Olanzapine and fluoxetine administration and coadministration increase rat hippocampal pregnenolone, allopregnanolone and peripheral deoxycorticosterone: implications for therapeutic actions.

Olanzapine and fluoxetine elevate the GABAergic neuroactive steroid allopregnanolone to physiologically relevant concentrations in rodent cerebral cortex. It is unknown if these agents also alter pregnenolone or deoxycorticosterone. Since olanzapine and fluoxetine in combination have clinical utility and may demonstrate synergistic effects, we investigated neuroactive steroid alterations following olanzapine, fluoxetine or coadministration. Male rats received IP vehicle, olanzapine, fluoxetine or the combination of both agents in higher-dose (0, 10, 20 or 10/20 mg/kg, respectively) and lower-dose (0, 5, 10 or 5/10 mg/kg, respectively) experiments. Pregnenolone and allopregnanolone levels in hippocampus were determined by gas chromatography/mass spectrometry. Peripheral deoxycorticosterone and other steroid levels were determined by radioimmunoassay. Olanzapine, fluoxetine or the combination increased hippocampal pregnenolone and serum deoxycorticosterone in both higher- and lower-dose experiments, and elevated hippocampal allopregnanolone in higher-dose conditions. No synergistic effects on pregnenolone or allopregnanolone were observed following olanzapine and fluoxetine coadministration compared to either compound alone. Pregnenolone and its sulfate enhance learning and memory in rodent models, and therefore pregnenolone elevations may be relevant to cognitive changes in psychotic and affective disorders. Since pregnenolone decreases have been linked to depression, it is possible that olanzapine- and fluoxetine-induced pregnenolone elevations may contribute to the antidepressant actions of these agents.

Subjective effects and changes in steroid hormone concentrations in humans following acute consumption of alcohol.

BACKGROUND: GABAA receptors are an important site of action of endogenous neurosteroids and an important mediator of several behavioral effects of alcohol. This study examined the effects of alcohol on plasma steroid hormone concentrations on the hypothesis that the endocrine effects mediate some of the subjective effects of alcohol. METHODS: Thirty-two healthy subjects (17 men) with no history of a substance use disorder participated in this human laboratory study. All subjects consumed three standard drinks of grain alcohol. Subjective measures and blood samples for steroid concentrations were collected at baseline and 40 min after alcohol consumption. RESULTS: Alcohol increased self-reported stimulation, alcohol liking, and desire for more alcohol. Alcohol also increased pregnenolone (PREG) and dehydroepiandrosterone (DHEA) concentrations, while it decreased progesterone (PROG) and allopregnanolone (ALLO) concentrations, as well as ALLO/PREG and PROG/PREG ratios. In men, the change in PREG concentration was significantly correlated with alcohol liking, while the alcohol-induced change in ALLO concentration correlated significantly with both alcohol liking and desire for more alcohol. DISCUSSION: These findings provide preliminary support for the hypothesis that endogenous neurosteroids mediate some of the subjective effects of alcohol. Efforts to replicate these findings should aim to specify more clearly the nature and time course of the effects.

Characterization of the ethanol-deprivation effect in substrains of C57BL/6 mice.

