α6L9′S bacterial artificial chromosome (BAC) transgenic mice were constructed as previously described (Drenan et al. 2008). Briefly, an ectopic BAC containing a mutant, L9′S allele of the mouse α6 nAChR subunit gene was introduced into fertilized FVB/N embryos and implanted into pseudopregnant Swiss-Webster surrogates. The insertion site within the mouse genome is unknown. Founder animals were back-crossed to C57BL/6 12–15 times prior to these studies. Although >90% of the genome is expected to be C57BL/6 following this many rounds of back-crossing, elimination of FVB/N allelic DNA immediately adjacent to the BAC insertion site is not likely. α-Conotoxin MII binding experiments showed that α6* nAChRs are faithfully expressed in their correct anatomical locations and are not overexpressed in α6L9′S mice (Drenan et al. 2010, 2008). Both male and female mice were used in the current experiments with ages ranging from 64 to 193 days old at the time of experimental procedures. Mice were group housed (unless otherwise indicated) and had free access to food (Rodent Lab Diet 5001, Purina Mills Inc., St. Louis, MO, USA) and water in the home-cage. Ambient temperature in the colony and experimental rooms was maintained at 21 ± 2°C. Efforts were made to minimize any pain and discomfort to the mice. All experiments were conducted in accordance with the guidelines for care and use of animals provided by the National Institutes of Health (NIH), and all procedures were approved by the Purdue University Institutional Animal Care and Use Committee.
Alcohol (95% v/v) was diluted with tap water (drinking studies) or saline (CPP studies) to obtain the desired concentrations (3%, 6%, 10% and 20% v/v). For the place conditioning study, alcohol was administered intraperitoneally (i.p.) in doses of 0.5 and 2.0 g/kg of body weight with injection volumes of 3.15 and 12.6 ml/kg (20% v/v), respectively.
Place conditioning apparatus
The place conditioning apparatus has been utilized in prior place conditioning studies with mice (e.g. Powers et al. 2010). The apparatus consisted of 12 identical Plexiglas boxes (43.2 × 1.6 × 25.4 cm) enclosed in separate ventilated sound- and light-attenuated chambers (76.2 × 50.8 ×20.3 cm). The floor of each box consisted of interchangeable halves with distinct floor textures. One floor texture (the Grid floor) consisted of 4 mm steel rods mounted 3.5 mm apart and the other floor texture (the Hole floor) was made up of perforated 16 gauge stainless steel with 6.4 mm holes on 9.5 mm staggered centres. Locomotor activity and side position (left or right) for each mouse was continuously monitored in each box with an open field activity frame (SmartFrame Low Density, Lafayette Instrument Co, Lafayette, IN, USA) that contained infrared photobeams along the length and width of each frame (internal frame dimensions: 24.1 × 45.7 cm). Ventilation fans in each box masked outside noise.
Twenty-four-hour continuous-access alcohol drinking
Mice [n = 35; 17 α6L9′S (9 male/8 female) and 18 non-Tg (10 male/8 female)] were singly housed for 7 days with access to two water-filled tubes (25 ml fitted with metal drinking spouts) in order to habituate them to the experimental environment. On the eighth day, one of the water tubes was replaced with an alcohol tube for each mouse. Alcohol concentration was increased every 4 days: days 1–4 (3%), days 5–8 (6%), days 9–12 (10%) and days 13–16 (20%). Every 2 days, mice were weighed, drinking solutions were replaced and bottle position was alternated to avoid the development of a side preference (Kamens et al. 2010). Evaporation/leakage was estimated from tubes placed on empty cages, and mean volume lost was subtracted from individual drinking values before analysis.
Drinking in the dark
The DID experiment was conducted as previously described (Rhodes et al. 2005). The same mice used in the 24-h continuous-access alcohol drinking study remained singly housed for 33 days with normal water bottles. Mice then received one water-filled drinking tube on the home cage for 7 days. On days 8 through 11, body weights were measured and the water tube for each mouse was replaced with a 20% v/v alcohol tube 2 h after lights off (lights off at 0945 h). Mice had access to alcohol for 2 h. Amount of alcohol consumed was recorded daily after each 2-h session.
The place conditioning procedure consisted of one pretest, eight conditioning sessions and one post-test. All phases of the experiment were conducted on consecutive days except that a 48-h break separated the first four and the second four conditioning sessions. Experimental groups were counterbalanced by genotype, sex, order of exposure to the conditioning stimulus (CS), box enclosure and floor position (i.e. left vs. right side of the box). All animals were transferred on a cart to the procedure room 30 min before the start of experimental procedures each day.
