Activation Of Muscarinic Acetylcholine Receptors Research Articles

Muscarinic acetylcholine receptor activation synergizes the knockdown and toxicity of GABA-gated chloride channel insecticides.

Pest management requires continual identification of new physiological targets and strategies to control pests affecting agriculture and public/animal health. We propose the muscarinic system as a target for agrochemicals because of its physiological importance. Unlike the muscarinic system, gamma-amino butyric acid (GABA) receptors are an established insecticide target. Here, we investigated target-site synergism using small molecule probes (agonist and antagonist) against the muscarinic system and their ability to enhance the toxicity of GABAergic insecticides in Drosophila melanogaster (Meigen). Oral delivery of pilocarpine (muscarinic agonist) enhanced the toxicity of dieldrin, fipronil, and lindane, resulting in synergist ratios (SRs) between 4-32-fold (orally delivered) or between 2-67-fold when insecticides were topically applied. The synergism between pilocarpine and the GABA-insecticides was greater than the synergism observed with atropine (muscarinic antagonist), and was greater, or comparable, to the synergism observed with the metabolic inhibitor piperonyl butoxide. In addition to lethality, pilocarpine increased the knockdown of lindane. The mechanism of synergism was also investigated in the central nervous system using extracellular electrophysiology, where pilocarpine (3μmo/L) lowered the half-maximal inhibitory concentration (IC50 ) of lindane from 1.3 (0.86-1.98) μmol/L to 0.17 (0.14-0.21) μmol/L and fipronil's IC50 from 2.2 (1.54-3.29) μmol/L to 0.56 (0.40-0.77) μmol/L. Convergence of the cellular function between the muscarinic and GABAergic systems enhanced the insecticidal activity of GABA receptor blocking insecticides through the modulation of the central nervous system (CNS). The future impact of the findings could be the reduction of the active ingredient needed in a formulation with the development of muscarinic synergists. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Oxytocin-Cholinergic Central Interaction: Implications for Non-Social Memory Formation

Oxytocin (OT) and vasopressin (AVP) are two closely related neuropeptides implicated in learning and memory processes, anxiety, nociception, addiction, feeding behavior and social information processing. Regarding learning and memory, OT has induced long-lasting impairment in different behaviors, while the opposite was observed with AVP. We have previously evaluated the effect of peripheral administration of OT or its antagonist (AOT) on the inhibitory avoidance response of mice and on the modulation of cholinergic mechanisms. Here, we replicate and validate those results, but this time through central administration of neuropeptides, considering their poor passage through the blood–brain barrier (BBB). When we delivered OT (0.10 ng/mouse) and its antagonist (0.10 ng/mouse) through intracerebroventricular (ICV) injections, the neuropeptide impaired and AOT enhanced the behavioral performance on an inhibitory avoidance response evaluated 48 h after training in a dose-dependent manner. On top of that, we investigated a possible central interaction between OT and the cholinergic system. Administration of anticholinesterases inhibitors with access to the central nervous system (CNS), the activation of muscarinic acetylcholine (Ach) receptors and the increase of evoked ACh release using linopirdine (Lino) (3–10 µg/kg, IP), reversed the impairment of retention performance induced by OT. Besides, either muscarinic or nicotinic antagonists with unrestricted access to the CNS reduced the magnitude of the performance-facilitating effect of AOT’s central infusion. We suggest that OT might induce a cholinergic hypofunction state, resulting in an impairment of IA memory formation, a process for which the cholinergic system is crucially necessary.

Autophagy Induced by Muscarinic Acetylcholine Receptor 1 Mediates Migration and Invasion Targeting Atg5 via AMPK/mTOR Pathway in Prostate Cancer.

