Supplemental Material
General methods
Selective breeding paradigm
A number of measures were taken to maximize initial genetic variation and to reduce inbreeding in the selectively-bred rat lines (for details see Stead et al., 2006). Briefly, the founding population for the selectively bred lines was composed of 60 male and 60 female Sprague-Dawley rats purchased from Charles River Laboratories and obtained from three different locations to increase genetic diversity (Kingston, NY, USA; Portage, MI, USA; and Saint-Constant, QC, Canada). Animals from each of the three colonies contributed equally to the first generation (S1) of selectively-bred rats. Behavioral testing for locomotor response to a novel environment (see below) was performed on adult rats between postnatal days 55 and 65. Males and females with the highest and lowest scores from locomotor testing were bred together to generate the bred high-responder (bHR) and bred low-responder (bLR) lines, respectively. Following completion of locomotor testing, rats were bred for the subsequent generation at 80-90 days of age.
A. Locomotor response to novelty
As previously described (Stead et al., 2006; Clinton et al., 2007), each generation of selectively-bred rats were screened for locomotor activity in test chambers made of clear acrylic (43 x 21.5 x 25.5 cm (high) and equipped with infrared photocell emitters mounted 2.3 and 6.5cm above the grid floor. The animals were exposed to the test chamber for the first time on the day of testing. Horizontal and rearing activity was monitored in 5-min intervals over a period of 60 min via a computer. All testing was performed between 0800 and 1130. Total locomotion scores for each rat were calculated by adding the total number of horizontal and rearing movements. bHR rats whose scores fell one standard deviation below the bHR group average and bLR rats whose scores fell one standard deviation above the bLR group average were not used for the present studies.
B. Pavlovian conditional approach
B1. Pavlovian conditioning chambers (food, US)
Med Associates (St. Albans, VT) test chambers (21.6 cm x 17.8 cm floor area, 12.7 cm high) located inside a sound-attenuating enclosure were used. Each chamber was equipped with a food receptacle, which was located in the center of the 21.6-cm-wide-wall, 3 cm above the stainless steel grid floor. An illuminated retractable lever (Med Associates) was located approximately 2.5 cm to the left or right of the food receptacle, 3 cm above the floor. The side of the lever with respect to the food receptacle was counter-balanced to eliminate any side bias. The lever required only a 10-gram force to operate, such that most contacts with the lever were detected and recorded. Operation of the pellet dispenser (Med Associates) delivered one 45-mg banana flavored food pellet (Bio-Serv®, Frenchtown, NJ) into the food receptacle. Head entries into the food receptacle were recorded each time the rat broke a photobeam located inside the receptacle.
B1. Pavlovian conditioning procedures (food US)
All training sessions were conducted between 1300 and 1800 hr. Banana-flavored food pellets were placed into the rats’ home cages for 2 days prior to training to familiarize the animals with this food (the US). Two pre-training sessions were conducted which consisted of the delivery of 50 food pellets that were randomly delivered on a variable 30-s schedule (25-min session) and it was determined whether the rats were reliably retrieving the food pellets. Following pre-training, Pavlovian training sessions were conducted as described in the main text.
B2. Jugular catheterization surgery for Pavlovian conditioning (cocaine, US)
Briefly, one end of silicone catheter was inserted into the external jugular vein and the other was passed subcutaneously to exit the back of the animal, where it was connected to a pedestal constructed from a 22 gauge cannula and connected to a piece of polyethylene mesh using dental cement. For 2 weeks following surgery, catheters were flushed daily with 0.1 ml of sterile saline containing gentamicin (10 mg/ml) to prevent occlusions and microbial buildup in the catheter. After the first 2 weeks post-surgery, rats received a lower concentration of gentamicin (0.1 mg/ml delivered in 0.1 ml sterile saline) on a daily basis. Before and after Pavlovian training began catheters were checked for patency by injecting 0.1 ml of the short-acting barbiturate sodium thiopental (i.v.; 20 mg/ml in sterile water). Rats that became ataxic within 5 s were considered to have patent catheters.
B2. Habituation sessions prior to Pavlovian conditioning (cocaine, US)
Rats were habituated to lever presentation prior to Pavlovian training in order to obtain a stable measure of baseline responding. Habituation sessions were initiated by activation of the house light and white noise generator, both of which remained on throughout the sessions. The first two habituation sessions consisted of 24 trials in which the illuminated lever was extended for 8 s and the infusion pump activated for 2.8 s (as during Pavlovian training). The ITI varied randomly with a mean interval of 120 s. Following the first two habituation sessions, rats underwent jugular catheterization surgery (described above), and after recovery from surgery rats were exposed to 8 additional habituation sessions. The habituation session was altered such that it now consisted of 14 trials with a mean ITI of 60 s. The last habituation session was conducted using the same paradigm described below (but with saline instead of cocaine) in order to obtain measures of baseline behavior comparable to that during the training session.
B2. Pavlovian conditioning procedures (cocaine US)
Pavlovian training sessions were conducted between 0800 and 1300. Rats were brought to the chambers and connected to infusion lines via their backport. Sessions began with the activation of the red house light and white noise generator. One session consisted of 6 trials (CS-US pairings) and each trial consisted of the 8 s presentation of the illuminated lever (CS) paired with the non-contingent intravenous injection of 0.5 mg/kg (weight of the salt, dissolved in 0.9% sterile saline) of cocaine (US). The infusion pump was activated upon insertion of the lever because of the delay involved with an injection, and the injection itself took 2.8 s.
The number of approaches and the latency to approach the lever was recorded with a plastic insert that was equipped with photocells aligned 1 cm in front of the extended lever. This modification was made to the test chambers because it had previously been found that when paired with the intravenous delivery of cocaine, animals approach and investigate the lever, but seldom come into contact with it, and therefore lever presses do not provide a good measure of approach behavior when cocaine is used as the US (Uslaner et al., 2006).
