The Use of a Novel Drinkometer System for Assessing Pharmacological Treatment Effects on Ethanol Consumption in Rats

Abstract

Background

There are numerous studies in the preclinical alcohol research field showing that pharmacological interventions and many other manipulations can influence ethanol (EtOH) consumption in a free-choice paradigm in rats. Most of these studies are based on 24-hour measurements. These studies provide a measure of the total amount of EtOH consumed per day, but do not provide information on the drinking patterns within this period of measurement. Here, we used a novel drinkometer system in combination with Fourier analysis to provide detailed information on drinking patterns.

Methods

Our automated drinkometer system measures fluid consumption by means of high-precision sensors attached to the drinking bottles in the home cage of the rat and thereby ameliorates several limitations of a classical lickometer-based drinkometer system. As an example of its application, we used the alcohol deprivation effect (ADE) model for relapse-like drinking and tested as a reference compound lamotrigine, which has a robust effect on the ADE. Fourier analysis was chosen as the main strategy for 24-hour drinking pattern recognition during water/EtOH drinking.

Results

Under baseline conditions, voluntary EtOH consumption in rats can be expressed as characteristic oscillations that follow diurnal activity and differ in their amplitude, depending on the EtOH concentration. This diurnal drinking rhythmicity was altered during a relapse condition. Furthermore, lamotrigine given during the ADE did not significantly affect the drinking frequency or the number of approaches to the EtOH bottles when compared to vehicle-treated animals. However, EtOH intake during a drinking approach was dramatically reduced.

Conclusions

The use of the drinkometer system and mathematical modeling allows the characterization of treatment effects on relapse-like drinking with a great level of detail. One use of such detailed information may lie in its translational predictability. For instance, owing to lamotrigine treatment's lack of effect on EtOH drinking frequency or the number of approaches to the EtOH bottles, this compound might not be effective in relapse prevention per se but may reduce hedonic EtOH effects and could therefore be used in alcohol-dependent patients if harm reduction is the primary goal of treatment.

In 1940, Curt Paul Richter reported that laboratory rats voluntarily consume ethanol (EtOH), although with high individual variability (Richter and Campbell, 1940). This discovery marked the beginning of animal research in the study of EtOH reinforcement processes. Subsequently, with an increasing trend in the past 20 years, thousands of studies on voluntary EtOH consumption in rodents have been conducted, permitting the deciphering of the genetic and neurochemical basis of EtOH reinforcement (Helinski and Spanagel, 2011; Spanagel, 2009). Studies of voluntary EtOH consumption in laboratory animals are crucial for the development of medication in the field of alcohol research; indeed, all accepted pharmacotherapies have been based on animal work of this nature (Spanagel and Kiefer, 2008).

In animal models of voluntary oral EtOH consumption, animals have concurrent access to water and either 1 or several EtOH solutions in their home cage. Monitoring of EtOH intake is usually performed by weighing water and EtOH bottles once every 24 hours. However, the effects of EtOH depend not only on the total amount of EtOH consumed by a rat or mouse within 24 hours but also on the time course and pattern of drinking, measured as the frequency of approaches to an EtOH solution as well as the amount consumed per drinking approach (Leeman et al., 2010). The present study proposes a more sophisticated approach for monitoring EtOH drinking behavior and testing putative antirelapse compounds in the home cage. We registered drinking patterns with a high temporal distribution in male Wistar rats subjected to a long-term voluntary EtOH consumption procedure repeatedly interrupted with abstinence phases. For this purpose, a novel drinkometer system was employed in the present study. The first drinkometer was published by Hill and Stellar (1951). Since then, drinkometers have been used repeatedly for studying EtOH drinking patterns in rodents (Files et al., 1992; Kampov-Polevoy et al., 2000; Pastor et al., 2010). Most drinkometer systems involve a lick-sensing circuit, which applies a voltage to a metal drinking spout through a resistor and detect the voltage reductions that occur when the animal's tongue touches the spout, which grounds the spout through the animal's feet. Such drinkometer circuits have several limitations; the current that passes through the tongue may be detected by the animal, or it may alter the taste of the fluid and thereby influence the rate of licking (Overton and Overton, 2007). Furthermore, the classical drinkometer cage has a metal grid floor, which does not provide ideal conditions for long-term observations. The automated home cage–based system we are using does not involve a lick-sensing circuit. Instead, number of drinking approaches is monitored by continuous ultrasensitive weighing of the bottles. With this system, we can circumvent the above-mentioned limitations.

