Ethanol alters local cellular levels of (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) independent of the adrenals in subcortical brain regions - PubMed (original) (raw)

Ethanol alters local cellular levels of (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) independent of the adrenals in subcortical brain regions

Jason B Cook et al. Neuropsychopharmacology. 2014 Jul.

Abstract

The neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a positive modulator of GABAA receptors synthesized in the brain, adrenal glands, and gonads. In rats, ethanol activates the hypothalamic-pituitary-adrenal axis and elevates 3α,5α-THP in plasma, cerebral cortex, and hippocampus. In vivo, these effects are dependent on both the pituitary and adrenal glands. In vitro, however, ethanol locally increases 3α,5α-THP in hippocampal slices, in the absence of adrenal influence. Therefore, it is not known whether ethanol can change local brain levels of 3α,5α-THP in vivo, independent of the adrenals. To directly address this controversy, we administered ethanol (2 g/kg) or saline to rats that underwent adrenalectomy (ADX) or received sham surgery and performed immunohistochemistry for 3α,5α-THP. In the medial prefrontal cortex (mPFC), ethanol increased 3α,5α-THP after sham surgery, compared with saline controls, with no ethanol-induced change in 3α,5α-THP following ADX. In subcortical regions, 3α,5α-THP was increased independent of adrenals in the CA1 pyramidal cell layer, dentate gyrus polymorphic layer, bed nucleus of the stria terminalis, and paraventricular nucleus of the hypothalamus. Furthermore, ethanol decreased 3α,5α-THP labeling in the nucleus accumbens shore and central nucleus of the amygdala, independent of the adrenal glands. These data indicate that ethanol dynamically regulates local 3α,5α-THP levels in several subcortical regions; however, the adrenal glands contribute to 3α,5α-THP elevations in the mPFC. Using double immunofluorescent labeling we determined that adrenal dependence of 3α,5α-THP induction by ethanol is not due to a lack of colocalization of 3α,5α-THP with the cholesterol transporters steroidogenic acute regulatory protein (StAR) or translocator protein (TSPO).

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Figures

Figure 1

Ethanol (2 g/kg, i.p.) effects on (3α,5α)-3-hydroxypregnan-20-one (3_α_,5_α_-THP) immunoreactivity in the mPFC of rats are dependent on the adrenal glands. (a) Ethanol increases 3_α_,5_α_-THP immunoreactivity in the mPFC following sham surgery, but not ADX, compared with the respective saline controls. (b) The red box indicates the location (3.20 mm relative to bregma) of representative photomicrographs. (c) Representative photomicrographs ( × 10) of 3_α_,5_α_-THP immunoreactivity in the mPFC of sham saline (_n_=8), sham ethanol (_n_=9), ADX saline (_n_=10), and ADX ethanol (_n_=11) animals. Ethanol or saline was administered 60 min before tissue fixation and collection. Data are expressed as the mean positive pixels/mm2±SEM. *P<0.01 compared with respective saline administration. ADX, adrenalectomy; mPFC, medial prefrontal cortex; Sham, sham adrenalectomy surgery.

Figure 2

ADX does not alter the effect of ethanol (2 g/kg, i.p.) on (3α,5α)-3-hydroxypregnan-20-one (3_α_,5_α_-THP) immunoreactivity in the CA1 hippocampus and polymorphic cell layer of the DG. (a) Ethanol increases 3_α_,5_α_-THP immunoreactivity in the CA1 pyramidal cell layer after sham surgery and ADX, compared with respective saline controls. (b) The red box indicates the location (−3.14 mm relative to bregma) of representative photomicrographs within the hippocampus. (c) Representative photomicrographs ( × 10) of 3_α_,5_α_-THP immunoreactivity in the CA1 pyramidal cell layer of sham saline (_n_=8), sham ethanol (_n_=9), ADX saline (n_=10), and ADX ethanol (n_=11) animals. (d) Ethanol increases 3_α,5_α_-THP immunoreactivity in the polymorphic cell layer of the DG following sham surgery or ADX, when compared with respective saline controls. (e) The red box indicates the location (−3.60 mm relative to bregma) of representative photomicrographs within the DG. (f) Representative photomicrographs ( × 10) of 3_α,5_α_-THP immunoreactivity in the polymorphic cell layer of the DG (oval). Ethanol or saline were administered 60 min before tissue fixation and collection. Data are expressed as the mean positive pixels/mm2±SEM. * indicates the main effect of ethanol treatment, P<0.0001. ADX, adrenalectomy; CA1, Cornu Ammonis area 1; DG, dentate gyrus; Sham, sham adrenalectomy surgery.

