E�ects of Intracerebroventricular Injection of OpioidPeptides Selective for m, d and k Receptors onDiscriminative Stimulus Properties ofPentazocine in the Rat

MAKOTO UKAI*, EIJI MORI and TSUTOMU KAMEYAMADepartment of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468, Japan

The present study was designed to investigate the e�ects of centrally administered morphine and opioid peptideson the discriminative stimulus properties of pentazocine in the rat. Rats were trained to discriminate 3 mg/kg (s.c.) ofpentazocine from vehicle in a shock avoidance paradigm. A 3 mg/kg (s.c.) dose of pentazocine produced stimuluse�ects in common with those induced by a training dose (3 mg/kg, s.c.) of pentazocine. Morphine (0.1±3 mg, i.c.v.)produced a dose-dependent increase in responding appropriate for pentazocine lever. The m-selective opioid receptoragonist [D-Ala2,NMePhe4,Gly-ol] enkephalin (DAMGO) (0.0003±0.03 mg, i.c.v.) generalized to pentazocine cue.[D-Pen2, L-Pen5] enkephalin (DPLPE) (3 and 10 mg, i.c.v.), a d-selective opioid receptor agonist, produced partialgeneralization to pentazocine cue. However, the k-selective opioid receptor agonist dynorphin A-(1±13) (3 and10 mg, i.c.v.) did not generalize to pentazocine cue. The pentazocine-like discriminative stimulus e�ects of morphine(3 mg, i.c.v.) and DAMGO (0.03 mg, i.c.v.) were fully reversed by intracerebroventricular injection of the m-selectiveopioid receptor antagonist b-funaltrexamine (5 mg, i.c.v.). These results suggest that m-opioid receptors play a majorrole in the discriminative stimulus e�ects of pentazocine, while d-opioid receptors only partially contribute to them.# 1998 John Wiley & Sons, Ltd.

Hum. Psychopharmacol. Clin. Exp. 13: 171±176, 1998.

KEY WORDS Ð pentazocine; morphine; opioid peptides; b-funaltrexamine; discriminative stimulus properties; rat

INTRODUCTION

Because the discriminative stimulus e�ects of drugscorrelate well with their subjective e�ects, drugdiscrimination procedure is often used to clarifythe mechanisms of drug dependence (Holtzman,1985). Pentazocine, a mixed-action opioid agonistantagonist, has a history of abuse. Previously, thediscriminative stimulus e�ects of pentazocine havebeen determined in the rat (Ukai et al., 1989a,b).For example, butorphanol, nalorphine, morphineand SKF10,047 produce stimulus e�ects in com-mon with pentazocine. These ®ndings are quitecompatible with those obtained by receptor bind-ing studies in which pentazocine possesses a�nityfor various opioid receptors (Leslie, 1987).

Furthermore, the indirect dopamine receptoragonist methamphetamine generalizes to pentazo-cine cue. In contrast, SCH23390, a dopamine D1receptor antagonist, but not sulpiride, a dopamineD2 receptor antagonist, has been shown toantagonize the discriminative stimulus e�ects ofpentazocine (Ukai et al., 1989a). Therefore, thediscriminative stimulus e�ects of pentazocine maybe mediated via two components such as opioidand non-opioid, particularly dopaminergic neuro-transmission. However, the involvement of opioidreceptors in the stimulus e�ects of pentazocine hasnot been clari®ed in detail, because the opioidreceptor agonists and antagonist used previouslyhave not fully been speci®c for opioid receptortypes. The selective opioid receptor agonists andantagonist should have been used to clarify thediscriminative stimulus e�ects of pentazocine.*Correspondence to: M. Ukai.

CCC 0885±6222/98/030171±06$17.50# 1998 John Wiley & Sons, Ltd.

