Neurosteroids act on recombinant human GABAA receptors.

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Correlation of aromatic hydroxylation of 11 beta-substituted estrogens with morphological transformation in vitro but not with in vivo tumor induction by these hormones.

In estrogen-induced cancer, catechol formation from administered steroids has been postulated to be a necessary event for estrogen activation and subsequent damage to cellular macromolecules. In the present study, this hypothesis has been tested using two homologous series of structurally related estrogens: estradiol, 11 beta-methylestradiol, 11 beta-ethylestradiol, 11 beta-methyl-17 alpha-ethinylestradiol, 11 beta-ethyl-17 alpha-ethinylestradiol, 11 beta-methoxy-17 alpha-ethinylestradiol, and 17 alpha-ethinylestradiol. In the Syrian hamster renal carcinoma model, only 11 beta-methylestradiol and 17 alpha-ethinylestradiol were weak carcinogens (2 of 20 and 2 of 24 hamsters with tumors, respectively). The other estrogens tested induced renal carcinoma within 6 to 9 months with an incidence in the 80-100% range. The tumor incidence in vivo did not correlate with the rates of catechol formation by hamster kidney microsomes in vitro. Compared to estradiol (relative rate, 100), catechol formation by the substituted estrogens was significantly lower, ranging from 48 (11 beta-methylestradiol) to 2 (11 beta-methoxy-17 alpha-ethinylestradiol). Kidney DNA of hamsters treated with the four 17 alpha-ethinyl estrogens, when analyzed by 32P postlabeling assay, contained the same set of covalently modified nucleotides the formation of which had previously been found to precede estrogen-induced renal carcinogenesis in vivo. In contrast, relative rates of catechol estrogen formation by BALB/c 3T3 microsomes correlated with induction of morphological transformation of BALB/c 3T3 cells and decreased in the following order: 11 beta-methylestradiol greater than 17 alpha-ethinylestradiol greater than or equal to estradiol greater than 11 beta-ethylestradiol greater than 11 beta-methoxy-17 alpha-ethinylestradiol. The hormonal potencies of several estrogen derivatives studied by various assays did not correlate with in vivo carcinogenic or in vitro cell-transforming activities. It is concluded from these experiments that in cell culture catechol formation and morphological transformation are directly related. In vivo, aromatic hydroxylation of administered estrogens did not correlate with the incidence of estrogen-induced renal carcinoma in Syrian hamsters.

Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self-administration in rats.

This study was designed to compare the effects of several neuroactive steroids with varying patterns of modulation of gamma-aminobutyric acid (GABA)(A) and NMDA receptors on operant self-administration of ethanol or water. Once stable responding for 10% (w/v) ethanol was achieved, separate test sessions were conducted in which male Wistar rats were allowed to self-administer ethanol or water following pre-treatment with vehicle or one of the following neuroactive steroids: (3beta,5beta)-3-hydroxypregnan-20-one (epipregnanolone; 5, 10, 20 mg/kg; n=12), (3alpha,5beta)-20-oxo-pregnane-3-carboxylic acid (PCA; 10, 20, 30 mg/kg n=10), (3alpha,5beta)-3-hydroxypregnan-20-one hemisuccinate (pregnanolone hemisuccinate; 5, 10, 20 mg/kg; n=12) and (3alpha,5alpha)-3-hydroxyandrostan-17-one hemisuccinate (androsterone hemisuccinate; 5, 10, 20 mg/kg; n=11). The effect of the 3beta-epimer of PCA, (3beta,5beta)-20-oxo-pregnane-3-carboxylic acid (10, 20, 30 mg/kg; n=9), on ethanol self-administration was also examined. The compounds were administered using a Latin-square design 45 min prior to the weekly test sessions. The effects of the 30 mg/kg dose of the steroidal hemisuccinates on ethanol intake were also examined 5 min after administration of these drugs. Both epipregnanolone and PCA attenuated ethanol self-administration. However, neither of the hemisuccinate compounds significantly altered this behavior. The steroidal hemisuccinates (30 mg/kg; n=7) were also tested 5 min before behavior testing and had no effect on ethanol intake 5 min after administration. The 3beta-epimer of PCA also failed to alter ethanol intake. None of the test compounds altered water intake. In electrophysiological studies, the effects of PCA and androsterone hemisuccinate on evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) was examined in brain slices of the amygdala. PCA had a stimulatory effect at concentrations of 5 and 25 muM. Androsterone hemisuccinate had no agonist activity. The ability of epipregnanolone and PCA to alter ethanol intake appears to be related to different inhibitory actions of these compounds on either GABA(A) or NMDA receptors, respectively. Thus, dual modulation of these systems by selected neuroactive steroids may offer potential for modifying the reinforcing effects of alcohol.