To assess the potential of antirelapse effects of pharmacological treatment, we used lamotrigine as a reference compound. Lamotrigine—which acts on voltage-gated Na⁺-channels (Tarnawa et al., 2007)—has a robust effect on relapse-like drinking in rats (Vengeliene et al., 2007). In rats that had long-term voluntary access to EtOH followed by deprivation for several weeks, the re-presentation of EtOH leads to relapse-like drinking, a temporal increase in EtOH intake over baseline drinking. This robust phenomenon is called the alcohol deprivation effect (ADE) (Sinclair and Senter, 1967; Spanagel and Hölter, 1999). In recent years, this model has become widely used for examining the efficacy of pharmacological agents in preventing compulsive EtOH consumption and relapse (Vengeliene et al., 2008, 2009).

One of the main objectives of this study was the quantitative assessment of the measurements obtained from the drinkometer system. For addressing pattern recognition within biologic processes, various mathematical algorithms can be applied to characterize temporal rhythmicity in time series (Noori, in press). For data analysis from the drinkometer system, Fourier analysis was applied, as it is a powerful tool for investigating patterns within time series and thus provides an appropriate approach for pattern recognition with regard to water/EtOH intake. As drinking behavior in rats has been shown to demonstrate circadian rhythmicity (Araujo and Marques, 1996), we used Fourier analysis to study fluid intake patterns in a 4-bottle free-choice paradigm during both baseline and relapse-like conditions and assessed the effects of lamotrigine treatment on these patterns. The major advantage of this approach is that it allows the estimation of a probability measure for the 24-hour occurrence of a maximal drinking peak based on the coefficients of the Fourier series. This probability measure and the coefficients of the Fourier series allow a quantitative investigation of relapse drinking and the effects of lamotrigine on drinking behavior.

Materials and Methods

Animals

Sixteen 2-month-old male Wistar rats (from our own breeding colony at the CIMH, Mannheim, Germany) were used. All animals were housed individually in standard rat cages (Eurostandard Type III; Ehret, Emmendingen, Germany) under a 12/12-hour artificial light/dark cycle (lights on at 7:00 am). Room temperature was kept constant (temperature: 22 ± 1°C, humidity: 55 ± 5%). Standard laboratory rat food (Ssniff, Soest, Germany) and tap water were provided ad libitum throughout the experimental period. Body weights were measured weekly. All experimental procedures were approved by the Committee on Animal Care and Use and carried out in accordance with the local Animal Welfare Act and the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Drugs

EtOH drinking solutions were prepared from 96% EtOH (Merck, Darmstadt, Germany) and then diluted with tap water. Lamotrigine (generously provided by GSK, Verona, Italy) was dissolved in polyethylene glycol 400 (PEG 400; Sigma-Aldrich Co., St. Louis, MO) and then diluted with water (aqua ad iniectabilia; Braun, Melsungen AG, Germany) to a final PEG 400 concentration of 20%. The solution was freshly prepared and injected as a volume of 5 ml/kg intraperitoneally (IP). Control experiments were performed following the administration of 20% PEG 400.

Drinkometer System

The drinkometer system has been developed together with TSE Systems (Bad Homburg, Germany). It enables continuous long-term monitoring of liquid consumption by amount and time in a standard rat home cage. The system is equipped with 4 drinking stations to allow liquid choice. The drinking station consists of a glass vessel containing the liquid and a high-precision sensor for weighing the amount of liquid removed from the glass vessel (Fig. 1). Spillage and evaporation are minimized using special bottle caps. All drinking stations are monitored by a computer. The system features ultra-high resolution—down to 0.01 g. The whole system is mounted to a custom-made free-swinging steel frame to avoid any environmental disturbances. The drinkometer system measures the weight of a vessel in 200-ms steps and saves it in 1-second steps. The normal sampling can be set with minimum 1-minute intervals. For this study, frequent monitoring of drinking patterns was required to elucidate treatment effect on circadian drinking behaviors as it is known that several clock genes modulate the effects of EtOH and vice versa (Perreau-Lenz and Spanagel, 2008; Spanagel et al., 2005); sampling was performed at 10-minute intervals.

Figure 1. A home cage–based drinkometer system. This automated system allows continuous monitoring of drinking approaches with the precision to measure fluid intake of down to 10 μl in a 4-bottle free-choice paradigm. The system measures fluid consumption by means of high-precision sensors attached to the drinking bottles in the home cage of the rat.

Long-Term EtOH Consumption with Repeated Deprivation Phases

After 2 weeks of habituation to the animal room, rats were given ad libitum access to tap water and 5, 10, and 20% EtOH solutions (v/v). The first 2-week deprivation period was introduced after 8 weeks of continuous EtOH availability. After this deprivation period, rats were given access to EtOH again. This access was further interrupted repeatedly with deprivation periods in a random manner (i.e., access to EtOH was randomly interrupted with 2- to 3-week deprivation phases).

