INTRODUCTION

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An important feature of -aminobutyric acid type A (GABA_A) receptors in rat brain is their plasticity in response to exposure to brief or long-lasting physiological stimuli or to long-term pharmacological treatments. The kinetic characteristics of the various binding sites located on the GABA_A receptor, as well as the density and function of these receptors in different areas of the rat brain, are affected by acute stressful stimuli (handling, foot shock, swimming), postnatal development, aging, kindling, and long-term administration of anxiolytic, hypnotic, or anticonvulsant drugs (1-6). Although recent studies have suggested that some of these changes may be mediated at the level of expression of receptor subunit genes (7-10), the chemical or molecular events that underlie such rapid, short-term, or persistent changes in the density and affinity of binding sites associated with GABA_A receptors, as well as in the function of the receptor complex, remain to be characterized.

Neurosteroids accumulate in the mammalian brain in a manner that is, at least in part, independent of the peripheral tissues (adrenal glands and gonads) that synthesize these compounds (11). Systemic administration of progesterone or its metabolites 3-hydroxy-5-pregnan-20-one (allopregnanolone, AP) and 3,21-dihydroxy-5-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC) induces anxiolytic, hypnotic, or anticonvulsant effects (for review see ref. 12) by enhancing the function of GABA_A receptors (12, 13). These observations suggest that endogenous variations in the plasma and brain concentrations of these compounds induced by physiological, pharmacological, or pathological conditions might play an important role in modulation of neuronal excitability; such modulation, in turn, might result in alterations in such characteristics as emotional state, sleep pattern, and seizure threshold. Indeed, several studies have indicated that these neuroactive steroids may play a physiological role in aggression, epilepsy, and premenstrual syndrome (14-16). Consistent with these data, changes in the plasma and brain concentrations of neuroactive steroids elicited either by physiological conditions (17, 18) or by pharmacological treatments (12, 19, 20) have been associated with alterations in the behavioral and neurochemical responses linked to central GABA_A receptors.

To clarify the role of the rapid effects of these neuroactive steroids on the GABA_A receptor complex in the physiological modulation of receptor activity, we now have evaluated the possible functional relation between the plasticity of GABA_A receptors and the changes in plasma and brain concentrations of progesterone, AP, and THDOC during pregnancy and after delivery in rats. Furthermore, we examined whether administration of finasteride, a selective blocker of 5-reductase (21), at a dose that markedly reduces the concentrations of AP and THDOC in plasma and brain was able to reverse the changes in GABA_A receptor function and structure elicited by pregnancy.

	METHODS

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Animals. Adult female Sprague-Dawley rats (200-250 g; Charles River Breeding Laboratories) were studied. The animals were housed under an artificial 12-hr-light, 12-hr-dark cycle with lights on at 0800 hr.

Steroid Extraction and Assay. Animals were killed either by guillotine (for plasma steroid measurements) or by focused microwave irradiation (70 W/cm² for 4 s) to the head (for brain steroid measurements). Blood was collected from the trunk into heparinized tubes and centrifuged at 900 g for 20 min, after which the plasma was frozen until assayed for steroids.

³⁶Cl Uptake. Membrane vesicles were prepared from rat cerebral cortex as described (23). Portions (100 µl) of the vesicle preparation (1.4-1.8 mg of protein) were preincubated at 30°C for 10 min, after which ³⁶Cl influx was initiated by the addition of 100 µl of a solution containing ³⁶Cl (2 µCi/ml) in the absence or presence of muscimol. Uptake was terminated after 5 s by the addition of ice-cold buffer (3.5 ml) and rapid vacuum filtration through Whatman GF/C filters (presoaked with 0.05% polyethyleneimine to reduce nonspecific binding of ³⁶Cl) in a filtration manifold. The filters were washed twice with 3.5 ml of ice-cold buffer. Net uptake was calculated by subtracting ³⁶Cl uptake observed in the absence of muscimol (basal) from that observed in its presence. Protein concentration was measured as described (24) with BSA as standard.

Probe Preparation. Total RNA was extracted from rat brain (25) and subjected to reverse transcription with SuperScript reverse transcriptase (Life Technologies, Gaithersburg, MD) in the presence of oligo(dT). The resulting cDNA (1-10 ng) then was subjected to PCR as previously described (25) with primers designed to amplify GABA_A receptor subunit cDNA sequences that exhibit the least degree of intersubunit homology. The primers for the 2L subunit were designed to encompass a region of DNA including the 24 bp that differ between 2L and 2S (nucleotides 832-1369) (26). The PCR generated two fragments (537 and 513 bp) corresponding respectively to part of the cDNA sequence of the 2L and 2S subunits of the GABA_A receptor. The two reaction products were separated by electrophoresis on a 1.8% low melting point agarose gel, visualized by staining with ethidium bromide, excised from the gel, purified and inserted into the pAMP1 cloning vector (Life Technologies). The nucleotide sequences of the PCR products were 100% identical to those of the respective previously determined subunit DNA sequences. These two different clones were used to generate two specific probes, one selective for the 2L and the other specific for the 2S mRNA subunits of the GABA_A receptor.

