Muscarinic Cholinergic Receptors Research Articles

P.1.c.047 Asenapine: long-term and dose-dependent effects on adrenergic and muscarinic cholinergic receptors in rat brain

The main class of atypical antipsychotic drugs (APDs) in current use includes the protypical atypical APD, clozapine, as well as aripiprazole, asenapine, iloperidone, lurasidone, olanzapine, quetiapine, risperidone, and ziprasidone. At clinically effective doses, these agents produce extensive blockade of serotonin (5-HT)2A receptors, direct or indirect stimulation of 5-HT1A receptors, and to a lesser extent, reduction in dopamine (DA) D2 receptor-mediated neurotransmission. This contrasts with typical APDs, for example haloperidol and perphenazine, which are mainly DA D2/D3 receptor antagonists and have weaker, if any, potency as 5-HT2A receptor antagonists. Some, but not all, atypical APDs are also effective 5-HT2C receptor inverse agonists or neutral antagonists, 5-HT6 or 5-HT7 receptor antagonists. This diverse action on 5-HT receptors may contribute to significant differences in efficacy and tolerability among the atypical APDs. There is considerable preclinical and some clinical evidence that effects on 5-HT receptors contribute to the low risk of producing extrapyramidal side effects, which is the defining characteristic of an atypical APD, the lack of elevation in plasma prolactin levels (with risperidone and 9-hydroxyrisperidone being exceptions), antipsychotic action, and ability to improve some domains of cognition in patients with schizophrenia. The serotonergic actions of the atypical APDs, especially 5-HT2A receptor antagonism, are particularly important to the differential effects of typical and atypical APDs to overcome the effects of acute or subchronic administration of N-methyl-d-aspartate (NMDA) receptor antagonists, such as phencyclidine, ketamine, and dizocipline (MK-801). 5-HT1A receptor stimulation and 5-HT6 and 5-HT7 receptor antagonism may contribute to beneficial effects of these agents on cognition. In particular, 5-HT7 receptor antagonism may be the basis for the pro-cognitive effects of the atypical APD, amisulpride, a D2/D3 receptor antagonist, which has no effect on other 5-HT receptor. 5-HT2C receptor antagonism appears to contribute to the weight gain produced by some atypical APDs and may also affect cognition and psychosis via its influence on cortical and limbic dopaminergic activity.

Cytotoxic Effects of 4-Hydroxynonenal In Pc12 Cells: Involvement of Neurotransmitter Receptors

Effect of 4-hydroxynonenal (HNE) was studied on the sensitivity of various neurotransmitter receptors in PC-12 cells. Initially, cytotoxicity profiling of HNE was carried out using HNE concentrations 0.1–50mM for 30min to 24h. The endpoints selected were, trypan blue dye exclusion, MTT, LDH release and neutral red uptake (NRU) assays. Significant cytotoxic responses were observed by minimum 2h of exposure, except for HNE at 50mM, where cytotoxicity was exerted even at 90min. HNE concentrations 10–50mM were found to be cytotoxic, 2–5mM cytostatic and 0.1-lmM non-cytotoxic. Neurotransmitter receptors studies were carried out using specific radioligands with selected doses of HNE (1,10,25 and 50mM for l-8h). Significant decrease in binding of 3 H-QNB, 3 H-Fluinitrazepam and 3 H-Ketanserin, known to label cholinergic-muscarinic, benzodiazepine and serotonin 5HT 2A receptors respectively was observed even at lh exposure with 25 and 50mM concentrations of HNE. Whereas, significant inhibition in binding of 3 H-Spiperone to DA-D2 receptor was started at 4h of exposure and continued till 8h. Specific binding with 3 H-Fluinitrazepam and 3 H-Ketanserin was reached to saturation at dose of 50mM for 4h and onwards. PC-12 cells have shown particular vulnerability to cytotoxic concentrations of HNE. Experimental HNE exposure provides an intriguing model of toxicant-cell interactions, which likely involved receptors in HNE neurotoxicity, lead neurodegeneration. doi: 10.5214/ans.0972.7531.2007.140401

Relationships among mercury, selenium, and neurochemical parameters in common loons (Gavia immer) and bald eagles (Haliaeetus leucocephalus)

