8°C), Exp. CO2 (+0.7%), core temperature (Tcore, +0.5°C) and HR (+54bpm). SCOP nanoinjections in rRPa produced similar activations of BAT during cold exposure, following a brain transection caudal to the hypothalamus, and during the blockade of glutamate receptors in rRPa. We conclude that a tonically active cholinergic input to the rRPa inhibits BAT SNA via activation of local mAChR. The inhibition of BAT SNA mediated by mAChR in rRPa does not depend on activation of GABA receptors in rRPa. The increase in BAT SNA following mAChR blockade in rRPa does not depend on the activity of neurons in the hypothalamus or on glutamate receptor activation in rRPa.

Linking muscarinic receptor activation to UPS-mediated object memory destabilization: Implications for long-term memory modification and storage

Consolidated memories can become destabilized during reactivation, resulting in a transient state of instability, a process that has been hypothesized to underlie long-term memory updating. Consistent with this notion, relatively remote memories, which are resistant to standard destabilization procedures, are reliably destabilized when novel information (i.e., the opportunity for memory updating) is present during reactivation. We have also shown that cholinergic muscarinic receptor (mAChR) activation can similarly destabilize consolidated object memories. Synaptic protein degradation via the ubiquitin proteasome system (UPS) has previously been linked to destabilization of fear and object-location memories. Given the role of calcium in regulating proteasome activity, we hypothesized that activation of cholinergic receptors, specifically M1 mAChRs, stimulates the UPS via inositol triphosphate receptor (IP3R)-mediated release of intracellular calcium stores to facilitate object memory destabilization. We present converging evidence for this hypothesis, which we tested using a modified spontaneous object recognition task for rats and microinfusions into perirhinal cortex (PRh), a brain region strongly implicated in object memory. We extend our previous findings by demonstrating that M1 mAChRs are necessary for novelty-induced object memory destabilization. We also show that proteasome inhibition or IP3R antagonism in PRh prevents object memory destabilization induced by novelty or M1 mAChR stimulation. These results establish an intracellular pathway linking M1 receptors, IP3Rs, and UPS activity to object memory destabilization and suggest a previously unacknowledged role for cholinergic signaling in long-term memory modification and storage.

Cracking the Betel Nut: Cholinergic Activity of Areca Alkaloids and Related Compounds.

The use of betel quid is the most understudied major addiction in the world. The neuropsychological activity of betel quid has been attributed to alkaloids of Areca catechu. With the goal of developing novel addiction treatments, we evaluate the muscarinic and nicotinic activity of the four major Areca alkaloids: arecoline, arecaidine, guvacoline, and guvacine and four structurally related compounds. Acetylcholine receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Both arecoline- and guvacoline-activated muscarinic acetylcholine receptors (mAChR), while only arecoline produced significant activation of nicotinic AChR (nAChR). We characterized four additional arecoline-related compounds, seeking an analog that would retain selective activity for a α4* nAChR, with diminished effects on mAChR and not be a desensitizer of α7 nAChR. We show that this profile is largely met by isoarecolone. Three additional arecoline analogs were characterized. While the quaternary dimethyl analog had a broad range of activities, including activation of mAChR and muscle-type nAChR, the methyl analog only activated a range of α4* nAChR, albeit with low potency. The ethyl analog had no detectable cholinergic activity. Evidence indicates that α4* nAChR are at the root of nicotine addiction, and this may also be the case for betel addiction. Our characterization of isoarecolone and 1-(4-methylpiperazin-1-yl) ethanone as truly selective α4*nAChR selective partial agonists with low muscarinic activity may point toward a promising new direction for the development of drugs to treat both nicotine and betel addiction. Nearly 600 million people use Areca nut, often with tobacco. Two of the Areca alkaloids are muscarinic acetylcholine receptor agonists, and one, arecoline, is a partial agonist for the α4* nicotinic acetylcholine receptors (nAChR) associated with tobacco addiction. The profile of arecoline activity suggested its potential to be used as a scaffold for developing new tobacco cessation drugs if analogs can be identified that retain the same nicotinic receptor selectivity without muscarinic activity. We report that isoarecolone is a selective partial agonist for α4* nAChR with minimal muscarinic activity and 1-(4-methylpiperazin-1-yl) ethanone has similar nAChR selectivity and no detectable muscarinic action.

