Background The 5α-reduced pregnane neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a potent positive modulator of GABAA receptors capable of modulating neuronal activity. In rats, systemic ethanol administration increases cerebral cortical and hippocampal levels of 3α,5α-THP, but the effects of ethanol on 3α,5α-THP levels in other brain regions are unknown. There is a large body of evidence suggesting that 3α,5α-THP enhances ethanol sensitivity, contributes to some behavioral effects of ethanol, and modulates ethanol reinforcement and motivation to drink. In the present study, we used immunohistochemistry (IHC) to determine ethanol-induced changes in cellular 3α,5α-THP expression in brain regions associated with ethanol actions and responses. Methods Male Wistar rats were administered ethanol (2g/kg) or saline intraperitoneally and after 60 minutes transcardially perfused. IHC was performed on free floating sections (3–4 sections/animal/brain region) using an affinity purified anti-3α,5α-THP primary antibody and immunoreactivity was visualized with 3,3′-diaminobenzidine. Results Ethanol significantly increased 3α,5α-THP immunoreactivity by 24±6% in the medial prefrontal cortex, 32±12% in the hippocampal CA1 pyramidal cell layer, 52±5% in the polymorph cell layer of the dentate gyrus, 44±15% in the bed nucleus of the stria terminalis, and by 36±6% in the paraventricular nucleus of the hypothalamus. In contrast, ethanol administration significantly reduced 3α,5α-THP immunoreactivity by 25±5% in the nucleus accumbens “shore” and 21±3% in the central nucleus of the amygdala. No changes were observed in the ventral tegmental area, dorsomedial striatum, granule cell layer of the dentate gyrus, or the lateral and basolateral amygdala. Conclusions The results suggest acute ethanol (2g/kg) produces divergent, brain region specific, effects on cellular 3α,5α-THP levels. Regional differences in the effects of ethanol suggest there may be regional brain synthesis of 3α,5α-THP independent of the adrenal glands and novel mechanisms that reduce cellular 3α,5α-THP. Regional differences in ethanol-induced changes in 3α,5α-THP levels likely contribute to ethanol effects on neuronal function in brain.
Neuronal nicotinic acetylcholine receptors (nAChRs) are implicated in the reinforcing effects of many drugs of abuse, including ethanol. The present study examined the efficacy of cytisine, a nAChR partial agonist, and lobeline, a putative nAChR antagonist, on the maintenance of ethanol drinking by HAD-2 rats. Adult male HAD-2 rats were given access to ethanol (15% and 30%, with ad lib water and food) 22 hr per day for 12 weeks, beginning at 60 days old, after which cytisine (0.0, 0.5 and 1.5 mg/kg) was tested for 3 consecutive days. The rats were given an 18 day wash-out period, and were then tested with lobeline (0.0, 1.0 and 5.0 mg/kg) for 3 consecutive days. Ethanol intake was measured at 1, 4 and 22 hours post-injection. Rats were injected i.p. just prior to lights out (1200 h). There was a significant main effect of cytisine treatment on the 2nd test day, with the 1.5 mg/kg dose significantly reducing ethanol intake at the 1 hr and 4 hr time-points, relative to saline, and the 0.5 mg/kg dose inducing a significant reduction at the 4 hr time-point. Conversely, lobeline treatment resulted in significant main effects of treatment for all 3 time points, within each test day, with the 5.0 mg/kg dose significantly reducing ethanol intake, relative to saline, at each time-point within each test day. These findings provide further evidence that activity at the nAChR influences ethanol intake and is a promising target for pharmacotherapy development for the treatment of alcohol dependence and relapse.
Enduring memories of sensory cues associated with drug intake drive addiction. It is well known that stressful experiences increase addiction vulnerability. However, it is not clear how repeated stress promotes learning of cue-drug associations, as repeated stress generally impairs learning and memory processes unrelated to stressful experiences. Here, we show that repeated social defeat stress in rats causes persistent enhancement of long-term potentiation (LTP) of NMDA receptor-mediated glutamatergic transmission in the ventral tegmental area (VTA). Protein kinase A-dependent increase in the potency of inositol 1,4,5-triphosphate-induced Ca2+ signaling underlies LTP facilitation. Notably, defeated rats display enhanced learning of contextual cues paired with cocaine experience assessed using a conditioned place preference (CPP) paradigm. Enhancement of LTP in the VTA and cocaine CPP in behaving rats both require glucocorticoid receptor activation during defeat episodes. These findings suggest that enhanced glutamatergic plasticity in the VTA may contribute, at least partially, to increased addiction vulnerability following repeated stressful experiences.DOI: http://dx.doi.org/10.7554/eLife.15448.001
Background The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP, allopregnanolone) has been studied during withdrawal from ethanol in humans, rats and mice. Serum 3α,5α-THP levels decreased and brain levels were not altered following acute ethanol administration (2 g/kg) in male C57BL/6J mice, however the effects of chronic intermittent ethanol (CIE) exposure on 3α,5α-THP levels have not been examined. Given that CIE exposure changes subsequent voluntary ethanol drinking in a time-dependent fashion following repeated cycles of ethanol exposure, we conducted a time-course analysis of CIE effects on 3α,5α-THP levels in specific brain regions known to influence drinking behavior. Methods Adult male C57BL/6J mice were exposed to four cycles of CIE to induce ethanol dependence. All mice were sacrificed and perfused at one of two time points, 8 hr or 72 hr following the final exposure cycle. Free floating brain sections (40 μm; 3-5 sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3α,5α-THP. Results Withdrawal from CIE exposure produced time-dependent and region-specific effects on immunohistochemical detection of 3α,5α-THP levels across cortical and limbic brain regions. A transient reduction in 3α,5α-THP immunoreactivity was observed in the central nucleus of the amygdala 8 hr after withdrawal from CIE (-31.4 ± 9.3). Decreases in 3α,5α-THP immunoreactivity were observed 72 hr following withdrawal in the medial prefrontal cortex (−25.0 ± 9.3%), nucleus accumbens core (−29.9 ± 6.6%), and dorsolateral striatum (−18.5 ± 6.0%), while an increase was observed in the CA3 pyramidal cell layer of the hippocampus (+42.8 ± 19.5%). Sustained reductions in 3α,5α-THP immunoreactivity were observed at both time points in the lateral amygdala (8 hr −28.3 ± 12.8%; 72 hr −27.5 ± 12.4%) and in the ventral tegmental area (8 hr −26.5 ± 9.9%; 72 hr −31.6 ± 13.8%). Conclusions These data suggest that specific neuroadaptations in 3α,5α-THP levels may be present in regions of brain that mediate anxiety, stress and reinforcement relevant to ethanol dependence. The changes that occur at different time points likely modulate neurocircuitry involved in ethanol withdrawal as well as the elevated drinking observed after CIE exposure.
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.
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