Ethanol craving plays a major role in relapse drinking behavior. Relapse and ethanol craving are an important focus for the treatment of alcoholism. The ethanol-deprivation effect (EDE) is a widely used animal model of alcohol craving. While the EDE is widely studied in rats, the molecular mechanisms underlying EDE are not clearly understood. The C57BL/6 inbred mouse strain is widely used for behavioral and molecular analyses of ethanol drinking but studies on the EDE have not been reported in this strain. In the present study, we characterized a simple behavioral protocol that rapidly and reliably induced EDE in C57BL/6 mice. Briefly, single-housed adult male C57BL/6NCrl and C57BL/6J mice were presented at the beginning of dark phase with two-bottle choice drinking containing either 10% wt/vol ethanol or tap water for 18 h/day, as well as food ad libitum. Following ethanol drinking for 4 days or 14 days, mice were deprived of ethanol for a period of 4 days. To study EDE, mice were reinstated with two bottles containing either ethanol (10% wt/vol) or water. Mice were exposed to single or multiple ethanol-deprivation cycles. Ethanol consumption (g/kg/18 h) and percent ethanol preference (% preference/18 hrs) was recorded for individual mice. C57BL/6NCrl mice consumed moderate amounts (4.78+/-0.63 g/kg) of ethanol but showed robust EDE after ethanol-drinking episodes (4 days or 14 days) as evidenced by increased ethanol consumption and ethanol preference following reinstatement of ethanol. While repeated ethanol deprivation in C57BL/6NCrl mice transiently increased ethanol consumption and ethanol preference, the magnitude of these behaviors was reduced as compared to the first deprivation cycle. In contrast, the C57BL/6J substrain consumed substantially higher levels (9.65+/-0.90 g/kg) of ethanol but did not show a clear EDE after single or multiple ethanol-deprivation cycles. In conclusion, we established a simple and reliable behavioral model to study EDE in C57BL/6NCrl mice. A reliable behavioral model to study EDE in inbred C57BL/6NCrl mice could greatly facilitate further studies on molecular mechanisms of ethanol craving behavior.

Regulation of native GABAA receptors by PKC and protein phosphatase activity.

RATIONALE AND OBJECTIVE: Protein kinase C (PKC) modulation of ionotropic receptors is a common mechanism for regulation of channel function. The effects of PKC and phosphatase activation on native gamma-aminobutyric acid (GABA(A)) receptors in adult brain are unknown. Previous studies of recombinant GABA(A) receptors have provided evidence that PKC activation inhibits receptor function, whereas other studies suggest that PKC either increases or does not alter GABA(A) receptor function. The present study explored (a) the effects of PKC and phosphatase activity on GABA-mediated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes and (b) the effect of PKC activity on muscimol-induced loss of righting reflex (LORR) in adult rats. METHODS: GABA(A) receptor function in vitro was measured by muscimol-induced (36)Cl(-) uptake into cerebral cortical synaptoneurosomes. The in vivo effect of PKC on GABA(A)-mediated function was measured by intracerebroventricular (i.c.v.) injection of 4-beta-phorbol-12,13-dibutyrate (PDBu) or calphostin C followed by determination of muscimol-induced LORR. RESULTS: Adenosine triphosphate (ATP) and PDBu produced a concentration-dependent and specific reduction in muscimol-stimulated (36)Cl(-) uptake that was blocked by the PKC inhibitor calphostin C. Both adenosine diphosphate and 4alphaPDBu were ineffective. Phosphatase inhibition produced similar inhibition of muscimol responses. Furthermore, i.c.v. administration of PDBu and calphostin C produced opposing effects on both the onset and the duration of muscimol-induced LORR in rats. CONCLUSIONS: The present study provides evidence that PKC activation reduces GABA(A) receptor function in native receptors both in vitro and in vivo. Phosphatase inhibitors decrease muscimol-mediated Cl(-) uptake in GABA(A) receptors demonstrating coordinated regulation of native receptors by PKC and phosphatases.

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.

Ethanol-induced elevation of 3 alpha-hydroxy-5 alpha-pregnan-20-one does not modulate motor incoordination in rats.

BACKGROUND: Ethanol administration elevates the levels of GABAergic neuroactive steroids in brain and contributes to some of its behavioral actions. In the present study, we investigated whether such elevation of GABAergic neuroactive steroids contributes to the motor incoordinating effects of ethanol. METHODS: Sprague-Dawley rats were administered ethanol (2 g/kg intraperitoneally) or saline, and the level of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) was measured across time in cerebral cortex and in various brain regions at the peak time by radioimmunoassay. To study whether increases in GABAergic neuroactive steroids are responsible for the motor incoordinating actions of ethanol, rats were subjected to chemical (5alpha-reductase inhibitor, finasteride) and surgical (adrenalectomy) manipulations before receiving ethanol (2 g/kg intraperitoneally) injections. The rats were then subjected to different paradigms to evaluate motor impairment including the Majchrowicz motor intoxication rating scale, Rotarod test, and aerial righting reflex task at different time points. RESULTS: The radioimmunoassay of 3alpha,5alpha-THP in different brain regions showed that ethanol increases 3alpha,5alpha-THP levels by 3- and 9-fold in cerebral cortex and hippocampus, respectively. There was no change in 3alpha,5alpha-THP levels in cerebellum and midbrain. The time course of 3alpha,5alpha-THP elevations in the cerebral cortex showed significant increases 20-min after ethanol injection with a peak at 60 min. In contrast, motor toxicity peaked between 5 and 10 min after ethanol injections and gradually decreased over time. Furthermore, adrenalectomy or pretreatment with finasteride (2 x 50 mg/kg, subcutaneously) did not reduce motor incoordinating effects of ethanol as assessed by the Majchrowicz intoxication rating score, Rotarod test, or aerial righting reflex task. CONCLUSIONS: Ethanol increases GABAergic neuroactive steroids in a time- and brain region-selective manner. The role of neuroactive steroids in alcohol action is specific for certain behaviors. Alcohol-induced deficits in motor coordination are not mediated by elevated neuroactive steroid biosynthesis.

