Contribution of supraspinal μ- and δ-opioid receptors to antinociception in the rat

Etrropearr Jatrnrnl of Pharntacoiogy, 205 (1991) 247-252 0 1991 Elsevier Science Publishers B.V. All righis reserved 00&t-2999/91,,503.50

Christine Miaskowski ‘, Yetunde 0. Taiwo 3**and Jon D. Levine Scbals of ’ ~tt~s~ttg, ’ Med~citteand .’ Betttistrf, Uttit,ers~~of Ca@mia

2+3

at San Frattcisfo, San Francisco, CA %fI#,

U.S.A.

Received IO June 1991, revised MS received 30 August 1991. accepted 10 September 1991

This study evaluated the co~trib~t~o~of suprmpinat opioid receptors to the ~~u~~~o~ of ~~~i~~icg~ti~~, in tire rat. 1.rr.v. ~drn~~~stratio~of a selective F- (DAMGO) and a selective S- (DPDPE), but not a selective K- (U50,4$8Hf opioid receptor agonist, produced significant dose-dependent increases in mechanical nociceptive thresholds. ICI 174,864,a &opioid receptor antagonist, completely blocked the antinociceptive effects produced by DPDPE ([I)-Pen’,D-Pen’~enkephalin) at a dose that had no effect on the increases in nociceptive thresholds produced by DAMGO ([D-Ala’,N-MePhe-‘,Gty”-ol]enkephaIin). The simultaneous i.c.v. ~drnjnis~ratji~~~ of a ~w-an~in~~ce~t~vedose of DAMGO or DPDPE given in combination with sequentially increasing doses of the other opioid agonist, produced synergy (i.e., a more than additive antinociceptive effect). at the lower doses tested. The results of these experiments provide evidence to support the suggestion that both supraspinal JL- and &opioid receptors contribute to the production of ant~nocicep~ion, in the rat.

~tin~iception~

Cr-Opioids;S-Opioids; K--0pioids:Intracerebroventricutar; Synergy

Previous studies have demonstrated that opioid agonists produce increases in nociceptive thresholds through actions at CL-,K- (Martin et ai,, $9761 and S(Lord et al., 1977) opioid receptors. However, controversy still exists as to the relative contribution of each of these opioid receptors, at supraspinal sites, to the production of antino~icepti~n (Fang et al., 1986; Czlonkowski et al., 1987; Heyman et al., 1988; Leighton et al., 1988; Milan et al,, 1989). Some studies (Fang et al., 1986; Heyman et at., 19%) have suggested that onfy supraspinal CLreceptors are involved in antinociception while others {Calca~netti et al., 1984; Galligan et al., 1984; Porreca et al., 1984; Heyman et al., 198% Porreca et al,, 1987; Kovacs et al., 1988) suggest that both pand &opioid receptors are involved. In addition, controversy also exists over the contribution of supraspinal K receptors in ~p~oid-induced anti~ociception (Piercey et al., 1982; Schmauss and Yaksh, 1983; Czlo~kowski et al,, 1987; Leighton et al., 1988; Takemori et al., 19881.

Correspondence to: J.D. Levine. Division of Rheumatology, U426/80x 0724, University of California, San Francisco, CA 941430724, U.S.A. Tel. 1.41.5.4765108, fax I.415476 4845. * Present address: The Procter and Gamble Company, Cincinaati. OH 452394707, U.S.A.

In order to further evaIuate the relative contribution of supraspinal CL-,&- and K-opioid receptors to antinociception, in the rat, we examined the effect of i.c.v. administration af the seiective S-opioid antagonist, ICI f74,864 (Cotton et al., 1984; Heyman et al., f98’7; Porreca et a., 1987), on i.c.v. p- (DAMGO, Handa et al., 19811 and S- (DPDPE, Mosberg et al., 19831 opioid-induced increases in nociceptive thresholds, as well as the antinocj~eptive effects produced by i.c_v. administration of a K- (U50,4SSH, VonVoigtIander et ai., 1983) opioid agonist. In addition, we evaluated the antino~i~eptive effects produced by the simuItaneo~s i.c.v. administration of the selective p- and S-opioid agonists.

