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. Based on 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 neurosteroid precursors. Notably, the present finding that ADX/GDX significantly increased acute EtOH withdrawal severity in female WSP and WSR mice provides additional evidence that gonadal and adrenal steroids contribute to the withdrawal profile in intact animals (Gililland and Finn, 2007
; O'Dell et al., 2004
). Taken in conjunction with previous work indicating that certain GABAergic neurosteroids are anticonvulsant during chronic EtOH withdrawal (e.g., Alele and Devaud, 2007
; Cagetti et al., 2004
; Devaud et al., 1996
; Finn et al., 2000
), the evidence suggests that peripherally-derived endogenous anticonvulsant steroids can play an important role in modulating the severity of acute EtOH withdrawal severity.
We chose to use an acute EtOH withdrawal model as it is thought to provide information on neuronal hyperexcitability and adaptation and is considered by some to model “hangover” in humans (Prediger et al., 2006
). Depending on the mouse genotype, total clearance time for a 4 g/kg dose of EtOH has been reported to range from 4.2 − 8.5 hours in intact male mice, with the appearance of increased anxiety-like behavior or HIC occurring after the high dose of EtOH had been eliminated (Prediger et al., 2006
; Gililland and Finn, 2007
). In the present study, the time to peak withdrawal ranged from 4.6 − 6.3 hours (see ), consistent with the reported clearance time for this high dose of EtOH. Since the initial suppression in HICs following EtOH injection was followed by an exacerbation of HIC scores, these changes in convulsive behavior are thought to reflect a state of rebound central nervous system hyperexcitability during acute withdrawal (Crabbe et al., 1991a
). Additionally, the initial suppression in HIC scores did not differ in WSP and WSR mice, consistent with early work indicating that the lines did not differ in loss of righting reflex (Crabbe and Kosobud, 1986
). Given the strong genetic correlation between acute and chronic EtOH withdrawal severity in inbred strains (Metten and Crabbe, 2005
) and in the WSP and WSR selected lines (Crabbe et al., 1991a
), examination of hyperexcitability following withdrawal from a single high EtOH dose may provide insight regarding neuroadaptation following chronic EtOH withdrawal.
The combined removal of both gonads and adrenals significantly increased EtOH withdrawal severity in female, but not in male, WSP and WSR mice, as measured by hourly HIC scores, AUC, and peak HIC scores. This result suggests that sex, rather than genotype, was a more important predictor of the impact of peripheral steroid removal on acute EtOH withdrawal severity. Notably, the present findings are consistent with recent expression profiling results in WSP and WSR mice during the early phase of chronic EtOH withdrawal (i.e., 8 hours following termination of EtOH vapor exposure; Hashimoto and Wiren, 2008
). Specifically, of the total EtOH-regulated genes that were identified, cluster analysis revealed that the transcriptional response correlated with sex rather than the selected withdrawal phenotype. The distinct signaling pathways and targeted classes of genes that were altered by EtOH suggested enhanced neurotoxicity in the female mice (Hashimoto and Wiren, 2008
). However, during protracted withdrawal (i.e., 21 days following termination of EtOH vapor exposure), the pattern of EtOH-regulated genes correlated with genotype rather than sex (Hashimoto et al., 2008
). In conjunction with the present findings, it is possible that a distinct neuradaptive response occurs in female versus male WSP and WSR mice during acute EtOH withdrawal and the early phase of chronic EtOH withdrawal. Put another way, both our data and the Hashimoto and Wiren data from early withdrawal do not suggest pleiotropic influences of genes associated with withdrawal HIC severity. However, the strongest evidence for this would require findings in both WSP-1 vs WSR-1 and WSP-2 vs WSR-2 lines of mice. While we studied only one pair of replicate lines, Hashimoto and Wiren (2008)
tested both genetic replicates and reported data collapsed across replicates.
While the effect of ADX/GDX in WSP and WSR mice differs from the results in B6 and D2 mice (Gililland and Finn, 2007
), the different pattern of the present versus previous results may be due to inherent differences in the two genetic models examined. That is, selected line differences are due to changes in the allelic frequencies for genes important for the selection phenotype (from an initial genetically heterogeneous population comprised of alleles from 8 inbred strains) whereas inbred strain differences are attributed to the allelic differences encapsulated by chance with each strain (> 20 generations of brother-sister matings eliminated all genetic variability within an inbred strain so that all members are essentially a clone of all others). Since the B6 and D2 inbred strains were only 2 of the 8 strains that comprised the genetically heterogenous foundation population for the generation of the WSP and WSR selected lines, it is likely that there will be similarities as well as differences in the genes contributing to acute and chronic EtOH withdrawal severity in the WSP and WSR selected lines versus the B6 and D2 inbred strains.
