Dopaminergic activity in the nucleus accumbens (NAc) has been thought to be central to the brain processes that mediate the reinforcing properties of drugs of abuse (Di Chiara and Imperato, 1988; Koob and Bloom, 1988). For example, in vivo microdialysis studies indicate that extracellular levels of dopamine ([DA]e) in the NAc are elevated during amphetamine and cocaine self-administration (Ranaldi et al, 1999; Pettit and Justice, 1989). The effects of the self-administration of opiates on these indices are less consistent with both increases (Wise et al, 1995) and no significant effect reported (Hemby et al, 1995, 1999). However, in the latter circumstance where chronic self-administered heroin was reported to not increase NAc [DA]e (Hemby et al, 1995, 1999), heroin self-administered in combination with cocaine on the descending limb of the dose-intake relationship significantly potentiated the effects of cocaine on NAc [DA]e (Hemby et al, 1999). This is in agreement with data showing acute response-independent heroin (Zernig et al, 1997) and buprenorphine (Brown et al, 1991) to enhance the effects of cocaine on NAc [DA]e. The purpose of the present experiments was: (1) to use an isobolographic analysis of rat self-administration of cocaine, heroin, and the combination of cocaine with heroin to determine the nature of the interaction between these two drugs, and (2) to use microdialysis to determine the effects of this drug combination on NAc [DA]e in rats self-administering low doses of cocaine, heroin, or cocaine/heroin combinations.
In all, 23 adult male Fischer F-344 90-150-day-old rats (Harlan, Indianapolis, IN) were used (six for the isobolographic analysis and 17 for the microdialysis experiment). The rats were housed in a temperature-controlled environment on a reversed 12-h light-dark cycle (lights on 1700-0500) with food and water available ad libitum except during experimental sessions. Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) revised in 1996.
Rats were anesthetized with pentobarbital (50 mg/kg, i.p. -- Abbott Laboratories, North Chicago, IL) after pretreatment with atropine methyl nitrate (10 mg/kg, i.p. -- Sigma, St Louis, MO) and penicillin G procaine (75 000 U, i.m. -- Wyeth Laboratories, Philadelphia, PA) and implanted with venous catheters placed in the right jugular vein using previously described methods (Weeks, 1962, 1972; Pickens and Dougherty, 1972). The catheter (a small piece of polyvinyl-chloride tubing) was inserted into the right posterior facial vein, guided into the right jugular vein until it terminated just outside the right atrium and anchored to muscle in the area of the vein. The other end of the catheter continued subcutaneously to the back where it exited between the scapulae through a polyethylene shoulder harness. The harness provided a point of attachment for the catheter to a needle-tubing leash that passed out the top of the animal chamber. A leak proof swivel (Brown et al, 1976) was used to attach the leash to the tubing leading to the infusion pump so that the animals had almost complete freedom of movement. The rats were allowed to recover for 7 days before the initiation of experimental procedures with programmed infusions (0.2 ml delivered over 6.2 s) of heparinized saline (1.7 U/ml) administered at hourly intervals in the home cage to maintain functional catheters. Patency of catheters was evaluated at regular intervals by delivering an intravenous infusion of methohexital (10 mg/kg -- Eli Lilly, Indianapolis, IN) and determining the latency for loss of stability or consciousness which occurs within 1-2 s.
In all, 17 rats were implanted with microdialysis guide cannula CMA11 (CMA/Microdialysis, North Chelmsford, MA) to terminate at the dorsal surface of the NAc (9.4 mm from lambda, ±1.7 mm lateral from the midline, and 5.0 mm ventral from the dura (König and Klippel, 1967)) using a stereotaxic (Stoelting, Wood Dale, IL). The guide cannulas were secured to the skull with stainless steel screws and dental acrylic cement and obturators (28 g) inserted to prevent blockage.
Cocaine hydrochloride and heroin hydrochloride were obtained from the Drug Supply Program of the National Institute on Drug Abuse. Pentobarbital was purchased from Abbott Laboratories (North Chicago, IL), sodium heparin from Elkin-Sinn (Cherry Hill, NJ) and methyl atropine nitrate from Sigma Chemical Co. (St Louis, MO).
