- Email: [email protected]
Cocaine reinforcement and extracellular dopamine overflow in rat nucleus accumbens: an in vivo microdialysis study
Brain Research, 498 (1989) 199-203
199
Elsevier BRES 23734
Cocaine reinforcement and extracellular dopamine overflow in rat nucleus accumbens: an in vivo microdialysis study Yasmin L. Hurd 1, Friedbert Weiss 2, George F. Koob 2, Nils-Erik And 1 and Urban Ungerstedt 1 IKarolinska Institute, Department of Pharmacology, S-104 Ol Stockholm (Sweden) and 2Scripps Clinic and Research Foundation, Department of Basic and Clinical Research, La Jolla, CA 92037 (U.S.A.)
(Accepted 13 June 1989) Key words: Cocaine self-administration; Dopamine release; Dopamine uptake; Microdialysis; Addiction
Addictive properties of cocaine have been suggested to be mediated by an interplay of depletion (craving) and re-elevation (reinforcement) of dopamine (DA) levels in limbic brain area. In this study, direct measurement of dopamine in the extracellular fluid of rats freely self-administering cocaine was evaluated using in vivo microdialysis. Acute cocaine administration was associated with enhanced accumulation of DA in the nucleus accumbens, correlated with enhanced locomotor activity. In contrast, the increased DA overflow observed in drug-naive animals was attenuated in animals self-administering cocaine who had previous regular repeated (9-day) exposure to the drug. The results suggest that the absolute amount of DA in the extracellular space is not the critical factor correlated with the self-administration behavior. Additionally, the results indicate that the reduced ability of cocaine to re-elevate DA to first-time drug use is not due to a reduction of DA in the tissue or reduced DA synthesis, but may instead be associated with alterations of release and reuptake processes. In an effort to understand the mechanism of drug addiction in man, the self-administration (SA) behavioral model has been frequently used for estimating the abuse liability of cocaine. In this behavioral model animals receive an intravenous (i.v.) injection of a drug after making a correct operant response 21"22. The development of a stable level of drug intake over time is generally used as an index of the drug's reinforcing properties. Results from experiments in which the SA behavior has been manipulated, by pharmacological agents 4"5 or selective neurotoxic brain lesions 13A4, suggest that the strong reward and dependence potential of cocaine is closely associated with enhanced activity in the dopamine ( D A ) terminals of limbic brain areas (primarily the nucleus accumbens (N.Acc)). While it is well established that cocaine influences D A transmission by enhancing vesicularly stored D A and/or by inhibiting the uptake of D A located in the synaptic microenvironment s'12"13"15, questions remain as to whether augmentation of D A in the
synaptic cleft is truly correlated to the reinforcing aspects of cocaine. To address some of these questions we have used the technique of in vivo microdialysis 2°,23~24, which allows the continuous evaluation of neurochemical changes occurring in restricted brain regions of freely moving animals. Specifically, in the present investigation, extracellular levels of D A , 3,4-dihydroxyphenylacetic acid ( D O P A C ) and homovanillic acid (HVA) were monitored in the N.Acc of rats responding to cocaine reinforcement during 3 h, limited-access, SA sessions. The experiments were designed to test the effect of cocaine in drug-naive rats ('acute') and in rats previously trained to self-administer cocaine ('repeated'). Tissue D A levels and estimates of D A synthesis were also assessed in an effort to establish the overall effect of cocaine on D A t r a n s m i s s i o n . Male S p r a g u e - D a w l e y rats (250-300 g) previously trained to lever-press for water reinforcement were surgically prepared with a chronic sialastic
Correspondence: Y.L. Hurd, Karolinska Institute, Department of Pharmacology, Box 60 400, S-104 01 Stockholm, Sweden.
