Cocaine Self-Administration Induces Dopamine-Independent Self-Administration of Sigma Agonists
Supplementary Material
Takato Hiranita, PhD1, Maddalena Mereu, PhD 1, Paul L. Soto, PhD 2, Gianluigi Tanda, PhD 1 and Jonathan L. Katz, PhD 1
1Psychobiology Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program, Department of Health and Human Services, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224
2Behavioral BiologyResearchCenter, 5510 Nathan Shock Drive, Suite 3000, JohnsHopkinsUniversityMedicalSchool, Baltimore, MD21224-6823
Detailed Methods
Subjects. Forty-two male Sprague-Dawley rats (Taconic Farms, Germantown, New York) weighing approximately 300 g at the start of the study, served as subjects. Subjects were acclimated to a temperature- and humidity-controlled vivarium for at least one week with food (Scored Bacon Lover Treats, BIOSERV, Frenchtown, NJ) and tap water unrestrictedly available in their home cages under a 12:12-h light:dark cycle with lights on at 07:00 hours. After acclimation, weights of rats were maintained at approximately 320 g by adjusting their daily food ration. Subjects were surgically implanted in the right external jugular vein with a chronic indwelling catheter that exited at the mid-scapular region of the animal’s back. Catheter patency was maintained over the course of the behavioral procedures, however, the subjects used of microdialysis studies of dopamine concentrations in the shell of the nucleus accumbenswere surgically re-implanted with left external jugular catheters. Catheter implantation was performed under anesthesia (ketamine 60.0 mg/kg, i.p. and xylazine 12.0 mg/kg, i.p.). Catheters were infused daily with 0.1 ml of a sterile saline solution containing heparin (30.0 IU/ml), penicillin G potassium (250,000 IU/ml) to minimize the likelihood of infection, and the formation of clots or fibroids. All animals were allowed to recover from surgery for approximately seven days before drug self-administration studies were initiated.Details of allocation of all the subjectsto individual experiments in the present study are supplied in Supplementary Table 1.
Care of the subjects was in accordance with the guidelines of the National Institutes of Health and the National Institute on Drug Abuse Intramural Research Program Animal Care and Use Program, which is fully accredited by AAALAC International.
Self-Administration Procedures. Experimental sessions were conducted with animals placed in operant-conditioning chambers (modified ENV-203, Med Associates, St. Albans, VT) that measured 25.5 x 32.1 x 25.0 cm, and were enclosed within sound-attenuating cubicles equipped with a fan for ventilation and white noise to mask extraneous sounds. On the front wall of each chamber were two response levers, 5.0 cm from the midline and 4.0 cm above the grid floor. A downward displacement of a lever with a force approximating 20 g defined a response, which always activated a relay mounted behind the front wall of the chamber producing an audible “feedback” click. Three light-emitting diodes (LEDs) were located in a row above each lever. A receptacle for the delivery of 45-mg food pellets via a pellet dispenser (Med Associates, Model ENV-203-20), was mounted on the midline of the front wall between the two levers and 2.0 cm above the floor. A syringe infusion pump (Model 22, Harvard Apparatus, Holliston, MA) placed above each chamber delivered injections of specified volumes from a 10 ml syringe. The syringe was connected by Tygon tubing to a single-channel fluid swivel (375 Series Single Channel Swivels, Plymouth Meeting, PA) which was mounted on a balance arm above the chamber. Tygon tubing from the swivel to the subject’s catheter was protected by a surrounding metal spring and completed the connection to the subject.
Experimental sessions were conducted daily with subjects placed in chambers for experimental sessions that lasted for 120 min. After sessions subjects were returned to their cages in the vivarium. Sessions started with the illumination of the LEDs above each lever. Each downward deflection of the right lever turned off the LEDs and activated the infusion pump for 10 sec (fixed-ratio or FR 1 schedule) followed by a 20-sec time-out period during which LEDs were off and responding had no scheduled consequences. Drug injections were cocaine (0.32 mg/kg/injection, n=6), PRE-084 (0.32 mg/kg/injection, n=6) or (+)-pentazocine (0.32 mg/kg/injection, n=6). After the time out, the LEDs were illuminated and responding again had the scheduled consequences. Responses on the left lever were recorded but had no scheduled consequences. This condition remained in effect for 28 experimental sessions. Acquisition of drug self-administration behavior was defined as less than 20% variation in response rates across three consecutive sessions.
