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Fentanyl and apomorphine: Asymmetrical generalization of discriminative stimulus properties
FENTANYL AND APOMORPHINE: ASYMMETRICAL GENERALIZATION OF DISCRIMINATIVE STIMULUS PROPERTIES F. C. COLPAERT, C. J. E. NIEMEGEERSand P. A. J. JANSSEN Department of Pharmacology, Janssen Pharmaceutics Research Laboratories, B-2340 Beerse, Belgium (Accepted 27 ~~~~ry 1976) Summary-One group of rats (n = 9) was trained to discriminate 0.16 mg/kg apomorphine from solvent and another group (n = 9) was trained to discriminate 0.04 mg/kg fentanyl from solvent. Stimulus generalization experiments were performed in both groups; apomorphine and fentanyl were used as agonists, whereas haloperidoi and naloxone were studied as potential antagonists. The data indicate that a narcotic action which indirectly increases dopaminergic activity in the brain accounts for a significant, though not critical, component of the di~riminative stimulus complex which constitutes the narcotic cue.
It has been shown that fentanyl (COLPAERT,LAL, NIE- discriminative stimulus properties of fentanyl and MEGEERS and JANSSEN,1975a; ~OLPAERT,NIEMEGEERS, a~morphine was also investigated. LAL and JANSSEN,1975b), morphine (G~ANIJT~OS and LAL, 1975; HILL, JONESand BELL,1971; HIRSCHHORN MATERIALSAND METHODS and ROSECRANS,1974; ROSECRANS,GDDDLOE,BENNETT and HIRSCHHORN,1973) and numerous other The materials and methods used in the present exnarcotic analgesic drugs (COLPAERTand NIEMEGEERS,periments have been extensively described elsewhere (CDLP~ERT,N~E&#EGEERS and JANSSEN,1976b). 1975; COLPAERT, NIEMEGEERS and JANSSEN,1976a) can produce a discrimi~ativc stimulus complex in laboraAnimals tory animals. The discriminative stimulus complex The experimental animals were 18 male Wistar rats, produced by narcotic analgesics is defined as the narcotic cue (COLPAERTet al., 1975a) and has been evi- weighing 210 & log at the beginning of the experiments. They were housed in individual living cages, denced by the ability of these drugs to differentially stored in a continuously illuminated and air condicontrol operant behaviour in rats. Central dopamine systems are presumed to be intioned room (21 + 1°C; relative humidity 65 + 5%). Tap water was continuously available but the access volved in various pharmacological actions of narcotic to dry powdered standard laboratory chow was analgesic drugs (LAL, 1975; LAL, GIANUTSOSand PURI, 1975). The finding that the dopamine receptor limited to 2 hr a day, as specified below. blocking neuroleptic haloperidol (JANSSEN, 1967) Materials reduces the self-administration of morphine in rats (HANSONand CLI~INI-VE~~, 1972; SCHWARTZand Standard animal test cages, fitted with two levers, MARCHOK,1974; SMITHand DAVIS, 1973), constitutes a food cup and a house light, and programmed by solid-state logic modules were used. direct evidence in favour of such involvement. The present study aimed to determine the possible Procedures role of central dopamine systems in the cuing action Nine rats (group I) were trained to discriminate of narcotic analgesic drugs. To this end, the stimulus properties of the narcotic analgesic fentanyl (JA~%SSEN,0.16 mg/kg apomorphine plus solvent from solvent; NIE~GEERS and DONY, 1963) were assessed in rats 9 other animals (group II) were trained to discrimitrained to discriminate the dopamine receptor agonist nate 0.04 mg/kg fentanyl plus solvent from solvent. apomorphine (ERNST,1967) from solvent. This seemed Irrespective of the training drug used (i.e. apomorto be of potential interest, as preliminary experiments phine or fentanyl), all procedures were exactly the had indicated that fentanyl was generalized with aposame for both groups of animals. morphine in such trained animals. Conversely, apoThe 18 rats were trained to press (fixed ratio 10) morphine was submitted for test in rats trained to for food (45 mg Noyes food pellets) on one of two discriminate fentanyl from solvent. The possible antalevers. Following the combined injection of solvent gonistic action of haloperidol and naloxone on the (60 min) and training drug (30 min), the rats were required to press one of the levers (drug lever) in order to get reinforcement. Upon combined solvent Key words: fentanyl, apomorphine, halopcridol, naloxone, dopamine, drug discrimination. (60 min) plus solvent (30 min) treatment, they were 541
F. C. COLPAERT, C. J. E. NIEMEGEERS and P. A. J. JANSSEN
542
required to press the opposite lever (solvent lever). In both cases, pressing the incorrect lever had no programmed consequences. These combined training treatments are symbolized as SD and SS respectively. The lever assignments were drug lever: left, solvent lever: right in one half of the animals, and drug lever: right, solvent lever: left in the other half. Every week, each rat was run in daily 15-min sessions on 5 consecutive days. Both injection treatments were given according to 2 weekly alternating sequences, i.e. SD-SS-SS-SD-SD, and SS-SD-SDSS-SS. The number of responses made on either lever before obtaining the first food pellet (and, thus, before having made 10 correct responses) was recorded. All responses emitted during the entire course of the 1%min sessions were also recorded (total responses). The training criterion consisted of 10 consecutive sessions on which a “first food pellet” score < 12 was recorded for each rat individually. Following training, test sessions took place on Wednesdays and Fridays. On the 3 remaining days of the week, the training conditions were continued to ensure reliable performance. Before test sessions, the animals were treated with the injection combination to be studied, and 30 min after the last injection were introduced into the test cage. The rats then were to select one of the two levers; i.e. the lever on which they first totalled 10 responses was considered as the selected lever and subsequent reinforcement was made contingent upon pressing (fixed ratio 10) the selected lever. One hr following any training/test session, the animals were allowed to feed freely for 2 hr. The entire