0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
200 jugular catheter (to allow for later self-administration of cocaine) under halothane anaesthesia. During the operation a brain guide cannula (Carnegie Medicin AB, Stockholm, Sweden) was also implanted; coordinates A +1.7, L +1.3, and V - 0 . 5 mm (so that a microdialysis probe (membrane: 0.5 x 2 mm; Carnegie Medicin AB, Sweden) would extend 7.3 mm below the dura into the N.Acc). Four days following surgery the rats were allowed 3 h access every day to a metal lever mounted on the side wall of a square Plexiglas operant chamber. Every depression of the lever resulted in an i.v. injection of a 100 ~1 cocaine hydrochloride solution (0.75 mg/ kg/injection) dissolved in 0.9% saline and delivered over a period of 4 s. Animals were designated 'acute', if they had no previous SA experience (and minimal exposure to the operant apparatus before dialysis testing) or 'repeated', if they had been previously trained to self-administer cocaine (9-10 days) and had developed a stable intake of cocaine (less than 10% change from the mean total intake over 3 consecutive days). Two control groups were also studied, an 'acute' saline and a 'repeated' group ('repeated' cocaine-saline) which received saline on the day of testing. Since rats in the 'acute' saline or 'repeated' cocaine-saline group were not expected to self-administer saline, these animals were yoked to a trained animal self-administering cocaine at a stable response rate. Thus, each lever-press by the trained animal produced an injection of saline for the yoked animal. 'Acute' cocaine rats were also yoked since their response rate of injecting cocaine was unstable (great variability between rats) and not comparable to the amount of cocaine administered by the 'repeated' animals. On the test day, the microdialysis probe was slowly inserted into the N.Acc via the guide cannula. The probe was continuously perfused (2/~l/min) with an artificial cerebrospinal fluid solution consisting of (in mM): Na ÷ 155, K ÷ 2.9, Ca 2÷ 1.1, Mg 2÷ 0.83, C1132.76 and D-glucose 5.9; pH 7.4. Perfusate samples were collected every 5 min into 0.3 ml glass vials (containing 10/~1 of 0.1 M perchloric acid) situated in a CMA/100 microfraction collector (Carnegie Medicin AB). The samples were subsequently placed into a refrigerated autoinjector (CMA/200, Carnegie Medicin AB) that was attached to a HPLC system with electrochemical detection (oxid. poten-
tial 0.75 V). Briefly, a microbore (250 x 1 mm) 5/~m reverse-phase column (Brownlee Labs, Santa Barbara, U.S.A.) was used with a mobile phase containing 0.15 M phosphate buffer, 14% methanol, 0.1 mM EDTA and 0.6 mM sodium-octyl-sulfate (pH = 3.8). Basal values were determined for at least 60 min prior to the actual start of the SA-microdialysis session. At the end of the SA-microdialysis session, the dialysis probe was removed and 90 min later the animal was administered the DOPA decarboxylase inhibitor, 3-hydroxybenzylhydrazine (NSD 1015:100 mg/kg, i.p.). 30 min following the NSD injection the animal was decapitated, the brain quickly removed and the right N.Acc and caudate-putamen dissected on ice and stored at -80 °C. Tissue levels of DOPA, DA and DOPAC, were subsequently determined following alumina batch adsorption and reversephase ion pair HPLC (see ref. 1). Histological verification of the location of the probe was performed on the left region of the brain. The data were analyzed using analysis of variance with NewmanKeuls post-comparison test. In 'acute' cocaine animals the concentration of extraceUular DA in the N.Acc gradually increased from the beginning of an administration session to a stable level 300% higher (F3A8 = 4.297; P < 0.05) than control animals ('acute' rats receiving saline; Fig. 1). The maintenance of DA at a stable level is consistent with results observed after administration (single injection) of potent DA uptake inhibitors 8. Drug-naive animals which directly controlled the amount of cocaine that was self-administered (data not shown) strongly enhanced extracellular DA concentrations similar to animals passively receiving the drug. The results from previous experiments 9 would lead us to speculate that the acute enhancement of DA overflow observed in this study is linearly related to the level of cocaine in the extracellular space 9. In contrast, intravenous SA of cocaine failed to substantially enhance the extracellular concentration of DA in 'repeated' rats that were directly selfadministering cocaine (-~1 injection every 5 min; Fig. 1). These results show that repeated exposure to cocaine diminishes the ability of self-administered cocaine to significantly re-elevate the extracellular concentration of DA to levels associated with firsttime drug use. Interestingly, animals ('repeated
201
cocaine-saline') which had previously self-administered cocaine but received saline during the SAdialysis session had comparable extraceUular D A values to those induced by 'repeated' animals selfadministering cocaine (Fig. 1). Thus, it appears that the absolute level of D A in the extracellular fluid in the N.Acc (supposedly influencing intracellular D A mechanisms such as synthesis, receptor activation, etc.) is not directly correlated with the SA behavior. It should, however, be noted that although the extraceUular concentration of D A induced in these 'repeated' groups were not significantly different from each other, there was a tendency for D A values in 'repeated' animals receiving cocaine to be slightly higher than those receiving saline. If indeed extracellular D A in the N. Ace plays a more influential role than the results indicate in mediating the reinforcing aspects of cocaine, it can be suggested that repeated use of cocaine reduces the 'threshold', i.e. the required amount of D A necessary to be rewarding. Thus, a small increase in D A may be sufficient to be stimulatory after repeated use since the development of receptor supersensitivity, presumed to be associated with repeated cocaine u s e 6" lo, would amplify the effect of subsequent cocaine self-administration. As alluded to above, a D A depletion hypothesis 2
0.100 -~
i /
IIIIllll
II I I I I I l l
II l i l l l
I I I II I I I I I
--10
~0
~10
I--1ACUTESALINE ~Z3ACUTECOCAINE REPEATEDCOC~NE-SAI.JNE REPEAI~EDCOCAINE
o.
- - i
DOPA DOPAC DA Fig. 2. Tissue concentrations o f D O P A , D O P A C and D A in the nucleus accumbens. 'Acute' (first exposure to cocaine o r
saline), 'repeated cocaine' (10th cocaine self-administration session) and 'repeated cocaine-saline' ('repeated cocaine' rats receiving saline on the test day) groups are shown. Values are expressed as mean + S.E.M. (n = 4--6). Statistical significance were made with analysis of variance and Newman-Keuls post-comparison test (**P < 0.01 compared with controls).
has been frequently referred to which suggests that the depletion of D A is the motivating factor for cocaine dependence. Reduction of D A in the synaptic region following repeated administration of cocaine has been hypothesized to underlie the dysphoric (craving) effects of the drug. Thus, an animal is motivated to self-administer cocaine in an effort to re-elevate synaptic concentrations of D A to levels associated with positive reinforcing euphoric
ANTERIOR CAUDATE-PUTAMEN
15000.
