Divergent Population-Specific Effects of Chronic Ethanol Exposure on Excitability and Inhibitory Transmission in Male and Female Rat Central Amygdala.

The central nucleus of the amygdala (CeA) is implicated in alcohol use disorder (AUD) and AUD-associated plasticity. The CeA is a primarily GABAergic nucleus that is subdivided into lateral and medial compartments with genetically diverse subpopulations. GABAA receptors are heteromeric pentamers with subunits conferring distinct physiological characteristics. GABAA receptor signaling in the CeA has been implicated in ethanol-associated plasticity; however, population-specific changes in inhibitory signaling and subunit expression remain unclear. Here, we combined electrophysiology with single-cell gene expression analysis of population markers and GABAA receptor subunits to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure. We found that chronic ethanol exposure and withdrawal produced global changes in GABAA receptor subunit expression at the transcript and protein levels, increased excitability in female CeA neurons, and increased inhibitory synaptic transmission in male CeA neurons. When we examined CeA neurons at the single-cell level we found heterogenous populations, as previously reported. We observed ethanol-induced increases in excitability only in somatostatin neurons in the CeA of females, decreases in excitability only in the protein kinase C delta (PKCd) population in males, and ethanol-induced increases in inhibitory transmission in male PKCd and calbindin 2-expressing CeA neurons. There were no population-specific differences in GABAA receptor (Gabr) subunits in males but reduced GabrA5 expression in female somatostatin neurons. Collectively, these findings suggest that defined CeA populations display differential ethanol sensitivity in males and females, which may play a role in sex differences in vulnerability to AUD or expression of AUD pathology.SIGNIFICANCE STATEMENT The CeA is involved in the effects of ethanol in the brain; however, the population-specific changes in CeA activity remain unclear. We used recordings of CeA neuronal activity and single-cell gene expression to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure and found sex- and population-specific effects of chronic ethanol exposure and withdrawal. Specifically, female CeA neurons displayed increased excitability in the somatostatin CeA population, whereas male CeA neurons displayed increased inhibitory control in both PKCd and calbindin populations and decreased excitability in the PKCd population. These findings identify CeA populations that display differential sensitivity to ethanol exposure, which may contribute to sex differences in vulnerability to alcohol use disorder.

Chronic Ethanol Exposure and Withdrawal Impair Synaptic GABAA Receptor-Mediated Neurotransmission in Deep-Layer Prefrontal Cortex.

BACKGROUND: The prefrontal cortex (PFC) acts as an integrative hub for the processing of cortical and subcortical input into meaningful efferent signaling, permitting complex associative behaviors. PFC dysfunction is consistently observed with ethanol (EtOH) dependence and is a core component of the pathology of alcohol use disorders in current models of addiction. While intracortical gamma-aminobutryric acid (GABA)ergic neurotransmission is understood to be essential for maintaining coordinated network activity within the cortex, relatively little is known regarding functional GABAergic adaptations in PFC during EtOH dependence. METHODS: In the present study, male and female (> postnatal day 60) Sprague-Dawley rats were administered EtOH (5.0 g/kg; intragastric gavage) for 14 to 15 consecutive days. Twenty-four hours after the final administration, animals were sacrificed and brains extracted for electrophysiological recordings of isolated GABAA receptor-mediated currents or analysis of GABAA receptor subunit protein expression in deep-layer PFC neurons. RESULTS: Chronic EtOH exposure significantly attenuated activity-dependent spontaneous GABAA receptor-mediated inhibitory postsynaptic current (IPSC) frequency with no effect on amplitude. Furthermore, analysis of IPSC decay kinetics revealed a significant enhancement of IPSC decay time that was associated with decrements in expression of the α1 GABAA receptor subunit, indicative of further impaired phasic inhibition. These phenomena occurred irrespective of neuron projection destination and sex. Based on previous observations by our laboratory of an epigenetic mechanism for EtOH-induced changes in cortical GABAA receptor subunit expression, the histone deacetylase inhibitor Trichostatin A was administered to water- and EtOH-exposed animals, and prevented EtOH-induced changes in spontaneous IPSC frequency, IPSC decay kinetics, and GABAA receptor subunit expression. CONCLUSIONS: Taken together, these results demonstrate that chronic EtOH exposure impairs synaptic inhibitory neurotransmission in deep-layer pyramidal neurons of the medial PFC in both male and female rats. These maladaptations occur in neurons projecting to numerous regions implicated in the sequelae of EtOH dependence, offering a mechanistic link between the manifestation of PFC dysfunction and negative affective states observed with extended consumption.

