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 - PubMed (original) (raw)

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

Jason B Cook et al. J Neurosci. 2014.

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 (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-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.

Keywords: P450scc; alcohol; allopregnanolone; neuroactive steroid; neurosteroid; ventral tegmental area.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.

The P450scc construct increases functional P450scc expression resulting in elevated pregnenolone levels. A, Model to enhance neuroactive steroid synthesis by overexpressing P450scc, which performs the limiting enzymatic reaction in steroidogenesis. B, P450scc construct consisting of the P450scc gene driven by the ubiquitous CBA promoter flanked by ITRs. C, Transient transfection of mouse (Ltk−) fibroblast cells increased P450scc by ∼10-fold (p < 0.001) 48 h post-transfection. Representative immunoblot probed for P450scc with β-actin used as a loading control: + indicates transfection with P450scc construct, − indicates control cells. D, Pregnenolone levels were increased in the cell media (p < 0.0001), measured by RIA; *p < 0.0001 compared with control.

Figure 2.

rAAV2–P450scc transduction in the NAc shell increases P450scc mRNA and protein expression dependent on the volume of virus infused. A, Representative photomicrograph (10×) of rAAV2-GFP infection efficiency in the NAc shell 1 week after 2 μl rAAV2-GFP infusion (green, GFP; red, Nissl stain). The red box indicates the location of the representative photomicrograph (+1.60 mm relative to bregma) within the NAc. B, The 4 μl infusion of rAAV2–P450scc significantly increased P450scc mRNA (p < 0.005) at 1 week postsurgery. Representative gel showing P450scc mRNA following 4 μl rAAV2–P450scc or rAAV2-GFP infusion in the NAc shell. β-Actin was used as a loading control. C, rAAV2–P450scc transduction increased protein levels of P450scc following both the 2 μl (p < 0.005) and 4 μl (p < 0.05) infusions at 1 week postsurgery. Representative immunoblot probed for P450scc following 4 μl rAAV2–P450scc or rAAV2-GFP infusion in the NAc shell. P450scc is the uppermost band that was increased following rAAV2–P450scc transduction. β-Actin was used as a loading control, but is not seen in the positive control (rat adrenal) due to the small amount of protein loaded (1 μg) compared with brain samples;*p < 0.05 compared with GFP control, **p < 0.005 compared with GFP control.

Figure 3.

rAAV2–P450scc transduction in the NAc increases P450scc mRNA but does not alter operant ethanol self-administration or cellular 3α,5α-THP. A, Representative photomicrograph (10×) of rAAV2-GFP infection efficiency in the NAc 1 week after 3 μl of rAAV2-GFP infusion (green, GFP; red, Nissl stain). The red box indicates the location of the representative photomicrograph (+1.60 mm relative to bregma) within the NAc. B, rAAV2–P450scc transduction in the NAc did not alter operant ethanol responding or water responding, compared with rAAV2-GFP controls. Ethanol responding over the 14 d of test sessions is collapsed in the bar graph. C, rAAV2–P450scc transduction in the NAc did not alter mean ethanol intake (g/kg) over the 14 d of test sessions, compared with rAAV2-GFP controls. D, rAAV2–P450scc transduction in the NAc did not alter total distance traveled (centimeters) in the open field test at 26 d following vector infusions. E, The infusion of rAAV2–P450scc (3 μl) significantly increased P450scc mRNA in the NAc at 4 weeks postsurgery. Representative gel showing P450scc mRNA level following 3 μl rAAV2–P450scc or rAAV2-GFP infusion in the NAc. β-Actin was used as a loading control. F, Infusion of rAAV2–P450scc (3 μl) in the NAc of Wistar rats did not alter 3α,5α-THP-positive cells in the NAc at 1 week postsurgery. G, Representative photomicrographs (10×) of cellular 3α,5α-THP immunoreactivity in the NAc 1 week following 3 μl rAAV2-GFP or rAAV2–P450scc infusion in the NAc. Baseline (BL) represents 1 week average of ethanol responding during the week before surgery; *p < 0.0001 compared with GFP control.

Figure 4.

rAAV2–P450scc transduction in the VTA produces long-term reductions in operant ethanol self-administration and increases 3α,5α-THP-positive cells. A, Representative photomicrograph (10×) of rAAV2-GFP infection efficiency in the VTA 1 week after 2 μl rAAV2-GFP infusion (green, GFP; red, Nissl stain). The red box indicates the location of the representative photomicrograph (−5.80 mm relative to bregma) within the VTA. B, rAAV2–P450scc transduction in the VTA reduced operant ethanol (p < 0.005) but not water responding over the 21 d of test sessions, compared with rAAV2-GFP controls. Mean ethanol responding over the 21 d of test sessions is collapsed in the bar graph. C, rAAV2–P450scc transduction in the VTA reduced mean ethanol intake (g/kg; p < 0.01) over the 21 d of test sessions, compared with rAAV2-GFP controls. D, rAAV2–P450scc transduction in the VTA did not alter total distance traveled (centimeters) in the open field test at 4 weeks following vector infusions. E, Infusion of rAAV2–P450scc (2 μl) in the VTA increased 3α,5α-THP-positive cells in the VTA (p < 0.005) at 4 weeks postsurgery. F, Representative photomicrographs (10×) of cellular 3α,5α-THP immunoreactivity in the VTA 4 weeks following 2 μl rAAV2-GFP or rAAV2–P450scc infusion in the VTA. Baseline (BL) represents 1 week average of ethanol responding during the week before surgery; *p < 0.01 and **p < 0.005 compared with control values.

Figure 5.

Localization of viral vector infusions was determined using GFAP immunofluorescence to visualize needle tracts. A, Representative image showing GFAP immunofluorescence (red) at the needle tract and GFP-positive cells (green) in the VTA 4 weeks following rAAV2-GFP infusion. The red box indicates the location of the representative image (−5.20 mm relative to bregma) within the VTA. B, Location within the VTA of rAAV2-GFP (green circles) or rAAV2–P450scc (black circles) infusions, relative to bregma.

Figure 6.

Confocal scanning microscopy revealed that 3α,5α-THP colocalizes with NeuN-positive neurons and TH-positive neurons, but not GFAP-positive astrocytes in the VTA of P rats. A, 3α,5α-THP (green) colocalizes with NeuN (red) in the VTA. B, 3α,5α-THP (green) does not colocalize with GFAP (red) in the VTA. C, 3α,5α-THP (green) colocalized with all TH (red)-positive cells examined in the VTA (294 cells). 3α,5α-THP is also located in TH-negative cells in the VTA (yellow arrows).

Figure 7.

Simplified schematic of potential mechanisms of rAAV2–P450scc transduction-induced effects on VTA neurons believed to regulate ethanol reinforcement and consumption or in which optical stimulation is rewarding. Transduction of TH+ neurons may reduce activity in cells that project to either the (A) NAc, mPFC, and/or the (B) LHb. Transduction of afferent projections from (C) PFC or PPTg/LDT may decrease activation of TH+ projection neurons. Transduction of (D) BNST afferent projections onto GABAergic neurons have the potential to influence rewarding and/or aversive behavior. Transduction of inhibitory interneurons in the VTA is not expected to have any effect since biosynthetic enzymatic machinery is absent in these cells, but (E) increased extracellular levels of 3α,5α-THP may act via GABAergic interneurons to increase activity of TH+ neurons.

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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 - PubMed (original) (raw)