0022-3565/99/2881-0274$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics JPET 288:274 –280, 1999
Vol. 288, No. 1 Printed in U.S.A.
Synergistic Elevations in Nucleus Accumbens Extracellular Dopamine Concentrations during Self-Administration of Cocaine/Heroin Combinations (Speedball) in Rats1 SCOTT E. HEMBY, CONCHITA CO, STEVEN I. DWORKIN and JAMES E. SMITH Center for the Neurobiological Investigation of Drug Abuse, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina Accepted for publication August 13, 1998
This paper is available online at http://www.jpet.org
ABSTRACT The abuse of cocaine/opiate combinations (speedball) represents a growing trend in illicit drug use. Delineation of neurobiological substrates mediating the reinforcing effects of the combination may increase our knowledge of reinforcement mechanisms and provide useful new information for the development of pharmacotherapies. Several studies suggest dopaminergic innervations of the nucleus accumbens (NAc) have a central role in the brain processes underlying drug reinforcement. The present study was undertaken to determine the relationship between the self-administration of cocaine/heroin combinations and NAc extracellular dopamine concentrations ([DA]e) using in vivo microdialysis and microbore high-pressure liquid chromatography. Rats were assigned randomly to one of three groups to self-administer i.v. cocaine (125, 250, and 500 mg/infusion; n 5 5), heroin (4.5, 9, and 18 mg/infusion; n 5 5), or cocaine/heroin combinations (125/4.5; 250/9, and 500/18 mg/
Users of cocaine and opiate combinations (termed “speedball”) represent a growing subset of the drug abuse population (Greberman and Wada, 1994). In addition to the illicit use of cocaine/heroin combinations, several studies report significant use of cocaine by patients in methadone and levoalpha-acetylmethadol (LAAM) maintenance treatment programs (Dunteman et al., 1992; Schottenfeld et al., 1997). Despite the prevalence of this problem, the underlying neurobiological substrates mediating the effects of speedball have been understudied. Several hypotheses have been set forth to explain the combined use of these substances, including 1) enhancement of the positive effects of either drug, 2) a reduction in the magnitude or duration of undesired side effects; 3) production of a positive feeling or state not availReceived for publication May 14, 1998. 1 This research was supported in part by the U.S. Public Health Service Research Grants DA-06634, DA-00114, and DA-03628. Parts of this study were presented at the College on Problems of Drug Dependence, 1995.
infusion; n 5 4) under a fixed ratio (FR) 10: 20-s time-out schedule of reinforcement/multicomponent dosing session. After stable rates of responding were engendered and maintained, microdialysis samples were collected in 10-min intervals during the self-administration session. Self-administration of cocaine/heroin combinations produced synergisitic elevations in NAc [DA]e (1000% baseline) compared with cocaine (400% baseline) and heroin (not significantly different from baseline levels). Neither the number of infusions nor the interinfusion intervals was significantly different between the groups across the self-administration session. Moreover, cocaine concentrations were not significantly different between the cocaine and cocaine/heroin groups. These results demonstrate that heroin interacts with cocaine to produce synergistic elevations in [DA]e, providing a neurochemical basis for understanding the abuse liability of cocaine/opiate combinations.
able with either drug alone, or 4) nonadditive effects even though the drugs are administered concurrently (Kosten et al., 1987; Foltin and Fischman, 1992; Hemby et al., 1996). The hypothesis of enhanced euphorigenic effects is paralleled in preclinical studies, demonstrating that cocaine and heroin potentiate the reinforcing effects of one another in the selfadministration paradigm (Mattox et al., 1997; Rowlett and Woolverton 1997). The question remains as to the neurobiological substrates mediating the reinforcing effects of cocaine/heroin combinations. The mesolimbic dopamine system is a critical substrate for the reinforcing effects of drugs of abuse (Wise and Bozarth, 1987). Administration of abused drugs appears to activate this pathway and stimulate dopamine neurotransmission in the nucleus accumbens (NAc) in humans, nonhuman primates, and rodents (Porrino, 1993; Lyons et al., 1996; Volkow et al., 1997), an effect associated with the abuse liability of these substances (Koob and Bloom, 1988). However, a growing body of literature suggests that the reinforcing effects of
ABBREVIATIONS: [DA]e, extracellular dopamine concentrations; NAc, nucleus accumbens; [COC], cocaine concentrations; FR, fixed ratio; ANOVA, analysis of variance; TO, time-out. 274
1999
Cocaine/Heroin Combinations and Neurochemical Synergism
cocaine are mediated by dopamine, whereas opiate receptors mediate the reinforcing effects of heroin and morphine (see for review, Koob and Bloom, 1988; Hemby et al., 1997b). For example, several laboratories have demonstrated that NAc extracellular dopamine concentration ([DA]e) is elevated during cocaine self-administration sessions in rodents as measured by in vivo microdialysis (Pettit and Justice, 1989; 1991; Wise et al., 1995b; Hemby et al., 1997a). The in vivo neurochemical data are complemented by numerous studies demonstrating that dopamine receptor antagonists increase responding maintained by high-unit doses of cocaine under fixed ratio (FR) schedules of reinforcement, effects that are interpreted generally as an attenuation of the reinforcing effects of the drug. In contrast, administration of dopamine antagonists does not affect heroin self-administration (see for review, Hemby et al., 1997b). The lack of direct dopaminergic involvement in heroin self-administration is complimented by a recent study demonstrating that NAc [DA]e was not elevated during heroin self-administration sessions (Hemby et al., 1995). Summarily, these studies indicate that cocaine and heroin self-administration are mediated by dopaminedependent and dopamine-independent mechanisms, respectively. Interestingly, Brown et al. (1991) have reported that acute experimenter-administered combinations of cocaine and buprenorphine (opiate receptor mixed agonist/antagonist) produced synergistic elevations in NAc [DA]e. These results suggest that coadministration of cocaine and heroin may produce similar effects, although the involvement of NAc dopamine in the reinforcing effects of the cocaine/heroin combinations has not been determined to date. The suggested role of dopamine neurons in drug reinforcement, combined with the reported increase in euphorigenic effects of cocaine/heroin combinations reported by humans suggests involvement of NAc [DA]e in cocaine/heroin selfadministration. The authors hypothesize that the potentiated euphorigenic effects in humans and the corresponding potentiation of reinforcing effects in animal models are based on an augmented neurochemical response in brain pathways underlying reinforcement processes. To this end, the effect of i.v. self-administered cocaine, heroin, and cocaine/heroin combinations were examined in [DA]e and cocaine concentrations ([COC]) in the NAc of the rat using in vivo microdialysis.