Cognitive enhancement and antipsychotic-like activity following repeated dosing with the selective M4 PAM VU0467154

Although selective activation of the M1 muscarinic acetylcholine receptor (mAChR) subtype has been shown to improve cognitive function in animal models of neuropsychiatric disorders, recent evidence suggests that enhancing M4 mAChR function can also improve memory performance. Positive allosteric modulators (PAMs) targeting the M4 mAChR subtype have shown therapeutic potential for the treatment of multiple symptoms observed in schizophrenia, including positive and cognitive symptoms when assessed in acute preclinical dosing paradigms. Since the cholinergic system has been implicated in multiple stages of learning and memory, we evaluated the effects of repeated dosing with the highly selective M4 PAM VU0467154 on either acquisition and/or consolidation of learning and memory when dosed alone or after pharmacologic challenge with the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR) antagonist MK-801. MK-801 challenge represents a well-documented preclinical model of NMDAR hypofunction that is thought to underlie some of the positive and cognitive symptoms observed in schizophrenia. In wildtype mice, 10-day, once-daily dosing of VU0467154 either prior to, or immediately after daily testing enhanced the rate of learning in a touchscreen visual pairwise discrimination task; these effects were absent in M4 mAChR knockout mice. Following a similar 10-day, once-daily dosing regimen of VU0467154, we also observed 1) improved acquisition of memory in a cue-mediated conditioned freezing paradigm, 2) attenuation of MK-801-induced disruptions in the acquisition of memory in a context-mediated conditioned freezing paradigm and 3) reversal of MK-801-induced hyperlocomotion. Comparable efficacy and plasma and brain concentrations of VU0467154 were observed after repeated dosing as those previously reported with an acute, single dose administration of this M4 PAM. Together, these studies are the first to demonstrate that cognitive enhancing and antipsychotic-like activity are not subject to the development of tolerance following repeated dosing with a selective M4 PAM in mice and further suggest that activation of M4 mAChRs may modulate both acquisition and consolidation of memory functions.

L-Satropane Prevents Retinal Neuron Damage by Attenuating Cell Apoptosis and Aβ Production via Activation of M1 Muscarinic Acetylcholine Receptor

ABSTRACTMuscarinic acetylcholine receptor (mAChR) agonists have been used to treat glaucoma due to their intraocular pressure-lowering effects. Recently, it has been reported that retinal mAChRs activation can also stimulate neuroprotective pathways.Purpose: In our study, we evaluated the potential neuroprotective effect of L-satropane, a novel mAChR agonist, on retinal neuronal injury induced by cobalt chloride (CoCl2) and ischemia/reperfusion (I/R).Methods: CoCl2-induced hypoxia injury in cultured cell models and I/R-induced retinal neuronal damage in rats in vivo were used to evaluate the abilities of L-satropane. In detail, we measured the occurrence of retinal pathological changes including molecular markers of neuronal apoptosis and Aβ expression.Results: Pretreatment with L-satropane protects against CoCl2-induced neurotoxicity in PC12 and primary retinal neuron (PRN) cells in a dose-dependent manner by increasing retinal neuron survival. CoCl2 or I/R-induced cell apoptosis by upregulating Bax expression and downregulating Bcl-2 expression, which resulted in an increased Bax/Bcl-2 ratio, and upregulating caspase-3 expression/activity was significantly reversed by L-satropane treatment. In addition, L-satropane significantly inhibited the upregulation of Aβ production in both retinal neurons and tissue. We also found that I/R-induced histopathological retinal changes including cell loss in the retinal ganglion cell layer (GCL) and increased TUNEL positive retinal ganglion cells in GCL and thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) were markedly improved by L-satropane. The effects of L-satropane were largely abolished by the nonselective mAChRs antagonist atropine and M1-selective mAChR antagonist pirenzepine.Conclusion: These results demonstrated that L-satropane might be effective in preventing retinal neuron damage caused by CoCl2 or I/R. The neuroprotective effects of L-satropane may be attributed to decreasing cell apoptosis and Aβ production through activation of M1 mAChR.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca2+ oscillations in bEND5 cells, 300 μM being the most effective dose to generate a prolonged Ca2+ burst. Pharmacological manipulation revealed that Ach-evoked Ca2+ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca2+ release via inositol-1,4,5-trisphosphate receptors (InsP3Rs) and store-operated Ca2+ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP3R1 and InsP3R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

