M2 Muscarinic Subtypes Research Articles

Neuronal muscarinic acetylcholine receptor subtype modulation of inspiratory bursting at hypoglossal motoneurons in developing neonatal mice in vitro

Muscarinic acetylcholine receptor modulation contributes to changes in hypoglossal motoneuron excitability through pre- and post-synaptic effects, an important mechanism for regulation of airway tone during sleep. Previous data indicate a net excitatory effect of muscarinic modulation of inspiratory bursting in neonatal mouse brainstem slices, yet a net inhibitory effect on inspiratory bursting in adult rats. We tested the hypothesis that a developmental shift in muscarinic modulation impacts hypoglossal motoneurons, using neuroanatomical and electrophysiological methods. Immunofluorescence confirmed hypoglossal motoneurons express M1, M2, M3, and M5 muscarinic subtypes. We observed maturational decreases in M1 and M5 labeling intensity, M3 labeling intensity generally decreased with postnatal (P) age, although there was a transient increase in expression at P17. M2 labeling intensity did not change, although distribution of M2 labeling with postnatal maturation may change. Next, using the in-vitro rhythmic slice preparation from CD1 mice of either sex, local application of muscarine (100 μM) into the hypoglossal nucleus was tested in mice P0-14. The net effect of applying muscarine results in excitatory output after local application into the rhythmic slice because each data point is a positive percent change from control. The magnitude of this potentiation appears to lessen as the mice age toward P14. To evaluate the mechanisms through which the magnitude of muscarinic potentiation of inspiratory bursting changes with postnatal maturation, we next assessed the role of excitatory and inhibitory muscarinic receptor subtypes. Blocking M1 locally (pirenzepine, 100 μM) significantly decreased muscarine-mediated potentiation of inspiratory bursting (to 72±14% of control muscarine response, n=9, P0-5). Preliminary data indicate bath application of 4-DAMP (M3 antagonist) attenuated muscarinic potentiation of inspiratory bursting (range: to 7%-25% of control burst response, n=3, P0-5). Modulating M5 locally with a positive allosteric modulator VU 0238429 (1000 μM) had little effect on burst amplitude (119±4% of control burst response, n=3, P0-6). Blocking M2 locally (methoctramine, 2μM) showed no effect on inspiratory burst potentiation after applying muscarine (181±65% vs 187±69%, n=7, P0-5). Bath application of methoctramine did not have a significant effect on inspiratory burst potentiation by muscarine (179±65% vs control 182±90%, n=10) in P0-5 mice while preliminary analysis suggests an increasing effect of blocking M2 with postnatal maturation (147±56% vs control 134±30%, n=4, P9-14). These data support excitatory muscarinic receptor subtypes contributing to the excitatory response early in postnatal maturation. Based on our neuroanatomical data, we speculate the increasing inhibitory response to muscarinic modulation with postnatal maturation is due to a decreased contribution of excitatory muscarinic receptor subtypes. NIH R15HL148870 (Revill) and NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy) 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.

The summary on non-reactivation cholinergic properties of oxime reactivators: the interaction with muscarinic and nicotinic receptors

Organophosphorus inhibitors (OP) of acetylcholinesterase (AChE) represent a group of highly toxic compounds. The treatment of OP intoxication is, however, insufficiently ensured. Currently, two main categories of drugs-anticholinergics and oxime reactivators- are employed as antidotes. Oximes have been reported to act at several levels of the cholinergic transmission, and among the non-reactivation effects, the interaction with cholinergic receptors stands out. This review addresses issues correlated with non-reactivating effects of oxime reactivators with a special focus on the muscarinic and nicotinic receptors, but involvement of other cholinergic structures such as AChE and choline uptake carriers are discussed too. It can be concluded that the oxime reactivators show a variation in their antagonistic effect on the muscarinic and nicotinic receptors, which is likely to be of significance in the treatment of OP poisoning. In vitro data reported oximes to exert higher efficacy on the muscarinic M2 subtype than on the AChE. However, this effect seemed to be subtype specific since the antagonistic M3 effect was lower. Also, and importantly, the antimuscarinic effect was larger than that on nicotinic receptors. Even though atropine showed a much higher muscarinic antagonism, it is supposed that non-reactivation properties of oxime reactivators play a significant role in the treatment of OP poisoning.

