Adult male Wistar rats (Charles River, Raleigh, NC), weighing 225–275 g at the beginning of the experiments, were housed in groups of 2–3 per cage in a temperature-controlled (22°C) vivarium on a 12 h/12 h light/dark cycle (lights on at 8:00 PM) with ad libitum access to food and water. All behavioral tests were conducted during the dark phase of the light/dark cycle. All procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.
Self-administration sessions were conducted in standard operant conditioning chambers (Med Associates, St. Albans, VT). The rats were first trained to self-administer alcohol using a modified (Walker and Koob, 2007
) sucrose-fading procedure (Samson, 1986
), in which 10% (w/v) alcohol was added to a sweet solution and then sweeteners were gradually removed from the experimental solution. Upon completion of this procedure, the animals were allowed to self-administer a 10% (w/v) alcohol solution and water on a fixed-ratio 1 (FR1) schedule of reinforcement (i.e., each operant response was reinforced with 0.1 ml of the solution). For the pharmacological tests with mifepristone, rats were trained to self-administer alcohol according to the following protocol that generated the same amount of baseline drinking but with less testing in preliminary studies. First, the rats were given free-choice access to alcohol (10% w/v) and water for 1 day in their home cages to habituate them to the taste of alcohol. Second, the rats were subjected to an overnight session in the operant chambers with access to one lever (right lever) that delivered water (FR1). Food was available ad libitum
during this training. Third, after 1 day off, the rats were subjected to a 2 h session (FR1) for 1 day and a 1 h session (FR1) the next day, with one lever delivering alcohol (right lever). All of the subsequent sessions lasted 30 min, and two levers were available (left lever: water; right lever: alcohol) until stable levels of intake were reached. Responding obtained with this procedure is equivalent to the sucrose-fading procedure (Samson, 1986
; Walker and Koob, 2007
). Upon completion of this procedure, the animals were allowed to self-administer a 10% (w/v) alcohol solution and water on an FR1 schedule of reinforcement (i.e., each operant response was reinforced with 0.1 ml of the solution).
Alcohol vapor chambers
The rats were made dependent by chronic, intermittent exposure to alcohol vapors as previously described (O’Dell et al., 2004
; Gilpin et al., 2008
). They underwent cycles of 14 h on (blood alcohol levels during vapor exposure ranged between 150 and 250 mg%) and 10 h off, during which behavioral testing for acute withdrawal occurred (i.e., 6–8 h after vapor was turned off when brain and blood alcohol levels are negligible; Gilpin et al., 2009
). In this model, rats exhibit somatic withdrawal signs and negative emotional symptoms reflected by anxiety-like responses and elevated brain reward thresholds (Schulteis et al., 1995
; Roberts et al., 2000
; Valdez et al., 2002
; Rimondini et al., 2003
; O'Dell et al., 2004
; Zhao et al., 2007
; Sommer et al., 2008
; Edwards et al., 2012
). Nondependent rats were not exposed to alcohol vapor. For protracted abstinence, the animals were tested at the same time of the day as for acute withdrawal but 3–6 weeks after the vapor was turned off.
Operant self-administration during alcohol vapor exposure
Behavioral testing occurred 2–4 times per week. The rats were tested for alcohol (and water) self-administration on an FR1 schedule of reinforcement for thirteen 30 min sessions. Operant self-administration on an FR1 schedule requires minimal effort by the animal to obtain the reinforcer and herein was considered a measure of intake. For five sessions, the rats were tested on a PR schedule, under which the number of lever presses necessary to obtain the next reinforcer progressively increased according to the following progression: 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 7, 7, 9, 9, 11, 11, 13, 13, etc. The PR session stopped after 90 min or when 15 min had elapsed without the rat obtaining a reinforcer. In this test, the workload (“price”) for the next alcohol reinforcer increases progressively until the rat reaches a “breakpoint” (i.e., a measure of motivation/compulsivity) beyond which it no longer responds for alcohol.
