The basal ganglia is a brain region rich in dopamine and the principal site where dopamine blocking agents, including neuroleptic drugs, exert their central side effects. Although it has been established that neuroleptics block dopamine receptors in the basal ganglia, their effects on messenger RNA (mRNA) transcription are not known. The availability of a panel of complementary DNA (cDNA) clones for messenger RNAs which are effected by neuroleptic treatment would be an important step toward understanding the action of neuroleptics. I propose to study changes in mRNA levels in basal ganglia from animals treated with neuroleptics and dopamine neurotoxins. The first phase of the proposed project is to isolate and characterize basal ganglia enriched mRNA using subtractive hybridization and cDNA cloning techniques. These studies in normal animals represent a novel approach to studying selective gene transcription in the basal ganglia. I will initially use tissue derived from bovine brains to develop expertise in cDNA subtractive hybridization. I will then use brain tissue from untreated African green monkeys in a series of identical experiments. In both cases, mRNA from cerebellar tissue will be used to subtract common messages. These studies will result in the isolation of a panel of cDNA clones enriched to basal ganglia relative to cerebellum, and will be the basis for subsequent studies on the effects of dopamine blocking agents. In the second phase, I will examine the effect of decreased dopamine (DA) levels on mRNA transcription rates in this region. Two methods will be used to deplete postsynaptic levels of DA. First, DA pathways will be blocked by chronic treatment with the neuroleptic, Haldol. In the second method, a selective neurotoxin called MPTP, will be administered. The use of these agents in conjunction with subtractive hybridization techniques presents a unique opportunity to study the effects of dopamine depletion on mRNA transcription within neurons of the basal ganglia. The third and final phase of this project will be to characterize basal ganglia specific mRNAs, particularly those altered by drug treatment. For these studies, I will use in situ hybridization and immunocytochemistry to compare the anatomical distribution of experimentally isolated cDNAs from basal ganglia. Within the basal ganglia, the distribution of cDNAs will be compared with known neurotransmitter substances and other cell type specific markers. My long range goal is to understand the molecular bases for neuropsychiatric diseases and this proposal represents a first step toward that end.
