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Home , Casomorphin, DADLE, Deltorphin, Kelatorphan, Morphiceptin, Morphine

The Journal of Neuroscience, June 1991, 17(6): 1565-l 576

(D-Ala*) II: D,-dependent Stereotypies and Stimulation of Release in the

R. Longoni,’ L. Spina,’ A. Mulas,’ E. Carboni,’ L. Garau,’ P. Melchiorri,2 and G. Di Chiaral ‘Institute of Experimental and Toxicology, University of Cagliari, 09100 Cagliari, Italy and 21nstitute of Medical Pharmacology, University of Rome “La Sapienza,” 00185 Rome, Italy

In order to investigate the relative role of central 6- and has been implicated in the actions of systemic . F- receptors in behavior, the effects of (D- -like opiates stimulate DA release preferentially in the Ala*)cleltorphin II, a natural Gopioid , and PL017, nucleus accumbens (Di Chiara and Imperato, 1988a) and elicit a beta- derivative specific for mu receptors, hypermotility sensitive to blockade by the DA D, an- were compared after local intracerebral and intraventricular tagonist SCH 23390 (Longoni et al., 1987a). Intra-accumbens administration. lntracerebral infusion of the two was infusion of opioid peptides elicits motor stimulation, but this done bilaterally in the limbic nucleus accumbens and in the action seems independent from DA, being resistant to classic ventral and dorsal caudate of freely moving rats DA-receptor antagonists (neuroleptics; Pert and Sivit, 1977; Ka- through chronic intracerebral cannulas. After intra-accum- livas et al., 1983). Moreover, the syndrome elicited by intra- bens infusion, the two peptides elicited marked but opposite accumbens opiates is biphasic, as motor stimulation is typically behavioral effects: while (o-Ala2)deltorphin II evoked - preceded by motor inhibition and catalepsy (Costa11et al., 1978). dependent motor stimulation characterized by locomotion, This biphasic character might be due to differential behavioral sniffing, and oral stereotypies, PLO17 elicited motor inhibi- properties of p- versus &receptor stimulation in the accumbens tion with rigidity and catalepsy. These effects were site spe- (Havemann and Kuschinsky, 1985; DaugC et al., 1988). How- cific because they could not be evoked from the ventral or ever, no clearcut demonstration exists for separate r/6 functions from the dorsal caudate. Low doses of (0.1 mg/ in the CNS, and some authors have even suggested that &re- kg, s.c.) blocked the effects of PLO17 but not those of (D- ceptors cooperate with p-receptors for functional expression Ala2)deltorphin II, which instead were reduced by high doses (Vaught and Takemori, 1979; Rothman and Westfall, 1982). of naloxone (1 .O mg/kg) and by the putative b-antagonist Clarification of this issue rests on the availability of potent and ; this failed to affect the catalepsy induced specific of, respectively, p- and b-opioid receptors. While by PLO1 7. Therefore, while (D-Ala*)deltorphin II effects were specific b- peptides have only been known for a few years b-mediated, PLO1 7 effects were p-mediated. Blockade (Chang et al., 1983), information about specific and high-in- of dopamine D, receptors by SCH 23390 abolished (D- trinsic-activity &agonists had been lacking until the recent avail- Ala*)deltorphin II effects, while blockade of dopamine D, ability of (D-Ala2)deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly- receptors by raclopride or by haloperidol was without effect. NH,), a peptide extracted from the skin of a South American Local application by reverse dialysis of (D-Ala*)deltorphin II (Erspamer et al., 1989); with this &agonist and with PLO 17 (5 PM) to the accumbens resulted in a naloxone-sensitive as a specific p-agonist (Blanchard et al., 1987), we have under- increase of extracellular dopamine concentrations; these ef- taken a study of the behavioral and biochemical effects of the fects could not be evoked from the caudate, nor by PLO17 selective stimulation of p- and &opioid receptors in the rat in the accumbens. lntracerebroventricular administration of nucleus accumbens. (o-Ala*)deltorphin II or of PLO17 elicited behavioral effects qualitatively similar to those obtained from the accumbens. Materials and Methods Animals. Male Spraque-Dawleyrats (CharlesRiver; 280-300 gm) were are known to influence behavior in a complex, often used.Animals were housedin groupsof six under constanttemperature (21°C)and humidity and a light-dark cycle of 12 hr (light at 7:OO).Food biphasic manner (Babbini and Davis, 1972; Domino et al., 1976; and water were available ad libitum. All experimentswere performed Costa11et al., 1978; Tortella et al., 1978). On the basis of local between 8:00 and 13:O0. intracerebral administration and electrophysiological studies Surgery and injection procedure. Rats were anesthetizedwith Equi- (Broekkamp et al., 1979; Kelley et al., 1980; Stinus et al., 1980; thesin (, 0.8 gm; , 4.25 gm; MgSO,, 2.12 gm; propylene glycol, 40 ml; 95%, 10 ml; H,O, 34 ml), placed Matthews and German, 1984; Latimer et al., 1987); and, more on a David Kopf stereotaxicapparatus, and implanted bilaterally with recently, of brain dialysis studies in freely moving rats (Di Chiara permanentcannulas fixed to the skull using acrylic cement.Guide can- and Imperato, 1988a,b; Di Chiara, 1990), limbic dopamine (DA) nulas (22-gaugestainless steel) were positioned 2 mm abovethe intended injection site. Coordinates were accumbens: A, 3.4; L, 2; V, 7.2; dorsal Received June 19, 1990; revised Nov. 5, 1990; accepted Dec. 28, 1990. caudate: A, 1.6; L, 3.2; V, 5 or 4; ventral caudate: A, 2.8; L, 3.5; V, This work wassupported by fundsfrom M.U.R.S.T. and C.N.R. (Sp4,Medicina 7.2; lateral ventricle: A, 0; L, 1.5; V, 3.2 (Pellegrinoet al., 1979).Stilets Preventiva and Comitato Tecnologico). were inserted in the guide cannulas, and the animals were housed in- Correspondence should be addressed to Professor Gaetano Di Chiara, Institute dividually for recovery. of Experimental Pharmacologyand Toxicology, University of Cagliari, Viale A. The were injected by an infusion pump in a volume of 0.5 or Diaz 182,09 100Cagliari, Italy. 1 pl of saline at a rate of 0.5 rl/min. Intracerebroventricular injections Copyright 0 1991Society for Neuroscience 0270-6474/91/l 11565-12$03.00/O were made bilaterally in a volume of 5 ~1 for each side. After infusion, 1566 Longoni et al. l Dopaminergic Effects of Deltorphin

