The prairie vole colony was originally established from eight pairs generously provided by Dr. Joseph Lonstein at Michigan State University in March 2005. These voles originated from a colony at Emory University, which was derived from field-caught prairie voles in Illinois. Diversity in our colony was maintained by the generous donations of prairie voles from Dr. Phillip Smith at Texas Tech University in November 2007, and from Dr. Karen Bales at the University of California at Davis in February 2008.
Prior to any experimentation, prairie voles were housed in same-sex sibling groups after weaning at around 21 days, and maintained on a diet of mixed rabbit chow (LabDiet Hi-Fiber Rabbit), corn (Nutrena Cleaned Grains), and oats (Grainland Select Grains). All experiments took place in the vole colony room at the Portland Veterans Affairs Medical Center (VAMC). All animals were kept on a 12:12 hour light-dark cycle. All animals had ad libitum access to food and water throughout each experiment. Animals were alcohol- and experimentally-naïve, except where noted in Experiments 1 and 4.
In Experiment 1, we also used alcohol-naïve mice of C57BL/6J (C57) background bred in our animal colony at OHSU that were housed at four to five per cage with water and food constantly available, and C57 mice purchased from Jackson Laboratories and housed five per cage for one week prior to testing.
All animal care, breeding, and testing procedures were in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and approved by the local Institutional Animal Care and Use Committees at the VAMC and OHSU, Portland, OR, USA.
Drinking solutions and drugs
Fluids were available from 25 ml glass tubes with metal sipper tubes attached with a rubber stopper. For 24-hour access experiments, the bottles were filled to 25 ml, and for the limited access experiment the bottles were filled to 10 ml. After the period of consumption, the bottles were carefully removed to avoid spillage, and remaining volumes were read to the nearest 0.2 ml.
The animals in each alcohol drinking experiment were given a two-bottle choice test, always with one bottle of tap water, and the other bottle containing one of the following solutions. Alcohol solutions (3%, 6%, or 10%) were made as volume/volume (v/v) concentrations from 95% ethanol and tap water. Saccharin and quinine concentrations were 0.05% and 0.0025%, respectively, weight/volume (w/v) in tap water. Each solution was made fresh every other day and stored in an airtight container, and the solutions were replaced in the drinking tube at the start of each consumption period.
Experiment 1 – Assessment of alcohol elimination rates in voles and mice
In order to test whether high alcohol intake in voles is not due to unusually high ethanol elimination rates, we compared blood ethanol concentration (BEC) in voles and C57 mice after an intraperitoneal (i.p.) injection of 2.5 g/kg of ethanol (20%, v/v). Twenty ethanol-naïve prairie voles (10 females and 10 males; weights 36.2 ± 1.4 g) with ages ranging from 83 to 109 days and 20 ethanol-naïve mice (9 females and 11 males; weights 22.8 ± 0.7 g; 72-103 days old) were euthanized either 30, 60, 120, or 180 minutes after the injection by an overdose of CO2, and trunk blood was collected.
In addition, a separate study of alcohol elimination rate was conducted in 35 voles (17 females and 18 males; weights 42.1 ± 1.0 g; 106-134 days old) and 40 mice (20 females and 20 males; weights 23.5 ± 0.6 g; 86 days old) that were not naïve to alcohol. Briefly, these animals were given continuous access to increasing concentrations of alcohol (3%, 6%, 10%) over 12 days in a two-bottle choice test with water as described in subsequent experiments, while half the animals were housed in pairs and the others were housed in isolation. Ethanol injections (2.5 g/kg) were given eleven days following the final access to drinking alcohol, and the animals were euthanized at the same relative times that the naïve animals were.
Blood samples were centrifuged at 5223 relative centrifugal force (RCF) for 10 minutes, after which serum was removed and stored at -20°C before processing. BEC was determined using an Analox Analyzer (Analox Instruments, Luneburg, MO, USA) and is reported in milligrams per deciliter (mg/dl).
Experiment 2 – Investigation of the effects of social separation on alcohol intake
The effect of social housing on alcohol intake was tested in 30 female (44.2 ± 1.2 g) and 32 male (44.3 ± 1.3 g) adult prairie voles, ranging from 68 to 85 days old on the first day of the experiment. First they were moved from their home cage into a new cage with one of the same-sex littermates, where they remained for five days, with water available from the drinking tubes. On the sixth day, the pairs were moved to new cages, where half of the pairs were kept together, and the other half of the pairs were separated into individual cages. In order to monitor drinking behavior of each subject, we created a cage that would house a pair of prairie voles in a manner that would allow each exclusive access to drinking solutions. A wire mesh divider down the center of the cage kept each of the paired voles in one half of the cage, where it had access to its own drinking tubes, but could still see, hear, smell and interact with the other vole through the mesh. The cage was approximately 26.7 × 26.7 × 13.3 cm, the mesh wire was less than 2 mm thick, and the distance between wires was 1.3 cm in the length dimension of the cage and 2.6 cm in the height dimension. The individual cages were approximately equal in size to one side of the mesh-divided cage (26.7 × 16.5 × 13.3 cm).