series of test treatments in the two groups (see Tables 1 and 2) was given to all rats of these groups and the sequence of test treatments was completely randomized. Drugs and doses All drugs (apomorphine hydrochloride, fentanyl citrate, haloperidol and naloxone hydrochloride) were freshly prepared as aqueous solutions, and were subcutaneously injected at a constant volume of 1 ml/100 g body weight. The selection of doses applied in the test treatments reported herein was based on preli~nary experiments in rats not participating to the present study. RESULTS Control
msults
rats of group I required a median number of 35 training sessions to meet the training criterion. Following this training, all rats were run 3 times a week on control sessions. Their performance under these control conditions was essentially the same as that previously observed in similarly trained animals (COLPAERT,NIE~GEERS, KUYPS and JANSSEN,197%). In short, all rats succeeded in reaching a highly significant (two-tailed P < 0.001; binominal test; SIEGEL, 1956) level of correct lever selection, and the standard apomorphine dose (0.16 mg/kg) significantly reduced total response output (Table 1). The 9 rats of group II required a median number of 33 training sessions to criterion and their performance likewise resembled that of similarly trained aniThe 9
Table 1. Results obtained from rats (n = 9) trained to discriminate 0.16 mg/kg apomorphine plus solvent from solvent (group 1) Treatment 60 min Compound
Response output
Lever selection 30 min
Dose (msiks)
Compound
Dose (m&z)
Drug lever selected
First food pellet
Total responses
% of total responses on selected lever
Response level
Solvent Solvent
~
Solvent Apomorphine
0.16
O/234 232/234
10 (10-10) 10 (10-10)
1264 (+46) 980 (k61)
100 (1OClOO) 100 (100-100)
100 79* (k4.1)
Solvent Solvent
-
Apomorphine Apomorphine
0.04 0.08
319 9/9
10 (10-10) 10 (10-10)
1158 (k62) 1238 (,90)
100 (100-100) 100 (99.7-100)
84* (k5.8) 86* (k4.7)
Apomorphine Apomorphine
0.16 0.16
O/9 9i9
10 (10-10) 10 (10-10)
926 (,109) 795 (,122)
99.8 (90.1-100) 100 (99.~100)
71* (t-7.3) 66* (&9.9)
Fentanyl Fentanyl Fentanyl
0.02 0.04 0.08
0/g 5/g 3/3
10 (lo--12) 10 (10-13) 10 (10-11)
842 (+200) 309 (&91) 27 (k2Of
100 (99.2-100) 99.3 (93.3-100) 99.2 (98.~100)
66* (rt13) 32* (k9.4) 2” (F1.3)
Fentanyl Fentanyl
0.04 0.04
O/7
10 (10-13) 10 (10-10)
404 (i 154) 99.3 (92.3-100) 1257 (+80) 100 (100-100)
28* (k9.8) 99 (k3.4)
Haloperidol Naloxone Solvent Solvent Solvent Haloperidol Naloxone
0.02 5.0 0.02 0.63
O/9
“Drug lever selected” represents the number of drug lever selections out of the number of rats that did respond. The term “response level” refers to the total responses made on SD-control or test sessions, expressed as a percentage of total responses on the most recently preceding SS control session. “First food pellet” and “% of total responses on selected lever” are expressed as the median and 95% confidence limits. “Total responses” and “response level” are expressed as the mean (k 1 S.E.M.). The asterisk denotes two-tailed P < 0.05 (Wilcoxon test; SIEGEL,1956). The control data (upper part) are based on 26 determinations in all 9 rats (26 SS- and 26 SD-control sessions); the test data (lower part) are based on 1 determination in these 9 animals.
Narcotic cue
mals (COLPAERTet al., 1975a). Correct lever selection was significant in each rat individually (P < 0.001). The standard fentanyl dose (0.04 mg/kg) attenuated response output significantly (Table 2). Test results
The test data obtained from rats (group I) trained to discriminate 0.16 mg/kg apomorphine plus solvent from solvent are summarized in Table 1. It is shown that the various apomorphine doses (0.04 to 0.16 mg/kg) produced a dose-related drug lever selection, the median effective dose (ED,,) value thus yielded (LITCHFIELDand WILCOXON,1949) being 0.044 mg/kg (95% confidence limits 0.032-0.061). At all doses, apomorphine significantly reduced responding for food reward. Haloperidol (0.02 mg/kg) but not naloxone (5.0 mg/kg) pretreatment (60 min) completely prevented the detection of the apomorphine cue otherwise (see control data) induced by the standard 0.16 mg/kg apomorphine dose (30 min). In both instances, responding was essentially similar to SDcontrol values, and differed significantly from SScontrols. Fentanyl (0.02-0.08 mg/kg) was generalized with apomorphine in a dose-related way (EDso 0.037 mg/kg ; confidence limits 0.025-0.056). Fentanyl also significantly reduced responding. One rat failed to respond after any fentanyl injection, and so did 5 other animals at the dose of 0.08 mg/kg. The generalization of fentanyl with apomorphine was antagonized by haloperidol (0.02 mg/kg) as well as by naloxone (0.63 mg/kg) pretreatment. In addition, naloxone, but not haloperidol, counteracted the response-reducing effect of fentanyl (0.04 mg/kg). After any test treatment neither the accuracy of lever selection (as indicated by the ‘“first food pellet” values), nor the consistency of further responding (as indicated by the values of “% of total responses on selected lever”) was significantly poorer than SS or SD control values.