Imaging of the muscarinic acetylcholine neuroreceptor in rats with the M2 selective agonist [18F]FP-TZTP

[(18)F]FP-TZTP is an M2 muscarinic subtype selective receptor-binding radiotracer used in vivo to image human and nonhuman primate brain following both bolus injection and infusion. In order to carry out repeated studies in rodents, the techniques developed for primates must be transferred to rodents with the same precision. This includes obtaining a metabolite-corrected input function. We compared bolus injection with constant infusion in rats that were awake or under isoflurane anesthesia. Brain-plasma and brain-blood distribution ratios were calculated by dividing brain (18)F concentrations, determined in vivo by positron emission tomography imaging with the Advanced Technology Laboratory Animal Scanner, ex vivo by direct counting in excised brain tissue or by quantitative autoradiography by the plasma or whole blood concentrations that had been corrected for metabolite contents. Blood volume constraints prevented adequate blood sampling to define a precise input function after bolus injection, thus preventing full kinetic analysis. Constant infusion, however, required fewer blood samples to define the input function, allowing calculation of distribution ratios, but complete equilibrium between plasma and tissue had not yet been reached after 120 min. Our results showed that the blood clearance and metabolism were too rapid to obtain a reproducible input function after bolus injection. The equilibrium distribution ratios did not lead to precise biochemical parameters, but the constant infusion was more suitable in that distribution ratios between tissue and plasma were statistically more precise. Constant infusion is the better approach for studying [(18)F]FP-TZTP by small animal imaging.

Muscarinic acetylcholine receptors present in human osteoblast and bone tissue

Acetylcholine is the predominant neurotransmitter in the neuromuscular junction, and a role in bone has been postulated. The expression of nicotinic receptors has been reported in osteoblasts, but the expression and function of muscarinic receptor in bone remain obscure. In this study, we investigated the expression and functional activities of muscarinic receptor subtypes in human osteoblast cell lines and animal and human bone tissue. The mRNA levels of muscarinic receptor subtypes were detected by reverse-transcription polymerase chain reaction. We found that muscarinic subtypes m1, m2, m3, m4, and m5 were expressed at different levels in human osteosarcoma HOS cells, rat femur, and human rib bone tissue; m1, m4, m5 were in cultured mouse femur bone cells and cultured mouse calvarial bone cells; m2, m3, m4 were in bovine bone. The mRNA of neuronal markers, light-, medium- and heavy-neurofilament, was not found in human bone tissues to exclude the possible contamination from neuronal tissue. Methacholine induced an elevation in cytosolic calcium concentration and proliferation in HOS cells. Both effects were blocked by atropine. We conclude that muscarinic receptor is present in bone tissue to evoke calcium signaling and modulate cell proliferation. Different muscarinic receptor subtypes are distributed in various parts of the animal skeletal system including the different species and bone portions. Bone remodeling involving osteoblast proliferation leads the possibilities that muscarinic receptor may play roles in bone remodeling.

Identification and characterization of the rat M1 muscarinic receptor promoter.

Five subtypes of the muscarinic receptor have been cloned from both the rat and human genomes. Although all five genes have the coding sequences in a single exon, their structures 5' of the initiation codon are largely uncharacterized, except for the M4 receptor. In the brain, muscarinic receptors mediate motor and memory function by interaction with their ligand acetylcholine. In addition, the M1 muscarinic subtype has been implicated in behavior, stress-adaptive cardiovascular reflexes, and blood pressure regulation. In the current study the M1 muscarinic receptor noncoding 5'-flanking region has been identified and characterized, including the promoter and two 5' noncoding exons located approximately 13-14 kb from the coding exon. Similar to the M4 muscarinic receptor gene the M1 promoter is GC-rich, contains no TATA box, but has two potential CAAT boxes and several putative binding sites for transcription factors such as SP1 and AP-1-3. The transcription initiation site was identified by RNase protection and primer extension. Promoter activity was confirmed in transient expression assays, using luciferase reporter constructs. A 0.89-kb fragment consisting of 480 bp of the promoter, exon 1, and part of intron 1 expressed luciferase activity in two M1 receptor-expressing cell lines (CCL-107 and CCL-147), whereas a longer fragment (1.5 kb) that extends into intron 2 demonstrated significantly increased luciferase activity. The constructs exhibited responses indicating the presence of functional glucocorticoid-, acute-phase-, and heat shock-responsive elements.