RNase Protection Assay. RNase protection assays were performed as previously described (25). Total RNA was extracted from rat cerebral cortex or hippocampus and quantified by measurement of absorbance at 260 nm, and portions of 25 µg were subjected to the assay with GABA_A receptor subunit and cyclophilin cRNA probes. RNA-RNA hybrids were subjected to electrophoresis on a sequencing gel containing 5% polyacrylamide and urea and were visualized by autoradiography. The relative amounts of GABA_A receptor subunit and cyclophilin (internal standard) mRNAs were evaluated by densitometry (Bio-Rad GS-700). The data were normalized by dividing the OD value of each subunit-protected fragment by that of the cyclophilin-protected fragment.

Statistical Analysis. The statistical significance of differences was assessed by ANOVA followed by Scheffe's test.

	RESULTS

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Steroid Concentrations in Plasma and Cerebral Cortex. The changes in the concentrations of pregnane steroids in rat cerebral cortex during pregnancy and after delivery, compared with the corresponding concentrations on the day of estrus (control values), are shown in Fig. 1. The concentration of progesterone was increased markedly (9-fold) on day 10 of pregnancy, reached a peak (12-fold) on day 15, was still higher (10-fold) than the estrus value on day 19, and returned to control values immediately before delivery (day 21). The concentrations of pregnenolone and AP were not significantly changed on day 10 of pregnancy, showed a maximal increase (+63 and +208%, respectively) on day 19, and had returned to control values on day 21. The concentration of THDOC in the cerebral cortex remained unchanged during the first 15 days of pregnancy, was significantly increased (+90%) on day 19, and had returned to control values on day 21. The cortical concentrations of all steroids during the postpartum period did not differ from the control values.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Changes in the concentrations of pregnenolone (A), progesterone (B), AP (C), and THDOC (D) in rat cerebral cortex (Left) and plasma (Right) during pregnancy and after delivery. Values are shown for estrus (E); for days 10, 15, 19, and 21 of pregnancy (P); and for 2 days postpartum (PP). Data are expressed as nanograms of steroid either per gram of cortical tissue or per milliliter of plasma, and are means ± SEM of values obtained from 8-10 rats. *, P < 0.05; **, P < 0.01 vs. estrus.

³⁶Cl Uptake. The functional state of the GABA_A receptor-coupled Cl channel during pregnancy and after delivery was investigated by measuring ³⁶Cl uptake by cortical membrane vesicles. As expected (23), muscimol (5 µM) stimulated (+125%) ³⁶Cl uptake by membrane vesicles prepared from female rats killed during estrus (Fig. 2). The stimulatory effect of muscimol remained unchanged on day 10 of pregnancy, but was reduced on day 15 (21%) and on day 19 (24%) of pregnancy compared with the estrus value. In contrast, the efficacy of muscimol in stimulating ³⁶Cl uptake was increased (+32%) 2 days after delivery, before returning to control values by 7 days after delivery. A similar pattern of stimulation also was observed in the presence of lower (1 µM) or higher (10 and 50 µM) concentrations of muscimol (Fig. 3).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effects of muscimol on 36Cl uptake by membrane vesicles prepared from the cerebral cortex of rats during pregnancy and after delivery. Membrane vesicles, prepared from rats on the indicated days, were incubated for 10 min at 30°C before the addition of 36Cl in the absence (basal value) or presence of 5 µM of muscimol. Uptake of 36Cl was terminated 5 s later. Data are expressed as the percentage increase in 36Cl uptake induced by 5 µM of muscimol relative to the basal value and are means ± SEM of six different experiments, each performed with two rats per group. *, P < 0.05 vs. estrus.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of muscimol concentration on 36Cl uptake by membrane vesicles prepared from the cerebral cortex of rats in estrus, on day 19 of pregnancy, or 2 days after delivery. Net uptake indicates the influx of 36Cl (nmol/mg of protein) in 5 s induced by muscimol at 1-50 µM (basal uptake was subtracted from all values). Data are means ± SEM from 4-7 different experiments, each performed with two rats per group. Basal 36Cl uptake values (nmol/mg of protein) were 11.2 ± 0.4 (estrus), 10.8 ± 0.5 (day 19 of pregnancy), and 11.7 ± 0.6 (2 days after delivery). *, P < 0.05 vs. corresponding estrus value.