Materials and Methods Subjects and Surgical Procedures. Male Fisher F-344 rats (90 –150 days; 275–350 g; SASCO, Lincoln, NE) were housed individually in acrylic cages in a temperature-controlled vivarium on a 12-h reversed light/dark cycle (lights on: 5:00 PM.) with food and water available ad libitum, except during experimental sessions. Self-administrations occurred during the dark phase of the cycle. Rats were pretreated with atropine sulfate (10 mg/kg, i.p.), and anesthesia was induced by administration of sodium pentobarbital (Nembutal, 40 mg/kg, i.p.). While anesthetized, rats were implanted with chronic indwelling venous catheters followed by implantation of guide cannulas, as described previously (Hemby et al., 1995; 1997a). The catheter was implanted into the right jugular vein to extend to the right atrium and was anchored to surrounding muscle. The opposite end of the catheter was guided s.c. to the back and threaded through a plastic oval backplate residing above the scapulae. The skin was sutured over the backplate. Two Teflon screws attached the backplate to a shoulder harness, which was attached to a spring
275
leash. The catheter was threaded through the shoulder harness and spring leash and was connected to a single fluid channel swivel. Following catheter implantation, rats were secured in a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA) and implanted unilaterally with 20-gauge (Plastics One Inc., Roanoke, VA) stainless steel guide cannulas, with the side of placement counter-balanced within groups. Cannula tips were aimed for the dorsal surface of the NAc (19.5 mm from l, 61.3 mm lateral from the midline, and 25.0 mm ventral from dura; Ko¨nig and Klippel, 1974). Guide cannulas were secured with skull screws and dental acrylic cement; obturators (28 gauge; Plastics One Inc.), cut flush with the bottom of the cannulas, and inserted to prevent blockage. Penicillin G procaine (75,000 U/0.25 ml, i.m.) was administered immediately after surgery. After surgery, rats were transferred immediately to their respective home cages where they received hourly infusions of heparinized saline (1.7 U/ml, 200 ml/h) to maintain functional catheters. Infusions of methohexital (100 ml, 10 mg/kg, i.v.) were administered as needed to assess catheter patency. The health of the rats was monitored daily by the experimenter and weekly by institutional veterinarians according to the guidelines issued by the Bowman Gray Animal Care and Use Committee and by the National Institutes of Health. Self-Administration. For self-administration sessions, rats were transferred to operant-conditioning chambers that were enclosed in sound-attenuated chambers containing an exhaust fan, an 8-ohm speaker, a tone source, a house light, and a 20-ml syringe pump attached to the outside. Extraneous noise was masked by the exhaust fan and by white noise delivered continually through the speaker. The front panel of the operant chamber contained a fixed lever centered between the side panels and positioned 2.5 cm above the floor; a cue light was located 6 cm above the lever. Levers required approximately 0.25 N to operate, and the cue light was covered with red translucent cover. A counterbalanced arm containing a single-channel liquid swivel was located 8.5 cm above the chamber and attached to the outside of the front panel. IBM-compatible computers were used for session programming and data collection (Med Associates, Inc., East Farfield, VT). Rats were assigned randomly to groups to self-administer cocaine, heroin, or cocaine/heroin combinations. Responding was engendered under a FR 1: time-out (TO) 20-s schedule of three 1-h components. The ratio was gradually increased to 10. Subjects were allowed to self-administer cocaine i.v. (125, 250, and 500 mg/infusion; n 5 5), heroin (4.5, 9, and 18 mg/infusion; n 5 5), or cocaine/heroin combinations (cocaine/heroin: 125/4.5, 250/9.0, and 500/18 mg/infusion; n 5 4). Each dose was available during a different component, and doses were presented in ascending order. The infusion volume for the first component was 50 ml infused over 1.4 s, and the volumes for the successive components were 100 ml for component two (infused over 2.8 s) and 200 ml for component three (infused over 5.6 s). Before each component, a 10-min blackout was followed by a priming infusion of the dose to be administered in the succeeding component. After an additional 10-min blackout period, the lever was activated, and the cue light above the lever was illuminated. Upon completion of the response requirement, a drug infusion was delivered, the lever light extinguished, a tone was generated, and the house light was illuminated. During the 20-s TO after the infusion, responses on the lever were recorded but had no scheduled consequence. A minimum of 10 days of stable responding (less than 10% variation in the number of infusions) at FR10 in all components was required before microdialysis procedures were initiated. In Vivo Microdialysis and High-Pressure Liquid Chromatography (HPLC) Procedures. The microdialysis procedures and probe construction have been described previously (Hemby et al., 1995; 1997a). Briefly, two pieces of fused silica were inserted into 6 mm of regenerated cellulose membrane (250 mm o.d., 5000 mol wt cutoff; Spectra/Por, Los Angeles, CA). For the present experiment, the active portion of the probes was 2 mm, defined by the distance
276
Hemby et al.