1.6

2.8

3.6

Figure 1. Schematic diagram of the location of the cannula tip in the ac- cumbens (A 3.6, closed circles), ventral caudate (A 2.8, open squares), and dor- sal caudate (A 1.6, open circles).

the cannulas were kept in place for additional 2 min to allow diffusion postures, they were tested for catalepsy by placing them on a vertical of the drugs. wire grid, an animal was considered cataleptic if it remained immobile Behavioral testing. Two days after surgery, each rat was placed in a on the grid for more than 20 sec. Locomotion was measured by counting Perspex cage (27 x 42 x 15 cm) with a steel grid on the bottom and the number of sectors entered by the rat with its forelegs. Rearing and habituated for 60 min. The bottoms of the cages were divided into four grooming were measured in terms of number of items observed during equal sectors. After the habituation period and while in the same cage, periods of 10 min. The other behavioral items were quantified in terms each animal was given a of saline or of drugs of percentage of time spent by the rat in performing each behavior during 15 min (naloxone, SCH 23390, raclopride, naltrindole) or 2 hr (halo- each IO-min period. The behavior ofeach rat was observed continuously peridol) before bilateral intracerebral or intracerebroventricular injec- for 90 min after the intracerebral administration, and on videotapes tion. Behavior was videotaped, and the following behavioral categories only one behavioral item was analyzed at one time. Rats were used only were distinguished: 1, horizontal locomotion associated with sniffing; once. 2, rearing; 3, confined sniffing; 4, licking of the floor; 5, biting of the Dialysis. The rats were implanted under with a floor (gnawing); 6, grooming. When rats showed immobility with frozen dialysis tube (AN-69 Hospal; 0.3-mm outer diameter) inserted trans- The Journal of Neuroscience, June 1991, 1 f(6) 1567