543
The test data obtained from rats (group II) trained to discriminate 0.04 mg/kg fentanyl plus solvent from solvent are summarized in Table 2. Fentanyl produced the narcotic cue (EDso 0.019 mg/kg; confidence limits 0.01550.025) as well as response reduction in a dose-related way. Halo~ridol(O.08 mg/kg) pretreatment failed to antagonize the narcotic cue. However, although the accuracy of lever selection was not affected, the y0 of total responses on the selected lever was significantly reduced. In contrast, naloxone (0.63 mg/kg) counteracted the narcotic cue as well as the response inhibition otherwise produced by the standard 0.04 mg/kg fentanyl injection. Increasing apomorphine doses (0.16-0.63 mg/kg) consistently produced solvent lever selection and reduced total response output; 5 out of the 9 rats tested did not respond at all following 0.63 mg/kg apomorphine. The combined haloperidol(O.08 mg/kg) plus fentanyI(O.04 mg/kg) treatment was the only instance in which the % of total responses on selected lever sign~cantly deteriorated. “First food pellet” values were always within the range of control observations. DISCUSSION
The present results on the intrinsic effects of apomorphine in rats trained to discriminate 0.16 mg/kg apomorphine from solvent yield an ED,, value of 0.044 mg/kg. This value is a quite accurate replication of earlier data (ED,, 0.066 mg/kg; COLPAERT et al., 1975~) in similarly trained rats, thus confirming the reliability and predictability of data produced by the method used (C~LPAERTet at., 1976b). The finding that fentanyl is generalized with apomorphine (Table 1) constitutes direct evidence for the hypothesis that narcotic analgesics exert central dopaminergic activity. This is further substantiated by the ability of the dopamine receptor blocking neuroleptic
Table 2. Results obtained on rats (n = 9) trained to discriminate 0.04 mg/kg fentanyl plus solvent from solvent
(group II) Treatment
Lever selection
Dose Compound Solvent Solvent -.------Solvent Solvent Waloperidol Naloxone Solvent Solvent Solvent
Response output
30 min
60 min (mg/kg)
-
Compound
Drug lever
(mg/kg)
selected
Solvent
Fentanyl ---__________ Fentanyl Fentanyl 0.08 0.63 -
Dose
0.04 -------_
First food pellet
% of total Total responses
responses on selected lever
O/180 10 (10-10) 1279 (f113) 180/180 10 (l&10) 868 (f133)
100 (10~100) 100 (100-100)
0.01 0.02
O/9 519
10 (l&10) 1278 (,143) 100 (99.9-100) 10 (1GlO) 1153 (+143) 100 (99.2-100)
Fentanyl Fentanyl
0.04 0.04
9J9 O/9
10 (l&10) 10 (l&10)
Apomorphine Apomorphine Apomorphine
0.16 0.3 1 0.63
O/9 O/9 O/4
10 (i&10) 10 (l&10) 10 (l%-t3)
The control data are based on 20 determinations these 9 animafs. See Table 1 for details.
197 (,33) 82.4*(69.2-9&l) 1287 (i 107) 100 (99.7-100) 901 (296) 610 (rt94f 8.5 (F52)
100 (99.9100) 100 ~1~1~) 100 (99.~1~)
Response level loo
71* (k8.4) 110 (f8.2) 96 (k7.4) l8* (f2.7) 91 (k4.3) 68* (i2.9) 54* (i6.4) 6* (f3.2)
in all 9 rats. The test data are based on 1 determination
in
544
F. C. COLPAERT,C. J. E. NIEMEGEERSand P. A. J. JANSSEN
halope~dol (JANSSEN, 1967) to antagonize this generalization. The specific narcotic antagonist naloxone (BLUMBERG and DAYTON,1972) not only antagonized the cuing effect of fentanyl, but also counteracted the response-decreasing effect of the drug. Therefore, the present data can be discussed in terms of current concepts of the mechanism of action of narcotic analgesic drugs. Naloxone possesses a high affinity for the presumed opiate receptor (PERT, PASTERNAK and SNYDER,1973; SIMON,HILLERand EDEMAN,1973) but exerts very little intrinsic pharmacological activity (BLUMBERGand DAYTON, 1972). This implies that naloxone may occupy the available opiate receptor sites or modify the receptor’s conformation, and thereby prevent the occurrence of any narcotic action (thus antagonizing fentanyl’s stimulus properties as well as its response-reducing effects). As the affinity of haloperidol for the opiate receptor is at least 1,000 times lower than that of fentanyl (LEYSEN,personal communication) the neuroleptic fails to counteract the response reduction produced by fentanyl. The fact that, in group I, haloperidol nevertheless antagonizes the cuing action of fentanyl suggests, then, that this cuing action is based on some biochemical action of fentanyl which is initiated by its binding to the opiate receptor, and which results in an increased dopaminergic activity in the brain. In the light of the finding that narcotic analgesics increase the concentration of dopamine’s metabolite, homovanillic acid, in the rat striatum (KUSCHINSKYand HORNYKIEWICZ,1972, 1974), it seems reasonable to suggest that this action may consist of, or result in, an increased dopamine biosynthesis in the presynaptic neurone. The dose-effect relationship of fentanyl in rats trained to discriminate 0.04 mg/kg fentanyl from solvent (EDse 0.019 mg/kg) is virtually identical to earlier data in similarly trained rats (ED,, 0.011 mg/kg; COLPAERTet al., 1975a). Despite the conclusion established above that narcotic analgesics increase central dopaminergic activity, the rats of group II did not generalize apomorphine with the standard