Male WSP and WSR mice from both genetic replicates were exposed to 72 hr ethanol vapor or air, and separate groups of mice were euthanized at 0, 4, or 8 hrs following removal from the inhalation chambers. Chronic ethanol vapor exposure produced equivalent BEC upon removal from the inhalation chamber. Mean ± SEM BEC was 1.10 ± 0.05 mg/ml for WSP and 1.14 ± 0.02 mg/ml for WSR mice. While there was no significant line or replicate difference in BEC, the interaction was significant [F(1,104) = 11.68, P = 0.001]. This was due to the fact that BECs were slightly higher in WSP-1 vs. WSP-2 mice and slightly lower in WSR-1 vs. WSR-2 mice (not shown).
The initial analysis of plasma ALLO levels revealed that there were no main effects of genetic replicate, nor were there interactions with genetic replicate. When the analysis was conducted, collapsed across genetic replicate, plasma ALLO levels were lower in WSP vs. WSR mice and were significantly influenced by treatment/time [F(3,145) = 3.24, P < 0.05]. The significant interaction between line and treatment/time [F(3,145) = 4.21, P < 0.01] was due to the chronic ethanol-induced decrease in plasma ALLO levels that occurred only in WSP mice (). Post-hoc tests confirmed this conclusion. Plasma ALLO levels in WSP mice were decreased by 71% in dependent animals and by 34% during peak withdrawal, whereas values were unchanged in WSR mice.
The effect of exposure to 72 hr ethanol vapor and withdrawal on (A) plasma ALLO, (B) cortical ALLO, and (C) plasma CORT levels in WSP and WSR male mice
In contrast, cortical ALLO levels () were decreased significantly in both WSP (by 50-54%) and WSR (by 37-46%) mice during ethanol withdrawal. As with plasma ALLO levels, there was no significant main effect of, or interaction with, genetic replicate. Subsequent analyses indicated that cortical ALLO levels were significantly altered by treatment/time [F(3,143) = 9.15, P < 0.001], with a significant interaction between line and treatment/time [F(3,143) = 2.78, P < 0.05]. The significant interaction appeared to be due to the fact that cortical ALLO levels were decreased significantly (by 40%) only in dependent WSR mice (i.e., 0 hr), whereas values were unchanged in WSP mice.
Plasma CORT levels () were significantly increased in dependent mice and during withdrawal, with the WSP mice exhibiting a slightly higher elevation during withdrawal when compared to the WSR mice. ANOVA confirmed that plasma CORT levels were significantly higher in WSP vs. WSR mice [F(1,152) = 4.19, P < 0.05] and were significantly increased by chronic ethanol exposure and withdrawal [F(3,152) = 22.23, P < 0.001]. Although the interaction between line and treatment/time was not significant, post-hoc tests were conducted based on our a priori hypothesis that plasma CORT levels would be increased to a greater extent in the WSP vs. WSR mice. Although absolute plasma CORT levels were higher during peak withdrawal in the WSP vs. WSR mice, the fold increase was similar in the lines (~ 6-fold), due to the slightly lower basal CORT levels in the air-exposed WSR mice.
The effect of chronic ethanol exposure and withdrawal on 5α-reductase-1 activity is depicted in . In general, there were some line differences in basal 5α-reductase activity, but the overall effects of treatment and time appeared to be similar in the lines. Hippocampal 5α-reductase activity () was higher in WSP vs. WSR mice [F(1,24) = 36.53, P < 0.001] and was decreased significantly by chronic ethanol exposure [F(2,24) = 6.58, P < 0.01]. The lack of significant interaction between line and treatment/time was due to the fact that hippocampal enzyme activity was decreased similarly in dependent WSP (↓ 34%) and WSR (↓ 40%) mice, and only slightly decreased in both lines during ethanol withdrawal (↓ 13% in WSP, ↓ 15% in WSR). Cortical 5α-reductase activity () also was higher in WSP vs. WSR mice [F(1,25) = 23.61, P < 0.001] and was decreased significantly during chronic ethanol withdrawal [F(2,25) = 11.30, P < 0.001]. The decrease in cortical enzyme activity during ethanol withdrawal was similar (↓ 50%) in both lines. Amygdala 5α-reductase activity () only was significantly influenced by line [F(1,25) = 21.22, P < 0.001] (WSP > WSR), whereas midbrain 5α-reductase activity () was slightly decreased by chronic ethanol exposure (↓ 20% in dependent WSP, ↓ 13% in dependent WSR), and adrenal 5α-reductase activity () was a bit more variable.
The effect of chronic ethanol exposure and withdrawal on 5α-reductase-1 activity in (A) hippocampus, (B) cortex, (C) amygdala, (D) midbrain, and (E) adrenals from WSP and WSR mice
The effect of chronic ethanol exposure and withdrawal on expression of the 5α-reductase-1 transcript is depicted in . Since transcript expression was normalized to the respective air control values, we only were able to analyze for treatment/time effects and for interaction with selected line. The overall pattern of the results suggests that there were some line differences in the effects of ethanol exposure and withdrawal on 5α-reductase expression. Hippocampal 5α-reductase mRNA levels () appeared to be lower in WSP than in WSR mice and to be decreased by ethanol exposure and withdrawal. . In dependent mice, 5α-reductase expression was reduced only in WSP mice (↓76%). During withdrawal, mRNA levels remained decreased in WSP mice (↓55%) versus the change in WSR mice (↓31%). However, there was a much more profound line difference in the regulation of the cortical 5α-reductase transcript by chronic ethanol exposure and withdrawal (). Cortical mRNA expression was significantly lower in WSP versus WSR mice [F(1,13) = 75.12, P < 0.001] and was significantly altered by treatment/time [F(2,13) = 9.18, P < 0.01]. The significant interaction between line and treatment/time [F(2,13) = 23.03, P < 0.001] confirmed the differential regulation of the 5α-reductase transcript in WSP and WSR mice. In WSP mice, cortical expression was significantly decreased (by 75%) in dependent animals and during ethanol withdrawal. In contrast, 5α-reductase mRNA levels were increased 2.76-fold in dependent WSR mice, with a further significant increase (↑4.3-fold) during ethanol withdrawal. Treatment did not significantly alter mRNA levels in the amygdala (not shown), midbrain (not shown), or adrenal (). Planned comparisons of mRNA levels in adrenal tissue indicated that 5α-reductase expression was decreased to a greater extent in the WSP line (↓55% in dependent mice, ↓67% during withdrawal), when compared to the WSR line (↓47% during withdrawal).
Chronic ethanol exposure and withdrawal differentially altered expression of the 5α-reductase-1 transcript in (A) hippocampus, (B) cortex, and (C) adrenals from WSP and WSR mice