Ethanol-induced reinforcement (i.e., elicitation of a pleasurable response to ingestion of ethanol), and the generation of craving for ethanol after chronic ingestion of ethanol, have been considered as phenomena which involve the neuronal systems projecting to an area of brain identified as the nucleus accumbens. The dopaminergic systems projecting to the nucleus accumbens and the post-receptor signal transduction systems (i.e., G proteins, adenylyl cyclases, protein kinase A) of cells receiving this dopaminergic input have been implicated in the reinforcing and craving phenomena associated with intake of cocaine, amphetamine and opiates. The data regarding ethanol's actions are more sparse. We will, therefore, identify with in situ hybridization techniques which adenylyl cyclases are expressed in the nucleus accumbens of rats. We will characterize the activity of these enzyme(s) in the presence and absence of ethanol in vitro and we will then detail the effects of chronic ethanol intake on the various components of the dopamine receptor-modulated cAMP signaling cascade in the nucleus accumbens. D1 and D2 dopamine receptors will be quantified by use of autoradiographic and membrane binding techniques, G protein alpha and beta gamma subunits will be examined with immunohistochemistry and quantitative Western blotting techniques, mRNA for specific adenylyl cyclases will be examined using RNAse protection assays and adenylyl cyclase proteins and their distribution will be measured with immunohistochemical and Western blotting techniques. The activity of adenylyl cyclase(s) under various stimulatory and inhibitory conditions and the activity of PKA will be examined in nucleus accumbens tissue obtained from control and ethanol-treated animals. Finally, to provide further insights into the relationship of the cAMP signalling cascade and inherent levels of ethanol consumption by animals, we will perform a phenotypic and genetic correlation analysis of the adenylyl cyclase systems in the nucleus accumbens and the alcohol preferences of HAD (alcohol preferring) and LAD (alcohol non-preferring) rats and their F1, F2 and backcross offspring. These later studies will be guided by our initial work with control and ethanol treated animals with regard to the optimal parameters of the adenylyl cyclase systems to be examined in the HAD and LAD rats and their offspring. Our studies, in total, should shed light on important components of ethanol's actions which may be intimately related to the addictive properties of ethanol as well as other drugs.
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