[show abstract][hide abstract] ABSTRACT: Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.
[show abstract][hide abstract] ABSTRACT: Recent studies with M3 muscarinic acetylcholine receptor (M3R) mutant mice suggest that drugs selectively targeting this receptor subtype may prove useful for the treatment of various pathophysiological conditions. Moreover, the use of M3R-based designer G protein-coupled receptors (GPCRs) has provided novel insights into how Gq-coupled GPCRs can modulate whole-body glucose homeostasis by acting on specific peripheral cell types. More recently, we succeeded in using X-ray crystallography to determine the structure of the M3R bound to the bronchodilating drug tiotropium, a muscarinic antagonist (inverse agonist). This new structural information should facilitate the development of orthosteric or allosteric M3R-selective drugs that are predicted to have considerable therapeutic potential.
Journal of Molecular Neuroscience 09/2013; · 2.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by Gs-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the Gq family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a Gq-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a Gq-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V1b vasopressin receptor, a Gq-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V1b receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of Gq-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.
[show abstract][hide abstract] ABSTRACT: The dorsal striatum has been implicated in reward-based decision making, but the role played by specific striatal circuits in these processes is essentially unknown. Using cell phenotype-specific viral vectors to express engineered G-protein-coupled DREADD (designer receptors exclusively activated by designer drugs) receptors, we enhanced Gi/o- or Gs-protein-mediated signaling selectively in direct-pathway (striatonigral) neurons of the dorsomedial striatum in Long-Evans rats during discrete periods of training of a high versus low reward-discrimination task. Surprisingly, these perturbations had no impact on reward preference, task performance, or improvement of performance during training. However, we found that transiently increasing Gi/o signaling during training significantly impaired the retention of task strategies used to maximize reward obtainment during subsequent preference testing, whereas increasing Gs signaling produced the opposite effect and significantly enhanced the encoding of a high-reward preference in this decision-making task. Thus, the fact that the endurance of this improved performance was significantly altered over time-long after these neurons were manipulated-indicates that it is under bidirectional control of canonical G-protein-mediated signaling in striatonigral neurons during training. These data demonstrate that cAMP-dependent signaling in direct-pathway neurons play a well-defined role in reward-related behavior; that is, they modulate the plasticity required for the retention of task-specific information that is used to improve performance on future renditions of the task.
Journal of Neuroscience 07/2013; 33(28):11668-11676. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The release of insulin from pancreatic β-cells is regulated by a considerable number of G protein-coupled receptors (GPCRs). During the past several years, we have focused on the physiological importance of β-cell M3 muscarinic acetylcholine receptors (M3Rs). At the molecular level, the M3R selectively activates G proteins of the Gq family. Phenotypic analysis of several M3R mutant mouse models, including a mouse strain that lacks M3Rs only in pancreatic β-cells, indicated that β-cell M3Rs play a key role in maintaining blood glucose levels within a normal range. Additional studies with transgenic M3R mouse models strongly suggest that strategies aimed at enhancing signaling through β-cell M3Rs may prove useful in the treatment of type 2 diabetes. More recently, we analyzed transgenic mice that expressed an M3R-based designer receptor in a β-cell-specific fashion, which enabled us to chronically activate a β-cell Gq-coupled receptor by a drug that is otherwise pharmacologically inert. Drug-dependent activation of this designer receptor stimulated the sequential activation of Gq, PLC, ERK1/2, and IRS2 signaling, thus triggering a series of events that greatly improved β-cell function. Most importantly, chronic stimulation of this pathway protected mice against experimentally induced diabetes and glucose intolerance, either induced by streptozotocin or by the consumption of an energy-rich, high-fat diet. Since β-cells are endowed with numerous receptors that mediate their cellular effects via activation of Gq-type G proteins, these findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.