On the day the animals were moved to new cages, they began a continuous access two-bottle choice test with water and 3% ethanol. The position of the alcohol bottle relative to the water bottle was switched daily to avoid the potential effects of side preference. The choice test consisted of four days at each concentration (3%, 6%, 10%), given in increasing order to all animals. For all choice tests, consumption from both tubes was monitored every 24 hours, and preference for the solution relative to total fluid intake was calculated, in addition to dose of ethanol consumed per body weight (g/kg). Voles were weighed on the first day of the experiment, and every third day throughout. Following the last day of the choice test with ethanol, the bottles were all switched to water for 24 hours, and then a two-bottle choice tastant test began with 0.05% saccharin and water for two days, followed by 0.0025% quinine and water for two days.
Experiment 3 – Investigation of a circadian pattern of fluid consumption using a lickometer system
In order to designate the best time of day for a procedure for limited access to alcohol, fluid consumption was monitored throughout the circadian cycle to determine whether there existed a peak period of consumption in prairie voles, as has been observed in mice and rats (Aalto, 1986
; Freund, 1970
), and utilized to achieve high alcohol intake (Rhodes et al., 2005
; Ryabinin et al., 2003
; Sharpe et al., 2005
). To examine drinking at regular short intervals without having to disturb the animals, we utilized a “lickometer” apparatus that would record the precise time of each lick on a drinking spout. The apparatus has been described previously (Ford et al., 2005). Briefly, it consisted of a raised, stainless steel rod floor beneath a four-sided Plexiglas box with a perforated lid for ventilation, nested inside a shoebox cage with bedding beneath the rod floor. Each animal had access to two drinking tubes, one containing water, and the other containing either saccharin or 10% ethanol, available through two holes in one side of the box. Modifications to the apparatus used by Ford et al. included the use of 50 ml conical polypropylene tubes fitted with a rubber stopper with a metal sipper tube, and the addition of a small Petri dish secured to the rod floor opposite the drinking tubes to contain food. The metal floor and sipper tubes created a circuit that was completed when the animal made contact with the sipper, which was recorded by a lickometer device (MED Associates, Inc., St. Albans, VT) interfaced to a computer with MED-PC IV software (MED Associates, Inc.) for collection of cumulative lick records. The tubes were filled at the start of each day, weighed, and secured to the cage. At the end of each session, the tubes were weighed again before refilling, to determine the amount of fluid that was consumed. Food was replenished at the start of each session. To avoid a potential entrainment of activity to the time of new food and fluid delivery, and to record a full 24 hours without interruption, each session started at a slightly different time each day (noted in ).
Fluid intake and the circadian cycle
In this experiment, 24 adult prairie voles (12 male, 12 female; 30.7 ± 0.9 g; 95-137 days old on the first day) were housed individually in the apparatus described. During the first four days, they had access to water and saccharin, followed by three days with only water available, and then four days with water and 10% ethanol available. The voles were weighed immediately before commencing the saccharin and ethanol experiments.
Preference for alcohol over water was calculated based on fluid consumption and recorded licks from each fluid, and alcohol dose consumed was calculated based on the weight of ethanol solution consumed.
Experiment 4 – Establishing a limited access two-bottle choice procedure
To determine whether prairie voles could voluntarily self-administer alcohol in quantities sufficient to produce substantial BECs and changes in neural activity as indicated by increased Fos immunoreactivity (IR) we established a two-hour limited-access procedure. The animals used here were the 26 of the same animals used in Experiment 2, and so were not naïve to alcohol, and continued to be pair- or singly-housed. In this study, begun 26 days after the last alcohol consumption, the voles were given a two-bottle choice test with 10% ethanol and water for two hours, starting at the onset of the light cycle (based on the results of Experiment 3), and repeated over four consecutive days. Preference for alcohol and dose consumed were calculated as described above, also subtracting the average volume missing from four control tubes in empty cages from the volume of fluid consumption for each solution.
Immediately after the end of the two-hour drinking session on the last day, animals were euthanized by CO2 inhalation, followed by decapitation. An additional 12 age-matched voles that were alcohol- and experimentally-naïve were euthanized at the same time. Trunk blood samples were collected for analysis of BEC, as described in Experiment 1. Brains were removed and fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours. Subsequently, brains were transferred to 20% sucrose in PBS with 0.1% sodium azide (NaN3) overnight, followed by 30% sucrose (in PBS with 0.1% NaN3) until slicing.