C. Impulsive behavior
C1. Delay-discounting procedures
Behavioral testing occurred between the hours of 1300 and 1700. On the first day of training, rats were brought to the chambers and received 50 banana pellets delivered on a random interval schedule with a mean ITI of 30 s to familiarize the rats with retrieving pellets from the food cup. This session lasted ≈25 min. Rats were then trained on a fixed ratio (FR) 1 schedule of reinforcement to lever press for the banana pellets. These sessions lasted 30 min, or until the rat reached a criterion of 50 presses/session. Alternating levers (i.e. left or right) were presented during each training session such that rats were exposed to 3 FR1 sessions with the left lever and 3 FR1 sessions with the right lever. By the end of this training phase all rats had reached a criterion of 50 presses in 30 min for both levers.
Rats were then trained on a simplified version of the delay-discounting task. These sessions consisted of 90 trials, each lasting 40 s. Each trial began with activation of the house light and the large cue light placed above the food cup. The rat was required to poke its nose into the food cup within 10 s, which resulted in the cue light being turned off and the presentation of the illuminated lever (either the right or the left, randomly selected). Initiation of a trial as a result of a head entry into the food cup ensured that the rat was centrally located between the two levers prior to each trial. If the rat failed to initiate the trial by placing its nose into the food cup within 10 s of activation of the cue light, the trial was aborted and the houselight and cue light turned off. Likewise, if the rat failed to press the lever within 10 s of presentation, the trial was aborted, the illuminated lever retracted, and the houselight turned off. If the rat successfully pressed the lever within the 10 s period, the lever was retracted, a single banana pellet was delivered into the food cup, and the large cue light above the food cup was activated. Once the rat retrieved the food pellet (or after 10 s had elapsed), the cue light was turned off and the house light was turned off 6 s later. The forced-choice training procedures were prolonged for this task to ensure that there were no group differences in successfully completing this simplified version of the task prior to moving to the test phase. Rats that did not complete at least 60 successful trials for 3 consecutive days were exposed to additional sessions for a maximum of 24 days. Once a rat reached stable levels of responding, they were then exposed to the training procedures just once every 5 days to ensure that their performance did not change. All of the rats underwent training for the last 5 sessions (days 20-24) at which time all were successfully meeting the criterion of at least 60 successfully completed trials.
Each test session of the delay-discounting task consisted of 60 trials (at 100-s intervals) which began with the houselight turned off and the levers retracted. Similar to the forced-choice training phase described above, at the onset of each trial the houselight and cue light above the food cup were activated and the rat was required to poke its nose into the food cup within 10 s to prevent abortion of the trial. Following successful head entry into the food cup, the cue light was turned off and either one (during forced-choice) or both levers (during free-choice) were extended. Forced choice trials were included to encourage the rat to sample from both levers. The ‘immediate’ and the ‘delay’ lever were right-left counterbalanced across animals. If the rat failed to press the lever within 10 s, the trial was aborted, houselight turned off and levers retracted. If the rat pressed the immediate lever, the lever was retracted, the cue light turned on and one food pellet was immediately delivered into the food cup. If the rat pressed the delay lever, the lever was retracted, the cue light activated, and four food pellets were delivered at the specified delay (see main text). As described above, after the rat retrieved the food pellet(s) or after 10 s had elapsed the cue light was turned off and the houselight was turned off 6 s later.
C2. Probabilistic choice procedures
Behavioral testing occurred between the hours of 1300 and 1700. The training phase for the probabilistic-choice task was the same as that described for the delay-discounting task (i.e. retrieval of food pellets from food cup followed by FR1 training for each lever, and forced-choice training). The forced-choice training procedures were prolonged for this task to ensure that there were no group differences in successfully completing this simplified version of the task prior to moving to the test phase. Rats that did not exhibit stable or peak levels of responding for 3 consecutive days were exposed to the forced-choice trials for a maximum of 28 days. Once a rat reached stable levels of responding, they were then exposed to the training procedures just once every 5 days to ensure that their performance did not change. All of the rats underwent training for the last 5 sessions (days 23-28) at which time all were successfully completing approximately 90% of the trials. Though it is noteworthy that bHR rats acquired this behavior at a faster rate than bLR rats, it is unlikely that these differences during training affected performance during the test sessions—especially since the data that were analyzed and reported were collected after weeks of exposure to the probabilistic-choice task (days 20-24).
Probabilistic-choice test sessions consisted of 80 trials (at 40-s intervals) which began with the houselight turned off and the levers retracted. Similar to the delay-discounting task, at the onset of each trial the houselight and cue light above the food cup were activated and the rat was required to poke its nose into the food cup within 10 s to prevent abortion of the trial. Following successful head entry into the food cup, the cue light was turned off and either one (during forced-choice trials) or both levers (during free-choice trials) were extended. One lever was designated the large/uncertain lever and the other the small/certain lever (right-left counterbalanced across animals). If the rat failed to press the lever within 10 s the trial was aborted, houselight turned off, and levers retracted. If the animal pressed the small/certain lever, the lever was retracted, the cue light turned on, and one food pellet was delivered into the food cup. If the rat pressed the large/uncertain lever, the lever was retracted, the cue light activated, and four food pellets were delivered into the food cup if a given probability was met (see main text).
C3. DRL training
The training phase for the DRL task began a few days after the delay-discounting task. Even though the rats had been trained previously for the delay-discounting procedure, they briefly underwent retraining prior to DRL testing to ensure that there were no bHR/bLR differences in operant behavior that might affect interpretation of the results. Rats were trained for FR1 responding for banana pellets for two 45-in sessions prior to the DRL task.