.e .e" ~A'A'A
llOeO•
I II ,,-responeerate
o - - o Aotnl~g~JNIE 0 - - 0 A~I11~COeA/H£ A - - A ns~.A'nm~ - s A t a ~ " - - AR I ~ eOCeNE
~o~
NUCLEUS ACCUMBENS
..A.,A
80 time (rain)
.I ~"A'A'%.A. ~'A,A.
110
t40
Fig. 1. Extracellular concentration o f D A in the nucleus accumbcns o f freely moving rats during cocaine microdialysis session. T h e effects of intravenous cocaine o r saline in 'acute' (first exposure) rats and those previously exposed (9 days) to cocaine ('repeated cocaine') are shown. 'Repeated cocainesaline' depict those animals with repeated exposure to cocaine but receiving saline on the test day. The response rate pattern f r o m a representative 'repeated cocaine' animal is also shown on the top axis o f the figure. Each vertical line indicates a lever-press resulting in an intravenous administration o f
cocaine (0.75 mg/kg/injection). Values are expressed as mean + group S.E.M. (n = 4-8).
r-~ Acu'rESALINE E23ACUTECOCAINE BB REPEATEDCOCAINE-SALINE REPEA'I'~)COCAINE
10000.
5000,
DOPA
DOPAC
DA
Fig. 3. Tissue concentrations of D O P A , D O P A C and D A in the anterior caudate-putamen. 'Acute' (first exposure to cocaine or saline), 'repeated cocaine' (10th cocaine selfadministration session) and 'repeated cocaine-saline' ('repeated cocaine' rats receiving saline on the test day) groups are shown. Values are expressed as mean _+ S.E.M. (n = 4-6). Statistical significance were made with analysis of variance and Newman-Keuls post-comparison test (*P < 0.05 compared with controls).
202 levels. However, the inability of 'repeated' animals injecting cocaine at regular interinjection intervals to significantly elevate extracellular D A is inconsistent with the idea that D A levels are re-elevated to initial 'reinforcing' levels. Nevertheless, when saline was administered to animals that had regularly selfadministered cocaine a subtle reduction of synaptic D A levels was noticeable. Simple regression analyses showed that the extracellular D A values in 'acute' and 'repeated' animals receiving cocaine were positively correlated (i.e. increased) to D A overflow with time while the D A overflow in the 'repeated cocaine-saline' animals decreased over time (r = -0. 703, P < 0.001). This suggests that the D A signal might indeed be diminished when the drug is not available to cocaine-dependent animals. The reduced ability of i.v. cocaine SA to elevate the extracellular level of D A after repeated use may be explained in terms of such factors as: (1) decreased D A synthesis 11'18"19, (2) decreased tissue levels of D A in the neurons 16'17, (3) increased metabolism of D A 17, (4) increased uptake of D A from the synapse 18"19and/or (5) decreased release of D A from the presynaptic terminals 3. The first two hypotheses were not compatible with the results obtained in this study. Tissue levels of D A and D O P A accumulation, an index of D A synthesis, were not significantly altered following 'acute' or 'repeated' cocaine treatments (Fig. 2). The only significant change in tissue D A levels was observed in the anterior caudate-putamen, where D A was significantly elevated (F3,15 = 6.688; P < 0.01; Fig. 3). Although no significant difference was observed for extracellular levels of D O P A C and H V A follow-
ing any cocaine treatment (figure not shown), tissue D O P A C levels in the N . A c c in animals acutely or repeatedly treated with cocaine were found to be significantly reduced compared with controls (F3.13 = 36.616; P < 0.01). This may indeed indicate that some compensatory events had occurred in maintaining normal extracellular D O P A C levels. It is expected that the amount of cocaine exposure, environmental cues associated with the use of cocaine, and time between the last cocaine injection (withdrawal phenomena) all influence the results of this study. Nevertheless, two components of cocaine effects of D A overflow can be hypothesized to exist: (1) an acute phase in which D A overflow is enhanced in the extraceUular fluid, most likely correlated to extracellular levels of cocaine and (2) a chronic phase in which modification of release and/or uptake mechanisms (rather than reduced synthesis or tissue levels) attenuates the efficacy of i.v. cocaine to augment extracellular D A levels in the N.Acc. While substantial evidence links the rewarding effects of cocaine to mesolimbic D A neurotransmission, it is apparent that cocaine reward does not depend on the absolute amount of enhanced D A overflow alone. It is possible that the N.Acc is not the only brain region 7 and D A not the only neurochemical substrate mediating reward and dependence effects of cocaine.
