Animals

All animal experiments were performed under approval of the Uppsala Animal Ethical Committee and following the principles of the Guide for the Care and Use of Laboratory Animals and the guidelines of the Swedish Legislation on Animal Experimentation (Animal Welfare Act SFS1998:56) and the European Communities Council Directive (86/609/EEC).

Pregnant female Wistar rats (RccHan: WI, gestation day 16) were sourced from Harlan Laboratories B.V. (Horst, The Netherlands) and single housed under standard conditions (22 °C, 50 ± 10 % humidity, 12 h light–dark cycle commencing at 06:00, ad libitum access to pellet food and tap water, masking background noise). This is the least sensitive phase during pregnancy and was chosen to minimise the influence of stress related to transit. No signs of negative impact from transport were noticed during acclimatisation in the animal facility, and the delivery was normal in all females. The litters were cross-fostered and mixed so each litter contained four female and six males. Only male offspring were used in the continuation of the experiment. Upon weaning (post-natal day (PND) 21), animals were group housed under standard conditions as described above. Treatment groups were randomised, and adolescent animals (PND 28) were exposed to either ethanol or water. Upon completion of adolescent treatments (PND 59), two divergent protocols were followed, detailed schematically in Fig. 1.

Fig. 1 Experimental outline showing the time points (weeks) for the behavioural and neurochemical analyses. Dopamine (DA) recordings were done in vivo with high-speed chronoamperometry. PR represents progressive ratio sessions with two different doses PR1 (0.1 mg/kg/infusion) and PR2 (0.05 mg/kg/infusion), 52 × 14 mm (300 × 300 DPI) Full size image

Drugs and solutions

Ethanol, Solveco Etanol A 96 % (Solveco AB, Rosersberg, Sverige), was diluted in tap water and d-amphetamine sulphate (Sigma-Aldrich, LLC, St. Louis, MO, USA) and diluted in sterile NaCl 9 mg/mL (Braun Melsungen AG, Melsungen, Germany). Sucrose food pellets (5-TUL, TestDiet, St. Louis, MO, USA) were used during the operant training. Anaesthesia agents were thiobutabarbital (Sigma-Aldrich, LSS, St. Louis, MO, USA), isoflurane (Forene Abbott, Solna, Sweden) or propofol (Braun Melsungen AG, Melsungen, Germany). For in vivo chronoamperometry, l-ascorbic acid, potassium chloride, sodium chloride, sodium phosphate and calcium chloride were purchased from Sigma-Aldrich, LLC (St. Louis, MO, USA) and diluted in Milli-Q water. For the post-operative care, buprenorphine (Schering-Plough, Brussels, Belgium), carpofen (Pfizer, Oy Animal Health, Helsinki, Finland) and amoxicillin (Ceva Animal Health, Dublin, Ireland) were used. For catheter maintenance, Heparin LEO (LEO Pharmaceuticals, Copenhagen, Denmark), sterile water (Braun Melsungen AG, Melsungen, Germany) and glycerol (Glycerol Unimedic AB, Matfors, Sweden) were used.

Ethanol exposure

The animals received intragastric administration of either water or ethanol (2 g/kg, 20 % w/v ethanol diluted with water) for three consecutive days per week. This drinking paradigm was chosen to mimic common episodic adolescent drinking patterns and because intermittent ethanol exposure with drug-free days in-between has been shown to be necessary to induce neurobiological alterations similar to those seen in the transition to habitual and compulsive drinking (Spanagel 2003). The dose and route of administration were chosen to achieve binge-like oral consumption according to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) definition of binge drinking (>0.08 g/dl in 2 h) (NIAAA Drinking Levels Defined). Unpublished results from other experiments in our laboratory as well as in published data from others (Walker and Ehlers 2009) have shown that 2 g/kg (intragastric) ethanol will produce blood alcohol concentrations reaching the binge criterion. Administrations were given at 09:00 on PND 28–30, 36–38, 43–45, 50–52, 57–59, followed by four days without treatment.

In vivo dopamine recordings

For animals that underwent ethanol exposure only (ethanol n = 9, water n = 8), dopamine recordings were taken between 11 and 12 weeks of age (corresponding to the age of the initiation of amphetamine self-administration for the second set of animals). For the animals that additionally underwent self-administration trials, dopamine recordings were conducted during weeks 18 and 19 (ethanol n = 5, water n = 4). In all cases, animals were drug-free for a minimum of 2 weeks before dopamine recordings commenced (Fig. 1).