Pharmacological Study

The pharmacological study using 5 mg/kg of lamotrigine—this was an optimal dose in our previous study (Vengeliene et al., 2007)—was introduced at the end of the seventh EtOH deprivation phase of 15-day duration. Thus, before drug treatment, all animals had 44 weeks of access to EtOH interrupted with several 2- to 3-week withdrawal phases. To study the effects of drug treatment on the expression on ADE, rats were divided into 2 groups (n = 8) in such a way that the mean baseline total EtOH intake, as well as the intake of every solution separately (i.e., water, and 5, 10, and 20% EtOH), was matched. Baseline drinking was monitored for 6 days. After the last day of baseline measurement, the EtOH bottles were removed from the cages, leaving the animals with free access to food and water for 15 days. Thereafter, each animal was subjected to a total of 5 IP injections (starting at 7 pm with 12-hour intervals) of either vehicle or 5 mg/kg lamotrigine. We administered the drug in 12-hour intervals because the half-life of plasma elimination of this compound in rat is 17.4 hours (Carles Large, personal communication). The EtOH bottles were reintroduced after the second injection (at ~9 am on the 16th day of EtOH deprivation), and the drinking was measured each 10 minutes for the following 6 days. Total daily EtOH intake (g/kg of body weight/d) was calculated from these data.

Data on daily total EtOH intake were analyzed using a 2-way analysis of variance (ANOVA) with repeated measures (factors were between subjects—treatment group, and within subjects—day). Whenever significant differences were found, post hoc Student–Newman–Keuls test was performed. The level of significance was p < 0.05.

Pattern Analysis

Pattern analysis was performed using data of the last 3 baseline drinking days and the first postabstinence drinking day when animals were receiving either vehicle or lamotrigine treatment. Our main strategy for detecting patterns within the drinkometer data sets was to characterize recurrent drinking events as Fourier series. The Fourier analysis provides a function with the most likelihood to describe the amount of water drinking or drinking of EtOH mixtures in the drinkometer system during the measurement interval L. Based on the frequencies and the Fourier coefficients , respectively, this approach provides approximate measures for drinking frequencies, drinking peak times, and peak intake in control groups and enables us to compare the drinking behavior of experimental groups based on these properties. In MATLAB (http://www.mathworks.com), we used the maximum likelihood estimation (MLE) to determine the Fourier coefficients of our model based on the drinking data sets. Using 10-minute drinking data for each EtOH mixture (5, 10, and 20%) and water, the MLE method suggests 2 sets of Fourier parameters {a_n, b_n} and {} and the frequencies for Fourier representations of averaged drinking amounts f(t) for the control group. for the group of animals treated with 5 mg of lamotrigine. We analyzed the differences between these 2 groups based on the direct coefficient comparisons , , and by the differences in the area under the curves (AUC) of each model function for each EtOH concentration c = {0, 5, 10, 20%}. While the AUC provides an approximate qualitative measure, we used arithmetic mean values as a quantitative measure for the average number of approaches to the drinking bottles. This further analysis was performed to confirm the findings of the Fourier analysis. Furthermore, the parameters and have been applied to classify considerable differences between the vehicle and lamotrigine administration throughout this study. In addition, we analyzed the Fourier coefficient variations using a 1-way ANOVA. The chosen level of significance was p < 0.05. Specifically, the parameters intake a₀, amplitude, and frequency () were compared within and between the vehicle- and lamotrigine-treated animals, describing the approximate mean of water/EtOH intake, the maximal peak of water/EtOH intake, and the number of maximal peak occurrences in 1 hour, respectively. By multiplying the obtained frequencies by 24, we obtained a probability measure for the occurrence of 1 maximal peak within the 24-hour intervals (a 24-hour periodic oscillatory pattern), which is more convenient for the biologic interpretation of the results.

Results

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.

Figure 2. Total daily ethanol (EtOH) intake (g/kg/d) before and after an EtOH deprivation period of 2 weeks. Arrows indicate the administration of either vehicle (n = 8) or 5 mg/kg of lamotrigine (n = 8). The measurement of EtOH intake in last 3 days is given as baseline drinking—“B” Data are presented as means ± SEM. *Significant differences from the vehicle treatment group, p < 0.05.

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).

