Central M2 and M4 muscarinic acetylcholine (ACh) receptors (mAChRs) are known to play key roles in many fundamental functions of the brain, including learning and memory, locomotor activity, and mood control. For example, whole body M2 receptor knockout (KO) mice displayed severe cognitive deficits in several learning and memory tasks and reduced synaptic plasticity in the hippocampus (Seeger et al. J. Neurosci. 24, 10117-10127, 2004). Analysis of the whole body M4 receptor KO mice showed that activation of M4 receptors counteracts the stimulatory effects of D1 dopamine receptor activation (Gomeza et al. Proc. Natl. Acad. Sci. USA 96, 10483-104888, 1999). Since striatal D1 dopamine receptors play an important role in the regulation of locomotor activity and D1 receptors in the nucleus accumbens are involved in mediating the rewarding effects of drug of abuse, these findings are of considerable therapeutic interest.[unreadable] In order to learn more about the localization of the M2 and M4 mAChRs involved in these important central processes, we decided to use Cre/loxP technology to generate a series of conditional mAChR mutant mice which lack M2 or M4 receptors only in specific regions of the brain. In the first step, we generated targeting constructs in which the M2 and M4 receptor coding sequences were surrounded by loxP sites. The targeting constructs were then extrapolated into mouse embryonic stem cells (ES cells). Properly targeted ES cell clones were identified by Southern blotting and PCR analysis. The properly targeted ES cells were then injected into blastocyst hosts, and the injected blastocysts were implanted into pseudopregnant mice (this work was done by Dr. Chuxia Deng's lab at NIDDK). The resulting chimeric mice were analyzed for germline transmission of the floxed M2 or M4 receptor allele. The neo cassette (which was contained in the targeting constructs and served as a positive selection marker during ES cell culture) was removed via matings with EIIa Cre mice which express Cre recombinase early in development (note that the neo gene was flanked by two loxP sites).[unreadable] The floxed M2 and M4 receptor mice were then crossed with transgenic mice that express Cre recombinase only in certain regions of the brain (e.g. forebrain, distinct regions of the hippocampus, D1 receptor-containing cells, etc.). The resulting mice are currently being studied by various biochemical, pharmacological, and behavioral techniques. The behavioral analysis of the mutant animals includes assays to assess learning and memory, locomotor activity, drug-seeking behavior, and several other important central functions. The analysis of the various mutant mouse strains is currently ongoing. The analysis of these newly generated mutant mice should lead to important new insights into the physiological and pathophysiological roles of these receptors. It is likely that these studies will identify novel therapeutic targets for the treatment of various important disorders of the CNS, including Alzheimers and Parkinsons disease and drug addiction.[unreadable] [unreadable] The following results were obtained in collaborative studies:[unreadable] [unreadable] 1. Electrophysiological studies with tissues from various mAChR KO mice showed[unreadable] 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.[unreadable] (Origlia et al., J. Physiol. 577, 829-840, 2006)[unreadable] [unreadable] 2. M5 mAChR KO mice showed reduced blood flow in the cerebral cortex, hippocampus and several other regions of the brain. Moreover, the M5 mAChR mutant mice also exhibited impairments in hippocampus-dependent memory, associated with deficits in hippocampal synaptic plasticity. The M5 receptor therefore represents an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.[unreadable] (Araya et al., Neurobiol. Dis. 24, 334-344, 2006)[unreadable] [unreadable] 3. Schizophrenic patients show decreased measures of sensorimotor gating, such as prepulse inhibition of startle (PPI). Behavioral studies showed that M5 mAChR KO mice displayed decreased PPI relative to wild-type mice, suggesting that M5 receptor activity plays a role in modulating sensorimotor gating. [unreadable] Thomsen et al., Psychopharmacology 192, 97-110, 2007.