mAChR Activation Research Articles

The Activation of Muscarinic Acetylcholine Receptors Protects against Neuroinflammation in a Mouse Model through Attenuating Microglial Inflammation.

Neuroinflammation is a critical factor that contributes to neurological impairment and is closely associated with the onset and progression of neurodegenerative diseases. In the central nervous system (CNS), microglia play a pivotal role in the regulation of inflammation through various signaling pathways. Therefore, mitigating microglial inflammation is considered a promising strategy for restraining neuroinflammation. Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and exhibit clear neuroprotective effects in various disease models. However, whether the activation of mAChRs can harness benefits in neuroinflammation remains largely unexplored. In this study, the anti-inflammatory effects of mAChRs were found in a neuroinflammation mouse model. The expression of various cytokines and chemokines was regulated in the brains and spinal cords after the administration of mAChR agonists. Microglia were the primary target cells through which mAChRs exerted their anti-inflammatory effects. The results showed that the activation of mAChRs decreased the pro-inflammatory phenotypes of microglia, including the expression of inflammatory cytokines, morphological characteristics, and distribution density. Such anti-inflammatory modulation further exerted neuroprotection, which was found to be even more significant by the direct activation of neuronal mAChRs. This study elucidates the dual mechanisms through which mAChRs exert neuroprotective effects in central inflammatory responses, providing evidence for their application in inflammation-related neurological disorders.

Muscarinic and nicotinic receptors stimulation by vagus nerve stimulation ameliorates trastuzumab-induced cardiotoxicity via reducing programmed cell death in rats

Despite its efficacy in human epidermal growth factor receptor 2 positive cancer treatment, trastuzumab-induced cardiotoxicity (TIC) has become a growing concern. Due to the lack of cardiomyocyte regeneration and proliferation in adult heart, cell death significantly contributes to cardiovascular diseases. Cardiac autonomic modulation by vagus nerve stimulation (VNS) has shown cardioprotective effects in several heart disease models, while the effects of VNS and its underlying mechanisms against TIC have not been found. Forty adult male Wistar rats were divided into 5 groups: (i) control without VNS (CSham) group, (ii) trastuzumab (4 mg/kg/day, i.p.) without VNS (TSham) group, (iii) trastuzumab + VNS (TVNS) group, (iv) trastuzumab + VNS + mAChR blocker (atropine; 1 mg/kg/day, ip, TVNS + Atro) group, and (v) trastuzumab + VNS + nAChR blocker (mecamylamine; 7.5 mg/kg/day, ip, TVNS + Mec) group. Our results showed that trastuzumab induced cardiac dysfunction by increasing autonomic dysfunction, mitochondrial dysfunction/dynamics imbalance, and cardiomyocyte death including apoptosis, autophagic deficiency, pyroptosis, and ferroptosis, which were notably alleviated by VNS. However, mAChR and nAChR blockers significantly inhibited the beneficial effects of VNS on cardiac autonomic dysfunction, mitochondrial dysfunction, cardiomyocyte apoptosis, pyroptosis, and ferroptosis. Only nAChR could counteract the protective effects of VNS on cardiac mitochondrial dynamics imbalance and autophagy insufficiency. Therefore, VNS prevented TIC by rebalancing autonomic activity, ameliorating mitochondrial dysfunction and cardiomyocyte death through mAChR and nAChR activation. The current study provides a novel perspective elucidating the potential treatment of VNS, thus also offering other pharmacological therapeutic promises in TIC patients.

Characterization of HCN channel subtypes at hypoglossal motoneurons throughout postnatal maturation in mice