2. Materials and methods The experiments were performed OR 260-280 g, male Sprague-Dawley rats (Bantin and ~n~~~, Fremont, CA). One week prior to the experiments, under pentobarbital anesthesia (65 mg/kg), a 22-gauge stainless steel tan&a was stereota~t~~al~y implanted into the third ventricle and was f?xed in pIace with acrylic dental cement. A 30-gauge stainless steel stylette was placed in the i.c.v. cannula to keep it patent. Rats that exhibited neurological deficits, following the surgical procedure, were eliminated from the study.

a~b-&[jtto ww-with&anal test (Randit L” yf~$ S&to, 19571 was used to measure mechanics! tn~&xptive threshold. The mechanical stimulus was ~~~~~~~ with a I&f3 Basils analgesymeter iSWelting, , fl) which generates 9 linearty increasing meree, applied by a dome-shaped plastic tip, to surface of the rat’s hindpaw. The nocicepti:r tb~~~d was defimd as the force in g at which the ~ni~l ~ithd~~~ ie paw. The rnts were trained in the test procedure, for 3 h daily. for five days prior to ~~t~ ~~!e~tio~ ITaiwo et a!., 19S9k on the day of the experiment, rats were first tested at 5 min intztvals for Z h. Baseline thresholds were defined as the mean of the fast six measurements prior to drug administration. The avetzge baseline threshold, in this _ap of rats was 89.6 f 14.2 g fmean F S.E.M.; p3 = 321. The effect of the opioid agonists on nociceptive tb~shold wns rested 15. 20 and 25 min after i.c.v. administration. at a time of peak effect of these agonists tGa!!igan et a!.. 1984 Leighton et a!.. 1958). The change in noeiceptive threshold. for each dose of drug, was caiculated as the average percentage change from basehne threshold of these three consecutive measurements. A cut-off of 5tlil g was used for the pawwithdrawal test and a value of 5tX! g assigned if an animal reached cut-off. Dose response curves for i.c.s. DAM90 ([DAla’&-MePhet.Gly “-o!]enkephalinl, DPDPE ([DPen’.Pe$]enkephslin) (Peninsula Laboratories, Belmont. CA1 and U~~.4~~~, fa generous gift from Dr. Robert Lahti, Upjohn, Kalamazoo, MI), as single agents. were determined by a~linistering sequentially increasing doses of each drug. in a volume of 1 ~1, at ,io min intetvais. We have shown that administration of a single high dose of an opioid produces an inc-;ase in nociceptive threshold of similar magnitude as when the same dose is administered as the last dose in an ascending series, as performed in these experiments unpublished results). Therefore, the sequentially lower doses. preceding the highest dose, do not appear to contribute to the antinociceptive effects produced, in these er+periments, by the highest dose of opioid agonist. The effect of administe~ng the setective S-receptor antagonist ICI 174.864 (N,N-dia!!y-Try-Aib-Aib-PheLeu-G!!z Cotton et al., 1984; Cambridge Research Biochemicals, Valley Stream. NY), at a dose (3 pg) which has been shown to be a selective antagonist at the S-opioid receptor (Porreca et a!., 1984). was tested with each sequentially increasing dose of DAMGO or DPDPE adntinistered into the third ventricle. A!! drugs, used in the study, were dissolved in saline. TO test if actuation of supraspinal p- and S-opioid receptors with selective opioid agonists, produced more

than additive antinociceptive effects, the following combinations were tested: (11 a low-antin~iceptive dose (i.e., a dose which produced approximately a 20% increase in nociceptive threshoId) of DPDPE 150 fg) was co-administered with each sequentially increasing dose of DAMGO and (21 a low-antinociceptive dose of DAMGO (50 fg> was co-administered with each sequentially increasing dose of DPDPE. The dose-dependent effects of the combination regimens were then compared with the dose-dependent effects of the agents administered alone. The number of rats, in each group, is indicated in the figure legends. The total duration of the experiments was 5.S h for the i.c.v. control studies, 4 h for the antagonist studies, and 4.5 h for the interaction studies. 2.2. Statistical art&m