fentanyl treatment. This asymmetrical generalization indicates that dopaminergic activity accounts for a significant though not critical component of the discriminative stimulus complex which constitutes the narcotic cue. The latter conclusion is further argued for by the following findings. First, in group II, haloperidol (0.08 mg/kg) pretreatment failed to antagonize the narcotic cue produced by 0.04 mg/kg fentanyl, but significantly deteriorated the “/:,of total responses on the selected lever. The latter effect is very remarkable in view of the fact that only pressing the selected lever (in this case the drug lever) was rewarded. This deterioration seems to be a specific effect, as haloperidol does not affect the mere ability underlying discriminative responding in similarly trained rats (COLPAERTet al., 1975a). Second, whereas the narcotic cue is produced at a fentanyl dose (ED,,) of 0.019 mg/kg in group II (see also COLPAERTrt al., 1975a), the same drug produces detectable dopaminergic activity only at a
dose approximately two times higher (EDSo 0.037 mg/kg; group I). The conclusion that the dopaminergic activity of narcotic analgesics constitutes a significant though not critical component of the narcotic cue may elucidate the relationship which possibly exists between the discriminative stimulus properties and the reinforcing action of these drugs. The self-administration of morphine (HANSON and CLIMINI-VENEMA, 1972; SCHWARTZ and MARCHOK, 1974; SMITH and DAVIS, 1973) as well as that of apomorphine (BAXTER, GLUCKMAN,STEIN and SCERNI,1974) and amphetamine (DAVKS and SMITH,1975; YOKELand WISE, 1975) can be effectively antagonized by the specific dopamine receptor blocking neuroleptics haloperidol (JANSSEN,1967) and pimozide (JANSSEN,NIEMEGEERS, SCHELLEKENS, DRESSE,LENAERTS, PINCHARD,SCHAPER, VAN NUETENand VERBRUGGEN,1968). From these studies, it can be inferred that both neuroleptic drugs effectively block the reinforcing stimuli maintaining the self-administration of morphine, apomorphine and amphetamine in rats. For one thing, this conclusion explains the therapeutic effectiveness of haloperido1 in narcotic dependence (KARKALASand LAL, 1973; LIXOMPTEand FRIEDMAN,1974) and amphetamine abuse (ANGRIST,LEE and GERSHON,1974). For another, it very strongly suggests that the reinforcing stimuli produced by narcotic analgesics are constituted by the dopaminergic action which the present study has shown to be a significant component of the narcotic cue. It is interesting to note that, were the latter suggestion valid, it would imply that rats trained to discriminate apomorphine from solvent may be reliable detectors of the reinforcing action of narcotic analgesic drugs. Acknowledgement-ale expert technical assistance of J. J. M. D. KUYPS is gratefully acknowledged.
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Narcotic COLPAERT, F. C., NIEMEGEERS,C. J. E., LAL, H. and JANSSEN, P. A. J. (1975b). Investigations on drug produced and subjectively experienced discriminative stimuli. 2. Loperamide, an antidiarrheal devoid of narcotic cue producing actions. L$e SC;. 16: 717-728. COI.PAERT. F. C., NIEMEGEERS, C. J. E. and JANSSEN, P. A. J. 1976a). The narcotic discriminative stimulus complex: relationship to analgesic activity. J. Pharm. PharIMUC.28: 183-187. COLPAERT, F. C., NIEMEGEERS, C. J. E. and JANSSEN, P. A. J. (1976 b). Theoretical and methodological considerations on drug discrimination learning. Psychophnrmacologia 46: 16%177. COLPAERT, F. C., NIEMEGEERS, C. J. E., KUYPS, J. J. M. D. and JANSSEN, P. A. J. (1975 c). Apomorphine as a discriminative stimulus, and its antagonism by haloperidol. Eur. J. Pharmac. 32: 383-386. DAVIS, W. M. and SMITH, S. G. (1975). Effect of haloperidol on (+)-amphetamine self-administration. J. Pharm. Pharmac. 27: 540-542. ERNST, A. M. (1967). Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats. Psychophaimacologia lo:-316-323. GIANUTSOS, G. and LAL, H. (1975). Effect of loperamide, haloperidol and methadone in rats trained to discriminate morphine from saline. Psychopharmacologia 41: 267-270. HANSON, H. M. and CLIMINI-VENEMA, C. A. (1972). Effects of haloperidol on self-administration of morphine in rats. Fedn Proc. Fedn Am. Sots exp. Biol. 31: 503. HILL, H. E., JONES, B. E. and BELL, E. C. (1971). State dependent control of discrimination by morphine and pentobarbital. Psychopharmacologia 22: 305-313. HIRSCHHORN, I. D. and ROSECRANS,J. A. (1974). Morphine and A9-tetrahydrocannabinol: tolerance to the stimulus effects. Psychopharmacologia 36: 243-253. JANSSEN, P. A. J. (1967). The pharmacology of haloperidol. Int. J. Neuropsychiat. 3: l&18. JANSSEN, P. A. J., NIEMEGEERS, C. J. E. and DONY, J. G. H. (1963). The inhibitory effect of fentanyl (R 4263) and other morphine-like analgesics on the warm water induced tail withdrawal reflex. ArzneimittekForsch. 13: 502-507. JANSSEN, P. A. J., NIEMEGEERS, C. J. E., SCHELLEKENS, K. H. L., DRESSE, A., LENAERTS, F. M., PINCHARD, A., SCHAPER, W. K. A., VAN NUETEN, J. M. and VERBRUGGEN, F. J. (1968). Pimozide, a chemically novel, highly potent and orally long-acting neuroleptic drug. Arzneimittel-Forsch. 18: 261-287.
