A. Leslie Morrow

The role of GABAA receptors in the acute and chronic effects of ethanol: a decade of progress

The past decade has brought many advances in our understanding of GABAA receptor-mediated ethanol action in the central nervous system. We now know that specific GABAA receptor subtypes are sensitive to ethanol at doses attained during social drinking while other subtypes respond to ethanol at doses attained by severe intoxication. Furthermore, ethanol increases GABAergic neurotransmission through indirect effects, including the elevation of endogenous GABAergic neuroactive steroids, presynaptic release of GABA, and dephosphorylation of GABAA receptors promoting increases in GABA sensitivity. Ethanol’s effects on intracellular signaling also influence GABAergic transmission in multiple ways that vary across brain regions and cell types. The effects of chronic ethanol administration are influenced by adaptations in GABAA receptor function, expression, trafficking, and subcellular localization that contribute to ethanol tolerance, dependence, and withdrawal hyperexcitability. Adolescents exhibit altered sensitivity to ethanol actions, the tendency for higher drinking and longer lasting GABAergic adaptations to chronic ethanol administration. The elucidation of the mechanisms that underlie adaptations to ethanol exposure are leading to a better understanding of the regulation of inhibitory transmission and new targets for therapies to support recovery from ethanol withdrawal and alcoholism.

Effects of ethanol on ion channels.

Ion channels play critical roles in nervous system function, from initiating rapid synaptic activity to propagation of action potentials. Studies have indicated that many of the effects of ethanol on the nervous system are likely caused by the actions of ethanol on ion channels. Ion channels are multimeric structures that gate ions through subtle changes in tertiary structure. Ethanol readily enters molecular sites within multimeric ion channels, modifying intermolecular forces and bonds that are important for the open-close-inactivation kinetic properties of channels. The diversity of channel composition caused by the multimeric structure results in subtypes of channels that have a spectrum of sensitivity to ethanol that translates into brain regional differences in ethanol sensitivity, in part caused by differences in ion channel subunit composition. Ethanol has been shown to affect both receptor-activated ion channels and voltage-gated ion channels. The acute intoxicating and incoordinating effects of ethanol are probably related to inhibition of subtypes of NMDA-glutamate receptor ion channels and potentiation of certain subtypes of GABAA receptor ion channels. Effects on these channels, as well as glycine, nicotinic cholinergic, serotonergic, and other ion channels, likely contribute to the euphoric, sedative, and other acute actions of ethanol. Changes in ion channel subunit composition, density, and properties probably also contribute to ethanol tolerance, dependence, withdrawal hyperexcitability, and neurotoxicity. A substantial number of studies have implicated glutamate NMDA receptor, GABAA, and L-type voltage-gated calcium channels in the adaptive changes in the brain during chronic ethanol exposure. The diversity of ion channels subunits, their prominent role in brain function, and ethanol action are likely to make them important contributors to alcoholism and alcohol abuse.

Bidirectional Alterations of GABAA Receptor Subunit Peptide Levels in Rat Cortex During Chronic Ethanol Consumption and Withdrawal

Abstract: The pharmacological properties of γ‐aminobutyric acidA (GABAA) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABAA receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABAA receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol‐dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ∼32% in both ethanol‐dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol‐induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABAA receptor subunit peptide levels are associated with ethanol dependence in rats. GABAA receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABAA receptor gene expression underlie the functional properties of GABAA receptors in ethanol‐dependent rats and those undergoing ethanol withdrawal.

Allopregnanolone levels and reactivity to mental stress in premenstrual dysphoric disorder.

BACKGROUND This study was designed to examine basal and stress-induced levels of the neuroactive progesterone metabolite, allopregnanolone, in women with premenstrual dysphoric disorder (PMDD) and healthy control subjects. Also, because evidence suggests that allopregnanolone negatively modulates the hypothalamic-pituitary-adrenal axis, plasma cortisol levels were examined. An additional goal was to investigate the relationship between premenstrual symptom severity and luteal phase allopregnanolone levels. METHODS Twenty-four women meeting prospective criteria for PMDD were compared with 12 controls during both the follicular and luteal phases of confirmed ovulatory cycles, counterbalancing phase at first testing. Plasma allopregnanolone and cortisol were sampled after an extended baseline period and again 17 min following the onset of mental stress. Owing to low follicular phase allopregnanolone levels, only luteal phase allopregnanolone and cortisol were analyzed. RESULTS During the luteal phase, PMDD women had significantly greater allopregnanolone levels, coupled with significantly lower cortisol levels, during both baseline and mental stress. Moreover, significantly more controls (83%) showed the expected stress-induced increases in allopregnanolone compared with PMDD women (42%). Premenstrual dysphoric disorder women also exhibited a significantly greater allopregnanolone/progesterone ratio than control subjects, suggesting alterations in the metabolic pathways involved in the conversion of progesterone to allopregnanolone. Finally, PMDD women with greater levels of premenstrual anxiety and irritability had significantly reduced allopregnanolone levels in the luteal phase relative to less symptomatic PMDD women. No relationship between symptom severity and allopregnanolone was observed in controls. CONCLUSIONS These results suggest dysregulation of allopregnanolone mechanisms in PMDD and that continued investigations into a potential pathophysiologic role of allopregnanolone in PMDD are warranted.