Rats were trained to intravenously self-administer cocaine, heroin, or cocaine/heroin combinations initially under a fixed ratio 1 (FR1) schedule of reinforcement, where one response on the lever was sufficient to deliver a drug infusion during daily sessions using a within-session dose-intake procedure (Martin et al, 1996). This procedure involved sessions that consisted of three 60-min trials where three different doses of the drug were made available for each of the three 60-min trials. A priming infusion of the dose that was available was given at the beginning of each of the three trials. The trials were separated by either 10- (microdialysis experiment) or 20- (isobolographic experiment) min time out (TO). The response requirement was raised to a final ratio of 2. Experimental chambers contained a house light, a stimulus light, response lever, and a tone source. The operant chambers were housed in sound- and light-attenuating cubicles and daily self-administration sessions were conducted 5 days/week. Each drug infusion was paired with a 20-s tone and light stimulus and the response lever was retracted during the TO periods and at the end of the session.
An evaluation of the interactions between self-administered cocaine and heroin and their combination was accomplished using a previously reported isobolographic procedure (Tallarida et al, 1997). Isobolographic analysis is a method that examines interactions between two drugs that produce monotonic dose-dependent effects and have similar maximal effects. However, the self-administration of cocaine and heroin have different maximal effects and both dose-intake curves are bitonic functions that have inverted U-shapes. For these reasons a single effect level was chosen that was achieved by both drugs for the analysis (15 infusions). Six male, Fischer 344 rats were trained to self-administer infusions of cocaine in combination with heroin in 4-h sessions using a within-session dosing procedure with exposure to three doses for 60 min each as described above for 5 days/week. The doses used for training were 166.5 μg/infusion of cocaine in combination with 4.5 μg/infusion of heroin, 333 μg/infusion of cocaine in combination with 9 μg/infusion of heroin, and 666 μg/infusion of cocaine in combination with 18 μg/infusion of heroin. These doses were chosen because 333 μg/infusion of cocaine and 9 μg/infusion of heroin maintain similar rates of self-administration (Martin et al, 1998). The dose was altered by varying the time of operation of the infusion pump and an increasing dose order of presentation was used. The initial response requirement was an FR1, which was raised to 10 over several sessions. When stable responding was obtained at FR10, saline and different cocaine/heroin dose combinations were substituted for the training doses on Tuesdays and Fridays if the behavior on the preceding sessions did not vary by more than 10% from the overall mean. The dose range of cocaine/heroin was sequentially lowered to 41.6/1.1, 83.3/2.3, and 166.5/4.5 μg/infusion, then to 10.4/0.3, 20.8/0.6, and 41.6/1.1 μg/infusion, and finally to 5.2/0.1, 10.4/0.3, and 20.8/0.6 μg/infusion to generate the remaining portion of the descending and ascending limbs of the dose-effect curve for the combination.
After the cocaine/heroin combination dose-effect curve was obtained, the same doses of cocaine and heroin that were used in the combinations were systematically substituted alone for the training doses of the combination. The rats were maintained at the training doses of each drug or combination and various doses for each drug were substituted for the training doses on Tuesdays or Thursdays, if the behavior on the preceding sessions did not vary by more than 10% from the overall mean. The animals were exposed to all dose ranges of cocaine, heroin, and speedball with a maximum of two alternate dose ranges per week, with saline extinction evaluated a minimum of twice. The dose range within a session was chosen such that there was one overlapping dose with previous sessions and the data from these overlapping doses were averaged together. In this manner, values for self-administered drug intake were obtained at various doses until both the ascending and descending limbs of the dose-intake relationship were determined for cocaine, heroin, and the cocaine/heroin combinations for each of the six rats.
In all, 17 rats were trained to intravenously self-administer cocaine (666, 333, and 166.5 μg/infusion; N=5), heroin (18, 9, and 4.5 μg/infusion; N=6), or cocaine/heroin combinations (cocaine/heroin -- 666/18, 333/9, and 166.5/4.5 μg/infusion; N=6) using an FR2 schedule and a within-session dose-intake procedure as outlined above. Self-administration sessions were 3½ in duration 5 days/week with 60 min access to each dose and a descending order of drug presentation with a 10-min TO between doses. When stable patterns of self-administration were obtained (the mean number of infusions delivered for each dose of cocaine did not vary by more than 10% of the mean), the doses of the drugs were decreased by 50% sequentially over the next 10 sessions until a range of doses was found for which the rats would only consistently self-administer the highest of the three available doses of cocaine (166.5, 83.3, and 41.6 μg/infusion; heroin -- 4.5, 2.3, and 1.1 μg/infusion; cocaine/heroin -- 166.5/4.5, 83.3/2.3, and 41.6/1.1 μg/infusion).
