A. Chistina Grobin

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.

Regional variations in the effects of chronic ethanol administration on GABAA receptor expression: potential mechanisms

Gamma-aminobutyric acid type A (GABA(A)) receptors in brain adapt to chronic ethanol exposure via changes in receptor function and subunit expression. The present review summarizes currently available data regarding changes in GABA(A) receptor subunit mRNA and peptide expression. Data are presented from various different brain regions and the variations between specific brain regions used to draw conclusions about mechanisms that may underlie GABA(A) receptor adaptations during chronic ethanol exposure. In the whole cerebral cortex, chronic ethanol exposure leads to a reduction of GABA(A) receptor alpha1 subunit mRNA and peptide levels and a near equivalent increase in alpha4 subunit mRNA and peptide levels. This observation is the primary support for the hypothesis that altered receptor composition is a mechanism for GABA(A) receptor adaptation produced by chronic ethanol exposure. However, other brain regions do not display similar patterns of subunit changes. Moreover, subregions within cortex (prefrontal, cingulate, parietal, motor, and piriform) exhibit patterns of changes in subunit expression that differ from whole cortex. Therefore, regional differences in GABA(A) receptor subunit expression are evident following chronic ethanol administration, thus suggesting that multiple mechanisms contribute to the regulation of GABA(A) receptor expression. These mechanisms may include the involvement of other neurotransmitter systems, endogenous steroids and second or third messenger cross-talk.

Clozapine markedly elevates pregnenolone in rat hippocampus, cerebral cortex, and serum: candidate mechanism for superior efficacy?

Clozapine demonstrates superior efficacy in patients with schizophrenia, but the precise mechanisms contributing to this clinical advantage are not clear. Clozapine and olanzapine increase the GABAergic neuroactive steroid (NS) allopregnanolone, and it has been hypothesized that NS induction may contribute to the therapeutic actions of these agents. Pregnenolone administration improves learning and memory in rodent models, and decreases in this NS have been associated with depressive symptoms in humans. These pregnenolone characteristics may be relevant to the actions of antipsychotics. We therefore investigated potential pregnenolone alterations in rat hippocampus and cerebral cortex following clozapine, olanzapine, and other second generation agents as a candidate NS mechanism contributing to antipsychotic efficacy. In the first set of experiments, intact, adrenalectomized, and sham-operated male rats received vehicle or clozapine (20 mg/kg) IP. In the second set, male rats received vehicle, olanzapine (5 mg/kg), quetiapine (20 mg/kg), ziprasidone (10 mg/kg) or aripiprazole (5 mg/kg) IP. Pregnenolone levels were determined by gas chromatography/mass spectrometry. Clozapine markedly elevates pregnenolone in rat hippocampus, cerebral cortex, and serum; hippocampal levels were strongly correlated with serum levels (r=0.987). Olanzapine also elevates pregnenolone levels, but to a lesser degree than clozapine. Pregnenolone induction may contribute to the clinical actions of clozapine and olanzapine.

3α-Hydroxy-5α-pregnan-20-one levels and GABAA receptor-mediated 36Cl− flux across development in rat cerebral cortex

Despite considerable evidence showing dramatic changes in the plasticity of GABAA receptors during neuronal development and studies showing a direct link between neurosteroid concentrations and alterations in GABAA receptor expression, little is known about the role of neurosteroids in GABAA receptor plasticity early in development. The relationship between changes in brain concentrations of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, 3α,5α-THP) and GABAA receptor function in the brain during early development was investigated in rats. The concentration in fetal forebrain of the pregnane metabolite 3α,5α-THP declined precipitously prior to parturition, before returning to normal (adult male) values on the day of birth (P0). Postnatal cortical 3α,5α-THP levels remain quite low (<2 ng/g) until postnatal day 10 (PD10) and PD14 when we found elevated cortical 3α,5α-THP levels (3.3±0.8 and 3.8±0.9 ng/g, respectively). These levels reverted to basal values by PD15 (0.56±0.4 ng/g). We examined GABAA receptor-mediated 36Cl− flux in cortex of PD7, PD12 and PD16 rat brain. We found a 32% reduction in the stimulation (apparent Emax) of 36Cl− uptake by muscimol in PD12 tissue relative to adult. The potentiating effects of 3α,21-dihydroxy-5α-pregnane-20-one (tetrahydrodeoxycorticosterone, THDOC) and flunitrazepam were decreased in PD12 tissue. These data provide a better understanding of potential contributions endogenous GABAergic neurosteroids may make to normal neuronal development.

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.

Neonatal neurosteroid administration alters parvalbumin expression and neuron number in medial dorsal thalamus of adult rats

The neuroactive steroid 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone) is a potent endogenous modulator of GABAA receptor function. A single neonatal allopregnanolone administration (10 mg/kg, i.p.) was previously shown to alter the localization of parvalbumin-positive neurons in the prefrontal cortex at maturity. Cells in the prefrontal cortex receive the majority of their inputs from the medial dorsal nucleus of the thalamus. We investigated whether neonatal allopregnanolone administration alters the neuronal population of the medial dorsal nucleus of the thalamus. We show that the number of parvalbumin-expressing neurons is increased while the total neuron number is decreased in the medial dorsal nucleus after allopregnanolone administration. EAAT3 (excitatory amino acid transporter type 3, the neuron-specific glutamate reuptake transporter) immunoreactivity was unchanged in adjacent sections. These findings suggest that neonatal allopregnanolone administration disrupts the normal development of the prefrontal cortex and medial dorsal thalamus, indicating that neurosteroid levels are important for proper development of thalamocortical systems and may play a role in neurodevelopmental disorders such as schizophrenia.