Increasing numbers of researchers discovered the expression of muscarinic acetylcholine receptor 1 in human cancers, while its function in human prostate cancer is still unclear. Our present study focused on CHRM1 to clarify its role in mediating autophagy in prostate cancer. We used immunohistochemistry, western blotting, and immunofluorescence experiments to observe the expression of muscarinic acetylcholine receptor 1 both in nude mice with subcutaneous tumors and in prostate cancer cells. The autophagy was observed through transmission electron microscopy, western blotting, quantitative real-time PCR, and immunofluorescence. After that, we used lentivirus to establish CHRM1 and Atg5 knockdown models. Then, the migration and invasion abilities after knocking down muscarinic acetylcholine receptor 1 and Atg5 were detected by transwell assays. In addition, the AMPK/mTOR pathway-related targets were detected by western blotting. We found that muscarinic acetylcholine receptor 1 was abundantly expressed both in vitro and in vivo in prostate cancer. The overexpression of muscarinic acetylcholine receptor 1 positively regulated migration and invasion in tumor cells as well as the activation of autophagy. Muscarinic acetylcholine receptor 1 was highly correlated with Atg5 and activated the AMPK/mTOR signaling pathway. Downregulation of Atg5 inhibited cell autophagy in prostate cancer cells and the migration and invasion of prostate cancer cells. Meanwhile, abnormal expressions of AMPK/mTOR pathway-related proteins were found. In conclusion, the present findings indicated that muscarinic acetylcholine receptor 1 is highly expressed in prostate cancer cells and promotes cell invasion and migration of prostate cancer. Autophagy is activated in prostate cancer cells and the activation of muscarinic acetylcholine receptor 1 positively regulates autophagy in prostate cancer cells. Moreover, muscarinic acetylcholine receptor 1 induces autophagy-mediated cell migration and invasion by targeting Atg5 in prostate cancer cells via AMPK/mTOR pathway, which uncovered that regulating muscarinic acetylcholine receptor 1, identified in this study, can be a promising solution for treating prostate cancer.

Expression of M3 muscarinic acetylcholine receptors in gastric cancer.

Introduction: Gastric cancer represents a real public health problem as far as incidence, aggressiveness and unfavorable prognosis are concerned. The autonomous nervous system might be one of the major factors involved in the onset, progression, and metastasis, both sympathetically and parasympathetically. The increased activation of the M3 muscarinic acetylcholine receptors (mAChRs) triggers pro-oncogenic mechanisms, especially at a gastric level, through the activation of the Hippo signaling pathway and the increase of the nerve growth factor. Patients, Materials and Methods: In this study, biopsy or postoperative gastric resection pieces have been evaluated by histopathological (HP) and immunohistochemical (IHC) examination in a group of 77 gastric patients and 23 patients without an oncological diagnosis. To quantify the IHC signal, also considering the HP aspect, light microscopy images were obtained. Results: The M3 mAChR expression analysis has been correlated with the different gastric adenocarcinoma differentiation degrees (G1–G3). M3 mAChR presence has been observed also in the non-malignant gastric tissue, but it was significantly increased in the tumor tissue. The highest receptor expression was recorded in patients with a poorly-differentiated (G3) adenocarcinoma, these expressions decreasing with the increase of the differentiation degree towards moderately-differentiated (G2) and well-differentiated (G1). Conclusions: Surgical or pharmacological parasympathetic activity inhibition could decrease the development and progression of gastric tumors and could improve the gastric cancer patient’s prognosis.

Characterizing the Contribution of Muscarinic Receptor Subtypes to Inspiratory Bursting Activity at Hypoglossal Motor Neurons of CD1 Mice

Obstructive sleep apnea (OSA) is characterized by cessation of breathing during sleep, due to repetitive episodic collapse of the airway or increased airway resistance. This loss of airway patency during sleeping conditions is controlled in part by the tongue, innervated by the hypoglossal (XII) motor nucleus. Decreased excitability of XII motor neurons (MNs) that innervate the tongue may occur due to activation of muscarinic acetylcholine receptors acting through G‐protein coupled signal transduction pathways. The five types of muscarinic acetylcholine receptors can be broadly categorized as excitatory or inhibitory. M1, M3, and M5 receptors are excitatory receptors coupled to Gq, whereas M2 and M4 receptors are inhibitory receptors coupled to Gi. XII MNs express M1, M2, M3, and M5 subtypes of muscarinic receptors, but the specific mechanisms of how cholinergic signaling modulates excitability of XII MNs are not well understood. The objective of this study was to investigate how the muscarinic receptor subtypes individually contribute to inspiratory activity at XII MNs. We tested the hypothesis that activation of M1 and M5 receptors potentiate, whereas activation of M2 receptors inhibit inspiratory bursting in XII MNs. Using medullary rhythmic slice preparations from neonatal CD1 mice (postnatal day P0‐5) local application of muscarine (30s, 100 μM) to the XII motor nucleus increased inspiratory burst amplitude to 180 ± 7% [n = 23]. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarine‐mediated potentiation of inspiratory bursting to 94 ± 15% and 72 ± 14% of control muscarine response [n = 9]. Activating M1 receptors locally with cevimeline (1 mM) for 30s and 60s increased burst amplitude to 116 ± 7% and 117 ± 7% of control muscarine response [n = 6]. Blocking M2 receptors locally with AF‐DX 116 (1 μM & 10 μM), the muscarine‐mediated effect was 101 ± 10% [n = 3] and 95 ± 10% [n = 3] of control muscarine‐mediated excitation. M2 receptor blockade was also tested by application of the M2 receptor antagonist, methoctramine (MTH). Local application of MTH (2 μM) had no effect on muscarine‐mediated potentiation of inspiratory bursting (171 ± 15% compared to control muscarine response of 177 ± 14% [n = 10]). Similarly, blocking M2 receptors with bath application of MTH (200 nM) had no effect on muscarine‐mediated potentiation of inspiratory bursting (166 ± 20% compared to 161 ± 14% control muscarine response) or on inspiratory burst period (10 ± 0.3% compared to 10 ± 0.4% control muscarine response) [n = 5]. Modulating M5 receptors locally with VU 0238429 (20 μM and 1000 μM) increased burst amplitude to 116% ± 5% and 119 ± 4% of control muscarine‐mediated excitation [n = 3]. Our data partially support a role of M1 receptors contributing to the muscarinic potentiation of inspiratory bursting in XII MNs. Future research will evaluate other muscarinic receptor agonists and antagonists, as well as determine how the muscarinic modulation of inspiratory bursting changes with postnatal maturation.

