Muscarinic Acetylcholine Receptor Knockout Mice Research Articles

Increased cocaine self-administration in M4 muscarinic acetylcholine receptor knockout mice

The reinforcing effects of cocaine are mediated by the mesolimbic dopamine system. Behavioral and neurochemical studies have shown that the cholinergic muscarinic M(4) receptor subtype plays an important role in regulation of dopaminergic neurotransmission. Here we investigated for the first time the involvement of M(4) receptors in the reinforcing effects of cocaine using chronic intravenous cocaine self-administration in extensively backcrossed M(4) receptor knockout (M(4) (-/-)) mice. We evaluated acquisition of cocaine self-administration in experimentally naïve mice. Both cocaine self-administration and food-maintained operant behavior were evaluated under fixed ratio 1 (FR 1) and progressive ratio (PR) schedules of reinforcement. In addition, cocaine-induced dopamine release and cocaine-induced hyperactivity were evaluated. M(4) (-/-) mice earned significantly more cocaine reinforcers and reached higher breaking points than their wild-type littermates (M(4) (+/+)) at intermediate doses of cocaine under both FR 1 and PR schedules of reinforcement. Under the PR schedule, M(4) (-/-) mice exhibited significantly higher response rates at the lowest liquid food concentration. In accordance with these results, cocaine-induced dopamine efflux in the nucleus accumbens and hyperlocomotion were increased in M(4) (-/-) mice compared to M(4) (+/+) mice. Our data suggest that M(4) receptors play an important role in regulation of the reward circuitry and may serve as a new target in the medical treatment of drug addiction.

Cholinergic responses of ophthalmic arteries in M3 and M5 muscarinic acetylcholine receptor knockout mice.

To determine the functional role of M(3) and M(5) muscarinic acetylcholine receptor subtypes in ophthalmic arteries using gene-targeted mice. Muscarinic receptor gene expression was quantified in murine ophthalmic arteries using real-time PCR. To test the functional relevance of M(3) and M(5) receptors, ophthalmic arteries from mice deficient in either subtype (M3R(-/-), M5R(-/-), respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal vessel diameter in response to muscarinic and nonmuscarinic receptor agonists were measured by video microscopy. With the use of real-time PCR, all five muscarinic receptor subtypes were detected in ophthalmic arteries. However, mRNA levels of M(1), M(3), and M(5) receptors were higher than those of M(2) and M(4) receptors. In functional studies, after preconstriction with phenylephrine, acetylcholine and carbachol produced concentration-dependent dilations of ophthalmic arteries that were similar in M5R(-/-) and wild-type mice. Strikingly, cholinergic dilation of ophthalmic arteries was almost completely abolished in M3R(-/-) mice. Deletion of either M(3) or M(5) receptor did not affect responses to nonmuscarinic vasodilators such as bradykinin or nitroprusside. These findings provide the first evidence that M(3) receptors are critically involved in cholinergic regulation of diameter in murine ophthalmic arteries.

Role of the M3 muscarinic acetylcholine receptor in β‐cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Diminished antigen-specific IgG 1 and interleukin-6 production and acetylcholinesterase expression in combined M 1 and M 5 muscarinic acetylcholine receptor knockout mice

Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M 5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca 2+-signaling and up-regulation of c-fos expression; moreover, M 1 mAChRs play a crucial role in the differentiation of CD8 + T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M 1 and M 5 mAChR knockout (M 1/M 5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG 1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG 1 were significantly lower in M 1/M 5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M 1/M 5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M 1/M 5 KO mice. These results suggest that M 1 and/or M 5 mAChRs are involved in regulating cytokine ( e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG 1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.