The inspiratory phase of breathing includes a stiffening of the upper airway to decrease airway resistance and is mediated by excitatory motor output from hypoglossal motoneurons (XII MNs). This excitation decreases from wakefulness to sleep. This decrease in excitation is in large part due to increasing acetylcholine levels, which activate muscarinic acetylcholine receptors (mAChRs) that have a net inhibitory effect in adult preparations. By contrast, activation of mAChRs in neonatal rodent preparations has a net effect of potentiating inspiratory burst amplitude. Therefore, characterizing changes in muscarinic effects at XII MNs across postnatal maturation will help elucidate the mechanism underlying the shift from muscarinic excitation to inhibition. mAChRs modulate several effector ion channels including the hyperpolarization activated cyclic nucleotide gated (HCN) channel. HCN channels give rise to I h, a mixed cation current activated at hyperpolarized membrane potentials. I h additionally shows upregulation at XII MNs with postnatal maturation. Since there are four HCN subtypes (HCN1-4), the reported changes in I h may be caused by changes in HCN channel subtype levels. HCN1 and HCN2 are most prominent in adult XII MNs, whereas the expression of HCN1-4 in neonates is unknown. Thus, the purpose of this research is to characterize changes in HCN gene and channel expression levels at XII MNs across postnatal maturation. We hypothesis that both HCN1 and HCN2 subtypes will increase with postnatal maturation, and that HCN2 has higher levels than HCN1. To test this hypothesis, we used neuroanatomical techniques to evaluate changes in expression patterns of the four HCN proteins at the XII nuclei across postnatal maturation in CD-1 mice. We performed double-labeled immunofluorescence experiments against HCN1 and HCN2 and co-labeled with choline acetyltransferase (ChAT) on 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0-P1, P4-P6, P9-P10, P12-P14, P17-P19, and adult. Images of the XII nuclei were collected using a confocal microscope. ImageJ was used to determine the average XII MN HCN subtype intensity across postnatal maturation. Preliminary data suggest that HCN1 (n=2) channels undergo a decrease in expression at P17-19 before a final increase in adulthood (P0-1= 97-100%, P4-6= 92-100%, P9-10= 85-95%, P12-14= 77-97%, P17-19= 61-67%, adult= 71-82%). By contrast, preliminary data suggest that HCN2 (n=2) expression intensity remained relatively consistent with a decrease in labeling intensity at P4-6 (P0-1= 97-100%, P4-6=62-84%, P9-10= 45-95%, P12-14= 67-90%, P17-19= 50-93%, adult= 75-100%). These modest changes in labeling intensity of HCN channel subtypes may not contribute to the observed shift of mAChR modulation at XII MNs from excitation to inhibition with postnatal maturation. Biomedical Sciences, College of Graduate Studies; Department of Physiology, College of Graduate Studies, Midwestern University; 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.

Contribution of Ih to Postnatal Maturation of Muscarinic Modulation of Inspiratory Bursting at Hypoglossal Motoneurons in Neonatal Mice

Activation of hypoglossal motoneurons (XII MNs) contributes to stiffening of the upper airway during inspiration. The magnitude of this excitation is decreased during sleep, likely due to a combination of withdrawal of noradrenaline and acetylcholine release at XII MNs. Acetylcholine acts at muscarinic acetylcholine receptors (mAChRs) and surprisingly mAChR activation has an excitatory effect on inspiratory bursting in neonatal preparations, which becomes inhibitory in adult preparations. mAChRs modulate several ion channels and characterizing muscarinic effect changes at the XII nucleus will help to explain the mechanism of the inhibitory shift over postnatal maturation. The hyperpolarization activated cyclic nucleotide gated (HCN) channel is positively modulated by mAChRs. HCN channels give rise to Ih, a mixed cation current activated at hyperpolarized membrane potentials that contribute to membrane depolarization. Ihincreases dramatically with postnatal maturation in XII MNs. Thus, we hypothesize that 1) the effects of muscarinic modulation of Ih increases with postnatal maturation, and 2) that muscarinic activation of Ih contributes to the net inhibitory component of muscarinic modulation of inspiratory bursting at XII motoneurons. To test our hypotheses, we used the rhythmic medullary slice preparation to test the functional effects of muscarinic modulation of Ih on inspiratory bursting in neonatal CD1 mice (postnatal day, P0-P4) in combination with pharmacological block of Ih with ZD7288 (25 μM, 150s). Local application of muscarine (100μM, 30s) in the XII motor nucleus increased inspiratory burst amplitude (189 ± 58% of baseline, n = 9). Contrary to our hypothesis, there was no statistically significant difference between inspiratory burst amplitude elicited by muscarine in the presence of ZD7288 (192 ± 47% of baseline, n = 9) versus with the aCSF vehicle control (174 ± 43% of baseline, p = 0.098). We next tested a higher ZD7288 concentration (100 μM) and longer application times (150s, 330s) in neonatal preparations. Preliminary data (n=2) indicate that ZD7288 may increase muscarinic potentiation of inspiratory burst amplitude at XII MNs (muscarinic potentiation (100μM, 30s): control — 112 ±65 % of baseline, aCSF vehicle control - 131 ± 38% of baseline; ZD7288 100μM, 150s - 148 ± 44% of baseline; ZD7288 100μM, 330s - 107 ± 72% of baseline). Future research will elucidate whether the Ih contribution to muscarinic modulation of inspiratory bursting increases with postnatal maturation. 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.