Strength of cholinergic tone dictates the polarity of dopamine D2 receptor modulation of striatal cholinergic interneuron excitability in DYT1 dystonia

Balance between cholinergic and dopaminergic signaling is central to striatal control of movement and cognition. In dystonia, a common disorder of movement, anticholinergic therapy is often beneficial. This observation suggests there is a pathological increase in cholinergic tone, yet direct confirmation is lacking. In DYT1, an early-onset genetic form of dystonia caused by a mutation in the protein torsinA (TorA), the suspected heightened cholinergic tone is commonly attributed to faulty dopamine D2 receptor (D2R) signaling where D2R agonists cause excitation of striatal cholinergic interneurons (ChIs), rather than the normal inhibition of firing observed in wild-type animals, an effect known as “paradoxical excitation”. Here, we provide for the first time direct measurement of elevated striatal extracellular acetylcholine (ACh) in a knock-in mouse model of human DYT1 dystonia (TorA∆E/+ mice), confirming a striatal hypercholinergic state. We hypothesized that this elevated extracellular ACh might cause chronic over-activation of muscarinic acetylcholine receptors (mAChRs) and disrupt normal D2R function due to their shared coupling to Gi/o-proteins. We tested this concept in vitro first using a broad-spectrum mAChR antagonist, and then using a M2/M4 mAChR selective antagonist to specifically target mAChRs expressed by ChIs. Remarkably, we found that mAChR inhibition reverses the D2R-mediated paradoxical excitation of ChIs recorded in slices from TorA∆E/+ mice to a typical inhibitory response. Furthermore, we recapitulated the paradoxical D2R excitation of ChIs in striatal slices from wild-type mice within minutes by simply increasing cholinergic tone through pharmacological inhibition of acetylcholinesterase (AChE) or by prolonged agonist activation of mAChRs. Collectively, these results show that enhanced mAChR tone itself is sufficient to rapidly reverse the polarity of D2R regulation of ChI excitability, correcting the previous notion that the D2R mediated paradoxical ChI excitation causes the hypercholinergic state in dystonia. Further, using a combination of genetic and pharmacological approaches, we found evidence that this switch in D2R polarity results from a change in coupling from the preferred Gi/o pathway to non-canonical β-arrestin signaling. These results highlight the need to fully understand how the mutation in TorA leads to pathologically heightened extracellular ACh. Furthermore the discovery of this novel ACh-dopamine interaction and the participation of β-arrestin in regulation of cholinergic interneurons is likely important for other basal ganglia disorders characterized by perturbation of ACh-dopamine balance, including Parkinson and Huntington diseases, l-DOPA-induced dyskinesia and schizophrenia.

A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation.

M3 Muscarinic Acetylcholine Receptors Inhibit Autophagy through Activation of mTORC1 in Human Neuroblastoma Cells.