HUMAN PSYCHOPHARMACOLOGY, VOL. 13, 171±176 (1998)

Opioids are thought to produce their e�ects byacting at three di�erent types of opioid receptors,such as m, d and k receptors (Pasternak, 1993;Dhawan et al., 1996). Selective agonists andantagonists have played a critical role in disting-uishing these opioid receptor types. For example,the opioid peptides such as [DD-Ala2,NMePhe4,Gly-ol] enkephalin (DAMGO), [DD-Pen2,LL-Pen5]enkephalin (DPLPE) and dynorphin A-(1±13)have been characterized as m-, d- and k-selectiveopioid receptor agonists, respectively (Pasternak,1993; Dhawan et al., 1996).

The present study was designed to fully examinethe involvement of opioid receptor types in thediscriminative stimulus e�ects of pentazocine.Furthermore, in an attempt to clarify the centralmediation of opioid receptors in the discriminativestimulus e�ects of pentazocine, opioids selectivefor receptor types were administered intracerebro-ventricularly.

MATERIALS AND METHODS

Animals

The subjects were male Fischer-derived rats (NihonCharles River Co., Atsugi, Kanagawa, Japan)weighing 210±230 g at the start of discriminationtraining. The rats were housed in a ventilatedcolony room where they had continuous access tofood and water. The lights in the room wereilluminated between 8:00 a.m. and 8:00 p.m.

Apparatus

The apparatus (25� 35� 32 cm) has beendescribed in detail elsewhere (Ukai and Holtzman,1988; Ukai et al., 1989b). Brie¯y, it has one`starting' lever on one wall and two `choice'response levers on the wall opposite to the startinglever. The choice response levers were separated bya clear Plexiglas partition, 5.0 cm wide, thatextended from the ceiling of the test chamber to1.0 cm above the grid ¯oor (Neuroscience Inc.,Tokyo, Japan). A constant-current shock genera-tor (SGC-001, Muromachi Kikai Co., Tokyo,Japan) delivered a scrambled electroshock to thegrid ¯oor of the chamber, which was housed withina ventilated, light-proof, and sound-attenuatingenclosure. Personal computers (PC-9801, UV2,NEC Corporation, Tokyo, Japan) were used tocontrol the schedule contingencies.

Procedure

Rats were trained to discriminate 3 mg/kg ofpentazocine from vehicle in a two-choicediscrete-trial avoidance paradigm (Ukai et al.,1989a,b; Ukai, 1996). The onset of a trial wassignalled by the simultaneous illumination of thehouse light and the presentation of white noise. Atthis time, the rat was required to press the `starting'lever mounted in one wall of the test chamber andthen to press one of the two `choice' leversmounted in the opposite wall. The ®rst startingresponse of the trial terminated the white noise andthe appropriate choice response extinguished thehouse light and ended the trial. Beginning 5 s(avoidance time) after the onset of the trial,2±3 mA shock was delivered to the grid ¯oor ofthe chamber every 3 s in 0.5±1 s pulse until thetwo-response chain was completed. The rat with-out making correct lever-press received electro-shock for a maximum time of 50 s (escape time).The intertrial interval was 50 s during which timethe chamber was dimly illuminated by a red light.Experimental sessions ended after 21 trials or30 min, whichever came ®rst. The ®rst trial of eachsession was considered a `warm-up' and wasexcluded from the data analysis. Training sessionswere conducted 6 days/week. Either pentazocine(3 mg/kg) or its vehicle was injected s.c. 30 minbefore each training session. Training continueduntil rats could reliably complete at least 18 of20 trials (i.e. 90 per cent, exclusive of the ®rst trial)on the appropriate choice level under bothconditions Once this criterion was met, a cannulawas implanted in the lateral cerebral ventricle ofthe rats. Rats were anesthetized with sodiumpentobarbital (50 mg/kg, i.p.). When necessary,chloral hydrate (200 mg/kg, i.p.) was used as anadjunct to anesthesia. The rats were placed in astereotaxic instrument (Narishige Inc., Tokyo,Japan) and a 22-gauge stainless steel guide cannula(No. C313G, Neuroscience Inc., Tokyo, Japan)was inserted into the left lateral ventricle (AP,ÿ0.8 mm relative to bregma; L, �1.4 mm; V,ÿ3.5 mm relative to dura) (Paxinos and Watson,1982; Ukai and Holtzman, 1988; Ukai et al., 1993,1995). After recovery from surgery, trainingsessions were resumed until stimulus control ofbehavior was reestablished (i.e. 18 of 20 trialscompleted on the appropriate choice lever).Drug test sessions were conducted provided therats satis®ed the performance criterion in twoconsecutive training sessions. During test sessions,