The role of GABAergic neuroactive steroids in ethanol action, tolerance and dependence.

This article reviews data on ethanol and neurosteroid interactions in the CNS. We discuss how GABAergic neurosteroids, including 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC, produced in response to systemic ethanol administration contribute to several of the effects of ethanol associated with modulation of GABA(A) receptors in rodents. There is an essential correlation between the time course of ethanol-induced 3alpha,5alpha-TH PROG production in the brain and specific behavioral and neural effects of ethanol. Furthermore, the anticonvulsant and inhibitory effects of ethanol on spontaneous neural activity were completely prevented by a key inhibitor of steroid biosynthesis. 3alpha,5alpha-TH PROG influences cognitive processing, spatial learning and memory and alters drinking behaviors in rats. Furthermore, ethanol induction of 3alpha,5alpha-TH PROG is diminished in tolerant and dependent animals. These effects are associated with increases in the sensitivity of GABA(A) receptors to neurosteroids and suggest an important role in ethanol withdrawal. Together, we suggest that 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC contribute to ethanol action and this interaction may represent a new mechanism of ethanol action. The identification of neurosteroid intermediaries involved in ethanol action may lead to important advances in the field and the development of novel therapeutics for alcoholism.

Radioimmunoassay of 3α-hydroxy-5α-pregnan-20-one in rat and human plasma

Abstract A radioimmunoassay for measuring 3α-hydroxy-5α-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3α-hydroxy-20-oxo-5α-pregnan-11α-yl carboxymethyl ether coupled to bovine serum albumin. 3α-Hydroxy-5α-pregnan-20-one was purified from ether extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3α-Hydroxy-5α-pregnan-20-one was detected in plasma from female rats on the day ofestrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.

Chronic ethanol and pentobarbital administration in the rat: Effects on GABAA receptor function and expression in brain

Chronic exposure of rats to ethanol significantly decrease GABAA receptor-mediated 36Cl- uptake in cerebral cortical synaptoneurosomes. Muscimol and pentobarbital stimulation as well as ethanol enhancement of muscimol-stimulated 36Cl- flux are significantly decreased following chronic ethanol inhalation. Repeated pentobarbital administration has a similar effect on muscimol and pentobarbital-stimulated 36Cl- uptake in cerebral cortical synaptoneurosomes. We have postulated that these adaptive response may be associated with an alteration of GABAA receptor gene expression. Chronic ethanol exposure resulted in a significant reduction in the levels of GABAA receptor alpha-subunit mRNAs. The most abundant mRNA species in the rat cerebral cortex were reduced 40-50% (4.4 Kb mRNA, 43%, 4.8 Kb mRNA, 47%). beta-Actin mRNA and poly(A)+ RNA levels were not significantly reduced following chronic ethanol exposure. Repeated pentobarbital administration had no effect on the level of the 4.4 and 4.8 Kb transcripts of alpha-subunit mRNAs in rat cerebral cortex. These data suggest that chronic ethanol exposure alters the level of mRNAs coding for the alpha-subunit of the GABAA receptor. This decrease may reflect an alteration of mRNA processing in the cell or an alteration in GABAA receptor gene expression.

