No abstract

Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain. To investigate the molecular identity of hippocampal, cortical, and striatal inhibitory muscarinic autoreceptors in a more direct manner, we used genetically altered mice lacking functional M2 and/or M4 mAChRs [knock-out (KO) mice]. After labeling of cellular ACh pools with [3H]choline, potassium-stimulated [3H]ACh release was measured in superfused brain slices, either in the absence or the presence of muscarinic drugs. The nonsubtype-selective muscarinic agonist, oxotremorine (0.1-10 microm), inhibited potassium-stimulated [3H]ACh release in hippocampal, cortical, and striatal slices prepared from wild-type mice by up to 80%. This activity was totally abolished in tissues prepared from M2-M4 receptor double KO mice. Strikingly, release studies with brain slices from M2 and M4 receptor single KO mice indicated that autoinhibition of ACh release is mediated primarily by the M2 receptor in hippocampus and cerebral cortex, but predominantly by the M4 receptor in the striatum. These results, together with additional receptor localization studies, support the novel concept that autoinhibition of ACh release involves different mAChRs in different regions of the brain.

A proper balance between striatal muscarinic cholinergic and dopaminergic neurotransmission is required for coordinated locomotor control. Activation of striatal muscarinic acetylcholine receptors (mAChRs) is known to modulate striatal dopamine release. To identify the mAChR subtype(s) involved in this activity, we used genetically altered mice that lacked functional M1-M5 mAChRs [knock-out (KO) mice]. In superfused striatal slices from wild-type mice, the non-subtype-selective muscarinic agonist oxotremorine led to concentration-dependent increases in potassium-stimulated [3H]dopamine release (by up to 60%). The lack of M1 or M2 receptors had no significant effect on the magnitude of these responses. Strikingly, oxotremorine-mediated potentiation of stimulated striatal [3H]dopamine release was abolished in M4 receptor KO mice, significantly increased in M3 receptor-deficient mice, and significantly reduced (but not abolished) in M5 receptor KO mice. Additional release studies performed in the presence of tetrodotoxin suggested that the dopamine release-stimulating M4 receptors are probably located on neuronal cell bodies, but that the release-facilitating M5 and the release-inhibiting M3 receptors are likely to be located on nerve terminals. Studies with the GABA(A) receptor blocker bicuculline methochloride suggested that M3 and M4 receptors mediate their dopamine release-modulatory effects via facilitation or inhibition, respectively, of striatal GABA release. These results provide unambiguous evidence that multiple mAChR subtypes are involved in the regulation of striatal dopamine release. These findings should contribute to a better understanding of the important functional roles that the muscarinic cholinergic system plays in striatal function.

Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate͞drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M 5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M 5 receptor-deficient mice (M5 ؊/؊ mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M 5 ؊/؊ mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M 5 ؊/؊ mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M 5 receptors. The finding that M 5 receptor activity modulates both morphine reward and withdrawal processes suggests that M 5 receptors may represent a novel target for the treatment of opiate addiction.T he M 5 muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M 1 -M 5 ) to be cloned (1, 2). Owing to the lack of ligands that can selectively stimulate or inhibit the M 5 receptor (3, 4), the physiological roles of this receptor subtype have remained obscure (5, 6). To address this issue, we recently used a gene-targeting approach to gen-Immunoprecipitation studies have shown that M 5 receptors are expressed at very low levels in the brain, representing less than 2% of the total muscarinic receptor population (M 1 -M 5 ) (8). Interestingly, M 5 receptor mRNA has been identified as the only muscarinic receptor mRNA in dopamine-containing neurons of the substantia nigra and the ventral tegmental area (VTA) (9, 10). It has therefore been proposed that M 5 receptors may play a role in modulating dopamine release from midbrain dopaminergic neurons (9, 10). Consistent with this hypothesis, we recently demonstrated that muscarinic agonist-induced increases in striatal dopamine release were reduced in M 5 Ϫ/Ϫ mice (7). Moreover, Forster et al. (11) recently reported that the sustained increase in dopamine levels in the nucleus accumbens (Acb) observed after electrical stimulation of the laterodorsal tegmental nucleus (12) is absent in M 5 Ϫ/Ϫ mice. Laterodorsal tegmental nucleus neurons represent the major source of cholinergic input to the dopamine-containing neurons of the VTA (13, 14) that project to the Acb and other limbic areas (15-17). It is therefore likely that activation of M 5 receptors express...