When stable patterns of FR2 self-administration were obtained at these low doses, the microdialysis session was initiated. Microdialysis probes were inserted through the previously implanted guide cannula approximately 18 h prior to the self-administration session during which microdialysis was to occur. The inlet tubing to the microdialysis probe was connected to a syringe containing artificial cerebrospinal fluid and one channel of a dual channel fluid swivel (Instech Laboratories, Plymouth Meeting, PA), while the other channel was used for intravenous cocaine infusions. Artificial cerebrospinal fluid (aCSF; 145 mM NaCl, 1.2 mM CaCl, 2.8 mM KCl, 1.2 mM MgCl, 5.4 mM D-glucose, and 1.25 mM NaHPO at a pH of 7.2) was perfused at a rate of 0.5 μl/min.
Microdialysis samples were collected into microcentrifuge tubes from the free end of the outlet tubing at 10-min intervals, 30-min pre-session, during the 210-min session, and 60-min post-session, and the samples immediately frozen on dry ice and stored at -70°C until analysis.
DA was measured in a 1.0 μl aliquot of each dialysate using high-pressure liquid chromatography with electrochemical detection (HPLC-EC) consisting of a syringe pump (model LC-260D; ISCO, Lincoln, NE) with an air-actuated injection valve (model ACI4UW; Valco) and a 1.0 μl sample loop, a microbore column (Spherisorb, ODS2, 5.0 μm, 0.5 mm i.d. × 100 mm), a dual glassy carbon working electrode (model PM; EG&G Princeton Applied Research, Princeton, NJ), a reference electrode (RE-1; Bioanalytical Systems Inc., W. Lafayette, IN), and an EC detector (model 400; EG&G Princeton Applied Research), with the applied potential set at +700 mV as referenced to Ag/AgC1. The mobile phase consisted of 20 mM citric acid, 46 mM NaHPO, 0.25 mM EDTA, 0.7 mM 1-decanesulfonic acid, 10 mM triethylamine, and 21% methanol (v/v), pH 5.4 (Hemby et al, 1997, 1999), and the flow rate of 15 μl/min resulted in a retention time of 5 min for DA. DA was quantified by comparing samples with standards of known concentration and the limit of detection was 0.5 fmol, which corresponded to a concentration of 0.5 nM.
Cocaine was measured in a 0.5 μl aliquot of each dialysate sample using HPLC and UV detection. The HPLC consisted of an SSI pump (model 222D; Scientific Systems, Inc., State College, PA), a Rheodyne injection valve (model 7520) with a 0.5 μl sample loop, a Spherisorb microbore column (0.5 × 100 mm, 3 μm C), and an LDC analytical variable wavelength detector (model 3200). The concentration of cocaine in the microdialysates was measured using UV absorbance at a wavelength of 235 nm. The mobile phase consisted of 50 mM NaHPO, 10 mM triethylamine, 0.1 mM EDTA, 22% acetonitrile, and 15% methanol, with pH adjusted to 5.6 and flow rate of 15 μl/min, that resulted in a retention time of 8 min (Hemby et al, 1997, 1999). The detection limit for cocaine was 100 fmol, which corresponded to 0.2 μM. Concentrations of cocaine in the dialysate were determined by comparing with known concentrations of standards.
Animals were killed by decapitation under pentobarbital anesthesia at the end of the microdialysis session and the brains were removed and frozen at -80°C. Frozen brains were warmed to -20°C and sections (20 μm) were taken at the cannula track and placement verified with microscopy following fixation and staining (Klüver and Barrera, 1953).
For isobolographic analysis, the ascending limb of the dose-effect curve was used since the descending limb likely results from multiple effects of the drug (Tallarida et al, 1997). Since there were differences in the maxima of the dose-effect curves for cocaine and heroin, the dose of drug that resulted in the self-administration of 15 infusions/hourly component was determined for cocaine and heroin alone or in combination (A) using a nonlinear curve fit of the ascending limb of the dose-effect curve for each individual animal using Prism software (GraphPad, San Diego, CA). Isobolograms were generated from the A values for cocaine and heroin alone and in combination. The interaction between cocaine and heroin was evaluated for synergism according to previously published methods by calculating the theoretical additive dose combination that would result in 15 infusions (Z) and comparing this value to the experimental value (Z) as described previously (Tallarida et al, 1997).
Microdialysis data were analyzed using a two-way ANOVA with drug (cocaine, heroin, or cocaine/heroin combinations) and dose component serving as the independent variables and either % of baseline or dialysate concentrations of [DA] as the dependent measure followed by post hoc analysis of differences between means using Bonferroni t-tests for multiple comparisons. Cocaine concentrations were analyzed by two-way ANOVA with drug (cocaine/heroin combination or cocaine) and session dose component serving as the independent variables and [Coc] as the dependent measure, followed by post hoc analysis of differences between means using Bonferroni t-tests for multiple comparisons. The ratios of [DA]/cocaine concentration were analyzed by one-way ANOVA.