WSP and WSR mice were selectively bred for high (WSP) or low (WSR) chronic ethanol withdrawal severity, as assessed using HICs. The present findings continue our efforts to test the hypothesis that the line difference in ethanol withdrawal severity is mediated, in part, by changes in the modulatory effects of the neurosteroid ALLO on GABAA
receptor function. Recent findings in male mice indicate that the anticonvulsant efficacy of ALLO (versus limbic convulsion endpoints) was significantly decreased during ethanol withdrawal in WSP mice, whereas sensitivity was unchanged in similarly treated WSR mice (Finn et al., 2006
). The present findings support the same line differences, and importantly, show that the change in sensitivity is more robust in female mice.
The age range of the female mice (38 – 62 days) suggests that mice were tested during late adolescence and early adulthood. Early work in 100 mice suggests that first stage of estrus occurred over a range of 28 – 49 days of age, with a median at the 35th
day (Engle and Rosasco, 1927
). The fact that 74% of the mice exhibited normal estrous cycles by day 38, with 89% exhibiting normal cycles by day 42, suggests that the majority of mice in the present study were sexually mature when tested. Additionally, age did not provide a significant adjustment for any of the PTZ seizure endpoints or percent change scores, nor did it change the pattern of any results, when age was included as a covariate. Thus, the age range of the animals did not significantly alter the present findings.
The reduced efficacy during withdrawal was observed following all ALLO doses (3.2 – 17 mg/kg) in female WSP mice against limbic convulsion endpoints, whereas male WSP mice exhibited reduced efficacy to only the 17-mg/kg ALLO dose, when measured by the percent change in threshold dose for onset to MC twitch (Finn et al., 2006
). A bigger difference between the present findings and those in male mice is that female WSP mice also exhibited reduced sensitivity to the anticonvulsant effect of ALLO against the brainstem convulsion endpoints. Specifically, the percent change in threshold dose for onset to THE was reduced following the 3.2 and 10 mg/kg ALLO doses in female WSP mice, but unchanged in male WSP mice (Finn et al., 2006
). This suggests that the plasticity of GABAA
receptors in limbic versus hindbrain convulsion circuits may be differentially altered during ethanol withdrawal in male versus female WSP mice. Regardless, since the reduced sensitivity to ALLO's anticonvulsant effect in the male WSP mice was accompanied by a rightward shift in the ability of ALLO to potentiate GABA-stimulated chloride uptake (Finn et al., 2006
), we presume that functional sensitivity of GABAA
receptors to ALLO also is decreased in female WSP mice during ethanol withdrawal.
In contrast to the results in WSP mice, sensitivity to the anticonvulsant effect of ALLO was either enhanced or unchanged in female WSR mice during ethanol withdrawal, depending on the dose and convulsion endpoint. Specifically, sensitivity to the anticonvulsant effect of the 10 mg/kg ALLO dose was enhanced versus the limbic convulsion endpoints, whereas sensitivity to the 3.2 mg/kg ALLO dose was enhanced versus the brainstem convulsion endpoints. Sensitivity to the other ALLO doses for each convulsion endpoint was unchanged during ethanol withdrawal, consistent with recent results in male WSR mice (Finn et al., 2006
). However, the enhanced sensitivity to the anticonvulsant effect of ALLO during ethanol withdrawal at selected doses is consistent with previous findings in male C57BL/6 mice (Finn et al., 2000
) and in male and female rats in which sensitivity to the anticonvulsant effect of GABAergic steroids such as ALLO, pregnanolone and alphaxalone was enhanced during ethanol withdrawal (e.g., Alele and Devaud, 2007
; Cagetti et al., 2004
; Devaud et al., 1996
). Taken in conjunction with the findings that functional sensitivity of GABAA
receptors to ALLO was unchanged in male WSR mice (Finn et al., 2006
) but was enhanced in male (Devaud et al., 1996
) and female rats (Alele and Devaud, 2007
) during ethanol withdrawal, it is possible that functional sensitivity of GABAA
receptors to ALLO might be enhanced during ethanol withdrawal in female WSR mice. Additional studies are necessary to confirm this assumption.
The ALLO dose range examined (3.2 – 17 mg/kg) is identical to that used in previous studies in male mice, where we identified genetic differences in the change in sensitivity to ALLO during ethanol withdrawal in seizure prone versus seizure resistant genotypes (Finn et al., 2000
). We reasoned that use of the same dose range would allow us to make comparisons across studies. While our earlier work documented that the 17-mg/kg dose of ALLO decreased rotarod performance and forelimb grip strength in air-exposed mice (Finn et al., 1997
), mice were tolerant to the ataxic and muscle relaxant effects of ALLO during ethanol withdrawal (Finn et al., 2000
). Importantly, the potential ataxic or sedative effects of the 17-mg/kg dose of ALLO did not confound our assessment of ALLO's anticonvulsant effect, as PTZ was infused until each animal exhibited each of the four readily identifiable convulsion endpoints.
