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.

Dopamine receptor signaling and current and future antipsychotic drugs.

All currently efficacious antipsychotic drugs have as part of their mechanism the ability to attenuate some or all of the signaling through the dopamine D(2) receptor. More recently, the dopamine D(1) receptor has been hypothesized to be a promising target for the treatment of negative and/or cognitive aspects of schizophrenia that are not improved by current antipsychotics. Although cAMP has been presumed to be the primary messenger for signaling through the dopamine receptors, the last decade has unveiled a complexity that has provided exciting avenues for the future discovery of antipsychotic drugs (APDs). We review the signaling mechanisms of currently approved APDs at dopamine D(2) receptors, and note that aripiprazole is a compound that is clearly differentiated from other approved drugs. Although aripiprazole has been postulated to cause dopamine stabilization due to its partial D(2) agonist properties, a body of literature suggests that an alternative mechanism, functional selectivity, is of primary importance. Finally, we review the signaling at dopamine D(1) receptors, and the idea that drugs that activate D(1) receptors may have use as APDs for improving negative and cognitive symptoms. We address the current state of drug discovery in the D(1) area and its relationship to novel signaling mechanisms. Our conclusion is that although the first APD targeting dopamine receptors was discovered more than a half-century ago, recent research advances offer the possibility that novel and/or improved drugs will emerge in the next decade.

Ethanol reduces GABAA alpha1 subunit receptor surface expression by a protein kinase Cgamma-dependent mechanism in cultured cerebral cortical neurons.

Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA(A) receptors composed of alpha1beta2/3gamma2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA(A) alpha1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) beta, gamma, and epsilon isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABA- and zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA(A) receptor alpha1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKCgamma with alpha1 subunits, whereas PKCbeta/alpha1 association and PKCepsilon/alpha1 colocalization were not altered by ethanol exposure. Selective PKCgamma inhibition by transfection of selective PKCgamma small interfering RNAs blocked ethanol-induced internalization of GABA(A) receptor alpha1 subunits, whereas PKCbeta inhibition using pseudo-PKCbeta had no effect. These findings suggest that ethanol exposure selectively alters PKCgamma translocation to GABA(A) receptors and PKCgamma regulates GABA(A) alpha1 receptor trafficking after ethanol exposure.

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.

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.

Role of acetaldehyde in ethanol-induced elevation of the neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one in rats.

BACKGROUND: Systemic ethanol administration increases neuroactive steroid levels that increase ethanol sensitivity. Acetaldehyde is a biologically active compound that may contribute to behavioral and rewarding effects of ethanol. We investigated the role of acetaldehyde in ethanol-induced elevations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) levels in cerebral cortex. METHODS: Male Sprague-Dawley rats were administered ethanol, and plasma acetaldehyde concentrations were measured by gas chromatography to determine relevant concentrations. Rats were then administered acetaldehyde directly, acetaldehyde plus cyanamide to block its degradation, or ethanol in the presence of inhibitors of ethanol metabolism, to determine effects on 3alpha,5alpha-THP levels in cerebral cortex. RESULTS: Ethanol administration (2 g/kg) to rats results in a peak acetaldehyde concentration of 6-7 microM at 10 minutes that remains stable for the duration of the time points tested. Direct administration of acetaldehyde eliciting this plasma concentration does not increase cerebral cortical 3alpha,5alpha-THP levels, and inhibition of ethanol-metabolizing enzymes to modify acetaldehyde formation does not alter ethanol-induced 3alpha,5alpha-THP levels. However, higher doses of acetaldehyde (75 and 100 mg/kg), in the presence of cyanamide to prevent its metabolism, are capable of increasing cortical 3alpha,5alpha-THP levels. CONCLUSIONS: Physiological concentrations of acetaldehyde are not responsible for ethanol-induced increases in 3alpha,5alpha-THP, but a synergistic role for acetaldehyde with ethanol may contribute to increases in 3alpha,5alpha-THP levels and ethanol sensitivity.

Hypothalamic-pituitary-adrenal axis modulation of GABAergic neuroactive steroids influences ethanol sensitivity and drinking behavior.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to elevations in gamma-aminobutyric acid (GABA)-ergic neuroactive steroids that enhance GABA neurotransmission and restore homeostasis following stress. This regulation of the HPA axis maintains healthy brain function and protects against neuropsychiatric disease. Ethanol sensitivity is influenced by elevations in neuroactive steroids that enhance the GABAergic effects of ethanol, and may prevent excessive drinking in rodents and humans. Low ethanol sensitivity is associated with greater alcohol consumption and increased risk of alcoholism. Indeed, ethanol-dependent rats show blunted neurosteroid responses to ethanol administration that may contribute to ethanol tolerance and the propensity to drink greater amounts of ethanol. The review presents evidence to support the hypothesis that neurosteroids contribute to ethanol actions and prevent excessive drinking, while the lack of neurosteroid responses to ethanol may underlie innate or chronic tolerance and increased risk of excessive drinking. Neurosteroids may have therapeutic use in alcohol withdrawal or for relapse prevention.

Systemic ethanol administration elevates deoxycorticosterone levels and chronic ethanol exposure attenuates this response.

Systemic ethanol administration is known to elevate levels of the GABAergic neuroactive steroid 3alpha,21-dihydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THDOC). 3alpha,5alpha-THDOC is synthesized from deoxycorticosterone (DOC) by metabolism in adrenals and brain. The present study investigated DOC levels in plasma and brain following ethanol administration to naïve and ethanol-exposed rats. Rats were administered ethanol (2 g/kg, i.p.) or saline and DOC levels were measured in plasma and brain regions by radioimmunoassay. Chronic ethanol-exposed rats were administered an ethanol challenge (2 g/kg, i.p.) following 15 days of ethanol liquid diet consumption. Ethanol administration markedly increased DOC levels in plasma, cerebral cortex, hippocampus, hypothalamus, cerebellum, and olfactory tubercle of naïve rats. Ethanol challenge produced an attenuated elevation of DOC in rat plasma and brain following chronic ethanol consumption for 2 weeks. These findings suggest that acute ethanol increases DOC levels in ethanol naïve rats and chronic ethanol consumption induces tolerance to ethanol-induced increases in DOC levels.