Effects of postnatal maturation on muscarinic acetylcholine receptor distribution in hypoglossal motorneurons

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. Hypoglossal motoneurons (XII MNs), which innervate the genioglossus muscle, receive less excitatory noradrenergic drive, and may be inhibited by activation of muscarinic acetylcholine receptors, during REM sleep. Indeed, preliminary data from intact adult rats during natural sleep implicated an inhibitory effect of muscarinic acetylcholine receptors on hypoglossal motoneurons. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at hypoglossal motoneurons. Our project examines the distribution of muscarinic acetylcholine receptors at XII MNs, and how that changes across postnatal maturation. We hypothesizes there would be an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) muscarinic receptors in XII MNs with postnatal maturation. We performed double‐labeled immunofluorescence experiments on perfused 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0‐P2, P3‐P5, P6‐P10, P11‐P13, P14‐P17, and adult against muscarinic targets M1, M2, M3, M5. Slices were co‐labeled with choline acetyltransferase (ChAT) and 4’,6‐diamidino‐2‐phenylindole (DAPI). Images of the hypoglossal motor nucleus (HMN) were collected using a confocal microscope. ImageJ was used to determine the average HMN muscarine receptor subtype intensity across postnatal maturation. Preliminary data (n = 1) indicate that M1 receptors showed a consistent decrease in labeling intensity in the HMN across development (P5=100%, P13=80%, P17=67%, adult = 40%), whereas M3 (n = 1) (P4= 90%, P9= 73%, P13=89%, P17= 100%, adult= 41%) showed a transitory increases in expression before a final decrease into adulthood. M5 (n = 1 or 2) expression shows a bimodal distribution of labeling intensity (P0‐2= 100%, P4‐6= 58‐99%, P7‐10 = 52‐70%, P11‐13= 35‐58 %, P14‐17 = 27 % adult= 17‐37 %). In contrast, M2 receptor expression intensity remained relatively high with small fluctuations into adulthood (n=2), (P0‐2 = 92‐94 %, P4‐6 = 100%, P7‐10 = 92‐95 %, P11‐13 = 78‐107 %, P14‐17 = 88‐107 %, adult = 73‐77 %). These data partly support our hypothesis that there would be a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors. We speculate that the decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift of muscarinic modulation from excitation to inhibition with postnatal maturation.