Fish-eating birds can be exposed to levels of dietary methylmercury (MeHg) known or suspected to adversely affect normal behavior and reproduction, but little is known regarding Hg's subtle effects on the avian brain. In the current study, we explored relationships among Hg, Se, and neurochemical receptors and enzymes in two fish-eating birds--common loons (Gavia immer) and bald eagles (Haliaeetus leucocephalus). In liver, both species demonstrated a wide range of total Hg (THg) concentrations, substantial demethylation of MeHg, and a co-accumulation of Hg and Se. In liver, there were molar excesses of Se over Hg up to about 50-60 microg/g THg, above which there was an approximate 1:1 molar ratio of Hg:Se in both species. However, in brain, bald eagles displayed a greater apparent ability to demethylate MeHg than common loons. There were molar excesses of Se over Hg in brains of bald eagles across the full range of THg concentrations, whereas common loons often had extreme molar excesses of Hg in their brains, with a higher proportion of THg remaining as MeHg compared with eagles. There were significant positive correlations between brain THg and muscarinic cholinergic receptor concentrations in both species studied; whereas significant negative correlations were observed between N-methyl-D-aspartic acid (NMDA) receptor levels and brain Hg concentration. There were no significant correlations between brain Se and neurochemical receptors or enzymes (cholinesterase and monoamine oxidase) in either species. Our findings suggest that there are significant differences between common loons and bald eagles with respect to cerebral metabolism and toxicodynamics of MeHg and Se. These interspecies differences may influence relative susceptibility to MeHg toxicity; however, neurochemical responses to Hg in both species were similar.

Central Anticholinergic Syndrome: A Case Report

Postoperative central anticholinergic syndrome (CAS) is caused by anticholinergic medications that cross the blood-brain barrier. Medications with central anticholinergic effects block muscarinic cholinergic receptors, resulting in a wide array of symptoms. Symptoms may range from coma to a highly agitated state. CAS may be underdiagnosed because of its varying presentation and lack of awareness. Differential diagnosis for the patient presenting with abnormal neurological signs and symptoms should include CAS after the exclusion of other potential causes. This case report details the occurrence of CAS in a patient in her 20's. A review of CAS including causes, signs and symptoms, incidence, differential diagnosis, and treatment is discussed.

Developmental co-exposure to methylmercury and PCB153 or PCB126 affects cerebral cholinergic muscarinic receptors at weaning and puberty in rats

Developmental co-exposure to methylmercury and PCB153 or PCB126 affects cerebral cholinergic muscarinic receptors at weaning and puberty in rats

Scopolamine infused into perirhinal cortex improves object recognition memory by blocking the acquisition of interfering object information.

In a previous study, we reported apparently paradoxical facilitation of object recognition memory following infusions of the cholinergic muscarinic receptor antagonist scopolamine into the perirhinal cortex (PRh) of rats. We attributed these effects to the blockade by scopolamine of the acquisition of interfering information. The present study tested this possibility directly by modifying the spontaneous object recognition memory task to allow the presentation of a potentially interfering object either before the sample phase or in the retention delay between the sample and choice phases. Presentation of an object between the sample and choice phases disrupted subsequent recognition of the sample object (retroactive interference), and intra-PRh infusions of scopolamine prior to the presentation of the irrelevant object prevented this retroactive interference effect. Moreover, presentation of an irrelevant object prior to the sample phase interfered proactively with sample object recognition, and intra-PRh infusions of scopolamine prior to the presentation of the pre-sample object prevented this proactive interference effect. These results suggest that blocking muscarinic cholinergic receptors in PRh can disrupt the acquisition of potentially interfering object information, thereby facilitating object recognition memory. This finding provides further, strong evidence that acetylcholine is important for the acquisition of object information in PRh.