Activation of Gq/11‐coupled muscarinic acetylcholine receptors (mAChRs) has been shown to promote the growth and survival of different types of cancers derived from epithelial and endothelial cells [1]. Previous studies in human neuroblastoma cells have reported that M3 mAChRs prevent apoptotic cell death induced by a number of cytotoxic agents [2,3]. However, there is little information on the effect of mAChRs on autophagy, an evolutionary conserved self‐degradation process that may act as a tumor‐suppressor pathway. In the present study we show that in human SH‐SY5Y neuroblastoma cells activation of M3 mAChRs inhibits autophagy triggered by serum and L‐glutamine removal from the growth medium. Cell exposure to either the cholinergic agonist carbachol (CCh) (30 μM) or the mAChR agonist oxotremorine‐M (100 μM) inhibited the formation of the autophagosome marker LC3‐II and increased the accumulation of the autophagy substrate p62. These effects were blocked by either atropine or the M3 mAChR‐preferring antagonist darifenacin, but not MT7, a selective M1 mAChR antagonist. The inhibitory effect of CCh on autophagy was prevented by blockade of Gq/11 with YM254890 and by inhibition of protein kinase C (PKC), and was mimicked by the PKC activator phorbol 12‐myristate 13‐acetate (PMA). Both CCh and PMA induced mTORC1 activation and enhanced the phosphorylation/inactivation of ULK1, a mTORC1 substrate and a key initiator of autophagy. Inhibition of mTORC1 by rapamycin prevented CCh‐induced ULK1 phosphorylation and autophagy inhibition. The data indicate that in SH‐SY5Y cells Gq/11‐coupled M3 mAChRs inhibit autophagy by stimulating the PKC‐mTORC1 signaling pathway. The attenuation of the self‐degradation process induced by autophagy may contribute to the growth promoting effects of M3 mAChRs in human neuroblastoma cells.Support or Funding InformationSupported by Regione Autonoma della Sardegna, L.R.7,‐2012‐CRP‐60567

Disease-Modifying Effects of M1 Muscarinic Acetylcholine Receptor Activation in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is the leading cause of dementia worldwide, and currently no disease-modifying therapy is available to slow or prevent AD, underscoring the urgent need for neuroprotective therapies. Selective M1 muscarinic acetylcholine receptor (mAChR) activation is an attractive mechanism for AD therapy since M1 mediates key effects on memory, cognition, and behavior and has potential for disease-modifying effects on Aβ formation and tau phosphorylation. To validate M1 as a neuroprotective treatment target for AD, the M1-selective agonist, VU0364572, was chronically dosed to 5XFAD mice from a young age preceding Aβ pathology (2 months) to an age where these mice are known to display memory impairments (6 months). Chronic M1 activation prevented mice from becoming memory-impaired, as measured by Morris water maze (MWM) testing at 6 months of age. Additionally, M1 activation significantly reduced levels of soluble and insoluble Aβ40,42 in the cortex and hippocampus of these animals, as measured by ELISA and immunohistochemistry. Moreover, soluble hippocampal Aβ42 levels were strongly correlated with MWM memory impairments and M1 activation with VU0364572 abolished this correlation. Finally, VU0364572 significantly decreased oligomeric (oAβ) levels in the cortex, suggesting one mechanism whereby VU0364572 may be exerting its neuroprotective effects is by reducing the available oAβ pool in the brain. These findings suggest that chronic M1 activation has neuroprotective potential for preventing memory impairments and reducing neuropathology in AD. M1 activation therefore represents a promising avenue for preventative treatment, as well as a promising opportunity to combine symptomatic and disease-modifying effects for early AD treatment.

Muscarinic Acetylcholine Receptors and M-Currents Underlie Efferent-Mediated Slow Excitation in Calyx-Bearing Vestibular Afferents.

