Adult (8–10 week), male C57BL/6J mice bred in house were used for immunohistochemistry, gene expression, and brain infusion experiments (n = 46). For consumption experiments, adult, male α4 knock-out (α4 KO) mice and their wild type (WT) litter mates (n = 45), as well as heterozygous Leu9′Ala knock-in mice and their WT litter mates (n = 42), all bred on site, were used. The genetic engineering of both α4 KO and Leu9′Ala mouse lines have been described previously (Ross et al., 2000
; Tapper et al., 2004
). Both lines have been back-crossed to a C57BL/6J background for at least nine generations. C57BL/6J mice were group housed four mice/cage and given food and water ad libitum
. For consumption experiments, mice were individually housed on a reversed 12 h light/dark cycle (lights on 10PM, off 10 AM) with ad libitum
access to food and water (except during experiments as described below). All experiments were conducted in accordance with the guidelines for care and use of laboratory animals provided by the National Research Council (National Research Council, 1996
), as well as with an approved animal protocol from the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.
Drugs and drinking solutions
Ethanol drinking solutions were prepared from 190 proof absolute anhydrous ethanol (Pharmco-Aaper) diluted to 2 % or 20 % ethanol (v/v) using tap water. Sucrose was dissolved in tap water to make a 10 % (w/v) concentration. Varenicline tartrate, a gift from Pfizer, and ethanol were dissolved in 0.9 % saline and administered via intraperitoneal (i.p.) injection at the indicated doses. For infusion of drug into the brain, varenicline was dissolved in artificial cerebrospinal fluid (aCSF containing, in mM: 126 NaCl, 2.5 KCl, 2 CaCl2
, 1 MgCl2
, 1.25 NaH2
, 26 NaHCO3
, 25 D-glucose). For immunohistochemistry and behavioral experiments, varenicline doses were chosen based on previous studies of varenicline effects on nicotine self-administration and DA turnover in addition to predicted therapeutic concentrations achieved in smokers’ brains (O’Connor et al., 2010
; Rollema et al., 2010
). Varenicline concentrations are reported as free base.
Adult (8–10 weeks), male, C57BL/6J mice were i.p. injected with saline for three days prior to the start of the experiment to habituate them to handling and to reduce c-Fos activation due to stress. Four groups of three mice were used in each of the following conditions: an i.p. injection of saline followed by an i.p. injection of saline, an i.p. injection of saline, followed by a 2.0 g/kg ethanol injection, an i.p. injection of 0.3 mg/kg varenicline followed by a saline injection, and 0.3 mg/kg varenicline i.p. injection followed by a 2.0 g/kg ethanol injection. The time between the first and second injections was 15 min. Ninety minutes after the second injection, all mice were deeply anesthetized with sodium pentobarbital (200 mg/kg, i.p.) and perfused transcardially with 10 ml of 0.1 M phosphate-buffered saline (PBS) followed by 10 ml of 4 % paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Brains were removed and post-fixed for 2 h with the same fixative and cryoprotected in sodium phosphate buffer containing 30 % sucrose until brains sank. VTA serial coronal sections (20 μm) were cut on a microtome (Leica Microsystems Inc.) and collected into a 24-well tissue culture plate containing 1 × PBS. Slices containing VTA were collected between −2.8 mm and −4.04 mm from bregma. After rinsing sections in PBS twice for 5 min, they were treated with 0.2 % Trition X-100 PBS (PBST) for 5 min followed by incubation in 2 % BSA/PBS for 30 min. Sections were washed with PBS once and then incubated in a cocktail of primary antibodies for tyrosine hydroxylase (TH, monoclonal, 1:250 dilution, Santa Cruz Biotechnology) and c-Fos (polyclonal, 1:400 dilution, Santa Cruz Biotechnology) in 2 % BSA/PBS overnight at 4°C. The sections were then washed with PBS three times for 5 min followed by incubation in secondary fluorescent labeled antibodies (goat anti- rabbit Alexa Fluor® 488 and goat anti-mouse Alexa Fluor ®594, 1:300 dilutions, Molecular Probes, Inc.) at room temperature in the dark for 30 min. After washing with PBS 5 times for 5 min/wash, sections were mounted on slides using VECTASHIELD® Mounting Medium (Vector laboratories, Inc.). The number of positive neurons was counted under a fluorescence microscope (Carl Zeiss MicroImaging Inc.) at a magnification of 400×. The intensity of fluorescence was quantified using a computer-associated image analyzer (Axiovision Rel. 4.6). Neurons were counted as signal-positive if intensities were at least 2 times higher than that of the average value of background (sections stained without primary antibodies). Experimenters were blind to drug treatment.