[show abstract][hide abstract] ABSTRACT: Muscarinic receptor-based designer receptors have emerged as powerful novel tools to study G-protein-coupled receptor (GPCR) signaling and physiology. These new designer GPCRs, which are most frequently referred to as DREADDs (designer receptors exclusively activated by designer drug), are unable to bind acetylcholine, the endogenous muscarinic receptor agonist, but can be activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound, with high potency and efficacy. The various DREADDs differ primarily in their G protein coupling preference. More recently, an arrestin-biased DREADD has also been developed. The expression of DREADDs in distinct tissues or cell types has enabled researchers to study the outcome of selective stimulation of distinct GPCR (or arrestin) signaling pathways in a temporally and spatially controlled fashion in vivo. In this review, we provide an up-to-date snapshot of where this field currently stands and which important novel insights have been gained using this new technology.
Trends in Pharmacological Sciences 06/2013; · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic β cell function are predicted to prove beneficial for the treatment of T2D. In the present study, we demonstrate that drug-mediated, chronic, and selective activation of β cell Gq signaling greatly improve β cell function and glucose homeostasis in mice. These beneficial metabolic effects were accompanied by the enhanced expression of many genes critical for β cell function, maintenance, and differentiation. By employing a combination of in vivo and in vitro approaches, we identified a novel β cell pathway through which receptor-activated Gq leads to the sequential activation of ERK1/2 and IRS2 signaling, thus triggering a series of events that greatly improve β cell function. Importantly, we found that chronic stimulation of a designer Gq-coupled receptor selectively expressed in β cells prevented both streptozotocin-induced diabetes and the metabolic deficits associated with the consumption of a high-fat diet in mice. Since β cells are endowed with numerous receptors that mediate their cellular effects via activation of Gq-type G proteins, our findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.
The Journal of clinical investigation 03/2013; · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Impaired lung function caused by decreased airway diameter (bronchoconstriction) is frequently observed whether body weight is abnormally high or low. That these opposite conditions affect the airways similarly suggests that the regulation of airway diameter and body weight are intertwined. We show here that, independently of its regulation of appetite, melanocortin pathway, or sympathetic tone, leptin is necessary and sufficient to increase airway diameter by signaling through its cognate receptor in cholinergic neurons. The latter decreases parasympathetic signaling through the M(3) muscarinic receptor in airway smooth muscle cells, thereby increasing airway diameter without affecting local inflammation. Accordingly, decreasing parasympathetic tone genetically or pharmacologically corrects bronchoconstriction and normalizes lung function in obese mice regardless of bronchial inflammation. This study reveals an adipocyte-dependent regulation of bronchial diameter whose disruption contributes to the impaired lung function caused by abnormal body weight. These findings may be of use in the management of obesity-associated asthma.
[show abstract][hide abstract] ABSTRACT: Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gα(olf) and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gα(s)-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal G(s) signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Lastly, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and non-invasive modulation of Gα(s) signaling in specific neuronal populations in vivo. This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gα(s) signaling in other neurobehavioral contexts.Neuropsychopharmacology accepted article preview online, 5 December 2012; doi:10.1038/npp.2012.251.
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 12/2012; · 6.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M(3) muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.
[show abstract][hide abstract] ABSTRACT: Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G-protein-coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic β cells. In this study, we examined the ability of SPL to modulate the activity of β-cell M(3) muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G-protein signaling 4 (RGS4) to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G-protein-coupled receptor-associated proteins may serve as novel targets for drug therapy aimed at improving β-cell function for the treatment of type 2 diabetes.-Ruiz de Azua, I., Nakajima, K.-I., Rossi, M., Cui, Y., Jou, W., Gavrilova, O., Wess, J. Spinophilin as a novel regulator of M(3) muscarinic receptor-mediated insulin release in vitro and in vivo.
The FASEB Journal 06/2012; 26(10):4275-86. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M(4) acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M(4) receptors could be a novel target for modulating psychostimulant effects of cocaine.
For the first time, we here addressed this issue by investigating the effects of a novel selective positive allosteric modulator of M(4) receptors, VU0152100, on cocaine-induced behavioral and neurochemical effects in mice.