Pretest (Day 1)
Initial unconditioned preference for each CS (Grid vs. Hole) was assessed 24-h prior to the start of the first conditioning trial. Subjects were injected (i.p.) with saline (at a volume equal to a 0.5 or 2.0 g/kg dose of alcohol, depending on the experiment) and placed in the testing box with free access to both floor types for 60 min.
Conditioning sessions (Days 2–11)
Mice underwent a differential place conditioning procedure in which they were randomly assigned to one of two conditioning subgroups within each experimental subgroup. Each conditioning subgroup received 5 min of exposure to either a Grid or Hole floor paired with alcohol treatment (4 CS+ conditioning sessions) and the other floor type paired with saline (4 CS− conditioning sessions) on alternating days, for a total of eight conditioning sessions. The floors and inside of the box were wiped with a damp sponge between each subject.
Separate experiments were run for each dose of alcohol (0.5 and 2.0 g/kg), but all procedures were identical. A total of 32 mice [17 α6L9′S (8 male/9 female) and 15 non-Tg (4 male/11 female)] were used for the 0.5 g/kg experiment, and 28 mice [17 α6L9′S (9 male/8 female) and 11 non-Tg (3 male/8 female)] were used for the 2.0 g/kg experiment. The low dose of alcohol (0.5 g/kg) was chosen because we hypothesized that α6L9′S mice would be more sensitive to alcohol reward, and a 0.5 g/kg dose is below the threshold for producing alcohol-induced CPP in C57BL/6 mice (e.g. Cunningham et al. 1992; Wrobel 2011). The 2.0 g/kg dose was used as a positive control.
Post-test (Day 12)
Mice were tested for CPP 24-h after their final conditioning session. As in the pretest, mice had free access to both floors in the same position for 60 min. Mice received an injection of saline (equal volume to a 0.5 or 2.0 g/kg dose of alcohol) directly before being placed in the apparatus to match cues during conditioning.
Brain slice preparation for electrophysiology
Brain slices were prepared as previously described (Engle et al. 2012). α6L9′S and non-Tg mice were genotyped at 21–28 days after birth. Mice were anesthetized with sodium pentobarbital (100 mg/kg; i.p.) followed by cardiac perfusion with oxygenated (95% O2/5% CO2), 4°C N-methyl-d-glucamine (NMDG)-recovery solution containing (in mm): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na+ ascorbate, 2 thiourea, 3 Na+ pyruvate, 10 MgSO4·7H2O, 0.5 CaCl2·2H2O (300–310 mOsm, pH 7.3–7.4). Brains were removed and retained in 4°C NMDG-recovery solution for 1 min. Coronal slices (250 µm) were cut with a microslicer (DTK-Zero 1; Ted Pella, Redding, CA, USA). Brain slices recovered for 12 min at 33°C in oxygenated NMDG-recovery solution, after which they were held until recording in HEPES holding solution containing (in mm): 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na+ ascorbate, 2 thiourea, 3 Na+ pyruvate, 2 MgSO4·7H2O, 2 CaCl2·2H2O (300–310 mOsm, pH 7.3–7.4). Coordinates for recordings in (substantia nigra pars compacta) SNc/VTA were within the following range: −3.8 to −2.9 mm from bregma, 4.0–4.8 mm from the surface, and 0.0–2.0 mm from the midline.
Immunohistochemistry and confocal microscopy
Transgenic mice expressing α6* nAChR subunits fused in-frame with green fluorescent protein (GFP) (α6GFP mice; 2 male/2 female) were anesthetized with sodium pentobarbital (100 mg/kg; i.p.) and transcardially perfused with 15 ml of ice-cold phosphate-buffered saline (PBS) followed by 25 ml of ice-cold 4% paraformaldehyde (PFA) in PBS. Brains were removed and post-fixed for 2 h at 4°C. Coronal sections (50 µm) were cut on a microslicer and collected into PBS. Sections were permeabilized (20 mm HEPES, pH 7.4, 0.5% Triton X-100, 50 mm NaCl, 3 mm MgCl2, 300 mm sucrose) for 1 h at 4°C, blocked [0.1% Triton X-100, 5% donkey serum in Tris-buffered saline (TBS)] for 1 h at room temperature and incubated overnight at 4°C in solutions containing primary antibodies (diluted in 0.1% Triton X-100, 5% donkey serum in TBS). Primary antibodies and final dilutions were as follows: 1:500 rabbit anti-GFP (A11122; Invitrogen, Carlsbad, CA, USA), 1:500 sheep anti-tyrosine hydroxylase (AB1542, Millipore, Temecula, CA, USA) and 1:500 mouse anti-GAD67 (MAB5406; Millipore). Sections were washed three times for 10 min each in TBST (0.1% Triton X-100 in TBS) followed by incubation at room temperature for 1 h with secondary antibodies (diluted in 0.1% Triton X-100, 5% donkey serum in TBS). Secondary antibodies and final dilutions were as follows: 1:1000 goat anti-rabbit Alexa 488 (A11008; Invitrogen), 1:1000 donkey anti-sheep Alexa 555 (A21436; Invitrogen) and 1:1000 donkey anti-mouse Alexa 555 (A31570; Invitrogen). Sections were then washed four times in TBST for 10 min each. All sections were mounted on slides and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA, USA), then imaged with a Nikon (Nikon Instruments, Melville, NY, USA) A1 laser-scanning confocal microscope system. Nikon Plan Apo × 10 air and × 60 oil objectives were used. Alexa 488 was excited with an argon laser at 488 nm, and Alexa 555 was excited with a yellow solid-state laser at 561 nm.