Proof-of-Concept Trial with the Neurosteroid Pregnenolone Targeting Cognitive and Negative Symptoms in Schizophrenia

The neurosteroid pregnenolone and its sulfated derivative enhance learning and memory in rodents. Pregnenolone sulfate also positively modulates NMDA receptors and could thus ameliorate hypothesized NMDA receptor hypofunction in schizophrenia. Furthermore, clozapine increases pregnenolone in rodent hippocampus, possibly contributing to its superior efficacy. We therefore investigated adjunctive pregnenolone for cognitive and negative symptoms in patients with schizophrenia or schizoaffective disorder receiving stable doses of second-generation antipsychotics in a pilot randomized, placebo-controlled, double-blind trial. Following a 2-week single-blind placebo lead-in, patients were randomized to pregnenolone (fixed escalating doses to 500 mg/day) or placebo, for 8 weeks. Primary end points were changes in BACS and MCCB composite and total SANS scores. Of 21 patients randomized, 18 completed at least 4 weeks of treatment (n=9/group). Pregnenolone was well tolerated. Patients receiving pregnenolone demonstrated significantly greater improvements in SANS scores (mean change=10.38) compared with patients receiving placebo (mean change=2.33), p=0.048. Mean composite changes in BACS and MCCB scores were not significantly different in patients randomized to pregnenolone compared with placebo. However, serum pregnenolone increases predicted BACS composite scores at 8 weeks in the pregnenolone group (rs=0.81, p=0.022). Increases in allopregnanolone, a GABAergic pregnenolone metabolite, also predicted BACS composite scores (rs=0.74, p=0.046). In addition, baseline pregnenolone (rs=−0.76, p=0.037), pregnenolone sulfate (rs=−0.83, p=0.015), and allopregnanolone levels (rs=−0.83, p=0.015) were inversely correlated with improvements in MCCB composite scores, further supporting a possible role for neurosteroids in cognition. Mean BACS and MCCB composite scores were correlated (rs=0.74, p<0.0001). Pregnenolone may be a promising therapeutic agent for negative symptoms and merits further investigation for cognitive symptoms in schizophrenia.

GABRA2 alleles moderate the subjective effects of alcohol, which are attenuated by finasteride

GABAA receptors are involved in the subjective effects of alcohol. Endogenous neuroactive steroids interact with GABAA receptors to mediate several behavioral effects of alcohol in rodents. Based on a haplotypic association of alcohol dependence with the gene encoding the GABAA receptor α-2 subunit (GABRA2), we examined whether GABRA2 alleles are associated with the subjective response to alcohol. We also examined whether finasteride (a 5-α steroid reductase inhibitor), which blocks the synthesis of some neuroactive steroids, reduces the subjective response to alcohol. In all, 27 healthy social drinkers (15 males) completed a randomized, double-blind, placebo-controlled study of high-dose finasteride. After being pretreated with study drug, subjects consumed three alcoholic drinks. Subjective effects were measured repeatedly over the ascending blood alcohol curve. To examine the moderating role of genetic variation in GABRA2, a single-nucleotide polymorphism that was informative in association studies was included as a factor in the analysis. Subjects homozygous for the more common A-allele (n=7) showed more subjective effects of alcohol than did individuals with one or two copies of the alcohol dependence-associated G-allele (n=20, including two homozygotes). Among the A-allele homozygotes, there was a greater reduction in several subjective effects during the finasteride session compared to the placebo session. These findings provide preliminary evidence that the risk of alcoholism associated with GABRA2 alleles may be related to differences in the subjective response to alcohol. The effects of finasteride provide indirect evidence for a mediating role of neuroactive steroids in some of the subjective effects of alcohol.

Olanzapine and clozapine increase the GABAergic neuroactive steroid allopregnanolone in rodents

The neuroactive steroid allopregnanolone is a potent γ-aminobutyric acid type A (GABAA) receptor modulator with anxiolytic and anticonvulsant effects. Olanzapine and clozapine also have anxiolytic-like effects in behavioral models. We therefore postulated that olanzapine and clozapine would elevate allopregnanolone levels, but risperidone and haloperidol would have minimal effects. Male rats received intraperitoneal olanzapine (2.5–10.0 mg/kg), clozapine (5.0–20.0 mg/kg), risperidone (0.1–1.0 mg/kg), haloperidol (0.1–1.0 mg/kg), or vehicle. Cerebral cortical allopregnanolone and peripheral progesterone and corticosterone levels were determined. Adrenalectomized animals were also examined. Both olanzapine and clozapine increased cerebral cortical allopregnanolone levels, but neither risperidone nor haloperidol had significant effects. Olanzapine and clozapine also increased serum progesterone and corticosterone levels. Adrenalectomy prevented olanzapine- and clozapine-induced elevations in allopregnanolone. Allopregnanolone induction may contribute to olanzapine and clozapine anxiolytic, antidepressant, and mood-stabilizing actions. Alterations in this neuroactive steroid may result in the modulation of GABAergic and dopaminergic neurotransmission, potentially contributing to antipsychotic efficacy.