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.

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 Receptor Stimulation Induces Autophagy in Human Glioblastoma Cancer Stem Cells via mTOR Complex-1 Inhibition.

Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.

A Developmental Switch in Cholinergic Mechanisms of Modulation in the Medial Nucleus of the Trapezoid Body.

The medial nucleus of the trapezoid body (MNTB) has been intensively investigated as a primary source of inhibition in brainstem auditory circuitry. MNTB-derived inhibition plays a critical role in the computation of sound location, as temporal features of sounds are precisely conveyed through the calyx of Held/MNTB synapse. In adult gerbils, cholinergic signaling influences sound-evoked responses of MNTB neurons via nicotinic acetylcholine receptors (nAChRs; Zhang et al., 2021) establishing a modulatory role for cholinergic input to this nucleus. However, the cellular mechanisms through which acetylcholine (ACh) mediates this modulation in the MNTB remain obscure. To investigate these mechanisms, we used whole-cell current and voltage-clamp recordings to examine cholinergic physiology in MNTB neurons from Mongolian gerbils (Meriones unguiculatus) of both sexes. Membrane excitability was assessed in brain slices, in pre-hearing (postnatal days 9-13) and post-hearing onset (P18-20) MNTB neurons during bath application of agonists and antagonists of nicotinic (nAChRs) and muscarinic receptors (mAChRs). Muscarinic activation induced a potent increase in excitability most prominently prior to hearing onset with nAChR modulation emerging at later time points. Pharmacological manipulations further demonstrated that the voltage-gated K+ channel KCNQ (Kv7) is the downstream effector of mAChR activation that impacts excitability early in development. Cholinergic modulation of Kv7 reduces outward K+ conductance and depolarizes resting membrane potential. Immunolabeling revealed expression of Kv7 channels as well as mAChRs containing M1 and M3 subunits. Together, our results suggest that mAChR modulation is prominent but transient in the developing MNTB and that cholinergic modulation functions to shape auditory circuit development.

A muscarinic, GIRK channel-mediated inhibition of inspiratory-related XII nerve motor output emerges in early postnatal development in mice.

In neonatal rhythmic medullary slices, muscarinic acetylcholine receptor (mAChR) activation of hypoglossal (XII) motoneurons that innervate the tongue has a net excitatory effect on XII inspiratory motor output. Conversely, during rapid eye movement sleep in adult rodents, XII motoneurons experience a loss of excitability partly due to activation of mAChRs. This may be mediated by activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Therefore, this study was designed to evaluate whether muscarinic modulation of XII inspiratory motor output in mouse rhythmic medullary slices includes GIRK channel-mediated inhibition and, if so, when this inhibitory mechanism emerges. Local pressure injection of the mAChR agonist muscarine potentiated inspiratory bursting by 150 ± 28% in postnatal day (P)0-P5 rhythmic medullary slice preparations. In the absence of muscarine, pharmacological GIRK channel block by Tertiapin-Q did not affect inspiratory burst parameters, whereas activation with ML297 decreased inspiratory burst area. Blocking GIRK channels by local preapplication of Tertiapin-Q revealed a developmental change in muscarinic modulation of inspiratory bursting. In P0-P2 rhythmic medullary slices, Tertiapin-Q preapplication had no significant effect on muscarinic potentiation of inspiratory bursting (a negligible 6% decrease). However, preapplication of Tertiapin-Q to P3-P5 rhythmic medullary slices caused a 19% increase in muscarinic potentiation of XII inspiratory burst amplitude. Immunofluorescence experiments revealed expression of GIRK 1 and 2 subunits and M1, M2, M3, and M5 mAChRs from P0 to P5. Overall, these data support that mechanisms underlying muscarinic modulation of inspiratory burst activity change postnatally and that potent GIRK-mediated inhibition described in adults emerges early in postnatal life.NEW & NOTEWORTHY Muscarinic modulation of inspiratory bursting at hypoglossal motoneurons has a net excitatory effect in neonatal rhythmic medullary slice preparations and a net inhibitory effect in adult animals. We demonstrate that muscarinic modulation of inspiratory bursting undergoes maturational changes from postnatal days 0 to 5 that include emergence of an inhibitory component mediated by G-protein-coupled inwardly rectifying potassium channels after postnatal day 3 in neonatal mouse rhythmic medullary slice preparations.