Differential effects of muscarinic receptor blockade in prelimbic cortex on acquisition and memory formation of an odor-reward task

The present experiments determined the consequences of blocking muscarinic cholinergic receptors of the prelimbic (PL) cortex in the acquisition and retention of an odor-reward associative task. Rats underwent a training test (five trials) and a 24-h retention test (two retention trials and two relearning trials). In the first experiment, rats were bilaterally infused with scopolamine (20 or 5 microg/site) prior to training. Although scopolamine rats showed acquisition equivalent to PBS-injected controls, they exhibited weakened performance in the 24-h retention test measured by number of errors. In the second experiment, rats were injected with scopolamine (20 microg/site) immediately or 1 h after training and tested 24 h later. Scopolamine rats injected immediately showed severe amnesia detected in two performance measures (errors and latencies), demonstrating deficits in retention and relearning, whereas those injected 1 h later showed good 24-h test performance, similar to controls. These results suggest that muscarinic transmission in the PL cortex is essential for early memory formation, but not for acquisition, of a rapidly learned odor discrimination task. Findings corroborate the role of acetylcholine in consolidation processes and the participation of muscarinic receptors in olfactory associative tasks.

Differential involvement of M1-type and M4-type muscarinic cholinergic receptors in the dorsomedial striatum in task switching

Previous experiments have demonstrated that the rat dorsomedial striatum is one brain area that plays a crucial role in learning when conditions require a shift in strategies. Further evidence indicates that muscarinic cholinergic receptors in this brain area support adaptations in behavioral responses. Unknown is whether specific muscarinic receptor subtypes in the dorsomedial striatum contribute to a flexible shift in response patterns. The present experiments investigated whether blockade of M1-type and/or M4-type cholinergic receptors in the dorsomedial striatum underlie place reversal learning. Experiment 1 investigated the effects of the M1-type muscarinic cholinergic antagonist, muscarinic-toxin 7 (MT-7) infused into the dorsomedial striatum in place acquisition and reversal learning. Experiment 2 investigated the effects of the M4-type muscarinic cholinergic antagonist, muscarinic-toxin 3 (MT-3) injected into the dorsomedial striatum in place acquisition and reversal learning. All testing occurred in a modified cross-maze across two consecutive sessions. Bilateral injections of MT-7 into the dorsomedial striatum at 1 or 2 μg, but not 0.05 μg impaired place reversal learning. Analysis of the errors revealed that MT-7 at 1 and 2 μg significantly increased regressive errors, but not perseverative errors. An injection of MT-7 2 μg into the dorsomedial striatum prior to place acquisition did not affect learning. Experiment 2 revealed that dorsomedial striatal injections of MT-3 (0.05, 1 or 2 μg) did not affect place acquisition or reversal learning. The findings suggest that activation of M1-type muscarinic cholinergic receptors in the dorsomedial striatum, but not M4-type muscarinic cholinergic receptors facilitate the flexible shifting of response patterns by maintaining or learning a new choice pattern once selected.

A Late Response on the Cholinergic Neurotransmitter System in Acute Massive Blood Loss

Objective: to study the impact of acute massive blood loss on the properties of muscarinic cholinergic receptors and catalytic parameters of cerebral acetylcholinesterase in albino rats in the late resuscitative periods. Materials and methods. Experiments were made on anesthetized non-inbred overanxious male rats exposed to 2-hour arterial hypotension (mean blood pressure 40—45 mm Hg). M-cholinergic receptor binding to the radioligand H3-quinuclidinyl benzilate was estimated by the method of Yamamura and Snyder (1974) and the catalytic properties of rat cerebral acetylcholinesterase on postresusci-tative day 40 were determined by the colorimetric technique (Ellman et al. 1961). Results. A response of m-cholinergic receptors to impaired cholinergic inflow has been found to appear as the higher efficiency of their performance, by increasing affinity and to be aimed at providing the normal functioning of synapses in cholinergic mediation deficiency. Conclusion. The findings suggest that this late hypercompensation of the cholinergic system in the postresuscitative period is excessive to the brain nerve cells of the rats experienced massive blood loss and resuscitation and, by causing nerve tissue structural changes, may result in the occurrence of new pathological processes.

Activation of muscarinic cholinergic receptors induces MCF-7 cells proliferation and angiogenesis by stimulating nitric oxide synthase activity

Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These receptors play key physiological roles and changes in their expression and/or function are involved in several diseases. We had previously demonstrated that mAChR expression is up regulated in three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. We here evidence that similarly to previous results obtained in mice, human breast tumor homogenates over expressed mAChR in comparison with normal breast tissue. Thus, to test the muscarinic actions on human breast adenocarcinoma cells we investigate the effect of CARB on MCF-7 cells proliferation and neovascular response. Particularly we observe that: CARB stimulates tumor cells proliferation, being 10-9 M the maximal effective dose for the muscarinic agonist. This action was due to M3 and M1 receptors activation being nitric oxide synthase (NOS) its effector enzyme via phospholipase C and protein kinase C signaling pathway. NOS1 and NOS3 isoforms are expressed in MCF-7 cells and its activation by CARB triggers nitric oxide synthesis and vascular endothelial growth factor expression increasing blood vessels formation induced by mammary tumor cells in vivo. We can conclude that non-neuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.

