Selected Line Difference in the Effects of Ethanol Dependence and Withdrawal on Allopregnanolone Levels and 5α-Reductase Enzyme Activity and Expression

Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR 97239, USA.
Alcoholism Clinical and Experimental Research (Impact Factor: 3.21). 10/2009; 33(12):2077-87. DOI: 10.1111/j.1530-0277.2009.01047.x
Source: PubMed


Allopregnanolone (ALLO) is a progesterone derivative that rapidly potentiates gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated inhibition and modulates symptoms of ethanol withdrawal. Because clinical and preclinical data indicate that ALLO levels are inversely related to symptoms of withdrawal, the present studies determined whether ethanol dependence and withdrawal differentially altered plasma and cortical ALLO levels in mice selectively bred for differences in ethanol withdrawal severity and determined whether the alterations in ALLO levels corresponded to a concomitant change in activity and expression of the biosynthetic enzyme 5alpha-reductase.
Male Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice were exposed to 72 hours ethanol vapor or air and euthanized at select times following removal from the inhalation chambers. Blood was collected for analysis of ALLO and corticosterone levels by radioimmunoassay. Dissected amygdala, hippocampus, midbrain, and cortex as well as adrenals were examined for 5alpha-reductase enzyme activity and expression levels.
Plasma ALLO was decreased significantly only in WSP mice, and this corresponded to a decrease in adrenal 5alpha-reductase expression. Cortical ALLO was decreased up to 54% in WSP mice and up to 46% in WSR mice, with a similar decrease in cortical 5alpha-reductase activity during withdrawal in the lines. While cortical gene expression was significantly decreased during withdrawal in WSP mice, there was a 4-fold increase in expression in the WSR line during withdrawal. Hippocampal 5alpha-reductase activity and gene expression was decreased only in dependent WSP mice.
These results suggest that there are line and brain regional differences in the regulation of the neurosteroid biosynthetic enzyme 5alpha-reductase during ethanol dependence and withdrawal. In conjunction with the finding that WSP mice exhibit reduced sensitivity to ALLO during withdrawal, the present results are consistent with the hypothesis that genetic differences in ethanol withdrawal severity are due, in part, to modulatory effects of GABAergic neurosteroids such as ALLO.