Relationship between symptom severity and steroid variation in women with premenstrual syndrome: study on serum pregnenolone, pregnenolone sulfate, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one.

The relationship between symptoms of premenstrual syndrome (PMS) and serum levels of pregnenolone (Pe), pregnenolone sulfate (PS), 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha- pregnan-20-one (5 alpha-THP), LH, 17 beta-estradiol (E2), and progesterone (P) was investigated during 2 consecutive menstrual cycles in 12 patients using daily measurements. Corresponding hormones were also measured during 1 cycle in 8 control women. Pe, PS, 5 alpha-DHP, and 5 alpha-THP showed a significant cyclicity within menstrual cycles and a high rate of correlation with P variation in both PMS patients and controls. No significant difference was found between PMS patients and controls in average serum concentrations of Pe, PS, 5 alpha-DHP, 5 alpha-THP, and LH during the luteal phase, whereas a significantly higher level of E2 and a lower level of P were observed in PMS patients. The variation in symptom scores was compared with that in hormone levels within each woman. The symptom peak showed a delay of 3-4 days after the serum P, Pe, 5 alpha-DHP, and 5 alpha-THP peaks. However, the plasma PS peak appeared on the same day or only 1 day before the symptom peak in PMS patients. When comparing the 2 cycles studied, more negative symptoms occurred in cycles with higher luteal phase E2, Pe, and PS concentrations, whereas higher luteal phase 5 alpha-DHP and 5 alpha-THP concentrations were associated with improved symptom ratings in PMS patients. These results suggest that the mentioned steroids are related to the severity of distressing symptoms in PMS patients.

Circulating levels of anxiolytic steroids in the luteal phase in women with premenstrual syndrome and in control subjects.

The levels of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and the epimeric 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone) were studied in women with prospectively confirmed premenstrual syndrome (n = 15) and in a group of asymptomatic control women (n = 12) during the luteal phase of the menstrual cycle. Single late luteal phase plasma samples were selected to make comparisons of plasma hormone levels between patients and controls in the following measures: allopregnanolone, pregnanolone, the ratio of allopregnanolone to pregnanolone, the ratio for each of these anxiolytic steroids to the parent compound progesterone, and the ratio of the sum of allopregnanolone and pregnanolone to progesterone. Differences in these measures were compared by analysis of variance. Additionally, correlations were performed among the various hormone measures and between the hormone measures and the symptom self-ratings. Analysis of variance showed no significant between group differences in the plasma levels of allopregnanolone, pregnanolone, and progesterone. Plasma levels of both allopregnanolone and pregnanolone were correlated with plasma progesterone levels. However, there were no significant correlations between the severity of mood and behavioral symptoms and plasma levels of progesterone, allopregnanolone, and pregnanolone. These data suggest that symptoms of premenstrual syndrome are not associated with a simple deficiency state of either progesterone or its anxiolytic steroid metabolites.

Circulating levels of allopregnanolone in humans: gender, age, and endocrine influences.

Allopregnanolone is a neuroactive steroid involved in modulating behavioral functions, stress, and neuroendocrine axes in rats. Changes in plasma allopregnanolone levels throughout the menstrual cycle have been reported in healthy women, but there exists no information on the possible gender or age-related changes or on the source(s) of circulating allopregnanolone. The aim of the present study was to assess serum allopregnanolone concentrations according to gender, menstrual cycle, age, and menopause in normal men and women; serum progesterone (P) and dehydroepiandrosterone (DHEA) levels were evaluated in the same specimens. In addition, the possible source of circulating allopregnanolone in fertile women was investigated by using stimulatory and inhibitory endocrine tests acting on the ovary and/or adrenal cortex. The present study included 189 fertile women, 112 postmenopausal women, and 46 men. Serum steroid levels were determined after extraction, using specific RIAs. Allopregnanolone levels in fertile women in the follicular phase were similar to those in age-matched men; no significant difference was found between fertile women in the follicular phase and postmenopausal women. The highest levels were found in fertile women during the luteal phase (P < 0.01). An age-related decrease was observed in men (P < 0.01), but not in women. P and DHEA levels were significantly higher in women than in men and were higher in fertile women than in postmenopausal women (P < 0.01). Both P and DHEA showed an age-related decrease in men and women (P < 0.01). Serum allopregnanolone and P, but not DHEA, significantly increased in response to a GnRH test, whereas corticotropin-releasing factor and ACTH tests elicited a significant increase in allopregnanolone, P, and DHEA levels (P < 0.01). The suppression of adrenal steroidogenesis by dexamethasone markedly reduced both allopregnanolone and DHEA serum levels (P < 0.01). In conclusion, the present study demonstrated that although men show an age-related decrease, serum allopregnanolone levels in women do not change with age and correlate with P levels during the menstrual cycle and in response to endocrine tests. Ovary and adrenal cortex may be major sources of circulating allopregnanolone in fertile women.

