3.1. 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 validity of cumulative licks might not be as high in female mice. Body weights (mean ± SEM) were unaffected by ALLO treatment, and ranged between 20.7 ± 0.3 to 21.3 ± 0.2 g during the dose-response assessment.
3.2. Baseline ethanol intake across drug treatment history
Two sub-chronic regimens of FIN and an ALLO dose-response assessment were evaluated within the same animals over a 3-month period. The baseline intakes immediately prior to treatment with 50 and then 100 mg/kg FIN were 3.34 ± 0.09 and 3.25 ± 0.09 g/kg/2-hrs, respectively. The baseline intakes during the intervening ALLO testing were 3.29 ± 0.08 g/kg/2-hrs. These observations indicated that limited access intake was quite stable across an extended number of days, and, importantly, that the various pharmacological manipulations conducted had no residual effect on the re-establishment of a subsequent drinking baseline.
3.3. ALLO dose response assessment
ALLO did not significantly alter the 10E dose (g/kg) consumed during the 2-hr access period (). However, the cumulative total of 10E licks was significantly altered as a function of ALLO dose [F(6,137) = 2.63; P < 0.05], with the 10 (P < 0.05), 14 and 20 mg/kg doses (P < 0.01 for each) reducing licks by 12–13% when compared to baseline. The unexpected disparity between ALLO’s influence on 10E licks versus ethanol dose prompted the evaluation of a measure to assess number of licks per unit of volume (licks/ml; see ). A significant main effect of ALLO treatment on the licks/ml value was determined [F(6,137) = 10.00; P < 0.001], with ALLO doses ranging from 10–20 mg/kg eliciting significant reductions in the number of licks per ml by 12–18% (P < 0.01 or P < 0.001). ALLO administration did not significantly alter water licks, total fluid intake, or ethanol preference.
Effects of ALLO on ethanol intake and bout microarchitecture
There was a significant main effect of ALLO dose on bout frequency [F(6,137) = 2.77; P < 0.05], but post-hoc tests revealed that no single dose was different from baseline measures. In contrast, the notable influence of ALLO dose on bout size [F(6,137) = 3.37; P < 0.01] and inter-bout interval [F(6,137) = 4.20; P < 0.001] both yielded significant pair-wise comparisons. The largest ALLO dose tested significantly diminished both the mean bout size (P < 0.01) by 19% and the inter-bout interval (P < 0.05) by 21% versus baseline values. Although not reaching statistical significance, a strong trend for 20 mg/kg ALLO to decrease bout size (P = 0.06) also was observed.
In order to evaluate ALLO’s impact on consumption onset, characteristics of the first drinking bout were analyzed separately (bottom of ). ALLO significantly altered both the size [F(6,137) = 3.78; P < 0.01] and rate [F(6,137) = 2.68; P < 0.05] of the first bout, with the 3.2 mg/kg dose augmenting first bout size by 48% (P < 0.05) and reducing first bout lick rate by 53% (P < 0.01). Investigation into the temporal distribution of licks within 20-min intervals throughout the drinking sessions () revealed a significant main effect of ALLO dose [F(6,132) = 2.63; P < 0.05] and session interval [F(5,110) = 19.62; P < 0.001], as well as a statistically significant dose x interval interaction [F(30,660) = 3.52; P < 0.001]. In order to dissect the lick pattern during consumption onset, the initial 20-min interval (in ) was further divided into two 10-min intervals as illustrated in panel B. This separate analysis likewise detected a significant influence of ALLO dose [F(6,132) = 4.17; P < 0.001] and a dose x interval interaction [F(6,132) = 5.75; P < 0.001]. During drinking onset, the highest ALLO dose (24 mg/kg) significantly blunted 10E licks during the initial 10-min of access (P < 0.01) whereas mid-range neurosteroid doses (i.e., 10 & 17 mg/kg) significantly enhanced 10E licks during minutes 10–20 (P < 0.001; P = 0.06 for the 14 mg/kg dose), when compared to baseline. The enhanced onset of 10E licks observed following 17 mg/kg ALLO extended into the early maintenance phase of the session (20–40 min interval; P < 0.01 versus baseline). In contrast, ALLO dose-dependently suppressed drinking throughout the 60–80 min interval (P < 0.05 for 14 and 24 mg/kg; P < 0.001 for 17 mg/kg), when compared to respective baseline 10E licks.
Effects of exogenous ALLO challenge on the temporal distribution of ethanol (10E) licks
3.4. FIN dose assessment
Seven days of treatment with 50 mg/kg FIN elicited a significant main effect of treatment session [F(16,352) = 7.02; P < 0.001] as well as a session x drug interaction [F(16,352) = 2.11; P < 0.01] for ethanol dose. However, its influence on g/kg intake was both modest and transient (P < 0.05 during treatment day #2; see ) when compared to VEH-treated mice. In contrast, 7 days of treatment with 100 mg/kg FIN produced a more robust and persistent effect on ethanol intake (), with significant effects of drug [F(1,21) = 5.79; P < 0.05] and session [F(16,336) = 4.14; P < 0.001] and a significant drug x session interaction [F(16,336) = 7.70; P < 0.001]. Ethanol dose was significantly reduced by 25–34% following 100 mg/kg FIN administration (see treatment days 1 – 7 in ; P < 0.05 in each case) when compared to the ethanol dose consumed by VEH-treated mice. Notably, the FIN-induced decrease in ethanol intake abruptly concluded with treatment cessation, as evidenced by the immediate return to baseline consumption levels on the first day of withdrawal (see withdrawal day 1 in ). Because 100 mg/kg FIN was markedly more efficacious in decreasing ethanol consumption than the 50-mg/kg dose, the remaining analyses reported for drinking bout measures and lick patterns pertain specifically to the 100 mg/kg dose.