between the ends of the fused silica. The inlet silica line was connected to a syringe filled with artificial cerebrospinal fluid, which was used as the perfusion medium. This artificial cerebrospinal fluid perfusate consisted of 145 mM NaCl, 1.2 mM CaCl2, 2.8 mM KCl, 1.2 mM MgCl2, 5.4 mM D-glucose, and 1.25 mM NaH2PO4 (pH 5 7.2). Approximately 15 h before the microdialysis session, microdialysis probes were inserted through previously implanted guide cannulas. The perfusion flow rate was 0.6 ml/min. Dialysate samples were collected in microcentrifuge tubes from the free end of the outlet silica line in 10-min intervals, immediately frozen on dry ice, and later stored at 280°C until analysis. Three microliters of the sample was assayed for dopamine using microbore HPLC with electrochemical detection, while the remaining 3 ml was assayed for cocaine using microbore HPLC with ultraviolet detection. The HPLC for dopamine analysis consisted of a syringe pump (model LC-260D; Isco, Lincoln, NE), an air-actuated injection valve (model ACI4UW; Valco Instruments, Houston, TX), with a 1.0-ml sample loop, a Spherisorb microbore column (0.5 mm i.d. 3 100 mm, 5 mm C18 silica), a dual glassy carbon working electrode (model PM; EG&G Princeton Applied Research, Princeton, NJ), a reference electrode (RE-1; Bioanalytical Systems Inc., West Lafayette, IN), and an electrochemical detector (model 400; EG&G Princeton Applied Research). Columns were packed in the laboratory with silica purchased from Phase Separations, Inc. (Norwalk, CT). The applied potential was 1700 mV as referenced to Ag/AgCl. The mobile phase consisted of 27.2 mM sodium phosphate-monobasic, 10% v/v methanol, 4.9 mM triethylamine, 13 mM disodium-EDTA, and 0.99 mM sodium octyl sulfate, with the pH adjusted to 5.75 with 0.1 N phosphoric acid. The flow rate of the mobile phase was 10 to 12 ml/min, and the detection limit for dopamine was 100 pM. Quantification of dopamine was achieved by comparing samples with standards of known concentration. The HPLC for cocaine analysis consisted of a HPLC pump (model 222D; Scientific Systems, Inc., State College, PA) adapted for microbore use, a Rheodyne injection valve (model 7520; Rohnert Park, CA) with a 0.5-ml sample loop, a Spherisorb microbore column (0.5 mm i.d. 3 100 mm, 3 mm C18), and an analytical variable wavelength detector (model 3200, Thermo Instruments, Inc., Riveria Beach, FL) customized for microbore chromatography. The wavelength was 235 nm, and the absorbance was 0.0001, full scale. The mobile phase consisted of 50 mM sodium phosphate monobasic, 10 mM triethylamine, 17% acetonitrile, and 10% methanol, with the pH adjusted to 5.6 with a flow rate of 25 ml/min. The detection limit for cocaine was 200 nM. Quantification of cocaine was achieved by comparing samples with standards of known concentration. Histology. Verification of guide cannula tracts and assessment of gliosis at the probe site were determined in all subjects. Brains were removed, and coronal stions (20 mm) were taken from 100 mm rostral to 100 mm caudal to the cannula tract. Sections were fixed in 4% paraformaldehyde and stained with cresylecht violet. Probe placements were verified by light microscopy in a “blind” manner to reduce experimenter bias of the results. Probe placements were within the NAc for all subjects in this study (Fig. 1). Drugs. Cocaine HCl and heroin HCl were kindly provided by the National Institute on Drug Abuse. Atropine sulfate was purchased from Sigma Chemical Co. (St. Louis, MO), sodium pentobarbital was purchased from Abbott Laboratories (North Chicago, IL), penicillin G procaine was purchased from Butler Company (Columbus, OH), and methohexital was purchased from Eli Lilly and Company (Indianapolis, IN). Cocaine doses are expressed as the weight of the salt, whereas heroin doses are expressed as the weight of the free base. Cocaine and heroin were dissolved in heparinized 0.9% saline. Data Analysis. Behavioral and microdialysis data were analyzed using a two-way repeated measures analysis of variance (ANOVA) with group and dose as the fixed effects and time as the repeated measure. For behavioral analysis, dependent measures included the number of infusions, interinfusion interval, postreinforcement pause (time elapsed between the end of the 20-s TO and the first response
Vol. 288
Fig. 1. Composite representation of microdialysis probe placements in the NAc for the cocaine (top), heroin (middle), and cocaine/heroin (bottom) groups. Bars indicate the location of the active area of the individual probes and are drawn to scale (2 mm 3 200 mm). The area of the NAc is identified by the dashed line. Sections are 9650 mm rostral to l according to the atlas of Ko¨nig and Klippel (1974).