0 locomotion sm rearing eZa gmomtng

k 10 20 30 40 50 60 70 80 90 nG 10 c

10 20 30 40 50 60 70 SO 90 minutes

60 I confined snlfflng 50 ma gnawlng + llcldng

60 50

40 Figure 2. Quantitative behavioral ef- 30 fects of different doses of (D- 60- Alaz)deltorphin II (0.5, 1.0, and 2.0 pg) 20 injected bilaterally in the nucleus ac- 50. cumbens. Nonstereotyped items (lo- -2 x40- 10 comotion, rearing, and grooming) were expressed as number of items per- 0 ;30- formed every 10 min. Stereotyped items 10 20 30 40 50 60 70 SO 90 (confined sniffing, gnawing&king) were expressed as percent of the observation time ( 10 min) spent in performing each item. The data are means + SEM ofthe results obtained in at least 10 different 10 20 30 40 50 60 70 80 SO minutes animals for each dose.

versally through the dorsal caudate (coordinates: A, 7.4; V, 5.5), or MA). (D-Ala2)deitorphin II was obtained as previously reported (Er- through the nucleus accumbens (coordinates: A, 9.6; V, 7.0; Konig and spamer et al., 1989). All drugs were dissolved in saline. Haloperidol Klippel, 1963). solution (Serenase, Janssen) was diluted in distilled water. The technique used to prepare and implant the dialysis probes was Statistics. The data were analyzed by one-way ANOVA for repeated essentially that already described (Imperato and Di Chiara, 1984, 1985, measures followed by Newman-Keuls multiple comparison test. Dif- 1986). Experiments were performed in freely moving rats 24 hr after ferences were considered as significant at p < 0.05. surgery. The Ringer’s solution used for perfusing the dialysis fibers had the following composition: 147 mM NaCl, 2.2 mM CaCl,, and 4.0 mM KCl. Samples (20 mitt, 40 ~1) of dialysate were injected in a high- Results performance liquid chromatograph equipped with a reverse-phase ODS Intra-accumbens (o-AlaZ)deltorphin II column (Supelcosil, Supelco Inc., Bellefonte, PA) and with an electro- chemical detector (BAS, Lafayette, IN) in order to quantitate DA, dihy- The bilateral intra-accumbens administration of (D- droxyphenylacetic (DOPAC), and homovanillic (HVA), according Alaz)deltorphin II (0.5-2.0 kg on each side) elicited a syndrome to the procedure already reported (Imperato and Di Chiara, 1984). of pure behavioral stimulation characterized by locomotion Histology. After behavioral testing, rats were given an overdose of (typically associated with sniffing), by rearing and grooming, Equithesin and perfused intracardially with saline followed by 10% formalin: the brains were removed, frozen, and sectioned in order to and by stereotyped behavior with confined sniffing, gnawing, determine the locations of the cannulas (Fig. 1). and licking (Fig. 2). The incidence of these behavioral items was DIWS. The followina drugs were used: (D-Ala*)-deltorphin II (Tyr-D- dose related. Ala-Phe-Glu-Val-Val-Gly-l?Hz), SCH 23390 (kindly donated by Dr. E. Locomotion increased after all the doses tested (0.5, 1, and Ongini, Essex, Italy), raclopride (ASTRA Lakemedel AB, Sodertalje, Sweden), PLO 17 (Peninsula Laboratories, CA), naloxone hydrochloride 2.0 pg, bilaterally); rearing and stereotypies (confined sniffing (Sigma Chemical Co., St. Louis, MO), and naltrindole (RBI, Natick, and licking/gnawing) reached a plateau at 1.O and 2.0 pg; con- 1568 Longoni et al. * Dopaminergic Effects of Deltorphin