Cardiovascular effects of antipsychotics

There is great concern over cardiovascular disease in the schizophrenic population owing to the high incidence of cardiovascular mortality. Increased cardiovascular mortality is related to lifestyle choices (e.g., smoking and sedentary lifestyle) and a high prevalence of comorbid medical conditions, including dyslipidemia, the metabolic syndrome and Type 2 diabetes. One factor that increases cardiovascular risk is the medications used to treat the core features of schizophrenia. Adverse cardiovascular effects of antipsychotic treatment have been recognized for many decades, especially tachycardia, orthostatic hypotension and rare instances of sudden death; but, since 2000, there has been a significant shift in the focus of risk perception. The older antipsychotic literature is replete with papers primarily concerned with the physiological consequences of muscarinic cholinergic antagonism, α1-adrenergic antagonism or receptors associated with cardiac conduction, but the current literature recognizes that, for most antipsychotic-exposed patients, the more significant cardiovascular burden of treatment is mediated by metabolic adverse effects such as weight gain, dyslipidemia and diabetes mellitus. The purpose of this review is to examine the cardiovascular risks of treatment with antipsychotic medications, elucidating relevant mechanisms and differences between various agents, especially for metabolic adverse effects seen with atypical antipsychotics.

Pre- and postsynaptic mechanisms in the clonidine- and oxymetazoline-induced inhibition of gastric motility in the rat

The inhibitory effect of clonidine (non-selective α 2-adrenoceptor agonist) and oxymetazoline (α 2A-adrenoceptor selective agonist) was compared on basal and stimulated gastric motor activity (gastric tone and contractions) using the balloon method in the rat. It was shown that oxymetazoline (0.2–1.7 μmol/kg, i.v.) decreased the basal motility, while clonidine (1.9–3.8 μmol/kg, i.v.) failed to affect it. When motility was stimulated centrally by insulin (5 IU/rat, i.v.), both clonidine (1.9–3.8 μmol/kg, i.v.) and oxymetazoline (0.1–3.4 μmol/kg, i.v.) inhibited the gastric motor activity. However, while the effect of clonidine was antagonized by the non-selective α 2-adrenoceptor antagonist yohimbine (5 μmol/kg, i.v.) and the α 2A-adrenoceptor selective antagonist BRL 44408 (3 μmol/kg, i.v.), the effect of oxymetazoline was only partially affected. Prazosin (α 1- and α 2B-adrenoceptor antagonist, 0.07–0.28 μmol/kg, i.v.) also failed to reverse the effect of oxymetazoline. Furthermore, when gastric motility was stimulated peripherally by activation of postsynaptic cholinergic muscarinic receptors by the combination of carbachol (0.14 μmol/kg, i.v.) and hexamethonium (37 μmol/kg, i.v.), clonidine (3.8 μmol/kg, i.v.) failed to affect the increased motor activity, however, oxymetazoline (0.8–3.4 μmol/kg, i.v.) exerted a pronounced inhibition. These results suggest that different mechanisms may be involved in the inhibitory effect of clonidine and oxymetazoline; while clonidine reduces the gastric motility by activation of presynaptic α 2-adrenoceptors, postsynaptic component in the effect of oxymetazoline has also been raised.

Anesthesia increases in vivo N-([18F]fluoroethyl)piperidinyl benzilate binding to the muscarinic cholinergic receptor

The in vivo binding of N-[18F]fluoroethyl-piperidinyl benzilate ([18F]FEPB) to the muscarinic cholinergic receptor was measured in awake and anesthetized rats. Studies were done using an equilibrium infusion technique to provide estimates of specific binding as distribution volume ratios. Anesthesia with either isoflurane or sodium pentobarbital produced a significant (65-90%) increase of radiotracer binding in receptor-rich brain regions (striatum, cortex, hippocampus) relative to awake controls. Pretreatment of anesthetized animals with the acetylcholinesterase inhibitor phenserine produced no further increases in radioligand binding, in contrast to the large (>70%) increases previously observed in awake animals following drug treatment. These studies demonstrate that anesthesia can produce significant changes in baseline biochemical measures that can obscure even very large effects of pharmacological challenges.

