Rahul T. Khisti

Neuroactive steroid 3α-hydroxy-5α-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 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) that contribute to specific behavioral actions of ethanol. The present study determined the effect of adrenalectomy and 5α-reductase type-1/type-2 enzyme inhibition, known to reduce neuroactive steroids, on ethanol-induced increases in cerebral cortical levels of 3α,5α-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 3α,5α-THP and plasma progesterone levels and reduced the duration of ethanol-induced loss of righting reflex. Prior systemic administration of 5α-dihydroprogesterone (10 or 15 mg/kg, i.p.), an immediate precursor of 3α,5α-THP, to adrenalectomized rats not only restored the ethanol-induced increases in cerebral cortical 3α,5α-THP levels but also reversed the effect of adrenalectomy on ethanol-induced loss of righting reflex. Prior administration of the 5α-reductase inhibitor finasteride (2×25, 2×75 or 2×150 mg/kg, s.c.) and the 5α-reductase type-1 inhibitor SKF-105,111 (50 mg/kg, i.p.) did not reduce ethanol-induced increases in the cerebral cortical levels of 3α,5α-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 3α,5α-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.

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.

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.

GABAergic neurosteroid modulation of ethanol actions

Summary: 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 anti-depressant, anxiolytic, spatial learning deficits and drug discriminatory actions in rodents. Thus ethanol-induced increases in neuroactive steroids represent a novel mechanism of ethanols 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.

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.

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.

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

Rationale and objectiveProtein 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 (GABAA) receptors in adult brain are unknown. Previous studies of recombinant GABAA receptors have provided evidence that PKC activation inhibits receptor function, whereas other studies suggest that PKC either increases or does not alter GABAA receptor function. The present study explored (a) the effects of PKC and phosphatase activity on GABA-mediated 36Cl− uptake in cerebral cortical synaptoneurosomes and (b) the effect of PKC activity on muscimol-induced loss of righting reflex (LORR) in adult rats.MethodsGABAA receptor function in vitro was measured by muscimol-induced 36Cl− uptake into cerebral cortical synaptoneurosomes. The in vivo effect of PKC on GABAA-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.ResultsAdenosine triphosphate (ATP) and PDBu produced a concentration-dependent and specific reduction in muscimol-stimulated 36Cl− uptake that was blocked by the PKC inhibitor calphostin C. Both adenosine diphosphate and 4αPDBu 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.ConclusionsThe present study provides evidence that PKC activation reduces GABAA receptor function in native receptors both in vitro and in vivo. Phosphatase inhibitors decrease muscimol-mediated Cl− uptake in GABAA receptors demonstrating coordinated regulation of native receptors by PKC and phosphatases.

Ethanol-induced elevation of 3α-hydroxy-5α-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.