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172 M. UKAI ET AL.

both choice levers were activated so that aresponse on either choice lever after the start-ing response terminated the trial. Test sessionsand training sessions were identical in all otheraspects.

Drugs and treatments

The following drugs were used: pentazocine(Yamanouchi Pharmaceutical Co., Tokyo, Japan);morphine hydrochloride (Shionogi PharmaceuticalCo., Osaka, Japan); DAMGO and DPLPE(Peninsula Laboratories Inc., Belmont, CA,USA); dynorphin A-(1±13) (Peptide InstituteInc., Osaka, Japan); b-funaltrexamine (b-FNA)(Research Biochemicals Inc., Natick, MA, USA).The doses of drugs were expressed in terms of thefree base. Pentazocine was dissolved in isotonicsaline (0.9 per cent NaCl, pH 6.4). Morphine,opioid peptides and b-FNA were dissolved insterile isotonic saline (Otsuka PharmaceuticalCo., Tokyo, Japan). Drugs were given 30 minbefore the session. Pentazocine was given s.c.,whereas the others i.c.v. Pentazocine was injectedin a volume of 1.0 ml/kg body weight. Intra-cerebroventricular injections were made by ®rstbackloading saline or the drug solutions into a28-gauge stainless steel injection cannula (C313I,Bioresearch Center Inc., Nagoya, Japan) con-nected to microsyringe by PE-20 tube. A volumeof 5 ml/rat was then administered at a rate of 1 ml/30 s. The injection cannula was left in place for atleast 1 min after injection.

Data analysis

The data were analyzed in terms of the numberof trials completed on the pentazocine-appropriatelever. Trials completed on the vehicle-appropriatelever were recorded but are not shown in the®gures. All of the animals used completed all trialsof every session. A dose of test drug wasconsidered to produce discriminative stimuluse�ects comparable to those produced by thetraining dose (3 mg/kg, s.c.) of pentazocine if arat completed at least 18 of 20 trials on thepentazocine-appropriate lever. A dose of test drugwas considered to antagonize stimulus e�ectscomparable to those produced by the trainingdose (3 mg/kg, s.c.) of pentazocine if a ratcompleted at least 18 of 20 trials on the vehicle-appropriate lever.

RESULTS

Stimulus-generalization curve for pentazocine

Pentazocine (0.3±3 mg/kg, s.c.) produced dose-dependent stimulus e�ects in common with atraining dose (3 mg/kg, s.c.) of pentazocine(Figure 1).

Figure 1. Dose-response curve for discriminative stimuluse�ects of pentazocine in the rat trained to discriminate 3 mg/kg of pentazocine from vehicle (V). Symbols denote themean+ S.E.M. of nine rats used. The true values of trials topentazocine lever were as follows; 0.2+ 0.2 for V, 0.9+ 0.4 forpentazocine 0.3 mg/kg, 13.8+ 3.1 for pentazocine 1 mg/kg, and19.7+ 0.2 for pentazocine 3 mg/kg. Data are reported as thenumber of trials of a 20-trial session completed on thepentazocine-appropriate lever. The remaining trials werecompleted on the vehicle-appropriate lever and are not shown.The dashed horizontal lines at 18 and 2 responses represent thecriteria for pentazocine- and vehicle-appropriate responding,respectively, during training sessions

Discriminative stimulus e�ects of morphineand DAMGO

Morphine (0.1±3 mg, i.c.v.) and DAMGO (0.0003±0.03 mg, i.c.v.) produced stimulus e�ects incommon with pentazocine, although 0.0003 and0.003 mg (i.c.v.) doses of DAMGO did not fullyproduce responding appropriate for pentazocinelever (Figure 2).

