mAChR Activation Research Articles

M1 muscarinic acetylcholine receptors: A therapeutic strategy for symptomatic and disease-modifying effects in Alzheimer's disease?

The M1 muscarinic acetylcholine receptor (mAChR) plays a crucial role in learning and memory processes and has long been identified as a promising therapeutic target for the improvement of cognitive decline in Alzheimer's disease (AD). As such, clinical trials with xanomeline, a mAChR orthosteric agonist, showed an improvement in cognitive and behavioral symptoms associated with AD. Despite this, the clinical utility of xanomeline was hampered by a lack of M1 receptor selectivity and adverse cholinergic responses attributed to activation of peripheral M2 and M3 mAChRs. More recently, efforts have focused on developing more selective M1 compounds via targeting the less-conserved allosteric binding pockets. As such, positive allosteric modulators (PAMs) have emerged as an exciting strategy to achieve exquisite selectivity for the M1 mAChR in order to deliver improvements in cognitive function in AD. Furthermore, over recent years it has become increasingly apparent that M1 therapeutics may also offer disease-modifying effects in AD, due to the modulation of pathogenic amyloid processing. This article will review the progress made in the development of M1 selective ligands for the treatment of cognitive decline in AD, and will discuss the current evidence that selective targeting of the M1 mAChR could also have the potential to modify AD progression.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

PKC and Ras are Involved in M1 Muscarinic Receptor-Mediated Modulation of AMPA Receptor GluA1 Subunit.

M1 muscarinic acetylcholine receptors (M1 mAChRs) have long been an attractive target for the treatment of Alzheimer's disease (AD), the most common cause of dementia in the elderly. M1 mAChR agonists show desirably preclinical activities; however, most have not gone further into late clinical trials due to ineffectiveness or side effects. Thus, to understand the signaling pathways involved in M1 mAChR-mediated memory improvement may be important for design of biased agonists with on-target therapeutic effects. M1 mAChRs are classically coupled to Gαq or ectopically to Gαs to activate multiple kinases such as protein kinase C (PKC), Ras and protein kinase A (PKA). Our previous studies have found that M1 mAChRs could improve learning and memory through modulating AMPA receptor GluA1 subunit via PKA-PI3K-Akt signaling. Here, we further investigated whether PKC and Ras were involved in M1 mAChR-mediated modulation of GluA1. We demonstrated the role of PKC and Ras in the signaling pathway, as both PKC inhibitors Ro-31-8425 or Gö6983 and Ras inhibitor salirasib abolished the membrane insertion of GluA1 and enhancement of its phosphorylation at Ser845 induced by M1 mAChRs in the primary cultured neurons and hippocampus in vivo. We further showed that PKC and Ras modulated PKA-PI3K-Akt signaling since the increases of PKA, Akt and mTOR activities by M1 mAChR activation were blocked by PKC and Ras inhibitors. These data demonstrated the detailed mechanism underlying M1 mAChR-mediated modulation of GluA1 through Gαq/11 coupling, broadening the knowledge of the downstream signaling after M1 mAChR-Gαq/11 coupling.

Synergy of Glutamatergic and Cholinergic Modulation Induces Plateau Potentials in Hippocampal OLM Interneurons.

Oriens-lacunosum moleculare (OLM) cells are hippocampal inhibitory interneurons that are implicated in the regulation of information flow in the CA1 circuit, inhibiting cortical inputs to distal pyramidal cell dendrites, whilst disinhibiting CA3 inputs to pyramidal cells. OLM cells express metabotropic cholinergic (mAChR) and glutamatergic (mGluR) receptors, so modulation of these cells via these receptors may contribute to switching between functional modes of the hippocampus. Using a transgenic mouse line to identify OLM cells, we found that both mAChR and mGluR activation caused the cells to exhibit long-lasting depolarizing plateau potentials following evoked spike trains. Both mAChR- and mGluR-induced plateau potentials were eliminated by blocking transient receptor potential (TRP) channels, and were dependent on intracellular calcium concentration and calcium entry. Pharmacological tests indicated that Group I mGluRs are responsible for the glutamatergic induction of plateaus. There was also a pronounced synergy between the cholinergic and glutamatergic modulation, plateau potentials being generated by agonists applied together at concentrations too low to elicit any change when applied individually. This synergy could enable OLM cells to function as coincidence detectors of different neuromodulatory systems, leading to their enhanced and prolonged activation and a functional change in information flow within the hippocampus.