of the next ratio), latency to the first reinforcer of each component, and ratio run time (time elapsed between the first and last response of the ratio). [DA]e and [COC] were the dependent measures for the microdialysis data. Post hoc analyses were conducted as needed using Fisher’s least significant difference test. The null hypothesis was rejected when P , .05.
Results Baseline [DA]e A schematic representation of probe placements from subjects included in this experiment is shown in Fig. 1. Histological examination revealed that probe placements for all subjects were located in the NAc, medial to the anterior commissure. Baseline values of dopamine were assessed in each subject by collecting three samples before the beginning of the self-administration session. All subsequent samples were expressed as the mean (6S.E.M.) of the percent variation of the mean baseline value. There were no statistically significant differences in baseline [DA]e between the groups (cocaine: 7.4 6 2.5 nM; heroin: 7.7 6 1.1 nM; cocaine/heroin: 9.3 6 1.7 nM). Self-Administration Behavioral Data. The selected doses of cocaine, heroin, and cocaine/heroin combinations engendered and maintained rates and patterns of responding typically observed
1999
Cocaine/Heroin Combinations and Neurochemical Synergism
Fig. 2. Individual self-administration records for each group during the self-administration test session. Each tic mark indicates a drug infusion after completion of 10 lever presses. Each 1-h component was preceded by a 10-min blackout period, an infusion of the dose available in the subsequent session, and another 10-min blackout period (vertical gray bars).
under FR schedules of reinforcement (Fig. 2; Hemby et al., 1995, 1996). The dose-effect curves (number of infusions and interinfusion intervals) for the groups self-administering cocaine and heroin were not significantly different from the group self-administering cocaine/heroin combinations (Fig. 3). However, for both measures, there was significant main
Fig. 3. Top panel, mean number (6S.E.M.) of infusions per component for the self-administration test session (first component, hatched column; second, open column; third, filled column). There was no significant difference in the number of infusions between groups for the session. However, there were significant dose-dependent effects characterized as decreases in the number of infusions with successive components for all groups (cocaine: F(2,12)57.32, P , .009; heroin: F(2,12)54.10, P , .045; cocaine/heroin: F(2,9)523.64, P , .001). Post hoc analyses revealed the following significant differences in the number of infusions for cocaine (component 1 . 2, 3), heroin (component 1. 3) and cocaine/heroin combinations (component 1. 2, 3). Bottom panel, mean interinfusion interval (6S.E.M) per component for the self-administration test session (first component, hatched column; second, open column; third, filled column). There was a main effect of drug on the interinfusion interval between components of the self-administration session [F(2,22)536.34; P , .001]. Significant dose-dependent increases in the interinfusion intervals were observed in the cocaine [F(2,12)516.90; P , .001] and cocaine/heroin groups [f(2,9)5122.95; P , .001]. Post hoc analyses revealed significant differences for cocaine (component 1 , 2,3) and cocaine/heroin combinations (component 1 , 2,3).