Cellular GABAergic Neuroactive Steroid (3α,5α)-3-Hydroxy-Pregnan-20-One (3α,5α-THP) Immunostaining Levels Are Increased in the Ventral Tegmental Area of Human Alcohol Use Disorder Patients: A Postmortem Study.

BACKGROUND: The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP; allopregnanolone) enhances GABAergic activity and produces subjective effects similar to ethanol (EtOH). The effect of chronic alcohol exposure on 3α,5α-THP concentrations has been studied in mouse, rat, and monkey limbic brain areas. Chronic EtOH exposure produced divergent brain region and cell-specific changes in 3α,5α-THP concentrations in animal studies. However, 3α,5α-THP levels in similar human brain regions have never been examined in individuals diagnosed with alcohol use disorder (AUD). Therefore, we used immunohistochemistry (IHC) to examine 3α,5α-THP levels in the ventral tegmental area (VTA), substantia nigra pars medialis (SNM), and amygdala of human postmortem brains of patients diagnosed with AUD compared with social drinkers. The effects of sex and liver disease on 3α,5α-THP concentrations were examined in the aforementioned brain regions. METHODS: Human postmortem brains of AUD patients and age-matched controls were obtained from the New South Wales Brain Tissue Resource Center. IHC was performed using anti-3α,5α-THP antibody on formalin-fixed paraffin-embedded brain sections to detect cellular 3α,5α-THP levels. Immunoreactivity was analyzed by pixel density/mm2 for the comparison between AUD patients and controls. RESULTS: 3α,5α-THP immunoreactivity was increased by 23.2 ± 9% in the VTA of AUD patients compared with age-matched controls (p = 0.014). Moreover, a 29.6 ± 10% increase in 3α,5α-THP immunoreactivity was observed in the SNM of male AUD patients compared with male controls (p < 0.01), but not in female subjects. 3α,5α-THP immunoreactivity in the VTA and SNM regions did not differ between noncirrhotic and cirrhotic AUD patients. A sex difference in 3α,5α-THP immunoreactivity (female 51 ± 18% greater than male) was observed among control subjects in the SNM, but no other brain region. 3α,5α-THP immunoreactivity in the basolateral amygdala and lateral amygdala was negatively correlated with the length of the tissue fixation time as well as the age of the subjects, precluding assessment of the effect of AUD. CONCLUSIONS: Cellular 3α,5α-THP levels in VTA are increased in human AUD patients, an effect that is likely independent of sex and liver disease. The differences between animal models and human studies should be factored into the interpretation of the physiological significance of elevated 3α,5α-THP levels in humans.

Voluntary ethanol consumption reduces GABAergic neuroactive steroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP) in the amygdala of the cynomolgus monkey.