Receptor Subtype Abundance as a Tool for Effective Intracellular Signalling

The regulation of heart function is one of the essentials for the survival of organism. Therefore, the effective mechanisms of regulation can minimize the energy requirements and improve the ability to react to different needs on time and appropriately. Two receptor types, beta-adrenoceptors and muscarinic receptors, with almost antagonistic function, are "basic regulators" of the heart parameters. It is relevant to mention that beside the main adrenoceptors and muscarinic receptors subtypes (beta(1)- and M(2)-subtype), other minor subtypes that regulate heart function, i.e. beta-, beta-adrenoceptors, alpha(1)-adrenoceptors and minor subtypes of muscarinic receptors (M(1), M(3) and M(5)) are present in the heart. In this regard is intriguing that just two catecholamines (adrenaline, noradrenaline) have many "targets"--receptors that differ so much in the functional consequences of their activation: while beta(1)- and beta(2)-adrenoceptors cause cardiostimulation, beta(3)-adrenoceptors are responsible for cardioinhibition and alpha(1)-adrenoceptors contribute to enhanced inotropy. Similarly, some data show that other muscarinic receptors than M(2) muscarinic subtype, are expressed in the heart and these minor subtype(s) can contribute to the heart regulation in similar way as beta(3)-adrenoceptors to the catecholamine action. Taken together, regulation of heart function through different receptor subtypes and using homologous and heterologous regulation can represent an effective tool for coping with permanently changing environmental conditions.

Quantitative structure–selectivity relationship for M2 selectivity between M1 and M2 of piperidinyl piperidine derivatives as muscarinic antagonists

Muscarinic M2 receptor antagonists with high subtype selectivity (M2/M1) will decrease the toxicity in central nervous system in treatment of AD. The exploration of quantitative structure–selectivity relationship (QSSR) to muscarinic M2 receptor antagonists will provide design information for drug with fewer side effects. In this paper, CoMFA models of p K i(M1), p K i(M2) and p[ K i(M2)/ K i(M1)] (p K i(M2) − p K i(M1)) were used to study the subtype selectivity (M2/M1) of piperidinyl piperidine derivatives as muscarinic M2 subtype receptor antagonists. The parameters of the three models are: 0.633, 0.636 and 0.726 for cross-validated r 2 ( r cv 2 ), 0.109, 0.204 and 0.09 for the Standard error of estimate (SD), respectively. The results show the model of p[ K i(M2)/ K i(M1)] is the best one for design of piperidinyl piperidine derivatives as muscarinic antagonists with high subtype selectivity (M2/M1).

Dehydroepiandrosterone sulfate and estrone sulfate reduce GABA‐recurrent inhibition in the hippocampus via muscarinic acetylcholine receptors

Several recent studies have established a role for estrogens in ameliorating specific neurodegenerative disorders, mainly those associated with the cholinergic neurons of the basal forebrain and their targets in the cortex and hippocampus. We have previously demonstrated that endogenous and exogenous application of the neurosteroid dehydroepiandrosterone sulfate (DHEAS) markedly reduces GABA-mediated recurrent inhibition and synchronizes hippocampal unit activity to theta rhythm (Steffensen (1995) Hippocampus 5:320-328). In this study, we evaluated the role of muscarinic receptors in mediating the effects of DHEAS and estrone sulfate (ES), the principal circulating estrogen in humans, on short-latency-evoked potential responses, paired-pulse inhibition (PPI), paired-pulse facilitation, and GABA interneuron activity in the dentate gyrus and CA1 subfields of the rat hippocampus. In situ microelectrophoretic application of the muscarinic M2 subtype cholinergic receptor agonist cis-dioxolane, DHEAS, and ES markedly reduced PPI in the dentate and CA1 that was blocked by the M2 receptor antagonist gallamine. Similar to DHEAS, microelectrophoretic administration of ES increased population spike amplitudes, without increasing excitatory transmission, but this effect was not blocked by gallamine. Microelectrophoretic application of cis-dioxolane and ES markedly increased the firing rate of dentate hilar interneurons and CA1 oriens/alveus interneurons and enhanced their synchrony to hippocampal theta rhythm. These findings suggest that select GABA-modulating neurosteroids and neuroactive estrogen sulfates alter septohippocampal cholinergic modulation of hippocampal GABAergic interneurons mediating recurrent, but not feedforward, inhibition of hippocampal principal cell activity.