277
effect of dose [infusions: F(2,22)526.13, P , .001; interinfusion interval: F(2,22)536.34, P , .001]. There was a significant dose-dependent effect on the number of infusions for cocaine [F(2,12)57.32; P , .009], heroin [F(2,12)54.10; P , .045], and cocaine/heroin combinations [F(2,9)524.39; P , .001]. The number of infusions was inversely proportional to the dose self-administered in all groups, characteristic of the descending limb of the dose-effect function. In contrast, interinfusion intervals were related linearly to the self-administered dose(s) for the cocaine [F(2,12)516.9; P , .001] and cocaine/heroin groups [F(2,9)5122.95; P , .001]. For postreinforcement pause, timed from the end of the TO period following each infusion until the first response of the subsequent ratio, there was a significant main effect of drug [F(2,10)55.89, P , .025] and dose [F(2,20)591.51, P , .001] as well as a significant drug 3 dose interaction [F(4,20)56.68, P , .0015]. Postreinforcement pauses were linearly related to dose(s) for the cocaine (109.1 6 34.9, 264.3 6 24.8, 543.3 6 17.0 s for 125, 250 and 500 mg/infusion; [F(2,12)568.56, P , .001]) and cocaine/heroin groups (144.8 6 16.0, 397.0 6 17.3, 636.7 6 35.4 s for 125/4.5; 250/9.0 and 500/18.0 mg/infusion; [F(2,9)5100.32, P , .001], indicating that the initiation of responding after each infusion was dependent on the dose infused. In addition, there was a significant main effect of dose on latency to the first reinforcer [F(2,22)53.55, P , .047] where the cocaine group exhibited the only significant dose-dependent effect [F(2,12)54.89, P , .03]. There was no significant effect on ratio run-time, measured as the elapsed time between the first and last response of the ratio, in any of the groups tested. These data indicate that responding, once initiated, was not differentially altered by the three doses investigated in each group. Microdialysis Data. NAc [DA]e were significantly different between all groups (Fig. 4, top panel) for the self-administration session [F(2,330)516.61, P , .001]. In addition, there was a significant drug X dose interaction[F(60,330)511.3, P , .001]. Post hoc analyses revealed significant differences between all groups; however, there were no significant differences in [DA]e between doses of the same drug. Self-administration of all of the cocaine/heroin combinations tested resulted in a greater than 2-fold increase in NAc [DA]e compared with cocaine alone and a greater than additive effect for the individual doses of cocaine and heroin combined. Both cocaine/heroin combinations and cocaine produced consistent large elevations in [DA]e compared with no change for heroin. NAc [DA]e were elevated approximately 1000% of baseline in the cocaine/heroin combination group and approximately 400% of baseline in the cocaine group, whereas [DA]e were not significantly different from baseline levels during the heroin self-administration session. Elevations observed in the cocaine/heroin and cocaine groups were sustained throughout the self-administration session and there were no significant differences between components for either group. During the hour following the end of the self-administration session, [DA]e declined from 1000% to approximately 200% for the cocaine/heroin group and from 400% to approximately 100% of baseline for the cocaine group. In both groups receiving cocaine, [COC] were detectable following the first priming infusion of the session and remained detectable throughout the session, with successively decreasing [COC] in the postsession samples (Fig. 4, bottom panel). There was no significant difference in [COC] between
278
Hemby et al.
Vol. 288
Fig. 4. Effects of cocaine (f), heroin (F), and cocaine/heroin combinations (Œ) on [DA]e (top panel) and [COC] (bottom panel) in the NAc during self-administration sessions. Data are expressed as mean percentage of baseline [DA]e 6 S.E.M. and mean [COC] 6 S.E.M. Samples were collected in 10-min intervals. The first three filled circles in the top panel represent baseline [DA]e samples collected before the self-administration session. Each 1-h component was preceded by a 10-min blackout period, an infusion of the dose available in the subsequent session, and another 10-min blackout period (vertical gray bars). After collection of the sixth sample in the final component, the response lever was retracted, and the response lever light was extinguished. Samples were collected for an additional 60 min (open symbols). Note the fifth and sixth [COC] samples for the cocaine/heroin group were below the limit of detection. There was a significant main effect of Drug on [DA]e for the session F(2,330) 5 16.61, P 5 .0005, and a significant interaction of drug over time F(60,330)511.3, P , .0001. Post hoc analyses revealed significant differences between all groups (P , .05). However, there was no significant difference in [COC] between the two groups during or after the self-administration session.
the cocaine and cocaine/heroin groups during the test session, suggesting that the synergistic neurochemical response to the cocaine/heroin combination was not the result of altered brain levels of cocaine.
