A multicomponent ethanol response battery across a cumulative dose ethanol challenge reveals diminished adolescent rat ethanol responsivity relative to adults

Abstract

Adolescence is a conserved developmental period associated with low alcohol responsivity, which can contribute to heavy drinking and development of an alcohol use disorder (AUD) later in life. To investigate ethanol responsivity between adolescent and adult rats, we developed an ethanol response battery (ERB) to assess acute ethanol responses across cumulative doses of ethanol during the rising phase of the blood ethanol curve. We tested the hypothesis that adolescent male and female rats would exhibit lower ethanol responsivity to a cumulative ethanol challenge relative to adults. Male and female adolescent (postnatal day [P]40) and adult (P85) Wistar rats underwent ERB assessment following consecutive doses of ethanol (i.e., 1.0, 1.0, and 1.0 g/kg) to produce cumulative ethanol doses of 0.0, 1.0, 2.0, and 3.0 g/kg. The ERB consisted of (1) the 6-point behavioral intoxication rating scale, (2) body temperature assessment, (3) tail blood collection, (4) accelerating rotarod assessment, (5) tilting plane assessment, and (6) loss of righting reflex (LORR) assessment. Across cumulative ethanol doses, adolescent and adult rats evidenced progressive changes in ERB measures. On the ERB, adolescent rats of both sexes evidenced (1) lower intoxication rating, (2) blunted hypothermic responses, particularly in females, (3) longer latencies to fall from the accelerating rotarod, and (4) less tilting plane impairment relative to adults despite comparable BECs. All adult rats, regardless of sex, displayed a LORR at the 3.0 g/kg cumulative ethanol dose while among the adolescent rats, only one male rat and no females showed the LORR. These data reveal decreased adolescent ethanol responsivity across body temperature, intoxication, balance, and coordination responses to a cumulative ethanol challenge as assessed using the novel ERB relative to adults. The results of this study suggest that adolescent-specific low ethanol responsivity may contribute to adolescent binge drinking and increased risk for development of an AUD.

Keywords: acute alcohol; adolescence; alcohol sensitivity; development; hypothermia.

FIGURE 1

Schematic of ethanol response battery (ERB) protocol. Ethanol-naïve male and female adolescent (P40) and adult (P85) Wistar rats were assessed on the ERB. Each trial of the ERB consisted of (1) the 6-point behavioral intoxication rating scale, (2) body temperature assessment, (3) tail blood collection, (4) accelerating rotarod assessment, (5) tilting plane assessment, and (6) loss of righting reflex (LORR) following the final ethanol dose. The ERB was conducted during a cumulative ethanol dose-response challenge. Following baseline ERB assessment, rats received ethanol doses (1.0, 1.0, 1.0 g/kg, i.p.) approximately 30 min apart for cumulative ethanol doses of 1.0, 2.0, and 3.0 g/kg) with the ERB initiated 15 min following each ethanol dose.

FIGURE 2

Body weight and blood ethanol concentration (BEC) assessment during the ethanol response battery (ERB) across the cumulative ethanol challenge. (A) Adolescent (P40) male subjects weighed significantly less than adult (P85) male subjects (t[10] = 21.5, p < 0.0001, Student’s t-test). (B) Adolescent (P40) female subjects weighed significantly less than adult (P85) female subjects (t[10] = 15.7, p < 0.0001, Student’s t-test). (C) Assessment of BECs 15 min after each ethanol dose across the cumulative ethanol challenge during ERB testing in male subjects reveal a dose-dependent increase in BECs (main effect of Dose: F(_2,20) = 102.3, p < 0.0001, repeated measures ANOVA) that did not differ as a function of age (main effect of Age: p = 0.15, repeated measures ANOVA). (D) Assessment of BECs in female subjects 15 min after each ethanol dose across the cumulative ethanol challenge revealed a dose-dependent increase in BECs (main effect of Dose: F(_2,20) = 85.0, p < 0.0001, repeated measures ANOVA) that was unaffected by age (main effect of Age: p = 0.96, repeated measures ANOVA). n = 6 subjects/age/sex. Data are presented as mean ±SEM. **p < 0.01.

FIGURE 3

Ethanol response battery (ERB) assessment revealed an adolescent-associated reduction in intoxication rating to a cumulative ethanol challenge relative to adults. (A) Across adolescent (P40) and adult (P85) male subjects, assessment of intoxication revealed that adolescents had lower intoxication rating scores at the 1.0 g/kg (U = 0.00, p = 0.002, Mann-Whitney test) and 3.0 g/kg (U = 3.00, p = 0.015, Mann-Whitney test) dose, but not at the 2.0 g/kg (U = 12.00, p = 0.394, Mann-Whitney test) dose relative to male adults. (B) Across adolescent (P40) and adult (P85) female subjects, assessment of intoxication revealed that adolescents had lower intoxication rating scores at the 1.0 g/kg (U = 0.00, p = 0.002, Mann-Whitney test), 2.0 g/kg (U = 0.00, p = 0.002, Mann-Whitney test), and 3.0 g/kg (U = 3.00, p = 0.015, Mann-Whitney test) doses relative to female adults. n = 6 subjects/age/sex. Data are presented as median with interquartile range. *p < 0.05, **p < 0.01.