GIRK channel as a versatile regulator of neurotransmitter release via L-type Ca2+ channel-dependent mechanism in the neuromuscular junction

G protein-gated inwardly rectifying potassium (GIRK) channels are one of the main regulators of neuronal excitability. Activation of GIRK channels in the CNS usually leads to postsynaptic inhibition. However, the function of GIRK channels in the presynaptic processes, notably neurotransmitter release form motor nerve terminals, is yet to be comprehensively understood. Here, using electrophysiological and fluorescent approaches, the role of GIRK channels in neurotransmitter release from frog motor nerve terminals was studied. We found that the inhibition of GIRK channels with nanomolar tertiapin-Q synchronized exocytosis events with action potential but suppressed spontaneous and evoked neurotransmitter release, as well as Ca2+ transient and membrane permeability for K+. The action of GIRK channel inhibition on evoked neurotransmission was prevented by selective antagonist of voltage-gated Ca2+ channels of L-type. Furthermore, the effects of muscarinic acetylcholine receptor activation on neurotransmitter release, Ca2+ transient and K+ channel activity were markedly modulated by inhibition of GIRK channels. Thus, at the motor nerve terminals GIRK channels can regulate timing of neurotransmitter release and be a positive modulator of synaptic vesicle exocytosis acting partially via L-type Ca2+ channels. In addition, GIRK channels are key players in a feedback control of neurotransmitter release by muscarinic acetylcholine receptors.

M<sub>3</sub> Muscarinic Acetylcholine Receptor Antagonist Darifenacin Protects against Pulmonary Fibrosis through ERK/NF-κB/miR-21 Pathway

Idiopathic pulmonary fibrosis is an untreatable lethal lung disease, which is related to the aberrant proliferation of fibroblasts. M3 muscarinic acetylcholine receptor (M3-mAChR) activation exerts proliferative effect on various kinds of cells. However, whether M3-mAChR inhibition has a protective effect on pulmonary fibrosis remains unexplored. A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Darifenacin was used to block M3-mAChR. Histological changes were observed using Masson’s Trichrome and hematoxylin and eosin (HE) staining. Hydroxyproline was measured by Hydroxyproline detection kit. Transforming growth factor β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, pulmonary fibroblasts were isolated from lungs of neonatal rat. After treatment, the cell viability, Hydroxyproline level was measured by MTT and Hydroxyproline detection kit respectively. The expression level of extracellular signal-regulated kinase (ERK), nuclear factor kappa-B (N-NF-κB), and microRNA-21 (miR-21) was detected by western blot or quantitative real-time PCR (qRT-PCR). Darifenacin relieved the fibrotic effects provoked by bleomycin. The expression level of hydroxyproline, TGF-β1 and TNF-α level was all downregulated after darifenacin treatment. In lung fibroblasts, darifenacin decreased cell viability and hydroxyproline level induced by bleomycin. Besides, phosphorylation-ERK and nuclear N-NF-κB protein level was downregulated, as well as miR-21 level. M3-mAChR antagonist darifenacin attenuates bleomycin-induced pulmonary fibrosis in rats, which may relate to the ERK/NF-κB/miRNA-21 signaling pathway.

Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression.

Age and circadian rhythm‐dependent effects of M1 muscarinic acetylcholine receptor positive allosteric modulators and donepezil on sleep‐wake architecture and arousal

AbstractBackgroundDegeneration of basal forebrain cholinergic signaling in aging and Alzheimer's disease (AD) is linked with abnormalities in arousal, sleep/wake architecture, and cognition. While acetylcholinesterase inhibitors (AChEIs) are approved for the treatment of AD, they produce dose‐limiting adverse effects due to nonselective activation of peripheral muscarinic acetylcholine receptors (mAChRs). Selectively targeting the M1 mAChR using M1 positive allosteric modulators (PAMs) has shown promise for boosting cognitive performance and arousal across preclinical species. We evaluate the effects of the M1 PAM VU0453595 on arousal and sleep/wake architecture in aged and young mice across both the active and inactive phases of the circadian cycle. We hypothesize that dosing an M1 PAM during the active phase in aged mice will produce a greater enhancement of arousal and wakefulness, normalizing deficits observed in aged mice.MethodUtilizing electroencephalography, we examined the effects of the M1 PAM VU0453595 in comparison with the AChEI Donepezil on sleep‐wake architecture and state dependent spectral power in young and aged C57/BL6 mice following dosing in either the active or inactive phases of the circadian cycle.ResultWhen compared to young mice, aged mice displayed several alterations in normal sleep‐wake architecture; in the inactive phase, reductions in rapid eye movement (REM) sleep, while in the active phase increases in non‐REM (NREM) sleep and decreases in wakefulness. Aged mice also displayed reductions in slow delta power during NREM sleep and increased delta power during wake. In young mice, dosing with both compounds increased wakefulness and arousal during wake in the inactive phase, with reduced effects in the active phase. In the aged mice VU0453595 increased arousal during wake in the inactive phase, and both wakefulness and arousal during wake when dosed in the active phase. Donepezil in contrast produced increased wakefulness in the inactive period, but reduced wakefulness in the active period and limited effects on arousal during wake.ConclusionThe M1 PAM VU0453595 produced increases in wakefulness and arousal in aged mice during the active phase, suggesting that selective M1 PAMs may be suitable for dosing during the active phase in aging and AD populations to normalize disruptions in wakefulness and arousal.