Clozapine, but not haloperidol, increases brain concentrations of neuroactive steroids in the rat.

The extrapyramidal side effects of typical antipsychotics, which are induced to a markedly reduced extent by clozapine, have been linked to a dysfunction of central gamma-aminobutyric acid (GABA)-mediated neurotransmission. The effects of clozapine on the brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, AP) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), two potent and endogenous positive allosteric modulators of GABA-mediated chloride current intensities at GABA(A) receptors, were compared with those of the typical antipsychotic haloperidol. A single administration of clozapine (1.25-20 mg/kg, IP), but not of haloperidol (0.1 or 0.5 mg/kg, IP), induced dose- and time-dependent increases in the concentrations of progesterone, AP, and THDOC in the cerebral cortex and striatum of rats. Clozapine (at 10 mg/kg, but not at lower doses) also increased the concentrations of these steroids as well as that of corticosterone in plasma in intact rats, but failed to increase the cortical concentrations of AP and THDOC in adrenalectomized-orchidectomized rats. An acute challenge with clozapine (10 mg/kg), administered 48 h after the termination of daily treatment with the same dose for 19 days, still increased the cortical concentrations of progesterone, AP, and THDOC. These results suggest that the clozapine-induced increases in neuroactive steroid concentrations in the brain may contribute to the atypical pharmacological profile of this antipsychotic drug.

Steroidogenesis in rat brain induced by short- and long-term administration of carbamazepine.

Although carbamazepine (CBZ) is used therapeutically in the treatment of various neurological and psychiatric conditions, its mechanism of action remains largely unknown. CBZ has now been shown to inhibit the binding of [(3)H]PK 11195 to peripheral benzodiazepine receptors (PBRs) in rat brain and ovary membranes in vitro with a potency (IC(50), approximately 60 microM) much lower than that of unlabeled PK 11195 (IC(50), approximately 2.0 nM). Administration of CBZ to rats induced dose (25 to 100 mg/kg, i.p.) and time (15 to 60 min) dependent increases in the concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone in both the cerebral cortex and plasma. CBZ also induced steroidogenesis in the brain of adrenalectomized-orchiectomized rats, suggesting that this effect is mediated in a manner independent of peripheral PBRs. The increase in brain concentrations of neuroactive steroids induced by a single injection of CBZ was associated with a marked protective effect against isoniazid-induced convulsions. In contrast, long-term administration of CBZ (50 mg/kg, twice a day for 30 days) induced tolerance to the anticonvulsant action of the drug. This same treatment, however, did not prevent the ability of a challenge dose of CBZ to stimulate steroidogenesis. These results indicate that CBZ-induced steroidogenesis might not be responsible for the anticonvulsant activity of this drug.

Isoniazid-induced inhibition of GABAergic transmission enhances neurosteroid content in the rat brain.

Isoniazid (375 mg/kg, s.c.), a drug that decreases GABAA receptor-mediated transmission, elicited a time-dependent increase of neuroactive steroid (pregnenolone, progesterone and allotetrahydrodeoxycorticosterone) concentrations in rat brain and plasma. This treatment also time-dependently increased the plasma concentration of corticosterone. Brain and plasma neuroactive steroid levels peaked between 40 and 120 min after isoniazid administration, respectively, and returned to control values by 5 hr. Acute foot shock stress mimicked the effect of isoniazid by increasing in a time-dependent manner the same neuroactive steroids both in brain and plasma. Abecarnil (0.3 mg/kg, i.p.), a beta-carboline derivative with anxiolytic properties, antagonized the effect of both isoniazid and foot shock on brain and plasma neuroactive steroids and on plasma corticosterone level. These data indicate that an inhibition of central GABAergic transmission enhances the concentrations of THDOC and its precursors pregnenolone and progesterone in the rat brain and plasma as well as the plasma levels of corticosterone. This finding suggests that GABA exerts a tonic inhibitory action on the mechanisms involved in the regulation of the synthesis and release of these neuroactive steroids in the central nervous system and plasma.