FIGURE 4

Ethanol response battery (ERB) assessment revealed a female-specific adolescent-associated reduction in hypothermic responsivity to a cumulative ethanol challenge relative to adults. (A) Body temperature assessment at baseline did not differ between adolescent and adult male subjects (t[10] = 0.0, p = 0.99, Student’s t-test). (B) Body temperature assessment at baseline did not differ between adolescent and adult female subjects (t[10] = 0.29, p = 0.78, Student’s t-test). (C) Assessment of body temperature following cumulative ethanol challenge dosing in male subjects revealed a dose-dependent hypothermic response across ethanol doses (main effect of Dose: F(_2,20) = 12.7, p = 0.003, repeated measures ANOVA) that was insignificantly blunted in adolescent male rats relative to adult males (main effect of Age: F(_1,20) = 2.4, p = 0.15, repeated measures ANOVA). (D) Assessment of body temperature following cumulative ethanol challenge dosing in female subjects revealed a dose-dependent hypothermic response across cumulative ethanol doses (main effect of Dose: F(_2,20) = 10.4, p = 0.001, repeated measures ANOVA) as well as a tread toward reduced ethanol-induced hypothermia in adolescent female subject rats relative to adult females (main effect of Age: F(_1,20) = 4.6, p = 0.056, repeated measures ANOVA). n = 6 subjects/age/sex. Data are presented as mean ±SEM.

FIGURE 5

Ethanol response battery assessment revealed an adolescent-associated reduced sensitivity to impairment on the rotarod across the cumulative ethanol challenge relative to adults. (A) Baseline performance on the rotarod did not differ between adolescent and adult male subjects (t[10] = 1.1, p = 0.32, Student’s t-test). (B) Baseline performance on the rotarod did not differ between adolescent and adult female subjects (t[10] = 0.38, p = 0.71, Student’s t-test). (C) Across adolescent (P40) and adult (P85) male subjects, assessment of rotarod performance revealed a dose-dependent impairment in time to remain on the rotarod across cumulative ethanol doses (main effect of Dose: F(_2,20) = 162.5, p < 0.0001, repeated measures ANOVA). Relative to adult male rats, adolescent males spent significantly more time on the rotarod across the cumulative ethanol challenge (main effect of Age: F(_1,20) = 4.8, p = 0.052, repeated measures ANOVA). (D) Across adolescent and adult female subjects, time on the rotarod was reduced across cumulative ethanol doses (main effect of Dose: F(_2,20) = 21.6, p < 0.0001, repeated measures ANOVA). Adolescent female rats, regardless of ethanol dose, were less impaired on the rotarod relative to adult females (main effect of Age: F(_1,20) = 13.0, p = 0.005, repeated measures ANOVA). n = 6 subjects/age/sex. Data are presented as mean ±SEM. *p ≤ 0.05, **p < 0.01.

FIGURE 6

Ethanol response battery assessment revealed an adolescent-associated reduced sensitivity to impairment on the tilting plane across the cumulative ethanol challenge relative to adults. (A) Baseline performance on the tilting plane did not differ between adolescent and adult male subjects (t[10] = 0.85, p = 0.42, Student’s t-test). (B) At baseline, adolescent female rats performed slightly better on the tilting plane than adult females (t[10] = 3.8, p = 0.003, Student’s t-test). (C) Assessment of tilting plane performance in male subjects revealed a dose-dependent reduction in angle of slide across cumulative ethanol doses (main effect of Dose: F(_2,20) = 344.4, p < 0.0001, repeated measures ANOVA) whereas adolescents, regardless of ethanol dose, were less impaired on the tilting plane than adults (main effect of Age: F(_1,20) = 5.5, p = 0.041, repeated measures ANOVA). (D) Assessment of tilting plane performance in female subjects revealed a dose-dependent reduction in angle of slide across cumulative ethanol doses (main effect of Dose: F(_2,20) = 40.1, p < 0.0001, repeated measures ANOVA) whereas adolescents were overall less impaired on the tilting plane relative to adults (main effect of Age: F(_1,20) = 37.4, p = 0.009, repeated measures ANOVA). Follow-up posthoc analysis of the significant Dose × Age interaction (F(_2,20) = 7.67, p = 0.003, repeated measures ANOVA) revealed less impairment in adolescent female rats relative to adult females at cumulative ethanol doses of 1.0 g/kg (p = 0.045, Šidák’s multiple comparisons test), 2.0 g/kg (p = 0.008, Šidák’s multiple comparisons test), and 3.0 g/kg (p = 0.013, Šidák’s multiple comparisons test). Data are presented as mean ±SEM. *p ≤ 0.05, **p < 0.01.

FIGURE 7

The ethanol response battery revealed an adolescent-associated insensitivity to loss of righting reflex (LORR) relative to adults. (A) Assessment of LORR in male rats following the final cumulative dose of ethanol (i.e., 3.0 g/kg) revealed that only one adolescent displayed LORR whereas all of the adults evidenced LORR (X ² (1, N = 12) = 8.6, p = 0.003, Pearson Chi Square). (B) Assessment of LORR in female rats following the final cumulative dose of ethanol (i.e., 3.0 g/kg) revealed that no adolescents displayed LORR whereas all of the adults evidenced LORR (X ² (1, N = 12) = 12, p = 0.0005, Pearson Chi Square). n = 6 subjects/age/sex. Data are presented as number of subjects with LORR. **p < 0.01.