Brains were sliced into 30 μm floating sections in 0.1% NaN3
in PBS. Slices containing the nucleus accumbens (NAc), the lateral septum (LS), the central nucleus of the amygdala (CeA), and the perioculomotor urocortin-containing neurons (pIIIu) were chosen for immunohistochemistry (IHC). These brain regions were selected for analysis because they most frequently showed changes in Fos IR following alcohol administration in previous rodent studies (Bachtell et al., 1999
; Sharpe et al., 2005
; Vilpoux et al., 2009
The Fos IHC protocol used here was based on previously published procedures in mice and rats (Ryabinin et al., 1999
). Endogenous peroxidase activity was quenched with 0.3% peroxide in PBS, followed by blocking with goat serum in PBS/Triton-X 100. The slices were incubated overnight with a primary rabbit polyclonal antibody to c-Fos (Santa Cruz Biotechnology, Santa Cruz, CA, 1:2000). Slices were subsequently incubated in biotinylated anti-rabbit antibody, made in goat (Vector Laboratory Inc., Burlingame, CA), ABC solution (Vector Laboratory Inc.), and diaminobenzidine (Thermo Scientific, Rockford, IL) to visualize the stain.
In the NAc, CeA, and pIIIu, Fos IR was quantified by counting the number of cells stained above background. Counting was performed manually by a trained experimenter blind to the identification of the samples. In the LS, little or no staining was observed in tissues, and so this area was not quantified.
For each day of drinking, preference for alcohol over water was calculated by dividing the volume of alcohol consumed by the total volume of fluid consumed. Additionally, g/kg consumed was calculated for each session by dividing the grams of alcohol consumed (the density of alcohol multiplied by the v/v concentration multiplied by the volume consumed) by the weight of the animal in kilograms.
Several of the voles occasionally chewed through the rubber stopper of the drinking tubes, leading to spillage of the fluids. This behavior has not been observed in mice using the same equipment in our lab. On these occasions, animals that chewed through the stopper were removed from analysis for that day, and will be referred to as outliers. In addition, statistical outliers defined as animals having intake of at least one fluid more than two standard deviations from the mean intake were removed from analysis for that day (this information is included in the Results section). However, these individuals were included in analysis of Fos and BECs, where consumption was not used as a dependent variable, and outliers for measures of Fos IR and BEC were not removed, based on the intra-experiment reliability of these measures. The statistical results obtained with exclusion of outliers never contradicted the results obtained with them in the analyses.
In Experiment 1, alcohol elimination rates were determined by a regression line, and the slopes and intercepts of prairie voles and mice were compared with an F test. Effects of sex, housing, and age were assessed as appropriate using a two-factor ANOVA with sex, housing, or age (old or young) as one factor, time of BEC assessment as the other between-subjects factor, and BEC as the dependent variable.
For Experiment 2, group differences in preference and g/kg were determined by two-factor repeated measures ANOVA, with sex and housing condition as between-subject factors, and alcohol concentration as the repeated measure. Preference and g/kg were each averaged across the four days of drinking at each concentration of alcohol and used for the repeated measures analyses. A two-factor repeated measures ANOVA, with sex and housing condition as between-subject factors, and alcohol concentration as the repeated measure, was also used to analyze water intake (g/kg). Body weights were monitored throughout the experiments, but were not affected by the experimental manipulations, as was expected, and are therefore not described here. The tastant tests for saccharin and quinine were analyzed by two-way ANOVA with sex and housing condition as independent variables, and saccharin and quinine preference as dependent variables. Where appropriate, Fisher's PLSD was used for post-hoc comparisons, and tests of simple effects were used to discover the basis of interaction effects.
The correlation of alcohol consumption between sibling partners was analyzed in Experiment 2 to determine whether individual members of a pair drank similar amounts. Separate correlations were performed for pair-housed animals and separated partners, using the average g/kg consumed from the 10% ethanol solution by one member of a pair as the X variable and g/kg consumed by the partner as the Y variable. Pearson's r was computed, and a threshold α = 0.05 level of significance was applied to the correlation. The same test was applied to consumption of saccharin and quinine.
In Experiment 3, the number of licks was determined for each solution during each hour using SoftCR for Windows (MED Associates, Inc.). A software error caused nearly all of the data from the second day of the saccharin consumption study to not be recorded. As a result of this and the irregular drinking pattern observed on the first day of the experiment, which was likely due to the novelty of the cage and the saccharin, only the third and fourth days of saccharin consumption were examined. All four days of ethanol consumption were analyzed, excepting only the first hour of the first day, where unusually high numbers of licks for both fluids were recorded.
A repeated measures ANOVA was used to analyze consumption separately for each fluid (saccharin, ethanol, or water), and for the total amount of fluid (saccharin or ethanol plus water) consumed during each period. First the statistical test was applied to each day separately, with each hour as the repeated measure. Then the data were collapsed across all days of consumption, and again analyzed with each hour as the repeated measure. Fisher's PLSD was used for post hoc comparisons. The preference ratios calculated from lick data and fluid data were compared by a Pearson correlation.
In Experiment 4, induction of Fos by alcohol consumption was compared to that of naïve animals for each brain area investigated, using the Mann-Whitney test, since the measures were not normally distributed. For pIIIu, which showed significant Fos induction, correlational analyses were performed to examine the relationship between Fos IR and preference or alcohol consumption (g/kg), using the Spearman rank r test. This nonparametric analysis was also used to examine the relationship between BEC and preference or alcohol consumption since neither Fos nor BEC data were normally distributed. Naïve animals were not included in the correlational analyses. An α level of 0.05 was used for all tests.