Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice

Abstract

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.Muscarinic acetylcholine receptors are known to regulate numerous fundamental physiological processes, including the muscarinic actions of acetylcholine on peripheral effector tissues and a multitude of central sensory, vegetative, and motor functions (1-4). In addition, disturbances in central muscarinic neurotransmission have been implicated in a variety of pathophysiological conditions, including Alzheimer's and Parkinson's diseases (1-4).Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic receptor subtypes referred to as M1-M5 (5-7). The M1-M5 receptors are prototypical members of the superfamily of G protein-coupled receptors. Although the odd-numbered muscarinic receptor subtypes (M1, M3, and M5) are selectively linked to G q͞11 proteins, the even-numbered receptors (M2 and M4) are preferentially coupled to G proteins of the G i͞o family (5-7).The M1-M4 receptors are widely expressed throughout the central nervous system and the body periphery (6,(8)(9)(10). Studies with subtype-selective antibodies and in situ mRNA hybridization experiments have shown that most brain regions express several different muscarinic receptor subtypes (8-10). Based on this observation, it has been extremely difficult to assign specific central functions to individual muscarinic receptor subtypes.In addition, the lack of muscarinic agonists and antagonists with pronounced subtype selectivity also has represented a major limitation in studying the physiological roles of the M1-M5 receptors (5-7). This problem is accentuated further in the case of in vivo studies in which the actual concentrations of drugs at their sites of action are difficult to determine because of pharmacokinetic factors.In the body periphery, muscarinic receptors mediate the well known functions of acetylcholine at parasympathetically innervated effector organs, including contraction of smooth muscle, stimulatio...

Dysregulated hippocampal acetylcholine neurotransmission and impaired cognition in M2, M4 and M2/M4 muscarinic receptor knockout mice

“…Previous studies with M2 and M4 receptorspecific antibodies have shown that inactivation of the M2 receptor gene did not lead to changed M4 receptor expression levels in the hippocampus and vice versa. 9,17 On the other hand, the increase in acetylcholine release produced by the nonselective muscarinic antagonist scopolamine was attenuated in M2-but not M4-KO mice. In agreement with the present findings, oxotremorine-induced reduction in stimulated [ 3 H]acetylcholine release from hippocampal slices was abolished in M2-KO animals but preserved in M4-KOs.…”

“…Antisense 14,15 and gene targeting technologies have emerged as powerful new tools in identifying the receptor subtypes involved in various muscarinic cholinergic functions. [16][17][18][19][20][21] Although pharmacological, neuroanatomical and clinical studies have suggested an important role of muscarinic receptors in synaptic plasticity and memory, the functions of the individual muscarinic receptor subtypes remain unclear. In this study, we have used M2 and M4 receptor single knockout (KO) as well as M2/M4 receptor double KO mice to investigate the functional importance of M2 and M4 receptors in the regulation of acetylcholine release in the hippocampus and in cognitive processes.…”

Towards a muscarinic hypothesis of schizophrenia

“…63,64 However, the M 5 -subtype may be relevant to schizophrenia as it is located in the brainstem and midbrain, where it has an effect on dopamine release. 65 Based on their functional activity, muscarinic receptors can be subdivided into two groups (M 1 , M 3 [66][67][68][69] A better understanding of the physiological role of the different subtypes of the muscarinic receptors has been gained from the study of knockout animals that lack one or more of these receptors; [70][71][72][73][74][75] for a review see Bymaster et al 76 ). Depending on the muscarinic receptor subtype involved, cholinergic activation can have different effects on the peripheral and central nervous function.…”

Molecular mechanisms that control initiation and termination of physiological depolarization-evoked transmitter release

“…1 suggest that a PTX-sensitive GPCR is involved in control of initiation and termination of ACh release. We first examined whether the effects of PTX are mediated by M 2 R by using NMJs of knockout mice lacking functional M 2 R (M 2 -KO) (23). Here, PTX had no effect on SR (Fig.…”