Statistical analysis of the dose-dependent effects of i.c.v. administration of DAMGO, DPDPE and US0,4SSH, as single agents, on nociceptive thresholds was performed using a one-way analysis of variance WNOVA). In order to determine if the opioid-induced increases in nociceptive thresholds for either the single agents or the combination regimens reached a plateau at the highest doses tested, post-hoc contrasts of the dose-dependent effects of DAMGO and DPDPE, as single agents, and for each of the combination regimens were done using the Scheffe post-hoc test (Melton and Tsokos, 1983; Cohen and Cohen, 1983; Sokal and Rohlf, 1987; Marascuilo and Serlin, 19881. The results of these post-hoc contrasts, comparing differences between dose effects, are presented in the results section, using successively larger integers to represent each successively higher dose in the cumulative dose-response curve (from 0 = the vehicle or low-antinociceptive dose up to 4 = the highest dose of the opioid agonist). A statistical analysis for synergistic interactions between the dose-dependent effects of single agents and combination regimens is not possible over the range of doses where the dose-dependent effects for either the single agent or the combination regimen have reached a plateau. Therefore, following the statistical procedure to determine the presence of a plateau for either the single agents or the combination regimens, a twofactor, repeated measures ANOVA was done using the portion of the dose-response curves below the plateau effect. If the dose-response curves are divergent, then a statistically significant interaction term, in a two-factor, repeated measures ANOVA, demonstrates that the difference between the two groups deviates from parallelism and therefore is more than additive, that is synergistic (Melton and Tsokos, 1983; Cohen and Cohen, 1983; Sokal and Rohlf, 1987; Marascuilo and

219

Serlin, 1988). Differences were considered statistically significant at the P < 0.05 level. 3. Results 3.1. Ad~~inistrat~o~~of p-, 5-, and u-opioid agonkts as singie agents

I.c.v. administration of sequentially increasing doses of DAMGO and DPDPE, as single agents, each produced statistically si~ficant, dose-dependent increases in nociceptive thresholds (F(4,28) = 33.09 and (F(4,28) = 21.85, respectively, both P < 0.001; fig. 11, using one-way ANOVA. Intracerebroventricular administration of sequentially increasing doses of W50,488H did not produce a significant increase in nociceptive threshold (Ff6,30) = 1.17, P > 0.05).

Fig. 2. Dose-dependence relationship for the effects of i.c.v. administration of sequentially increasing doses of DAMGG fN = 8) and DPDPE (N = 81, in the presence and absence of ICI 174.864 (bth N = 8) on paw-withdrawal nociceptive thresholds.

3.3. Co-administratiart of DPDPE (50 fs) with sequentialIy increasi~lgdoses

of DAMGO (fig. 3AI

3.2. Antagonist studies ICI 174,864 markedly antagonized the effects of the selective &opioid agonist, DPDPE while it did not significantly effect the increases in nociceptive thresholds produced by the h-agonist, DAMGO (fig. 2). When the effects of DPDPE plus ICI 174,864 were compared with the effects of DPDPE administered as a single agent, ANOVA revealed significant main effects of group and dose (F(1,14) = 21.66 and R3,42) = 31.55, respectively, both P < 0.001) as we!1 as a significant group X dose interaction (F;‘(3,42)= 22.86, P < 0.001). However, there was no significant group effect (F(1,14) = 0.66, P > 0.05) or group X dose interaction (F3,42) = 0.56, P > 0.05) when the effects produced by DAMGO plus ICI 174,864 were compared with the effects produced by DAMGO administered as a single agent.

Post-hoc contrasts comparing the dose-dependent increases in nociceptive thresholds produced by the A.

o-owro

*-*lWW+-@W 150 r 125 .

T 125 O-ODAbtGO Cl-UDPDPE A-AU50.488H

150

100 75

8 za

50

125

25 0 -25

k!

5 fg

0.5 P1

50 Pll

3 “9

Fig. 1. Dose-dependence relationship for the i.c.v. administration of sequentially increasing doses of DAMGO (N = 8). DPDPE (N = 8). and U50,488H (N = 16) on paw-withdrawal nociceptive thresholds. In this and all subsequent figures, responses are graphed as percentage change from baseline threshold after administration of the various agents. Each point in the figure represents the mean k S.E.M. Some error bars are contained within the symbols.

1

5 fP

OAF4

(0)

0f

121

=n9 (3)

5ng (‘1

Fig. 3. (A) The effects of administering sequentially increasing doses of DAMGO (N = 8) and a low-ant nociceptive dose (50 fg) of DPDPE co-administered with each sequentially increasing dose of DAMGO (N = 16) on paw-withdrawal nociceptive thresholds. (B) The effects of administering sequentially increasing doses of DPDPE (N = 8) and a low-antinociceptive dose (50 fg) of DAMGO co-administered with each sequentially increasing doses of DPDPE (N = 16) on paw-withdrawal nociceptive thresholds. The integers below the doses on the x-axis are the values reported in the results section for the dose-dependent effects of the single agent and the combination regimen.