Discriminative stimulus e�ects of DPLPE

DPLPE (1±10 mg, i.c.v.) produced dose-dependentresponding appropriate for pentazocine lever, butits e�ects were only partial (Figure 3).

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Discriminative stimulus e�ectsof dynorphin A-(1±13)

Three and 10 mg (i.c.v.) doses of dynorphinA-(1±13) did not completely produce stimuluse�ects in common with pentazocine (Figure 4).

E�ects of b-FNA

The pentazocine-like discriminative stimulus e�ectsof morphine (3 mg, i.c.v.) and DAMGO (0.03 mg,i.c.v.) were almost completely reversed by pretreat-ment with b-FNA (Figure 5).

DISCUSSION

The discriminative stimulus e�ects of pentazocinein the rat used in this study were establishedafter 35 sessions of training. The discriminativestimulus e�ects of pentazocine in the rat weredose-dependent similar to those in squirrelmonkeys under shock-avoidance schedule (Whiteand Holtzman, 1982). Intracerebroventricularinjection of morphine (3 mg, i.c.v.) and them-selective opioid receptor agonist DAMGO(0.03 mg, i.c.v.) generalized to pentazocine cue.

Figure 2. Discriminative stimulus e�ects of graded doses of morphine and[DD-Ala2,NMePhe4,Gly-ol] enkephalin (DAMGO) in the rat trained to discriminate3 mg/kg of pentazocine (P) from vehicle (V). Symbols denote the mean+S.E.M. offour rats used. The true values of trials to pentazocine lever were as follows;19.8+ 0.3 for P, 0.5+ 0.3 for V, 0.3+ 0.3 for morphine 0.1 mg, 6.5+ 3.8 formorphine 0.3 mg, 10.5+ 4.7 for morphine 1 mg, 19.3+ 0.5 for morphine 3 mg,0.5+ 0.3 for DAMGO 0.0003 mg, 9.3+ 3.5 for DAMGO 0.003 mg, and 19.8+ 0.3for DAMGO 0.03 mg. Other details are the same as in Figure 1

Figure 3. Discriminative stimulus e�ects of graded doses of[DD-Pen2,LL-Pen5] enkephalin (DPLPE) in the rat trained todiscriminate 3 mg/kg of pentazocine (P) from vehicle (V).Symbols denote the mean+S.E.M. of four rats used. The truevalues of trials to pentazocine lever were as follows; 19.8+ 0.3for P, 0.5+ 0.3 for V, 0.8+ 0.5 for DPLPE 1 mg, 8.8+ 4.2 forDPLPE 3 mg, and 13.5+ 3.8 for DPLPE 10 mg. Other detailsare the same as in Figure 1

Figure 4. Discriminative stimulus e�ects of graded doses ofdynorphin A-(1±13) (DYN) in the rat trained to discriminate3 mg/kg of pentazocine (P) from vehicle (V). Symbols denotethe mean+S.E.M. of four rats used. The true values of trials topentazocine lever were as follows; 19.5+ 0.3 for P, 0 for V,0.5+ 0.5 for DYN 3 mg, and 6.3+ 4.4 for DYN 10 mg. Otherdetails are the same as in Figure 1

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174 M. UKAI ET AL.