Stimulation of vestibular efferent neurons excites calyx and dimorphic (CD) afferents. This excitation consists of fast and slow components that differ >100-fold in activation kinetics and response duration. In the turtle, efferent-mediated fast excitation arises in CD afferents when the predominant efferent neurotransmitter acetylcholine (ACh) activates calyceal nicotinic ACh receptors (nAChRs); however, it is unclear whether the accompanying efferent-mediated slow excitation is also attributed to cholinergic mechanisms. To identify synaptic processes underlying efferent-mediated slow excitation, we recorded from CD afferents innervating the turtle posterior crista during electrical stimulation of efferent neurons, in combination with pharmacological probes and mechanical stimulation. Efferent-mediated slow excitation was unaffected by nAChR compounds that block efferent-mediated fast excitation, but were mimicked by muscarine and antagonized by atropine, indicating that it requires ACh and muscarinic ACh receptor (mAChR) activation. Efferent-mediated slow excitation or muscarine application enhanced the sensitivity of CD afferents to mechanical stimulation, suggesting that mAChR activation increases afferent input impedance by closing calyceal potassium channels. These observations were consistent with suppression of a muscarinic-sensitive K+-current, or M-current. Immunohistochemistry for putative M-current candidates suggested that turtle CD afferents express KCNQ3, KCNQ4, and ERG1-3 potassium channel subunits. KCNQ channels were favored as application of the selective antagonist XE991 mimicked and occluded efferent-mediated slow excitation in CD afferents. These data highlight an efferent-mediated mechanism for enhancing afferent sensitivity. They further suggest that the clinical effectiveness of mAChR antagonists in treating balance disorders may also target synaptic mechanisms in the vestibular periphery, and that KCNQ channel modulators might offer similar therapeutic value.SIGNIFICANCE STATEMENT Targeting the efferent vestibular system (EVS) pharmacologically might prove useful in ameliorating some forms of vestibular dysfunction by modifying ongoing primary vestibular input. EVS activation engages several kinetically distinct synaptic processes that profoundly alter the discharge rate and sensitivity of first-order vestibular neurons. Efferent-mediated slow excitation of vestibular afferents is of considerable interest given its ability to elevate afferent activity over an extended time course. We demonstrate for the first time that efferent-mediated slow excitation of vestibular afferents is mediated by muscarinic acetylcholine receptor (mAChR) activation and the subsequent closure of KCNQ potassium channels. The clinical effectiveness of some anti-mAChR drugs in treating motion sickness suggest that we may, in fact, already be targeting the peripheral EVS.

A unifying hypothesis for M1 muscarinic receptor signalling in pyramidal neurons.

Phasic release of acetylcholine (ACh) in the neocortex facilitates attentional processes. Acting at a single metabotropic receptor subtype, ACh exerts two opposing actions in cortical pyramidal neurons: transient inhibition and longer-lasting excitation. Cholinergic inhibitory responses depend on calcium release from intracellular calcium stores, and run down rapidly at resting membrane potentials when calcium stores become depleted. We demonstrate that cholinergic excitation promotes calcium entry at subthreshold membrane potentials to rapidly refill calcium stores, thereby maintaining the fidelity of inhibitory cholinergic signalling. We propose a 'unifying hypothesis' for M1 receptor signalling whereby inhibitory and excitatory responses to ACh in pyramidal neurons represent complementary mechanisms governing rapid calcium cycling between the endoplasmic reticulum, the cytosol and the extracellular space. Gq -coupled M1-type muscarinic acetylcholine (ACh) receptors (mAChRs) mediate two distinct electrophysiological responses in cortical pyramidal neurons: transient inhibition driven by calcium-dependent small conductance potassium ('SK') channels, and longer-lasting and voltage-dependent excitation involving non-specific cation channels. Here we examine the interaction of these two cholinergic responses with respect to their contributions to intracellular calcium dynamics, testing the 'unifying hypothesis' that rundown of inhibitory SK responses at resting membrane potentials (RMPs) reflects depletion of intracellular calcium stores, while mAChR-driven excitation acts to refill those stores by promoting voltage-dependent entry of extracellular calcium. We report that fidelity of cholinergic SK responses requires the continued presence of extracellular calcium. Inhibitory responses that diminished after repetitive ACh application at RMPs were immediately rescued by pairing mAChR stimulation with subthreshold depolarization (∼10mV from RMPs) initiated with variable delay (up to 500ms) after ACh application, but not by subthreshold depolarization preceding mAChR stimulation. Further, rescued SK responses were time-locked to ACh application, rather than to the timing of subsequent depolarizing steps, suggesting that cholinergic signal transduction itself is not voltage-sensitive, but that depolarization facilitates rapid cycling of extracellular calcium through the endoplasmic reticulum to activate SK channels. Consistent with this prediction, rescue of SK responses by subthreshold depolarization required the presence of extracellular calcium. Our results demonstrate that, in addition to gating calcium release from intracellular stores, mAChR activation facilitates voltage-dependent refilling of calcium stores, thereby maintaining the ongoing fidelity of SK-mediated inhibition in response to phasic release of ACh.

