Sex differences in acute ethanol withdrawal severity after adrenalectomy and gonadectomy in Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice
Introduction
Fluctuations in endogenous steroid hormones can affect behaviors through fast-acting membrane receptors (e.g., Purdy et al., 1992, Rupprecht and Holsboer, 1999; Seyle, 1942). These steroids have been termed neurosteroids due to their de novo synthesis in neural tissue and high levels in the brain, and several are potent positive modulators of γ-aminobutyric acidA (GABAA) receptors (Belelli and Lambert, 2005, Follesa et al., 2006, Purdy et al., 1992). Neurosteroids enhance GABAergic inhibition, an action mimicked by acute ethanol (EtOH) administration (reviewed in Biggio et al., 2007, Criswell and Breese, 2005, Grobin et al., 1998).
EtOH administration can increase neurosteroid concentrations to biologically and physiologically relevant levels (e.g., Barbaccia et al., 1999, Finn et al., 2004b, VanDoren et al., 2000). This steroidogenic effect of EtOH was able to modulate EtOH's anxiolytic, antidepressant, and anticonvulsant effects (Hirani et al., 2002, Hirani et al., 2005, VanDoren et al., 2000). Additionally, EtOH's steroidogenic effect was due primarily to synthesis in the periphery, since it was blocked by removal of the peripheral sources of neurosteroid precursors (specifically, the adrenals and gonads; O'Dell et al., 2004). Importantly, EtOH produced both a direct and an indirect effect on GABAA receptor function, with the indirect effect due to the ability of EtOH to promote brain steroidogenesis via a local action that was independent of the hypothalamic–pituitary–adrenal axis (Sanna et al., 2004).
Recent findings suggest that EtOH-induced fluctuations in neurosteroids are seizure protective, since acute EtOH withdrawal severity, measured by handling-induced convulsions (HICs), was significantly increased in adrenalectomized (ADX) and gonadectomized (GDX) mice from two inbred strains that differ in EtOH withdrawal severity (Gililland and Finn, 2007). In DBA/2J (D2) mice, which generally exhibit high acute and chronic EtOH withdrawal (Crabbe, 1998, Crabbe et al., 1983, Roberts et al., 1992), removal of the adrenals significantly increased acute withdrawal severity in males, whereas removal of adrenals and gonads was required to increase withdrawal in female D2 mice when compared with values in intact animals (i.e., SHAM surgery). In C5BL/6J (B6) mice, which exhibit mild EtOH withdrawal (e.g., Crabbe, 1998), only removal of the adrenals was required to increase acute EtOH withdrawal in male B6 mice over values in the SHAM animals, but there was no effect of either surgery in female B6 mice (Gililland and Finn, 2007). The inbred strain difference in the effect of ADX and GDX on acute withdrawal severity is consistent with the idea that genetic differences in EtOH withdrawal severity are due in part to modulatory effects of GABAergic neurosteroids (reviewed in Finn et al., 2004a). Moreover, the fact that acute EtOH withdrawal severity was increased in the ADX and GDX animals suggests that an EtOH-induced increase in an anticonvulsant steroid (or steroids) contributes to the withdrawal response in intact animals.
To further examine genetic differences in the contribution of peripheral sources of GABAergic neurosteroids during acute EtOH withdrawal, we investigated lines of mice that were selectively bred for either severe (Withdrawal Seizure-Prone; WSP) or minimal (Withdrawal Seizure-Resistant; WSR) chronic EtOH withdrawal HICs (Crabbe et al., 1985). The WSP mice exhibit greater than a 10-fold more severe withdrawal than WSR mice after 72 h of equivalent EtOH vapor exposure (Crabbe et al., 1985) and following the administration of a single high dose of EtOH (i.e., acute withdrawal; Crabbe et al., 1991a, Crabbe et al., 1991b). The WSP and WSR lines also differ during chronic EtOH withdrawal in endogenous levels of, and sensitivity to, the GABAergic 5α-reduced progesterone (PROG) metabolite allopregnanolone (ALLO; Beckley et al., 2008, Finn et al., 2004a, Follesa et al., 2006). Specifically, endogenous ALLO levels and sensitivity to ALLO were significantly reduced in WSP versus WSR mice during EtOH withdrawal (Beckley et al., 2008, Finn et al., 2006, Finn et al., 2004a). On the basis of these data and the recent findings in B6 and D2 mice (Gililland and Finn, 2007), the purpose of the present study was to determine whether the removal of the gonads and adrenals would differentially alter acute EtOH withdrawal severity in the WSP and WSR selected lines. We hypothesized that the removal of the peripheral sources of neurosteroids would increase acute withdrawal severity in both selected lines, but that the effect would be more prominent in the WSP line. This would suggest that GABAergic compounds produced in the adrenals and gonads play a crucial role in seizure protection during withdrawal from acute EtOH exposure, and that differences in genotype and steroid manipulation contribute to EtOH withdrawal severity.
Section snippets
Subjects
The WSP/WSR selection has produced two independent pairs of selected lines with similar characteristics, both of which were generated initially from HS/Ibg mice (Crabbe et al., 1985). The WSP and WSR mice used in this study were from the replicate-1 line (WSP-1 and WSR-1), At the time of testing, mice were from selected generation 26 (filial generations 108–110). Sexually mature, 8–12-week-old drug naïve, male and female WSP and WSR mice were bred in the Veterinary Medical Unit at the Veterans
Results
The time course for the change in HICs following administration of a high EtOH dose is shown in Fig. 1 for all groups. Details of the analysis of the hourly HIC data are not presented, as similar results were found with the analysis of AUC. As depicted in Fig. 1, hourly HIC scores were significantly higher in WSP versus WSR mice and were significantly elevated in the ADX/GDX versus SHAM animals in WSP females (hours 2 and 8–24), WSP males (hours 1, 11, and 12), and WSR females (hours 7, 8,
Discussion
Since it was first postulated that steroids produced peripherally (rather than in the brain) were important in regulating convulsions (e.g., Pericic et al., 1999), there has not been much direct evidence for their importance in EtOH withdrawal-related phenotypes. On the basis of the recent findings in B6 and D2 mice (Gililland and Finn, 2007), the purpose of the present experiments was to pursue further the idea that acute EtOH withdrawal is worsened in animals that have been depleted of
Acknowledgments
This research was supported by USPHS grants AA10760 (J.C.C.) and AA12439 (D.A.F.) from the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and VA Merit Review grants awarded to D.A.F. and J.C.C. Ms. Kaufman is supported by F31 AA017019 from NIAAA. We thank Michelle Tanchuck for expert technical assistance.
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