Neuroactive steroids such as (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) enhance the gamma-aminobutyric acid (GABA)-ergic effects of ethanol and modulate excessive drinking in rodents. Moreover, chronic ethanol consumption reduces 3α,5α-THP levels in human plasma, rat hippocampus and mouse limbic regions. We explored the relationship between 3α,5α-THP levels in limbic brain areas and voluntary ethanol consumption in the cynomolgus monkey following daily self-administration of ethanol for 12 months and further examined the relationship to hypothalamic-pituitary-adrenal (HPA) axis function prior to ethanol exposure. Monkeys were subjected to scheduled induction of ethanol consumption followed by free access to ethanol or water for 22 h/day over 12 months. Immunohistochemistry was performed using an anti-3α,5α-THP antibody. Prolonged voluntary drinking resulted in individual differences in ethanol consumption that ranged from 1.2 to 4.2 g/kg/day over 12 months. Prolonged ethanol consumption reduced cellular 3α,5α-THP immunoreactivity by 13 ± 2 percent (P < 0.05) in the lateral amygdala and 17 ± 2 percent (P < 0.05) in the basolateral amygdala. The effect of ethanol was most pronounced in heavy drinkers that consumed ≥3 g/kg ≥ 20 percent of days. Consequently, 3α,5α-THP immunoreactivity in both the lateral and basolateral amygdala was inversely correlated with average daily ethanol intake (Spearman r = -0.87 and -0.72, respectively, P < 0.05). However, no effect of ethanol and no correlation between drinking and 3α,5α-THP immunoreactivity were observed in the basomedial amygdala. 3α,5α-THP immunoreactivity following ethanol exposure was also correlated with HPA axis function prior to ethanol exposure. These data indicate that voluntary ethanol drinking reduces amygdala levels of 3α,5α-THP in non-human primates and that amygdala 3α,5α-THP levels may be linked to HPA axis function.

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

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.

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.

Altered GABAA receptor expression and seizure threshold following acute ethanol challenge in mice lacking the RIIß subunit of PKA.

Ethanol causes pathological changes in GABAA receptor trafficking and function. These changes are mediated in part by ethanol activation of protein kinase A (PKA). The current study investigated the expression of the GABAA α1 and α4 subunits and the kinase anchoring protein AKAP150, as well as bicuculline-induced seizure threshold, at baseline and following acute injection of ethanol (3.5 g/kg IP) in a mouse line lacking the regulatory RIIß subunit of PKA. Whole cerebral cortices were harvested at baseline, 1 h, or 46 h following injection of ethanol or saline and subjected to fractionation and western blot analysis. Knockout (RIIß-/-) mice had similar baseline levels of PKA RIIα and GABAA α1 and α4 subunits compared to wild type (RIIß+/+) littermates, but had deficits in AKAP150. GABAA α1 subunit levels were decreased in the P2 fraction of RIIß-/-, but not RIIß+/+, mice following 1 h ethanol, an effect that was driven by decreased α1 expression in the synaptic fraction. GABAA α4 subunits in the P2 fraction were not affected by 1 h ethanol; however, synaptic α4 subunit expression was increased in RIIß+/+, but not RIIß-/- mice, while extrasynaptic α4 and δ subunit expression were decreased in RIIß-/-, but not RIIß+/+ mice. Finally, RIIß knockout was protective against bicuculline-induced seizure susceptibility. Overall, the results suggest that PKA has differential roles in regulating GABAA receptor subunits. PKA may protect against ethanol-induced deficits in synaptic α1 and extrasynaptic α4 receptors, but may facilitate the increase of synaptic α4 receptors.

Ethanol administration produces divergent changes in GABAergic neuroactive steroid immunohistochemistry in the rat brain.

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 (EtOH) administration increases cerebral cortical and hippocampal levels of 3α,5α-THP, but the effects of EtOH on 3α,5α-THP levels in other brain regions are unknown. There is a large body of evidence suggesting that 3α,5α-THP enhances EtOH sensitivity, contributes to some behavioral effects of EtOH, and modulates EtOH reinforcement and motivation to drink. In this study, we used immunohistochemistry (IHC) to determine EtOH-induced changes in cellular 3α,5α-THP expression in brain regions associated with EtOH actions and responses. METHODS: Male Wistar rats were administered EtOH (2 g/kg) or saline intraperitoneally and after 60 minutes transcardially perfused. IHC was performed on free-floating sections (3 to 4 sections/animal/brain region) using an affinity purified anti-3α,5α-THP primary antibody, and immunoreactivity was visualized with 3,3'-diaminobenzidine. RESULTS: EtOH significantly increased 3α,5α-THP immunoreactivity by 24 ± 6% in the medial prefrontal cortex, 32 ± 12% in the hippocampal Cornu Ammonis area 1 (CA1) pyramidal cell layer, 52 ± 5% in the polymorph cell layer of the dentate gyrus (DG), 44 ± 15% in the bed nucleus of the stria terminalis, and 36 ± 6% in the paraventricular nucleus of the hypothalamus. In contrast, EtOH 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 DG, or the lateral and basolateral amygdala. CONCLUSIONS: The results suggest acute EtOH (2 g/kg) produces divergent, brain region specific, effects on cellular 3α,5α-THP levels. Regional differences in the effects of EtOH 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 EtOH-induced changes in 3α,5α-THP levels likely contribute to EtOH effects on neuronal function in brain.