The rats were then maintained on an FR1 schedule until stable levels of alcohol self-administration were reattained, and the alcohol solution was adulterated with increasing concentrations of quinine (0.0005, 0.001, 0.0025, and 0.005 g/L) presented between-sessions (one concentration per session). This test measures the persistence of animals to consume alcohol despite the aversive bitter taste of quinine that was added to the alcohol solution and was considered herein a measure of compulsive intake.
Finally, the rats were tested for saccharin (0.004%, w/v) self-administration under an FR1 schedule for five 30 min sessions to determine whether the effects were specific for alcohol or generalized to other types of reward. A submaximal rewarding saccharin concentration was chosen based on previous studies (Vendruscolo et al., 2010
) to prevent reaching a “ceiling effect” in any group and maintain similar response rates as alcohol.
Brains from dependent and nondependent rats were collected and snap-frozen with isopentane for measurements of GR and MR mRNA levels during acute alcohol withdrawal (24 h after the vapor was turned off). This time point was chosen because we were interested in more stable dysregulations of gene expression and to avoid any transient effects caused by earlier withdrawal. Importantly, the escalation of alcohol consumption has been demonstrated in rats at 2–8 h and 24 h of withdrawal and 2–7 weeks post-vapor (Valdez et al., 2002
; Rimondini et al., 2003
; O’Dell et al., 2004
; Sommer et al., 2008
; Gilpin et al., 2008
). Brains from a separate cohort of animals were dissected 3 weeks after the vapor was turned off to investigate whether changes in GR mRNA levels could be detected during protracted alcohol abstinence. The brains were sliced on a cryostat, and bilateral punches (300 µm thickness, 2 mm diameter) were collected from the prefrontal cortex (PFC), nucleus accumbens (NAc), bed nucleus of the stria terminalis (BNST), amygdala, and hippocampus. Because we expected that smaller gene expression changes might occur during protracted alcohol abstinence, we dissected subregions of some of the regions used above for GR quantification from 300 µm cryostat-cut slices (Cuello and Carson, 1983
Quantitative nuclease protection array
The expression of GR and MR mRNA during acute alcohol withdrawal was assessed using quantitative nuclease protection assays (qNPA, High Throughput Genomics, Tucson, AZ). This assay was used because it allowed us to quantitatively measure several stress/reward-related genes at the same time. The primer sequences were the following: GR (position 85, GACTTTTATAAAAGCCTGAGGGGAGGAGCTACA GTCAAGGTTTCTGCATC; position 318, GGGGCTGTATATGGGAGAGACAGAAA CAAAAGTGATGGGGAATGACTTGG; position 1474, CCAGCATGCCGCTATCGG AAATGTCTTCAGGCTGGAATGAACCTTGAAGC; position 1734, CACACTCAACA TGTTAGGTGGGCGTCAAGTGATTGCAGCAGTGAAATGGC), MR (position 346, CCTCTCCATCCTCATTGCCGATCAGCCAGTATTGGACTTGCTGGTAGCGG; position 1764, GTCAAGCAAGCACTCATGTTCAGGCGCCTCTTTTAAAGGGAACC CCACGG; position 3380, CTGGGAATGCCAAACCCCTTTACTTTCACAGAAAGTG ACGGGAGATACCG; position 4508, CCCGCGTGGGAAGTGTTCGTGAGACTCTA GTGCGCAGCTGTGGCTTCTGC). RNA was extracted and purified from brain tissue using the PureLinkTM RNA Mini Kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Two hundred nanograms of total RNA were diluted in lysis buffer (up to 25 µl) that contained the cDNA riboprobes complementary to targeted RNA and added to a 96-well plate. The plate was then heated at 95°C for 15 min (denaturation) and incubated for 16 h at 60°C for hybridization of the probes to RNA. The single-strand nucleic acids (unbound RNA and probes) were eliminated by S1-nuclease digestion for 90 min at 50°C. The enzymatic reaction was terminated by adding 10 µl of S1 stop solution, followed by incubation at 95°C for 15 min to inactivate the S1 nuclease and hydrolyze bound RNA. Ten microliters of neutralizing solution were added to hydrolyze the DNA:RNA heteroduplexes and degrade the RNA, leaving the sample with the selected probe only. The samples were then transferred for RNA quantification to an “ArrayPlate,” in which 16 spots, each containing a linker to specifically capture each probe, had been printed into each well, and incubated at 50°C for 24 h to allow for probe hybridization to the ArrayPlate. After several washes, 40 µl of detection linker solution was added to each well, and the ArrayPlate was incubated at 60°C for 90 min to allow the detection linker to hybridize to the ArrayPlate. The plate was washed for another cycle and incubated at 37°C for 30 min after the addition of the detection enzyme (40 µl). This step was followed by another wash and the addition of horseradish peroxidase chemiluminescent substrate. The chemiluminescent signal from each well of the ArrayPlate was quantified and reported by SuperCapella Imager.
