Members of the muscarinic acetylcholine receptor family (M1-M5) have central roles in the regulation of many fundamental physiological functions. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R-/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake, are significantly reduced in M3R-/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R-/- mice. However, M3R-/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.

Members of the muscarinic acetylcholine receptor family are thought to play key roles in the regulation of a large number of important functions of the CNS. However, the precise roles of the individual muscarinic receptor subtypes in modulating these processes are not well understood at present, primarily because of the lack of ligands with sufficient receptor subtype selectivity. To investigate the behavioral significance of the M 1 muscarinic receptor (M 1 R), which is abundantly expressed in the forebrain, we subjected M 1 receptor-deficient mice Ϫ/Ϫ mice showed slight performance deficits in auditory-cued fear conditioning and in an eight-arm radial maze, most likely because of the hyperactivity phenotype displayed by the M 1 R Ϫ/Ϫ mice. Our results indicate that M 1 muscarinic receptors play an important role in the regulation of locomotor activity but appear to be less critical for cognitive processes, as generally assumed.

The M5 muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M1-M5) 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 extracerebral 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. M olecular cloning studies have revealed the existence of five molecularly distinct muscarinic acetylcholine receptor subtypes (M 1 -M 5 ) (1, 2). During the past decade, considerable progress has been made in delineating the physiological roles of the M 1 -M 4 muscarinic receptors (3, 4). In contrast, the physiological relevance of the M 5 receptor subtype, which is the most recent member of the muscarinic receptor family to be cloned (5, 6), remains unknown at present (7,8). However, expression of the cloned M 5 muscarinic receptor gene in cultured mammalian cells has shown that the encoded receptor protein is functional and efficiently couples to G proteins of the G q family, similar to the M 1 and M 3 receptor subtypes (5-8).Immunoprecipitation and in situ mRNA hybridization studies have demonstrated the presence of M 5 receptor protein͞mRNA in different areas of the brain including hippocampus, hypothalamus, and distinct midbrain regions (substantia nigra pars compacta and ventral tegmental area) (9-11). However, M 5 receptors are expressed at very low levels, representing less than 2% of the total muscarinic receptor population (M 1 -M 5 ) expressed in the brain (9). Interestingly, the use of highly sensitive reverse transcriptase-PCR (RT-PCR) techniques suggests that M 5 receptors are expressed in all major brain regions (12). More recently, M 5 receptors also have been detected in several peripheral tissues or cells including peripheral blood l...