1 And6n, N.-E., Golembiowsha-Nikintin, K. and Thornstrfm, U., Selective stimulation of dopamine and noradrenaline autoreceptors by B-HT 920 and B-HT 933, respectively, Naunyn-Schmiedeberg's Arch. Pharmacol., 321 (1982) 100-104. 2 Dackis, C.A. and Gold, M.S., New concepts in cocaine addiction: the dopamine depletion hypothesis, Neurosci. Behav. Rev., 9 (1985) 469-477. 3 Dembeic, D., Inhibition of potassium-stimulated release of [3H]dopamine from rat striata as a result of prior exposure to cocaine, nomifensine, or mazindol, Neurochem. Res., 5 (1980) 345-349. 4 de la Garza, R. and Johanson, C.E., Effects of haloperidol and physostigmine on self-administration of local anesthetics, Pharmacol. Biochem. Behav., 17 (1980) 1295-1299. 5 deWit, H. and Wise, R.A., Blockade of cocaine reinforcement in rats with dopamine receptor blocker pimozide but
not with the noradrenergic blockers phentolamine and phenoxybenzamine, J. Psychol., 31 (1977) 195-203. Goeders, N.E. and Kuhar, M.J., Chronic cocaine administration induces opposite changes in dopamine receptors in the striatum and nucleus accumbens, Alcohol Drug Res., 7 (1987) 207-216. Goeders, N.E. and Smith, J.E., Reinforcing properties of cocaine in the medial prefrontal cortex: primary action on presynaptic dopaminergic terminals, Pharmacol. Biochem. Behav., 25 (1986) 191-199. Hurd, Y.L. and Ungerstedt, U., In vivo neurochemical profile of DA uptake inhibitors and releasers in rat caudate-putamen, Eur. J. Pharmacol., in press. Hurd, Y.L., Kehr, J. and Ungerstedt, U., In vivo microdialysis as a technique to monitor drug transport: correlation of extracellular cocaine levels and dopamine overflow in the rat brain, J. Neurochem., 51 (1988) 1314-1416.
We are extremely grateful to Robert Lintz and Maria Grabowska-And6n for their excellent technical assistance. The study was supported by grants from the Swedish Medical Research Council (B8814X-03574-17B) and N I D A grant D A 04398.
6
7
8 9
203
10 Memo, M., Pradhan, A. and Hanbauer, I., Cocaineinduced supersensitivity of striatal dopamine receptors: role of endogenous calmodulin, Neuropharmacology, 20 (1981) 1145-1150. 11 Missale, C., Castelletti, L., Govoni, S., Spano, P.F., Trabucchi, M. and Hanbauer, I., Dopamine uptake is differentially regulated in rat striatum and nucleus accumbens, J. Neurochem., 45 (1985) 51-56. 12 Moore, K., Chiueh, CC. and Zeldes, G., Release of neurotransmitters from the brain in vivo by amphetamine, methylphenidate and cocaine. In E.H. Ellinwood and M.M. Kilbey (Eds.), Cocaine and Other Stimzdunts, Wiley, London, 1977, pp. 143-160. 13 Petit, H.O., Ettenberg, A., Bloom, F.E. and Koob, G.F., Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats, Psychopharmacology, 84 (1984) 167-173. 14 Roberts, D.C., Koob, F., Klonoff, P. and Fibiger, H.C., Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens, Pharm. Biochem. Behav., 12 (1980) 781-787. 15 Ross, S.B. and Renyi, A.L., Uptake of some tritiated sympathomimetic amines by mouse brain cortex in vitro, Acta Pharmacol. Toxicol., 24 (1966) 297-309. 16 Roy, S.N., Bhattacharyya, A.K., Pradhan, S. and Pradhan, S.N., Behavioural and neurochemical effects of repeated administration of cocaine in rats, Neuropharmacology, 17 (1978) 559-564. 17 Taylor, D. and Ho, B.T., Neurochemical effects of cocaine
18
19
20
21
following acute and repeated injection, J. Neurosci. Res., 3 (1977) 95-101. Trulson, M.E., Joe, J.C., Babb, S. and Raese, J.D., Chronic cocaine administration depletes tyrosine hydroxylase immunoreactivity in the mesolimbic dopamine system in rat brain: quantitative light microscopic studies, Brain Rex Bull., 19 (1987) 39-45. Trulson M. and Ulissey, M.J., Chronic cocaine administration decreases dopamine synthesis rate and increases [3H]spiroperidol binding in rat brain, Brain Res. Bull., 19 (1987) 35-38. Ungerstedt, U. and Pycock, C., Functional correlates of dopamine neurotransmission, Bull. Schweiz Akad. Med. Wiss., 1278 (1974) l-5. Van Ree, J.M., Slangen, J.L. and De Wied, D., Intravenous self-administration of drugs in rats, J. Pharmacol.
Exp. Ther., 204 (1978) 547-557. 22 Weeks, J.R., Experimental morphine
addiction: method for automatic intravenous injections in unrestrained rats, Science, 138 (1962) 143-144. 23 Westerink, B.H.C., Damsa, G., De Vries, J.B. and Horn, A.S., Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems, Life Sci., 41 (1987) 1763-1776. 24 Zetterstriim, T., Sharp, T., Marsden, C. and Ungerstedt, U., In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after d-amphetamine, J. Neurochem., 41 (6) (1983) 1769-1773.