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KARKALAS, J. and LAL, H. (1973). A comparison of haloperidol with methadone in blocking heroin-withdrawal symptoms. Int. Pharmacopsychiat. 8: 248-251. KUSCHINSKY, K. and HORNYKIEWICZ, 0. (1972). Morphine catalepsy in the rat: relation to striatal dopamine metabolism. Eur. J. Pharmac. 19: 199-122. KUSCHINSKY, K. and HORNYKIEWICZ, 0. (1974). Effects of morphine on striatal dopamine metabolism: possible mechanism of its opposite effect on locomotor activity in rats and mice. Eur. J. Pharmac. 26: 41-50. LAL, H. (1975). Narcotic dependence, narcotic action and dopamine receptors. Life Sci. 17: 483496. LAL, H., GIANUT& G. and PURI, S. K. (1975). A comparison of narcotic analgesics with neurolentics on behavioral measures of dopaminergic activiti. Life Sci. 17: 29-34. LECOMPTE, G. and FRIEDMAN, J. J. (1974). Chemotherapy in the treatment of heroin addiction: an alternative to methadone. J. Drug Issues 4: 332-341. LITCHFIELD. J. T. and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmat. e?cp. Ther. 96: 99-113. PERT, C. B., PASTERNAK, G. and SNYDER, S. H. (1973). Opiate agonists and antagonists discriminated by receptor binding in brain. Science 182: 1359-1361. ROSECRANS, J. A., GCIODLOE, M. H., BENNETT, G. I. and HIRSCHHORN, I. D. (1973). Morphine as a discriminative cue: effects of amine depletors and naloxone. Eur. J. Pharmac. 21: 252-256. SCHWARTZ, A. S. and MARCHOK, P. L. (1974). Depression of morphine-seeking behaviour by dopamine inhibition. Nature, Lond. 248: 257-258. SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences, 312 pp. McGraw-Hill, New York. SIMON, E. J., HILLER, J. M. and EDELMAN, I. (1973). Stereospecific binding of the potent narcotic analgesic [3H] etorphine to rat-brain homogenate. Proc. natn. Acad. Sci. U.S.A. 70: 1947-1949. SMITH, S. G. and DAVIS, W. M. (1973). Haloperidol effects on morphine self-administration: testing for pharmacological modification of the primary reinforcement mechanism. Psychol. Rec. 23: 215-221. YOKEL, R. A. and WISE, R. A. (1975). Increased lever pressing for amphetamine after pimozide in rats: implications for a dopamine theory of reward. Science 187: 547-549.
It has been shown that fentanyl (COLPAERT,LAL, NIE- discriminative stimulus properties of fentanyl and MEGEERS and JANSSEN,1975a; ~OLPAERT,NIEMEGEERS, a~morphine was also investigated. LAL and JANSSEN,1975b), morphine (G~ANIJT~OS and LAL, 1975; HILL, JONESand BELL,1971; HIRSCHHORN MATERIALSAND METHODS and ROSECRANS,1974; ROSECRANS,GDDDLOE,BENNETT and HIRSCHHORN,1973) and numerous other The materials and methods used in the present exnarcotic analgesic drugs (COLPAERTand NIEMEGEERS,periments have been extensively described elsewhere (CDLP~ERT,N~E&#EGEERS and JANSSEN,1976b). 1975; COLPAERT, NIEMEGEERS and JANSSEN,1976a) can produce a discrimi~ativc stimulus complex in laboraAnimals tory animals. The discriminative stimulus complex The experimental animals were 18 male Wistar rats, produced by narcotic analgesics is defined as the narcotic cue (COLPAERTet al., 1975a) and has been evi- weighing 210 & log at the beginning of the experiments. They were housed in individual living cages, denced by the ability of these drugs to differentially stored in a continuously illuminated and air condicontrol operant behaviour in rats. Central dopamine systems are presumed to be intioned room (21 + 1°C; relative humidity 65 + 5%). Tap water was continuously available but the access volved in various pharmacological actions of narcotic to dry powdered standard laboratory chow was analgesic drugs (LAL, 1975; LAL, GIANUTSOSand PURI, 1975). The finding that the dopamine receptor limited to 2 hr a day, as specified below. blocking neuroleptic haloperidol (JANSSEN, 1967) Materials reduces the self-administration of morphine in rats (HANSONand CLI~INI-VE~~, 1972; SCHWARTZand Standard animal test cages, fitted with two levers, MARCHOK,1974; SMITHand DAVIS, 1973), constitutes a food cup and a house light, and programmed by solid-state logic modules were used. direct evidence in favour of such involvement. The present study aimed to determine the possible Procedures role of central dopamine systems in the cuing action Nine rats (group I) were trained to discriminate of narcotic analgesic drugs. To this end, the stimulus properties of the narcotic analgesic fentanyl (JA~%SSEN,0.16 mg/kg apomorphine plus solvent from solvent; NIE~GEERS and DONY, 1963) were assessed in rats 9 other animals (group II) were trained to discrimitrained to discriminate the dopamine receptor agonist nate 0.04 mg/kg fentanyl plus solvent from solvent. apomorphine (ERNST,1967) from solvent. This seemed Irrespective of the training drug used (i.e. apomorto be of potential interest, as preliminary experiments phine or fentanyl), all procedures were exactly the had indicated that fentanyl was generalized with aposame for both groups of animals. morphine in such trained animals. Conversely, apoThe 18 rats were trained to press (fixed ratio 10) morphine was submitted for test in rats trained to for food (45 mg Noyes food pellets) on one of two discriminate fentanyl from solvent. The possible antalevers. Following the combined injection of solvent gonistic action of haloperidol and naloxone on the (60 min) and training drug (30 min), the rats were required to press one of the levers (drug lever) in order to get reinforcement. Upon combined solvent Key words: fentanyl, apomorphine, halopcridol, naloxone, dopamine, drug discrimination. (60 min) plus solvent (30 min) treatment, they were 541