ALLO's efficacy as an anticonvulsant in the air- and ethanol-exposed mice was fairly dose-dependent, exhibiting a graded dose-response function for the increase in threshold dose of PTZ for onset to MC twitch and THE (that may not have been readily apparent due to the scaling of the y-axes in Figures and ). Although ALLO levels were not measured in the present study, earlier work has documented that administration of the 10 and 17 mg/kg doses of ALLO increased plasma levels to approximately 200 ng/ml and 300 ng/ml, respectively, in both air- and ethanol-exposed mice (Finn et al., 2000
). Since ALLO concentrations below 10 μM are selective for activity at GABAA
receptors (discussed in Finn et al., 2004a
; Rupprecht and Holsboer, 1999
), the present findings most likely represent actions at GABAA
receptors, rather than a contribution from effects at serotonin type 3 (5-HT3
), nicotinic or sigma receptors.
Ethanol withdrawal significantly increased basal sensitivity to PTZ in female WSP and WSR mice, measured by the significant decrease in the threshold dose for onset to RB clonus and THE. This finding is similar to that reported earlier in male WSP and WSR mice (Finn and Crabbe, 1999
) and male rats (Kokka et al., 1993
) during ethanol withdrawal. Increased sensitivity to (+)bicuculline also has been reported in male and female rats (e.g., Devaud et al., 1995
) during ethanol withdrawal. However, subtle statistical differences in basal sensitivity to PTZ during ethanol withdrawal were apparent in the comparison of data in female and male mice from the WSR and WSP lines. In particular, ethanol withdrawal significantly decreased the threshold dose of PTZ to elicit RB seizures and THE in male WSP, female WSP, and female WSR mice, while it produced a non-significant decreased in male WSR mice. Regardless, the overall pattern of altered PTZ sensitivity across convulsion endpoints in both male and female WSR (and WSP) mice indicates that both sexes experienced increased seizure susceptibility during ethanol withdrawal, as evidenced by the significant main effects of air versus ethanol vapor exposure. Taken in conjunction with the sex difference in basal PTZ sensitivity (i.e., PTZ threshold dose in air controls was higher in female than in male WSP and WSR mice), it is likely that the greater shift in seizure risk during ethanol withdrawal in the female WSR mice was due to their higher basal PTZ threshold dose in the air control treatment. Overall, these results are consistent with previous work and indicate that the increase in basal sensitivity of GABAA
receptors to antagonist ligands during ethanol withdrawal (reviewed in Morrow, 1995
) appears to generalize across sexes and genotypes. However, the time course for these changes differs in male and female rats (Alele & Devaud, 2007
; Devaud and Chadda, 2001
), with male rats exhibiting increased sensitivity to (+)bicuculline for a longer duration during ethanol withdrawal than female rats (3 days versus 1 day, respectively).
The present study was conducted in intact female mice, since recent findings indicated that ovariectomy altered the sex difference in ethanol withdrawal-related responses (Alele & Devaud, 2007
). Although estrous cycles were not assessed in the current experiment, hormone levels suggest that females may have been in the late diestrus or early proestrus phase of the estrous cycle, based on the high estradiol and low progesterone levels. A recent study utilizing multiple phases of ethanol exposure and withdrawal in C3H/Hecr mice reported that intermittent ethanol exposure interrupted estrous cycles, resulting in vaginal cell samples consistent with diestrus (Veatch et al., 2007
). Since estradiol and progesterone levels were sufficiently variable to suggest that estrous cycles may not have been suppressed, it is possible that procedural or genetic differences might explain the variation between studies in estrous-cycle suppression during ethanol exposure. Another possibility is that the hormone measurements were affected by the seizure induction procedure, since blood was collected after PTZ infusion. Nonetheless, plasma estradiol or progesterone levels were not consistently correlated with all convulsion endpoints, and the extremely low r2
values indicated that the variation in convulsion endpoints could not be explained by the variation in hormone levels. Thus, it is unlikely that any potential estrous cycle-related differences in treatment groups contributed to the present findings.