Chronic intermittent ethanol exposure and withdrawal alters (3α,5α)-3-hydroxy-pregnan-20-one immunostaining in cortical and limbic brain regions of C57BL/6J mice.

BACKGROUND: The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP; allopregnanolone) has been studied during withdrawal from ethanol (EtOH) in humans, rats, and mice. Serum 3α,5α-THP levels decreased, and brain levels were not altered following acute EtOH 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 EtOH drinking in a time-dependent fashion following repeated cycles of EtOH 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 4 cycles of CIE to induce EtOH dependence. All mice were sacrificed and perfused at 1 of 2 time points, 8 or 72 hours following the final exposure cycle. Free-floating brain sections (40 µm; 3 to 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 hours after withdrawal from CIE (-31.4 ± 9.3%). Decreases in 3α,5α-THP immunoreactivity were observed 72 hours 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 hours -28.3 ± 12.8%; 72 hours -27.5 ± 12.4%) and in the ventral tegmental area (8 hours -26.5 ± 9.9%; 72 hours -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 EtOH dependence. The changes that occur at different time points likely modulate neurocircuitry involved in EtOH withdrawal as well as the elevated drinking observed after CIE exposure.

Acute, but not repeated, cocaine exposure alters allopregnanolone levels in the midbrain of male and female rats.

RATIONALE: Multiple psychiatric disorders are associated with altered brain and serum levels of neuroactive steroids, including the endogenous GABAergic steroid, allopregnanolone. Clinically, chronic cocaine use was correlated with decreased levels of pregnenolone. Preclinically, the effect of acute cocaine on allopregnanolone levels in rodents has had mixed results, showing an increase or no change in allopregnanolone levels in some brain regions. OBJECTIVE: We hypothesized that cocaine acutely increases allopregnanolone levels, but repeated cocaine exposure decreases allopregnanolone levels compared to controls. METHODS: We performed two separate studies to determine how systemic administration of 15 mg/kg cocaine (1) acutely or (2) chronically alters brain (olfactory bulb, frontal cortex, dorsal striatum, and midbrain) and serum allopregnanolone levels in adult male and female Sprague-Dawley rats. RESULTS: Cocaine acutely increased allopregnanolone levels in the midbrain, but not in olfactory bulb, frontal cortex, or dorsal striatum. Repeated cocaine did not persistently (24 h later) alter allopregnanolone levels in any region in either sex. However, allopregnanolone levels varied by sex across brain regions. In the acute study, we found that females had significantly higher allopregnanolone levels in serum and olfactory bulb relative to males. In the repeated cocaine study, females had significantly higher allopregnanolone levels in olfactory bulb, frontal cortex, and serum. Finally, acute cocaine increased allopregnanolone levels in the frontal cortex of females in proestrus, relative to non-proestrus stages. CONCLUSION: Collectively these results suggest that allopregnanolone levels vary across brain regions and by sex, which may play a part in differential responses to cocaine by sex.

Neurosteroid [3α,5α]-3-hydroxy-pregnan-20-one enhances IL-10 production via endosomal TRIF-dependent TLR4 signaling pathway.