Though analysis of AUC and peak HIC scores can provide insight as to overall differences in acute EtOH withdrawal between ADX/GDX and SHAM groups, examination of the HIC scores each hour can give hints regarding the manner in which withdrawal was changing (i.e., increase in magnitude or duration of withdrawal, precipitation in onset of withdrawal). ADX/GDX produced a slight increase in the magnitude of withdrawal in female WSR mice (↑ peak withdrawal, ; ↑ HIC scores, ), whereas ADX/GDX increased the duration of withdrawal in female WSP mice (no change in peak withdrawal, but sustained ↑ HIC scores, ), when compared with the withdrawal profile in respective SHAM animals. These data suggest that removal of peripherally derived steroids increased withdrawal severity following a single high dose of EtOH in both female WSP and WSR mice, but that there were slight differences in the manner by which withdrawal was increased.
In contrast, there was no effect of surgical status in the male WSR mice (), consistent with the lack of effect of ADX/GDX on acute withdrawal severity in these animals. In the WSP mice, hourly HIC scores only were elevated in the ADX/GDX versus SHAM animals at hrs 1, 11 and 12 (), suggesting that there was a transient increase in the duration of withdrawal in male WSP mice. These results in male WSP mice were somewhat surprising, given that our recent work suggested that chronic EtOH withdrawal rendered male WSP mice much more sensitive than naïve mice to the proconvulsant effect of pharmacologically decreasing endogenous ALLO levels (Gililland-Kaufman et al., 2008
) and that chronic withdrawal produced a persistent decrease in endogenous ALLO levels in conjunction with a decreased sensitivity to an exogenous ALLO challenge in WSP versus WSR mice (Finn et al., 2004a
). While these results suggest that WSP mice would be more sensitive to the removal of peripherally derived steroids (including ALLO) on acute EtOH withdrawal severity, it is possible that the ADX/GDX surgery represented a greater physiological challenge to WSP mice that negatively impacted their survival rate (see Results). Although speculative, it is possible that the WSP male mice that survived the ADX/GDX surgery were physiologically less sensitive to steroid manipulations, which may have contributed to the lack of effect of surgical status on acute withdrawal severity. However, more research is needed to determine the degree of hormone sensitivity in WSP male mice.
Another possible explanation for the present findings is that there were sex differences in the effect of ADX/GDX on ethanol metabolism. While we did not examine this possibility in the present study, we recently determined that there were no sex or strain differences in the effect of ADX/GDX on ethanol metabolism, when compared with values in respective SHAM animals (Gililland and Finn, 2007
). Additionally, ADX/GDX did not produce a significant change in the time to achieve peak withdrawal in the present study (), which might imply a difference in EtOH metabolism. These findings suggest that the increase in acute EtOH withdrawal following ADX/GDX in female WSP and WSR mice was not due to an indirect effect of surgical status on EtOH metabolism.
The mechanism(s) for the overall sex, but not genotype, differences in the effects of surgical status on withdrawal severity following a single high dose of EtOH are not known. With the proviso that ADX/GDX is removing all peripheral steroids, we measured 2 adrenal and/or gonadal steroids with documented proconvulsant or anticonvulsant properties (CORT, PROG), to determine the relative contribution of these steroids to the acute EtOH withdrawal response (i.e., by comparing the change in steroid levels with the change in acute withdrawal severity in ADX/GDX versus SHAM animals). It has been known for decades that PROG has anticonvulsant properties (Seyle, 1942
), and that this anticonvulsant effect primarily was due to its 5α-reduction to ALLO (Frye et al., 2002
; Kokate et al., 1999
). Additionally, the anticonvulsant action of ALLO and other GABAergic steroids during EtOH withdrawal has been well-documented (e.g., Alele and Devaud, 2007
; Cagetti et al., 2004
; Devaud et al., 1996
; Finn et al., 2000
). Although we were unable to measure testosterone levels in the present study, it has been shown to exhibit proconvulsant and anticonvulsant properties, depending on its metabolism (discussed in Reddy, 2004b
). When testosterone was aromatized into 17β-estradiol, it was proconvulsant (Reddy, 2004b
). However, when testosterone was reduced to 5α-dihydrotestosterone and 3α-androstanediol, it was anticonvulsant (Frye and Reed, 1998
; Reddy, 2004a
). Finally, the excitatory versus inhibitory effects of CORT are mediated by mineralocorticoid (MR) and glucocorticoid (GR) receptors in the brain, with predominant MR activation increasing excitatory hippocampal output (discussed in De Kloet et al., 1998
). Relevant to the present study, activation of MR exerted a proconvulsant effect against pentylenetetrazol-induced convulsions in WSP mice (Roberts et al., 1993
), and acute EtOH withdrawal severity was significantly increased by acute and chronic administration of CORT in WSP mice (Roberts et al., 1991
). However, even though the proconvulsant effect of exogenous administration of CORT is fairly well-established, it should be noted that deoxycorticosterone (DOC, precursor to CORT) has been reported to have anticonvulsant properties due to its 5α-reduction to GABAergic metabolites (Reddy and Rogawski, 2002