The Regulation Effect of α7nAChRs and M1AChRs on Inflammation and Immunity in Sepsis.

The inflammatory storm in the early stage and immunosuppression in the late stage are responsible for the high mortality rates and multiple organ dysfunction in sepsis. In recent years, studies have found that the body's cholinergic system can spontaneously and dynamically regulate inflammation and immunity in sepsis according to the needs of the body. Firstly, the vagus nerve senses and regulates local or systemic inflammation by means of the Cholinergic Anti-inflammatory Pathway (CAP) and activation of α7-nicotinic acetylcholine receptors (α7nAChRs); thus, α7nAChRs play important roles for the central nervous system (CNS) to modulate peripheral inflammation; secondly, the activation of muscarinic acetylcholine receptors 1 (M1AChRs) in the forebrain can affect the neurons of the Medullary Visceral Zone (MVZ), the core of CAP, to regulate systemic inflammation and immunity. Based on the critical role of these two cholinergic receptor systems in sepsis, it is necessary to collect and analyze the related findings in recent years to provide ideas for further research studies and clinical applications. By consulting the related literature, we draw some conclusions: MVZ is the primary center for the nervous system to regulate inflammation and immunity. It coordinates not only the sympathetic system and vagus system but also the autonomic nervous system and neuroendocrine system to regulate inflammation and immunity; α7nAChRs are widely expressed in immune cells, neurons, and muscle cells; the activation of α7nAChRs can suppress local and systemic inflammation; the expression of α7nAChRs represents the acute or chronic inflammatory state to a certain extent; M1AChRs are mainly expressed in the advanced centers of the brain and regulate systemic inflammation; neuroinflammation of the MVZ, hypothalamus, and forebrain induced by sepsis not only leads to their dysfunctions but also underlies the regulatory dysfunction on systemic inflammation and immunity. Correcting the neuroinflammation of these regulatory centers and adjusting the function of α7nAChRs and M1AChRs may be two key strategies for the treatment of sepsis in the future.

5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis.

5-diphosphoinositol pentakisphosphate (5-IP7) is a signalling metabolite linked to various cellular processes. How extracellular stimuli elicit 5-IP7 signalling remains unclear. Here we show that 5-IP7 in β cells mediates parasympathetic stimulation of synaptotagmin-7 (Syt7)-dependent insulin release. Mechanistically, vagal stimulation and activation of muscarinic acetylcholine receptors triggers Gαq-PLC-PKC-PKD-dependent signalling and activates IP6K1, the 5-IP7 synthase. Whereas both 5-IP7 and its precursor IP6 compete with PIP2 for binding to Syt7, Ca2+ selectively binds 5-IP7 with high affinity, freeing Syt7 to enable fusion of insulin-containing vesicles with the cell membrane. β-cell-specific IP6K1 deletion diminishes insulin secretion and glucose clearance elicited by muscarinic stimulation, whereas mice carrying a phosphorylation-mimicking, hyperactive IP6K1 mutant display augmented insulin release, congenital hyperinsulinaemia and obesity. These phenotypes are absent in mice lacking Syt7. Our study proposes a new conceptual framework for inositol pyrophosphate physiology in which 5-IP7 acts as a GPCR second messenger at the interface between peripheral nervous system and metabolic organs, transmitting Gq-coupled GPCR stimulation to unclamp Syt7-dependent, and perhaps other, exocytotic events.

Metaplastic Reinforcement of Long-Term Potentiation in Hippocampal Area CA2 by Cholinergic Receptor Activation.