199
Elsevier BRES 23734
Cocaine reinforcement and extracellular dopamine overflow in rat nucleus accumbens: an in vivo microdialysis study Yasmin L. Hurd 1, Friedbert Weiss 2, George F. Koob 2, Nils-Erik And 1 and Urban Ungerstedt 1 IKarolinska Institute, Department of Pharmacology, S-104 Ol Stockholm (Sweden) and 2Scripps Clinic and Research Foundation, Department of Basic and Clinical Research, La Jolla, CA 92037 (U.S.A.)
(Accepted 13 June 1989) Key words: Cocaine self-administration; Dopamine release; Dopamine uptake; Microdialysis; Addiction
Addictive properties of cocaine have been suggested to be mediated by an interplay of depletion (craving) and re-elevation (reinforcement) of dopamine (DA) levels in limbic brain area. In this study, direct measurement of dopamine in the extracellular fluid of rats freely self-administering cocaine was evaluated using in vivo microdialysis. Acute cocaine administration was associated with enhanced accumulation of DA in the nucleus accumbens, correlated with enhanced locomotor activity. In contrast, the increased DA overflow observed in drug-naive animals was attenuated in animals self-administering cocaine who had previous regular repeated (9-day) exposure to the drug. The results suggest that the absolute amount of DA in the extracellular space is not the critical factor correlated with the self-administration behavior. Additionally, the results indicate that the reduced ability of cocaine to re-elevate DA to first-time drug use is not due to a reduction of DA in the tissue or reduced DA synthesis, but may instead be associated with alterations of release and reuptake processes. In an effort to understand the mechanism of drug addiction in man, the self-administration (SA) behavioral model has been frequently used for estimating the abuse liability of cocaine. In this behavioral model animals receive an intravenous (i.v.) injection of a drug after making a correct operant response 21"22. The development of a stable level of drug intake over time is generally used as an index of the drug's reinforcing properties. Results from experiments in which the SA behavior has been manipulated, by pharmacological agents 4"5 or selective neurotoxic brain lesions 13A4, suggest that the strong reward and dependence potential of cocaine is closely associated with enhanced activity in the dopamine ( D A ) terminals of limbic brain areas (primarily the nucleus accumbens (N.Acc)). While it is well established that cocaine influences D A transmission by enhancing vesicularly stored D A and/or by inhibiting the uptake of D A located in the synaptic microenvironment s'12"13"15, questions remain as to whether augmentation of D A in the
synaptic cleft is truly correlated to the reinforcing aspects of cocaine. To address some of these questions we have used the technique of in vivo microdialysis 2°,23~24, which allows the continuous evaluation of neurochemical changes occurring in restricted brain regions of freely moving animals. Specifically, in the present investigation, extracellular levels of D A , 3,4-dihydroxyphenylacetic acid ( D O P A C ) and homovanillic acid (HVA) were monitored in the N.Acc of rats responding to cocaine reinforcement during 3 h, limited-access, SA sessions. The experiments were designed to test the effect of cocaine in drug-naive rats ('acute') and in rats previously trained to self-administer cocaine ('repeated'). Tissue D A levels and estimates of D A synthesis were also assessed in an effort to establish the overall effect of cocaine on D A t r a n s m i s s i o n . Male S p r a g u e - D a w l e y rats (250-300 g) previously trained to lever-press for water reinforcement were surgically prepared with a chronic sialastic
Correspondence: Y.L. Hurd, Karolinska Institute, Department of Pharmacology, Box 60 400, S-104 01 Stockholm, Sweden.