To investigate the effect of VU0152100 on the acute reinforcing effects of cocaine, we use an acute cocaine self-administration model. We used in vivo microdialysis to investigate whether the effects of VU0152100 in the behavioral studies were mediated via effects on dopaminergic neurotransmission. In addition, the effect of VU0152100 on cocaine-induced hyperactivity and rotarod performance was evaluated.
We found that VU0152100 caused a prominent reduction in cocaine self-administration, cocaine-induced hyperlocomotion, and cocaine-induced striatal dopamine increase, without affecting motor performance. Consistent with these effects of VU0152100 being mediated via M(4) receptors, its inhibitory effects on cocaine-induced increases in striatal dopamine were abolished in M(4) receptor knockout mice. Furthermore, selective deletion of the M(4) receptor gene in dopamine D(1) receptor-expressing neurons resulted in a partial reduction of the VU0152100 effect, indicating that VU0152100 partly regulates dopaminergic neurotransmission via M(4) receptors co-localized with D(1) receptors.
These results show that positive allosteric modulators of the M(4) receptor deserve attention as agents in the future treatment of cocaine abuse.
[show abstract][hide abstract] ABSTRACT: Muscarinic acetylcholine (ACh) receptors (mAChRs; M(1)-M(5)) regulate the activity of an extraordinarily large number of important physiological processes. We and others previously demonstrated that pancreatic β-cells are endowed with M(3) mAChRs which are linked to G proteins of the G(q) family. The activation of these receptors by ACh or other muscarinic agonists leads to the augmentation of glucose-induced insulin release via multiple mechanisms. Interestingly, in humans, ACh acting on human β-cell mAChRs is released from adjacent α-cells which express both choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT), indicative of the presence of a non-neuronal cholinergic system in human pancreatic islets. In order to shed light on the physiological roles of β-cell M(3) receptors, we recently generated and analyzed various mutant mouse models. Specifically, we carried out studies with mice which overexpressed M(3) receptors or mutant M(3) receptors in pancreatic β-cells or which selectively lacked M(3) receptors or M(3)-receptor-associated proteins in pancreatic β-cells. Our findings indicate that β-cell M(3) receptors play a key role in maintaining proper insulin release and whole body glucose homeostasis and that strategies aimed at enhancing signaling through β-cell M(3) receptors may prove useful to improve β-cell function for the treatment of type 2 diabetes (T2D).
[show abstract][hide abstract] ABSTRACT: Muscarinic receptors have long been known as crucial players in hippocampus-dependent learning and memory, but our understanding of the cellular underpinnings and the receptor subtypes involved lags well behind. This holds in particular for the hippocampal CA3 region, where the mechanisms of synaptic plasticity depend on the type of afferent input. Williams and Johnston (Williams S, Johnston D. Science 242: 84-87, 1988; Williams S, Johnston D. J Neurophysiol 64: 1089-1097, 1990) demonstrated muscarinic depression of mossy fiber (MF) long-term potentiation (LTP) through a presynaptic site of action and Maeda et al. (Maeda T, Kaneko S, Satoh M. Brain Res 619: 324-330, 1993) proposed a bidirectional modulation of MF LTP by muscarinic receptor subtypes. Since then, this issue, as well as muscarinic regulation of plasticity at associational/commissural (A/C) fiber-CA3 synapses has remained largely neglected, not least because of the lack of highly selective ligands for the different muscarinic receptor subtypes. In the present study, we performed field potential and whole cell recordings from the hippocampal CA3 region of M(2) receptor knockout mice to determine the role of M(2) receptors in short-term and long-term plasticity at A/C and MF inputs to CA3 pyramidal cells. At the A/C synapse, M(2) receptors promoted short-term facilitation and LTP. Unexpectedly, M(2) receptors mediated the opposite effect on LTP at the MF synapse, which was significantly reduced, most likely involving a depressant effect of M(2) receptors on adenylyl cyclase activity in MF terminals. Our data demonstrate that cholinergic projections recruit M(2) receptors to redistribute the gain of LTP in CA3 pyramidal cells in an input-specific manner.