For the cocaine self-administration group, responses on the left rather than right lever produced injections for the next seven sessions, with all other conditions as in the first 28 sessions. During the subsequent nine sessions, injections and accompanying stimulus changes were discontinued (extinction) with other aspects of the sessions unchanged. Finally, responses on the left lever again produced cocaine injections for five sessions under the FR 1 schedule as described above (reacquisition).
During the initial 28 sessions with PRE-084 or (+)-pentazocine, responding was not maintained by either drug. Subsequently the PRE-084 group was studied with 5 different doses (0.03-1.0 mg/kg/inj) for 14 sessions each, after which they were allowed to self-administer cocaine (0.32 mg/kg/inj) for 14 sessions under the FR 1 schedule as described above. The (+)-pentazocine group was immediately changed to cocaine self-administration under the FR 1 schedule. After cocaine self-administration, all of the subjects were returned to the FR 1 schedule of PRE-084 or (+)-pentazocine self-administration, and the subsequent series of sessions (change in active lever, extinction, reacquisition) for both groups was as described for the cocaine group.
A separate group of subjects (n=6) were trained with food reinforcement (45 mg food pellets, Bioserv, Rodent Purified Diet, Dustless Precision Pellets, Frenchtown, NJ) to press the right lever under an FR 1-response schedule of reinforcement. All aspects of the schedule for food delivery were similar to those for drug self-administration except that food pellets rather than injections maintained responding. Similar to the study of drug self-administration, this study of food reinforcement had the periods of acquisition of lever-pressing, alternating the lever on which responses produced food, extinction, and reacquisition. After the reacquisition phase, the subjects were catheterized, allowed to respond again for five sessions with food reinforcement, and subsequently allowed to self-administer PRE-084 (0.32 mg/kg/inj) for 28 sessions.
The self-administration procedure was slightly modified to pharmacologically assess mechanisms of the reinforcing effects of PRE-084 and to compare them to those of cocaine. Subjects from the above described cocaine self-administration experiments and several experimentally naïve subjects (N=18) were trained until their cocaine (0.32 mg/kg/inj) self-administration was consistent from one session to the next under the FR 1 schedule described above. Subsequently, the FR value was increased to five and the session was divided into five 20-min components, each preceded by a 2-min time-out period. This arrangement (Hiranita et al, 2009) allowed the assessment of a range of self-administered doses in a single session. By adjusting infusion volumes and durations, the drug dose per injection was incremented in the five sequential components in an ascending dose-order as follows: no injection (also referred to as extinction, or EXT, because responses had no scheduled consequences), 0.03, 0.10, 0.32, and 1.0 mg/kg/inj for cocaine. Infusion volumes and durations were respectively 0, 5.6, 18.0, 56.0, 180 μl and 0, 0.32, 1.0, 3.2 10.0 sec, based on a body weight of 0.32 kg. A sample injection of the drug at the corresponding dose occurred independently of responding at the end of the time-out period that preceded each component except the first.
Training continued until responding was maintained with less than 20% variation in response rates across three consecutive sessions. With these stable performances the effects on cocaine self-administration (0.03, 0.10, 0.32, and 1.0 mg/kg/inj) of presession i.p. injections of the DA receptor antagonists (SCH 39166, L-741,626 or haloperidol) or the preferential σ1R antagonist (BD1063) were assessed. These drugs were also examined in the same rats with PRE-084 (0.03, 0.10, 0.32, and 1.0 mg/kg/inj) substituted for cocaine under otherwise identical conditions. The effects of pre-session treatments on cocaine self-administration were separated by a minimum of 72 hours. The antagonists were studied with a mixed order of drugs and doses.
In-vivo Microdialysis Procedures. Subjects were male Sprague–Dawley rats from self-administration experiments shown in figure 3a and 3c (open triangles up). Rats were implanted with microdialysis probes under a mixture of ketamine and xylazine (60.0 and 12.0 mg/kg i.p., respectively) anesthesia. Rats were placed in a stereotaxic apparatus, the skull was exposed, and a small hole was drilled to expose the dura. Rats were then implanted with a concentric dialysis probe (see below) aimed at the NAc shell either in the right or in the left brain side, as described previously (Tanda et al 2005; Tanda et al 2008). Coordinates were from the rat brain atlas of Paxinos and Watson (1987) [uncorrected coordinates: Anterior = + 2.0 mm from bregma, Lateral = ± 1.0 mm from bregma, Vertical = -7.9 mm from dura]. After the surgery, rats were allowed to recover overnight in hemispherical CMA-120 cages (CMA/Microdialysis AB, Solna, Sweden). Concentric dialysis probes were prepared with AN69 hollow fibers (Hospal Dasco, Bologna, Italy). Briefly, two 4-cm pieces of silica fused capillary tubes (the inlet and outlet tubing of the probes) were inserted into a 6-mm capillary (0.25 mm external diameter) dialyzing fiber (closed by a drop of glue on the other side) with the inlet tubing set at about 0.1 mm from the closed end of the fiber and the outlet set at about 1.8 mm from the inlet tip. The open end of the dialysis membrane was then glued, and the protruding two silica fused capillary tubes were inserted and glued into a 22-G stainless steel needle (2.4 mm length). The needle was attached to a CMA/10 clip (CMA/Microdialysis AB) and mounted in a stereotaxic holder. The exposed dialyzing surface of the fibers, i.e., the portion not covered by glue, was limited to the lowest 1.8 mm of the probes.
