Supplemental Information Saunders et al.
A cocaine context renews drug seeking preferentially in a subset of individuals
Benjamin T. Saunders, Elizabeth G. O’Donnell, Elyse L. Aurbach, and Terry E. Robinson
Male Sprague-Dawley rats (Harlan, IN) weighing 250-300 g upon arrival were housed individually on a 12-hr light/12-hr dark cycle (lights on at 0800) in a climate-controlled colony room. Water and food were available ad libitum. After arrival rats were given one week to acclimate to the colony room before testing began, during which the experimenters repeatedly handled them.
Pavlovian training and self-administration testing were conducted in standard (22x18x13 cm) test chambers (Med Associates Inc., St. Albans, VT, USA) located inside sound-attenuating cabinets. A ventilating fan masked background noise.
Pavlovian conditioned approach training: Chambers had a food cup located in the center of one wall, 3 cm above a stainless steel grid floor. Head entries into the food cup were recorded by breaks of an infrared photobeam located inside the magazine. A retractable lever illuminated from behind was located 2.5 cm to the left or right of the food cup, approximately 6 cm above the floor. The location of the lever with respect to the food cup was counterbalanced across rats. On the wall opposite the food cup, a red house light remained illuminated throughout all sessions.
Self-administration: For these sessions, the food cup and lever were removed and replaced with two nose-poke ports located 3 cm above the floor on the wall opposite the house light. A nose poke into the active port, detected by an infrared photobeam inside the hole, resulted in an intravenous cocaine infusion, delivered by a syringe pump through a tube connected to the rat’s catheter back port. The syringe pump was located outside the sound-attenuating chamber so it would not provide an auditory cue. The infusion tube was suspended into the chamber via a swivel mechanism, allowing the rat free movement. Active and inactive nose-poke ports were counterbalanced across rats. All measures were recorded using Med Associates software.
Cocaine-context conditioning: In Experiment 1 (conducted in the fall of 2010 by E.L.A.) the test chambers were constructed with black acrylic (67.5 length x 33 width x 60.5 cm tall). Cameras (SPE-57, CCTV Specialty Bullet Cameras, Lake Worth, FL, USA) were mounted 12 cm directly above the open ceiling of the chambers to record video. Video was analyzed using Topscan (Clever Sys., Inc., Reston, VA). Locomotor activity was measured as chamber crossovers. In Experiment 2 (conducted in 2012 by B.T.S. and E.G.O.) the test chambers were clear acrylic chambers (43.5 length x 21.5 width x 21 cm tall). A smaller (23 long x 6 wide x 21 cm tall) clear acrylic insert was positioned in the center of the larger chamber, which restricted each rat’s movement to the perimeter of the chamber. To record locomotor activity, we surrounded each chamber with a rectangular strip (47 long x 28 cm wide) containing 5 photocell pairs distributed along the longer side of the chamber. These photocells recorded movement within the chamber as total photocell beam breaks and chamber crossovers.
For Experiments 3 and 4 (conducted by B.T.S. and E.G.O.), following Pavlovian training, rats were prepared with intravenous catheters as described previously under ketamine hydrochloride (100 mg/kg i.p.) and xylazine (10 mg/kg i.p.) anesthesia. Following surgery, catheters were flushed daily with 0.2 ml sterile saline containing 5 mg/ml gentamicin sulfate (Vedco, MO) to minimize infection and prevent occlusions. At the end of self-administration training, catheter patency was tested by intravenous injection of 0.2 ml sodium thiopental (20 mg/ml in sterile water, Hospira, IL). Only rats that became ataxic within 5-10 s were considered to have patent catheters and included in the analyses. For Experiment 4, after receiving an intravenous catheter, rats were positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The skull of each rat was leveled and chronic guide cannulae (22 gauge stainless steel; Plastics One) were inserted bilaterally 2 mm above the target site in the NAc core (relative to Bregma: anterior +1.8 mm; lateral +1.6 mm; ventral -5.0 mm). Guide cannulae were secured with skull screws and acrylic cement, and wire stylets (28 gauge, Plastics One) were inserted to prevent occlusion. After surgery, all rats received antibiotic and carprofen (5 mg/kg) for pain. Rats were given 7 days to recover from surgery before testing commenced.
