Does tolerance to ethanol-induced ataxia explain the sensitized response to ethanol?

Under conditions of repeated exposure to ethanol, a sensitized locomotor stimulant response develops in some strains of mice. It has been hypothesized that the sensitized response is a consequence of tolerance development to the sedative/incoordinating effects of ethanol. Conversely, ethanol-induced sensitization and tolerance may be independent effects of repeated ethanol exposure. A published study in C57BL/6J by DBA/2J recombinant inbred strains concluded that the two phenomena are not genetically related and thus perhaps mechanistically distinct. To extend evaluation beyond the genetic variance found in C57BL/6J and DBA/2J mice and examine phenotypic associations, we simultaneously measured ethanol-induced sensitization and tolerance in a genetically diverse panel of 15 standard inbred mouse strains and a genetically heterogeneous stock that was produced by the intercrossing of eight inbred mouse strains. Changes in activity counts and ataxia ratio across repeated ethanol treatments indexed sensitization and tolerance, respectively. Photocell beam breaks provided the measure of activity, and foot slip errors corrected for activity in a grid test provided a measure of coordination. The results were strain and individual dependent. The genetic correlation between magnitude of sensitization and tolerance was not significant in the panel of inbred strains, but when individual data were correlated, without regard to strain, there was a significant correlation. This relationship was also significant in the genetically heterogeneous population of mice. However, magnitude of tolerance explained only 10% of the variance in sensitization among individuals of the inbred strain population, whereas it explained 44% of the variance among individuals of the eight-strain cross. When repeated exposures to ethanol were disassociated from the test apparatus, this relationship in the eight-strain cross disappeared. Furthermore, days to peak sensitization and tolerance across days did not perfectly mirror each other. Overall, our data do not support shared genetic mechanisms in sensitization and tolerance development but suggest a partial relationship among individuals that could be related to drug–environment associations.

Analysis of DBA/2J data alone from Experiment 1.
Because we used DBA/2J as a mouse model for high sensitivity to EtOH-induced sensitization, we separately analyzed the DBA/2J strain data from Experiment 1 (Supplementary Figures S6).
Locomotor activity counts.To determine differences between the CS and CE groups within the DBA/2J strain for locomotor activity (Supplementary Figure S6A), repeated measures ANOVA was performed across test days.There was a significant day by treatment group interaction (F[6,108]=6.8,p<0.001).Further interrogation of this interaction revealed no significant difference in baseline activity (day 2) between the CS and CE groups and no significant difference on day 3 between the CS and CE groups.However, the CE group had higher locomotor activity levels on test days 5, 7, 9 and 11, when compared to the CS group.The difference on day 11 supports between-group sensitization.In the within-group repeated measures ANOVA across days, there was no significant effect of day for the CS group; whereas the effect of day was significant in the CE group (F[6,54]=7.3,p<0.001).In the CE group, there was greater locomotor activity on day 11 when compared to all other days which supports within-group sensitization.See Supplementary Figure S6A for significant mean differences.
Grid test error counts.To determine differences between the CS and CE groups within the DBA/2J strain for foot slip errors (Supplementary Figure S6B), repeated measures ANOVA was performed across test days.There was a significant day by treatment group interaction (F[6,108]=28.9,p<0.001).Further interrogation of this interaction revealed no significant difference in baseline errors (day 2) between the groups.There were significantly more foot slip errors in CE than CS group mice on days 3, 5, 7, and 9. On day 11, when CS group mice received EtOH, there was no significant difference in the number of foot slip errors between the CS and CE groups.Repeated measures ANOVA for errors in the CS group found a significant main effect of day (F[6,54]=160.8,p<0.001), due to a larger number of errors on day 11.Repeated measures ANOVA for errors within the CE group also found a significant main effect of day (F[6,54]=26.7,p<0.001), with an increase in errors on day 3 compared to baseline, and elevated errors over the course of testing.See Supplementary Figure S6B for significant mean differences.
Grid test ataxia ratio.To determine differences between the CS and CE groups within the DBA/2J strain for ataxia ratio (Supplementary Figure S6C), repeated measures ANOVA was performed across test days.There was a significant day by treatment group interaction (F[6,108]=37.4,p<0.001).Further interrogation of this interaction revealed no significant difference in baseline ataxia ratio (day 2) between the groups.However, there was significantly greater ataxia in CE compared to CS group mice on days 3, 5, 7, 9 and 11.Repeated measures ANOVA for ataxia ratio in the CS group found a main effects of day (F[6,54]= 49.5, p<0.001), due to a larger ataxia ratio on day 11.Repeated measures ANOVA for ataxia ratio within the CE group also found a significant main effect of day (F[6,54]=28.7,p<0.001), with an increase in errors on day 3 decreasing across treatments in support of tolerance.Data are shown in Supplemental Figure 6, panel C. See Supplementary Figure S6C for significant mean differences.

Blood EtOH concentration (BEC).
There was no difference between treatment groups for BEC on day 11 in the DBA/2J mice (mean ± SE for CE = 2.51 ± 0.06 mg/ml and CS = 2.66 ± 0.07 mg/ml).
Activity counts across days in the chronic saline (CS) and chronic EtOH (CE) groups for each strain.Data shown are means ± SE.N per strain per group = 9-10.
Number of errors across days in the chronic saline (CS) and chronic EtOH (CE) groups for each strain.Data shown are means ± SE.N per strain group per group = 9-10.
Ataxia ratio across days in the chronic saline (CS) and chronic EtOH (CE) groups for each strain.Shown are means ± SE.N per strain per group = 9-10.SupplementaryFigure S4.Mean EtOH-induced error phenotypes in 15 inbred mouse strains.(A) Solid bars represent day 3 -day 2 error counts for the chronic EtOH (CE) group.Open bars in the inset represent day 11 -day 2 data for the chronic saline (CS) group.Strain order for the CS data is the same as that listed along the x-axis.The r represents the Pearson's correlation between the CE and CS groups for error counts after acute ethanol administration.(B) Change in errors (day 3 -day 3) with repeated ethanol exposure in the CE group.(C) Error counts on day 11 were corrected for baseline errors.Open bars represent the CS group data that received ethanol for the first time on day 11; solid bars represent the CE group data that received EtOH for the fifth time on day 11.Shown are means ± SE. ***p<0.001for the difference between the CS and CE groups within a given strain.
. Mean blood ethanol concentration (BEC) in 15 inbred mouse strains.Blood samples were collected from mice in experiment 1, immediately following testing on day 11.Solid bars represent BEC (mg/mL) for the chronic EtOH (CE) treatment group, which received EtOH for the fifth time on day 11.Open bars represent BEC for the chronic saline (CS) treatment group which received EtOH for the first time on day 11.Shown are means ± SE. *p<0.05 for the main effect of group; ### p<0.001 for the main effect of strain.
locomotor activity, error counts and ataxia ratio for DBA/2J mice from Experiment 1. (A) Mean locomotor activity in CS and CE treatment groups.(B) Mean number of foot slip errors in the CS and CE treatment groups.(C) Mean ataxia ratio in the CS and CE groups.See Table1in main manuscript for treatment schedule.Shown are means ± SE. *p<0.05,**p<0.01,***p<0.001for the difference between CE and CS groups for that test day; +++p<0.001for the acute response to EtOH (CS group day 11 vs day 2; CE group day 3 vs day 2); ###p<0.001for significant sensitization or tolerance in CE group mice (day 11 vs day 3).