Based on the above, we reasoned that ADX/GDX would significantly decrease PROG and CORT levels, and that the change in acute withdrawal severity in ADX/GDX versus SHAM animals would point to the relative contribution of these steroids to the withdrawal profile. That is, an increase in acute withdrawal severity in the ADX/GDX animals would suggest that an anticonvulsant steroid (such as PROG or DOC) was important for maintaining the withdrawal profile in intact animals, whereas a decrease in acute withdrawal severity in ADX/GDX animals would suggest the opposite (i.e., a proconvulsant steroid was important, such as CORT). The results indicated that PROG and CORT levels were decreased in the ADX/GDX WSP mice by 72% and 71% in males and by 70% and 81% in females, respectively. However, in the WSR mice, PROG and CORT levels were decreased in the ADX/GDX animals by 31% and 25% in males and by 96% and 83% in females, respectively. Even with the proviso that the steroid levels were measured following a high dose of EtOH and scoring for withdrawal over a 24 hour period, WSR males did not exhibit the marked decrease in steroid levels following ADX/GDX that was apparent in WSR females as well as in WSP males and females. Though a study by Croft and colleagues (Croft et al., 2008
) indicates that 24 hours after acute ethanol administration is a sufficient time for both blood and brain CORT concentrations to return to baseline levels in intact male TO strain mice, it is still possible that in WSR male mice, the 24 hour time point may not be sufficient for levels to return to baseline. Therefore, future studies may want to examine the 48 hour time point. Since organ removal was confirmed in all animals, these results in WSR mice, in conjunction with the lack of effect of ADX/GDX on acute withdrawal severity, are consistent with previous work indicating that WSR mice are more resistant to steroid manipulations (Finn et al., 2004a
; Roberts et al., 1991
The fact that all ADX/GDX female groups tested experienced a significant increase in HICs over values in SHAM animals suggests that endogenous anticonvulsant steroids are important modulators of the behavioral effects of acute EtOH withdrawal and that the major source of these steroids is the adrenals (and gonads) rather than the brain. Since withdrawal was unaffected by ADX/GDX in the male mice, it is unlikely that testosterone or its metabolites were contributing to the present findings. Thus, the results in female mice are consistent with the idea that removal of PROG (or DOC) and their respective 5α-reduced GABAergic metabolites contributed to the increase in acute withdrawal severity in the ADX/GDX animals. However, the relative involvement of a single or multiple GABAergic steroids to the present findings is unclear.
The present findings revealed that PROG levels were decreased significantly by ADX/GDX in all but male WSR mice, and that they were higher in female than in male WSR, but not WSP SHAM mice. Based on the low PROG levels in SHAM female mice, it is likely that the mice were in estrus, though estrous cyclicity was not monitored due to the short time frame of the experiment and the robust manipulation of the animals involved. Although we were unable to measure ALLO levels in the present studies, it is likely that the significant reduction in PROG levels in the ADX/GDX mice would correspond to a marked decrease in ALLO levels. Additionally, there is evidence that female mice have higher circulating ALLO levels than male mice (Finn et al., 2004b
). Removing the main peripheral sources of PROG would produce a concomitant reduction in the synthesis of ALLO and other GABAergic steroids, which could contribute to the increase in HICs during acute withdrawal in the ADX/GDX female mice. Consistent with this idea, plasma PROG levels were significantly negatively correlated with acute withdrawal-related AUC in the female mice. Thus, the increase in acute EtOH withdrawal severity in the female ADX/GDX mice is consistent with the idea that endogenous anticonvulsant steroids (such as ALLO) may contribute to seizure protection during EtOH withdrawal.
Our previous work provides strong evidence that reduced sensitivity to the anticonvulsant effect of ALLO (Beckley et al., 2008
; Finn et al., 2006
) and to the ability of ALLO to potentiate GABAA
receptor function during chronic EtOH withdrawal (Finn et al., 2006
) represents a correlated response to selection in male and female WSP and WSR mice (discussed in Crabbe et al., 1990
). That is, some of the genes influencing chronic EtOH withdrawal severity also affect sensitivity to the anticonvulsant effect of ALLO, which is a metabolite of PROG, during EtOH withdrawal. Since the line difference in ALLO sensitivity during EtOH withdrawal was even greater in the female mice, it is possible that the hormonal environment in female WSP mice is such that changes in endogenous anticonvulsant steroid levels can have a more pronounced effect on GABAA
receptor function (and acute and chronic EtOH withdrawal severity) than in WSP males, similar to what we observed in the present study.
In conclusion, the present findings have further corroborated the evidence that withdrawal from an acute high EtOH dose can be modulated by anticonvulsant steroids produced in the periphery. Based on these findings, it can be postulated that these anticonvulsant steroids contribute to the neuroadaptation and neuroexcitability produced by high doses of EtOH, with sex and genotype differences in the magnitude of the effect. Given that acute and chronic EtOH withdrawal may be under the control of a common group of genes (Crabbe et al., 1991a
; Metten and Crabbe, 2005
), the examination of acute withdrawal-related hyperexcitability can provide insight regarding neuroadaptation following chronic EtOH withdrawal. Thus, the finding that certain peripherally-derived GABAergic anticonvulsant steroids can modulate the severity of alcohol withdrawal provides information that can be important for the treatment of alcohol dependence as well as for understanding mechanism(s) contributing to the development of physical dependence and the expression of withdrawal.