Synthesis and Muscarinic M2 Subtype Antagonistic Activity of Unnatural ent‐Himbacine and an Enantiomeric Pair of (2′S,6′R)‐Diepihimbacine.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthetic studies on himbacine, a potent antagonist of the muscarinic M2 subtype receptor. Part 2: Synthesis and muscarinic M2 subtype antagonistic activity of the novel himbacine congeners modified at the C-3 position of lactone moiety

With an aim to disclose the convergency and flexibility of our previously explored synthetic route to natural himbacine 1, and moreover, to clarify some novel aspects of the structure–activity relationships of 1, we prepared various structural types of novel himbacine congeners, 3-demethylhimbacine (3-norhimbacine) 2 and 4-epi-3-demethylhimbacine (4-epi-3-norhimbacine) 4-epi-2 and their enantiomers (ent-2 and ent-4-epi-2), 11-methylhimbacine 3, and 3-epihimbacine 4 in optically pure forms by employing our methodology. All of the synthesized congeners correspond to the compounds modified at the C-3 position of γ-lactone moiety involved in 1. Among these congeners, 3-demethylhimbacine (3-norhimbacine) 2 was found to exhibit more potent muscarinic M2 receptor binding affinity than natural 1.

Synthesis and muscarinic M2 subtype antagonistic activity of unnatural ent-himbacine and an enantiomeric pair of (2′S,6′R)-Diepihimbacine

The title three compounds ent-1, 6, and ent-6 were synthesized by coupling the chiral sulfone 4 or ent-4 with the chiral piperidinaldehyde 5 or ent-5, which were readily prepared following the synthetic routes previously established by the novel total synthesis of natural himbacine 1. Their muscarinic M2 subtype binding affinity was evaluated in comparison to that of 1, disclosing that the stereochemistry of both the tricyclic moiety and the piperidine part of 1 plays crucial roles in its potent activity.

A new enzymic method for the isolation and culture of human bladder body smooth muscle cells.

Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method.

Atrophy of Rat Exorbital Lacrimal Glands Induced by Atropine.

To assess the morphological changes of the lacrimal gland after cholinergic suppression, we performed light- and transmission-electron microscopic examinations on the exorbital lacrimal glands in rats after intragastric intubation of atropine, a nonselective antimuscarinic compound with a high affinity for M3 muscarinic subtype receptors. In addition, areas of acinar cells were measured by an image processor. The weights of the lacrimal gland were decreased at hours 36 and 48 after atropine single administration at 250 mg/kg. Areas of acinar cells, measured by an image processor, were also decreased. Electron-microscopic examination demonstrated decreased number of secretory granules in the apical cytoplasm of acinar cells in all rats on days 7 and 28 of repeated atropine treatment. No changes were evident in other cytoplasmic organelles. Four- and even thirteen-week repeated administration studies revealed no progression in lacrimal acinar cell atrophy. The present findings demonstrate that an antimuscarinic compound that has high affinity for M3 muscarinic subtype receptors induces lacrimal gland acinar cell atrophy caused by a decrease in secretory granules, and that the repeated administration is not associated with any progression or any other changes.