Hippocampal CA2, an inconspicuously positioned area between the well-studied CA1 and CA3 subfields, has captured research interest in recent years because of its role in social memory formation. However, the role of cholinergic inputs to the CA2 area for the regulation of synaptic plasticity remains to be fully understood. We show that cholinergic receptor activation with the nonselective cholinergic agonist, carbachol (CCh), triggers a protein synthesis-dependent and NMDAR-independent long-term synaptic depression (CCh-LTD) at entorhinal cortical (EC)-CA2 and Schaffer collateral (SC)-CA2 synapses in the hippocampus of adult male Wistar rats. The activation of muscarinic acetylcholine receptors (mAChRs) is critical for the induction of CCh-LTD with the results suggesting an involvement of M3 and M1 mAChRs in the early facilitation of CCh-LTD, while nicotinic AChR activation plays a role in the late maintenance of CCh-LTD at CA2 synapses. Remarkably, we find that CCh priming lowers the threshold for the subsequent induction of persistent long-term potentiation (LTP) of synaptic transmission at EC-CA2 and the plasticity-resistant SC-CA2 pathways. The effects of such a cholinergic-dependent synaptic depression on subsequent LTP at EC-CA2 and SC-CA2 synapses have not been previously explored. Collectively, the results demonstrate that CA2 synaptic learning rules are regulated in a metaplastic manner, whereby modifications triggered by prior cholinergic stimulation can dictate the outcome of future plasticity events. Moreover, the reinforcement of LTP at EC inputs to CA2 following the priming stimulus coexists with concurrent sustained CCh-LTD at the SC-CA2 pathway and is dynamically scaled by modulation of SC-CA2 synaptic transmission.SIGNIFICANCE STATEMENT The release of the neuromodulator acetylcholine is critically involved in processes of hippocampus-dependent memory formation. Cholinergic afferents originating in the medial septum and diagonal bands of Broca terminating in the hippocampal area CA2 might play an important role in the modulation of area-specific synaptic plasticity. Our findings demonstrate that cholinergic receptor activation induces an LTD of synaptic transmission at entorhinal cortical- and Schaffer collateral-CA2 synapses. This cholinergic activation-mediated LTD displays a bidirectional metaplastic switch to LTP on a future timescale. This suggests that such bidirectional synaptic modifications triggered by the dynamic modulation of tonic cholinergic receptor activation may support the formation of CA2-dependent memories given the increased hippocampal cholinergic tone during active wakefulness observed in exploratory behavior.

The Combined Treatment with Chemotherapeutic Agents and the Dualsteric Muscarinic Agonist Iper-8-Naphthalimide Affects Drug Resistance in Glioblastoma Stem Cells.

Background: Glioblastoma multiforme (GBM) is characterized by heterogeneous cell populations. Among these, the Glioblastoma Stem Cells (GSCs) fraction shares some similarities with Neural Stem Cells. GSCs exhibit enhanced resistance to conventional chemotherapy drugs. Our previous studies demonstrated that the activation of M2 muscarinic acetylcholine receptors (mAChRs) negatively modulates GSCs proliferation and survival. The aim of the present study was to analyze the ability of the M2 dualsteric agonist Iper-8-naphthalimide (N-8-Iper) to counteract GSCs drug resistance. Methods: Chemosensitivity to M2 dualsteric agonist N-8-Iper and chemotherapy drugs such as temozolomide, doxorubicin, or cisplatin was evaluated in vitro by MTT assay in two different GSC lines. Drug efflux pumps expression was evaluated by RT-PCR and qRT-PCR. Results: By using sub-toxic concentrations of N-8-Iper combined with the individual chemotherapeutic agents, we found that only low doses of the M2 agonist combined with doxorubicin or cisplatin or temozolomide were significantly able to counteract cell growth in both GSC lines. Moreover, we evaluated as the exposure to high and low doses of N-8-Iper downregulated the ATP-binding cassette (ABC) drug efflux pumps expression levels. Conclusions: Our results revealed the ability of the investigated M2 agonist to counteract drug resistance in two GSC lines, at least partially by downregulating the ABC drug efflux pumps expression. The combined effects of low doses of conventional chemotherapy and M2 agonists may thus represent a novel promising pharmacological approach to impair the GSC-drug resistance in the GBM therapy.

Pulmonary Microcirculation during Experimental Pulmonary Thromboembolism under Conditions of Activation and Blockade of Muscarinic Acetylcholine Receptors.

Changes in pulmonary microcirculation were studied in isolated perfused rabbit lungs during modelling pulmonary thromboembolism under conditions of acetylcholine infusion against the background of treatment with M1 acetylcholine receptor blocker pirenzepine or blockade of muscarinic acetylcholine receptors with atropine. In the first case, the increase in pulmonary artery pressure was less pronounced than in case of atropine treatment. In response to pulmonary embolism after acetylcholine infusion against the background of pirenzepine pretreatment, the capillary hydrostatic pressure and postcapillary resistance did not change, while after atropine treatment, these parameters increased. In case of pulmonary embolism after acetylcholine infusion combined with selective blockade of M1 muscarinic acetylcholine receptors, the capillary filtration coefficient increased to a greater extent, than in the control and after blockade of muscarinic acetylcholine receptors.