In Experiment 4, before the renewal test session, rats received a microinjection of vehicle (0.9% sterile saline) or flupenthixol (15 µg in saline; Sigma, St. Louis, MO), a relatively nonspecific DA receptor antagonist. Flupenthixol was chosen in order to antagonize all actions of endogenous DA near the injection site, to assess the general (i.e., not specific to a particular receptor) function of DA in context renewal. The flupenthixol dose was chosen based on previous studies , for which flupenthixol has no nonspecific effects on locomotion, and, importantly, goal-tracking conditioned approach behavior is not impaired , suggesting that the expression of vigorous behavior is unaffected. Note that in the current study we were interested in understanding the role of dopamine in the nucleus accumbens core, generally, in cocaine context renewal, and not in assessing the full dose-response function of flupenthixol’s effects, which would require replicating these studies at several other doses. Intracerebral microinjections were made through 28-gauge injector cannulae (Plastics One) lowered to the injection site in the NAc core (ventral -7.0 mm relative to skull), 2 mm below the ventral tip of the guide cannulae. During infusions rats were gently held by the experimenter. All infusions were administered bilaterally at a volume of 0.5 µl/side, delivered over 90 s using a syringe pump (Harvard Apparatus, Holliston, MA) connected to microinjection cannulae via PE-20 tubing. After infusions, the injectors were left in place for 60 s to allow for drug diffusion before being withdrawn and replaced with wire stylets. Rats received a microinjection of saline approximately 5 days before the renewal test, to acclimate them to the injection procedure. All microinjections were given approximately 10-15 min before rats were placed in the test chamber, to account for delay drug actions .
Experimental design and procedures
Pavlovian training: Pavlovian training procedures were the similar to those described previously . For two days prior to the start of training, 10 banana-flavored pellets (45 mg, BioServe, #F0059; Frenchtown, NJ) were placed in the home cages to familiarize the rats with this food. Approximately one week after arrival, rats were placed in the test chambers, with the lever retracted, and trained to retrieve pellets from the food cup by receiving 25 45-mg banana pellets on a variable time (VT) 30-sec schedule. All rats retrieved the pellets and began Pavlovian training the next day. Each trial consisted of insertion (and simultaneous illumination) of the lever (CS) into the chamber for 8 s, after which time the lever was retracted and a single food pellet (US) was immediately delivered into the adjacent food cup. Each training session consisted of 25 trials in which CS-US pairings occurred on a variable time (VT) 90-s schedule (the time between CS presentations varied randomly between 30 and 150 s). Lever deflections, food cup entries during the 8-s CS period, latency to the first lever deflection, latency to first food cup entry during the CS period, and food cup entries during the inter-trial interval were measured.
Quantification of behavior using an index of Pavlovian conditioned approach (PCA): For some analyses rats were classed into three groups: (1) Those who preferentially interacted with the lever (“sign-trackers”, STs), (2) those who preferentially interacted with the food cup during lever presentation (“goal-trackers”, GTs), and (3) those who had no clear preference for the lever or food cup (“intermediates”, INs). The extent to which behavior was lever (CS) or food-cup directed was quantified using a composite index that incorporated three measures of Pavlovian conditioned approach: (1) the probability of either deflecting the lever or entering the food cup during each CS period [P(lever) - P(food cup)]; (2) the response bias for contacting the lever or the food cup during each CS period [(#lever deflections - #food-cup entries) / (#lever deflections + #food-cup entries)]; and (3) the latency to contact the lever or the food cup during the CS period [(lever deflection latency - food-cup entry latency) / 8]. Thus, the Pavlovian conditioned approach index (PCA Index) score consisted of [(Probability difference score + Responses bias score + Latency difference score) / 3]. This formula produces values on a scale ranging from -1.0 to +1.0, where scores approaching -1.0 represent a strong food cup-directed bias and scores approaching +1.0 represent strong lever-directed bias. The average PCA Index score for days 4 and 5 of training was used to class rats. Rats were designated STs if they obtained an average index score of +0.3 or greater, and as GTs if they obtained a score of -0.3 or less. The remaining rats within the middle score range were classed as INs. Note that INs were excluded from further study because we wanted to focus on those animals that differed markedly on their propensity to attribute incentive salience to reward cues . It is important to point out that the number of rats classified as STs and GTs varies across different cohorts, which may in part be due to genetic differences between rats from different providers , which means that the final group sizes are not necessarily equal. A total of 105 STs and 66 GTs were used in the experiments (see also Figure S1).
Cocaine context conditioning replication (Experiment 2)
Given the small N of the GT groups in our first context conditioning study, we conducted a replication, using a different apparatus to measure locomotion. Following Pavlovian conditioning, a separate cohort of rats (N=27) were first habituated to the photocell conditioning chambers in two 40-min sessions, during which we recorded locomotor activity (photocell beam breaks). Next, rats were assigned to cocaine paired and unpaired conditioning groups, matched based on locomotor response on the second day of habituation. Four groups were tested: GT-Unpaired (n=5), GT-Paired (n=5), ST-Unpaired (n=8), ST-Paired (n=9). Next, five conditioning sessions were conducted. Rats in the paired groups received an i.p. injection of cocaine (10 mg/kg) before being placed in the photocell chambers for each 40-min session. Paired rats then received a saline injection in their home cage 40 min after the end of the conditioning session. Unpaired rats received the opposite injection schedule. On day 6, all rats received an injection of saline before a 40-min test session. A computer recorded total photocell beam breaks.