The results are in accordance with those in squirrelmonkeys and rats (Kuhn et al., 1976; Hirschhorn,1977; White and Holtzman, 1982; Ukai et al.,1989b). The discriminative stimulus e�ects ofpentazocine were almost fully antagonized by them-selective opioid receptor antagonist b-FNA,suggesting that the discriminative stimulus e�ectsof pentazocine are mediated through m-opioidreceptors. Furthermore, b-FNA (5 mg, i.c.v.)almost fully antagonized the pentazocine-likediscriminative stimulus e�ects of morphine, imply-ing that the generalization of morphine to penta-zocine cue is mediated through m-opioid receptorsin the brain. In fact, the discriminative stimuluse�ects of morphine generalize to DAMGO, whilethe generalization is antagonized with b-FNA(5 mg, i.c.v.) (Locke and Holtzman, 1986; Ukaiand Holtzman, 1988). The d-selective opioidreceptor agonist DPLPE or the k-selective opioidreceptor agonist dynorphin A-(1±13) does notgeneralize to morphine cue (Ukai and Holtzman,1988). Thus, the present data support that thediscriminative stimulus e�ects of morphine aremediated through m-opioid receptors.

In contrast, DPLPE (10 mg, i.c.v.) showedpartial generalization to pentazocine cue. Becausethe animals treated with a 10 mg (i.c.v.) dose ofDPLPE were highly sensitive to external stimuli,such as sound and touch, we could not inject more

than a 10 mg (i.c.v.) dose of DPLPE. The analgesicpotency of DPLPE has been demonstrated to beone-third of morphine (Galligan et al., 1984). Inthe present study, morphine (3 mg, i.c.v.) fullygeneralized to pentazocine cue, while a 10 mg(15.5 nmol, i.c.v.) dose of DPLPE equivalent to a3 mg (10.5 nmol, i.c.v.) dose of morphine inanalgesic potency did not generalize to pentazocinecue. Therefore, d-opioid receptors do not fullycontribute to the discriminative stimulus e�ects ofpentazocine. However, the dopamine D1 receptorantagonist SCH23390 has been reported to inhibitthe discriminative stimulus e�ects of pentazocine(Ukai et al., 1989a). In addition, because thedopamine D1 receptor agonist SKF38393-inducedbehavioral responses are augmented by DPLPE(Toyoshi et al., 1992), d-opioid receptor agonistswould play a facilitating role in the discriminativestimulus e�ects of pentazocine.

The k-selective opioid receptor agonist dynor-phin A-(1±13) did not generalize to pentazocinecue. This result is consistent with that by usingU-50,488H (unpublished observation). Although itis well known that the analgesic e�ects ofpentazocine involve k-opioid receptors, the presentdata demonstrate that the discriminative stimuluse�ects of pentazocine are not associated withk-opioid receptors.

In conclusion, the discriminative stimulus e�ectsof pentazocine related to the liability of psychicdependence exclusively result from the activationof m-opioid receptors, while d-opioid receptors areonly partially involved in it.

ACKNOWLEDGEMENT

This research was supported in part by a Grant-in-Aid for Scienti®c Research from the Ministry ofEducation, Science, Sports and Culture, Japan.

REFERENCES

Dhawan, B. N., Cesselin, F., Raghubir, R., Reisine, T.,Bradley, P. B., Portoghese, P. S. and Hamon, M.(1996). International Union of Pharmacology. XII.Classi®cation of opioid receptors. PharmacologicalReviews, 48, 567±592.

Galligan, J. J., Mosberg, H. I., Hurst, R., Hruby, V. J.and Burks, T. F. (1984). Cerebral d opioid receptorsmediate analgesia but not the intestinal motility e�ectsof intracerebroventricularly administered opioids.Journal of Pharmacology and Experimental Thera-peutics, 229, 641±648.