Expression Levels of GABA_A Receptor 2L Subunit mRNA. The GABA_A receptor subunit mRNA expression in the cerebral cortex and hippocampus of rats in estrus, during pregnancy (day 19), and after delivery (day 2) was measured by RNase protection assay. The expression level of transcripts encoding for the 2L subunit in both the cerebral cortex and hippocampus was decreased by 25% on day 19 of pregnancy, but had returned to control values by the second day after delivery (Table 1). No significant changes were apparent for the expression of mRNAs encoding the 1, 2, 3, 4, 1, 2, 3, or 2S subunits of the GABA_A receptor in either the cerebral cortex or hippocampus (data not shown).

View this table: [in this window] [in a new window]   Table 1.   Effects of pregnancy and delivery on the amount of GABAA receptor 2L subunit mRNA in the cerebral cortex and hippocampus

Effects of Finasteride on Neurosteroid and GABA_A Receptor Expression. To clarify the roles of AP and THDOC in the changes in GABA_A receptor structure and function during pregnancy, we investigated the effects of finasteride administration (a specific 5-reductase inhibitor; ref. 21), on both muscimol-stimulated ³⁶Cl uptake and the amount of 2L subunit mRNA. A single administration of this drug (25 mg/kg, s.c.) reduced the concentrations of AP and THDOC both in estrus rats (65% and 60% in the cerebral cortex and plasma, respectively) and in rats on day 19 of pregnancy (80% and 55% in the cerebral cortex and plasma, respectively) (Table 2). The finasteride-induced inhibition of AP and THDOC production lasted longer than 19 hr, although at this time the brain AP and THDOC concentrations had begun to return to pretreatment values.

View this table: [in this window] [in a new window]   Table 4.   Effects of subchronic treatment with finasteride on muscimol-stimulated 36Cl uptake by cerebrocortical membrane vesicles of estrus and pregnant rats

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of subchronic treatment with finasteride on 2L subunit mRNA expression in the hippocampus of estrus and pregnant rats. Finasteride (25 mg/kg, s.c.) or vehicle was injected daily from day 12 to day 18 of pregnancy, and rats were killed on day 19. Estrus rats were similarly treated with finasteride for 7 days. Data are expressed as a percentage of the estrus value and are means ± SEM of 12-14 animals for each group. *, P < 0.01 vs. estrus.

	DISCUSSION

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We have shown that the changes in GABA_A receptor responsiveness to muscimol and subunit mRNA expression that occur in rat brain during pregnancy and after delivery are related to fluctuations of the brain cortical concentrations of AP. Whereas the kinetics of changes in the concentrations of progesterone, AP, and THDOC were similar in plasma, they markedly differed in the cerebral cortex where the surge of AP was slower than that of progesterone and reached a peak on day 19 of pregnancy, at the time when the GABA_A receptor responsiveness and the 2L subunit expression were maximally decreased. In contrast, brain progesterone concentration followed its plasma levels, being maximally increased between days 12 and 15 and remaining at a plateau up to day 19. A similar dissociation between progesterone and AP concentrations also has been detected in human cerebrospinal fluid (27).

The present results suggest that in the brain of pregnant rats the synthesis/accumulation of AP is not simply a function of the plasma progesterone concentration. Moreover, the greater decline of AP concentration induced by finasteride in the brain of pregnant dams, compared with their plasma and to the brain of estrus rats (Table 2), invites us to speculate that the rate of synthesis of AP in the brain cortex is greater than in plasma in pregnant rats, and increased in the latter compared with estrus rats. Henceforth, it might inferred that by inhibiting the AP synthesis and observing its decline at short times after finasteride administration, one may be enable to measure the AP turnover rate, as a better functional index than the changes in concentrations. One possible explanation for the different rates of AP decline in pregnant and estrus rats is that steroid hormones or other agents may regulate the activity or expression of either 5-reductase or 3-hydroxysteroid oxidoreductase in brain during pregnancy therefore selectively changing rates of AP synthesis. Indeed, estradiol has been shown to regulate 3-hydroxysteroid oxidoreductase activity in rat brain (28). Thus, one may suggest a dynamic regulation of steady state: for instance, a high plasma level of progesterone may be more economically maintained with an inhibition of progesterone metabolism by progesterone. Such a mechanism can be studied by measuring turnover rates at high or low steady state.

Measurements of ³⁶Cl uptake by cerebrocortical membrane vesicles revealed a progressive decrease in the stimulatory effect of muscimol that peaked during the last third of pregnancy, with a sudden increase immediately before delivery and a further increase 2 days after delivery. Because the tone of GABAergic transmission is a function of GABA turnover rate and release, and denervation is characterized by receptor supersensitivity, it might be inferred that the aforementioned changes in GABA_A receptor responsiveness to muscimol reflect to some extent a decreased and an increased GABAergic inhibitory activity during pregnancy and after delivery, respectively.