The atypical antipsychotic olanzapine disturbs depotentiation by modulating mAChRs and impairs reversal learning

Antipsychotic medication is an essential component for treating schizophrenia, which is a serious mental disorder that affects approximately 1% of the global population. Olanzapine (Olz), one of the most frequently prescribed atypical antipsychotics, is generally considered a first-line drug for treating schizophrenia. In contrast to psychotic symptoms, the effects of Olz on cognitive symptoms of schizophrenia are still unclear. In addition, the mechanisms by which Olz affects the neural circuits associated with cognitive function are unknown. Here we show that Olz interrupts depotentiation (reversal of long-term potentiation) without disturbing de novo LTP (long-term potentiation) and LTD (long-term depression). At hippocampal SC-CA1 synapses, inhibition of NMDARs (N-methyl-d-aspartate receptors), mGluRs (metabotropic glutamate receptors), or mAChRs (muscarinic acetylcholine receptors) disrupted depotentiation. In addition, co-activation of NMDARs, mGluRs, and mAChRs reversed stably expressed LTP. Olz inhibits the activation of mAChRs, which amplifies glutamate signaling through enhanced NMDAR opening and Gq (Gq class of G protein)-mediated signal transduction. Behaviorally, Olz impairs spatial reversal learning of mice in the Morris water maze test. Our results uncover a novel mechanism underpinning the cognitive modulation of Olz and show that the anticholinergic property of Olz affects glutamate signaling and synaptic plasticity.

The effect of coactivation of muscarinic and nicotinic acetylcholine receptors on LTD in the hippocampal CA1 network

The neuromodulator acetylcholine (ACh) is considered to have a crucial effect on sensory inputs in the process of learning and memory, and ACh activates muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors. Meanwhile in a hippocampal CA1 network including inhibitory connections, long-term potentiation (LTP) or long-term depression (LTD) is induced by the application of positive timing of the spike timing-dependent plasticity (STDP) protocol, while LTD is induced by negative timing protocol. In the previous study, the influence of ACh on LTD induced by the negative timing protocol application in the interneuron-blocked CA1 network was reported. However, the responsibility of mAChR and nAChR on pyramidal neuron and interneuron on STDP induction is still unclear. In order to clarify the role of AChRs in LTD, positive or negative timing protocol was applied in the interneuron-activated CA1 network in the presence of eserine. Consequently, the LTD induced by the positive timing protocol was switched to LTP, and the LTD by negative timing protocol was shifted toward potentiation when ACh was effective. The STDP facilitation was more effectively brought by mAChR activation on pyramidal neuron than nAChR, while mAChR on interneuron had a potential to down regulate the facilitation. These findings suggest that the direction (LTD/LTP) of STDP is determined by the activation of mAChR not only on pyramidal neuron but also on interneuron, and the magnitude of STDP is sensitively fine-tuned by nAChR. Therefore, the modulation of synaptic plasticity induced by the coactivation of mAChR and nAChR might be an important stage in integrating ACh and sensory inputs in the hippocampal CA1 network.