Antinociceptive Effect of Spirocyclopiperazinium Salt Compound DXL-A-24 and the Underlying Mechanism.

The antinociceptive effects of spirocyclopiperazinium salt compound DXL-A-24 on neuropathic pain and chemical-stimulated pain were investigated in this study. After the administration of DXL-A-24, the paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT) were increased in rats suffering from neuropathic pain (chronic constriction injury, CCI) on days 1, 3, 5, 7 and 14 after surgery, and pain responses were inhibited in mice stimulated with chemicals (formalin or acetic acid). In the analysis of antinociceptive targets, the effect of DXL-A-24 was blocked by a peripheral nicotinic acetylcholine receptor (nAChR) antagonist (hexamethonium, Hex) or α7 nAChR antagonist (methyllycaconitine, MLA) in the formalin test. Meanwhile, the effect of DXL-A-24 was also blocked by a peripheral muscarinic acetylcholine receptor (mAChR) antagonist (atropine methylnitrate, Amn) or M4 mAChR antagonist (tropicamide, TRO). The antinociceptive signalling pathway was explored using molecular biology methods in ipsilateral dorsal root ganglions (DRGs) of CCI rats after the administration of DXL-A-24 for 7days. Western blot analyses showed that the increased levels of phosphorylation of calcium/calmodulin-dependent protein kinaseII alpha (CaMKIIα) and cAMP response element-binding protein (CREB) were eliminated, and the qRT-PCR assay showed that the increase in the expression of Tumor necrosis factor alpha (TNF-α) mRNA was reduced. Meanwhile, immunofluorescence staining revealed that the increase in calcitonin gene related peptide (CGRP) expression was inhibited by the administration of DXL-A-24, and the effect was blocked by MLA or TRO. In conclusion, DXL-A-24 exerts significant antinociceptive effects on neuropathic pain and chemical-stimulated pain. The antinociceptive effect of DXL-A-24 is probably attributed to the activation of peripheral α7 nAChR and M4 mAChR, the subsequent inhibition of the CaMKIIα/CREB signalling pathway, and finally the inhibition of TNF-α and CGRP expression.

Hippocampus-dependent fear conditioning is not sensitized by muscarinic receptor activation following systemic injection of pilocarpine

The regulation of muscarinic acetylcholine receptors (mAChR) critically influences emotional outcomes. Previous researches indicate that a single systemic injection of pilocarpine – a mAChR agonist – displays long-term defensive behaviors in rats evaluated in distinct unconditioned tests up to 3 months following treatment. However, it is not clear whether these effects share underlying behavioral phenotypes involved in conditioned responses. With this in mind, we examined whether mAChR activation modulates contextual fear conditioning (CFC) and/or hippocampal synaptic plasticity. Adult male Wistar rats were injected with pilocarpine (150 mg/kg) and behaviorally evaluated in the CFC test or followed by synaptic plasticity (LTP/LTD) investigation in CA1 stratum radiatum of hippocampal slices. There was no difference between groups in the quantification of freezing behavior during the test period (24 h after treatment) besides a decrease of freezing 1 month later. Similarly, no changes were observed in rats conditioned 24 h later and tested 1 month after. Synaptic plasticity investigation following short- or long-term treatment revealed no differences between control and treated subjects. In summary, our results show that hippocampus-dependent fear behavior and memory consolidation mediated by hippocampal cholinergic inputs are not sensitive to activation of mAChR by a systemic nonconvulsant dose of pilocarpine. Therefore, we suggest that the long-term defensive behaviors and anxiogenic-like features displayed by pilocarpine observed in rats are mediated by different underlying mechanisms and or set of synapses.

Anti-inflammatory and antioxidant effects of muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus

Recently we found that acute treatment with Oxotremorine (Oxo), a non-selective mAChRs agonist, up-regulates heat shock proteins and activates their transcription factor heat shock factor 1 in the rat hippocampus. Here we aimed to investigate: a) if acute treatment with Oxo may regulate pro-inflammatory or anti-inflammatory cytokines and oxidative stress in the rat hippocampus; b) if chronic restraint stress (CRS) induces inflammatory or oxidative alterations in the hippocampus and whether such alterations may be affected by chronic treatment with Oxo. In the acute experiment, rats were injected with single dose of Oxo (0.4 mg/kg) and sacrificed at 24 h, 48 h and 72 h. In the CRS experiment, the rats were exposed for 21 days to the CRS and then were treated with Oxo (0.2 mg/kg) for further 10 days. The acute Oxo treatment showed an ability to significantly reduce reactive oxygen species (ROS), singlet oxygen (1O2), pro-inflammatory cytokines levels (IL-1β and IL-6) and phosphorylated NF-κB-p65. Acute Oxo treatment also increased superoxide dismutase (SOD)-2 protein levels and stimulated SOD activity. No differences were detected in the anti-inflammatory cytokine levels, including IL-10 and TGF-β1. In the group of rats exposed to the CRS were found increased hippocampal IL-1β and IL-6 levels, together with a reduction of SOD activity level. These changes produced by CRS were counteracted by chronic Oxo treatment. In contrast, the upregulation of ROS and 1O2 levels in the CRS group was not counteracted by chronic Oxo treatment. The results revealed a hippocampal anti-inflammatory and antioxidant effect of Oxo treatment in both basal conditions and anti-inflammatory in the CRS rat model.

Clothianidin, a neonicotinoid insecticide, activates α4β2, α7 and muscarinic receptors to induce in vivo dopamine release from rat striatum

Clothianidin (CLO) is a neonicotinoid insecticide that produces toxic effects in experimental animals and humans. These effects are associated primarily to its action as a nicotinic agonist, acting on insect and vertebrate nicotinic acetylcholine receptors (nAChRs), but little is known about the mechanisms of action on the mammalian nervous system. In the rat striatum, CLO induces increases in the dopamine overflow in a concentration-dependent manner. In the present study, we evaluate, using in vivo brain microdialysis in adult Sprague-Dawley rats, the participation of specific nAChRs and muscarinic cholinergic receptors (mAChRs) on CLO-induced striatal dopamine release. We investigate the effects of selective antagonists of α4β2 heteromeric, β2 subunit, α7 nAChRs, and of broad-spectrum antagonist of mAChRs (atropine) on CLO-induced dopamine release. Intrastriatal administration of antagonists of α4β2 N-n-decilonicotinium iodide (NDNI), and of α7 methylcaconitine (MLA) significantly decreased the CLO-induced dopamine overflow in a concentration-dependent form, whereas pretreatment with the antagonist of β2 subunit DHβE not having effect. Pretreatment with the muscarinic antagonist atropine also blocked the increases in the extracellular dopamine levels. Taken together, these results suggest that the stimulatory effect of CLO on in vivo dopamine from rat striatum depends on the activation of α4β2 present in dopaminergic terminals and α7 nAChRs subtypes expressed in glutamatergic terminals in the striatum. On the other hand, the CLO-induced dopamine release also appears to involve the activation of mAChRs.

Striatal, Hippocampal, and Cortical Networks Are Differentially Responsive to the M4- and M1-Muscarinic Acetylcholine Receptor Mediated Effects of Xanomeline.

Preclinical and clinical data suggest that muscarinic acetylcholine receptor activation may be therapeutically beneficial for the treatment of schizophrenia and Alzheimer’s diseases. This is best exemplified by clinical observations with xanomeline, the efficacy of which is thought to be mediated through co-activation of the M1 and M4 muscarinic acetylcholine receptors (mAChRs). Here we examined the impact of treatment with xanomeline and compared it to the actions of selective M1 and M4 mAChR activators on in vivo intracellular signaling cascades in mice, including 3′-5′-cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation and inositol phosphate-1 (IP1) accumulation in the striatum, hippocampus, and prefrontal cortex. We additionally assessed the effects of xanomeline on hippocampal electrophysiological signatures in rats using ex vivo recordings from CA1 (Cornu Ammonis 1) as well as in vivo hippocampal theta. As expected, xanomeline’s effects across these readouts were ...

Role and mechanism of muscarinic acetylcholine receptor in the regulation of submandibular gland secretion

