Male-outbred Lister hooded rats (Charles River, Kent, UK), weighing 180–200 g at the start of the experiment, were housed in pairs in polycarbonate cages (L = 40 cm, W = 25 cm, H = 18 cm) and maintained under a reversed 12-h light/dark cycle (lights on at 7.00 p.m.) at a constant temperature (21 ± 1 °C), with free access to laboratory chow (SDS) and water. The experimental procedures were conducted in accordance with the UK 1986 Animals (Scientific Procedures) Act (project licence PPL 80/1767).
Instrumental training and testing took place in 12 operant conditioning chambers (29.5 × 32.5 × 23.5 cm; Med Associates, Georgia, VT) equipped with two 4-cm wide retractable levers that were mounted in the intelligence panel 12 cm apart and 8 cm from the grid floor. Above each lever was a cue light (2.5 W, 24 V), and a house light (2.5 W, 24 V) was located at the top of the opposite wall. A dipper delivered 0.04 ml of a 20 % (w/v) sucrose solution to a recessed magazine (3.8 cm2 and 5.5 cm from the grid floor) situated between the levers. Entry into this magazine was detected by the interruption of an infrared source. The floor of the chamber was covered with a metal grid with bars 1 cm apart and connected to a shock generator and scrambler (Campden Instruments, UK), which delivered 0.5-mA foot shocks. The grid was located 8 cm above an empty tray. The testing chamber was placed within sound- and light-attenuating housing equipped with a ventilation fan that also screened external noise. Silastic tubing shielded with a metal spring extended from each animal’s intravenous catheter to a liquid swivel (Stoelting, Wood Dale, IL) mounted on an arm fixed outside the operant conditioning chamber. Tygon tubing extended from the swivel to a Razel infusion pump (Semat Technical, UK) located adjacent to the housing. The operant conditioning chambers were controlled by software written in C++ using the Whisker control system (Cardinal et al. 2000).
Rats were anaesthetised with ketamine hydrochloride (100 mg/kg, intraperitoneal (i.p.); Ketaset) and xylazine (9 mg/kg, i.p.; Rompun) then implanted with a single catheter (CamCaths, Cambridge, UK) in the right jugular vein. The tubing ran subcutaneously over the shoulder, and the mesh end of the catheter was sutured subcutaneously on the dorsum. After surgery, rats were single housed and kept so for the rest of the experiment. To prevent infection, rats were treated post-surgically for 7 days with 10 mg/kg Baytril subcutaneously (Genus Express, Bury St. Edmunds, UK) for 7 days (Caine et al. 1992). Rats were subsequently limited to 20 g food per day provided following daily experimental sessions.
In experiment 1, rats were assigned to one of six groups: whether they were trained for sucrose or cocaine and whether the taking or the seeking response was punished after training. These latter groups were also distinguished by whether rats had the availability to concomitantly nose poke for sucrose thereby yielding the “sucrose taking” (n = 12), the “cocaine taking” (n = 18), the “cocaine taking + sucrose” (n = 16), the “sucrose seeking” (n = 12), “cocaine seeking” (n = 15) and the “cocaine seeking + sucrose” (n = 19) groups. In experiment 2, rats were assigned to one of four groups: whether they had short (ShA) or long (LgA) access to cocaine following seeking-taking task training and whether they had the availability to concomitantly nose poke for sucrose thereby yielding the “ShA” (n = 17), the “ShA + sucrose” (n = 31) the “LgA” (n = 20) and the “LgA + sucrose” (n = 37) groups.
Acquisition of the taking response
Behavioural training began 7–10 days after surgery for the cocaine groups and 1 week after arrival for the sucrose groups. For all rats, each session began with the insertion of the taking lever (left/right counterbalanced). Responding was reinforced under a fixed ratio (FR) 1 schedule. Each lever press resulted in either sucrose (0.2 ml of a 20 % sucrose solution, which was delivered by presenting the dipper five times during 5 s at the rate of one presentation per second) or cocaine (0.25-mg/kg infusion of cocaine at a rate of 0.1 ml/5 s) and was accompanied by retraction of the taking lever, offset of the house light and illumination of the stimulus light above the lever for 20 s. The sessions terminated after either 30 reinforcers or 40 min for the sucrose groups or 2 h for the cocaine groups. Training of the taking response continued for five to seven sessions.
Training of the seeking-taking chain
Each cycle of the seeking-taking-chained schedule started with the insertion of the seeking lever with the taking lever retracted, and the first press on the seeking lever initiated a random interval (RI) schedule. The RI parameter was progressively increased from 2 to 120 s. The first lever press after the RI had elapsed and terminated the first link of the chain, resulting in the retraction of the seeking lever and insertion of the taking lever to initiate the second link. One press on the taking lever was followed by the drug or sucrose reinforcement accompanied by the same stimulus events as during the training of the taking response. There followed a time-out period in which neither the lever was available, but the houselight was illuminated and the stimulus light was off. Thereafter, the seeking lever was reinserted to start the next cycle of the schedule. For the sucrose group, the time out was kept to 20 s, but for cocaine-trained animals, this time-out period was progressively increased across five consecutive daily sessions from 20 s to 10 min after each cocaine infusion over five consecutive sessions of training. Consequently, all the rats were responding on a heterogeneous chained (tandem FR1 RI120-s) FR1 TO schedule allowing a maximum of 11 reinforcements.