Effects of sub-chronic FIN administration and its subsequent withdrawal on daily ethanol dose (g/kg) consumed
Commensurate with the observed reductions in ethanol dose, FIN-treated mice significantly diminished the total number of 10E licks by 32–37% when compared to their within-group baseline level. This statistically significant outcome [F(4,48) = 39.01; P < 0.001] was apparent with both acute (treatment days 1–3; P < 0.001) and sub-chronic (treatment days 4–7; P < 0.001) FIN administration (). A more subtle, yet significant, decrease in 10E licks (P < 0.001) was detected during early withdrawal from FIN, an effect that dissipated during late FIN withdrawal (days 4–7 post-treatment). However, preference for 10E was unaltered by FIN treatment.
Effects of FIN treatment (100 mg/kg) and withdrawal on ethanol intake and bout microarchitecture
Water intakes were typically at or below the level of detection (i.e., 0.05 ml), and were not significantly altered by FIN treatment or its subsequent withdrawal (). The slight difference in water intake between the VEH control and baseline values, which was at the minimal detectable limit, was likely due to the fact that the FIN and VEH treatment groups were balanced for ethanol (rather than water) intake.
The effect of FIN treatment on total fluid intake (10E plus water) was similar to that for 10E (reported above), no doubt due to the fact that this measure primarily was comprised of 10E intake. Total fluid intake was significantly reduced by FIN treatment [F(4,48) = 32.28; P < 0.001] during treatment days 1–3 (P < 0.001), treatment days 4–7 (P < 0.001), and withdrawal days 1–3 (P < 0.05) when compared to within-group baseline values.
Based on the suppression of total fluid intake following FIN treatment, we analyzed home cage water intake during the 22 hr between limited access sessions (see Inter-Session Water Intake in ). Inter-session water intake was significantly increased during the 7 days of FIN treatment (P < 0.001), suggesting that mice were able to compensate for the decrease in total fluid consumption that occurred during the limited access session. No change in inter-session water intake was observed in VEH-treated mice during FIN treatment (data not shown).
Examination of 10E bout dynamics revealed that the overall decline in drinking that resulted from FIN treatment was primarily attributable to a marked attenuation in bout frequency [F(4,48) = 44.21; P < 0.001] and the corresponding increase in inter-bout interval [F(4,48) = 15.62; P < 0.001]. In contrast, mean bout size, bout duration, and lick rate were each unaffected by FIN exposure. First bout dynamics were likewise unchanged by FIN treatment (), suggesting that the blockade of ALLO biosynthesis selectively impinged on the maintenance, but not the onset, of drinking during the limited access sessions.
Significant overall effects of FIN treatment phase [F(4,48) = 36.65; P < 0.001] and session interval [F(5,60) = 70.18; P < 0.001], and a statistically significant phase x interval interaction [F(20,240) = 2.07; P < 0.01] were determined for the temporal distribution of 10E licks throughout the drinking sessions (). Subsequent simple main effect analyses for each session interval revealed a significant influence of FIN treatment phase on all 20-min intervals [F(4,48) = 3.38 – 10.07; P < 0.05 for 20–40, P < 0.01 for 60–80 min, and P < 0.001 for 0–20, 40–60, 80–100 min intervals], except 100–120 min, when compared to baseline measures. Despite the significant main effect of FIN treatment phase during the initial 20-min of 10E access, post-hoc comparisons of each treatment phase versus baseline values were not significant (). Furthermore, there was no significant effect of FIN treatment phase on 10E licks when the initial 20-min of access was further delineated and analyzed as two 10-min bins (). Consistent with the absence of detectable differences in first bout dynamics as a function of FIN treatment and withdrawal (), these temporal lick observations collectively suggested that FIN exhibited little impact on drinking onset. In contrast, FIN treatment days 1–3 (and treatment days 4–7 to a lesser degree) reliably suppressed licking behavior during minutes 20–100 versus baseline, resulting in a blunted maintenance of consumption. Just as the g/kg dose consumed exhibited a rapid recovery to baseline levels upon FIN withdrawal (), lick patterns were almost completely restored by withdrawal days 4–7 ().
Effects of FIN treatment (100 mg/kg) and withdrawal on the temporal distribution of ethanol (10E) licks
3.5. Brain ALLO levels following acute FIN challenge
As noted above, this analysis was conducted in FIN naive mice, to avoid the potential confound of treatment history on the ability of an acute FIN challenge to suppress ALLO levels. In this subset of mice (n = 10), ALLO concentrations were 5.11 ± 0.57 ng/g (n = 4), 3.61 ± 0.45 ng/g (n = 3), and 2.80 ± 0.24 ng/g (n = 3) for the animals pretreated with VEH, 50 mg/kg FIN, or 100 mg/kg FIN, respectively. A one-way ANOVA for ALLO concentrations revealed a significant influence of FIN dose [F(2,7) = 6.20; P < 0.05]. Acute treatment with 100 mg/kg FIN significantly attenuated brain ALLO levels by 45% (P < 0.05) whereas 50 mg/kg FIN elicited a strong trend for a decrease in ALLO by 31% (P = 0.06) when compared to VEH-treated mice.