Background: Previous studies demonstrated the inhibitory effect of allopregnanolone (3α,5α-THP) on the activation of inflammatory toll-like receptor 4 (TLR4) signals in RAW264.7 macrophages and the brains of selectively bred alcohol-preferring (P) rats. In the current study, we investigated the impact of 3α,5α-THP on the levels of IL-10 and activation of the TRIF-dependent endosomal TLR4 pathway. Methods: The amygdala and nucleus accumbens (NAc) of P rats, which exhibit innately activated TLR4 pathways as well as RAW264.7 cells, were used. Enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were used to ascertain the effects of 3α,5α-THP on the TRIF-dependent endosomal TLR4 pathway and endosomes were isolated to examine translocation of TLR4 and TRIF. Additionally, we investigated the effects of 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) on the levels of IL-10 in RAW264.7 macrophages. Finally, we examined whether inhibiting TRIF (using TRIF siRNA) in RAW264.7 cells altered the levels of IL-10. Results: 3α,5α-THP administration facilitated activation of the endosomal TRIF-dependent TLR4 pathway in males, but not female P rats. 3α,5α-THP increased IL-10 levels (+13.2 ± 6.5%) and BDNF levels (+21.1 ± 11.5%) in the male amygdala. These effects were associated with increases in pTRAM (+86.4 ± 28.4%), SP1 (+122.2 ± 74.9%), and PI(3)K-p110δ (+61.6 ± 21.6%), and a reduction of TIRAP (-13.7 ± 6.0%), indicating the activation of the endosomal TRIF-dependent TLR4 signaling pathway. Comparable effects were observed in NAc of these animals. Furthermore, 3α,5α-THP enhanced the accumulation of TLR4 (+43.9 ± 11.3%) and TRIF (+64.8 ± 32.8%) in endosomes, with no significant effect on TLR3 accumulation. Additionally, 3α,5α-THP facilitated the transition from early endosomes to late endosomes (increasing Rab7 levels: +35.8 ± 18.4%). In RAW264.7 cells, imiquimod (30 µg/mL) reduced IL-10 while 3α,5α-THP and 3α,5α-THDOC (0.1, 0.3, and 1.0 µM) restored IL-10 levels. To determine the role of the TRIF-dependent TLR4 signaling pathway in IL-10 production, the downregulation of TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%). TRIF (-62.9 ± 28.2%) in RAW264.7 cells led to a reduction in IL-10 levels (-42.3 ± 8.4%) and 3α,5α-THP (1.0 µM) no longer restored the reduced IL-10 levels. Conclusion: The results demonstrate 3α,5α-THP enhancement of the endosomal TLR4-TRIF anti-inflammatory signals and elevations of IL-10 in male P rat brain that were not detected in female P rat brain. These effects hold significant implications for controlling inflammatory responses in both the brain and peripheral immune cells.

Pharmacological administration of 3α,5α-THP into the nucleus accumbens core increases 3α,5α-THP expression and reduces alcohol self-administration.

BACKGROUND: Alcohol affects multiple circuits in the brain, mainly disrupting the delicate balance between inhibitory γ-aminobutyric acid (GABA) transmission and excitatory glutamate signaling in brain areas involved in reward circuits. These include the amygdala, nucleus accumbens (Acb), and ventral tegmental area (VTA). This action impairs circuits that regulate behavioral control of craving and alcohol seeking and intake. Studies in both rodent models and postmortem human brain of patients with alcohol use disorder (AUD) have highlighted the association between the loss of GABAergic inhibition and the development of addiction. The neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) is a potent positive modulator of GABAA receptors. Chronic alcohol consumption reduces 3α,5α-THP levels, resulting in decreased GABA inhibition. We previously demonstrated that enhancing neurosteroid biosynthesis by overexpression of the cholesterol side-chain cleavage enzyme P450scc decreased alcohol intake in male alcohol-preferring rats (P-rats). While most of the evidence of alcohol-induced alterations comes from studies in male subjects, some data show that females are more vulnerable to alcohol's effects than males. METHODS: In this study, we investigated the ability of 3α,5α-THP direct infusions in two brain regions that contribute to alcohol reinforcement, the VTA and Acb core (AcbC), to regulate alcohol self-administration in female P-rats. RESULTS: Administration of 3α,5α-THP into the AcbC increased 3α,5α-THP-positive cell expression in this area and reduced alcohol self-administration. By contrast, 3α,5α-THP infusion into the VTA did not significantly affect alcohol self-administration, though trends for a reduction were found. CONCLUSIONS: Our results show that local increases in 3α,5α-THP in the AcbC may alter mesolimbic activity that drives a reduction in alcohol self-administration.