Journal of Neurophysiology 04/2012; 108(1):91-100. · 3.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.
[show abstract][hide abstract] ABSTRACT: Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non-neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells.
RT-PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments.
Transcripts encoding ChAT and OCT1-3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase in the transepithelial ion current (EC₅₀: 23.3 µM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na⁺-channel inhibitor amiloride. The Cl⁻-channel inhibitor niflumic acid or the K⁺-channel blocker Ba²⁺ attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene-deficient animals revealed the participation of muscarinic receptor subtypes M₁ and M₃.
The presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways.
British Journal of Pharmacology 02/2012; 166(4):1388-402. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.
The FASEB Journal 02/2012; 26(2):604-16. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Muscarinic acetylcholine (ACh) receptors (mAChRs; M₁-M₅) regulate the activity of an extraordinarily large number of important physiological processes. During the past 10-15 years, studies with whole-body M₁-M₅ mAChR knockout mice have provided many new insights into the physiological and pathophysiological roles of the individual mAChR subtypes. This review will focus on the characterization of a novel generation of mAChR mutant mice, including mice in which distinct mAChR genes have been excised in a tissue- or cell type-specific fashion, various transgenic mouse lines that overexpress wild-type or different mutant M₃ mAChRs in certain tissues or cells only, as well as a novel M₃ mAChR knockin mouse strain deficient in agonist-induced M₃ mAChR phosphorylation. Phenotypic analysis of these new animal models has greatly advanced our understanding of the physiological roles of the various mAChR subtypes and has identified potential targets for the treatment of type 2 diabetes, schizophrenia, Parkinson's disease, drug addiction, cognitive disorders, and several other pathophysiological conditions.
[show abstract][hide abstract] ABSTRACT: The neurotransmitter dopamine plays important roles in modulating cognitive, affective, and motor functions. Dysregulation of dopaminergic neurotransmission is thought to be involved in the pathophysiology of several psychiatric and neurological disorders, including schizophrenia, Parkinson's disease and drug abuse. Dopaminergic systems are regulated by cholinergic, especially muscarinic, input. Not surprisingly, increasing evidence implicates muscarinic acetylcholine receptor-mediated pathways as potential targets for the treatment of these disorders classically viewed as "dopamine based". There are five known muscarinic receptor subtypes (M(1) to M(5)). Due to their overlapping expression patterns and the lack of receptor subtype-specific ligands, the roles of the individual muscarinic receptors have long remained elusive. During the past decade, studies with knock-out mice lacking specific muscarinic receptor subtypes have greatly advanced our knowledge of the physiological roles of the M(1)-M(5) receptors. Recently, new ligands have been developed that can interact with allosteric sites on different muscarinic receptor subtypes, rather than the conventional (orthosteric) acetylcholine binding site. Such agents may lead to the development of novel classes of drugs useful for the treatment of psychosis, drug abuse and Parkinson's disease. The present review highlights recent studies carried out using muscarinic receptor knock-out mice and new subtype-selective allosteric ligands to assess the roles of M(1), M(4), and M(5) receptors in various central processes that are under strong dopaminergic control. The outcome of these studies opens new perspectives for the use of novel muscarinic drugs for several severe disorders of the CNS.
ACS Chemical Neuroscience 01/2012; 3(2):80-89. · 3.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: The anterolateral cell group of the bed nucleus of the stria terminalis (BNST(ALG)) serves as an important relay station in stress circuitry. Limbic inputs to the BNST(ALG) are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNST(ALG). Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNST(ALG). Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analog, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired-pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M(2) receptor-preferring antagonists, and was absent in M(2)/M(4) receptor knockout mice, suggesting that presynaptic M(2) receptors mediate the effect of ACh. Immunoelectron microscopy studies further revealed the presence of M(2) receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M(2) receptors might be an important modulator of the stress circuit and hence a novel target for drug development.