2.1 Subjects
One-hundred and sixteen male Sprague-Dawley rats (Charles River, QC) weighing between 250 and 300g at the beginning of each experiment were individually housed in standard rat cages (polycarbonate; 50.5 x 48.5 x 20 cm) with standard environmental enrichment. Upon arrival, rats were given 1 week of acclimatization to the facility and were maintained on a 12-h reverse light/dark schedule (lights off 7:00 AM, on 7:00 PM). All behavioral testing was conducted during the dark period. Rats had access to 25 g per day of standard rat chow and water ad libitum in their home cages. All procedures were approved by the Animal Care Committee at the University of Guelph and were performed in accordance with recommendations provided by the Canadian Council on Animal Care and ARRIVE guidelines.
2.2 Surgery
Rats in Study 2 were surgically implanted with IV silastic catheters (Fisher Scientific, Whitby, ON) in the right jugular vein under general anesthesia induced by isoflurane (4% induction, 2% maintenance). Meloxicam (5 mg/kg subcutaneous, SC, Ontario Veterinary College, Guelph, ON) was administered approximately 30 minutes before and 24 h after surgery. Rats were given atropine sulfate (10 mg/kg SC, Ontario Veterinary College, Guelph, ON) prior to surgery and received a small injection of Lidocaine (2.0% - 0.05 ml) at the sites of incision. Depocillin (300,000 IU, 0.1 ml/rat intramuscular, Ontario Veterinary College, Guelph, ON) was administered immediately following surgery. The catheter was secured to the vein with silk sutures and was passed SC to the back of the rat, approximately 3 cm posterior from the front shoulder blades where it exited into a connector (a modified 22-gauge cannula; Plastics One, Roanoke, VA) secured to surgical mesh with dental cement. A plastic blocker was placed over the opening of the cannula when not in use. Catheters were flushed daily with saline and every second day with 0.10 ml of a saline–heparin solution (15 IU/ml Heparin, Ontario Veterinary College, Guelph, ON). Rats were given at least 7 days to recover from surgery before behavioural testing began.
2.3 Apparatus
2.3.1 Operant chambers
Twenty Plexiglas operant chambers (model ENV-008CT, Med Associates, Georgia, VT) were each enclosed in larger sound-attenuating plywood cabinets (model ENV-018M, Med Associates). Each operant chamber contained a house light (28 V), and two levers, one retractable (active) and one stationary (inactive), located 10 cm apart and 8 cm above the floor. The active lever entered and remained extended during the entire duration of all sessions, and all responses were recorded. In addition, presses on this lever activated a white light located 3 cm above the lever that served as a discrete CS that was paired with heroin delivery in Study 2. The inactive lever served to control for non-specific lever presses; responses on this lever had no consequence but were recorded. Presses on the active lever also activated infusion pumps (Razel Scientific Instruments, Stanford, CT) positioned outside the sound-attenuating cabinets that were used to deliver IV heroin infusions in Study 2.
2.3.2 Object location
The object location memory task assesses the ability of rats to discriminate between familiar and novel locations of objects placed in an open field (Ennaceur et al., 1997). The apparatus consisted of an open box (70 cm x 70 cm x 60 cm) made of white corrugated plastic. The floor was black and two walls opposite from each other were covered with two distinct patterns. Objects used varied in height, size, and texture. An overhead camera was used to record object exploration of rats while in the apparatus.
2.4 Procedures
2.4.1 Conditioning by passive injections of heroin
For each of the 6 Pavlovian conditioning sessions in Study 1, rats were injected with 1 mg/kg heroin and immediately confined in operant chambers for 1 h. The operant chamber was used as a contextual CS to enhance methodological consistency with Study 2. The number of conditioning sessions was selected based on previous findings indicating that such CS elicits approach behavior and modulates memory consolidation when experienced post-training (Francis et al., 2022; Rizos et al., 2005; Wolter et al., 2020).
2.4.2 Self-administration
In Study 2, rats were placed in the same operant chambers as Study 1, attached to the infusion lines, and allowed to self-administer 0.05 mg/kg/inf heroin on a fixed ratio 1 (FR1) schedule for a total of 11 daily 3 h-sessions. Each session began with activation of the house light, entry of the retractable lever, and illumination of the discrete light CS above the active lever for 30 s. If a rat responded on the active lever during this first period, it received a 150 µl infusion of heroin. Similarly, subsequent presses on the active lever led to heroin infusions and simultaneous illumination of the discrete light CS for 5 s. Responses on the inactive lever were without consequences.
For the 12th 3 h-session of self-administration, the schedule of reinforcement was changed to a variable ratio 20 (VR20; heroin infusion obtained, on average, after 20 presses on the active lever). This schedule was chosen because a shift from FR1 to VR20 can generate a DA-dependent prediction error and activate memory stabilization processes (Exton-McGuinness et al., 2014, 2015; Reichelt et al., 2013). The range of active lever presses (1 – 58) required to achieve an average of 20 responses x inf was based on responses made during the 11th self-administration session.