Deleterious Interaction between the Neurosteroid (3α,5α)3-Hydroxypregnan-20-One (3α,5α-THP) and the Mu-Opioid System Activation during Forced Swim Stress in Rats.

The neurosteroid 3α,5α-THP is a potent GABAA receptor-positive modulator and its regulatory action on the HPA axis stress response has been reported in numerous preclinical and clinical studies. We previously demonstrated that 3α,5α-THP down-regulation of HPA axis activity during stress is sex-, brain region- and stressor-dependent. In this study, we observed a deleterious submersion behavior in response to 3α,5α-THP (15 mg/kg) during forced swim stress (FSS) that led us to investigate how 3α,5α-THP might affect behavioral coping strategies engaged in by the animal. Given the well-established involvement of the opioid system in HPA axis activation and its interaction with GABAergic neurosteroids, we explored the synergic effects of 3α,5α-THP/opiate system activation in this behavior. Serum ß-endorphin (ß-EP) was elevated by FSS and enhanced by 3α,5α-THP + FSS. Hypothalamic Mu-opiate receptors (MOP) were increased in female rats by 3α,5α-THP + FSS. Pretreatment with the MOP antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 2 mg/kg, IP) reversed submersion behavior in males. Moreover, in both males and females, CTAP pretreatment decreased immobility episodes while increasing immobility duration but did not alter swimming duration. This interaction between 3α,5α-THP and the opioid system in the context of FSS might be important in the development of treatment for neuropsychiatric disorders involving HPA axis activation.

Brexanolone therapeutics in post-partum depression involves inhibition of systemic inflammatory pathways.

BACKGROUND: Brexanolone has rapid, long-lasting, and remarkable efficacy in the treatment of post-partum depression (PPD). We test the hypothesis that brexanolone inhibits proinflammatory modulators and macrophage activation in PPD patients, which may promote clinical recovery. METHODS: PPD patients (N = 18) provided blood samples before and after brexanolone infusion according to the FDA-approved protocol. Patients were unresponsive to prior treatment before brexanolone therapy. Serum was collected to determine neurosteroid levels and whole blood cell lysates were examined for inflammatory markers and in vitro responses to the inflammatory activators lipopolysaccharide (LPS) and imiquimod (IMQ). FINDINGS: Brexanolone infusion altered multiple neuroactive steroid levels (N = 15-18), reduced levels of inflammatory mediators (N = 11) and inhibited their response to inflammatory immune activators (N = 9-11). Specifically, brexanolone infusion reduced whole blood cell tumor necrosis factor-α (TNF-α, p = 0.003), and interleukin-6 (IL-6, p = 0.04) and these effects were correlated with HAM-D score improvement (TNF-α, p = 0.049; IL-6, p = 0.02). Furthermore, brexanolone infusion prevented LPS and IMQ-induced elevation of TNF-α (LPS: p = 0.02; IMQ: p = 0.01), IL-1ß (LPS: p = 0.006; IMQ: p = 0.02) and IL-6 (LPS: p = 0.009; IMQ: p = 0.01), indicating inhibition of toll-like receptor (TLR)4 and TLR7 responses. Finally, inhibition of TNF-α, IL-1ß and IL-6 responses to both LPS and IMQ were correlated with HAM-D score improvements (p < 0.05). INTERPRETATION: Brexanolone actions involve inhibition of inflammatory mediator production and inhibition of inflammatory responses to TLR4 and TLR7 activators. The data suggest that inflammation plays a role in post-partum depression and that inhibition of inflammatory pathways contributes to the therapeutic efficacy of brexanolone. FUNDING: The Foundation of Hope, Raleigh, NC and UNC School of Medicine, Chapel Hill.

(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

Medial prefrontal cortex-basolateral amygdala circuit dysfunction in chronic alcohol-exposed male rats.