Figure 3. The Fourier representations of the averaged 10-minute intake of water (ml/kg) (A), 5% ethanol (EtOH) (g/kg) (B), 10% EtOH (g/kg) (C), and 20% EtOH (g/kg) (D) during the last day of baseline measurements and during the first day of postabstinence drinking in the vehicle-treated animal group. During the alcohol deprivation effect (ADE), a dramatic increase in the frequency of drinking approaches to both 10 and 20% EtOH solutions (C, D) represents the loss of the normal 24-hour drinking pattern relative to water and 5% EtOH (A, B).

Table 1. The Model Parameters Derived from the Fourier Coefficients Describe the Mean Water/Ethanol (EtOH) Intake During 10-Minute Intervals (Intake: in Milliliter per kg of Body Weight for Water and in Grams of Pure EtOH per Kilogram of Body Weight for EtOH Solutions), the Maximal Peak of Water/EtOH Intake (Amplitude: in ml/kg for Water and in g/kg for EtOH Solutions), and the Number of Maximal Intake-Peak Occurrences in an Hour (Frequency). The Extension of the Latter Parameter to 24-Hour Cycles Provides a Measure for the Existence of 24-Hour Drinking Patterns within Our Analysis. The Maximum Peak/24-H Values Denote the Probability of the Occurrence of the Maximum Drinking Peak in a 24-Hour Interval

Treatment	Fourier coefficients	Water	5% EtOH	10% EtOH	20% EtOH
The table shows the Fourier coefficients calculated for the last 3 days of baseline drinking (baseline), the first postabstinence day (ADE, day 1), and the average of the successive 2 postabstinence days (ADE, days 2 to 3) in vehicle and lamotrigine-treated rats. Vehicle and lamotrigine treatments were performed during the first 3 postabstinence days. n.p.—no 24-hour drinking pattern could be detected. ^a Indicates significant differences from the baseline condition, ^b Indicates significant difference from the vehicle control group, and ^c Indicates significant differences from both the baseline condition and the vehicle control group, p < 0.05.
Baseline
	Intake	0.181	0.002	0.005	0.004
	Amplitude	0.415	0.004	0.016	0.016
	Frequency/1 h	0.044	0.044	0.043	0.044
	Max peak/24 h	1	1	1	1
Alcohol deprivation effect (ADE), day 1
Vehicle	Intake	0.045a	0.006a	0.006	0.004
	Amplitude	0.170a	0.021a	0.018	0.027
	Frequency/1 h	0.044	0.040	0.633a	0.918a
	Max peak/24 h	1	1	14 (n.p.)a	21 (n.p.)a
Lamotrigine	Intake	0.299c	0.003b	0.003b	0.002c
	Amplitude	1.441c	0.014a	0.008c	0.006c
	Frequency	0.035	0.068	0.648a	0.920a
	Max peak/24 h	1	1	15 (n.p.)a	22 (n.p.)a
ADE, Days 2 to 3
Vehicle	Intake	0.120	0.010a	0.007	0.004
	Amplitude	0.304	0.015a	0.023	0.014
	Frequency	0.044	0.048	0.069	0.415a
	Max peak/24 h	1	1	1–2 (n.p.)	10 (n.p.)a
Lamotrigine	Intake	0.254b	0.004b	0.001c	0.002b
	Amplitude	0.480	0.005b	0.008c	0.012
	Frequency	0.046	0.021c	0.019c	0.016c
	Max peak/24 h	1	0.5 (n.p.)c	0.5 (n.p.)c	0.4 (n.p.)c

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).

During the subsequent 2 postabstinence days, diurnal drinking patterns of more concentrated EtOH solutions (10 and 20%) were still altered but not as pronounced as during the first ADE day (Table 1), but the patterns recovered completely during the postabstinence days 4 to 6 (data not shown).

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.

Discussion

In the present study, we obtained detailed information on the drinking patterns following abstinence and lamotrigine treatment by means of a novel drinkometer system and subsequent Fourier analysis. Results of the Fourier analysis showed that water/EtOH drinking patterns in rats during baseline drinking conditions can be expressed as characteristic oscillations that follow a diurnal activity. Depending on the EtOH concentration, these drinking oscillations differ in their amplitude. However, the probability that an animal will drink at a particular time point during its active (dark) phase remains a characteristic feature of standard drinking behavior and does not depend on the drinking fluid offered. This probability refers to the occurrence of 1 maximum drinking peak within a 24-hour period and may be used as a key representation of the diurnal control over drinking behavior in general.