Atropine, a muscarinic cholinergic receptor antagonist increases serotonin, but not dopamine levels in discrete brain regions of mice

We investigated the effects of atropine, a muscarinic acetylcholine (ACh) receptor antagonist, on the level of serotonin in discrete brain regions, the nucleus raphe dorsalis (NRD), nucleus caudatus putamen (NCP), cerebral cortex and the cerebellum. Biogenic amines were assayed employing HPLC electrochemistry in these regions 30 min following different doses of atropine (5, 10, 25mg/kg; i.p.), and at various time points (15, 30, 60, 120 min) after 25mg/kg of the drug. The cholinergic receptor antagonist caused a dose-dependent alteration in the level of serotonin in NRD, but the increase was not dose-dependent for other regions studied. The metabolite of serotonin, 5-hydroxyindoleacetic acid was unaffected. Atropine did not affect the levels of dopamine or its metabolites dihydroxyphenyl acetic acid and homovanillic acid. The present study suggests significant effect of this antimuscarinic agent on the synthesis of serotonin in the central serotoninergic pathways, which may have clinical relevance.

Flurbiprofen, A Cyclooxygenase Inhibitor, Reduces the Brain Arachidonic Acid Signal in Response to the Cholinergic Muscarinic Agonist, Arecoline, in Awake Rats

Cholinergic muscarinic receptors, when stimulated by arecoline, can activate cytosolic phospholipase A(2) (cPLA(2)) to release arachidonic acid (AA) from membrane phospholipid. This signal can be imaged in the brain in vivo using quantitative autoradiography following the intravenous injection of radiolabeled AA, as an increment in a regional brain AA incorporation coefficient k*. Arecoline increases k* significantly in brain regions having muscarinic M(1,3,5) receptors in wild-type but not in cyclooxygenase (COX)-2 knockout mice. To further clarify the roles of COX enzymes in the AA signal, in this paper we imaged k* following arecoline (5 mg/kg i.p.) or saline in each of 81 brain regions of unanesthetized rats pretreated 6 h earlier with the non-selective COX inhibitor flurbiprofen (FB, 60 mg/kg s.c.) or with vehicle. Baseline values of k* were unaffected by FB treatment, which however reduced by 80% baseline brain concentrations of prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)), eicosanoids preferentially derived from AA via COX-2 and COX-1, respectively. In vehicle-pretreated rats, arecoline increased the brain PGE(2) but not TXB(2) concentration, as well as values for k* in 77 of the 81 brain regions. FB-pretreatment prevented these arecoline-provoked changes. These results and those reported in COX-2 knockout mice suggest that the AA released in brain following muscarinic receptor-mediated activation is lost via COX-2 to PGE(2) but not via COX-1 to TXB(2), and that increments in k* following arecoline largely represent replacement by unesterified plasma AA of this loss.

Recovery of Oligomers and Cooperativity When Monomers of the M2 Muscarinic Cholinergic Receptor Are Reconstituted into Phospholipid Vesicles

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.

Role of muscarinic cholinergic receptors in the control of the intensity of nonquantal acetylcholine release from rat motor nerve endings

Role of muscarinic cholinergic receptors in the control of the intensity of nonquantal acetylcholine release from rat motor nerve endings

Perinatal co-exposure to methylmercury and PCB153 or PCB126 in rats alters the cerebral cholinergic muscarinic receptors at weaning and puberty