Changes in GABAA receptor gamma 2 subunit gene expression induced by long-term administration of oral contraceptives in rats.

The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.

GABA(B) receptor-mediated increase of neurosteroids by gamma-hydroxybutyric acid.

Among the pharmacological actions of gamma-hydroxybutyric acid (GHB), some may involve GABA(A) receptor-mediated mechanisms. GHB, however, fails to directly interact with sites for agonists and modulators on the GABA(A) receptor complex. We hypothesized that, in vivo, GHB may interfere with GABA(A) receptor function by altering the brain concentrations of the neurosteroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, AP) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), positive allosteric modulators of GABA-gated chloride currents. In male Wistar rats, GHB dose-dependently (75-1000 mg/kg, i.p.) increased AP, THDOC and their precursors pregnenolone and progesterone in brain cortex and hippocampus. The increases of AP (4-5 fold) and THDOC (3-4 fold) elicited by 300 mg/kg GHB peaked between 30 and 90 min and abated by 180 min. The selective GABA(B) receptor antagonist SCH 50911 (50 mg/kg, i.p.) prevented the action of GHB, while the GABA(B) receptor agonist baclofen (5-10 mg/kg) mimicked it. NCS-382 (50 mg/kg, i.p.), the purported selective antagonist of the GHB receptor, failed to antagonize GHB, but at 300 mg/kg increased brain cortical neurosteroids to the same extent as 300 mg/kg GHB; coadministration of GHB and NCS-382, however, failed to yield an additive effect. These results strongly suggest that GHB, via a GABA(B) receptor-mediated mechanism, increases the brain concentrations of neurosteroids, whose properties as amplifiers of the GABA-gated chloride conductances may play a role in the GABA(A) receptor-mediated pharmacological actions of GHB.

Synthesis, metabolism, and pharmacological activity of 3 alpha-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes.

Certain 3 alpha-hydroxy steroids have recently been shown to bind to the gamma-aminobutyric acid (GABA) receptor gated chloride ion channel with high affinity and to potentiate the inhibitory effects of GABA when measured both in vitro and in vivo. In the present study, a series of natural and synthetic 3 alpha-hydroxy steroids were tested for their ability to potentiate GABA-receptor-mediated chloride ion (Cl-) uptake into cerebral cortical synaptoneurosomes. The naturally occurring metabolites 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC) were found to be the most active in augmenting GABAA-receptor-mediated Cl- uptake. Pharmacological activity was reduced in the corresponding isomers with the 5 beta-pregnane configuration and by some, but not all, modifications of the side chain. The ability of these steroids to potentiate muscimol-stimulated Cl- uptake is lost by acetylation at C3, introduction of unsaturation at C9(11), inversion to the 3 beta-hydroxy isomer, or inversion of configuration at C17. A facile procedure is reported for the synthesis of unlabeled and tritium-labeled allopregnanolone and allotetrahydroDOC. The 9 alpha,11 alpha,12 alpha-3H-labeled derivatives of allopregnanolone and allotetrahydroDOC were used to identify the distribution and metabolic products of these active steroids. Uptake of the more hydrophobic [3H]allopregnanolone into brain was significantly greater than that of [3H]allotetrahydroDOC. The principal 3H-labeled metabolites recovered from brain were the 3-ketone derivatives of allopregnanolone and allotetrahydroDOC, which are both inactive on GABA-receptor-mediated Cl- flux. Molecular modeling of the active steroids based on quantitative structure-activity relationships provides evidence to support the stereospecificity of the binding interactions and suggests that there may be more than one type of steroid binding site associated with the GABAA-receptor-mediated chloride ionophore.

The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations.