Muscarinic acetylcholine receptors (mAChRs), including M1-M5 subtypes, are classic receptors in regulating water, ion, and solute transport in salivary gland. Our work focuses on the studies on the expression pattern and function of mAChR in the submandibular gland (SMG), and the underlying mechanism involved in the mAChR-regulated secretion, together with the effect of parasympathectomy on the salivary secretion. Microvascular autotransplantation of SMG into the temporal fossa provides a continuous and endogenous source of fluids, and is currently an effective method for treating severe keratoconjunctivitis sicca. By using RT-PCR, Western blotting, and immunofluorescence, our data demonstrated that the expression of M1 and M3 subtypes were decreased in latent period in rabbit SMG autotransplantation model, whereas carbachol stimulation promoted the salivary secretion, as well as M1 and M3 expressions. By contrast, mAChRs were hypersensitive in epiphora SMGs, whereas atropine gel and botulinum toxin A application significantly inhibited the hypersecretion in both animal models and patients. Furthermore, the possible intracellular signal molecules involved in the mAChR-modulated salivary secretion were explored. Activation of mAChR upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediated water secretion through transcellular pathway, and led to AQP5 trafficking from lipid rafts to non-lipid microdomain. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the mAChR-regulated AQP5 content. mAChR activation also modulated the expression, distribution, and function of tight junction proteins, and increased paracellular permeability. ERK1/2/β-arrestin2/clathrin/ubiquitin signaling pathway was responsible for the mAChR-regulated downregulation of tight junction molecule claudin-4. Cytoskeleton filamentous actin (F-actin) was also involved in the distribution and barrier function of epithelial tight junctions. Besides, endothelial tight junctions were opened by mAChR agonist-evoked salivation in the mice. Furthermore, parasympathetic denervation increased resting salivary secretion in the long terminrats and minipigs. Taken together, our work demonstrated that mAChR regulated saliva secretion via transcellular and paracellular pathways in SMG epithelium as well as tight junction opening in SMG endothelium. Modulation of mAChR might be a promising strategy to ameliorate SMG dysfunction.

Roles of the M4 acetylcholine receptor in the basal ganglia and the treatment of movement disorders.

Acetylcholine (ACh) released from cholinergic interneurons acting through nicotinic and muscarinic acetylcholine receptors (mAChRs) in the striatum have been thought to be central for the potent cholinergic regulation of basal ganglia activity and motor behaviors. ACh activation of mAChRs has multiple actions to oppose dopamine (DA) release, signaling, and related motor behaviors and has led to the idea that a delicate balance of DA and mAChR signaling in the striatum is critical for maintaining normal motor function. Consistent with this, mAChR antagonists have efficacy in reducing motor symptoms in diseases where DA release or signaling is diminished, such as in Parkinson's disease and dystonia, but are limited in their utility because of severe adverse effects. Recent breakthroughs in understanding both the anatomical sites of action of ACh and the mAChR subtypes involved in regulating basal ganglia function reveal that the M4 subtype plays a central role in regulating DA signaling and release in the basal ganglia. These findings have raised the possibility that sources of ACh outside of the striatum can regulate motor activity and that M4 activity is a potent regulator of motor dysfunction. We discuss how M4 activity regulates DA release and signaling, the potential sources of ACh that can regulate M4 activity, and the implications of targeting M4 activity for the treatment of the motor symptoms in movement disorders. © 2019 International Parkinson and Movement Disorder Society.

Acetylcholine Release Inhibits Distinct Excitatory Inputs Onto Hippocampal CA1 Pyramidal Neurons via Different Cellular and Network Mechanisms.

In hippocampal CA1, muscarinic acetylcholine (ACh) receptor (mAChR) activation via exogenous application of cholinergic agonists has been shown to presynaptically inhibit Schaffer collateral (SC) glutamatergic inputs in stratum radiatum (SR), and temporoammonic (TA) and thalamic nucleus reuniens (RE) glutamatergic inputs in stratum lacunosum-moleculare (SLM). However, steady-state uniform mAChR activation may not mimic the effect of ACh release in an intact hippocampal network. To more accurately examine the effect of ACh release on glutamatergic synaptic efficacy, we measured electrically evoked synaptic responses in CA1 pyramidal cells (PCs) following the optogenetic release of ACh in genetically modified mouse brain slices. The ratio of synaptic amplitudes in response to paired-pulse SR stimulation (stimulus 2/stimulus 1) was significantly reduced by the optogenetic release of ACh, consistent with a postsynaptic decrease in synaptic efficacy. The effect of ACh release was blocked by the M3 receptor antagonist 4-DAMP, the GABAB receptor antagonist CGP 52432, inclusion of GDP-β-S, cesium, QX314 in the intracellular patch clamp solution, or extracellular barium. These observations suggest that ACh release decreased SC synaptic transmission through an M3 muscarinic receptor-mediated increase in inhibitory interneuron excitability, which activate GABAB receptors and inwardly rectifying potassium channels on CA1 pyramidal cells. In contrast, the ratio of synaptic amplitudes in response to paired-pulse stimulation in the SLM was increased by ACh release, consistent with presynaptic inhibition. ACh-mediated effects in SLM were blocked by the M2 receptor antagonist AF-DX 116, presumably located on presynaptic terminals. Therefore, our data indicate that ACh release differentially modulates excitatory inputs in SR and SLM of CA1 through different cellular and network mechanisms.