Alcohol is a commonly used drug that can produce alcohol use disorders (AUDs). Few individuals with AUDs receive treatment and treatment options are complicated by issues with effectiveness and compliance. Alcohol has been shown to differentially affect specific brain regions and an improved understanding of circuit-specific dysregulation caused by alcohol is warranted. Previous work has implicated both the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA) in alcohol-associated plasticity, however studies directly examining the impact of alcohol exposure on this circuit are lacking. The current study employed an optogenetic strategy to investigate the prelimbic mPFC to BLA circuit and changes in circuit activity following chronic intragastric ethanol exposure in male Sprague Dawley rats. We observed monosynaptic connections with light-evoked stimulation of mPFC terminals in the BLA with efficacy and short latency. We also found that mPFC-BLA projections are primarily glutamatergic under basal inhibitory control, with a lesser population of GABAergic projections. We examined optically-evoked glutamate currents in the BLA using repeated trains of stimulation that displayed accommodation, or a reduction in evoked current amplitude over repeated stimulations. We found that following chronic ethanol exposure mPFC-BLA glutamatergic connections were dysregulated such that there were decreases in overall function, notably in synaptic strength and accommodation, with no change in probability of evoked glutamate release. The lesser GABAergic component of the mPFC-BLA circuit was not altered by chronic ethanol exposure. Collectively these data indicate that mPFC-BLA circuitry is a significant target of alcohol-associated plasticity, which may contribute to pathological behavior associated with AUDs.

Sex- and subtype-specific adaptations in excitatory signaling onto deep-layer prelimbic cortical pyramidal neurons after chronic alcohol exposure.

Long-term alcohol use results in behavioral deficits including impaired working memory, elevated anxiety, and blunted inhibitory control that is associated with prefrontal cortical (PFC) dysfunction. Preclinical observations demonstrate multiple impairments in GABAergic neurotransmission onto deep-layer principal cells (PCs) in the prelimbic cortex that suggest dependence-related cortical dysfunction is the product of elevated excitability in these cells. Despite accumulating evidence showing alcohol-induced changes in interneuron signaling onto PCs differ between sexes, there is limited data explicitly evaluating sex-specific ethanol effects on excitatory signaling onto deep-layer PCs that may further contribute to deficits in PFC-dependent behaviors. To address this, we conducted electrophysiological and behavioral tests in both male and female Sprague-Dawley rats to evaluate the effects of chronic ethanol exposure. Among our observations, we report a marked enhancement in glutamatergic signaling onto deep-layer PCs in male, but not female, rats after alcohol exposure. This phenomenon was furthermore specific to a sub-class of PC, sub-cortically projecting Type-A cells, and coincided with enhanced anxiety-like behavior, but no observable deficit in working memory. In contrast, female rats displayed alcohol-induced facilitation in working memory performance with no change in expression of anxiety-like behavior. Together, these results suggest fundamental differences in alcohol effects on cell activity, cortical sub-circuits, and PFC-dependent behaviors across male and female rats.

(3α,5α)3-hydroxypregnan-20-one (3α,5α-THP) regulation of hypothalamic and extrahypothalamic corticotropin releasing factor (CRF): Sexual dimorphism and brain region specificity in Sprague Dawley rats.

CRF is the main activator of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. CRF neurons are found mainly in the hypothalamus, but CRF positive cells and CRF1 receptors are also found in extrahypothalamic structures, including amygdala (CeA), hippocampus, NAc and VTA. CRF release in the hypothalamus is regulated by inhibitory GABAergic interneurons and extrahypothalamic glutamatergic inputs, and disruption of this balance is found in stress-related disorders and addiction. (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP), the most potent positive modulator of GABAA receptors, attenuates the stress response reducing hypothalamic CRF mRNA expression and ACTH and corticosterone serum levels. In this study, we explored 3α,5α-THP regulation of hypothalamic and extrahypothalamic CRF mRNA and peptide expression, in male and female Sprague Dawley rats, following vehicle or 3α,5α-THP administration (15 mg/kg). In the hypothalamus, we found sex differences in CRF mRNA expression (females +74%, p < 0.01) and CRF peptide levels (females -71%, p < 0.001). 3α,5α-THP administration reduced hypothalamic CRF mRNA expression only in males (-50%, p < 0.05) and did not alter CRF peptide expression in either sex. In hippocampus and CeA, 3α,5α-THP administration reduced CRF peptide concentrations only in the male (hippocampus -29%, p < 0.05; CeA -62%, p < 0.01). In contrast, 3α,5α-THP injection increased CRF peptide concentration in the VTA of both males (+32%, p < 0.01) and females (+26%, p < 0.01). The results show sex and region-specific regulation of CRF signals and the response to 3α,5α-THP administration. This data may be key to successful development of therapeutic approaches for stress-related disorders and addiction.