GABAergic agents prevent alpha-melanocyte stimulating hormone induced anxiety and anorexia in rats.

Alpha-melanocyte stimulating hormone (alpha-MSH) is a hypothalamic peptide believed to play a tonic inhibitory role in feeding and energy homeostasis. Systemic administration of alpha-MSH is known to produce anorexia and anxiety. Since synaptic contacts between gamma-aminobutyric acid (GABA)ergic terminals and alpha-MSH neurons in the hypothalamus have been reported, the present work was undertaken to refine our knowledge on the role of GABAergic systems in anxiety and anorexia induced by intracerebroventricular (icv) administration of alpha-MSH in rats. The anxiety was assessed by elevated plus maze, and spontaneous food consumption was monitored during dark cycle. Prior administration of diazepam and muscimol that promote the function of GABA(A) receptors reversed the anxiogenic response and decreased food intake elicited by alpha-MSH. In contrast, bicuculline, the GABA(A) receptor antagonist, not only enhanced the effects of alpha-MSH but also prevented the influence of GABAergic drugs on alpha-MSH-induced anorexia and anxiety. These findings suggest that alpha-MSH-induced anxiety and anorexia are due to its negative influence on GABAergic system.

Ethanol rapidly induces steroidogenic acute regulatory protein expression and translocation in rat adrenal gland.

Acute ethanol exposure increases GABAergic neuroactive steroids in plasma and brain by releasing these steroids or their precursors from the adrenal glands. The present study showed that ethanol administration rapidly increases the expression of steroidogenic acute regulatory protein (StAR) in the cytosolic and mitochondrial fractions of adrenal glands. The increased StAR protein expression paralleled increases in plasma pregnenolone, progesterone and corticosterone levels. The rapid synthesis and translocation of StAR protein in adrenals likely represent the mechanism of ethanol-induced increases in neuroactive steroids.

Neuroactive steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one modulates ethanol-induced loss of righting reflex in rats.

Systemic ethanol administration elevates plasma and brain levels of GABAergic neuroactive steroids, including 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) that contribute to specific behavioral actions of ethanol. The present study determined the effect of adrenalectomy and 5alpha-reductase type-1/type-2 enzyme inhibition, known to reduce neuroactive steroids, on ethanol-induced increases in cerebral cortical levels of 3alpha,5alpha-THP and hypnotic effects in male rats. Systemic ethanol administration to male rats increases plasma levels of progesterone and corticosterone similar to acute stress, indicating release of these steroids from adrenal glands. Adrenalectomy markedly reduced the elevation of cerebral cortical 3alpha,5alpha-THP and plasma progesterone levels and reduced the duration of ethanol-induced loss of righting reflex. Prior systemic administration of 5alpha-dihydroprogesterone (10 or 15 mg/kg, i.p.), an immediate precursor of 3alpha,5alpha-THP, to adrenalectomized rats not only restored the ethanol-induced increases in cerebral cortical 3alpha,5alpha-THP levels but also reversed the effect of adrenalectomy on ethanol-induced loss of righting reflex. Prior administration of the 5alpha-reductase inhibitor finasteride (2 x 25, 2 x 75 or 2 x 150 mg/kg, s.c.) and the 5alpha-reductase type-1 inhibitor SKF-105,111 (50 mg/kg, i.p.) did not reduce ethanol-induced increases in the cerebral cortical levels of 3alpha,5alpha-THP at hypnotic doses of ethanol. Furthermore, these drugs did not alter the duration of loss of righting reflex. However, significant correlations between cerebral cortical 3alpha,5alpha-THP levels and the duration of loss of righting reflex were obtained regardless of finasteride administration. These results demonstrate the contributory role of neuroactive steroids in the ethanol-induced loss of righting reflex and the source of ethanol-induced elevation of GABAergic neuroactive steroids. Ethanol-induced increases in neurosteroids could be pertinent to the etiology of sleep-related disorders associated with alcoholism.