Decreased prepulse inhibition and increased sensitivity to muscarinic, but not dopaminergic drugs in M5 muscarinic acetylcholine receptor knockout mice

Schizophrenic patients show decreased measures of sensorimotor gating, such as prepulse inhibition of startle (PPI). In preclinical models, these measures may be used to predict antipsychotic activity. While current antipsychotic drugs act largely at dopamine receptors, the muscarinic acetylcholine receptors offer promising novel pharmacotherapy targets. Of these, the M(5) receptor gene was recently implicated in susceptibility to schizophrenia. Due to the lack of selective ligands, muscarinic receptor knockout mice have been generated to elucidate the roles of the five receptor subtypes (M(1)-M(5)). Here, we used M(5) receptor knockout (M(5)-/-) mice to investigate the involvement of M(5) receptors in behavioral measures pertinent to schizophrenia. We tested the hypothesis that disruption of M(5) receptors affected PPI or the effects of muscarinic or dopaminergic agents in PPI or psychomotor stimulation. We measured PPI in M(5)-/-, heterozygous and wild-type mice without drugs, and with clozapine (0.56-3.2 mg/kg) or haloperidol (0.32-3.2 mg/kg) alone, and as pretreatment to D: -amphetamine. In addition, we evaluated locomotor stimulation by the muscarinic antagonist trihexyphenidyl (0.56-56 mg/kg) and by cocaine (3.2-56 mg/kg). The M(5)-/- mice showed decreased PPI relative to wild-type mice, and clozapine appeared to reduce this difference, while haloperidol increased PPI regardless of genotype. The M(5)-/- mice also showed more locomotor stimulation by trihexyphenidyl than wild-type mice, while cocaine had similar effects between genotypes. These data suggest that disruption of the M(5) receptor gene affected sensorimotor gating mechanisms, increased sensitivity to clozapine and to the psychostimulant effects of muscarinic antagonists without modifying the effect of dopaminergic drugs.

Muscarinic acetylcholine receptor knockout mice show distinct synaptic plasticity impairments in the visual cortex

In the present report, we focused our attention on the role played by the muscarinic acetylcholine receptors (mAChRs) in different forms of long-term synaptic plasticity. Specifically, we investigated long-term potentiation (LTP) and long-term depression (LTD) expression elicited by theta-burst stimulation (TBS) and low-frequency stimulation (LFS), respectively, in visual cortical slices obtained from different mAChR knockout (KO) mice. A normal LTP was evoked in M(1)/M(3) double KO mice, while LTP was impaired in the M(2)/M(4) double KO animals. On the other hand, LFS induced LTD in M(2)/M(4) double KO mice, but failed to do so in M(1)/M(3) KO mice. Interestingly, LFS produced LTP instead of LTD in M(1)/M(3) KO mice. Analysis of mAChR single KO mice revealed that LTP was affected only by the simultaneous absence of both M(2) and M(4) receptors. A LFS-dependent shift from LTD to LTP was also observed in slices from M(1) KO mice, while LTD was simply abolished in slices from M(3) KO mice. Using pharmacological tools, we showed that LTP in control mice was blocked by pertussis toxin, an inhibitor of G(i/o) proteins, but not by raising intracellular cAMP levels. In addition, the inhibition of phospholipase C by U73122 induced the same shift from LTD to LTP after LFS observed in M(1) single KO and M(1)/M(3) double KO mice. Our results indicate that different mAChR subtypes regulate different forms of long-term synaptic plasticity in the mouse visual cortex, activating specific G proteins and downstream intracellular mechanisms.

Functional Activity of the M2 and M4 Receptor Subtypes in the Spinal Cord Studied with Muscarinic Acetylcholine Receptor Knockout Mice