Discussion Dopaminergic projections to the NAc are known to play an important role in the reinforcing effects of abused drugs (Wise and Bozarth, 1987). The present study was undertaken to compare in vivo changes in NAc [DA]e during self-administration sessions of cocaine, heroin, and cocaine/heroin combinations. Self-administration of cocaine and cocaine/heroin combinations produced substantial elevations in NAc [DA]e during the self-administration session that were paralleled by similar elevations in [COC] in the groups self-administering cocaine and cocaine/heroin combinations. Differences in [DA]e between the cocaine and cocaine/heroin groups can not be attributed to alterations in the bioavailability of cocaine since the [COC] were not significantly different between the two groups. This point is supported by a previous study in humans which reported that the disposition of cocaine or morphine was not affected by the presence of the other drug (Foltin and Fishman, 1992). To our knowledge, this is the first published report on in vivo neurochemical changes during the self-administration of cocaine/opiate combinations and the first report of synergisitic elevations in NAc [DA]e for self-administered illicit drug combinations. The results from
the cocaine self-administration session confirm previous studies (Pettit and Justice, 1989, 1991; Wise et al., 1995b; Hemby et al., 1997a) and extend them by demonstrating elevations in [DA]e for multiple doses of the drug presented in the same session. In contrast, heroin self-administration failed to significantly alter NAc [DA]e at the doses tested, in confirmation of a previous study (Hemby et al., 1995). It should be noted that the selected doses of heroin were shown to reliably engender and maintain responding under the present experimental conditions. These results obtained in the present study are inconsistent with the hypothesis proposed by Wise and Bozarth (1987). Mounting evidence suggests the reinforcing effects of opiates are mediated by opiate receptors postsynaptic to dopamine terminals in the NAc (Van Ree and Ramsey, 1987; Hemby et al., 1997b). The present results demonstrate that self-administered heroin and cocaine interact in a synergistic manner to elevate NAc [DA]e. The neurochemical data from cocaine and cocaine/heroin combination self-administration sessions support a substantial body of pharmacological data indicating a role for dopamine in drug reinforcement (see for review, Hemby et al., 1997b). Systemic and central administration of selective dopamine receptor antagonists increase rates of cocaine selfadministration maintained under FR schedules of reinforcement and decrease break points under progressive ratio schedules, indicative of a decrease in the reinforcing effects of cocaine. Similarly, selective destruction of presynaptic dopamine terminals in the NAc produce extinction-like responding in rats trained to self-administer cocaine (Roberts et al., 1980; Pettit et al., 1984). The pharmacological relevance of the synergistic elevations in [DA]e observed in the present study is offered by a recent study in which pretreatment with the D2 receptor-selective antagonist eticlopride increased the self-administration of cocaine/heroin combinations (Hemby et al., 1996). Eticlopride exerted similar effects on cocaine self-administration but was not effective in altering heroin self-administration. In contrast, naltrexone pretreatment increased cocaine/heroin and heroin self-administration, but had no effect on cocaine self-administration. The increase in self-administration after antagonist administration is considered a compensatory response to the receptor antagonism, such that more drug is available to compete with the antagonist at the receptor. These data indicate that the self-administration of cocaine/heroin combinations is dependent on both dopamine and opiate receptor mechanisms. The lack of change in NAc [DA]e during the heroin selfadministration confirms a previous study from our laboratory (Hemby et al., 1995). This finding is supported by other studies demonstrating that pharmacological and neurochemical manipulations that alter the functional integrity of the mesolimbic dopamine pathway do not affect the acquisition or maintenance of opiate self-administration (Pettit et al., 1984, Gerrits and Van Ree, 1996). Furthermore, several studies have shown that heroin self-administration in rats is not altered by systemic (Ettenberg et al., 1982; Van Ree and Ramsey, 1987; Hemby et al., 1996) or intra-NAc (Van Ree and Ramsey, 1987) administration of dopamine receptor antagonists. The reinforcing effects of heroin appear to be mediated by opiate receptors in the NAc, inasmuch as intra-NAc administration of either opiate receptor antagonists (Koob et al., 1984; Corrigall and Vaccarino, 1988) or agents that pro-
1999
Cocaine/Heroin Combinations and Neurochemical Synergism
duce excitotoxic lesions (Zito et al., 1985) alter i.v. heroin self-administration in a manner consistent with this premise. Collectively, these results support the hypothesis that heroin self-administration is mediated in a dopamineindependent manner. In contrast to the present findings, one group has reported NAc [DA]e to be increased during heroin self-administration sessions (Kiyatkin et al., 1993; Wise et al., 1995a). However, the differences between these studies are probably due to a number of contributing factors, including analytical procedures, self-administration procedures, and behavioral histories of the rats (see for review, Hemby et al., 1997b). As previously discussed, a significant volume of pharmacological data support the present neurochemical data on heroin self-administration and indicate that opiate reinforcement is mediated in a dopamine-independent manner. In light of the contrasting effects of cocaine and heroin self-administration on [DA]e, self-administration of cocaine/ heroin combinations produced synergistic elevations in [DA]e. However, the question remains as to the functional significance of the observed elevation. The neurochemical effects could arguably be ascribed to the reinforcing effects of the combination or to increased general motor activity. Overt behavioral changes, such