Chronic ethanol exposure produces tolerance to elevations in neuroactive steroids: mechanisms and reversal by exogenous ACTH.

Acute ethanol administration increases potent GABAergic neuroactive steroids, specifically (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) and (3α,5α)-3,21-dihydroxypregnan-20-one. In addition, neuroactive steroids contribute to ethanol actions. Chronic ethanol exposure results in tolerance to many effects of ethanol, including ethanol-induced increases in neuroactive steroid levels. To determine the mechanisms of tolerance to ethanol-induced increases in neuroactive steroids, we investigated critical signaling molecules that are required for acute ethanol effects. Male Sprague-Dawley rats were administered ethanol via liquid diet for 2 weeks and steroid levels, adrenocorticotrophic hormone (ACTH) and adrenal steroidogenic acute regulatory (StAR) protein expression were measured. Chronic ethanol exposure elicits tolerance to ethanol-induced elevation of serum ACTH and the steroids pregnenolone and progesterone. Surprisingly, chronic ethanol exposure does not result in tolerance to ethanol-induced increases in adrenal StAR protein. However, ethanol-induced StAR phosphorylation is decreased when compared to acute ethanol administration. A separate group of rats exposed to chronic ethanol diet were subsequently challenged with ethanol (2 g/kg) and exhibited a blunted elevation of serum ACTH and progesterone as well as cerebral cortical and hippocampal 3α,5α-THP. Administration of ACTH with the ethanol challenge restored the elevation of serum ACTH and progesterone as well as cerebral cortical 3α,5α-THP levels to those observed in ethanol-naïve rats. Thus, chronic ethanol exposure disrupts ACTH release, which results in tolerance to ethanol-induced increases in neuroactive steroid levels. Loss of the ethanol-induced increases in neuroactive steroids may contribute to behavioral tolerance to ethanol and influence the progression towards alcoholism.

Ethanol induction of steroidogenesis in rat adrenal and brain is dependent upon pituitary ACTH release and de novo adrenal StAR synthesis.

The mechanisms of ethanol actions that produce its behavioral sequelae involve the synthesis of potent GABAergic neuroactive steroids, specifically the GABAergic metabolites of progesterone, (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP), and deoxycorticosterone, (3alpha,5alpha)-3,21-dihydroxypregnan-20-one. We investigated the mechanisms that underlie the effect of ethanol on adrenal steroidogenesis. We found that ethanol effects on plasma pregnenolone, progesterone, 3alpha,5alpha-THP and cortical 3alpha,5alpha-THP are highly correlated, exhibit a threshold of 1.5 g/kg, but show no dose dependence. Ethanol increases plasma adrenocorticotropic hormone (ACTH), adrenal steroidogenic acute regulatory protein (StAR), and adrenal StAR phosphorylation, but does not alter levels of other adrenal cholesterol transporters. The inhibition of ACTH release, de novo adrenal StAR synthesis or cytochrome P450 side chain cleavage activity prevents ethanol-induced increases in GABAergic steroids in plasma and brain. ACTH release and de novo StAR synthesis are independently regulated following ethanol administration and both are necessary, but not sufficient, for ethanol-induced elevation of plasma and brain neuroactive steroids. As GABAergic steroids contribute to ethanol actions and ethanol sensitivity, the mechanisms of this effect of ethanol may be important factors that contribute to the behavioral actions of ethanol and risk for alcohol abuse disorders.