Chronic Effects of Traumatic Brain Injury on Hippocampal Vesicular Acetylcholine Transporter and M2Muscarinic Receptor Protein in Rats

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may be due to presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and M2receptors can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2muscarinic subtype receptor protein levels at four time points: 1 day, 1 week, 2 weeks, and 4 weeks following injury. Rats were anesthetized and surgically prepared for controlled cortical impact injury (4 m/s, 2.5- to 2.9-mm depth) and sham surgery. Animals were sacrificed and coronal sections (35 μm thick) were cut through the dorsal hippocampus for VAChT and M2immunohistochemistry. Semiquantitative measurements of VAChT and M2protein in hippocampal homogenates from injured and sham rats were assessed using Western blot analysis. Immunohistochemistry showed no obvious changes in VAChT and M2immunoreactivity at 1 day and 1 week postinjury. At 2 and 4 weeks postinjury, an increase in hippocampal VAChT protein and a corresponding loss of hippocampal M2protein was observed compared to sham controls. Consistent with these results, Western blot analyses at 4 weeks postinjury demonstrated a 40–50% increase in VAChT and a 25–30% decrease in M2. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI, by upregulating the storage capacity and subsequent release of ACh and downregulating presynaptic inhibitory receptors.

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats.

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats. 2. Competition binding studies with uterine membrane preparations and [3H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pKi) for each antagonist as follows; oestrogen-treated - atropine (7.98) > or = himbacine (7.83) > methoctramine (7.52) > or = hexahydrosiladiphenidol (HHSiD; 7.32) > or = 5,11-dihydro- 11-[[[2-[2 - [(dipropylamino)methyl] - 1 piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido-[2,3-b][1,4]- benzodiazepin-6-one (AF-DX 384; 7.10)> 11 -[[2- [(diethylamino)methyl]-1-piperidinyl]-acetyl]5,11-dihydro-6H-pyridol+ ++]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant atropine (8.05)> or =methoctramine (7.95)> or =himbacine (7.71)> or =HHSiD (7.52)> or =AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18). 3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD2=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA2 estimates were: oestrogen-treated - atropine (9.42)> himbacine (8.73) HHSiD (8.68) methoctramine (8.49)> or =AF-DX 384 (7.91)> or =AF-DX 116 (7.36)> or =pirenzepine (7.26); late pregnant atropine (9.48)>himbacine (8.37)> or = HHSiD (8.22) > or =methoctramine (8.01) > or =AF-DX 116 (7.73)> or = AF-DX 384 (7.44)> or = pirenzepine (6.92). 4. The relative pKi estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M2 subtypes. In functional studies pA2 values indicated the additional presence of muscarinic M3 receptor or, possibly an atypical receptor subtype. The similarity between pKi and pA2 estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

Pharmacology of Muscarinic Receptor Subtypes Constitutively Activated by G Proteins

We have examined the effects of raising G protein concentration on the pharmacology of a series of agonist and antagonist ligands at the m1, m3, and m5 muscarinic subtypes using a functional assay. Overexpression of G(alpha q) induced constitutive activity of these receptors. The constitutive activity was reversed completely by every muscarinic antagonist tested, which indicates that they are all negative antagonists (inverse agonists). The potencies of antagonists for reversing G protein-induced activity and agonist-induced activity were identical, suggesting the same mechanism of action. Overexpression of G(alpha q) increased the potencies of every tested agonist and the efficacies of all partial agonists. The fold-gains in potency were positively correlated with ligand efficacy with the most efficacious agonists displaying the greatest potency gains. In addition, the efficacies of partial agonists approached those of full agonists. Constitutive activity of receptors has been explained by allosteric models in which receptors exist in spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and antagonists, respectively. In this context, drug efficacy and potency are interrelated because they both depend on the same parameters, namely the absolute and relative affinities of a compound for receptors in active and inactive states and the ratio and concentrations of receptors in active and inactive states. All of our data are consistent with this model, in which raising G protein levels favors formation of the active conformation of receptors. Based on our findings, regulation of G protein concentration may be an important means of controlling receptor activity in vivo. These results define the functional relationship between G protein levels and muscarinic receptor pharmacology.