Only for experiment 2, animals were subsequently given access to cocaine over 1 h (ShA and ShA + sucrose) or 6 h (LgA and LgA + sucrose) daily for 14 sessions according to the same protocol as described for the acquisition of the taking response (i.e. no seeking component) to allow for the emergence of escalation in the LgA groups.
All eight cocaine groups received three further sessions on this seeking-taking chained schedule. During these sessions, the rats in the cocaine taking + sucrose, the cocaine seeking + sucrose, the ShA + sucrose and the LgA + sucrose groups were also trained to nose poke into the magazine for 0.04 ml of a 20 % sucrose solution, which was delivered under an RI schedule, the parameter of which was progressively increased to 60 s.
All rats received a further four sessions of training under the seeking-taking chain to establish a baseline against which to assess the effects of punishment. During each punishment session, half of the cycles contained no punishment and were identical to those in baseline training. In the remaining cycles, either the taking response or the seeking response was punished: (i) punishment of taking responding, performance of the taking response delivered a .5-s foot shock rather than reinforcement for the sucrose taking, the cocaine taking and the cocaine taking + sucrose groups; (ii) punishment of seeking responding, the first response that met the RI requirement in the seeking link delivered the .5-s foot shock and led to a direct transition to the TO period without the taking link for the sucrose seeking, the cocaine seeking and the cocaine seeking + sucrose groups. The reinforced and punished cycles were presented randomly within each session for eight daily sessions of punishment (Pelloux et al. 2007). In experiment 2, only the seeking responding was punished according to the procedure described above.
For experiment 1, the effect of punishment of sucrose or cocaine seeking or taking was assessed by the number of cycles completed. A cycle is considered completed when cocaine is injected or shock is presented. Therefore, in all cases, this measure includes completion of the seeking cycle. A three-way mixed analyses of variance (ANOVA) between performance over the 4 days of baseline and 8 days under intermittent punishment of sucrose taking, the sucrose seeking, the cocaine taking and the cocaine seeking groups evaluated the effect of punishment, assessed by a within-subject variable of sessions and the between-subjects factors of the type of reinforcement (sucrose vs cocaine) and locus of punishment contingency (seeking vs taking). Additionally, the impact of an alternative reinforcer on punishment efficacy was evaluated through a three-way mixed ANOVA between the performance under baseline and intermittent punishment of the cocaine seeking, the cocaine taking, the cocaine seeking + sucrose and the cocaine taking + sucrose groups. Session was the within-subject factor. The between-subject variables of contingency contrasted the effects of punishment of the seeking and taking responses, whereas those of condition evaluated the effect of the availability to concomitantly nose poke for sucrose.
The effects of punishment on responding were further investigated. Within the 4 days of baseline and 8 days of punishment sessions, the total times taken to initiate the seeking link, to complete the seeking link, and to press the taking lever by the cocaine seeking, the cocaine taking, the cocaine seeking + sucrose and the cocaine taking + sucrose groups were analysed using a four-way ANOVA with contingency and condition as between-subject factors and sessions and step of the schedule as within-subject factors. Three-way ANOVAs were then performed in the cocaine taking and cocaine taking + sucrose groups with condition as the between-subject factor and sessions and step of the schedule as within-subject factors.
The impact of contingency on concomitant nose poke responding for sucrose was assessed over the 4 days of baseline and 8 days of punishment in the cocaine taking + sucrose and cocaine seeking + sucrose groups using a two-way mixed ANOVA contrasting the within-subject variable of session.
The level of investment in cocaine over sucrose-related behaviours was computed as the number of seeking or taking responses for cocaine as a proportion of the total number of motivated responses made for cocaine and sucrose across, respectively, the seeking or taking period of the schedule. This ratio was analysed in the cocaine taking + sucrose and cocaine seeking + sucrose groups through a two-way mixed ANOVA contrasting the within-subject variable of session and step of schedule and with punishment contingency as a between-subject factor.
For experiment 2, the first hour of drug self-administration was compared by mixed two-way ANOVA with session as the within-subjects factor and group as the between-subjects factor. The impact of cocaine self-administration history on baseline performance for cocaine, in the presence or absence of an alternative, was evaluated by conducting a two-way ANOVA of the averaged number of seeking responses over the 4 days of baseline with the variable of cocaine history (ShA vs LgA) and the variable of sucrose contrasting the presence or not of concomitant sucrose reinforcement. We conducted a three-way mixed ANOVA between performance over the 4 days of baseline and 8 days under intermittent punishment, assessed by a within-subject variable of session, the effect of cocaine history (ShA and LgA) and the impact of the condition of the option for alternative reinforcement. Within the ShA + sucrose and LgA + sucrose, the impact of cocaine reinforcement history on concomitant nose poke responding for sucrose was assessed over the 4 days of baseline and 8 days of punishment through two-way mixed ANOVA.
All post hoc analyses were conducted using Tukey’s honestly significant difference (HSD) tests.