1. This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro-cortical content of 3 alpha-hydroxy-5 alpha-pregnan-20 one (allopregnanolone, AP), a potent endogenous positive modulator of 7-aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. 2. An acute depletion of the cerebral content of GABA or an attenuation of GABAA receptor-mediated transmission by systemic injections of isoniazid (375 mg kg-1, s.c.) or FG 7142 (15 mg kg-1, i.p.) induced a transient increase in the cerebro-cortical and plasma concentrations of AP in handling-habituated (not stressed) rats. 3. Two stress paradigms, handling in naive rats and mild foot shock in handling-habituated rats, that reduce central GABAergic tone mimicked the effects of isoniazid and FG 7142 on cortical AP content; foot shock in handling-habituated rats, but not handling in naive animals, also increased plasma AP. Isoniazid, FG 7142, and foot shock also each increased the concentrations of the AP precursors, pregnenolone and progesterone, in both brain and plasma of handling-habituated rats, whereas handling in naive rats increased the concentrations of these steroids only in brain. 4. Pretreatment of handling-habituated rats with the anxiolytic beta-carboline derivative abecarnil, a positive allosteric modulator of GABAA receptors, which per se failed to affect the AP concentration in brain or plasma, prevented the increase in brain and plasma AP induced by foot shock or isoniazid. 5. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. 6. These observations indicate that inhibition of GABAergic transmission, induced by foot shock or pharmacological manipulations, results in an increase in the concentrations of AP in brain and plasma, possibly via a modulation of hypothalamic-pituitary-adrenal (HPA) axis. 7. Given that AP enhances GABAA receptor function with high efficacy and potency, an increase in brain AP concentration may be important in the fine tuning of the GABA-mediated inhibitory transmission in the central nervous system.

2-Phenyl-imidazo[1,2-a]pyridine derivatives as ligands for peripheral benzodiazepine receptors: stimulation of neurosteroid synthesis and anticonflict action in rats.

Selective activation of peripheral benzodiazepine receptors (PBRs) in adrenal cells and brain oligodendrocytes promotes steroidogenesis. Three 2-phenyl-imidazo[1,2-a]pyridine derivatives (CB 34, CB 50 and CB 54) have now been investigated with regard to their selectivity for PBRs and their ability to stimulate central and peripheral steroidogenesis in rats. The three CB compounds (10(-10)-10(-4) M) potently inhibited the binding of the PBR ligand [3H]-PK 11195 to brain and ovary membranes in vitro, without substantially affecting [3H]-flunitrazepam binding to central benzodiazepine receptors. These compounds (10(-7)-10(-4) M) also had little or no marked effects on GABA-evoked Cl- currents in voltage-clamped Xenopus oocytes expressing human alpha1beta2gamma2S GABA(A) receptors. In addition, they failed to affect ligands binding to GABA(B), D1/D2 dopamine, muscarinic acetylcholine, N-methyl-D-aspartic acid and opiate receptors. Intraperitoneal administration of CB compounds (3-50 mg kg(-1)) induced a dose-dependent increase in the concentrations of neuroactive steroids in plasma and brain. The brain concentrations of pregnenolone, progesterone, allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC) showed maximal increases in 96+/-3, 126+/-14, 110+/-12 and 70+/-13% above control, respectively, 30 to 60 min after injection of CB 34 (25 mg kg(-1)). CB 34 also increased the brain concentrations of neuroactive steroids in adrenalectomized-orchiectomized rats, although to a lesser extent than in sham-operated animals, suggesting that CB compounds stimulate brain steroidogenesis independently of their effects on peripheral tissues. The increase in brain and plasma neurosteroid content induced by CB 34 was associated with a marked anticonflict effect in the Vogel test. Our results indicate that the three CB compounds tested are specific and potent agonists at peripheral benzodiazepine receptors, and that they stimulate steroidogenesis in both the brain and periphery.

Functional correlation between allopregnanolone and [35S]-TBPS binding in the brain of rats exposed to isoniazid, pentylenetetrazol or stress.

