Ethanol intake patterns in female mice: Influence of allopregnanolone and the inhibition of its synthesis

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Abstract

The neurosteroid allopregnanolone (ALLO) is a positive modulator of GABAA receptors that exhibits a psychopharmacological profile similar to ethanol (i.e., anxiolytic, sedative-hypnotic). Based on research suggesting that manipulation of ALLO levels altered ethanol self-administration in male rodents, the current studies determined whether exogenous ALLO administration or the inhibition of its synthesis in vivo modulated ethanol intake patterns in female C57BL/6J mice. Lickometer circuits collected temporal lick records of ethanol (10%, v/v) and water consumption during daily 2 h limited access sessions. Following the establishment of stable ethanol intake, studies examined the effect of an acute ALLO challenge (3.2–24.0 mg/kg) or a 7-day blockade of ALLO production with finasteride (FIN; 50 or 100 mg/kg) on ethanol intake in a within-subjects design. In contrast to results in male mice, ethanol dose (g/kg), ethanol preference and most of the bout parameters were unaltered by ALLO pretreatment in female mice. Ethanol intake in females also was recalcitrant to 7-day treatment with 50 mg/kg FIN, whereas 100 mg/kg FIN significantly reduced the ethanol dose consumed by 35%. The FIN-attenuated ethanol intake was attributable to a significant decrease in bout frequency (up to 45%), with lick patterns indicating reduced maintenance of consumption throughout the 2-h session. FIN also produced a dose-dependent decrease in brain ALLO levels. In conjunction with data in male mice, the present findings indicate that there are sex differences in the physiological regulation of ethanol intake patterns by GABAergic neurosteroids.

Introduction

The neurosteroid allopregnanolone (ALLO, 3α-hydroxy-5α-pregnan-20-one) positively modulates type-A γ-aminobutyric acid (GABAA) receptors, the major inhibitory neurotransmitter receptor in the nervous system. Like ethanol (Grobin et al., 1998), increasing concentrations of ALLO induce sedation in laboratory rodents (e.g., Gasior et al., 1999) and humans (Söderpalm et al., 2004, Timby et al., 2006); although in some cases the sedative effect may differ between men and women (van Broekhoven et al., 2007). Neurosteroids have received increased attention in recent years, due to the demonstration that ALLO can modulate sexual behavior (Frye and Rhodes, 2005b), learning and memory (Johansson et al., 2002), maternal behavior (Mann, 2006), psychosis-like behaviors (Ugale et al., 2004), anxiety-like behaviors (Finn et al., 2003, Finn et al., 2006a, Gulinello and Smith, 2003), seizure protection (Frye and Rhodes, 2005a, Finn et al., 2006b) and feeding behaviors (Chen et al., 1996, Reddy and Kulkarni, 1999; but see Fudge et al., 2006).

Given that the GABAA receptor complex is a shared site for ALLO and some of ethanol's neurobiological effects, it is not surprising that ALLO is implicated in diverse lines of ethanol research. For example, low dose ethanol (i.e., 1.5 g/kg) potentiated the effect of ALLO on motor impairment (Vanover et al., 1999), whereas ethanol-induced loss of righting reflex was potentiated by ALLO (Członkowska et al., 2000). Both ethanol (2 g/kg) and ALLO (10–17 mg/kg) produce locomotor stimulation in mice (Palmer et al., 2002), and a quantitative trait locus has been identified on mouse chromosome 2 that contributes to the locomotor-stimulant effects of both ethanol and ALLO (Palmer et al., 2006). Further, recent evidence has demonstrated that ALLO's interaction with the GABAA receptor may be necessary for some of ethanol's pharmacological effects. ALLO facilitates inhibitory transmission by increasing the decay time of miniature inhibitory post-synaptic currents of neuronal GABAA receptors, with brain regional differences in sensitivity (e.g., Belelli and Herd, 2003). Additional electrophysiological studies demonstrated that ALLO was necessary for ethanol's enhancement of GABAergic neurotransmission in substantia nigra neurons (Criswell et al., 1999), and that the combination of ALLO and ethanol could enhance GABAergic neurotransmission at concentrations that were ineffective when administered separately (Akk et al., 2007). Thus, data suggest that the interaction between ALLO and ethanol extends beyond the possibility that individual genes exert pleiotropic control over the effects of these substances, but rather that endogenous ALLO levels may exert a permissive action for some of the behavioral (and electrophysiological) effects of ethanol.

An additional consideration is that acute ethanol administration can increase cortical ALLO levels to pharmacologically active concentrations (e.g., Barbaccia et al., 1999, VanDoren et al., 2000), but there are sex and species differences in this effect. For instance, ethanol increases endogenous ALLO levels in male but not female C57BL/6J mice (Finn et al., 2004c), male and female rats (Morrow et al., 1999) and in male and female adolescents (Torres and Ortega, 2003, Torres and Ortega, 2004). However, ethanol decreased ALLO levels in adult men and caused no change in adult women (Pierucci-Lagha et al., 2006). In terms of the physiological relevance, ethanol has both a direct (e.g., Mihic et al., 1997) and an indirect effect on GABAA receptor function, with the indirect effect involving the biosynthesis of GABAergic neurosteroids (Sanna et al., 2004).

