Ovariectomy has minimal effects on neuroadaptations associated with ethanol dependence in female rats

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Abstract

We previously found gender selective alterations in gene expression for GABAA and NMDA receptors associated with the development of ethanol dependence. Males and females have a differing hormonal environment, including steroid hormone derivatives (neuroactive steroids) that exert effects at GABAA and NMDA receptors. Therefore, we explored whether the removal of ovarian steroids would alter gender differences in response to chronic ethanol exposure. We found that ovariectomy reduced ethanol drinking levels by 15%, comparable to earlier observations between intact female and male rats. However, investigation of the effects of chronic ethanol exposure on intact versus ovariectomized female rats uncovered few differences in chronic ethanol-induced alterations in selected GABAA or NMDA receptor subunit peptide levels. In general, findings for both groups of females were similar to previous observations. There was no reduction in GABAA receptor α1 subunit levels in cerebral cortex in either intact or ovariectomized female rats, in contrast to the significant reduction observed in male rats. In addition, both intact and ovariectomized female rats had increased levels of the NMDA NR1 subunit in cerebral cortex and hypothalamus, but not in hippocampus, whereas ethanol dependent male rats displayed significant increases in the NR1 subunit only in hippocampus. Radioligand binding analysis with [35S]TBPS found no differences in modulation of the GABAA receptor by neuroactive steroids between ethanol dependent male, intact female or ovariectomized female rats. Seizure susceptibility was not different between intact or ovariectomized female rats during ethanol withdrawal. We did observe differential effects on brain allopregnanolone and plasma corticosterone levels between ethanol dependent intact and ovariectomized female rats, suggesting that ovarian steroids influence HPA axis adaptations to prolonged ethanol exposure. Overall, these data suggest that ovarian steroids do not significantly impact the gender selective alterations of GABAA and NMDA receptors associated with ethanol dependence.

Introduction

There are many differences between men and women in patterns of drinking, physiological responses and pathological responses to alcohol, as well as different manifestations of alcohol abuse, dependence and alcoholism. Men have significantly higher rates of alcohol dependence than women across most age groups (Hilton and Clark, 1991), whereas women are quicker to suffer pathological consequences of alcohol abuse and dependence (Ashley et al., 1977, Harper et al., 1990, Urbano-Marquez et al., 1995). While many of the sex differences in alcohol drinking, dependence and alcoholism can be ascribed to societal or environmental factors, inherent gender differences in physiology may also play a role. Males and females operate under a different hormonal milieu that could influence their responses to ethanol. These differences may result from organizational influences of gonadal steroids laid down during development. Alternatively, activational effects of hormones may be involved — in that the presence of gonadal steroids influences responses to ethanol intake. For example, female rats display a larger and more prolonged increase in corticosterone and adrenocorticotrophic hormone (ACTH) release than do males, following acute ethanol administration (Rivier, 1993, Ogilvie and Rivier, 1996).

Certain steroid hormone derivatives exert effects at GABAA or NMDA receptors rather than via classical steroid mechanisms. These compounds are termed neuroactive steroids. The majority of neuroactive steroids possess selective actions at GABAA receptors. Allopregnanolone (3α-hydroxy-5α-pregnan-20-one) is a progesterone derivative that has been well studied. This neuroactive steroid has potent anxiolytic and anticonvulsant effects, believed to result from its interactions at GABAA receptors (Bitran et al., 1991, Wieland et al., 1991, Finn and Gee, 1994). Other neuroactive steroids, such as pregnenolone and dehydroepiandrosterone (DHEA), exert effects at NMDA receptors as well as GABAA receptors (Farb and Gibbs, 1996, Baulieu and Robel, 1998). Most neurosteroids are present at different levels in females compared to males, in both humans and rats (Paul and Purdy, 1992, Corpéchot et al., 1993, Labrie et al., 1997). This suggests that there may be gender differences in the inherent regulation of GABAA and NMDA receptors.

Ethanol is believed to exert many of its effects by interactions at GABAA and NMDA receptors. Acute ethanol is incoordinating, anxiolytic, anticonvulsant and sedative/hypnotic, strikingly similar to many effects of positive modulators of GABAA receptors and NMDA receptor antagonists. Numerous investigations have provided evidence that ethanol enhances GABAergic neurotransmission and inhibits glutamatergic neurotransmission (see Crews et al., 1996, Eckardt et al., 1998, Grobin et al., 1998 for review). Due to their overlapping sites of action, there may be important interactions between ethanol and neuroactive steroids in their modulation of GABAA or NMDA receptors.

Following chronic ethanol administration, animals (including humans) become dependent on, and tolerant to ethanol. This is manifested as a reduced behavioral response to doses of ethanol that were initially intoxicating. In addition, a withdrawal syndrome is uncovered following abrupt removal of ethanol after prolonged exposure. Neuroadaptations of GABAA and NMDA receptors are believed to play a role in the development of ethanol dependence and withdrawal. Subunit selective alterations in GABAA and NMDA receptor gene expression have been observed following chronic ethanol exposure (Mhatre et al., 1993, Mhatre and Ticku, 1994, Devaud et al., 1995b, Devaud et al., 1997, Follesa and Ticky, 1995, Snell et al., 1996). It has been proposed that these alterations in gene expression lead to the assembly of new populations of GABAA and NMDA receptors containing different subunits than in the normal state. As pharmacological properties of these receptors are conferred by subunit composition, ethanol dependence may be associated with the expression of GABAA and NMDA receptors, displaying an altered functional responsiveness to ethanol.

We have previously found gender differences in the effects of chronic ethanol exposure on GABAA and NMDA receptor subunit expression (Devaud et al., 1998, Devaud and Morrow, 1999), pointing to the need for a more thorough characterization of the influence of gender on neuroadaptations to chronic ethanol exposure. If neuroactive steroid and ethanol interactions at GABAAand NMDA receptors have a role in responses to chronic ethanol exposure, then removal of neuroactive steroid precursors (such as progesterone) should alter the effects of prolonged ethanol exposure on these receptors. Therefore, the present study investigated whether neuroadaptations of GABAA and NMDA receptors associated with ethanol dependence differed between intact and ovariectomized female rats.

Section snippets

Experimental procedures

All experimental procedures were conducted under a protocol approved by the Idaho State University Animal Welfare Committee and met IACUC guidelines.

Results

We have previously found gender selective responses to ethanol dependence in male and female rats (Devaud et al., 1995a, Devaud et al., 1995b, Devaud et al., 1996, Devaud et al., 1998, Devaud and Morrow, 1999, Devaud et al., 1999). The most prominent differences were observed at the molecular level — in the determinations of the effects of chronic ethanol exposure on GABAA and NMDA receptor subunit peptide levels. We hypothesized that these gender differences could result from the differing

Discussion

This series of investigations was undertaken to determine the influence of ovarian steroids on responses to chronic ethanol exposure in female rats. We had previously found gender selective effects of several brain adaptations associated with the development of ethanol dependence. As certain steroid derivatives are neuroactive and exert significant, physiological effects at GABAA and/or NMDA receptors, we hypothesized that removal of some of these steroids (those of ovarian origin) could

Acknowledgments

The steroid assays were conducted by Tejas Patel at the University of North Carolina. This research was supported by PHS award AA00191 to L.L.D.

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