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    • "We suspected that the air exposure in the present study produced a greater activation of the hypothalamic-pituitary-adrenal (HPA) axis than that in our prior work, which was confirmed by our analysis of plasma CORT concentrations. In the present study, basal CORT levels were ~26 μg/dl; these values are over 5-fold higher than those in our prior work (<5 μg/dl for both WSP and WSR mice; Tanchuck et al. 2009) and are associated with a moderate degree of stress. Ethanol withdrawal did produce a further activation of the HPA axis in the present study, measured by the 100 % increase in plasma CORT concentrations, but this increase was much lower than what we observed in our prior work (~6-fold increase in both WSP and WSR mice; Tanchuck et al. 2009). "
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    ABSTRACT: Rationale The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acidA receptors (GABAARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal. Objectives Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABAAR sensitivity to ALLO in Withdrawal Seizure-Prone (WSP) but not in Withdrawal Seizure-Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABAAR-active steroids is not known. Methods After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of ten neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. Results We quantified levels of nine neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of five neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed five neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. Conclusions Due to lower basal levels of some GABAAR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABAAR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABAAR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.
    Psychopharmacology 05/2014; 231(17). DOI:10.1007/s00213-014-3618-y · 3.88 Impact Factor
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    • "Although ALLO levels were not measured in the present studies, we believe that it is unlikely that the brain regional differences were due to a differential effectiveness of FIN to alter ALLO biosynthesis. Recent study has found that EtOH withdrawal did not significantly decrease midbrain 5a-reduc- tase levels in WSP mice, using both quantitative reverse transcription polymerase chain reaction (Tanchuck et al., 2009) and in situ hybridization (Roselli et al., 2011) techniques, suggesting that FIN would be equally effective at inhibiting 5a-reductase activity in air-and EtOH-exposed mice in both brain regions. Also, other studies show that infusions of 10 lg FIN into the VTA or hippocampus reduced ALLO levels by 60 to 80% (Frye and Vongher, 2001; Rhodes and Frye, 2005a,b). "
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    ABSTRACT: Background: Allopregnanolone (ALLO) is a potent positive modulator of γ-aminobutyric acidA receptors (GABAA Rs) that affects ethanol (EtOH) withdrawal. Finasteride (FIN), a 5α-reductase inhibitor that blocks the formation of ALLO and other GABAergic neurosteroids, alters EtOH sensitivity. Recently, we found that Withdrawal Seizure-Prone mice from the first genetic replicate (WSP-1) exhibited behavioral tolerance to the anticonvulsant effect of intrahippocampal ALLO during EtOH withdrawal and that intrahippocampal FIN significantly increased EtOH withdrawal severity. The purpose of this study was to determine whether neurosteroid manipulations in the substantia nigra reticulata (SNR) and ventral tegmental area (VTA) produced effects during EtOH withdrawal comparable to those seen with intrahippocampal ALLO and FIN. Methods: Male WSP-1 mice were surgically implanted with bilateral guide cannulae aimed at the SNR or VTA at 2 weeks prior to EtOH vapor or air exposure for 72 hours. Initial studies examined the anticonvulsant effect of a single ALLO infusion (0, 100, or 400 ng/side) at a time corresponding to peak withdrawal in the air- and EtOH-exposed mice. Separate studies examined the effect of 4 FIN infusions (0 or 10 μg/side/d) during the development of physical dependence on the expression of EtOH withdrawal. Results: ALLO infusion exerted a potent anticonvulsant effect in EtOH-naïve mice, but a diminished anticonvulsant effect during EtOH withdrawal. Administration of FIN into the SNR exerted a delayed proconvulsant effect in EtOH-naïve mice, whereas infusion into the VTA increased EtOH withdrawal duration. Conclusions: Activation of local GABAA Rs in the SNR and VTA via ALLO infusion is sufficient to exert an anticonvulsant effect in naïve mice and to produce behavioral tolerance to the anticonvulsant effect of ALLO infusion during EtOH withdrawal. Thus, EtOH withdrawal reduced sensitivity of GABAA Rs to GABAergic neurosteroids in 2 neuroanatomical substrates within the basal ganglia in WSP-1 male mice.
    Alcoholism Clinical and Experimental Research 12/2012; 37(5). DOI:10.1111/acer.12027 · 3.21 Impact Factor
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    • "In a subsequent study (Bergeson et al., 2003), an epistatic interaction between the chromosomal 13 region where Srd5a1 was located and a region on chromosome 11 that contained several genes for GABA A receptor subunits (α1, α6, γ2) suggested that an interaction between Srd5a1 and GABA A receptor subunit genes might occur. Consistent with this idea, ethanol withdrawal significantly reduced endogenous ALLO levels (Tanchuck et al., 2009) as well as the sensitivity of GABA A receptors to ALLO, measured biochemically or behaviorally following systemic or microinjection (Finn et al., 2006; Gililland-Kaufman et al., 2008), in WSP mice. Thus, it was possible that the modulatory effects of ALLO on ethanol withdrawal severity in WSP mice reflected a balance between local concentration of ALLO at GABA A receptors and the concomitant change in sensitivity of the GABA A receptors to ALLO that were occurring during ethanol withdrawal. "
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    ABSTRACT: Several lines of evidence suggest that fluctuations in endogenous levels of the γ-aminobutyric acid (GABA)ergic neurosteroid allopregnanolone (ALLO) represent one mechanism for regulation of GABAergic inhibitory tone in the brain, with an ultimate impact on behavior. Consistent with this idea, there was an inverse relationship between ALLO levels and symptoms of anxiety and depression in humans and convulsive activity in rodents during alcohol withdrawal. Our recent studies examined the activity and expression of 5α-reductase (Srd5a1), the rate-limiting enzyme in the biosynthesis of ALLO, during alcohol withdrawal in mice selectively bred for high chronic alcohol withdrawal (Withdrawal Seizure-Prone [WSP]) and found that Srd5a1 was downregulated in the cortex and hippocampus over the time course of dependence and withdrawal. The purpose of the present studies was to extend these findings and more discretely map the regions of Srd5a1 expression in mouse brain using radioactive in situ hybridization in WSP mice that were ethanol naïve, following exposure to 72h ethanol vapor (dependent) or during peak withdrawal. In naïve animals, expression of Srd5a1 was widely distributed throughout the mouse brain, with highest expression in specific regions of the cerebral cortex, hippocampus, thalamus, hypothalamus, and amygdala. In dependent animals and during withdrawal, there was no change in Srd5a1 expression in cortex or hippocampus, which differed from our recent findings in dissected tissues. These results suggest that local Srd5a1 mRNA expression in WSP brain may not change in parallel with local ALLO content or withdrawal severity.
    Alcohol (Fayetteville, N.Y.) 09/2011; 45(8):763-72. DOI:10.1016/j.alcohol.2011.08.002 · 2.01 Impact Factor
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