## 2 Materials and Methods  
### Study Design  
The study was accepted by the Local Ethics Committee for Animal Experiments at the Warsaw University of Life Sciences, SGGW-WULS, Poland (for the study protocol: 21/2007 and for conducted analysis: 26/2007). The study was conducted in a group of 18 male Wistar rats, that was homogen, with an identical age of 20 days while beginning acclimatization and 30 days while beginning animal experiment, and with an initial body mass of \(92.76\pm 9.32\) at the age of 20 days, being obtained from the Mossakowski Medical Research Centre of the Polish Academy of Sciences (Warsaw, Poland). Since this moment rats were housed individually at constant room temperature (\(23\pm 2\)\({}^{\circ}\)C), humidity of 50-55%, in a 12-h light/12-h dark cycle and were provided ad libitum a standard laboratory diet (Labofeed H, Morawski Animal Feed Manufacturer, Kcynia, Poland). For animal model studies it is important if animals are housed individually or in a group, as both individual housing (due to social isolation) and housing in a group (due to social competition within the group, which is influenced by factors such as the size of groups, the group composition, space available, and the despotism of the dominant individual) may be a stressors [29]. This problem is especially important in the case of studies analyzing the influence of alcohol intake, as the alcohol intake may be influenced by a stress which is experienced by animal [30]. At the same time, for such studies, it is crucial to monitor carefully the individual alcohol intake, along with the decision to choose the model of individual housing, rather than housing in groups, as both options may influence the level of stress and resultant alcohol intake [31]. Such an approach was applied in the number of studies of various authors analyzing alcohol intake which housed their rats individually [32; 33; 34; 35]. In such situations, in order to reduce the influence of isolation on a stress that is experienced by animal, the acclimatization period should be applied. Moreover, if rats are housed individually while being juvenile, this is the other factor which may reduce their stress [36]. Based on the experiences by other researchers, such model was applied in order to reduce the level of stress, as animals were housed individually while being juvenile, and the acclimatization period was applied. Moreover, during all the experimental procedures, as well as during acclimatization period, animals were in the same room and remained in olfactory, auditory, and visual contact with their conspecifics, which is the other aspect that may reduce their stress [37]. The scheme of the study is presented in Figure 1. Before the experiment, there was a period of acclimatization, when rats received dry red wine (Sophia Sakar, Cabernet Sauvignon, Domain Menada, Stara Zagora, and Bulgaria, 11% alc. vol.) diluted in tap water to increasing concentration of ethanol (2%, 4%, 6%, 8%, and 10% (vol./vol.) ethanol solution), while the concentration was increased each second day. Figure 1: The scheme of the conducted study of the induced red wine consumption in the group of young male Wistar rats. After period of acclimatization, during the experimental period, rats received identical dry red wine (Sophia Sakar, Cabernet Sauvignon, Domain Menada, Stara Zagora, and Bulgaria, 11% alc. vol.) 10% (vol./vol.) ethanol solution, as in the last stage of acclimatization, as in the previously published studies [38; 39]. The wine was chosen on the basis of its characteristics, with the aim to find the wine which is broadly available and relatively cheap, but also characterized by a simple flavor, which results from the fact that for adolescents abusing alcohol, a price is among the most important determinants of its consumption.