Figure 3

ADX does not alter the effect of ethanol (2 g/kg, i.p.) on (3α,5α)-3-hydroxypregnan-20-one (3_α_,5_α_-THP) immunoreactivity in the BNST and PVN of the hypothalamus. (a) Ethanol increases 3_α_,5_α_-THP immunoreactivity in the BNST after sham surgery and ADX, when compared with respective saline controls. (b) The red box indicates the location (−0.26 mm relative to bregma) of representative photomicrographs within the BNST. (c) Representative photomicrographs ( × 10) of 3_α_,5_α_-THP immunoreactivity in the BNST of sham saline (_n_=8), sham ethanol (_n_=9), ADX saline (n_=10), and ADX ethanol (n_=11) animals. (d) Ethanol increases 3_α,5_α_-THP immunoreactivity in the PVN of the hypothalamus after sham surgery and ADX, when compared with respective saline controls. (e) The red box indicates the location (−1.80 mm relative to bregma) of representative photomicrographs within the PVN. (f) Representative photomicrographs ( × 10) of 3_α,5_α_-THP immunoreactivity in the PVN. Ethanol or saline were administered 60 min before tissue fixation and collection. Data are expressed as the mean positive pixels/mm2±SEM. * indicates the main effect of ethanol treatment, P<0.0001. ADX, adrenalectomy; BNST, bed nucleus of the stria terminalis; PVN, paraventricular nucleus; Sham, sham adrenalectomy surgery.

Figure 4

ADX does not alter the effect of ethanol (2 g/kg, i.p.) on (3α,5α)-3-hydroxypregnan-20-one (3_α_,5_α_-THP) immunoreactivity in the NAc shore (core/shell border) or CeA. (a) Ethanol decreases 3_α_,5_α_-THP immunoreactivity in the NAc shore after sham surgery and ADX, when compared with respective saline controls. (b) The red box indicates the location (1.60 mm relative to bregma) of representative photomicrographs. (c) Representative photomicrographs ( × 10) of 3_α_,5_α_-THP immunoreactivity in the NAc shore of sham saline (_n_=8), sham ethanol (_n_=9), ADX saline (n_=10), and ADX ethanol (n_=11) animals. (d) Ethanol decreases 3_α,5_α_-THP immunoreactivity in the CeA after sham surgery and ADX, when compared with respective saline controls. (e) The red box indicates the location (−2.56 mm relative to bregma) of representative photomicrographs. (f) Representative photomicrographs ( × 10) of 3_α,5_α_-THP immunoreactivity in the CeA of sham saline (_n_=8), sham ethanol (_n_=9), ADX saline (_n_=10), and ADX ethanol (_n_=11) animals. Ethanol or saline were administered 60 min before tissue fixation and collection. Data are expressed as the mean positive pixels/mm2±SEM. * indicates the main effect of ethanol treatment P<0.001, and ** indicates the main effect of ethanol treatment, P<0.0001 compared with respective saline administration. ADX, adrenalectomy; CeA, central nucleus of the amygdala; NAc, nucleus accumbens; Sham, sham adrenalectomy surgery.

Figure 5

(3α,5α)-3-hydroxypregnan-20-one (3_α_,5_α_-THP) colocalizes with StAR in the mPFC, CA1 pyramidal cells of the hippocampus, and NAc, whereas 3_α_,5_α_-THP colocalization with TSPO is observed in mPFC and CA1 hippocampus, but is minimal in NAc. In the CA1 hippocampus and NAc, TSPO is also located in cells that are not 3_α_,5_α_-THP-labeled. (a) In the mPFC, 3_α_,5_α_-THP (green) colocalizes with StAR (red, top panel) and TSPO (red, bottom panel). (b) In the CA1 hippocampus, 3_α_,5_α_-THP (green) colocalizes with StAR (red, top panel) and TSPO (red, bottom panel). Note the presence of TSPO labeling in a cell that is not labeled for 3_α_,5_α_-THP (yellow arrow). (c) In the NAc, 3_α_,5_α_-THP (green) colocalizes with StAR (red, top panel), but colocalization with TSPO is rarely observed (red, bottom panel). Note the presence of TSPO labeling in cells that are not labeled for 3_α_,5_α_-THP (yellow arrows). CA1, Cornu Ammonis area 1; mPFC, medial prefrontal cortex; NAc, nucleus accumbens; StAR, steroidogenic acute regulator protein; TSPO translocator protein.

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Ethanol alters local cellular levels of (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) independent of the adrenals in subcortical brain regions - PubMed (original) (raw)