GABAergic neurosteroid modulation of ethanol actions.

Systemic administration of ethanol elevates plasma and cerebral cortical GABAergic neuroactive steroids. The increase in neurosteroids is responsible for specific behavioural and electrophysiological actions of ethanol in rodents. This article recapitulates the current knowledge of the novel interaction between ethanol and neurosteroids and addresses the potential mechanism for ethanol-induced increase in brain neurosteroid levels. Ethanol-induced increase in the cortical neurosteroid content is modified by neurosteroid biosynthesis inhibitors and completely prevented by adrenalectomy in male rats. In line with this, adrenalectomy prevented the anticonvulsant and hypnotic effects of acute ethanol administration. It is speculated that acute ethanol administration might resemble acute stress and increase neuroactive steroids due to activation of hypothalamic-pituitary adrenal axis. Ethanol-induced increases in neuroactive steroids might be responsible for the antidepressant, anxiolytic, spatial learning deficits and drug discriminatory actions in rodents. Thus ethanol-induced increases in neuroactive steroids represent a novel mechanism of ethanol's action, responsible for several pharmacological and behavioural actions of ethanol. The development of new therapeutic strategies for alcoholism may arise based on the novel interaction between ethanol and neurosteroids in the brain.

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one affects dopamine-mediated behavior in rodents.

RATIONALE: The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) has been previously shown to induce catalepsy in mice that is modified by GABAergic, dopaminergic, adenosinergic and serotonergic agents. In light of the interaction of this endogenous neurosteroid with GABAergic and dopaminergic transmission, there is potential interest in the possible role of 3alpha,5alpha-THP in psychotic disorders. OBJECTIVE: This study assessed the effect of 3alpha,5alpha-THP in certain dopamine-mediated behavioral paradigms that are widely used to predict antipsychotic-like activity. METHODS: 3alpha,5alpha-THP (1-8 microg per animal, i.c.v.), the classic neuroleptic (dopamine receptor antagonist) haloperidol (0.25 mg/kg, i.p.), and the benzodiazepine diazepam (7 mg/kg, i.p.) were injected into different groups of animals, and their behavior was screened using the following animal tests: conditioned avoidance response, apomorphine-induced climbing, and amphetamine-induced motor hyperactivity. Separate groups of mice that received 3alpha,5alpha-THP (1-8 microg per animal, i.c.v.) were screened for catalepsy. Furthermore, the effect of a sub-cataleptic dose (0.1 microg per mouse, i.c.v.) of 3alpha,5alpha-THP, either alone or in combination with the GABA(A) receptor antagonist picrotoxin (0.8 mg/kg, i.p.) was measured on haloperidol-induced catalepsy. RESULTS: 3alpha,5alpha-THP like haloperidol reduced conditioned avoidance, apomorphine-induced cage climbing and amphetamine-induced motor hyperactivity. Diazepam only affected conditioned avoidance. 3alpha,5alpha-THP also induced dose-dependent catalepsy. Furthermore, sub-cataleptic doses of 3alpha,5alpha-THP potentiated haloperidol-induced catalepsy. This potentiation was blocked by prior treatment with the GABA(A) receptor antagonist picrotoxin. CONCLUSION: These findings suggest that 3alpha,5alpha-THP, by its action at the GABA(A) receptors, increases GABAergic tone leading to a behavioral profile similar to that of dopamine receptor antagonists.