Discovery of Selective M4 Muscarinic Acetylcholine Receptor Agonists with Novel Carbamate Isosteres.

It has been hypothesized that selective muscarinic acetylcholine receptor (mAChR) M4 subtype activation could provide therapeutic benefits to a number of neurological disorders while minimizing unwanted cholinergic side effects observed due to nonselective mAChR activation. Given the high sequence and structural homology of the orthosteric binding sites among mAChRs, achieving M4 subtype-selective activation has been challenging. Herein, we describe the discovery of a series of M4 subtype-selective agonists bearing novel carbamate isosteres. Comparison of the isosteres' electrostatic potential isosurface sheds light on key structural features for M4 subtype-selective activation. The identified key features were further illustrated in a proposed receptor-agonist interaction mode.

Optical Control of Cardiac Function with a Photoswitchable Muscarinic Agonist.

Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination, and offers new opportunities for intravital imaging and control of cardiac function.

M1 Muscarinic Receptors Modulate Fear-Related Inputs to the Prefrontal Cortex: Implications for Novel Treatments of Posttraumatic Stress Disorder

BackgroundThe prefrontal cortex (PFC) integrates information from multiple inputs to exert top-down control allowing for appropriate responses in a given context. In psychiatric disorders such as posttraumatic stress disorder, PFC hyperactivity is associated with inappropriate fear in safe situations. We previously reported a form of muscarinic acetylcholine receptor (mAChR)–dependent long-term depression in the PFC that we hypothesize is involved in appropriate fear responding and could serve to reduce cortical hyperactivity following stress. However, it is unknown whether this long-term depression occurs at fear-related inputs. MethodsUsing optogenetics with extracellular and whole-cell electrophysiology, we assessed the effect of mAChR activation on the synaptic strength of specific PFC inputs. We used selective pharmacological tools to assess the involvement of M1 mAChRs in conditioned fear extinction in control mice and in the stress-enhanced fear-learning model. ResultsM1 mAChR activation induced long-term depression at inputs from the ventral hippocampus and basolateral amygdala but not from the mediodorsal nucleus of the thalamus. We found that systemic M1 mAChR antagonism impaired contextual fear extinction. Treatment with an M1 positive allosteric modulator enhanced contextual fear extinction consolidation in stress-enhanced fear learning–conditioned mice. ConclusionsM1 mAChRs dynamically modulate synaptic transmission at two PFC inputs whose activity is necessary for fear extinction, and M1 mAChR function is required for proper contextual fear extinction. Furthermore, an M1 positive allosteric modulator enhanced the consolidation of fear extinction in the stress-enhanced fear-learning model, suggesting that M1 positive allosteric modulators may provide a novel treatment strategy to facilitate exposure therapy in the clinic for the treatment of posttraumatic stress disorder.

Cholinergic modulation of hippocampal calcium activity across the sleep-wake cycle.

Calcium is a critical second messenger in neurons that contributes to learning and memory, but how the coordination of action potentials of neuronal ensembles with the hippocampal local field potential (LFP) is reflected in dynamic calcium activity remains unclear. Here, we recorded hippocampal calcium activity with endoscopic imaging of the genetically encoded fluorophore GCaMP6 with concomitant LFP in freely behaving mice. Dynamic calcium activity was greater in exploratory behavior and REM sleep than in quiet wakefulness and slow wave sleep, behavioral states that differ with respect to theta and septal cholinergic activity, and modulated at sharp wave ripples (SWRs). Chemogenetic activation of septal cholinergic neurons expressing the excitatory hM3Dq DREADD increased calcium activity and reduced SWRs. Furthermore, inhibition of muscarinic acetylcholine receptors (mAChRs) reduced calcium activity while increasing SWRs. These results demonstrate that hippocampal dynamic calcium activity depends on behavioral and theta state as well as endogenous mAChR activation.

Muscarinic and Nicotinic Modulation of Neocortical Layer 6A Synaptic Microcircuits Is Cooperative and Cell-Specific

Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4β2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4β2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.