Our previous research demonstrated that postabstinence drinking is characterized not only by an increase in EtOH consumption but also by higher motivation for EtOH, as illustrated by higher breakpoints in lever pressing under a progressive ratio schedule (Spanagel and Hölter, 2000), as well as a preference shift toward more concentrated EtOH solutions (Hölter et al., 1998). The present study complements our earlier findings. We show that EtOH abstinence introduced after long-term intermittent drinking experience dramatically increased the drinking frequency of 10 and 20% EtOH solutions. In addition, we measured a complete loss of normal diurnal patterns of 10 and 20% EtOH consumption. Thus, the probability of the occurrence of the maximum drinking peak in a 24-hour interval could not be established anymore. Increased drinking frequency and loss of diurnal drinking rhythmicity could be interpreted as an abstinence-induced increase in EtOH “wanting.” However, the amount of EtOH consumed during a drinking approach remained similar to that measured under the baseline drinking conditions, suggesting that abstinence does not change “liking” of more concentrated EtOH solutions. One important subject in addiction research is the dissociation between drug “wanting” and “liking” (Robinson and Berridge, 2003). The loss of a normal diurnal drinking pattern caused by increased EtOH “wanting” during the ADE could be interpreted as a loss of control over EtOH-taking behavior.

In line with our previous report (Vengeliene et al., 2007), this study demonstrates that repeated administration of lamotrigine reduces daily EtOH intake during the ADE in a 4-bottle free-choice paradigm in rats. Our Fourier analysis further shows that on the first postabstinence day, drinking frequency in lamotrigine-treated animals was significantly increased when compared to baseline condition but was not different from that seen in vehicle-treated animals. It is believed that neural systems responsible for EtOH “wanting” are different from systems that mediate hedonic effects of EtOH (Spanagel, 2009) and that the most advantageous medication for addiction would be the one that reduces compulsive “wanting” to take EtOH. In our study, lamotrigine treatment dramatically reduced EtOH intake during the ADE. However, especially during the first ADE day, the EtOH drinking frequency or number of approaches to the EtOH bottles was not affected by lamotrigine treatment. In contrast, the amount of EtOH consumed during a drinking approach was dramatically reduced. Considering that lamotrigine treatment started at the end of abstinence phase, this could be interpreted in a way that lamotrigine has no effect on EtOH “wanting” but rather on EtOH “liking.” It has also been demonstrated that compounds with anti-glutamatergic properties, such as the anticonvulsant drugs topiramate and lamotrigine, potentiate EtOH-induced sedation/hypnosis (Chen and Holmes, 2009). The dose used in our study was relatively low, and it has been shown that this dose does not affect the locomotor activity of an animal (Vengeliene et al., 2007). However, we cannot rule out the possibility that lamotrigine treatment increased the sedative effect of EtOH and thereby reduced the amount of EtOH consumed by an animal during a drinking approach.

Further mathematical approaches, especially stochastic methods such as Markov chains, would provide an alternative approach to analyze the drinkometer data. Markov chain methods generate sequences of random values to accurately reflect probability distributions, assuming that all future steps depend only on the current state of the system. Increases in EtOH consumption during postabstinence drinking (ADE) could be interpreted as difficulties in controlling EtOH-taking behavior in terms of its termination or levels of use, representing a switch from normal to compulsive drug taking. Mathematical models of nonlinear hysteresis could provide a potential framework for in silico investigations of this behavioral switch. The appropriateness of the hysteresis models for addressing drug-related neurobiological switches has already been shown in previous studies (Noori, in press). Furthermore, the loss of control over EtOH intake could be assessed by estimating the loss of predictability of drinking behavior of an animal. Thus, drug treatment might have an effect in restoring such predictability, which would mean a regained control over the behavior.

In summary, here we demonstrate that the use of a home cage–based drinkometer system in combination with Fourier analysis provides detailed information on EtOH drinking patterns and treatment effects. The information retrieved from our analysis indicates that EtOH drinking in rats can be expressed as characteristic oscillations that follow a diurnal activity but are altered or even lost after deprivation. The combined use of such a drinkometer system and Fourier analysis or other mathematical modeling approaches will help to elucidate EtOH drinking patterns predictive for the development of addictive behavior. It will also provide a much deeper insight into experimental manipulation of drinking behavior, including environmental, pharmacological, and genetic manipulations. Most importantly, it provides translational predictivity for the human situation, as in the case of lamotrigine in the current study. Because of a lack of effect on the EtOH drinking frequency or number of approaches to the EtOH bottles, this compound might not be effective in relapse prevention but may reduce EtOH “liking,” as it dramatically reduced the amount of EtOH consumed during such a drinking approach.

Acknowledgments

We would like to thank Sabrina Koch for her technical assistance and Dr. Rick Bernardi for correcting the English. This work was supported by the Bundesministerium für Bildung und Forschung (NGFN Plus; FKZ: 01GS08152, FKZ: 01GS08155 see under www.ngfn-alkohol.de and Spanagel et al., 2010; FKZ: 01GS08151).