Cortical 3α-hydroxy-5α-pregnan-20-one levels after acute administration of Δ9-tetrahydrocannabinol, cocaine and morphine

RationaleThe neuroactive steroid, 3α-hydroxy-5α-pregane-20-one (allopregnanolone) is a potent modulator of GABAA receptor function. Moreover, pharmacologically relevant concentrations of allopregnanolone are found in brain during physiological conditions (stress, pregnancy and menstrual cycle) and pharmacological challenge (ethanol, fluoxetine, olanzapine). Enhanced levels of neurosteroids are thought to contribute to the therapeutic effects of fluoxetine and various effects of ethanol via GABAA receptors. Moreover, neurosteroids influence rewarding effects of ethanol in some models and modulate activation of the hypothalamic pituitary adrenal (HPA) axis. Thus, it is possible that enhanced allopregnanolone levels are involved in the effects of abused drugs.ObjectivesTo determine if other abused drugs elicit alterations in brain neurosteroid levels, Δ9-tetrahydrocannabinol (Δ9-THC), cocaine and morphine were administered to male rats.MethodsCortical brain tissue and plasma were collected and analyzed for steroid concentrations using radioimmunoassays.ResultsΔ9-THC (5 mg/kg, IP) elevated cortical allopregnanolone levels to pharmacologically active levels, while morphine (15 mg/kg, SC) produced a small but significant increase. Cocaine (30 mg/kg, IP) did not alter allopregnanolone levels, nor did lower doses of Δ9-THC or morphine. Plasma progesterone levels were elevated in both Δ9-THC and cocaine-treated animals.ConclusionsSome, but not all, drugs of abuse produce increases in cortical allopregnanolone levels. In addition, increases in plasma steroid precursor levels do not always translate into increases in brain allopregnanolone levels.

3α-Hydroxy-5α-pregnan-20-one exposure reduces GABAA receptor α4 subunit mRNA levels

Abstract To examine the direct effects of neurosteroids on γ-aminobutyric acid type A (GABA A ) receptor expression, we exposed developing neuronal cells (P19) in vitro to 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP, allopregnanolone). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed a concentration-dependent decrease in GABA A receptor α4 subunit mRNA expression that reversed 24 h after steroid withdrawal. These data suggest that variations in neurosteroid levels regulate the pattern of GABA A receptor subunit expression and may alter the trophic effects of GABA.

Fluorescence imaging of GABAA receptor-mediated intracellular [Cl−] in P19-N cells reveals unique pharmacological properties

This study describes the pharmacological properties of GABAA receptors expressed in P19-N cells using fluorescence imaging of intracellular chloride with 6-methoxy-N-ethylquinolinium iodide (MEQ). We show that application of the GABA agonist, muscimol (10-200 microM), produces time- and concentration-dependent increases in intracellular [Cl-] that are blocked by bicuculline. Diazepam (10 microM) and pentobarbital (1 mM) potentiate muscimol-stimulation. These receptors exhibit novel pharmacological properties. The neurosteroid, 3alpha-hydroxy-5alpha-pregnane-20-one (1-10 microM) exhibited weak potency in enhancement of muscimol-stimulation. Ethanol (50 and 100 mM) exhibited high efficacy on muscimol responses, a 4- to 5-fold potentiation, respectively, of muscimol (10 microM) alone. GABA and muscimol allosterically modulated specific binding of [3H]flunitrazepam to differentiated P19 cells. Modulation of GABAA receptor mediated increases in intracellular [Cl-] demonstrated stability in response magnitude from 7 to 15 days following removal of retinoic acid. In concert, GABAA receptor subunit mRNA and protein expression patterns in these neuron-like cells were stable over the same period. Using RT-PCR we determined that differentiated P19 cells lack gamma1, gamma2L, alpha6 and delta subunit mRNAs while expressing alpha1, alpha2, alpha3, alpha4, alpha5, beta1, beta2, beta3, gamma2S and gamma3. Furthermore, subunit specific antibody immunocytochemical labeling of cells with a neuronal morphology indicated the presence of alpha1, alpha2, alpha4, and gamma2 subunits (the only subunits tested). Therefore, P19-N cells should prove useful to researchers in need of a model cell culture system in which to study function and regulation of neuronal GABAA receptors.

Perinatal flunitrazepam exposure causes persistent alteration of parvalbumin-immunoreactive interneuron localization in rat prefrontal cortex.

GABA regulates proliferation via GABAA receptors during development of the neocortex. We recently demonstrated that the endogenous GABAA receptor modulator allopregnanolone plays a role in regulating normal neurodevelopment in prefrontal cortex. Benzodiazepine exposure during early development produces marked behavioral changes in adult rats. To determine if exposure to benzodiazepines during development alters GABAergic interneurons in prefrontal cortex (PFC), rat pups were exposed to flunitrazepam (2.5 mg/kg) on postnatal day (P) 2 and assayed for parvalbumin- and calbindin-immunoreactivity on P80. The ratio of parvalbumin labeled cells in deep vs. superficial layers increased five-fold; calbindin-immunoreactivity and total cell number were not altered. These data are consistent with altered distribution of a subset of interneurons after benzodiazepine exposure and suggest a role for GABAA receptor modulation in normal development of GABAergic systems in PFC.