Acetylcholinesterase inhibition protects against trastuzumab-induced cardiotoxicity through reducing multiple programmed cell death pathways

BackgroundTrastuzumab (Trz)-induced cardiotoxicity (TIC) is one of the most common adverse effects of targeted anticancer agents. Although oxidative stress, inflammation, mitochondrial dysfunction, apoptosis, and ferroptosis have been identified as potential mechanisms underlying TIC, the roles of pyroptosis and necroptosis under TIC have never been investigated. It has been shown that inhibition of acetylcholinesterase function by using donepezil exerts protective effects in various heart diseases. However, it remains unknown whether donepezil exerts anti-cardiotoxic effects in rats with TIC. We hypothesized that donepezil reduces mitochondrial dysfunction, inflammation, oxidative stress, and cardiomyocyte death, leading to improved left ventricular (LV) function in rats with TIC.MethodsMale Wistar rats were randomly assigned to be Control or Trz groups (Trz 4 mg/kg/day, 7 days, I.P.). Rats in Trz groups were assigned to be co-treated with either drinking water (Trz group) or donepezil 5 mg/kg/day (Trz + DPZ group) via oral gavage for 7 days. Cardiac function, heart rate variability (HRV), and biochemical parameters were evaluated.ResultsTrz-treated rats had impaired LV function, HRV, mitochondrial function, and increased inflammation and oxidative stress, leading to apoptosis, ferroptosis, and pyroptosis. Donepezil co-treatment effectively decreased those adverse effects of TIC, resulting in improved LV function. An in vitro study revealed that the cytoprotective effects of donepezil were abolished by a muscarinic acetylcholine receptor (mAChR) antagonist.ConclusionsDonepezil exerted cardioprotection against TIC via attenuating mitochondrial dysfunction, oxidative stress, inflammation, and cardiomyocyte death, leading to improved LV function through mAChR activation. This suggests that donepezil could be a novel intervention strategy in TIC.

Cholinergic Stimulation Modulates the Functional Composition of CA3 Cell Types in the Hippocampus.

The functional heterogeneity of hippocampal CA3 pyramidal neurons has emerged as a key aspect of circuit function. Here, we explored the effects of long-term cholinergic activity on the functional heterogeneity of CA3 pyramidal neurons in organotypic slices obtained from male rat brains. Application of agonists to either AChRs generally, or mAChRs specifically, induced robust increases in network activity in the low-gamma range. Prolonged AChR stimulation for 48 h uncovered a population of hyperadapting CA3 pyramidal neurons that typically fired a single, early action potential in response to current injection. Although these neurons were present in control networks, their proportions were dramatically increased following long-term cholinergic activity. Characterized by the presence of a strong M-current, the hyperadaptation phenotype was abolished by acute application of either M-channel antagonists or the reapplication of AChR agonists. We conclude that long-term mAChR activation modulates the intrinsic excitability of a subset of CA3 pyramidal cells, uncovering a highly plastic cohort of neurons that are sensitive to chronic ACh modulation. Our findings provide evidence for the activity-dependent plasticity of functional heterogeneity in the hippocampus.SIGNIFICANCE STATEMENT The large heterogeneity of neuron types in the brain, each with its own specific functional properties, provides the rich cellular tapestry needed to account for the vast diversity of behaviors. By studying the functional properties of neurons in the hippocampus, a region of the brain involved in learning and memory, we find that exposure to the neuromodulator acetylcholine can alter the relative number of functionally defined neuron types. Our findings suggest that the heterogeneity of neurons in the brain is not a static feature but can be modified by the ongoing activity of the circuits to which they belong.