Dopamine recordings were conducted using carbon fibre microelectrodes (SF1A, 30 μm outer diameter, 150 μm length, Quanteon, LLC, Nicholasville, KY, USA). A high-speed chronoamperometric protocol was utilised (550 mV, 1 Hz sampling rate, 200 ms total) via a FAST16-mkII recording system (Quanteon). Electrode-pipette assemblies were prepared and calibrated immediately prior to in vivo recordings as previously described (Gerhardt and Hoffman 2001; Littrell et al. 2012). Briefly, electrodes were coated with Nafion (Sigma-Aldrich, LLC, St. Louis, MO, USA) and calibrated to cumulative additions of ascorbic acid and dopamine (ascorbic acid 250 μM, dopamine 2 μM steps) applied to a bath of 0.05 M phosphate-buffered saline. Electrodes used displayed a detection limit of 0.0237 ± 0.0037 μM and a selectivity of 3,864.35 ± 881.16 for dopamine over ascorbic acid. Responses to dopamine were linear, with an average correlation coefficient (R 2) of 0.827 ± 0.025 and an average reduction/oxidation ratio of 0.628 ± 0.011 that is indicative of specific dopamine detection (Gerhardt and Hoffman 2001). After calibration, a micropipette filled with isotonic potassium chloride solution (120 mM KCl, 29 mM NaCl, 2.5 mM CaCl 2 , pH 7.2–7.4) was affixed with the tip 150–200 μm from the recording site of the electrode.

Animals were anaesthetized via intraperitoneal injection of 125 mg/kg thiobutabarbital and body temperature maintained with a thermostatic heating pad (Gaymar Industries, Inc., Orchard Park, New York). The electrode-pipette assembly was stereotaxically carefully placed in the dorsolateral striatum (AP +1.0, ML +3.0, DV −4.2 mm) and an Ag/AgCl reference electrode placed in the brain contralaterally and remote from working electrode-recording site.

After surgery and allowing 1 h for the stabilisation of electrode and surrounding tissue, 100 nl of potassium chloride solution was locally ejected using pressure ejection (PicoSpritzer III, Parker Hannifin Corporation, Pine Brook, NJ, USA; ejection pressure <22 psi, ejection time <2 s), and resultant local dopamine release was detected by the electrode as a peak of rising dopamine concentration. Potassium chloride ejections were repeated every 10 min until three successive consistent dopamine releases were recorded for use as baseline reference peaks. Five minutes after the third reference peak was evoked, a single 2-mg/kg dose of amphetamine was injected via tail vein. Five minutes post-injection of amphetamine, dopamine release was evoked again, and this was then repeated every 10 min until 55 min post-drug. Upon termination of recording, animals were sacrificed, then the brain removed and frozen for subsequent histological identification of electrode location.

Self-administration

Rats from both the ethanol and water groups were subjected to assessment of self-administration behaviour followed by in vivo chronoamperometry (Fig. 1), the latter procedure is described above.

Operant chamber apparatus

Self-administration training and testing was conducted in sound-attenuated operant chambers (MED Associates Inc., Vermont, USA) equipped with two stimulus lights above two retractable stainless steel levers. A white house light placed on the wall opposite the levers was on during the entire session. A ventilating fan operated throughout the sessions and served as a masking noise. Intravenous solutions were delivered using an infusion pump (PHM-100, 3.33 rpm; Med Associates Inc.), and a 10-mL plastic syringe placed in the pump was connected to the implanted catheter through CoEx tubing (Harvard Apparatus, Kent, UK) and protected by a flexible metal leash (CamCaths, Ely, UK). Experiments were run and data collected by a PC with the MED-PC software (MedPC IV, MED Associates Inc., Vermont USA). The experiments were conducted in the same boxes for both training and test sessions, and ethanol- and water-treated animals were processed simultaneously throughout all phases of the self-administration procedure.