In the last few decades, combined exposure to methylmercury (MeHg) and polychlorinated biphenyls (PCBs) from fish and seafood, and their potentially interactive effects on neurodevelopment, have been giving increasing cause for concern. We examined the combined effects of MeHg and either a non-dioxin PCB (PCB153) or a dioxin-like PCB (PCB126) congener on the developing brain cholinergic muscarinic receptors (MRs). These receptors are known to play a major role in many central functions including higher cognitive processes and the modulation of extrapyramidal motor activity. MRs in pup rat brains diminished following prenatal and lactational exposure, from gestational day [GD]7 to postnatal day [PND]21, to MeHg (0.5 mg/kg body weight [bw]/day), PCB153 (5 mg/kg bw/day), and PCB126 (100 ng/kg/day), alone or in combination. Total MR density, as well as M1, M2, and M3 receptor subtypes of the weanling and pubertal rats, were affected in a brain-area-, gender-, time- and compound-dependent fashion. MeHg decreased (by 15–20%) the total MR density in a delayed (PND36) manner in the cerebral cortex of both genders, and early (at weaning) in the cerebellum of both genders, with the effect lasting until puberty (in males only). MeHg decreased the ACh M1- and M3-immunopositive neurons in the cerebral cortex and also increased the M2-immunopositive Bergmann glia in the cerebellum. PCB153 also induced a delayed (PND36) decrease (of 20%) in total MR number in the cerebellum of the male offspring and in the cerebral cortex of both genders. The latter effect was coupled with a decrease in ACh M1- and ACh M3-immunopositive neuron populations. PCB126 decreased (by 30–40%) total MR density in a gender-dependent manner, males being more sensitive than females. The effect was evident early (at PND21) and lasted until puberty in the cerebellum, while it was observed later (at PND36) in the cerebral cortex. The M1 and M3 receptors were similarly affected by PCB126. Co-exposure to MeHg and either PCB153 or PCB126 had the same effect on the cerebral MRs as exposure to each compound alone. The results rule out additive or synergistic interactions between MeHg and PCB153 or PCB126 on MRs in the brain areas examined. Some early-onset changes persisted until puberty, while other modifications became manifest only at the advanced time point (PND36), when the brain levels of total Hg, PCB153, and PCB126 had declined. These data support the ability of MeHg and PCBs to induce delayed neurotoxicity after developmental exposure.

Cholesterol as a determinant of cooperativity in the M 2 muscarinic cholinergic receptor

M 2 muscarinic receptor extracted from Sf9 cells in cholate–NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[ 3H]methylscopolamine (NMS) is only 50% of that for [ 3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [ 3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588–604]. In contrast, M 2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [ 3H]NMS at either 30 or 4 °C, although there was a time-dependent inactivation during incubation with [ 3H]NMS at 30 °C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M 2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 °C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [ 3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [ 3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M 2 muscarinic receptor.

Identification of the neurotransmitters involved in modulation of transmitter release from the central terminals of the locust wing hinge stretch receptor

The flight motor system of the locust represents a model preparation for the investigation of neuromodulation. At the wing hinges are stretch receptors important in generating and controlling the flight motor pattern. The forewing stretch receptor (fSR) makes direct cholinergic synapses with depressor motor neurons (MN) controlling that wing, including the first basalar MN (BA1). The fSR/BA1 synapse is modulated by muscarinic cholinergic receptors located on gamma-aminobutyric acid (GABA)-ergic interneurons (Judge and Leitch [1999a] J. Comp. Neurol. 407:103-114; Judge and Leitch [1999b] J. Neurobiol. 40:420-431). However, electrophysiology has shown that fSR/BA is also modulated by biogenic amines (Leitch et al. [2003] J. Comp. Neurol. 462:55-70). We have used electron microscopic immunocytochemistry (ICC) to identify the neurotransmitters in neurons presynaptic to the fSR and to determine the relative proportion of these different classes of modulatory inputs. Approximately 55% of all inputs to the fSR are glutamate-IR, indicating that glutamatergic neurons may also play an important role in presynaptically modulating the fSR terminals. Anti-GABA ICC confirmed that over 40% of inputs to the fSR are GABA-IR (Judge and Leitch [1999a] J. Comp. Neurol. 407:103-114). Labelling sections with an antioctopamine antibody revealed neurons containing distinctive large, electron-dense granules, which could reliably be used to identify them. Aminergic neurons that modulate the synapse may have very few morphologically recognizable synaptic outputs. Although putative octopaminergic processes were found in close contact to horseradish peroxidase-filled fSR profiles, no morphologically recognizable synaptic inputs to the fSR were evident. Collectively, these data suggest that most inputs to the fSR are from either glutamatergic or GABAergic neurons.