F. C. COLPAERT, C. J. E. NIEMEGEERS and P. A. J. JANSSEN
542
required to press the opposite lever (solvent lever). In both cases, pressing the incorrect lever had no programmed consequences. These combined training treatments are symbolized as SD and SS respectively. The lever assignments were drug lever: left, solvent lever: right in one half of the animals, and drug lever: right, solvent lever: left in the other half. Every week, each rat was run in daily 15-min sessions on 5 consecutive days. Both injection treatments were given according to 2 weekly alternating sequences, i.e. SD-SS-SS-SD-SD, and SS-SD-SDSS-SS. The number of responses made on either lever before obtaining the first food pellet (and, thus, before having made 10 correct responses) was recorded. All responses emitted during the entire course of the 1%min sessions were also recorded (total responses). The training criterion consisted of 10 consecutive sessions on which a “first food pellet” score < 12 was recorded for each rat individually. Following training, test sessions took place on Wednesdays and Fridays. On the 3 remaining days of the week, the training conditions were continued to ensure reliable performance. Before test sessions, the animals were treated with the injection combination to be studied, and 30 min after the last injection were introduced into the test cage. The rats then were to select one of the two levers; i.e. the lever on which they first totalled 10 responses was considered as the selected lever and subsequent reinforcement was made contingent upon pressing (fixed ratio 10) the selected lever. One hr following any training/test session, the animals were allowed to feed freely for 2 hr. The entire
series of test treatments in the two groups (see Tables 1 and 2) was given to all rats of these groups and the sequence of test treatments was completely randomized. Drugs and doses All drugs (apomorphine hydrochloride, fentanyl citrate, haloperidol and naloxone hydrochloride) were freshly prepared as aqueous solutions, and were subcutaneously injected at a constant volume of 1 ml/100 g body weight. The selection of doses applied in the test treatments reported herein was based on preli~nary experiments in rats not participating to the present study. RESULTS Control
msults
rats of group I required a median number of 35 training sessions to meet the training criterion. Following this training, all rats were run 3 times a week on control sessions. Their performance under these control conditions was essentially the same as that previously observed in similarly trained animals (COLPAERT,NIE~GEERS, KUYPS and JANSSEN,197%). In short, all rats succeeded in reaching a highly significant (two-tailed P < 0.001; binominal test; SIEGEL, 1956) level of correct lever selection, and the standard apomorphine dose (0.16 mg/kg) significantly reduced total response output (Table 1). The 9 rats of group II required a median number of 33 training sessions to criterion and their performance likewise resembled that of similarly trained aniThe 9
Table 1. Results obtained from rats (n = 9) trained to discriminate 0.16 mg/kg apomorphine plus solvent from solvent (group 1) Treatment 60 min Compound
Response output
Lever selection 30 min
Dose (msiks)
Compound
Dose (m&z)
Drug lever selected
First food pellet
Total responses
% of total responses on selected lever
Response level
Solvent Solvent
~
Solvent Apomorphine
0.16
O/234 232/234
10 (10-10) 10 (10-10)
1264 (+46) 980 (k61)
100 (1OClOO) 100 (100-100)
100 79* (k4.1)
Solvent Solvent
-
Apomorphine Apomorphine
0.04 0.08
319 9/9
10 (10-10) 10 (10-10)
1158 (k62) 1238 (,90)
100 (100-100) 100 (99.7-100)
84* (k5.8) 86* (k4.7)
Apomorphine Apomorphine
0.16 0.16
O/9 9i9
10 (10-10) 10 (10-10)
926 (,109) 795 (,122)
99.8 (90.1-100) 100 (99.~100)
71* (t-7.3) 66* (&9.9)
Fentanyl Fentanyl Fentanyl
0.02 0.04 0.08
0/g 5/g 3/3
10 (lo--12) 10 (10-13) 10 (10-11)
842 (+200) 309 (&91) 27 (k2Of
100 (99.2-100) 99.3 (93.3-100) 99.2 (98.~100)
66* (rt13) 32* (k9.4) 2” (F1.3)
Fentanyl Fentanyl
0.04 0.04
O/7
10 (10-13) 10 (10-10)
404 (i 154) 99.3 (92.3-100) 1257 (+80) 100 (100-100)
28* (k9.8) 99 (k3.4)
Haloperidol Naloxone Solvent Solvent Solvent Haloperidol Naloxone
0.02 5.0 0.02 0.63
O/9
“Drug lever selected” represents the number of drug lever selections out of the number of rats that did respond. The term “response level” refers to the total responses made on SD-control or test sessions, expressed as a percentage of total responses on the most recently preceding SS control session. “First food pellet” and “% of total responses on selected lever” are expressed as the median and 95% confidence limits. “Total responses” and “response level” are expressed as the mean (k 1 S.E.M.). The asterisk denotes two-tailed P < 0.05 (Wilcoxon test; SIEGEL,1956). The control data (upper part) are based on 26 determinations in all 9 rats (26 SS- and 26 SD-control sessions); the test data (lower part) are based on 1 determination in these 9 animals.