Experiments were performed on freely moving rats in the same hemispherical cages in which they recovered from surgery. About 22 hours after implant, probes were connected to fluid swivels (375/ D/22QM; Instech, Plymouth Meeting, PA, USA), and Ringer’s solution (147.0 mM NaCl, 2.2 mM CaCl2, and 4.0 mM KCl) was delivered by a 1.0 ml syringe operated by a BAS Bee Syringe Pump Controller (BAS, West Lafayette, IN, USA) through the dialysis probes at a constant flow rate of 1 μl/min. Collection of dialysate samples started after 30 min, and 10 μl samples were taken every 10 min and immediately analyzed, as detailed below. After stable DA values (less than 10% variability) were obtained for at least three consecutive samples (after about 1 hour), rats were injected with increasing doses of PRE-084, 0.32, 1.0, 3.2, 10 mg/kg i.v, spaced 1 hour apart. Dialysate samples were injected without purification into a HPLC apparatus equipped with an MD 150 mm X 3.2 mm column, particle size 3.0 μm (ESA, Chelmsford, MA, USA), and a coulometric detector (5200a Coulochem II, or Coulochem III, ESA) to quantify DA. The oxidation and reduction electrodes of the analytical cell (5014B; ESA) were set at +125 mV and -125 mV, respectively. The mobile phase, containing 100 mM NaH2PO4, 0.1 mM Na2EDTA, 0.5 mM n-octyl sulfate, and 18% (v/v) methanol (pH adjusted to 5.5 with Na2HPO4), was pumped by an ESA 582 (ESA, Chelmsford, MA, USA) solvent delivery module at 0.50 ml/min. Assay sensitivity for DA was 2 fmoles/sample.
At the end of each experiment, rats were sacrificed by pentobarbital overdose. Brains were removed and left to fix in 4% formaldehyde in saline solution. Brains were then cut on a vibratome 1000 Plus (The Vibratome Company, St. Louis, MO, USA) in serial coronal slices based on Paxinos and Watson (1987). All of the probes were located inside the NAc shell have with placements shown diagrammatically in Supplementary Figure 1.
Drugs. The drugs used in the present study were as follows: (-)-cocaine hydrochloride (Sigma-Aldrich, St. Louis, MO), PRE-084 (Tocris, Ballwin, MO), (+)-pentazocine succinate (National Institute on Drug Abuse), BD1063 (Tocris), SCH 39166 (Tocris), L-741,626 (Sigma-Aldrich) and haloperidol (Sigma-Aldrich). 0.9% NaCl was used as vehicle for all compounds except L-741,626 and haloperidol. L-741,626 and haloperidol were dissolved in ethanol and diluted to ≤6% ethanol (v/v) and ≤25% polyethylene glycol (v/v) in sterile water. Drugs used were administered intravenously (cocaine, PRE-084, and (+)-pentazocine) or intraperitoneally (BD 163, SCH 39166, L-741,626 and haloperidol). BD1063 was administered at 5 min before sessions. All dopamine receptor antagonists were administered at 30 min before sessions. The chemical identities of drugs used are as follows: BD1063; 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride; L-741,626: (±)-3-[4-(4-chlorophenyl)-4-hydroxypiperidinyl]-methylindole; (+)-pentazocine: 2-dimethylallyl-5,9-dimethyl-2'-hydroxybenzomorphan succinate; PRE-084: 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride; SCH 39166: (6aS-trans)-11-chloro-6,6a,7,8,9,13bhexahydro-7-methyl-5H-benzo[d]naphth[2,1-b]azepin-12-ol hydrobromide.
