Elsevier

Neuropharmacology

Volume 42, Issue 3, March 2002, Pages 325-336
Neuropharmacology

Changes in GABAA receptor γ2 subunit gene expression induced by long-term administration of oral contraceptives in rats

https://doi.org/10.1016/S0028-3908(01)00187-3Get rights and content

Abstract

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 γ-aminobutyric acid type A (GABAA) receptor γ2L and γ2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various α and β 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 GABAA 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 GABAA receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABAA receptor gene expression in the rat cerebral cortex.

Introduction

In contrast to the direct genomic actions of other steroid hormones, the progesterone metabolites 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) and 3α,21-dihydroxy-5α-pregnan-20-one (allotetrahydrodeoxycorticosterone) rapidly affect the excitability of neurons through direct modulation of the activity of γ-aminobutyric acid type A (GABAA) receptors (Majewska, 1992; Lambert et al., 1995). The systemic administration of these compounds induces marked anticonvulsant, anxiolytic and sedative–hypnotic effects in animals (Bitran et al., 1991; Kokate et al., 1994; Concas et al., 1996), suggesting that changes in the concentrations of endogenous steroids (neurosteroids) in the brain might contribute to the physiological regulation of brain function. Indeed, long-lasting changes in the concentrations of progesterone and its metabolites induced either in vitro or pharmacologically in vivo (Yu et al., 1996; Smith et al., 1998a, Smith et al., 1998b; Fenelon and Herbison, 2000; Follesa et al., 2000) have been shown to affect GABAA receptor gene expression and activity in cultured neurons or the brain, respectively. Furthermore, fluctuations in the concentrations of these steroids induced by physiological conditions such as pregnancy, delivery and lactation also modulate GABAA receptor plasticity (Fenelon and Herbison, 1996; Concas et al., 1998; Follesa et al., 1998).

Given that GABAA receptors participate in the regulation of a variety of psychophysiological phenomena, including anxiety, sleep, depression, seizures and sexual function, these observations have also suggested that changes in the synthesis of neurosteroids might also contribute to various neurological and psychiatric disorders. Indeed, the plasma and cerebrospinal fluid concentrations of allopregnanolone have been shown to be selectively decreased in individuals with major depression and treatment with antidepressant drugs resulted in the normalization of these concentrations (Uzunova et al., 1998; Strohle et al., 1999). Moreover, plasma concentration of allopregnanolone appears to correlate with a feeling of well-being in women with premenstrual syndrome (Wang et al., 1996).

Since their introduction in 1960 as a reliable approach to birth control, oral contraceptives (OCs) have become some of the most widely prescribed drugs. The success of these agents has resulted from their high efficacy, the rapid reversibility of their effects, and their ease of use. Because they are used mostly by young, healthy women, much attention had been paid to the side effects of OCs. In addition to the well-characterized gynecological and cardiovascular effects of OCs, their use in some women is also associated with headaches, decreased libido and episodes of affective disorders such as depression or mood changes (Herzberg et al., 1971). The incidence of mild to moderate depressive syndrome among women taking OCs is ∼10 to 40%, with those who have experienced a previous depressive episode most at risk for this adverse side effect (Epperson et al., 1999).

Estrogens and progestins, the hormones combined in OC pills, suppress ovulation by preventing the pulsatile secretion of the gonadotropins follicle-stimulating hormone and luteinizing hormone from the pituitary, which in turn results in a persistent reduction in the synthesis of endogenous steroids (estrogens and progesterone) (Lobo and Stanczyk, 1994).

We have now investigated the effects of the long-term administration of OCs, and the associated reduction in the peripheral production of progesterone, on the brain and plasma concentrations of allopregnanolone and of its precursors progesterone and pregnenolone in rats and humans. Given that allopregnanolone positively modulates GABAA receptor function (Majewska, 1992; Lambert et al., 1995) and that long-term physiological (Fenelon and Herbison, 1996; Concas et al., 1998; Follesa et al., 1998) or pharmacological (Yu et al., 1996; Smith et al., 1998a, Smith et al., 1998b; Fenelon and Herbison, 2000; Follesa et al., 2000) exposure to this steroid derivative regulates the expression of specific GABAA receptor subunit genes, we also examined whether OC treatment affects the expression of these genes in the cerebral cortex or the behavior of rats. Moreover, the effects of OCs in ovariectomized (OVX) rats were also evaluated. Preliminary results of this work were presented in abstract form at the Second International Congress on Hormones, Brain and Neuropsychopharmacology (Concas et al., 2000).

Section snippets

Animals

Adult female Sprague-Dawley rats with body masses of 200 to 225 g were obtained from Charles River (Como, Italy). After arrival at the animal facility, rats were acclimatized to the new housing conditions for at least 1 week. The animals were housed six per cage under an artificial 12 h light, 12 h dark cycle (light on from 08.00 to 20.00 hours) at a constant temperature of 22±2°C and a relative humidity of 65%. They had free access to water and standard laboratory food throughout the

Effects of OCs on neurosteroid concentrations in rat cerebral cortex and plasma

The daily administration of the combination of EE (0.030 mg) and LNG (0.125 mg) for 6 weeks resulted in a marked decrease in the concentration of pregnenolone [−41% P<0.01; Fig. 1(A)], progesterone [−74%, P<0.001; Fig. 1(B)] and allopregnanolone [−79%, P<0.001; Fig. 1(C)] in the cerebral cortex of rats killed 24 h after the last treatment. This effect was still apparent 1 week after discontinuation of drug treatment but was no longer statistically significant after 2 weeks. In contrast, the

Discussion

We have shown that long-term treatment with OCs markedly decreases brain and plasma concentrations of pregnenolone, progesterone and allopregnanolone in rats and women, and induces changes in the expression of specific GABAA receptor subunit genes and anxiety-like behavior in rats.

In addition to its effects on peripheral steroidogenic tissues (Lobo and Stanczyk, 1994), the combination of EE and LNG induced a pronounced and persistent decrease in the concentrations of pregnenolone and its

Acknowledgements

We thank Professor Werner Sieghart for providing antibody against the GABAA receptor γ2 subunit. This research was supported by Grant 2001058727 from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Projects of National Relevance, D.M. 12 Novembre 2001 n. 235).

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