Laser Capture Microdissection (LCM)
Adult, male C57BL/6J mice were i.p. injected with saline for three days prior to the start of the experiment to habituate them to handling and to reduce c-Fos activation due to stress. On the experiment day, mice were i.p. injected with 2.0 g/kg ethanol and decapitated 90 min later. The brain was removed, snap-frozen in dry ice-cooled 2-methylbutane (−60° C) and stored at −80° C. Coronal serial sections (10 μm) of the VTA were cut using a cryostat (Leica Microsystems Inc.) and mounted on pre-cleaned glass slides (Fisher Scientific). The sections were immediately placed in a slide box on dry ice until completion of sectioning followed by storage at −80°C.
A quick immunofluorescence double-staining protocol for TH and c-Fos was used to identify TH and c-Fos immunopositive neurons. First, frozen sections were allowed to thaw for 30 seconds then immediately fixed in cold acetone for 4 min. Slides were then washed in PBS, incubated with a cocktail of primary antibodies for mouse anti-TH and rabbit anti-c-Fos (Santa Cruz Biotechnology, Inc., 1:50 dilutions) for 10 min, washed in PBS once followed by incubation in secondary fluorescent-labeled antibodies (Molecular Probes, Inc., a cocktail of goat anti-mouse Alex Fluor 594® and goat anti-rabbit Alex Fluor 488®, 1:100 dilution) for 10 min. The slides were washed in PBS once, then subsequently dehydrated in a graded ethanol series (for 30 s each in 70 % ethanol, 95 % ethanol, 100 % ethanol, and once for 5 min in xylene). Slides were allowed to dry for 5 min. All antibodies were diluted in DEPC-treated PBS containing 2 % BSA and 0.2 % Triton X-100. All ethanol solutions and xylene were prepared fresh to preserve RNA integrity.
The Veritas™ Microdissection System Model 704 (Arcturus Bioscience, Inc.) was used for LCM. Approximately 800 to 1400 TH-immunopositive neurons (including c-Fos-immunopositive and c-Fos-immunonegative) were cut from the VTA in each animal. Five to seven different mice were used per treatment. Neurons were captured on CapSure® Macro LCM caps (Arcturus Bioscience, Inc.) for mRNA isolation.
Total RNA was extracted from individual replicate samples using a Micro Scale RNA Isolation Kit (Ambion, Inc.). RNA samples extracted from DAergic neurons were reverse-transcribed into cDNA using TaqMan® Gene Expression Cells-to-CT™ Kit (Ambion, Inc.). PCR reactions were set up in 10-μl reaction volumes using TaqMan Gene Expression Assays (ABI, Inc.). GAPDH was used as an internal control gene to normalize gene expression levels. PCR was performed using an ABI PRISM 7500 Sequence Detection System. Negative controls with no reverse transcriptase were performed for all Taqman Assays. All reactions were performed in triplicate. Relative amplicon quantification was calculated as the difference between Ct values of GAPDH and that of the gene of interest. Relative gene expression differences between c-Fos immunopositive neurons and c-Fos immunonegative neurons were calculated using the 2−ΔΔCt method.