Although corticosterone levels were measured upon the termination of PTZ-induced convulsions, values were significantly higher in the ethanol- versus air-exposed mice. Thus, ethanol withdrawal produced a comparable increase in corticosterone concentrations in the female WSP and WSR mice. This finding is consistent with earlier work indicating that chronic ethanol exposure and withdrawal activates the HPA axis in male and female rats (e.g., Rivier, 1993
) and in male WSP and WSR mice (Finn, unpublished). The negative correlation between plasma corticosterone levels and PTZ threshold dose for three of the four convulsion endpoints suggests that increases in corticosterone levels were associated with decreased PTZ doses to elicit a convulsion (i.e., increase PTZ sensitivity). This finding could be related to the reported proconvulsant effect of corticosteroids (reviewed in Roberts and Keith, 1995
). However, pre-treatment with ALLO decreased plasma corticosterone levels in female WSP mice (in both air- and ethanol-exposed), with variable results in WSR mice (no effect in air-exposed, decrease following 3.2 and 10 mg/kg ALLO in ethanol-exposed). This finding is consistent with an earlier parallel study that was conducted in male C57BL/6 and DBA/2 mice (Finn et al., 2000
). The ability of ALLO pre-treatment to decrease plasma corticosterone levels may be due to the anxiolytic effect of this steroid (e.g., Akwa et al., 1999
; Bitran et al., 1991
; Finn et al., 1997
) and/or the ability of ALLO to decrease the stress-induced increase in HPA axis activation (Patchev et al., 1996
The WSR and WSP selected lines can be used to examine genetically correlated traits (Crabbe et al., 1990
). In male mice (Finn et al., 2006
) tolerance to ALLO's anticonvulsant effect and reduced sensitivity of GABAA
receptors to ALLO was observed in both replicate lines of WSP mice during ethanol withdrawal, providing strong evidence that the change in sensitivity to ALLO during ethanol withdrawal is a correlated response to selection. The present findings indicate that similar results were found in female mice, although only one of the two replicate lines was tested in the current experiment. Collectively, the results in male and female WSP and WSR mice are consistent with the hypothesis that selection for sensitivity to ethanol withdrawal severity is genetically associated with tolerance to the anticonvulsant effect of ALLO during ethanol withdrawal. These findings suggest that genes related to the sensitivity of GABAA
receptors to ALLO may play an important role in genetic susceptibility to ethanol withdrawal-induced convulsions.
In both male and female WSP and WSR mice, the most consistent line difference in the change in sensitivity to ALLO during withdrawal was observed versus the limbic convulsion endpoints. While microinjection of ALLO or pregnanolone into the hippocampus, amygdala and lateral septum can produce anticonvulsant (Finn et al., 2005
; Martin-Garcia and Pallares, 2005a
) and anxiolytic (Akwa et al., 1999
; Bitran et al., 1999
, Martin-Garcia and Pallares, 2005b
) effects, preliminary data indicates that male WSP mice exhibit tolerance to the anticonvulsant effect of intra-hippocampal ALLO during ethanol withdrawal (Finn et al., 2005
). Thus, the hippocampus may be one of several brain regions in the limbic neuroanatomical circuit underlying PTZ-induced convulsions whereby line differences in GABAA
receptors exist. Certainly, chronic ethanol exposure and withdrawal significantly alters the expression and peptide levels of several GABAA
receptor subunits in the hippocampus of male rats (↓ α1, ↑ α4, ↑ γ2, ↓ δ; Cagetti et al., 2003
; Grobin et al., 2000
; Matthews et al., 1998
), but the relevance of these changes in expression to GABAA
receptor subunit assembly or trafficking, composition of synaptic versus extra-synaptic receptors, or phosphorylation state of GABAA
receptors (see Kumar et al., 2004
; Liang et al., 2004
) to the present findings is not known. Additional factors influencing GABAA
receptor function are the rapid diffusion of hippocampal receptors from extra-synaptic to synaptic domains (Thomas et al., 2005
) and GABAA
receptor associated proteins (Chen and Olsen, 2007
). Taken in conjunction with the finding that the effects of ALLO on GABAergic transmission in the amydgala may depend on neural network activity (Wang et al., 2007
), it is possible that multiple mechanisms affect GABAA
receptor sensitivity to ALLO within a coordinated limbic convulsion circuit and thereby contribute to the tolerance to the anticonvulsant effect of ALLO in WSP mice during ethanol withdrawal.
In conclusion, the present findings and previous work indicate that cross-tolerance to ALLO is a correlated response to selection in male and female WSP and WSR mice. In other words, the results suggest that some of the genes that confer reduced sensitivity of GABAA
receptors to ALLO during ethanol withdrawal may impart increased severity of ethanol withdrawal. Taken in conjunction with the finding that other seizure-prone genotypes, such as the DBA/2 inbred strain (Finn et al., 2000
) also exhibit cross-tolerance to ALLO during ethanol withdrawal, an understanding of the GABAergic neural differences that underlie alcohol withdrawal severity may lead to improved therapies for the treatment of alcohol dependence and withdrawal.