Viral vector mediated overexpression of cytochrome P450 side chain cleavage in the VTA produces long-term reductions on ethanol self-administration

Neuroactive steroids are endogenous neuromodulators synthesized in the brain, adrenal glands, and gonads. Systemic administration of endogenous and synthetic neuroactive steroids can alter ethanol self-administration in rodents. We have hypothesized that neuroactive steroids alter ethanol self-administration by modulating mesolimbic circuitry. In the current study, a previously characterized adeno-associated serotype 2 viral vector (AAV2) expressing the mitochondrial cytochrome P450 side chain cleavage (P450scc) enzyme was used to produce long-term increases in P450scc expression. P450scc converts cholesterol to pregnenolone, which is the rate-limiting enzymatic reaction in neurosteroidogenesis. Overexpression of P450scc should allow us to investigate how sustained increases in steroidogenesis, isolated to specific brain regions, affect ethanol self-administration. The P450scc expressing vector (AAV2-P450scc) or control green fluorescent protein (GFP) expressing vector (AAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA, 2uL/hemisphere) or nucleus accumbens (NAc, 3uL/hemisphere) of alcohol preferring (P) rats (n=7-8/group) previously trained to self-administer ethanol. Following viral vector injection the animals were given 1 week to recover from surgery before operant self-administration sessions resumed. P450scc overexpression in the VTA reduced ethanol responding by 25% compared to controls (2-way ANOVA, p<0.01) over the 3 week test period. The reduction in responding following injection was due in part to a persistent reduction in responses (28%, 2-way ANOVA w/Bonferoni post-test, p<0.001) during the first 5 minutes of operant sessions over 3 weeks of testing. In contrast, P450scc overexpression in NAc did not significantly alter long-term ethanol self-administration. General locomotor activity was not altered by vector administration in VTA or NAc. P450scc overexpression did not increase allopregnanolone-like immunoreactivity in NAc; however, vector delivery in the VTA produced a 34% increase in allopregnanolone positive cells in the VTA, which did not reach statistical significance. These results provide evidence that P450scc overexpression, and presumably increased steroidogenesis, in the VTA reduces ethanol reinforcement. Investigating how neuroactive steroids modulate ethanol reinforcement may lead to the development of new therapeutic strategies for treating alcoholism.