Given these findings, one line of research has sought to determine whether manipulation of ALLO levels altered voluntary ethanol consumption. ALLO and other GABAergic neurosteroids are behaviorally reinforcing, at least under some conditions (Finn et al., 1997a, Finn et al., 1997b, Rowlett et al., 1999, Sinnott et al., 2002a), as is ethanol (e.g., Nizhnikov et al., 2006, Corbit and Janak, 2007, Cunningham and Patel, 2007). And, several results are consistent with the hypothesis that increases or decreases in ALLO levels could produce bi-directional effects on ethanol self-administration. For example, acute administration of ALLO increased operant responding for ethanol in male rats (Janak et al., 1998) and limited access ethanol preference drinking in male but not female C57BL/6 mice (Sinnott et al., 2002b). In contrast, administration of the GABAA receptor partial agonist/antagonist epipregnanolone (O’Dell et al., 2005) and depletion of 5α-reduced neurosteroids such as ALLO with finasteride (FIN; Ford et al., 2005a) decreased ethanol self-administration in rodents. Taken in conjunction with the finding that the GABAA receptor agonist muscimol decreased operant responding for ethanol (Janak and Gill, 2003), it is likely that the distinction between the effects of ALLO versus muscimol on ethanol intake reflects differences in activation of GABAA receptors by a direct agonist (e.g., muscimol) versus a positive allosteric modulator (e.g., ALLO). However, it also should be noted that recent work testing an expanded dose range of ALLO with lickometer circuits demonstrated a bimodal effect of ALLO on operant ethanol self-administration (Ford et al., 2007b) and limited access ethanol drinking (Ford et al., 2005b), with low doses increasing ethanol intake [e.g., 50 ng intracerebroventricular (ICV) or 3.2 mg/kg intraperitoneal (IP)] and high doses decreasing ethanol intake (400 ng ICV or 17 and 24 mg/kg IP). Thus, it also is possible that the effects of muscimol on ethanol intake are comparable to the high dose effects of ALLO.

Endogenous ALLO levels are higher in females than in males, with levels in female rodents fluctuating from 10–30 nM during the estrous cycle and increasing to 100 nM during pregnancy (Paul and Purdy, 1992, Finn and Gee, 1994, Concas et al., 1998). With regard to the effects of ethanol and ALLO in female C57BL/6 mice, female mice were resistant to the ability of ethanol injection or consumption to increase brain ALLO levels (Finn et al., 2004c) and to the ability of a narrow dose range of ALLO to modulate limited access ethanol intake (Sinnott et al., 2002b). Based on these findings, we reasoned that higher basal ALLO levels in female mice, in conjunction with the plasticity of GABAA receptors during the estrous cycle, might render female C57BL/6 mice less sensitive to the modulatory effects of ALLO on ethanol intake (i.e., the dose response curve in females would be shifted to the right of that in males). Thus, the purpose of the present experiments was to further examine the effects of ALLO on ethanol drinking behavior in female C57BL/6 mice by administering an expanded ALLO dose range to increase neural concentrations, and by administering the 5α-reductase inhibitor FIN (which inhibits production of the ALLO precursor, 5α-dihydroprogesterone) to decrease its neural concentrations (see Finn et al., 2006a for review). Since we were interested in the effects of these manipulations on the pattern of ethanol intake, an additional goal was to study the microstructure of ethanol bouts with the use of lickometers, an experimental method that allows for the analysis of self-administration behavior at a fine or microstructure level by recording individual licks (e.g., Boughter et al., 2007).

Section snippets

Animals

Twenty-four female C57BL/6J mice between 8 and 13 weeks of age were obtained from a colony at the Veterinary Medical Unit of the Portland Veterans Administration Medical Center. This colony is re-established every three generations with mice purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were individually housed in lickometer chambers (see below) and acclimated to a reverse light/dark schedule (12 h/12 h; lights off at 09:00 h) for 2–3 weeks. All mice were provided ad libitum access

Predictive validity of cumulative licks for drinking patterns

A significant positive correlation between ethanol dose (g/kg) and total ethanol licks was determined (r = 0.621, P < 0.001, n = 284) throughout the ALLO dose-response time course. A significant correlation between 10E volume depleted and ethanol licks was similarly noted (r = 0.625, P < 0.001, n = 284). The correlative strength between these variables was less than that previously documented in male mice under an identical consumption procedure (see Ford et al., 2005b), suggesting that the predictive

Summary of findings

Recent work in the laboratory has validated the use of lickometers to examine the microarchitecture of ethanol drinking patterns following exogenous administration of ALLO (Ford et al., 2005b, Ford et al., 2007b) or FIN (Ford et al., 2005a) in male C57BL/6 mice. Significant dose- and time-dependent effects were revealed that were not readily apparent when only total 2-h ethanol intake was considered. Thus, we reasoned that use of lickometers in the present studies would allow us to

Conclusions

Female C57BL/6 mice were less sensitive to the modulatory influence of exogenous ALLO (3.2–24 mg/kg) or FIN (50 mg/kg) on ethanol drinking patterns, when compared with recent findings in male rodents. Differences in ALLO modulation within female versus male mice suggest that the physiological regulation of ethanol intake and underlying consumption patterns are sex-specific, and that variations in therapeutic intervention (i.e., GABAergic manipulation) according to sex may be necessary for the

Conflict of interest

None.

Acknowledgements

We thank the Portland Alcohol Research Center (AA10760, Dr. John Crabbe, PI) for supplying the female C57BL/6 mice. The current studies were supported by funding from the Department of Veterans Affairs (VA Merit grant to DAF) and NIAAA (AA12439 to DAF). MMF is supported by a KO1 award from NIAAA (AA016849). EHB is supported by a pre-doctoral NRSA from NIMH (F31 MH081560).

Role of funding source: Funding for these studies was provided by the Department of Veterans Affairs (VA Merit Review Grant

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