!&J&W contrasts comparing the dose-dependent increases in nociceptive tbr~s~o~ds produced by the sIngIs agent (i.e.. PDPE) and the combination regimen are as foliows: (11 i.c.v. PDPE effects of dose AMGO + i.c.v. = I and (91 tow-dose i.c.v. FE effects of dose 2 < 3 = 1 (all dara demonstrate a plateau effect at the hvo highest dews of opioid agonist administered either as a singEe agent or as part Of the combination regimen. Based on the resuirs of the post-hoc contrasts a hvofactor. repeated measures ANOVA comparing the effc‘cts of the first three doses of DPDPE as a single agent to the DAMGO and DPDPE combination revealed sFatisticaIJy significant main effects of group and dose (F(!.X! = Is.05 and W.44) = 48.47. respectively, bath P < ~.~~~~ as well as a significant group x dose inEeraction ~l=G!-!~ = 3.12. P < 0.09.

~~~tro~~rs~ still esists with respect to the relative ~~~t~~b~tio~ of supraspinal ,u and 6 receptors to opiuced antinociception (Heyman et al., 1988). ata s~~~ort~~g the exclusive involvement of supraspinal g-opioid receptors in antinociceptive mechanisms has been provided by some studies (Fang et al.. 1986: Heyman et al.. 1986). In contrast, other studies using the selective b-opioid agonist, DPDPE (Galcagnetti et al.. 1984: Galligan et al., 1984; Porreca et al.. 1984; Kovacs et al., 1988) and the selective S-antagonist. ICI 174,864 (Heyman et al., 1987; Porreca et a!.. 1987). have provided evidence for a contribution of supraspinal 8. as well as. p receptors in antinociception. In thi we demonstrated that i.c.v. administration of 0 and DPDPE produced dose-de-

pendent increases in mechanical nociceptive threshold in the rat: that JGJ 174.S64 antagonized the antinocicaptive effect of the S-agonist, DPDPE, but trot that of the ~-agonist, DA~G~: and that SinlUJFaneOUsi.c.v. adnli~~stration of DAMGO and DPDPE can produce antinociceptive synergy. These data provide support for the . Jggestion that both g- and fi-opioid receptors contribute to supraspimd antinociceptive effects in the rat. While the mechanism underlying the antin~ice~ tive synergy produced by i.c.v. administration of w- and Li-opioid agonists is unknown, our findings are compatibJc with the SUggeStiOnthat Fwo poptdations of opiate receptors. on the same or different neurons, can interact by enhancing each others activity to produce a synergistic interaction. Our findings contrast with work by Schuman et al. (19861, who demonstrated, using a thermal nociceptive test, that i.c.v. coadministration of DPDPE with morphine reduced the antinociceptive effects of morphine. At the present time, it is difficult to explain why differerces in antinociceptive responses were observed by the various investigators. Perhaps some of the differences relate to the specific supraspinal site injected (Jensen and Yaksh, 19861, the dose of agonist administered, or the nature of the nociceptive stimulus used in the experiments (Kuraishi et al., 1983). Additional studies, using a variety of nociceptive tests, across a large dose range of agonist, and using selective antagonists at each site are needed to resolve this controversy. The post-hoc contrasts of the antinociceptive doseresponse effects produced by DAMGO and DPDPE, as single agents, as well as the combination regimens. consistently demonstrated a plateau of their antinociceptive effects at the highest doses tested. This plateau for the antinociceptive effects of supraspinal administration of receptor selective opioids was not observed, using the same antinociceptive model, when the same opioids were administered at the spinal site (Miaskowski et al., 1990; Sutters et al., 1990). The lack of a spinal plateau occurred despite the fact that intrathecal administration of the opioids produced larger increases in nociceptive thresholds. Thus, plateau effects for opioid-induced antinociception appears to be a property of antinociceptive circuitry activated by opioid receptors located at supraspinal sites. We found no antinociceptive effect with i.c.v. administration of the selective K-agonist, U50,4SSH, even at a dose of 5 pg (unpublished results). While several studies have concluded that K-agonists produce their antinociceptive effects through activation of spinal rather than supraspinal receptors (Piercey et al., 1982; Schmauss and Yaksh, 1983; Takemori et al., 19881, others have described increases in nociceptive thresholds, when U50,4SSH was administered at supraspinal sites (Czlonkowski et al., 1987; Leighton et al., 1988).

