Project 1 will explore how typical and atypical neuroleptic drugs affect protein phosphorylation pathways in striatum and prefrontal cortex. It is anticipated that typical and atypical neuroleptics share some, but not all, intracellular signaling targets. The similarities could reflect the common ability of these compounds to counteract positive symptoms, whereas the differences could reflect their differential actions on negative symptoms and extrapyramidal side effects. Aim I will establish a comprehensive survey of protein phosphorylation pathways regulated by neuroleptics in cortex and striatum. Previous work has shown that DARPP-32 is implicated in the actions of neuroleptics. Up until the present time, it has not been possible to distinguish between the biochemical regulation of DARPP-32 in specific neuronal cell types of striatum. These neurons are morphologically indistinguishable and are intermixed anatomically. The goal of Aim II is to obtain a more detailed understanding of the specific cell population where neuroleptics act. Novel BAC transgenic mouse technology will be used to overexpress epitope-tagged DARPP-32, RCS and mGluR5 in striatonigral, striatopallidal or cortical neurons. Pull down experiments followed by immunoblotting or mass spectrometry from these animals will allow cell-specific analyses of phosphorylation events. DARPP-32 KO mice show altered responses to neuroleptics and psychotomimetics. The goal of Aim III is to define neuronal circuitries in which DARPP-32 mediates actions of neuroleptics. The Cre/loxP technology will be used to generate mice lacking DARPP-32 in striatonigral, striatopallidal or cortical neurons. The mouse lines will be studied in terms of their responsivity to neuroleptics in biochemical, behavioural and electrophysiological assays, finally, in Aim IV, studies on the cellular biology of novel proteins interacting with metabotropic glutamate, serotonin and muscarinic receptors will be conducted. Thus, the proposed studies should provide a detailed knowledge on how neuroleptics affect protein phosphorylation pathways in defined neuronal circuitries.