Data analysis. Response rates were determined by dividing responses by elapsed time, excluding time outs after drug injections or food presentations. The significance of effects on response rates was assessed by ANOVA. A Bonferroni t-test was used to compare a series of response rate on drug injections, or food reinforcement. For studies involving dose-effect curves for drug self-administration, a post-hoc Bonferroni t-test was used for all pairwise comparisons. A two-way repeated-measures ANOVA was used to assess the effects of successive response rates during drug self-administration or food presentation (factors were session and lever: right or left). A two-way, repeated-measures ANOVA was also used to assess the drug-dose effects (factors were drug-dose and lever: right or left) or effects of pre-session treatment with antagonists on self-administration (factors were drug-dose and component: no injection or doses of cocaine or PRE-084). For all analyses, the criterion for significance was set at p<0.05.
The in-vivo microdialysis results were expressed as a percentage of basal DA values, which were calculated as means of the three consecutive samples immediately preceding the test drug or saline injection. All results are presented as group means (±SEM). Dose-effect curves were analyzed using standard ANOVA and linear regression techniques, from which ED50 values (doses producing 50% of maximal effect) were calculated from points on the linear part of the curves (Snedecor and Cochran, 1967). To assess whether cocaine experience altered the effects of PRE-084 data were analyzed by standard parallel-line bioassay techniques (Finney, 1964). The relative-potency value obtained represents the dose of PRE-084 in subjects with cocaine experience equal to 1 mg/kg PRE-084 in subjects without cocaine experience. A relative potency value with 95% confidence limits that include 1.00 is not significant.
Results
A separate group of experimentally-naïve rats was trained to lever-press with food reinforcement under an FR 1-response schedule (Fig. S2). Following that acquisition, the active lever was switched from the right to the left which produced a corresponding change in the lever on which responses were emitted. Pellet deliveries were subsequently discontinued (extinction) with a resulting decrease in response rates to low levels. When food pellets were again delivered after each response responding was re-acquired (Fig. S2). A two-way repeated-measures ANOVA indicated significant effects on response rates (p values ≤ 0.006) of session number (F49,245=12.8), right or left lever (F1,245=20.5), and their interaction (F49,245=28.3).
Supplementary Figure Legends
Supplementary Figure 1:Drawings of forebrain sections, based on those of Paxinos and Watson (1998), showing the microdialysis probe tracks. All of the dialyzing portions of the microdialysis probes were within the boundaries of theNAC shell.
Supplementary Figure 2: Reinforcing effects of food pellets in experimentally naïve rats. Each point represents the mean ±SEM in 6 subjects. Food reinforcement readily increased rates of responding (right lever) when each response produced a food pellet, without increasing rates of responding on the alternate (left) lever which had no scheduled consequences. When food pellets were available only for responses on the previously inactive (left) lever, responding switched to that lever. When food pellets were no longer available responding on both levers decreased to low levels. When food was again available for responses on the left lever, responding increased on that lever. *p<0.05, **p<0.01, ***p<0.001, compared with responding on the inactive lever (Bonferroni t-test). A two-way repeated-measures ANOVA indicated significant effects on response rates (p values ≤ 0.006) of session number (F49,245=12.8), right or left lever (F1,245=20.5), and their interaction (F49,245=28.3).
Supplementary Table 1: The experimental conditions for each group of subjects
Group / Initial Training / Experiment / Results shown1 / Cocaine / 1. Comparison of acquisition of cocaine and σ-agonist self-administration; cocaine group / Figure 1a
2. Antagonist treatments on cocaine self administration / Figures 4a-e
2 / Cocaine / Antagonist treatments on PRE-084 self administration / Figures 4f and 4j
3 / Cocaine / Antagonist treatments on PRE-084 self administration / Figures 4g-i
4 / PRE-084 / 1. Comparison of acquisition of cocaine and σ-agonist self-administration; PRE-084 group / Figures 1b and 2c
2. Acquisition of cocaine self-administration after exposure to PRE-084 / Figures 2a and 3c
3. PRE-084 self-administration after cocaine self-administration / Figures 3a, 3c and 2c
4. Measurement an extracellular dopamine concentration / Figure 5
5 / PRE-084 / 1. Dose-effect curve for PRE-084 self-administration before cocaine exposure / Figure 3c
2. Measurement an extracellular dopamine concentration / Figure 5
6 / (+)-Pentazocine / 1. Acquisition of (+)-pentazocine self-administration / Figure 1c
2. Acquisition of cocaine self-administration after exposure to (+)-pentazocine / Figure 2b
3. (+)-Pentazocine self-administration after cocaine self-administration / Figure 3b
7 / Food / 1. Acquisition of responding maintained by food presentation under FR 1 / Supplementary Figure 1
2. PRE-084 substitution for food under FR 1 / Figures 3d and 3e