Anti‐inflammatory cholinergic system modulates LPS‐induced acute lung injury

Lung cholinergic system suing acetylcholine (ACh) as a neurotransmitter regulates lung inflammation and is believed to play an anti-inflammatory role via activation of nicotinic (nAChR) and/or muscarinic (mAChR) acetylcholine receptors expressed in lung tissue. Therefore, studies that evaluate the effects of lung cholinergic deficiency are relevant to understand the multiple actions of ACh in acute lung injury (ALI). Aim To evaluate whether VAChT deficiency and treatment with agonist α-7 nAChR affects lung alterations observed in an experimental model of ALI. Methods Wild-Type (WT) mice and mice with decreased expression of VAChT KDHOM, which is correlated with 70% less of ACh release, received PNU-282987 (specific α7nAChR agonist,10 mg/kg) 6 hours after LPS (1mg/kg) intratracheal instillation. We evaluated lung inflammation focusing on inflammation in bronchioalveolar lavage fluid (BALF). We also compared the expression levels of α-7 and α-4 nAChRs in lung tissue using radioligand binding assays. Results LPS treatment induced higher increase in inflammatory cells in BALF in VAChT KDHOM compared to WT mice. The activation α-7 nAChR by treatment with PNU-282987 reduced the total cells, macrophages, and neutrophils recovered in BALF (p<0.001 for all comparisons) in both VAChT-KDHOM and WT that received LPS. The LPS induced upregulate α-7 nAChR levels in WT mice but not in the VAChT KDHOM mice. However, treatment with PNU-282987 induced increase in α-7 nAChR levels in LPS-treated VAChT-KDHOM mice. Unlike α-7 nAChR, α-4 nAChR expression was not changed by VAChT reduction, LPS-induce, and PNU-282987 treatment. Conclusion Our preliminary results suggest that α-7 nAChR upregulation contributes to the anti-inflammatory effect of lung cholinergic system. Unlike WT mice, VAChT-KDHOM mice did not upregulate α-7 nAChR in response to inflammatory insult (LPS treatment) resulting in higher inflammation in VAChT-KDHOM mice than WT mice. Furthermore, treatment of VAChT-KDHOM mice with PNU-282987 resulted in increase in α-7 nAChR expression in lung tissue and reduction of LPS-induced lung inflammation. Experiments to determine mAChRs expression in WT vs. VAChT-KDHOM mice are ongoing.

Control of Neuronal Excitability by Cell Surface Receptor Density and Phosphoinositide Metabolism.

Phosphoinositides are members of a family of minor phospholipids that make up about 1% of all lipids in most cell types. Despite their low abundance they have been found to be essential regulators of neuronal activities such as action potential firing, release and re-uptake of neurotransmitters, and interaction of cytoskeletal proteins with the plasma membrane. Activation of several different neurotransmitter receptors can deplete phosphoinositide levels by more than 90% in seconds, thereby profoundly altering neuronal behavior; however, despite the physiological importance of this mechanism we still lack a profound quantitative understanding of the connection between phosphoinositide metabolism and neuronal activity. Here, we present a model that describes phosphoinositide metabolism and phosphoinositide-dependent action potential firing in sympathetic neurons. The model allows for a simulation of activation of muscarinic acetylcholine receptors and its effects on phosphoinositide levels and their regulation of action potential firing in these neurons. In this paper, we describe the characteristics of the model, its calibration to experimental data, and use the model to analyze how alterations of surface density of muscarinic acetylcholine receptors or altered activity levels of a key enzyme of phosphoinositide metabolism influence action potential firing of sympathetic neurons. In conclusion, the model provides a comprehensive framework describing the connection between muscarinic acetylcholine signaling, phosphoinositide metabolism, and action potential firing in sympathetic neurons which can be used to study the role of these signaling systems in health and disease.

Rhythmogenic networks are potently modulated by activation of muscarinic acetylcholine receptors in the rodent spinal cord.

