Muscarinic receptors, activated by acetylcholine, play critical roles in the functional regulation of medium spiny neurons in the striatum. However, the muscarinic receptor signaling pathways are not fully elucidated due to their complexity. In this study, we investigated the function of muscarinic receptors in the striatum by monitoring DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M-r 32 kDa) phosphorylation at Thr34 (the PKA-site) using mouse striatal slices. Treatment of slices with a non-selective muscarinic receptor agonist, oxotremorine (10 mu M), rapidly and transiently increased DARPP-32 phosphorylation. The increase in DARPP-32 phosphorylation was completely abolished either by a dopamine D-1 receptor antagonist (SCH23390), tetrodotoxin, genetic deletion of M5 receptors, muscarinic toxins for M1 and M4 receptors, or 6-hydroxydopamine lesioning of dopaminergic neurons, whereas it was enhanced by nicotine. Analysis in D-1-DARPP-32-Flag/D-2-DARPP-32-Myc transgenic mice revealed that oxotremorine increases DARPP-32 phosphorylation selectively in D-1-type/striatonigral, but not in D-2-type/striatopallidal, neurons. When D-1 and D-2 receptors were blocked by selective antagonists to exclude the effects of released dopamine, oxotremorine increased DARPP-32 Thr34 phosphorylation only in D-2-type/striatopallidal neurons. This increase required activation of M1 receptors and was dependent upon adenosine A(2A) receptor activity. The results demonstrate that muscarinic receptors, especially M5 receptors, act at presynaptic dopaminergic terminals, regulate the release of dopamine in cooperation with nicotinic receptors, and activate D-1 receptor/DARPP-32 signaling in the striatonigral neurons. Muscarinic M1 receptors expressed in striatopallidal neurons interact with adenosine A(2A) receptors and activate DARPP-32 signaling. (C) 2012 Elsevier Ltd. All rights reserved.