251

One difference between those studies that showed a supraspinal K-opioid effect, and those that demonstrated no supraspinal K effect, is that the doses of U50,488H used in those studies that showed an antinociceptive effect (Czlonkowski et al., 1987; Leighton et al., 19881, were relatively larger (ranging from 33 to 100 pg) than the doses used in the present study. Perhaps, as with the p- and b-opioid agonists, differences in antinociceptive effects may be explained

partially by the specific anatomical site of drug injection, the type of nociceptive stimulus used, or the intensity of the no~iceptive stimulus (MilIan, 1989; Millan et al., 1989). In summa~, this study provides evidence to support the suggestion that, in the rat, both CL-and S-opioid receptors contribute to supraspinai antin~iceptive mechanisms. Acknowledgements We would like to thank Drs. Steven M. Paul and Lewis B. Sheiner for statistical consultation. This work was supported by NIH Grant DE08973 and Grants from the American Nurses’ and Rita Allen Foundations. C. Miaskowski is the 1990 Sterling Drug Scholar.

References Calcagnetti, D.J., F.J. Helmstetter and M.S. Fanselow, 1984, Analgesia produced by centrally administered DAGO, DPDPE, and U50,488H in the formalin test, Eur. J. Pharmacol. 153, 117. Cohen, J. and P. Cohen, 1983. Applied Multiple Regression/ Correlation Analysis for the Behavioral Sciences (Lawrence Erlbaum Associates, Publishers, Hillsdale, New Jersey) p. 301. Cotton, R., M.G. Giles, L. Miller, J.S. Shaw and D. Timms. 1984, ICI 174,864: a highly selective antagonist for the opioid delta-receptor, Eur. J. Pharmacol. 97, 331. Czlonkowski, A., M.J. Millan and A. Herz, 1987, The selective kappa-opioid agonist, U50,488H, produces antinociception in the rat via a supraspincl action, Eur. J. Pharmacol. 142, 183. Fang, F.G., H.L. Fields and N.M. Lee, 1986, Action at the mu receptor is sufficient to explain the supraspinal analgesic effect of opiates. J. Pharmacol. Exp. Ther. 238, 1039. Galligan, J-J., H-1. Mosberg. R. Hurst, V.J. Hruby and T.F. Burks, 1984, Cerebral delta opioid receptors mediate analgesia but not the intestinal motility of intracerebroventricularly administered opioids, J. Pharmacol. Exp. Ther. 229. 641. Handa, B.K., AC. Lane, J.A.H Lord, B.A. Morgan, M.J. Rance and C.F.C. Smith, 1981, Analogues of beta-LPH,,_, possessing selective agonist activity at mu-opiate receptors, Eur. J. Pharmacol. 70, 531. Heyman, J.S., R.J. Koslo, H.I. Moaberg, R.J. Tallarida and F. Porreca, 1986, Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in viva: studies with receptor selective agonists, Life Sci. 39, 1795. Heyman, J.S., S.A. Mulvaney, H.I. Mosberg and F. Porreca, 1987, Opioid delta-receptor involvement in supraspinal and spinal antinociception in mice, Brain Res. 420, 100. Heyman, J.S., J.L. Vaught, R.B. Raffa and F. Porreca, 1988, Can supraspinal delta-opioid receptors mediate antinociception?, Trends Pharrnacol. Sci. 9, 134.