0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
200 jugular catheter (to allow for later self-administration of cocaine) under halothane anaesthesia. During the operation a brain guide cannula (Carnegie Medicin AB, Stockholm, Sweden) was also implanted; coordinates A +1.7, L +1.3, and V - 0 . 5 mm (so that a microdialysis probe (membrane: 0.5 x 2 mm; Carnegie Medicin AB, Sweden) would extend 7.3 mm below the dura into the N.Acc). Four days following surgery the rats were allowed 3 h access every day to a metal lever mounted on the side wall of a square Plexiglas operant chamber. Every depression of the lever resulted in an i.v. injection of a 100 ~1 cocaine hydrochloride solution (0.75 mg/ kg/injection) dissolved in 0.9% saline and delivered over a period of 4 s. Animals were designated 'acute', if they had no previous SA experience (and minimal exposure to the operant apparatus before dialysis testing) or 'repeated', if they had been previously trained to self-administer cocaine (9-10 days) and had developed a stable intake of cocaine (less than 10% change from the mean total intake over 3 consecutive days). Two control groups were also studied, an 'acute' saline and a 'repeated' group ('repeated' cocaine-saline) which received saline on the day of testing. Since rats in the 'acute' saline or 'repeated' cocaine-saline group were not expected to self-administer saline, these animals were yoked to a trained animal self-administering cocaine at a stable response rate. Thus, each lever-press by the trained animal produced an injection of saline for the yoked animal. 'Acute' cocaine rats were also yoked since their response rate of injecting cocaine was unstable (great variability between rats) and not comparable to the amount of cocaine administered by the 'repeated' animals. On the test day, the microdialysis probe was slowly inserted into the N.Acc via the guide cannula. The probe was continuously perfused (2/~l/min) with an artificial cerebrospinal fluid solution consisting of (in mM): Na ÷ 155, K ÷ 2.9, Ca 2÷ 1.1, Mg 2÷ 0.83, C1132.76 and D-glucose 5.9; pH 7.4. Perfusate samples were collected every 5 min into 0.3 ml glass vials (containing 10/~1 of 0.1 M perchloric acid) situated in a CMA/100 microfraction collector (Carnegie Medicin AB). The samples were subsequently placed into a refrigerated autoinjector (CMA/200, Carnegie Medicin AB) that was attached to a HPLC system with electrochemical detection (oxid. poten-
tial 0.75 V). Briefly, a microbore (250 x 1 mm) 5/~m reverse-phase column (Brownlee Labs, Santa Barbara, U.S.A.) was used with a mobile phase containing 0.15 M phosphate buffer, 14% methanol, 0.1 mM EDTA and 0.6 mM sodium-octyl-sulfate (pH = 3.8). Basal values were determined for at least 60 min prior to the actual start of the SA-microdialysis session. At the end of the SA-microdialysis session, the dialysis probe was removed and 90 min later the animal was administered the DOPA decarboxylase inhibitor, 3-hydroxybenzylhydrazine (NSD 1015:100 mg/kg, i.p.). 30 min following the NSD injection the animal was decapitated, the brain quickly removed and the right N.Acc and caudate-putamen dissected on ice and stored at -80 °C. Tissue levels of DOPA, DA and DOPAC, were subsequently determined following alumina batch adsorption and reversephase ion pair HPLC (see ref. 1). Histological verification of the location of the probe was performed on the left region of the brain. The data were analyzed using analysis of variance with NewmanKeuls post-comparison test. In 'acute' cocaine animals the concentration of extraceUular DA in the N.Acc gradually increased from the beginning of an administration session to a stable level 300% higher (F3A8 = 4.297; P < 0.05) than control animals ('acute' rats receiving saline; Fig. 1). The maintenance of DA at a stable level is consistent with results observed after administration (single injection) of potent DA uptake inhibitors 8. Drug-naive animals which directly controlled the amount of cocaine that was self-administered (data not shown) strongly enhanced extracellular DA concentrations similar to animals passively receiving the drug. The results from previous experiments 9 would lead us to speculate that the acute enhancement of DA overflow observed in this study is linearly related to the level of cocaine in the extracellular space 9. In contrast, intravenous SA of cocaine failed to substantially enhance the extracellular concentration of DA in 'repeated' rats that were directly selfadministering cocaine (-~1 injection every 5 min; Fig. 1). These results show that repeated exposure to cocaine diminishes the ability of self-administered cocaine to significantly re-elevate the extracellular concentration of DA to levels associated with firsttime drug use. Interestingly, animals ('repeated
201
cocaine-saline') which had previously self-administered cocaine but received saline during the SAdialysis session had comparable extraceUular D A values to those induced by 'repeated' animals selfadministering cocaine (Fig. 1). Thus, it appears that the absolute level of D A in the extracellular fluid in the N.Acc (supposedly influencing intracellular D A mechanisms such as synthesis, receptor activation, etc.) is not directly correlated with the SA behavior. It should, however, be noted that although the extraceUular concentration of D A induced in these 'repeated' groups were not significantly different from each other, there was a tendency for D A values in 'repeated' animals receiving cocaine to be slightly higher than those receiving saline. If indeed extracellular D A in the N. Ace plays a more influential role than the results indicate in mediating the reinforcing aspects of cocaine, it can be suggested that repeated use of cocaine reduces the 'threshold', i.e. the required amount of D A necessary to be rewarding. Thus, a small increase in D A may be sufficient to be stimulatory after repeated use since the development of receptor supersensitivity, presumed to be associated with repeated cocaine u s e 6" lo, would amplify the effect of subsequent cocaine self-administration. As alluded to above, a D A depletion hypothesis 2
0.100 -~
i /
IIIIllll
II I I I I I l l
II l i l l l
I I I II I I I I I
--10
~0
~10
I--1ACUTESALINE ~Z3ACUTECOCAINE REPEATEDCOC~NE-SAI.JNE REPEAI~EDCOCAINE
o.
- - i
DOPA DOPAC DA Fig. 2. Tissue concentrations o f D O P A , D O P A C and D A in the nucleus accumbens. 'Acute' (first exposure to cocaine o r
saline), 'repeated cocaine' (10th cocaine self-administration session) and 'repeated cocaine-saline' ('repeated cocaine' rats receiving saline on the test day) groups are shown. Values are expressed as mean + S.E.M. (n = 4--6). Statistical significance were made with analysis of variance and Newman-Keuls post-comparison test (**P < 0.01 compared with controls).
has been frequently referred to which suggests that the depletion of D A is the motivating factor for cocaine dependence. Reduction of D A in the synaptic region following repeated administration of cocaine has been hypothesized to underlie the dysphoric (craving) effects of the drug. Thus, an animal is motivated to self-administer cocaine in an effort to re-elevate synaptic concentrations of D A to levels associated with positive reinforcing euphoric
ANTERIOR CAUDATE-PUTAMEN
15000.