Electrical stimulation of the spinal cord is a potent means for activating mammalian stepping in the absence of the descending control from the brain. Previously, we have shown that stimulation of pain delivering (Aδ) sacrocaudal afferents (SCA) has a powerful capacity to activate the sacral and lumbar rhythmogenic networks in the neonatal rodent spinal cord. Relatively little is known about the neural pathways involved in activation of the locomotor networks by Aδ afferents, on their mechanism of action and on the possibility to modulate their activity. We have shown that elevation of the endogenous level of acetylcholine at the sacral cord by blocking cholinesterase could modulate the SCA-induced locomotor rhythm in a muscarinic receptor-dependent mechanism. Here, we review these and more recent findings and report that controlled stimulation of SCA in the presence of muscarine is a potent activator of the locomotor network. The possible mechanisms involved in the muscarinic modulation of the locomotor rhythm are discussed in terms of the differential projections of sacral relay neurons, activated by SCA stimulation, to the lumbar locomotor rhythm generators, and to their target motoneurons. Altogether, our studies show that manipulations of cholinergic networks offer a simple and powerful means to control the activity of locomotor networks in the absence of supraspinal control. Cover Image for this issue: https://doi.org/10.1111/jnc.15079.

Mecamylamine inhibits seizure-like activity in CA1-CA3 hippocampus through antagonism to nicotinic receptors.

Cholinergic modulation of hippocampal network function is implicated in multiple behavioral and cognitive states. Activation of nicotinic and muscarinic acetylcholine receptors affects neuronal excitability, synaptic transmission and rhythmic oscillations in the hippocampus. In this work, we studied the ability of the cholinergic system to sustain hippocampal epileptiform activity independently from glutamate and GABA transmission. Simultaneous CA3 and CA1 field potential recordings were obtained during the perfusion of hippocampal slices with the aCSF containing AMPA, NMDA and GABA receptor antagonists. Under these conditions, spontaneous epileptiform discharges synchronous between CA3 and CA1 were recorded. Epileptiform discharges were blocked by addition of the calcium-channel blocker Cd2+ and disappeared in CA1 after a surgical cut between CA3 and CA1. Cholinergic antagonist mecamylamine abolished CA3-CA1 synchronous epileptiform discharges, while antagonists of α7 and α4β2 nAChRs, MLA and DhβE, had no effect. Our results suggest that activation of nicotinic acetylcholine receptors can sustain CA3-CA1 synchronous epileptiform activity independently from AMPA, NMDA and GABA transmission. In addition, mecamylamine, but not α7 and α4β2 nAChRs antagonists, reduced bicuculline-induced seizure-like activity. The ability of mecamylamine to decrease hippocampal network synchronization might be associated with its therapeutic effects in a wide variety of CNS disorders including addiction, depression and anxiety.

An acetylcholine-dopamine interaction in the nucleus accumbens and its involvement in ethanol's dopamine-releasing effect.

Alcohol use disorder is a chronic, relapsing brain disorder causing substantial morbidity and mortality. Cholinergic interneurons (CIN) within the nucleus accumbens (nAc) have been suggested to exert a regulatory impact on dopamine (DA) neurotransmission locally, and defects in CIN have been implied in several psychiatric disorders. The aim of this study was to investigate the role of CIN in regulation of basal extracellular levels of DA and in modulation of nAc DA release following ethanol administration locally within the nAc of male Wistar rats. Using reversed in vivo microdialysis, the acetylcholinesterase inhibitor physostigmine was administered locally in the nAc followed by addition of either the muscarinic acetylcholine (ACh) receptor antagonist scopolamine or the nicotinic ACh receptor antagonist mecamylamine. Further, ethanol was locally perfused in the nAc following pretreatment with scopolamine and/or mecamylamine. Lastly, ethanol was administered locally into the nAc of animals with accumbal CIN‐ablation induced by anticholine acetyl transferase‐saporin. Physostigmine increased accumbal DA levels via activation of muscarinic ACh receptors. Neither scopolamine and/or mecamylamine nor CIN‐ablation altered basal DA levels, suggesting that extracellular DA levels are not tonically controlled by ACh in the nAc. In contrast, ethanol‐induced DA elevation was prevented following coadministration of scopolamine and mecamylamine and blunted in CIN‐ablated animals, suggesting involvement of CIN‐ACh in ethanol‐mediated DA signaling. The data presented in this study suggest that basal extracellular levels of DA within the nAc are not sustained by ACh, whereas accumbal CIN‐ACh is involved in mediating ethanol‐induced DA release.