Sucrose training

Food restriction was initiated 48 h following the last intragastric pre-treatment session and was maintained throughout the self-administration training period to motivate food-seeking behaviour. Animal weights were carefully monitored and were not allowed to decrease more than 15 % from commencement of food restriction. After 2 days on food restriction, the training to self-administer 45 mg sucrose food pellets on a fixed ratio-1 (FR1) schedule was initiated. Each session started when the house light illuminated and the retractable levers were extended. A press on the active lever resulted in retraction of the lever and illumination of the stimulus light above the lever during a 10-s time-out period. The criteria for fulfilled self-administration training were accomplishment of 100 active lever presses within 30 min and a specificity >0.85 for the active lever.

Surgery

Intravenous catheters (CamCaths, Ely, UK) were implanted into the right jugular vein under isoflurane anaesthesia. Rats were administered post-operative analgesia (buprenorphine, 0.06 mg/kg s.c.; carpofen 5 mg/kg s.c.) and antibiotic (amoxicillin, 0.5 mL/kg s.c.). Catheters were flushed with a heparin solution (50 U/mL) before and after every session, and a heparinized glycerol lock solution (50:50 heparin/glycerol) was used over weekends. Catheter patency was tested before the start and at the end of the study with an infusion of the short-acting anaesthetic agent propofol.

Intravenous amphetamine self-administration

After a minimum of 4 days of recovery from surgery, rats (ethanol n = 8, water n = 8) were allowed to self-administer amphetamine on a daily 60 min FR3 schedule of reinforcement. Each session started when the house light was turned on and the retractable levers were extended. Three responses on the active lever resulted in an intravenous infusion of amphetamine (0.1 mg/kg/infusion), and a 10-s time-out period was initiated when both levers were retracted and a white stimulus light above the active lever was turned on. The lever designated to be the active lever was switched between sucrose training and intravenous self-administration. The maximum number of rewards during the 60-min baseline sessions was set to 20. A press on the inactive lever had no programmed consequences but was recorded by the software.

The rats underwent five baseline sessions of 60 min FR3 amphetamine (0.1 mg/kg/infusion) before the operant requirements were switched to the progressive ratio (PR) format. Under this schedule of reinforcement, the response requirement started at 1 and escalated for each drug infusion delivered according to following scheme: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, 737 and 901 (see Richardson and Roberts 1996). The PR schedule was tested at two doses (0.1 and 0.05 mg/kg per infusion) for two consecutive days each (see Fig. 1). The sessions ended when 1 h had passed since the last reward or after a maximum session time of 4 h. The breakpoint was defined as the total number of infusions during the session. Additionally, to test the dose–response function on a FR schedule, the unit dose of amphetamine (0, 0.025, 0.05 or 0.1 mg/kg per infusion) was varied, and each dose was tested for three consecutive 90 min FR3 sessions (Fig. 1). The first session of each dose in PR and dose–response trials was considered an acclimatisation session, and data collected during this session was not used in the statistical analysis.

Data analysis

Dopamine analysis

The main parameters examined from dopamine oxidation currents, i.e. the peak area, the maximal amplitude (μM) of evoked peaks and the time taken for dopamine concentration to decline to 20 % of the maximum for each peak, T80 (seconds), were analysed with the FAST analysis software (version 5.2; Quanteon, KY, USA). The amplitude is a measurement of dopamine release; T80 is the uptake measure, whereas the peak area encompasses both the dopamine release and reuptake of dopamine. The chronoamperometric recordings of amplitude and T80 allow analysis of both the release and reuptake inhibition action of amphetamine.

For comparison of reference peaks, the raw values of the above parameters for the first three consecutive consistent dopamine releases obtained were compared via a repeated measures ANOVA to examine the influence of time, adolescent treatment group and time-group effects (Statistica 10; StatSoft Inc., Tulsa, OK, USA).

For analysis of amphetamine challenge, mean baseline values of parameters were obtained from the three reference peaks for each animal and subsequent values described as a percentage of this baseline. Subsequently, repeated measures ANOVA were used to examine time, adolescent treatment group and time-group effects. Where time-group differences were observed (p < 0.05), the Tukey´s HSD post hoc test was applied.

Self-administration

Student’s t test was used to compare the PR trials and sucrose pellet operant training. The parameters tested during operant training were the number of days until the rats had fulfilled the training criteria and the specificity for the active lever (number of presses on the active lever/total number of lever presses) when the criteria were fulfilled. The analysis of amphetamine self-administration behaviour on FR schedules was done with repeated measures ANOVA.