.e .e" ~A'A'A
llOeO•
I II ,,-responeerate
o - - o Aotnl~g~JNIE 0 - - 0 A~I11~COeA/H£ A - - A ns~.A'nm~ - s A t a ~ " - - AR I ~ eOCeNE
~o~
NUCLEUS ACCUMBENS
..A.,A
80 time (rain)
.I ~"A'A'%.A. ~'A,A.
110
t40
Fig. 1. Extracellular concentration o f D A in the nucleus accumbcns o f freely moving rats during cocaine microdialysis session. T h e effects of intravenous cocaine o r saline in 'acute' (first exposure) rats and those previously exposed (9 days) to cocaine ('repeated cocaine') are shown. 'Repeated cocainesaline' depict those animals with repeated exposure to cocaine but receiving saline on the test day. The response rate pattern f r o m a representative 'repeated cocaine' animal is also shown on the top axis o f the figure. Each vertical line indicates a lever-press resulting in an intravenous administration o f
cocaine (0.75 mg/kg/injection). Values are expressed as mean + group S.E.M. (n = 4-8).
r-~ Acu'rESALINE E23ACUTECOCAINE BB REPEATEDCOCAINE-SALINE REPEA'I'~)COCAINE
10000.
5000,
DOPA
DOPAC
DA
Fig. 3. Tissue concentrations of D O P A , D O P A C and D A in the anterior caudate-putamen. 'Acute' (first exposure to cocaine or saline), 'repeated cocaine' (10th cocaine selfadministration session) and 'repeated cocaine-saline' ('repeated cocaine' rats receiving saline on the test day) groups are shown. Values are expressed as mean _+ S.E.M. (n = 4-6). Statistical significance were made with analysis of variance and Newman-Keuls post-comparison test (*P < 0.05 compared with controls).
202 levels. However, the inability of 'repeated' animals injecting cocaine at regular interinjection intervals to significantly elevate extracellular D A is inconsistent with the idea that D A levels are re-elevated to initial 'reinforcing' levels. Nevertheless, when saline was administered to animals that had regularly selfadministered cocaine a subtle reduction of synaptic D A levels was noticeable. Simple regression analyses showed that the extracellular D A values in 'acute' and 'repeated' animals receiving cocaine were positively correlated (i.e. increased) to D A overflow with time while the D A overflow in the 'repeated cocaine-saline' animals decreased over time (r = -0. 703, P < 0.001). This suggests that the D A signal might indeed be diminished when the drug is not available to cocaine-dependent animals. The reduced ability of i.v. cocaine SA to elevate the extracellular level of D A after repeated use may be explained in terms of such factors as: (1) decreased D A synthesis 11'18"19, (2) decreased tissue levels of D A in the neurons 16'17, (3) increased metabolism of D A 17, (4) increased uptake of D A from the synapse 18"19and/or (5) decreased release of D A from the presynaptic terminals 3. The first two hypotheses were not compatible with the results obtained in this study. Tissue levels of D A and D O P A accumulation, an index of D A synthesis, were not significantly altered following 'acute' or 'repeated' cocaine treatments (Fig. 2). The only significant change in tissue D A levels was observed in the anterior caudate-putamen, where D A was significantly elevated (F3,15 = 6.688; P < 0.01; Fig. 3). Although no significant difference was observed for extracellular levels of D O P A C and H V A follow-
ing any cocaine treatment (figure not shown), tissue D O P A C levels in the N . A c c in animals acutely or repeatedly treated with cocaine were found to be significantly reduced compared with controls (F3.13 = 36.616; P < 0.01). This may indeed indicate that some compensatory events had occurred in maintaining normal extracellular D O P A C levels. It is expected that the amount of cocaine exposure, environmental cues associated with the use of cocaine, and time between the last cocaine injection (withdrawal phenomena) all influence the results of this study. Nevertheless, two components of cocaine effects of D A overflow can be hypothesized to exist: (1) an acute phase in which D A overflow is enhanced in the extraceUular fluid, most likely correlated to extracellular levels of cocaine and (2) a chronic phase in which modification of release and/or uptake mechanisms (rather than reduced synthesis or tissue levels) attenuates the efficacy of i.v. cocaine to augment extracellular D A levels in the N.Acc. While substantial evidence links the rewarding effects of cocaine to mesolimbic D A neurotransmission, it is apparent that cocaine reward does not depend on the absolute amount of enhanced D A overflow alone. It is possible that the N.Acc is not the only brain region 7 and D A not the only neurochemical substrate mediating reward and dependence effects of cocaine.