Time course of context renewal: To understand the pattern of context renewal in GTs and STs who were re-exposed to the cocaine context following extinction in an alternate context, we analyzed the time course of responding across the 2-hr renewal session. Note that the pattern of results was similar for rats in Experiment 3 and the vehicle rats in Experiment 4, so the data were collapsed. We found that not only was the magnitude of responding different for GTs and STs in renewal, the pattern of responding also differed, as indicated by a significant session time by group interaction (F(11,38) = 2.321, p = 0.027). GTs maintained elevated responding throughout the session, relative to STs, whose responding quickly dropped to close to zero (Figure S4).
Figure S1. PCA Index score distributions for individual rats used in Experiments 1-4. Each bar represents the PCA Index score for an individual rat. Rats classified at goal-trackers (GTs) have scores ranging from -0.3 to -1.0. Rats classified at sign-trackers (STs) have scores ranging from +0.3 to +1.0.
Figure S2. Schematic of context renewal experimental design. Following Pavlovian training, STs and GTs were assigned to one of two contexts in either the Cocaine context group, or the Alternate context group.
Figure S3. Acquisition of cocaine self-administration behavior. Experiment 4. A) Mean (± SEM) number of active and inactive responses made across training infusion criteria (IC) for STs (n=25) and GTs (n=29). B) Mean (± SEM) time required to complete self-administration sessions across training IC for STs and GTs. C) Extinction training. Mean (± SEM) number of active responses made for STs and GTs extinguished in either the cocaine training context (COC groups: GTs n=14, STs n=9) or a novel, alternate context (ALT groups: GTs n=15, STs n=16).
Figure S4. Time course of cocaine context renewal. A) Mean (± SEM) number of active nose pokes made across 10-min intervals of 2-hr renewal session in the cocaine context, for GTs (n=19) and ST (n=21) who received extinction training in an alternate context. Note that data from rats in Experiment 3 and vehicle rats from Experiment 4 are included. *p < 0.05. **p < 0.01.
Figure S5. Location of microinjection tips within the NAc core relative to Bregma for rats used in Experiment 4. Square symbols represent STs and circles represent GTs.
Crombag HS, Badiani A, Maren S, Robinson TE (2000). The role of contextual versus discrete drug-associated cues in promoting the induction of psychomotor sensitization to intravenous amphetamine. Behavioural Brain Research 116(1): 1-22.
Di Ciano P, Cardinal RN, Cowell RA, Little SJ, Everitt BJ (2001). Differential involvement of NMDA, AMPA/kainate, and dopamine receptors in the nucleus accumbens core in the acquisition and performance of Pavlovian approach behavior. J Neurosci 21(23): 9471-9477.
Fitzpatrick CJ, Gopalakrishnan S, Cogan ES, Yager LM, Meyer PJ, Lovic V, et al (2013). Variation in the form of Pavlovian conditioned approach behavior among outbred male Sprague-Dawley rats from different vendors and colonies: Sign-tracking vs. goal-tracking. PLoS One 8(10): e75042.
Flagel SB, Watson SJ, Robinson TE, Akil H (2007). Individual differences in the propensity to approach signals vs goals promote different adaptations in the dopamine system of rats. Psychopharmacology (Berl) 191(3): 599-607.
Ito R, Hayen A (2011). Opposing roles of nucleus accumbens core and shell dopamine in the modulation of limbic information processing. J Neurosci 31(16): 6001-6007.
Lovic V, Saunders BT, Yager LM, Robinson TE (2011). Rats prone to attribute incentive salience to reward cues are also prone to impulsive action. Behav Brain Res 223(2): 255-261.
Meyer PJ, Lovic V, Saunders BT, Yager LM, Flagel SB, Morrow JD, et al (2012). Quantifying individual variation in the propensity to attribute incentive salience to reward cues. PLoS ONE 7(6): e38987.
Saunders BT, Robinson TE (2010). A cocaine cue acts as an incentive stimulus in some but not others: Implications for addiction. Biol Psychiatry 67(8): 730-736.
Saunders BT, Robinson TE (2011). Individual variation in the motivational properties of cocaine. Neuropsychopharmacology 36(8): 1668-1676.
Saunders BT, Robinson TE (2012). The role of dopamine in the accumbens core in the expression of Pavlovian-conditioned responses. Eur J Neurosci 36(4): 2521-2532.