Differential hypothalamic–pituitary–adrenal activation of the neuroactive steroids pregnenolone sulfate and deoxycorticosterone in healthy controls and alcohol-dependent subjects

Ethanol and the neuroactive steroids have interactive neuropharmacological effects and chronic ethanol administration blunts the ethanol-induced increase in neuroactive steroid levels in rodent plasma and brain. Few studies have explored neuroactive steroid regulation in alcohol-dependent human subjects. In fact, the regulation of adrenal neuroactive steroids has not been well defined in healthy controls. We thus explored the regulation of two neuroactive steroids, pregnenolone sulfate (PREG-S) and deoxycorticosterone, by pharmacological challenges to the hypothalamic-pituitary-adrenal (HPA) axis in healthy controls and one-month abstinent alcohol-dependent patients with co-occurring nicotine dependence. Plasma levels of PREG-S and deoxycorticosterone were measured by radioimmunoassay in controls and alcohol-dependent patients after challenges of naloxone, ovine corticotrophin releasing hormone (oCRH), dexamethasone, cosyntropin, and cosyntropin following high-dose dexamethasone. In addition, basal diurnal measures of both hormones were obtained. PREG-S plasma levels in healthy controls were increased by cosyntropin challenge (with and without dexamethasone pretreatment) and decreased by dexamethasone challenge. However, PREG-S concentrations were not altered by naloxone or oCRH challenges, suggesting that PREG-S is not solely regulated by hypothalamic or pituitary stimulation. Deoxycorticosterone, in contrast, is regulated by HPA challenge stimulation in a manner similar to cortisol. Alcohol-dependent patients had a blunted PREG-S response to cosyntropin (with and without dexamethasone pretreatment). Furthermore, the time to peak deoxycorticosterone response following oCRH was delayed in alcohol-dependent patients compared to controls. These results indicate that plasma PREG-S and deoxycorticosterone levels are differentially regulated by HPA axis modulation in human plasma. Further, alcohol-dependent patients show a blunted PREG-S response to adrenal stimulation and a delayed deoxycorticosterone response to oCRH challenge

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

Altered GABAA Receptor Expression and Seizure Threshold Following Acute Ethanol Challenge in Mice Lacking the RIIβ 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 RIIβ 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 (RIIβ−/−) mice had similar baseline levels of PKA RIIα and GABAA α1 and α4 subunits compared to wild type (RIIβ+/+) littermates, but had deficits in AKAP150. GABAA α1 subunit levels were decreased in the P2 fraction of RIIβ−/−, but not RIIβ+/+, 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 RIIβ+/+, but not RIIβ−/− mice, while extrasynaptic α4 expression was decreased in RIIβ−/−, but not RIIβ+/+ mice. Finally, RIIβ 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

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 Activation of Protein Kinase A Regulates GABAA Receptor Subunit Expression in the Cerebral Cortex and Contributes to Ethanol-Induced Hypnosis

Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking, and synaptic excitability. Both protein kinase C (PKC) and A (PKA) are involved in regulation of γ-aminobutyric acid type A (GABAA) receptors through phosphorylation. However, the role of PKA in regulating GABAA receptors (GABAA-R) following acute ethanol exposure is not known. The present study investigated the role of PKA in the effects of ethanol on GABAA-R α1 subunit expression in rat cerebral cortical P2 synaptosomal fractions. Additionally, GABA-related behaviors were examined. Rats were administered ethanol (2.0–3.5 g/kg) or saline and PKC, PKA, and GABAA-R α1 subunit levels were measured by western blot analysis. Ethanol (3.5 g/kg) transiently increased GABAA-R α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, lower ethanol doses (2.0 g/kg) had no effect on GABAA-R α1 subunit levels, although PKA type II regulatory subunits RIIα and RIIβ were increased at 10 and 60 min when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABAA-R α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABAA-R α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex (LORR) duration. This effect appears to be mediated in part by GABAA-R as increasing PKA activity also increased the duration of muscimol-induced LORR. Overall, these data suggest that PKA mediates ethanol-induced GABAA-R expression and contributes to behavioral effects of ethanol involving GABAA-R

Simultaneous quantification of GABAergic 3α,5α/3α,5β neuroactive steroids in human and rat serum

The 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3α,5α- and 3α,5β-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3α,5α-THP (+1488%, p<0.001), (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC, +205%, p<0.01), (3α,5α)-3-hydroxyandrostan-17-one (3α,5α-A, +216%, p<0.001), (3α,5α,17β)-androstane-3,17-diol (3α,5α-A-diol, +190%, p<0.01). (3α,5β)-3-hydroxypregnan-20-one (3α,5β-THP) and (3α,5β)-3-hydroxyandrostan-17-one (3α,5β-A) were not altered, while (3α,5β)-3,21-dihydroxypregnan-20-one (3α,5β-THDOC) and (3α,5β,17β)-androstane-3,17-diol (3α,5β-A-diol) were increased from undetectable levels to 271 ± 100 and 2.4 ± 0.9 pg ± SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3α,5α-THP (+1806%, p<0.0001), 3α,5β-THP (+575%, p<0.001), 3α,5α-THDOC (+309%, p<0.001). 3α,5β-THDOC levels were increased by 307%, although this increase was not significant because this steroid was detected only in 3/16 control subjects. Levels of 3α,5α-A, 3α,5β-A and pregnenolone were not altered. This method can be used to investigate the physiological and pathological role of neuroactive steroids and to develop biomarkers and new therapeutics for neurological and psychiatric disorders

Reduction of circulating and selective limbic brain levels of (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) following forced swim stress in C57BL/6J mice

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, and GABAergic neuroactive steroids contribute to homeostatic regulation of this circuitry. Acute forced swim stress (FSS) increases plasma, cortical, and hypothalamic (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) levels in rats. However, there have not been systemic investigations of acute stress on changes in plasma and brain levels of 3α,5α-THP in mouse models

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 β-endorphin (β-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

(3&alpha;,5&alpha;)3-Hydroxypregnan-20-one (3&alpha;,5&alpha;-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 (3&alpha;,5&alpha;)3-hydroxypregnan-20-one (3&alpha;,5&alpha;-THP). We previously showed that 3&alpha;,5&alpha;-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3&alpha;,5&alpha;-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 3&alpha;,5&alpha;-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 3&alpha;,5&alpha;-THP attenuated the CORT response only in females after RS. 3&alpha;,5&alpha;-THP reduced GR levels in male and females, but differently between stressors. 3&alpha;,5&alpha;-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3&alpha;,5&alpha;-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: 3&alpha;,5&alpha;-THP regulated the HPA axis at different levels, depending on sex