Narcotic cue
mals (COLPAERTet al., 1975a). Correct lever selection was significant in each rat individually (P < 0.001). The standard fentanyl dose (0.04 mg/kg) attenuated response output significantly (Table 2). Test results
The test data obtained from rats (group I) trained to discriminate 0.16 mg/kg apomorphine plus solvent from solvent are summarized in Table 1. It is shown that the various apomorphine doses (0.04 to 0.16 mg/kg) produced a dose-related drug lever selection, the median effective dose (ED,,) value thus yielded (LITCHFIELDand WILCOXON,1949) being 0.044 mg/kg (95% confidence limits 0.032-0.061). At all doses, apomorphine significantly reduced responding for food reward. Haloperidol (0.02 mg/kg) but not naloxone (5.0 mg/kg) pretreatment (60 min) completely prevented the detection of the apomorphine cue otherwise (see control data) induced by the standard 0.16 mg/kg apomorphine dose (30 min). In both instances, responding was essentially similar to SDcontrol values, and differed significantly from SScontrols. Fentanyl (0.02-0.08 mg/kg) was generalized with apomorphine in a dose-related way (EDso 0.037 mg/kg ; confidence limits 0.025-0.056). Fentanyl also significantly reduced responding. One rat failed to respond after any fentanyl injection, and so did 5 other animals at the dose of 0.08 mg/kg. The generalization of fentanyl with apomorphine was antagonized by haloperidol (0.02 mg/kg) as well as by naloxone (0.63 mg/kg) pretreatment. In addition, naloxone, but not haloperidol, counteracted the response-reducing effect of fentanyl (0.04 mg/kg). After any test treatment neither the accuracy of lever selection (as indicated by the ‘“first food pellet” values), nor the consistency of further responding (as indicated by the values of “% of total responses on selected lever”) was significantly poorer than SS or SD control values.
543
The test data obtained from rats (group II) trained to discriminate 0.04 mg/kg fentanyl plus solvent from solvent are summarized in Table 2. Fentanyl produced the narcotic cue (EDso 0.019 mg/kg; confidence limits 0.01550.025) as well as response reduction in a dose-related way. Halo~ridol(O.08 mg/kg) pretreatment failed to antagonize the narcotic cue. However, although the accuracy of lever selection was not affected, the y0 of total responses on the selected lever was significantly reduced. In contrast, naloxone (0.63 mg/kg) counteracted the narcotic cue as well as the response inhibition otherwise produced by the standard 0.04 mg/kg fentanyl injection. Increasing apomorphine doses (0.16-0.63 mg/kg) consistently produced solvent lever selection and reduced total response output; 5 out of the 9 rats tested did not respond at all following 0.63 mg/kg apomorphine. The combined haloperidol(O.08 mg/kg) plus fentanyI(O.04 mg/kg) treatment was the only instance in which the % of total responses on selected lever sign~cantly deteriorated. “First food pellet” values were always within the range of control observations. DISCUSSION
The present results on the intrinsic effects of apomorphine in rats trained to discriminate 0.16 mg/kg apomorphine from solvent yield an ED,, value of 0.044 mg/kg. This value is a quite accurate replication of earlier data (ED,, 0.066 mg/kg; COLPAERT et al., 1975~) in similarly trained rats, thus confirming the reliability and predictability of data produced by the method used (C~LPAERTet at., 1976b). The finding that fentanyl is generalized with apomorphine (Table 1) constitutes direct evidence for the hypothesis that narcotic analgesics exert central dopaminergic activity. This is further substantiated by the ability of the dopamine receptor blocking neuroleptic
Table 2. Results obtained on rats (n = 9) trained to discriminate 0.04 mg/kg fentanyl plus solvent from solvent
(group II) Treatment
Lever selection
Dose Compound Solvent Solvent -.------Solvent Solvent Waloperidol Naloxone Solvent Solvent Solvent
Response output
30 min
60 min (mg/kg)
-
Compound
Drug lever
(mg/kg)
selected
Solvent
Fentanyl ---__________ Fentanyl Fentanyl 0.08 0.63 -
Dose
0.04 -------_
First food pellet
% of total Total responses
responses on selected lever
O/180 10 (10-10) 1279 (f113) 180/180 10 (l&10) 868 (f133)
100 (10~100) 100 (100-100)
0.01 0.02
O/9 519
10 (l&10) 1278 (,143) 100 (99.9-100) 10 (1GlO) 1153 (+143) 100 (99.2-100)
Fentanyl Fentanyl
0.04 0.04
9J9 O/9
10 (l&10) 10 (l&10)
Apomorphine Apomorphine Apomorphine
0.16 0.3 1 0.63
O/9 O/9 O/4
10 (i&10) 10 (l&10) 10 (l%-t3)
The control data are based on 20 determinations these 9 animafs. See Table 1 for details.