M1 muscarinic receptors facilitate hippocampus-dependent cognitive flexibility via modulating GluA2 subunit of AMPA receptors

Cognitive flexibility is an important aspect of executive function. The cholinergic system, an important component of cognition, has been shown to modulate cognitive flexibility mainly through the striatum and prefrontal cortex. The role of M1 muscarinic receptors (M1 mAChRs), an important therapeutic target in the cholinergic system, in hippocampus-dependent cognitive flexibility is unclarified. In the present study, we demonstrated that selective activation of M1 mAChRs promoted extinction of initial learned response and facilitated acquisition of reversal learning in the Morris water maze, a behavior test that is mainly dependent on the hippocampus. However, these effects were abolished in GluA2 mutant mice with deficiency in phosphorylation of Ser880 by protein kinase C (PKC). Further long-term depression (LTD) in the hippocampal CA1 area induced by M1 mAChR activation was shown to be dependent on AMPA receptor subunit GluA2 but not GluA1. M1 mAChRs increased GluA2 endocytosis through phosphorylation of Ser880 by PKC. Inhibition of PKC blocked M1 mAChR-mediated LTD, memory switching and reversal learning facilitation. Moreover, the slow memory extinction observed in GluA2 mutant mice and PKC inhibitor-treated mice appeared to affect the consolidation and retrieval of reversal learning. Thus, these results demonstrate that M1 mAChRs mainly facilitate acquisition in spatial reversal learning and further elucidate that such an effect is dependent on the phosphorylation of GluA2 by PKC. The study helps clarify the role of M1 mAChRs in cognitive flexibility and may prompt the earlier prevention of cognitive inflexibility.

Striatal, Hippocampal, and Cortical Networks Are Differentially Responsive to the M4- and M1-Muscarinic Acetylcholine Receptor Mediated Effects of Xanomeline.

Preclinical and clinical data suggest that muscarinic acetylcholine receptor activation may be therapeutically beneficial for the treatment of schizophrenia and Alzheimer's diseases. This is best exemplified by clinical observations with xanomeline, the efficacy of which is thought to be mediated through co-activation of the M1 and M4 muscarinic acetylcholine receptors (mAChRs). Here we examined the impact of treatment with xanomeline and compared it to the actions of selective M1 and M4 mAChR activators on in vivo intracellular signaling cascades in mice, including 3'-5'-cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation and inositol phosphate-1 (IP1) accumulation in the striatum, hippocampus, and prefrontal cortex. We additionally assessed the effects of xanomeline on hippocampal electrophysiological signatures in rats using ex vivo recordings from CA1 (Cornu Ammonis 1) as well as in vivo hippocampal theta. As expected, xanomeline's effects across these readouts were consistent with activation of both M1 and M4 mAChRs; however, differences were observed across different brain regions, suggesting non-uniform activation of these receptor subtypes in the central nervous system. Interestingly, despite having nearly equal in vitro potency at the M1 and the M4 mAChRs, during in vivo assays xanomeline produced M4-like effects at significantly lower brain exposures than those at which M1-like effects were observed. Our results raise the possibility that clinical efficacy observed with xanomeline was driven, in part, through its non-uniform activation of mAChR subtypes in the central nervous system and, at lower doses, through preferential agonism of the M4 mAChR.

Metabotropic Acetylcholine and Glutamate Receptors Mediate PI(4,5)P2 Depletion and Oscillations in Hippocampal CA1 Pyramidal Neurons in situ

The sensitivity of many ion channels to phosphatidylinositol-4,5-bisphosphate (PIP2) levels in the cell membrane suggests that PIP2 fluctuations are important and general signals modulating neuronal excitability. Yet the PIP2 dynamics of central neurons in their native environment remained largely unexplored. Here, we examined the behavior of PIP2 concentrations in response to activation of Gq-coupled neurotransmitter receptors in rat CA1 hippocampal neurons in situ in acute brain slices. Confocal microscopy of the PIP2-selective molecular sensors tubbyCT-GFP and PLCδ1-PH-GFP showed that pharmacological activation of muscarinic acetylcholine (mAChR) or group I metabotropic glutamate (mGluRI) receptors induces transient depletion of PIP2 in the soma as well as in the dendritic tree. The observed PIP2 dynamics were receptor-specific, with mAChR activation inducing stronger PIP2 depletion than mGluRI, whereas agonists of other Gαq-coupled receptors expressed in CA1 neurons did not induce measureable PIP2 depletion. Furthermore, the data show for the first time neuronal receptor-induced oscillations of membrane PIP2 concentrations. Oscillatory behavior indicated that neurons can rapidly restore PIP2 levels during persistent activation of Gq and PLC. Electrophysiological responses to receptor activation resembled PIP2 dynamics in terms of time course and receptor specificity. Our findings support a physiological function of PIP2 in regulating electrical activity.