2.4.3 Object location memory task
Prior to receiving heroin in Studies 1 (passive injections) and 2 (self-administration), rats were exposed to the object location apparatus for a single, 10 min habituation session and to the operant chambers for a single, 1 h habituation session. In both studies, animals were then tested for object location memory at key stages of the conditioning/self-administration period. All tests included a sample and a choice phase. During the sample phase, rats were allowed to explore two identical objects positioned in adjacent corners of the apparatus for a total of 180 s, until 25 s of total object exploration was reached, or whichever came first. Immediately following the sample phase, rats were exposed to heroin or the heroin CS (see below), and then returned to their home cages where they remained undisturbed for 72 h. After this retention delay, they were placed back in the open field for the choice phase with one of the sample objects moved to a new location. This retention interval was chosen as a “suboptimal” condition in which drug naïve rats do not typically express object memory (Wolter et al., 2019, 2020). Object exploration was defined as the nose pointed directly at the object within 2 cm and/or touching the object with the nose. Time spent investigating objects were scored by an experimenter blind to experimental group allocations.
2.4.4 Tests of object memory in Study 1
Panel A of Figure 1 illustrates how the object location task was employed to explore the effects of SCH23390 (SCH) on memory modulation by heroin Pavlovian conditioning and by drug-free exposure to the heroin CS. Test 1 explored whether the 1st conditioning session could modulate object location memory consolidation in drug-naïve animals (n = 52), and no SCH was administered. Following 5 additional conditioning sessions, the same animals were re-tested (Test 2) to assess the effects of SCH (0, 0.05, 0.10 mg/kg, n = 12 per group) on memory modulation by the 6th session of conditioning. For this test, a subset of rats did not receive SCH (No SCH group; n = 16) because they were randomly selected to proceed to Test 3. This final test assessed the effect of SCH (0 and 0.10 mg/kg, n = 8 per group) on memory modulation by post-sample exposure (for 1 h) to the heroin CS in the absence of the drug.
Using a separate group of rats (n = 8) that was not conditioned, it was verified whether post-training SCH (within-group design, 3 tests, 0, 0.05, 0.10 mg/kg randomized using a Latin Square protocol) could block object location memory by itself using a 24 h retention period; this delay is sufficiently short for drug-naïve rats to display object memory (Winters et al., 2004, 2008).
2.4.5 Test of object memory in Study 2
Panel B of Figure 1 illustrates how the object location task was employed to explore whether the modulatory action of self-administered heroin on memory changes over the course of self-administration, and whether the predicted memory effect caused by a change in reinforcement schedule could be blocked by SCH. Therefore, Tests 1 and 2 assessed, in the same group of rats (n = 40), the effect of the 1st and 6th sessions of heroin self-administration on consolidation of object memory. For the final Test (Test 3), a subset of rats were not injected with SCH (No SCH; n = 20) and self-administered heroin on a VR20 schedule. The remaining rats received SCH or vehicle (0 mg/kg, n = 10, or 0.10 mg/kg, n = 10) prior to the VR20 self-administration session.
Using a separate group of rats (FR1 group, n = 16) similarly trained to self-administer heroin, it was verified whether post-sample exposure to the 12th session of heroin self-administration could modulate object location memory if the FR1 schedule was not changed.
2.5 Drugs
Heroin (Diacetylmorphine hydrochloride, Toronto Research Chemicals, Toronto, ON) and SCH23390 (Sigma-Aldrich, Oakville, ON) were dissolved in 0.9% physiological saline. For Study 1, heroin was injected at 1 mg/kg because this dose can enhance object location memory (Francis et al., 2022). For Study 2, heroin was self-administered IV at 0.05 mg/kg/infusion and a volume of 150 µl/infusion because of previous self-administration studies in our laboratory (Cummins & Leri, 2008; Leri & Stewart, 2001, 2002). SCH23390 was injected at 0, 0.05 or 0.10 mg/kg because when administered alone, rats display object memory when tested using a 24 h retention delay (de Lima et al., 2011). Finally, SCH23390 was administered immediately post-training followed by a 15 min delay (de Lima et al., 2011) before exposure to heroin or to the heroin CS.
2.6 Data analysis
Analysis of object location memory involved the calculation of a discrimination ratio (DR) in the first minute of the choice phase using the formula: [(time exploring object in novel location – time exploring object in familiar location)/total exploration time] (Dix & Aggleton, 1999). A score of 0 indicated equal exploration of both objects, while a positive score indicated more time spent investigating the object in the novel location. A sample DR was also calculated using an if/then scenario: (if “the right object is in a novel location” during the choice phase, then [(right object exploration – left object exploration)/total exploration of both objects]. A minimum exploration time was not used in these calculations. For all tests, total object exploration on choice was also analyzed to rule out possible motor effects of drug treatments administered 72 h prior. Because this variable was never significantly different between groups, data are not shown, and statistical analyses not reported.
When possible, the data of study Tests common to all animals were combined and analyzed as a single group. Paired t-tests, one, two and three-factor mixed repeated measures ANOVA followed by post hoc comparisons or multiple simple comparisons, when appropriate, were performed using Statistical Package for the Social Sciences (V28 for Mac, SPSS Inc., IBM) with an alpha = 0.05, unless corrected using the Bonferroni method. In cases where the assumption of sphericity was violated, the Greenhouse-Geisser (GG) corrected P value was used.