197 (,33) 82.4*(69.2-9&l) 1287 (i 107) 100 (99.7-100) 901 (296) 610 (rt94f 8.5 (F52)
100 (99.9100) 100 ~1~1~) 100 (99.~1~)
Response level loo
71* (k8.4) 110 (f8.2) 96 (k7.4) l8* (f2.7) 91 (k4.3) 68* (i2.9) 54* (i6.4) 6* (f3.2)
in all 9 rats. The test data are based on 1 determination
in
544
F. C. COLPAERT,C. J. E. NIEMEGEERSand P. A. J. JANSSEN
halope~dol (JANSSEN, 1967) to antagonize this generalization. The specific narcotic antagonist naloxone (BLUMBERG and DAYTON,1972) not only antagonized the cuing effect of fentanyl, but also counteracted the response-decreasing effect of the drug. Therefore, the present data can be discussed in terms of current concepts of the mechanism of action of narcotic analgesic drugs. Naloxone possesses a high affinity for the presumed opiate receptor (PERT, PASTERNAK and SNYDER,1973; SIMON,HILLERand EDEMAN,1973) but exerts very little intrinsic pharmacological activity (BLUMBERGand DAYTON, 1972). This implies that naloxone may occupy the available opiate receptor sites or modify the receptor’s conformation, and thereby prevent the occurrence of any narcotic action (thus antagonizing fentanyl’s stimulus properties as well as its response-reducing effects). As the affinity of haloperidol for the opiate receptor is at least 1,000 times lower than that of fentanyl (LEYSEN,personal communication) the neuroleptic fails to counteract the response reduction produced by fentanyl. The fact that, in group I, haloperidol nevertheless antagonizes the cuing action of fentanyl suggests, then, that this cuing action is based on some biochemical action of fentanyl which is initiated by its binding to the opiate receptor, and which results in an increased dopaminergic activity in the brain. In the light of the finding that narcotic analgesics increase the concentration of dopamine’s metabolite, homovanillic acid, in the rat striatum (KUSCHINSKYand HORNYKIEWICZ,1972, 1974), it seems reasonable to suggest that this action may consist of, or result in, an increased dopamine biosynthesis in the presynaptic neurone. The dose-effect relationship of fentanyl in rats trained to discriminate 0.04 mg/kg fentanyl from solvent (EDse 0.019 mg/kg) is virtually identical to earlier data in similarly trained rats (ED,, 0.011 mg/kg; COLPAERTet al., 1975a). Despite the conclusion established above that narcotic analgesics increase central dopaminergic activity, the rats of group II did not generalize apomorphine with the standard fentanyl treatment. This asymmetrical generalization indicates that dopaminergic activity accounts for a significant though not critical component of the discriminative stimulus complex which constitutes the narcotic cue. The latter conclusion is further argued for by the following findings. First, in group II, haloperidol (0.08 mg/kg) pretreatment failed to antagonize the narcotic cue produced by 0.04 mg/kg fentanyl, but significantly deteriorated the “/:,of total responses on the selected lever. The latter effect is very remarkable in view of the fact that only pressing the selected lever (in this case the drug lever) was rewarded. This deterioration seems to be a specific effect, as haloperidol does not affect the mere ability underlying discriminative responding in similarly trained rats (COLPAERTet al., 1975a). Second, whereas the narcotic cue is produced at a fentanyl dose (ED,,) of 0.019 mg/kg in group II (see also COLPAERTrt al., 1975a), the same drug produces detectable dopaminergic activity only at a
dose approximately two times higher (EDSo 0.037 mg/kg; group I). The conclusion that the dopaminergic activity of narcotic analgesics constitutes a significant though not critical component of the narcotic cue may elucidate the relationship which possibly exists between the discriminative stimulus properties and the reinforcing action of these drugs. The self-administration of morphine (HANSON and CLIMINI-VENEMA, 1972; SCHWARTZ and MARCHOK, 1974; SMITH and DAVIS, 1973) as well as that of apomorphine (BAXTER, GLUCKMAN,STEIN and SCERNI,1974) and amphetamine (DAVKS and SMITH,1975; YOKELand WISE, 1975) can be effectively antagonized by the specific dopamine receptor blocking neuroleptics haloperidol (JANSSEN,1967) and pimozide (JANSSEN,NIEMEGEERS, SCHELLEKENS, DRESSE,LENAERTS, PINCHARD,SCHAPER, VAN NUETENand VERBRUGGEN,1968). From these studies, it can be inferred that both neuroleptic drugs effectively block the reinforcing stimuli maintaining the self-administration of morphine, apomorphine and amphetamine in rats. For one thing, this conclusion explains the therapeutic effectiveness of haloperido1 in narcotic dependence (KARKALASand LAL, 1973; LIXOMPTEand FRIEDMAN,1974) and amphetamine abuse (ANGRIST,LEE and GERSHON,1974). For another, it very strongly suggests that the reinforcing stimuli produced by narcotic analgesics are constituted by the dopaminergic action which the present study has shown to be a significant component of the narcotic cue. It is interesting to note that, were the latter suggestion valid, it would imply that rats trained to discriminate apomorphine from solvent may be reliable detectors of the reinforcing action of narcotic analgesic drugs. Acknowledgement-ale expert technical assistance of J. J. M. D. KUYPS is gratefully acknowledged.
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