Patch clamp electrophysiology
Patch clamp electrophysiology was carried out as previously described (Engle et al. 2012) using tissue collected from adult mice (10 α6L9′S mice and 5 non-Tg mice). Each recorded cell within a slice was treated as a separate experiment, and each animal yielded between one and six recorded cells. Patch clamp experiments were not segregated by sex. A single slice was transferred to a 0.8 ml recording chamber (Warner Instruments, Hamden, CT, USA; RC-27L bath with PH-6D heated platform), and slices were superfused throughout the experiment with standard recording artificial cerebrospinal fluid (ACSF) (1.5–2.0 ml/min) containing (in mm): 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 24 NaHCO3, 12.5 glucose, 2 MgSO4·7H2O and 2 CaCl2·2H2O (300–310 mOsm, pH 7.3–7.4). Cells were visualized with an upright microscope (FN-1; Nikon) using infrared or visible differential interference contrast (DIC) optics. Patch electrodes were constructed from Kwik-Fil borosilicate glass capillary tubes (1B150F-4; World Precision Instruments, Inc., Sarasota, FL, USA) using a programmable microelectrode puller (P-97; Sutter Instrument Co., Novato, CA, USA). The electrodes had tip resistances of 4.5–8.0 MΩ when filled with internal pipette solution (pH adjusted to 7.25 with Tris base, osmolarity adjusted to 290 mOsm with sucrose) containing: 135 mm K+ gluconate, 5 mm ethyleneglycoltetraacetic acid (EGTA), 0.5 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, 2 mm Mg-ATP (adenosine triphosphate), and 0.1 mm guanosine triphosphate (GTP). Whole-cell recordings were taken at 32°C with an Axopatch 200B amplifier, a 16-bit Digidata 1440A A/D converter, and pCLAMP 10.3 software (all Molecular Devices; Sunnyvale, CA, USA). Data were sampled at 5 kHz and low-pass filtered at 1 kHz. The junction potential between the patch pipette and the bath solution was nulled immediately prior to gigaseal formation. Series resistance was uncompensated.
Ventral tegmental area DAergic neurons were identified as previously described (Drenan et al. 2008). These neurons typically exhibit broad spikes (>2 milliseconds), slow tonic firing (1–6 Hz) in coronal slice preparation, expression of significant Ih currents at hyperpolarized potentials, and significant sag responses in current clamp mode following injection of hyperpolarizing current. To examine the function of somatic nAChRs, ACh was locally applied using a Picospritzer III (General Valve, Fairfield, NJ, USA). Using a high-resolution micromanipulator (Sutter Instruments, Novato, CA, USA), the pipette tip was moved within 20–40 µm of the recorded cell 1–2 seconds before drug application. Acetylcholine-containing ACSF recording solution was then puffed at 10–20 psi for 250 milliseconds. Immediately after the application, the glass pipette was retracted.
Behaviours were analysed using analysis of variance (anova) with the significance level set at P ≤ 0.05. Between-group factors included genotype and sex. Within-group factors included concentration phase (3%, 6%, 10% and 20%), day, floor type (Grid or Hole), conditioning session (1–8), trial (4: one CS+ conditioning session and one CS− conditioning session), trial type (CS+ and CS−), block (2-day drinking averages), test (pretest and post-test), time bin (first and last 30 min) or min, where applicable. Only significant interactions including genotype are reported and followed-up with lower-order anovas and t-tests, where applicable. Preference test data were averaged into 30 min time bins before analysis since treatment effects on the expression of CPP can change over the course of the 60-min preference test (e.g. Chester et al. 1998; Powers et al. 2010). Pearson correlations between CS+ time and locomotor activity during the post-test were conducted because locomotor activity can affect the expression of CPP (Gremel & Cunningham 2007). Concentration–response curves for ACh-elicited inward currents in VTA neurons were derived by fitting mean peak current values for each ACh concentration to the Hill equation.