as increased motor activation or stereotypy, were not observed in the cocaine/heroin combination group compared with the cocaine and heroin groups. Similarly, there was no evidence of differences in measures of operant responding (responding during the time-out period after infusions, the amount of time required to complete the ratio, the latency to the first response after an infusion, etc.). Therefore, the elevated [DA]e observed during the cocaine/ heroin self-administration did not produce significant differences in the behavioral effects of cocaine or heroin alone. Under the present experimental procedures, combined doses of cocaine and heroin did not produce changes in the total number of infusions obtained or in the pattern of responding compared with the doses of cocaine and heroin alone. Previously, the authors and others have reported a leftward shift in the dose-effect function when cocaine and heroin were combined (Hemby et al., 1996; Rowlett and Woolverton, 1997), suggesting that the cocaine/heroin combinations were more potent than either drug alone. The difference in the present results and those published previously (Hemby et al., 1996) is probably due to the manner in which responding was engendered and/or the dose combinations chosen to be studied in the respective experiments. Experiments have been initiated to further test the hypothesis that relative reinforcing efficacy and/or potency is greater for the combination than for either drug alone (e.g., progressive ratio schedules and choice procedures). Due to the paucity of pharmacological data on cocaine/ heroin self-administration, the apparent mechanism by which heroin augments the dopaminergic response of cocaine remains unclear. The administration of cocaine decreases whereas morphine increases the firing rate of mesolimbic dopaminergic neurons (Matthews and German, 1984; Einhorn et al., 1988). Cocaine inhibits the reuptake of dopamine, resulting in increased extracellular concentrations (Harris and Baldessarini, 1973). In turn, elevated dopamine levels activate autoreceptors on the dopamine cell bodies, leading to hyperpolarization and decreased cell firing (Aghajanian and Bunney, 1974). In contrast, heroin activates m opiate receptors on g-aminobutyric acid (GABA) interneurons in the ven-
279
tral tegmental area, resulting in hyperpolarization of these neurons and a concomitant disinhibition of dopamine cell firing (Johnson and North, 1992). Increased dopaminergic cell firing results in increased uptake, a voltage-dependent process which occurs during the repolarization phase of the action potential (Rudnick and Clark, 1993). The synergistic effect of cocaine/heroin combinations on NAc [DA]e could result from the increased firing of dopamine neurons by opiates and the direct effect of cocaine on dopamine reuptake. However, the lack of significant increase in [DA]e during heroin self-administration alone suggests that the synergy is not additive and that the specific mechanism requires further investigation. The neurochemical synergy is significant as the magnitude of such elevations in [DA]e cannot be obtained with cocaine alone with the procedures used, without inducing significant seizure activity. Further studies are warranted to determine the physiological and pharmacological bases of the synergistic neurochemical effect of cocaine/ heroin self-administration. The present result that cocaine and heroin interact to produce a synergistic effect on a neurotransmitter relevant to reinforcement provides a neurochemical basis to support the hypothesis that the combination of cocaine and heroin produce an effect not available with either drug alone. The relationship of the synergisitic elevations in NAc [DA]e to alterations in the reinforcing efficacy/potency of the combination remains to be studied. The present data also suggest a neurochemical basis for the significant incidence of cocaine use among individuals in methadone or LAAM maintenance programs (Dunteman et al., 1992; Schottenfeld et al., 1997). The development of pharmacological adjuncts for substance abuse treatment is based primarily on a fundamental understanding of the neuropharmacological and neurochemical basis of the subjective and reinforcing effects. Improved pharmacological treatments for clinical intervention of cocaine/ heroin combination abuse may result from increased understanding of the neurobiological substrates mediating the behavioral effects of the drug combination. Acknowledgments
We appreciate the comments and suggestions of Charles O’Brien, Gary Aston-Jones, Sam Deadwyler, and Linda Porrino in the preparation of the manuscript. References Aghajanian GK and Bunney BS (1974) Pre- and post-synaptic feedback mechanisms in central dopaminergic neurons, in Frontiers Of Neurology And Neuroscience Research (Seeman P and Brown GM eds) pp 4 –11, University of Toronto Press, Toronto, Canada. Brown EE, Finlay JM, Wong JTF, Damsma G and Fibiger HC (1991) Behavioral and neurochemical interactions between cocaine and buprenorphine: Implications for the pharmacotherapy of cocaine abuse. J Pharmacol Exp Ther 256:119 –126. Corrigall WA and Vaccarino FJ (1988) Antagonist treatment in nucleus accumbens or periaqueductal grey affects heroin self-administration. Pharmacol Biochem Behav 30:443– 450. Dunteman GH, Condelli WS and Fairbank JA (1992) Predicting cocaine use among methadone patients: Analysis of findings from a national study. Hosp Commun Psychiatry 43:608 – 611. Einhorn LC, Johansen PA and White FJ (1988) Electrophysiological effects of cocaine in the mesoaccumbens dopamine system: studies in the ventral tegmental area. J Neurosci. 8:100 –112. Ettenberg, A, Pettit HO, Bloom FE and Koob GF (1982) Heroin and cocaine selfadministration in rats: mediation by separate neural systems. Psychopharmacology 78:204 –209. Foltin RW and Fischman MW (1992) The cardiovascular and subjective effects of intravenous cocaine and morphine combinations in humans. J Pharmacol Exp Ther 261:623– 632. Gerrits MA and Van Ree JM (1996) Effect of nucleus accumbens dopamine depletion on motivational aspects involved in initiation of cocaine and heroin selfadministration in rats. Brain Res 713:114 –124.
280
Hemby et al.
