Prairie voles were housed and tested in our breeding colony room at the Portland Veterans Affairs Medical Center Veterinary Medical Unit. The animals were housed under a 12:12 light-dark cycle, weaned at 21 days and housed with the entire litter until sex was determined at approximately 40 days of age and animals were housed with same-sex siblings. While the 12:12 cycle is not standard for vole studies which commonly use the 14:10 cycle, the 12:12 matches cycles that are typically used in other rodents and that we used in our previous work (Anacker et al., 2011
; Anacker and Ryabinin, 2010
). Only animals that had been housed with 1–3 siblings prior to testing were used in these experiments. Animals were given ad libitum
access to water and a diet of mixed rabbit chow (LabDiet Hi-Fiber Rabbit), corn (Nutrena Cleaned Grains) and oats (Grainland Select Grains) throughout the experiments. All animals were experimentally naïve.
In Experiment 1, we tested the hypothesis that one vole alters its alcohol intake to match that of its drinking partner when their drinking levels differ. For this, 81 prairie voles aged 69–100 days at the start of testing, weighing 39.8 ± 0.8 g (mean ± SEM), were tested in the first phase of the experiment. In this first phase, voles were weighed and then moved into individual housing, where they were given continuous access to two 25 mL glass cylinders fitted with a metal sipper tube and rubber stopper. One bottle contained tap water, and the other contained 10% ethanol (diluted volume/volume with tap water from 95% ethanol) for four days. During this time, the volume of fluid was monitored and refilled every 24 hours, and the position of the bottles was rotated daily to avoid a bias due to side preference.
Each animal’s preference for alcohol (volume of alcohol divided by total fluid volume consumed) and dose of alcohol (g/kg body weight) was assessed daily and categorized as high, medium, or low, based on the criteria presented in . Both preference and dose were normally distributed. The specific criteria were chosen because in preliminary tests they yielded approximately equal numbers of high, medium, and low drinkers. After four days of baseline drinking in isolation, each animal was then categorized by subtracting the number of ‘low’ scores for preference and dose from the number of ‘high’ scores. Animals receiving a positive number were labeled ‘high drinkers’ while those receiving negative numbers were labeled ‘low drinkers.’ Following categorization from baseline intake, 60 high- or low-drinking prairie voles were used for the study (30 female, 30 male), while the remaining ‘medium drinkers’ were not tested further.
Criteria for alcohol drinking level group assignment
For the second phase of the experiment, high drinkers were paired with low drinkers of the same sex, which were strangers to each other. Pairs were determined as follows, in order to achieve similar relative differences in alcohol intake between the high drinker and low drinker when compared between pairs. The highest drinker of the high group was paired with the highest drinker of the low group, the second-highest high with the second-highest low, and so on, until the lowest of the high drinkers was paired with the lowest of the low drinkers. The exceptions to this rule were when the pairing would lead to siblings being paired, and in such cases, the closest ranking animals would be switched. Using this method for pairing, there was a relatively similar difference in the drinking levels of the paired animals for each pair.
The paired animals were each housed on one side of a cage with a wire mesh divider that allowed the voles to interact but gave each exclusive access to its own drinking tubes. This housing was identical to our previously validated procedure showing that the voles’ alcohol drinking behavior is similar when they are housed in non-divided cages and in cages divided by a mesh (Anacker et al., 2011
). The pairs were housed together for four days, with the relative positions of the high and low drinkers within the cage determined randomly. During this period, each animal again had continuous access to 10% alcohol and water. The position of the bottles for each member of a pair was identical (for example, ethanol bottles on the left for both animals on day 1 of pair-housing, and ethanol bottles on the right on day 2). Animals were monitored daily for preference and intake, in order to determine whether any changes in drinking level occurred for each animal.
In the third phase of the experiment, the voles were placed in isolation and drinking was monitored with the same two-bottle choice test for four more days, in order to determine whether any changes in drinking level that occurred during the paired period persist in a subsequent isolation.
In Experiment 2, we tested whether any changes in drinking would occur when paired animals started with similar drinking levels, in order to show that the results of Experiment 1 were due specifically to the presence of the partner drinking at a different level, and not due to the development of an aversion to alcohol that could occur independent of the partner’s drinking. The same procedure was performed for the first phase of the experiment, using 51 animals, 71–112 days old, weighing 38.4 ± 0.7 g. Of these, 42 voles (20 female and 22 male) were classified as high and low drinkers and used in the second phase. In this experiment, high drinkers were paired with high drinkers, and low with low, and otherwise the second and third phases of the experiment were performed with the same methods as described for Experiment 1.
Experiment 3 was conducted just as Experiment 1, except that a saccharin solution (0.05%, weight/volume of tap water) was self-administered instead of alcohol, in order to test whether the effects of Experiment 1 were specific to alcohol or would extend to another rewarding substance. We have previously tested saccharin consumption in prairie voles and found that they show a high preference for this substance at the same concentration used here, and that consumption of saccharin is not correlated between partners as was seen for alcohol consumption (Anacker et al., 2011
). In this experiment, 39 animals from 67–101 days old, weighing 34.6 ± 1.2 g were used for the first phase, and 34 were labeled high or low for saccharin consumption and went on to the second phase. The criteria for categorization are presented in .
Criteria for saccharin drinking level group assignment
Since daily alcohol intake for individual animals differ due to common variability as well as the position of the bottle (some voles show a notable side preference), average intake was calculated across the four days at each phase of the experiments. Then, a repeated measures ANOVA was applied to the data from each experiment, using sex and baseline drinking category (high or low) as between subjects factors, the average drinking throughout each of the three four-day sessions (housing type: isolation 1, paired, or isolation 2) as the repeated measure, with dose of alcohol (g/kg) or alcohol preference as the dependent measures. When warranted by a significant main effect or interaction, the data were further assessed by Fisher’s PLSD or tests for simple effects. A few animals were eliminated from statistical analysis of alcohol preference, or both preference and dose, due to faulty (leaky) tubes; corresponding degrees of freedom are reported in the results for each test.