Gene delivery can be used to test hypothesis on brain mechanisms of alcoholism as well as providing a potential new therapeutic approach to drug abuse. Both human and animal data have implicated decreased dopamine (DA)and/or serotonin (5-hydroxytryptamine-5HT) in the nucleus accumbens (NAc) and other forebrain areas with high alcohol drinking behaviors. The hypothesis to be tested in this proposal is that viral vectors enhancing levels of the biogenic amines, DA and/or 5HT in specific brain regions will alter responses to ethanol, particularly high alcohol drinking behavior. Levels of DA and 5HT will be selectively increased in specific brain regions by constructing vectors containing synthetic enzymes using the adeno-associated virus (AAV) vector system. This approach prepares a therapeutic agent-drug that contains less than 5% of the original viral genome and the enzymes tyrosine hydroxylase (TH), aromatic amino acid decarboxylase( AADc) and tryptophan decarboxylase.( TRYPH). Preliminary studies have shown that the AAV vector is not neurotoxic and can express TH-AADC in brain to levels that raise the concentration of brain DA sufficient to partially restore motor abnormalities in models of Parkinson's disease. Expression is known to occur for at least several months in many brain regions. These preliminary studies indicate the AAV- Th-AADC vector can be used to increase DA levels in brain.
The aims of this proposal are: 1. To investigate the expression of AAV-tyrosine hydroxylase-aromatic amino acid decarboxylase (AAV-TH-AADC) vectors in various brain regions including the ventral tegmental area (VTA) , NAc, and forebrain. Variables will include vector load and brain region(s) of expression. Preliminary studies have indicated that a AAV-lacZ vector is expressed in NAc. The AAV-lacZ vector will be used to investigate specific cell sites of expression and to serve as a """"""""vehicle"""""""" control for AAV vectors containing the TH-AADC.Th-AADC expression will be determined using a FLAG epitope included in the vector for immunohistochemical distinction from endogenous enzyme, total TH immunohistochemistry, total TH activity, the content of DA and DA metabolites as well as PCR of viral DNA. 2. To determine the effects of AAV-Th-AADC vector induced increased dopamine in nucleus accumbens and/or VTA on ethanol behaviors, particularly high ethanol drinking behaviors in P and NP rats. 3. Develop an AAV-Tryptophan hydroxylase-aromatic amino acid decarboxylase vector (AAV-TRYPH-AADC) to enhance 5HT levels in NAc, VTA, Raphe, forebrain and other areas of interest. Variables will include vector load and brain region(s) of expression. 4. Determine the effects of increased 5HT synthesis in NAc, forebrain and/or Raphe on behaviors as in Specific Aim 2.
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