Muscarinic modulation increases excitability inspiratory and non-inspiratory hypoglossal motoneurons in mice postnatal days 0-5

Hypoglossal motoneurons (XII MNs) innervate the tongue and their activity normally contributes to stiffening of the airway to maintain airway patency. During sleep, there is normal reduction in airway tone. The specific mechanisms that contribute to decreased airway tone during sleep remain to be completely resolved, although research from adult rodents suggests activation of muscarinic acetylcholine receptors (mAChRs) is an important contributor by inhibiting XII MNs. Conversely, data from neonatal rodents, have shown that muscarinic modulation leads to an overall excitatory effect in XII MNs. Therefore, the overarching objective of this research project is to uncover the mechanisms through which muscarinic modulation can influence the excitability of XII MNs across postnatal maturation. Rhythmic medullary slices from neonatal mice (postnatal day 0-5) were used for whole cell electrophysiology experiments to test the effects of activating mAChRs at XII MNs in both non-inspiratory and inspiratory XII MNs. Data are reported as mean±SD. We hypothesized that mAChR activation at XII MNs would increase excitability of non-inspiratory and inspiratory XII MNs, and potentiate inspiratory bursting in inspiratory XII MNs. Starting at -60 mV, muscarinic modulation depolarized non-inspiratory and inspiratory XII MNs similarly (3.6±2.6 vs 4.6±2.1 mV, n=12, p>0.05), and had no effect on input resistance (CTL vs Musc; non-insp: 120±23 vs 128±33 MΩ; inspiratory: 140±28 vs 137±32 MΩ; n =12; p>0.05). Muscarinic modulation can increase the hyperpolarization-activated cation current (I h ), but we observed no difference in the sag potential in response to -150 (CTL vs Musc; non-insp: 2.3±2.3 vs 3.8±3.0 mV; inspiratory: 2.7±2.8 vs 4.9±2.8 mV; n =12; p > 0.05), -300 (CTL vs Musc; non-insp: 9.0±7.8 vs 11.7±8.0 mV; inspiratory: 9.9±6.4 vs 12.9±7.5 mV; n=12; p>0.05), and -500 pA (CTL vs Musc; non-insp: 21.9±15.0 vs 23.6±14.2 mV; inspiratory: 22.0±9.5 vs 23.5±9.5 mV; n=12; p>0.05) current steps from -45 mV in non-inspiratory compared to inspiratory XII MNs. Muscarinic modulation significantly increased the firing frequency after muscarine application for inspiratory cells with the current step injections 100 pA (CTL vs Musc; 14.7±6.8 vs 21.1±6.4 Hz, n=9 and 10; p<0.05) and 175 pA (CTL vs Musc; 19.5±6.4 vs 25.0±9.4 Hz, n=11 and 8; p<0.05). However, muscarinic modulation did not affect the slope of firing rate/injected current relationship for non-inspiratory and inspiratory XII MNs (CTL vs Musc; non-insp: 0.16±0.12 vs 0.15±0.04 Hz/pA, n=9; inspiratory: 0.12±0.02 vs 0.16±0.07 Hz/pA, n=11 and 12; p>0.05). Finally, muscarinic modulation significantly increased inspiratory burst amplitude (CTL vs Musc; 15.2±3.1 vs 18.3±4.5 mV; n=3; p<0.05), and a trend to increase inspiratory burst area (CTL vs Musc; 4260±779 vs 5354±3063 mV ms, n=3; p=0.08). These data indicated that muscarinic modulation has similar effects on non-inspiratory and inspiratory XII MNs early in postnatal growth. Funding: NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of Muscarinic Acetylcholine Receptors in Intestinal Epithelial Homeostasis: Insights for the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is an intestinal disorder that causes prolonged inflammation of the gastrointestinal tract. Currently, the etiology of IBD is not fully understood and treatments are insufficient to completely cure the disease. In addition to absorbing essential nutrients, intestinal epithelial cells prevent the entry of foreign antigens (micro-organisms and undigested food) through mucus secretion and epithelial barrier formation. Disruption of the intestinal epithelial homeostasis exacerbates inflammation. Thus, the maintenance and reinforcement of epithelial function may have therapeutic benefits in the treatment of IBD. Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors for acetylcholine that are expressed in intestinal epithelial cells. Recent studies have revealed the role of mAChRs in the maintenance of intestinal epithelial homeostasis. The importance of non-neuronal acetylcholine in mAChR activation in epithelial cells has also been recognized. This review aimed to summarize recent advances in research on mAChRs for intestinal epithelial homeostasis and the involvement of non-neuronal acetylcholine systems, and highlight their potential as targets for IBD therapy.