Reverse transcription and quantitative PCR
Given that GR mRNA levels and not MR mRNA levels were differentially expressed in dependent and nondependent rats during acute alcohol withdrawal, we only measured GR mRNA levels during protracted alcohol abstinence. Total RNA was extracted using the PicoPure RNA Isolation kit (Applied Biosystems, Foster City, CA) and treated with DNase I (Qiagen, Valencia, CA). Concentrations were determined using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, Carlsbad, CA). cDNA was reverse-transcribed from total RNA using iScript cDNA (Bio-Rad, Hercules, CA) in the presence of Oligo (dT) and random primers according to the manufacturer’s instructions. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using a SYBR Green-based detection system (iQ SYBR Green Supermix, Bio-Rad Laboratories, Hercules, CA). Reactions were performed on laser-equipped thermal cyclers to detect changes in fluorescence in real time, and cDNA concentrations of Nr3c1 were calculated according to the relative quantification (ddCt) method, corrected for differences in PCR efficiency, and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh), cyclophilin A (Ppia), or β-actin (Actb). The following primers were used: Nr3c1 primer pair 1 (forward, 5’ TACAAAGATTGCAGG TATCCTATGA 3’; reverse, 5’ ACTCTTGGCTCTTCAGACCTTC 3’), primer pair 2 (forward, 5’ GCACCAGCTATCAGAAGACC 3’; reverse, 5’ GCTCTACACCAGTTAG GACG 3’), Ppia (forward, 5’ TATCTGCACTGCCAAGACTGAGTG 3’; reverse, 5 ’ CTTCTTGCTGGTCTTGCCATTCC 3’), Actb (forward, 5’ AGATTACTGCCCTGGCT CCT 3’; reverse, 5’ CAGTGAGGCCAGGATAGAGC 3’).
Mifepristone (RU38486) treatment
To investigate the functional role of GRs in the escalation of alcohol self-administration during alcohol vapor exposure, the rats were trained to self-administer alcohol as described above and subcutaneously implanted with mifepristone pellets (a GR/progesterone receptor antagonist; 150 mg; Innovative Research of America, Sarasota, FL) or placebo pellets for chronic release (21 days). The mifepristone dose was chosen based on previous studies (Schneider et al., 2003
; Nephew et al., 2008
) and adjusted for bodyweight. Twenty-four hours later, alcohol vapor exposure and behavioral testing began. To investigate the functional role of GRs in escalated alcohol self-administration during protracted alcohol abstinence, additional groups of dependent and nondependent rats were subcutaneously implanted with mifepristone or placebo pellets 1 week after the vapor was turned off. Behavioral testing began 1 week after pellet implantation. Three animals from this experiment were excluded: one mifepristone-treated dependent rat and one placebo-treated nondependent rat that showed rejection of the pellet and one placebo-treated dependent rat outlier.
The data are expressed as mean and standard error of the mean (SEM). The data were analyzed using analysis of variance (ANOVA) with or without repeated measures, with session as the within-subjects factor and group (dependent vs. nondependent; low vs. high) and treatment (mifepristone vs. placebo pellet) as between-subjects factors. When appropriate, post hoc comparisons were performed using Fisher’s Least Significant Difference (LSD) test. Glucocorticoid receptor and MR mRNA levels were compared using Student’s t-test. The accepted level of significance for all tests was p ≤ 0.05.