Muscarinic receptor subtypes in subpopulations of human blood mononuclear cells as analyzed by RT-PCR technique

In this study we have analysed the expression of mRNA encoding the m1–m5 mAChR subtypes in human blood mononuclear cells and subpopulations of lymphocytes using reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Total RNA was extracted from human blood mononuclear cells, T cells, monocytes, EB virus transformed B cells and from two leukemic cell lines and analysed by RT-PCR. Our results indicate that mRNAs for the m3, m4 and m5 muscarinic subtypes are expressed in mononuclear cells and purified T cells while m1 and m2 mRNAs were not detected in these cells. No m1–m5 subtype mRNA was detected in B cells and monocytes, indicating absence of muscarinic receptors in these cells. The expression of muscarinic subtypes in the leukemic T cell line, Peer, and the promyelocytic leukemic cell line HL-60 was different from peripheral mononuclear cells. Both m3 and m5 subtypes were expressed in Peer cells but not the m4 subtype, whereas the m4 and m5 subtypes were detected in HL-60 cells.

Central muscarinic cholinergic M1 and M2 receptor changes in congenital ornithine transcarbamylase deficiency.

Congenital ornithine transcarbamylase (OTC) deficiency results in neuropathologic damage to the cerebral cortex, basal ganglia, and thalamus. However, the precise nature of the cell loss, as well as the pathophysiologic mechanisms responsible for it, have not been fully elucidated. In the present study, densities of the M1 and M2 subclasses of muscarinic cholinergic binding sites were assessed using quantitative receptor autoradiography in the brains of sparse-fur (spf) mice with congenital OTC deficiency and in age-matched CD-1 controls. Densities of binding sites for the muscarinic M1 subtype ligand [3H]pirenzepine were reduced by 24-54% (p < 0.01) in frontal cortex, caudate/ putamen, and hippocampal CA1 and CA2 areas. Since muscarinic M1 sites are localized presynaptically, their selective loss, together with a previous report of reduced activities of the presynaptic cholinergic enzyme choline acetyltransferase, confirms that loss of cholinergic neurons is an important feature of congenital OTC deficiency. Densities of binding sites for the predominantly postsynaptic muscarinic M2 subtype ligand 3H-AFDX 384 were increased by up to 60% (p < 0.01) in cerebral cortex, hippocampus, globus pallidus, as well as thalamic and hypothalamic structures of OTC-deficient mice. Increased M2 sites in the cerebral cortex, hippocampus, and thalamus are most likely the result of up-regulation of these sites after the loss of the presynaptic neuron. These findings support the presence of a central muscarinic cholinergic lesion in congenital OTC deficiency.

Muscarinic receptor subtypes in the medial preoptic area and sleep-wake cycles.

To clarify which muscarinic receptor subtype(s) mediate changes in sleep and cortical temperature (Tcort) induced by carbachol microinjections into the medial preoptic area (MPA), pirenzepine, tripitramine and +/- p < > -fluorohexahydro-sila-difenidol (p-F-HHSiD), which are highly selective muscarinic M1, M2 and M3 antagonists, respectively, were microinjected into the MPA of rats. Whereas pirenzepine (3.45 and 7.08 nmol) and p-F-HHSiD (3.90 and 7.80 nmol) were without effect, tripitramine (0.67 and 3.37 nmol) enhanced wakefulness, decreased slow wave and desynchronized sleep, and raised Tcort with the higher dose. The data suggest that in the MPA only M2 muscarinic subtypes may be functionally important in mediating the cholinergic effects on sleep and thermoregulation.

Involvement of M2 and non-M2 muscarinic receptors in hippocampal theta rhythm induced by carbachol infusion into the septum of the rat

Binding and autoradiographic studies have shown the presence of a rather high density of M2 muscarinic subtype receptors and the apparent absence or low density of the M1 subtype in the septum. We tested the hypothesis that, in the urethaneanesthetized rat, septal M2 receptors are involved in the generation of the hippocampla theta (ϑ) rhythm induced by itraseptal administration of carbachol, a potent cholinomimetic agent. Carbachol-induced ϑ was blocked by local infusion of the unspecific muscarinic antagonist agent, atropine (20 μg (29.55 nM)), given 10 min prior to carbachol. The intraseptal administration of low to high doses of gallamine (range: 20–180 μg (22.43–201.90 nM)), a specific M2 antagonist which displays high affinity for the septal region, resulted in significant changes in the electrophysiological characteristics of carbachol-induced ϑ but failed to abolish this rhythm. It is suggested that the latter may have resulted from a combined activation of both M2 and non-M2 receptors at septal level.