Muscarinic receptor activation overrides boundary conditions on memory updating in a calcium/calmodulin-dependent manner.

Long-term memory storage is a dynamic process requiring flexibility to ensure adaptive behavioural responding in changing environments. Indeed, it is well established that memory reactivation can "destabilize" consolidated traces, leading to various forms of updating. However, the neurobiological mechanisms rendering long-term memories labile and modifiable remain poorly described. Moreover, boundary conditions, such as the age or strength of the memory, can reduce the likelihood of this destabilization; yet, intuitively, these most behaviourally influential of memories should also be modifiable under appropriate conditions. Here, we provide evidence that salient novelty at the time of memory reactivation promotes integrative updating of resistant object memories in rats. Furthermore, blockade of muscarinic acetylcholine receptors (mAChRs; with pirenzepine) or disruption of calcium/calmodulin (Ca2+/CaM) with KN-93, a Ca2+/CaM-binding molecule that inhibits calcium/calmodulin-dependent protein kinase II (CaMKII) activation, in perirhinal cortex (PRh) prevented novelty-induced destabilization and updating of resistant object memories. Finally, PRh M1 mAChR activation (with CDD-0102A) was sufficient to destabilize resistant object memories for updating, and this effect was blocked by KN-93, possibly via inhibition of CaMKII activity. Thus, mAChRs and activation of CaMKII appear to interact as part of a mechanism to override boundary conditions on resistant object memories to ensure integrative modification with novel information. These findings therefore have important implications for understanding the dynamic nature of long-term memory storage and potential treatments for conditions characterized by maladaptive and inflexible memories.

Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.

Muscarinic acetylcholine receptor-dependent and NMDA receptor-dependent LTP and LTD share the common AMPAR trafficking pathway

The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). Long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission in the hippocampus are also induced by mAChR. An AMPA receptor (AMPAR) trafficking model for hippocampal neurons has been proposed to simulate N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity in the early phase. In this study, we demonstrated the validity of the hypothesis that the mAChR-dependent LTP/LTD shares a common AMPAR trafficking pathway associated with NMDAR-dependent LTP/LTD. However, unlike NMDAR, Ca2+ influx into the spine cytosol occurs owing to the Ca2+ stored inside the ER and is induced via the activation of inositol 1,4,5-trisphosphate (IP3) receptors during M1 mAChR activation. Moreover, the AMPAR trafficking model implies that alterations in LTP and LTD observed in Alzheimer's disease could be attributed to age-dependent reductions in AMPAR expression levels.

Cell-type-specific synaptic modulation of mAChR on SST and PV interneurons.

The muscarinic acetylcholine receptor (mAChR) antagonist, scopolamine, has been shown to have a rapid antidepressant effect. And it is believed that GABAergic interneurons play a crucial role in this action. Therefore, characterizing the modulation effects of mAChR on GABAergic interneurons is crucial for understanding the mechanisms underlying scopolamine's antidepressant effects. In this study, we examined the effect of mAChR activation on the excitatory synaptic transmissions in two major subtypes of GABAergic interneurons, somatostatin (SST)- and parvalbumin (PV)-expressing interneurons, in the anterior cingulate cortex (ACC). We found that muscarine, a mAChR agonist, non-specifically facilitated the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in both SST and PV interneurons. Scopolamine completely blocked the effects of muscarine, as demonstrated by recovery of sESPCs and mEPSCs in these two types of interneurons. Additionally, individual application of scopolamine did not affect the EPSCs of these interneurons. In inhibitory transmission, we further observed that muscarine suppressed the frequency of both spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in SST interneurons, but not PV interneurons. Interestingly, scopolamine directly enhanced the frequency of both sIPSCs and mIPSCs mainly in SST interneurons, but not PV interneurons. Overall, our results indicate that mAChR modulates excitatory and inhibitory synaptic transmission to SST and PV interneurons within the ACC in a cell-type-specific manner, which may contribute to its role in the antidepressant effects of scopolamine.

Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage (V 1/2) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized V 1/2 in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents.SIGNIFICANCE STATEMENT Vestibular afferents express a diverse complement of ion channels. In vitro studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Vagus nerve stimulation exerts cardioprotection against doxorubicin-induced cardiotoxicity through inhibition of programmed cell death pathways.

The aberration of programmed cell death including cell death associated with autophagy/mitophagy, apoptosis, necroptosis, pyroptosis, and ferroptosis can be observed in the development and progression of doxorubicin-induced cardiotoxicity (DIC). Vagus nerve stimulation (VNS) has been shown to exert cardioprotection against cardiomyocyte death through the release of the neurotransmitter acetylcholine (ACh) under a variety of pathological conditions. However, the roles of VNS and its underlying mechanisms against DIC have never been investigated. Forty adults male Wistar rats were divided into 5 experimental groups: (i) control without VNS (CSham) group, (ii) doxorubicin (3mg/kg/day, i.p.) without VNS (DSham) group, (iii) doxorubicin + VNS (DVNS) group, (iv) doxorubicin + VNS + mAChR antagonist (atropine; 1mg/kg/day, ip, DVNS + Atro) group, and (v) doxorubicin + VNS + nAChR antagonist (mecamylamine; 7.5mg/kg/day, ip, DVNS + Mec) group. Our results showed that doxorubicin insult led to left ventricular (LV) dysfunction through impaired cardiac autonomic balance, decreased mitochondrial function, imbalanced mitochondrial dynamics, and exacerbated cardiomyocyte death including autophagy/mitophagy, apoptosis, necroptosis, pyroptosis, and ferroptosis. However, VNS treatment improved cardiac mitochondrial and autonomic functions, and suppressed excessive autophagy, apoptosis, necroptosis, pyroptosis, and ferroptosis, leading to improved LV function. Consistent with this, ACh effectively improved cell viability and suppressed cell cytotoxicity in doxorubicin-treated H9c2 cells. In contrast, either inhibitors of muscarinic (mAChR) or nicotinic acetylcholine receptor (nAChR) completely abrogated the favorable effects mediated by VNS and acetylcholine. These findings suggest that VNS exerts cardioprotective effects against doxorubicin-induced cardiomyocyte death via activation of both mAChR and nAChR.

The M1 muscarinic acetylcholine receptor regulates the surface expression of the AMPA receptor subunit GluA2 via PICK1.

Muscarinic acetylcholine receptors (mAChRs) have been shown to play significant roles in the regulation of normal cognitive processes in the hippocampus, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are also involved in these processes. This study aims to explore the mAChR-mediated regulation of AMPARs GluA2 trafficking and to reveal the key proteins and the signaling cascade involved in this process. Primary hippocampal neurons, as cell models, were treated with agonist 77-LH-28-1 and antagonist VU0255035, Fsc231, and APV. C57BL/6J male mice were stereotactically injected with 77-LH-28-1 and Fsc231 to obtain hippocampal slices. The trafficking of GluA2 was detected by surface biotinylation and immunostaining. Activation of M1 mAChRs promoted endocytosis and decreased the postsynaptic localization of the AMPA receptor subunit GluA2 and that phosphorylation of GluA2 at Ser880 was increased by M1 mAChR activity. Fsc231 blocked the endocytosis and postsynaptic localization of GluA2 induced by 77-LH-28-1 without affecting the phosphorylation of Ser880. PICK1 was required for M1 mAChR-mediated GluA2 endocytosis and downstream of phosphorylation of GluA2-Ser880, and the PICK1-GluA2 interaction was essential for M1 mAChR-mediated postsynaptic expression of GluA2. Taken together, our results show a functional correlation of M1 mAChRs with GluA2 and the role of PICK1 in their interplay. The schematic diagram for the modulation of GluA2 trafficking by M1 mAChRs. Activation of M1 mAChRs induces PKC activation, and the interaction of PICK1-GluA2 determines the endocytosis and postsynaptic localization of GluA2.