1 And6n, N.-E., Golembiowsha-Nikintin, K. and Thornstrfm, U., Selective stimulation of dopamine and noradrenaline autoreceptors by B-HT 920 and B-HT 933, respectively, Naunyn-Schmiedeberg's Arch. Pharmacol., 321 (1982) 100-104. 2 Dackis, C.A. and Gold, M.S., New concepts in cocaine addiction: the dopamine depletion hypothesis, Neurosci. Behav. Rev., 9 (1985) 469-477. 3 Dembeic, D., Inhibition of potassium-stimulated release of [3H]dopamine from rat striata as a result of prior exposure to cocaine, nomifensine, or mazindol, Neurochem. Res., 5 (1980) 345-349. 4 de la Garza, R. and Johanson, C.E., Effects of haloperidol and physostigmine on self-administration of local anesthetics, Pharmacol. Biochem. Behav., 17 (1980) 1295-1299. 5 deWit, H. and Wise, R.A., Blockade of cocaine reinforcement in rats with dopamine receptor blocker pimozide but
not with the noradrenergic blockers phentolamine and phenoxybenzamine, J. Psychol., 31 (1977) 195-203. Goeders, N.E. and Kuhar, M.J., Chronic cocaine administration induces opposite changes in dopamine receptors in the striatum and nucleus accumbens, Alcohol Drug Res., 7 (1987) 207-216. Goeders, N.E. and Smith, J.E., Reinforcing properties of cocaine in the medial prefrontal cortex: primary action on presynaptic dopaminergic terminals, Pharmacol. Biochem. Behav., 25 (1986) 191-199. Hurd, Y.L. and Ungerstedt, U., In vivo neurochemical profile of DA uptake inhibitors and releasers in rat caudate-putamen, Eur. J. Pharmacol., in press. Hurd, Y.L., Kehr, J. and Ungerstedt, U., In vivo microdialysis as a technique to monitor drug transport: correlation of extracellular cocaine levels and dopamine overflow in the rat brain, J. Neurochem., 51 (1988) 1314-1416.
We are extremely grateful to Robert Lintz and Maria Grabowska-And6n for their excellent technical assistance. The study was supported by grants from the Swedish Medical Research Council (B8814X-03574-17B) and N I D A grant D A 04398.
6
7
8 9
203
10 Memo, M., Pradhan, A. and Hanbauer, I., Cocaineinduced supersensitivity of striatal dopamine receptors: role of endogenous calmodulin, Neuropharmacology, 20 (1981) 1145-1150. 11 Missale, C., Castelletti, L., Govoni, S., Spano, P.F., Trabucchi, M. and Hanbauer, I., Dopamine uptake is differentially regulated in rat striatum and nucleus accumbens, J. Neurochem., 45 (1985) 51-56. 12 Moore, K., Chiueh, CC. and Zeldes, G., Release of neurotransmitters from the brain in vivo by amphetamine, methylphenidate and cocaine. In E.H. Ellinwood and M.M. Kilbey (Eds.), Cocaine and Other Stimzdunts, Wiley, London, 1977, pp. 143-160. 13 Petit, H.O., Ettenberg, A., Bloom, F.E. and Koob, G.F., Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats, Psychopharmacology, 84 (1984) 167-173. 14 Roberts, D.C., Koob, F., Klonoff, P. and Fibiger, H.C., Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens, Pharm. Biochem. Behav., 12 (1980) 781-787. 15 Ross, S.B. and Renyi, A.L., Uptake of some tritiated sympathomimetic amines by mouse brain cortex in vitro, Acta Pharmacol. Toxicol., 24 (1966) 297-309. 16 Roy, S.N., Bhattacharyya, A.K., Pradhan, S. and Pradhan, S.N., Behavioural and neurochemical effects of repeated administration of cocaine in rats, Neuropharmacology, 17 (1978) 559-564. 17 Taylor, D. and Ho, B.T., Neurochemical effects of cocaine
18
19
20
21
following acute and repeated injection, J. Neurosci. Res., 3 (1977) 95-101. Trulson, M.E., Joe, J.C., Babb, S. and Raese, J.D., Chronic cocaine administration depletes tyrosine hydroxylase immunoreactivity in the mesolimbic dopamine system in rat brain: quantitative light microscopic studies, Brain Rex Bull., 19 (1987) 39-45. Trulson M. and Ulissey, M.J., Chronic cocaine administration decreases dopamine synthesis rate and increases [3H]spiroperidol binding in rat brain, Brain Res. Bull., 19 (1987) 35-38. Ungerstedt, U. and Pycock, C., Functional correlates of dopamine neurotransmission, Bull. Schweiz Akad. Med. Wiss., 1278 (1974) l-5. Van Ree, J.M., Slangen, J.L. and De Wied, D., Intravenous self-administration of drugs in rats, J. Pharmacol.
Exp. Ther., 204 (1978) 547-557. 22 Weeks, J.R., Experimental morphine
addiction: method for automatic intravenous injections in unrestrained rats, Science, 138 (1962) 143-144. 23 Westerink, B.H.C., Damsa, G., De Vries, J.B. and Horn, A.S., Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems, Life Sci., 41 (1987) 1763-1776. 24 Zetterstriim, T., Sharp, T., Marsden, C. and Ungerstedt, U., In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after d-amphetamine, J. Neurochem., 41 (6) (1983) 1769-1773.