1. The relation between changes in the cerebral cortical concentration of allopregnanolone and gamma-aminobutyric acid (GABA) type A receptor function after intracerebroventricular injection of this neurosteroid was investigated in male rats. 2. Intracerebroventricular administration of allopregnanolone (1.25 to 15 micrograms) produced a maximal increase (100 fold at the highest dose) in cortical allopregnanolone concentration within 5 min; the concentration remained significantly increased at 15 and 30 min, before returning to control values by 60 min. 3. The same treatment induced a rapid and dose-dependent decrease in the binding of t-[35S]-butylbicyclophosphorothionate ([35S]-TBPS) to cerebral cortical membranes measured ex vivo, an effect mimicked by the benzodiazepine midazolam but not by the 3 beta-hydroxyepimer of allopregnanolone. The time course of changes in [35S]-TBPS binding paralleled that of brain allopregnanolone concentration. 4. In a dose-dependent manner, allopregnanolone both delayed the onset of convulsions and inhibited the increase in [35S]-TBPS binding to cortical membranes induced by isoniazid. The potency of allopregnanolone in inhibiting [35S]-TBPS binding in isoniazid-treated rats was approximately four times that in control animals. 5. The ability of allopregnanolone to decrease [35S]-TBPS binding in isoniazid-treated rats also correlated with its anticonvulsant activity against pentylenetetrazol-induced seizures as well as its inhibitory effect on the increase in [35S]-TBPS binding induced by foot shock. 6. The results indicate that the in vivo administration of allopregnanolone enhances the function of GABAA receptors in rat cerebral cortex and antagonizes the inhibitory action of stress and drugs that reduce GABAergic transmission.

Aging is associated with changes in allopregnanolone concentrations in brain, endocrine glands and serum in male rats.

OBJECTIVE: Allopregnanolone is a potent neuroactive steroid hormone produced in the brain and in peripheral endocrine glands. The present study investigated possible age-related variations in allopregnanolone content in brain areas, endocrine glands and serum of male rats. DESIGN: Wistar male rats were categorized into 5 groups (6 rats in each) according to age: 6, 12, 16, 18 and 20 months respectively. METHODS: Allopregnanolone content in acidic homogenates of brain cortex, hypothalamus, pituitary, adrenals and gonads was measured by a specific radioimmunoassay. Serum allopregnanolone, corticosterone and testosterone were also assayed by radioimmunoassay. RESULTS: Brain cortex allopregnanolone content decreased significantly with age, while hypothalamic allopregnanolone content remained constant until 18 months and increased significantly at 20 months. Pituitary content showed a significant age-related reduction. Adrenal allopregnanolone content remained constant until 18 months, and was significantly higher at 20 months. Testis and serum allopregnanolone contents showed significant age-related increases. Serum testosterone levels showed an age-related decrease, while no age-related variation in serum corticosterone was found. CONCLUSIONS: The present study showed a significant impact of aging on allopregnanolone contents in brain, endocrine glands and serum, showing an age-related decrease in brain cortex and pituitary, and an age-related increase in testes, adrenals and serum.

Discriminative stimulus effects of ethanol and 3 alpha-hydroxy-5 alpha-pregnan-20-one in relation to menstrual cycle phase in cynomolgus monkeys (Macaca fascicularis).

The present study was designed to characterize the discriminative stimulus effects of ethanol and the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in nonhuman primates as a function of menstrual cycle phase. Female cynomolgus monkeys (Macaca fascicularis) were trained in a two-lever procedure to discriminate 1.0 g/kg ethanol (IG, 30 min pretreatment) from water using food reinforcement. A cumulative dosing procedure was used to assess changes in the potency of ethanol and an endogenous anxiolytic steroid in the follicular versus the luteal phase of the menstrual cycle. Plasma progesterone and allopregnanolone levels were determined within 24 h of testing to verify phase of menstrual cycle. The monkeys were more sensitive to the discriminative stimulus effects of ethanol and the ethanol-like effects of the endogenous neuroactive steroid allopregnanolone during the luteal phase of the menstrual cycle. These findings suggest that changes in the endogenous levels of ovarian-derived progesterone and allopregnanolone alter sensitivity to the discriminative stimulus effects of ethanol.

Comparison of the neurophysiological effects of allopregnanolone and ethanol in rats.

