Following the reintroduction of EtOH solutions after a period of abstinence, the vehicle-treated group showed a typical increase in EtOH consumption, indicating the occurrence of an ADE (Fig. 2). A 2-way ANOVA for repeated measures revealed a significant increase in daily EtOH intake after a deprivation phase as compared to baseline drinking, factor day: F(3, 42) = 8.1, p < 0.0001. This increase was seen only in the vehicle-treated animal group (Fig. 2). In lamotrigine-treated animals, daily EtOH intake on the first post-deprivation day was similar to that seen during baseline drinking but dropped below baseline levels from the second day onward. A 2-way ANOVA displayed a significant difference in EtOH intake between vehicle- and lamotrigine-treated animal groups, factor treatment group: F(1, 14) = 33.0, p < 0.0001, and a significant treatment group × day interaction effect, F(3, 42) = 11.8, p < 0.0001, showing that the treatment of rats with lamotrigine was capable of abolishing the expression of ADE.
For the Fourier analysis, 10-minute drinking data blocks were used, which provided detailed information on the water and EtOH consumption in terms of patterns of the drinking behavior over a 24-hour period. Our analysis shows that under baseline conditions, water/EtOH consumption followed a stable oscillatory pattern with clearly defined characteristics such as amplitude and frequency (Table 1, Fig. 3). Additionally, the analysis of the baseline drinking data revealed that during a 24-hour period, there is a probability of occurrence of a single maximum intake peak regardless of the fluid (Table 1). This peak seems to occur during the onset of the active (dark) phase of a rat (Fig. 3).
Cumulative calculations of water/EtOH intake shows that following reintroduction of the EtOH solutions after a period of abstinence, control vehicle-treated rats increased their EtOH intake, whereas water intake dropped down below baseline drinking levels (data not shown). Subsequent Fourier analysis revealed that the period of abstinence also caused considerable alterations in the characteristics of drinking oscillations during the first postabstinence day. These alterations were characterized by a dramatic increase in the frequency of drinking approaches for both 10% EtOH, F(1, 6) = 103.41, p < 0.00001, and 20% EoTH, F(1, 6) = 540.47, p < 0.000001, solutions, although both the amplitude and the amount of EtOH consumed during a 10-minute interval remained similar to that measured under the baseline drinking conditions (Table 1, Fig. 3). In fact, the probability of occurrence of 21 maximum drinking peaks for 20% EtOH solution within the first 24 hours of reexposure to EtOH was detected. On the other hand, animals maintained a stable diurnal pattern of water and 5% EtOH consumption monitored as unchanged frequency of drinking oscillations and pattern-defining peak drinking frequencies (the maximum peak/24 h).
More detailed analysis of the effects of lamotrigine on the ADE showed that the cumulative consumed amount of each EtOH solution was reduced compared to vehicle-treated animals (data not shown). Fourier analysis further revealed that although water intake in lamotrigine-treated animals was increased, factor treatment group: F(1, 6) = 88.95, p < 0.001 and F(1, 6) = 17.81, p < 0.05 for the first ADE day and for the 2 successive ADE days, respectively, a normal diurnal pattern of drinking oscillations of unchanged frequency and a single maximum peak of water intake probability was retained (Table 1). Differences in water consumption between the 2 treatment groups during the first post-abstinence days were also reflected in the number of approaches to the water bottle, factor treatment group: F(1, 6) = 7.70, p < 0.05 and F(1, 6) = 15.25, p < 0.001 for the first ADE day and for the 2 successive ADE days, respectively. EtOH consumption in lamotrigine-treated animals measured during 10-minute intervals was significantly reduced for all 3 EtOH solutions during the first postabstinence day, factor treatment group: F(1, 6) = 14.4, p < 0.01, F(1, 6) = 6.75, p < 0.05, and F(1, 6) = 6.0, p < 0.05 for 5, 10, and 20% EtOH, respectively, as well as during the 2 successive ADE days, factor treatment group: F(1, 6) = 5.4, p < 0.05, F(1, 6) = 11.07, p < 0.01, and F(1, 6) = 7.56, p < 0.05 for 5, 10, and 20% EtOH, respectively (Table 1). Contrarily, both drinking frequency and the number of actual approaches to the EtOH bottles during the first ADE day were not different in the lamotrigine treatment group when compared to the vehicle group (p = 0.43 and p = 0.21, p = 0.65 and p = 0.37, and p = 0.35 and p = 0.15 for the frequency and number of approaches of the for 5, 10, and 20% EtOH, respectively) (Table 1). Further injections of lamotrigine on ADE days 2 and 3 were then accompanied by a reduction in frequency of drinking oscillations, factor treatment group: F(1, 6) = 10.93, p < 0.05, F(1, 6) = 47.51, p < 0.001, and F(1, 6) = 68.5, p < 0.0001 for 5, 10, and 20% EtOH, respectively, and number of approaches, factor treatment group: F(1, 6) = 4.5, p < 0.05 and F(1, 6) = 5.87, p < 0.05 for 10 and 20% EtOH, respectively.