Figure 5. Pentazocine-like discriminative stimulus e�ects ofmorphine (3 mg), DAMGO (0.03 mg) and their combinationswith b-funaltrexamine (5 mg) in the rat trained to discriminate3 mg/kg of pentazocine (P) from vehicle (V). b-FNA:b-funaltrexamaine (5 mg) alone. n Ð n: morphine; s Ð s:DAMGO. Symbols denote the mean+ S.E.M. of four ratsused. The true values of trials to pentazocine lever were asfollows; 19.8+ 0.3 for P of morphine and DAMGO, 0.5+ 0.3for V of morphine and DAMGO, 0.3+ 0.3 for b-FNA alone ofmorphine, 0 for b-FNA alone of DAMGO, 19.3+ 0.5 formorphine alone, 19.8+ 0.3 for DAMGO alone, 0 for morphineor DAMGO plus b-FNA. Other details are the same as inFigure 1

# 1998 John Wiley & Sons, Ltd. HUMAN PSYCHOPHARMACOLOGY, VOL 13, 171±176 (1998)

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Hirschhorn, I. D. (1977). Pentazocine, cyclazocine, andnalorphine as discriminative stimuli. Psychopharma-cology, 54, 289±294.

Holtzman, S. G. (1985). Drug discrimination studies.Drug and Alcohol Dependence, 14, 263±282.

Kuhn, D. M., Greenberg, I. and Appel, J. B. (1976).Stimulus properties of the narcotic antagonist penta-zocine: similarity to morphine and antagonism bynaloxone. Journal of Pharmacology and ExperimentalTherapeutics, 196, 121±127.

Leslie, F. M. (1987). Methods used for the study ofopioid receptors. Pharmacological Reviews, 39,197±249.

Locke, K. W. and Holtzman, S. G. (1986). Behaviorale�ects of opioid peptides selective for m or d receptors.I. Morphine-like discriminative stimulus e�ects.Journal of Pharmacology and Experimental Thera-peutics, 238, 990±996.

Pasternak, G. W. (1993). Pharmacological mechanism ofopioid analgesics. Clinical Neuropharmacology, 16,1±18.

Paxinos, G. and Watson, C. (1982). The Rat Brainin Stereotaxic Coordinates. Academic Press, Sydney.

Toyoshi, T., Ukai, M. and Kameyama, T. (1992).Combination of a d opioid receptor agonist but nota m opioid receptor agonist with the D1-selectivedopamine receptor agonist SKF38393 markedlypotentiates di�erent behaviors in mice. EuropeanJournal of Pharmacology, 213, 25±30.

Ukai, M. (1996). Drug discrimination and in vivopharmacology of drugs. Methods and Findings

in Experimental and Clinical Pharmacology, 18,59±65.

Ukai, M. and Holtzman, S. G. (1988). Morphine-like discriminative stimulus e�ects of opioid pep-tides: possible modulatory role of D-Ala2-D-Leu5-enkephalin (DADL) and dynorphin A-(1±13).Psychopharmacology, 94, 32±37.

Ukai, M., Mori, E. and Kameyama, T. (1993). Cocaine-like discriminative stimulus properties of thed-selective opioid receptor agonist [DD-Pen2,LL-Pen5]enkephalin in the rat. European Journal ofPharmacology, 231, 143±144.

Ukai, M., Mori, E. and Kameyama, T. (1995). E�ectsof centrally administered neuropeptides on discrim-inative stimulus properties of cocaine in the rat.Pharmacology Biochemistry and Behavior, 51,705±708.

Ukai, M., Nakayama, S., Hamada, T. and Kameyama,T. (1989a). Blockade of dopamine D1 receptors bySCH23390 antagonizes discriminative stimulus e�ectsof pentazocine. Brain Research, 496, 377±379.

Ukai, M., Nakayama, S., Hamada, T. and Kameyama,T. (1989b). Discriminative stimulus properties ofthe mixed agonist-antagonist pentazocine in therat using two-choice discrete-trial avoidance para-digm. Pharmacology Biochemistry and Behavior, 33,355±359.

White, J. M. and Holtzman, S. G. (1982). Properties ofpentazocine as a discriminative stimulus in the squirrelmonkey. Journal of Pharmacology and ExperimentalTherapeutics, 223, 396±401.

# 1998 John Wiley & Sons, Ltd. HUMAN PSYCHOPHARMACOLOGY, VOL 13, 171±176 (1998)

176 M. UKAI ET AL.