RATIONALE: The central nervous system actions of allopregnanolone (3 alpha-hydroxy-5 alpha-pregnan-20-one) and ethanol are at least partially mediated by modulation of gamma-aminobutyric acid (GABA)-A receptors. Although ethanol and allopregnanolone have similar behavioral effects, their macro-electrophysiological profiles have not been directly compared. OBJECTIVE: The purpose of this study was to compare the effects of allopregnanolone and ethanol on the electroencephalogram (EEG) and event-related potentials (ERPs). METHODS: Male Wistar rats were implanted with cortical and amygdalar electrodes. The rats were then administered allopregnanolone (0.0-10 mg/kg), ethanol (0.0-1.0 g/kg), or a combination of the two before recording. RESULTS: Allopregnanolone and ethanol had similar effects on ERPs. When administered alone, both decreased cortical P1-N1 ERP amplitude by 25-50% and N1 amplitude in the amygdala by 75-80%. Combined administration of ethanol (0.50 g/kg) and allopregnanolone (5.0 mg/kg), doses which were ineffective alone, decreased N1 amplitude in the amygdala by 60%. Allopregnanolone and ethanol had dissimilar EEG effects. Allopregnanolone increased high frequency power in the cortex and amygdala by 25-30%. Ethanol decreased cortical and amygdalar power in the same high frequency bands by 25-45%. Allopregnanolone, but not ethanol, also shifted cortical frequency in the 32- to 50-Hz band. Combined administration of allopregnanolone and ethanol had no effect on EEG power but enhanced allopregnanolone's effect on cortical frequency. CONCLUSIONS: These data suggest that allopregnanolone's macro-electrophysiological profile resembles barbiturates and benzodiazepines more than ethanol. Further, the interactions of allopregnanolone and ethanol appear complex, with multiple effects observed (enhancement or reversal) depending on the neurophysiological variable assessed.

Ethanol-like discriminative stimulus effects of the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one in female Macaca fascicularis monkeys.

The present study was designed to characterize the discriminative stimulus effects of ethanol and the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in non-human primates. Female cynomolgus monkeys (Macaca fascicularis) were trained in a two-lever procedure to discriminate 1.0 g/kg ethanol (IG, 30 min pretreatment) from water using food reinforcement. Consistent with previous results in a variety of species, pentobarbital (0.56-17 mg/kg, IG) resulted in a dose-dependent substitution for the discriminative stimulus effects of ethanol, with an average ED50 value of 1.9 mg/kg. Administration of allopregnanolone (0.3-5.6 mg/kg, IV) also produced complete substitution for the discriminative stimulus effects of ethanol, with an ED50 value of 1.0 mg/kg. Plasma allopregnanolone levels 35 min following the administration of 3.0 mg/kg allopregnanolone ranged from 33 to 69 ng/ml. The ethanol-like discriminative stimulus effects of 1.0 mg/kg allopregnanolone (IV) were present for 60 min, with a return to complete water-appropriate responding at 90 min post-treatment. The results indicate that the endogenous neuroactive steroid allopregnanolone produces subjective effects in cynomolgus monkeys that are similar to ethanol. These findings suggest that changes in the endogenous levels of allopregnanolone could alter sensitivity to the subjective effects of ethanol.

Opposite effects of short- versus long-term administration of fluoxetine on the concentrations of neuroactive steroids in rat plasma and brain.

RATIONALE: Recent preclinical and clinical studies have shown that selective serotonin re-uptake inhibitors modulate neurosteroid synthesis in an opposite manner. OBJECTIVES: The action of long-term administration of fluoxetine was investigated on the peripheral and central concentrations of 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-TH PROG) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (of 3alpha,5alpha-TH DOC), progesterone, and pregnenolone in rats. We also investigated the effect of chronic treatment with fluoxetine on the foot-shock stress-induced increase in the plasma and brain concentrations of these steroids. METHODS: Fluoxetine was administered acutely (20 mg/kg) or chronically (10 mg/kg, once daily for 15 days). Steroids were extracted from plasma and brain, separated and purified by means of high-performance liquid chromatography, and quantified by means of radioimmunoassay. RESULTS: A single dose of fluoxetine (20 mg/kg, i.p.) induced in 20 min significant increases in the cerebral cortical and plasma concentrations of 3alpha,5alpha-TH PROG (+96% and +13%, respectively), 3alpha,5alpha-TH DOC (+129 and +31%, respectively), progesterone (+111 and +58%, respectively), and pregnenolone (+151 and +59%, respectively). In addition, the plasma concentration of corticosterone was also significantly increased (+24%) after acute administration of fluoxetine. In contrast, long-term administration of fluoxetine reduced the basal concentrations of these various steroids (ranging from -22 to -43%), measured 48 h after the last drug injection, in both brain and plasma. A challenge injection of fluoxetine (20 mg/kg, i.p.), however, was still able to increase the concentrations of steroids in both the brain and plasma of rats chronically treated with this drug. Acute foot-shock stress increased the cortical and plasma concentrations of steroids in rats chronically treated with fluoxetine to extents similar to those apparent in control rats. CONCLUSIONS: A repetitive increase in the brain concentrations of neuroactive steroids may contribute to the therapeutic action of fluoxetine.