Drinking in the dark (DID)
Ethanol consumption was measured using a similar Drinking in the Dark (DID) procedure as previously described (Hendrickson et al, 2009
). Animals were singly housed in experimental chambers for 1 week prior to the beginning of the DID sessions. The mice received a 15-ml graduated water bottle fitted with a one-hole rubber stopper with a stainless steel double-ball-bearing sipper tube which was sealed with Parafilm to prevent leakage. For the first three nights, two hours after the lights were off, mice were i.p. injected with saline immediately before their water bottle was replaced with the ethanol bottle (2 % or 20 %), and allowed to drink for two hours. This procedure was used to acclimatize the mice to the experimental conditions and allow them to reach a baseline of ethanol intake prior to drug administration. On the fourth night, the mice received their first dose of drug just prior to placement of the ethanol bottle. The amount of ethanol consumed was recorded after each two-hour session and converted to g/kg per each animal’s ethanol consumption and body weight. The mice were given 2 days of rest (no injections or ethanol) and then began the saline injection/ethanol consumption assay for two to three days or until a stable ethanol intake was reached. Once the baseline returned, a second, higher dose of drug was administered prior to the ethanol bottle being placed in the cage. In this design, all mice in one group drink a single concentration of ethanol throughout the experiment, but receive two doses of drug, 4–5 days apart, with the lower concentration of drug first. The baseline value immediately prior to the first drug exposure is shown in all figures. There was no significant difference in baseline ethanol intake between doses in any of the experiments (data not shown). For control experiments, mice received 10 % sucrose for two hours instead of ethanol.
C57BL/6J mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) (VEDCO). The surgical area was shaved and disinfected. Mice were placed in a stereotaxic frame (Stoelting Co) with mouse adaptor and a small incision was cut in the scalp to expose the skull. Using bregma and lambda as landmarks, the skull was leveled in the coronal and sagittal planes. For cannula placement, holes were drilled in the skull at the anteroposterior (AP, in reference to bregma) and mediolateral (ML) coordinates that correspond to either the anterior VTA (−2.6 mm AP, ± 0.5 mm ML) or posterior VTA (−3.4 mm AP, ± 0.5 mm ML) based on “The Mouse Brain in Stereotaxic Coordinates” (Paxinos and Franklin, 2000
). Stainless steel unilateral guide cannulae (−4.0 mm dorsal ventral, ± 0.5 mm ML, Plastics One) with dummy cannulaes in place, were inserted into the brain and secured to the skull with cerebond (Plastics One). Mice were allowed to recover for at least 3 days before behavioral testing.
Intra-VTA infusions and DID
Two hours after the lights were turned off, mice were anesthetized with 2 % isoflurane via a nose cone adaptor at a flow rate of 800 ml/L. Once anesthetized, an infusion cannula designed to reach −4.5 mm below the skull was inserted into the guide cannula and vehicle, 10 pmol, 100 pmol, or 1000 pmol varenicline was infused at a rate of 1 μl/min for 45 s and a total volume of 0.75 μl. Immediately after infusion, mice were placed back into their home cages and monitored until awake, ~45 s. During this time, the home cage water bottle was removed and replaced with a bottle containing 20 % ethanol, which was left in place for 2 hours as previously described for the DID experiments. Before the start of each experiment, mice were infused with vehicle daily until a stable baseline of ethanol intake was reached. After completion of behavioral experiments, mice were culled and brains were removed and frozen on dry ice. Coronal sections (20 μm) were cut with a cryostat (Leica Microsystems Inc.). Sections were mounted and stained with neutral red (Sigma) to visualize cannula placement. A total of 6 mice were excluded from analysis due to incorrect cannula placement.
Data were analyzed using Two-Way ANOVA with drug treatment and either genotype or brain region as variables as indicated followed by Bonferroni post hoc tests. Data were analyzed using Graphpad software (Graphpad Software, Inc.). Paired T-tests were used to analyze fold expression of qRT-PCR data. Results were considered significant at p < 0.05. All data are expressed as means ± standard errors of means (SEM).