Jensen, T.S. and T.L. Yaksh. 1986, III. Comparison of the antinociceptive action of mu and delta opioid rece;stor ligands i,n the periaqueductal gray matter, medial and paramediat ventral medulla in the rat as studied by microinjection technique, Brain Res. 372, 301. Kovacs, G.L., N. Nyolczas, M. Krivan and K. Gulya, 1988, Analgesic and tolerance-inducing effects of the highly selective delta opiod agonist [D-PenZ.D-Pen’] enkephalin in mice. Eur. J. Pharrnaco!. 150, 347. Kuraishi. Y.. Y. Harada, S. Artani. M. Satoh and H. Takagi, 1983, Separate involvement uf the spinal noradrenergic and serotonergiC Systems in morphine analgesia: the differences in mechanical and thermal algesic tests, Brain Res. 273, 242. Leighton. G.E.. R.E. Rodriquez. R.G. Hill and J. Hugh,es,19%. Kappaopioid agonists produce antinociception after i.v. and i.c.v. but not intrathecal administration in the rat, Br. 1. Ptarmacol. 93, 553. Lord. J.A.H., A.A. Waterfield, J. Hughes and H.W. Kosterlitz. 1977, Endogenous opioid peptides: multiple agonists and receptors, Nature (London). 267, 495. Marascuilo. L.A. and R.C. Serlin, 1988, Statistical Methods for the Social and Behavioral Sciences (W.H. Freeman and Company, New York) p. 448. Martin. W.R., C.G. Eades. J.A. Thompson. R.E. Huppler and P.E. Gilbert, 1976, The effects of morphine- and nalorphine-like drugs in the non-dependent and morphine-dependent chronic spinal dog, I. Pharmacol. Exp. Ther. 197,517. Melton, J.S. and J.O. Tsokos, 1983, Statistical Methods in Biological and Health Sciences (McGraw-Hill Book Company, New York) p. 271. Miaskowski. C.. Y.O. Taiwo and J.D. Levine, 1990, K- and S-opioid agonists synergize to produce potent analgesia. Brain Res. 509, 165. Millan, M.J., 1989, Kappa-opioid receptor-mediated antinociception in the rat. 1. Comparative actions of mu- and kappa-opioids against noxious thermal, pressure and electrical stimuli, J. Pharmacol. Exp. Ther. 251. 334. Millan. M.J.. A. Czlonkowski, A. Lipkowski and A. Herz, 1989, Kappa-opioid receptor-mediated antinociception in the rat. II. Supraspinal in addition to spinal sites of action, J. Pharmacol. Exp. Ther. 251. 342. Mosberg, H.I., R. Hurst, V.I. Hruby, K. Gee, H.I. Yamamura, J.J. Galligan and T.F. Burks, 1983, Bis-penicillamine enkephalins possess highly improved specificity toward delta opioid receptors, Proc. Natl. Acad. Sci. (U.S.A.). 80, 5871. Piercey, M.F., R.A. Lahti, L.A. Schroeder, F.J. Einspahr and C. Barsuhn. 1982, U-50,488H, a pure kappa receptor agonist with spinal analgesic loci in the mouse, Life Sci. 31, 1197. Porreca, F.. H.I. Mosberg, R. Hurst, V.J. Hruby and T. Bcrks, 1984, Roles of mu, delta. and kappa opioid receptors in spinal and supraspinal mediation of gastrointestinal transit effects and hotplate analgesia in the mouse. J. Pharmacol. Exp. Ther. 23tc. 341. Porreca, F., J.S. Heyman. H.I. Mosberg. J.R. Omnaas and J.L. Vaught, 1987. Role of mu and delta receptors in the supraspinal and spinal analgesic e”fects of [D-Pen’,D-Per?]enkephalin in the mouse, J. Pharmacol. Exp. Ther. 241, 393. Randall, L.O. and J.J. Selitto, 1957, A method for measurement of analgesic activity on inflamed tissue. Arch. Int. Pharmacodyn. 111,409. Schmauss. C. and T.L. Yaksh, 1983 In vivo studies on spinal opiate receptor systems mediating antinociception. II. Pharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat, J. Pharmacol. Exp. Ther. 228. I. Schuman, C., D. Gmerek. H. Mosberg. J. Woods, K. Rice and A. Jacobson, 1986, Delta opiate receptors in thermal analgesia, The Pharmacologist 28, 97.

ia&+!. RR

.wd F J R&StPf.I*“. fntnxhrctia~ to Bitolalrslic~ t\V.H. f.~~p.u~y. New \“ixk) p. tXS. !&ask~mxli~Y.0. Taiwr and J.D. Levine. IWO. sumxx. I;.:\.. ~\na&ew synergy and m1pr0w.l ttmtor tuncti0n produced by ~~~~.~~~~~~~ of r-d- 2nd ~-h.qU&. Brain Res. 5.30. -Xl. T~UWX.Y‘.O.. TJ. C&err-e and J.D. Levine. IW9. fiie contribution tF&niRg 10 wnsitivitv in the rwciceptive pw-\vithdraaal test. ta Rcs. x0. E5S. Frwu?“M &

Takemtrri. A.E.. B.Y. Ho, J.S. Naeseth and P.S. Portophese. 19X8, Norbinaltorphamine. a highly selective kappa-opioid antagonist in analgesic and receptor binding assays. J. Pharmacol. Exp. 752 ‘Ther. 246 .A. VonVoigtlander. P.F., R.A. Lahti and J.H. Ludens, 1983. U-C0.488H: a selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Esp. Ther. 221. 7.