Overexpression of the Steroidogenic Enzyme Cytochrome P450 Side Chain Cleavage in the Ventral Tegmental Area Increases 3α,5α-THP and Reduces Long-Term Operant Ethanol Self-Administration

Abstract

Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3␣,5␣)-3-hydroxypregnan-20-one (3␣,5␣-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3␣,5␣-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3␣,5␣-THP-positive cells in the VTA reduces ethanol reinforcement. 3␣,5␣-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

A Randomized, Double-Blind, Placebo-Controlled Trial of Pregnenolone for Bipolar Depression

“…Finally, orally administered pregnenolone has a complicated metabolism and may not be the most efficient method of increasing levels of pregnenolone and other neuroactive steroids in the brain. In the future, synthetic (eg, viral genedelivery approaches that modulate neurosteroidogenic enzyme expression in specific brain regions show promise in rodent models (Cook et al, 2014) neurosteroid derivatives might provide a more direct and receptor-specific method of delivering neuroactive steroids to the human brain.…”

Neurosteroidogenesis Today: Novel Targets for Neuroactive Steroid Synthesis and Action and Their Relevance for Translational Research

“…As a result, cholesterol is converted to soluble pregnenolone. Since this is a rate-limiting step, overexpression of this side chain cleavage enzyme would generally elevate neuroactive steroid levels (161). Pregnenolone can be further oxidized to progesterone under the catalysis of 3β-hydroxysteroid dehydrogenase or exit the mitochondrion without active transport.…”

“…In agreement, preclinical studies have found that administration of endogenous (epiallopregnanolone) or synthetic (3α,5β-20-oxo-pregnane-3-carboxylic acid) neuroactive steroids, as well as of the precursor pregnenolone, reduced ethanol self-administration in alcohol-preferring rats (204, 205). Likewise, recombinant adeno-associated serotype 2 vector mediated over-expression of P450 side chain cleavage (P450scc; the rate limiting enzyme in steroid synthesis) in the ventral tegmental area of alcohol preferring rats reduced ethanol reinforcement and consumption (161). This effect was long-lasting and was associated with an increase in 3α,5α-THP immunoreactivity in this brain area, suggesting that GABAergic neuroactive steroids may contribute to ethanol reinforcement.…”

Analytical challenges for measuring steroid responses to stress, neurodegeneration and injury in the central nervous system

“…Seven years after a first report, immunohistochemistry continues to be used to study the regulation of allopregnanolone levels in specific brain cells [47][48][49][50]. In these studies, the specificity of the affinity purified allopregnanolone antibody was determined by RIA prior to immunohistochemical analysis, which is inappropriate because RIA lack sufficient specificity.…”