The changes in GABA_A receptor sensitivity to muscimol during pregnancy and after delivery were associated with changes in the expression of 2L subunit mRNA that was decreased in the cerebral cortex and hippocampus during pregnancy and returned to control values 2 days after delivery. The observation that the amounts of 1, 2, 3, 4, 1, 2, 3, and 2S subunit mRNAs were not significantly affected by pregnancy or delivery suggests that the changes in 2L subunit mRNA expression are specific. However, it will be interesting to evaluate whether the 1 subunit expression was changed, because the presence of this subunit is associated with an increased efficacy of neurosteroids on GABA_A receptors (29). The 2 subunit has been shown to be essential for the modulatory action of benzodiazepines (30), and the probability of Cl channel opening during agonist occupation of the receptor is limited when this subunit is not present (31). Thus, it is possible that changes in the expression of genes encoding 1 and 2 receptor subunits may contribute to the changes in the efficacy of muscimol in enhancing ³⁶Cl uptake during pregnancy and after delivery.

Several studies have indicated that "in vivo" or "in vitro" long-term treatment with neuroactive steroids decrease the efficacy of GABA and its allosteric modulators on GABA_A receptors (25, 32, 33). Therefore, our data suggest that the increase in brain AP concentrations during the last third of pregnancy can be compared with a long-term administration of high doses of this neuroactive steroid, whereas the marked fall in its concentration at the end of pregnancy and after delivery is similar to a sudden discontinuation of such treatment. As recently shown (34), the induction of progesterone withdrawal results in a decrease in the total hippocampal GABA_A receptor-mediated current associated with an increased expression of 4 GABA_A receptor subunit in the hippocampus. We did not observe this latter change in our experimental model. However, the reason for this apparent discrepancy may be related to the substantial difference in the experimental systems because the complex hormonal changes associated with pregnancy cannot be replicated by the administration of a single hormone. Nevertheless, both the pharmacological data (34) and our physiological study indicate that the plasticity of GABA_A receptors is functionally related to fluctuations in endogenous steroid concentrations.

This latter conclusion is supported by the finding that a treatment with finasteride, an inhibitor of 5-reductase, the enzyme that converts progesterone to the AP precursor 5-dihydroprogesterone from days 12 to 18 of pregnancy prevented the AP surge in brain cortex as well as abolished the decreases in both the sensitivity of GABA_A receptors to the action of muscimol and the expression of 2L subunit mRNA normally observed during pregnancy. Moreover, a shorter treatment (from days 12 to 14) with finasteride induced a similar effect on GABA_A receptor responsiveness measured on day 15 of pregnancy. The greater decline in AP brain concentrations, compared with plasma, elicited by finasteride treatment in pregnant dams was unexpected, given the reported higher affinity of this compound for type 2 (peripheral steroidogenic tissues) than for type 1 (brain) 5-reductase (21). Although 5-reductase type 2 has not been detected in adult rat brain tissue ex vivo, the observation that the expression of this isozyme is transiently and androgen-dependently increased (male > female) during embryonic development (35) suggests that one should test whether the marked increases in the concentrations of certain steroid hormones during pregnancy might affect the expression of 5-reductase type 2 in brain. However, because the doses of finasteride used by us were elevated and repeated for several days, the susceptibility of 5-reductase type 1 to inhibition by finasteride also might have been reached in our experiments.

It remains to be determined whether the changes in GABA_A receptor function and subunit expression during pregnancy are because of direct or indirect (genomic) actions of neurosteroids. The oxidation of AP and THDOC, which may occur in vivo, yields 5-dihydroprogesterone (5-DHP) and 5-dihydrodeoxycorticosterone (5-DHDOC), both of which bind to and activate the cytosolic progesterone receptor (36). However, a GABA_A receptor subunit expression regulated by progesterone receptors occupied by 5-DHP and 5-DHDOC appears unlikely. Indeed, the high brain concentration of progesterone during pregnancy, which are finasteride resistant, would be expected to preclude an action of 5-DHP or 5-DHDOC at progesterone receptors. This conclusion cannot exclude a genomic action of progesterone on other transcription regulatory proteins operative in the regulation of transcription or translation of mRNAs encoding for GABA_A receptor subunits during pregnancy. However, the finasteride-induced reversal of the increases in AP brain concentrations and of the changes in GABA_A receptor responsiveness to the agonist and 2L subunit gene expression that occur during pregnancy suggest that these latter changes are the consequence of a AP action at the steroid recognition site on the GABA_A receptor. Accordingly, previous studies have shown that progesterone metabolites may modulate GABA_A receptor subunit gene expression by a similar mechanism (37).

In conclusion, our data demonstrate a functional relation between the fluctuations in the cerebrocortical concentrations of neurosteroids and GABA_A receptor plasticity during pregnancy and after delivery.