Greberman SB and Wada K (1994) Social and legal factors related drug abuse in the United States and Japan. Public Health Rep 109:731–737. Harris JE and Baldessarini RJ (1973) Uptake of (3H)-catecholamines by homogenates of rat corpus striatum and cerebral cortex: effects of amphetamine analogues. Neuropharmacology 12:669 – 679. Hemby SE, Co C, Koves TR, Smith JE and Dworkin SI (1997a) Differences in extracellular dopamine concentrations in the nucleus accumbens during responsedependent and response-independent cocaine administration in the rat. Psychopharmacology 133:7–16. Hemby SE, Dworkin SI and Johnson BA (1997b) Neuropharmacological basis of drug reinforcement, in Drug Addiction and Its Treatment: Nexus of Neuroscience and Behavior (Johnson BA and Roache JD eds) pp 137–169, Raven Press, New York. Hemby SE, Martin TJ, Co C, Dworkin SI and Smith JE (1995) The effects of intravenous heroin administration on extracellular nucleus accumbens dopamine concentrations as determined by in vivo microdialysis. J Pharmacol Exp Ther 273:591–598. Hemby SE, Smith JE and Dworkin SI (1996) The effects of eticlopride and naltrexone on cocaine, heroin, and cocaine/heroin combination self-administration. J Pharmacol Exp Ther 277:247–258. Johnson SW and North RA (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483– 488. Kiyatkin EA, Wise RA and Gratton A (1993) Drug- and behavior-associated changes in dopamine-related electrochemical signals during intravenous heroin selfadministration in rats. Synapse 14:60 –72. Ko¨nig JFR and Klippel RA (1974) The Rat Brain: A Stereotaxic Atlas Of The Forebrain And Lower Parts Of The Brainstem Kreiger Publishing, Huntington, NY. Koob GF and Bloom FE (1988) Cellular and molecular mechanisms of drug dependence. Science 242:715–723. Koob GF, Pettit HO, Ettenberg A and Bloom FE (1984) Effects of opiate antagonists and their quaternary derivatives on heroin self-administration in the rat. J Pharmacol Exp Ther 229:481– 486. Kosten TR, Rounsaville BJ and Kleber HD (1987) A 2.5-year follow-up of cocaine use among treated opioid addicts. Have our treatments helped? Arch Gen Psychiatry 44:281–284. Lyons D, Friedman DP, Nader MA and Porrino LJ (1996) Cocaine alters cerebral metabolism within the ventral striatum and limbic cortex of monkeys. J Neuroscience 16:1230 –1238. Matthews RT and German DC (1984) Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neurons by morphine. Neuroscience 11:617– 625. Mattox AJ, Thompson SS and Carroll ME (1997) Smoked heroin and cocaine base (speedball) combinations in rhesus monkeys. Exp Clinical Psychopharmacology 5:113–118. Pettit HO, Ettenberg A, Bloom FE and Koob GF (1984) Destruction of dopamine in
Vol. 288 the nucleus accumbens selectively attenuates cocaine but not heroin selfadministration in rats. Psychopharmacology 84:167–173. Pettit HO and Justice JB Jr (1989) Dopamine in the nucleus accumbens during cocaine self-administration as studied by in vivo microdialysis. Pharmacol Biochem Behav 34:899 –904. Pettit HO and Justice JB Jr (1991) Effect of dose on cocaine self-administration behavior and dopamine levels in the nucleus accumbens. Brain Res 539:94 –102. Porrino LJ (1993) Functional consequences of acute cocaine treatment depend on route of administration. Psychopharmacology 112:343–351. Roberts DCS, Koob GF, Klonoff P and Fibiger HC (1980) Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens. Pharmacol Biochem Behav 12:781–787. Rowlett JK and Woolverton WL (1997) Self-administration of cocaine and heroin combinations by rhesus monkeys responding under a progressive-ratio schedule. Psychopharmacology 133:363–371. Rudnick G and Clark J (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters. Biochim Biophys Acta 1144:249 –263. Schottenfeld RS, Pakes JR, Oliveto A, Ziedonis D and Kosten TR (1997) Buprenorphine vs methadone maintenance treatment for concurrent opioid dependence and cocaine abuse. Arch Gen Psychiatry 54:713–720. Van Ree JM and Ramsey N (1987) The dopamine hypothesis of opiate reward challenged. Eur J Pharmacol 134:239 –243. Volkow ND, Wang GJ, Fischman MW, Foltin RW, Fowler JS, Abumrad NN, Vitkun S, Logan J, Gatley SJ, Pappas N, Hitzemann R and Shea CE (1997) Relationship between subjective effects of cocaine and dopamine transporter occupancy. Nature (London) 386:827– 830. Wise RA and Bozarth MA (1987) A psychomotor stimulant theory of addiction. Psych Rev 94:469 – 492. Wise RA, Leone P, Rivest R and Leeb K (1995a) Elevations of nucleus accumbens dopamine and DOPAC levels during intravenous heroin self-administration. Synapse 21:140 –148. Wise RA, Newton P, Leeb K, Burnette B, Pocock D and Justice JB Jr (1995b) Fluctuations in nucleus accumbens dopamine concentration during intravenous cocaine self-administration in rats. Psychopharmacology 120:10 –20. Zito KA, Vickers G and Roberts DCS (1985) Disruption of cocaine and heroin self-administration following kainic acid lesions of the nucleus accumbens. Pharmacol Biochem Behav 23:1029 –1036.
Send reprint requests to: Dr. Scott E. Hemby, Yerkes Regional Primate Research Center, Department of Pharmacology, 95113 Rolling Research Center, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. E-mail:
[email protected]