Stimulation of spinal muscarinic acetylcholine receptors (mAChRs) produces potent analgesia. Both M(2) and M(4) mAChRs are coupled to similar G proteins (G(i/o) family) and play a critical role in the analgesic action of mAChR agonists. To determine the relative contribution of M(2) and M(4) subtypes to activation of G(i/o) proteins in the spinal cord, we examined the receptor-mediated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in M(2) and M(4) subtype knockout (KO) mice. Basal [(35)S]GTPgammaS binding in the spinal cord was similar in the wild-type controls, M(2) and M(4) single-KO, and M(2)/M(4) double-KO mice. The spinal [(35)S]GTPgammaS binding stimulated by either muscarine or oxotremorine-M was not significantly different among three groups of wild-type mouse strains. In M(2) single-KO and M(2)/M(4) double-KO mice, the agonist-stimulated [(35)S]GTPgammaS binding was completely abolished in the spinal cord. Furthermore, the agonist-stimulated [(35)S]GTPgammaS binding in the spinal cord of M(4) single-KO mice was significantly reduced ( approximately 15%), compared with that in wild-type controls. On the other hand, the spinal [(35)S]GTPgammaS binding stimulated by a mu-opioid agonist was not significantly different between wild-type and M(2) and M(4) KO mice. This study provides complementary new evidence that M(2) is the most predominant mAChR subtype coupled to the G(i/o) proteins in the spinal cord. Furthermore, these data suggest that a small but functionally significant population of M(4) receptors exists in the mouse spinal cord. The functional activity of these M(4) receptors seems to require the presence of M(2) receptors.

Quantitative analysis of binding parameters of [ 3H] N-methylscopolamine in central nervous system of muscarinic acetylcholine receptor knockout mice

We have studied binding parameters ( K d, B max) of [ 3H] N-methylscopolamine ([ 3H]NMS) in various brain regions and spinal cord of wild-type (WT) and muscarinic acetylcholine receptor (mAChR) subtype (M 1–M 5) knockout (KO) mice. In the M 1–M 4 KO mice, the number of [ 3H]NMS binding sites ( B max) was decreased throughout the central nervous system (CNS) with significant regional differences. Our results collectively suggest that M 1 receptor was present in a relatively high density in the cerebral cortex and hippocampus, and the densities of M 1 and M 4 subtypes were highest in the corpus striatum. M 2 receptor appeared to be the major subtype in the thalamus, hypothalamus, midbrain, pons-medulla, cerebellum and spinal cord. These findings may contribute significantly not only to the further understanding of the physiological roles of mAChR subtypes in the central cholinergic functions, but also to the development of selective therapeutic agents targeting specific subtype.

Muscarinic agonist-mediated increases in serum corticosterone levels are abolished in m(2) muscarinic acetylcholine receptor knockout mice.

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions in the central and peripheral nervous system. Due to the lack of receptor subtype-selective ligands, however, the physiological roles of individual muscarinic receptor subtypes remain to be determined. In this study, we examined the effects of the muscarinic M(2)/M(4) receptor-preferring agonist [5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.2.1]-octane (BuTAC) on serum corticosterone levels in M(2) and M(4) receptor single knockout (KO) and M(2,4) receptor double KO mice. Responses were compared with those obtained with the corresponding wild-type (WT) mice. BuTAC (0.03-0.3 mg/kg s.c.) dose dependently and significantly increased serum corticosterone concentrations in WT mice to 5-fold or greater levels compared with vehicle controls. In muscarinic M(2) and M(2,4) KO mice, however, BuTAC had no significant effect on corticosterone concentrations at doses of 0.1, 0.3, and 1 mg/kg s.c. In both WT and muscarinic M(4) KO mice increases in serum corticosterone concentrations induced by BuTAC (0.1 and 0.3 mg/kg) were not significantly different and were blocked by scopolamine. In summary, the muscarinic M(2,4)-preferring agonist BuTAC had no effect on corticosterone levels in mice lacking functional muscarinic M(2) receptors. These data suggest that the muscarinic M(2) receptor subtype mediates muscarinic agonist-induced activation of the hypothalamic-pituitary-adrenocortical axis in mice.

Cholinergic dilation of cerebral blood vessels is abolished in M(5) muscarinic acetylcholine receptor knockout mice.

The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice). M5R(-/-) mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M(5) receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M(5) receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M(5) receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R(-/-) mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R(-/-) mice. Our findings provide direct evidence that M(5) muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M(5) receptors may represent an attractive therapeutic target.

Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice.

Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.