c7 locomotion 150 100 h4 rearing i I confined sniffing m gnawing + licking c E $ 100 .- / 2 ii i Fi 86 50 z * M 50 2 I / . /

0 I 0-c I , 0.5 1 Naloxone Naloxone Naltrindole 0.1 PL9 0.1 mg/Kg 1 mg/Kg 6 w/Kg Intro-accumbens PLO 17 01(1 on each side.)

Figure 3. Effect of naloxone (0.1 or 1 mg/kg, s.c.) and naltrindole (6 Figure 5. Dose-responsecurve for catalepsyinduced by bilateral in- mg/kg,s.c.) on the behavioralsyndrome elicited by 2 pg(n-.Ala2)deltorphin fusion in the accumbensof the p-agonist PL017. Data are means f II infused bilaterally in the accumbens15 min after naloxone or nal- SEM of the results obtained in five animals at each dose level. trindole. Controls were administered saline subcutaneously15 min be- fore (n-Ala2)deltorphin II. Data are means of the results obtained in five animals and are expressedas percent of the values obtained in controls. *, P < 0.05 as comparedto saline controls. leptogenic doses (Morelli and Di Chiara, 1985) of the prefer- ential D, antagonist haloperidol (0.2 mg/kg, s.c.; Fig. 4, Table 1) or of the specific D, antagonist raclopride (3.0 mg/kg, s.c.; fined sniffing occupied the first half of the time course, while Hillegart and Ahlenius, 1987; Fig. 4, Table 1). oral stereotypies (licking and gnawing) occupied the second half. Increasing the dose from 1 to 2 pg resulted in a shift from lower- Intracaudate (o-Ala2)deltorphin II (confined sniffing) to higher-intensity sterotypies (licking/gnaw- As shown in Table 2, infusion of 0.5-2.0 rg (o-Ala*)deltorphin ing). II in the dorsal or in the ventral caudate elicited behavioral As shown in Figure 3 and Table 1, rather high doses of nal- effects not different from saline. oxone (1 mg/kg, s.c.) were necessary to prevent the effects of 1 or 2 pg (D-Ala*)deltorphin II; lower doses of naloxone (0.1 mg/ Intra-accumbens PLO1 7 kg, s.c.) were ineffective. Naltrindole (6 mg/kg, s.c.), a reportedly In contrast with the &agonist (D-Ala2)deltorphin II, the selective selective centrally acting nonpeptide d-antagonist (Portoghese et peptidic r-agonist PLO17 (0. l-l pg), infused bilaterally in the al., 1988), significantly reduced the deltorphin syndrome (Table accumbens, elicited a syndrome of motor inhibition with rigidity 1, Fig. 3). The behavioral syndrome induced by intra-accum- and catalepsy whose duration was dependent from the dose bens (D-Ala2)deltorphin II (1 or 2 pg, bilaterally) was prevented administered (Fig. 5). The catalepsy was phenomenologically by low doses ofthe potent antagonist of D, receptors SCH 23390 similar to neuroleptic catalepsy rather than to typical lead-pipe (0.025 and 0.05 mg/kg, s.c.; Fig. 4, Table 1) but not by cata- rigidity (see Di Chiara et al., 1979). No motor stimulation was observed during the 2 hr of observation. Naloxone, in low doses (0.1 mg/kg, s.c.), reversed the catalepsy induced by intra-ac- 150 0 locomotion cumbens PLO17 (0.5 cLgbilaterally; Fig. 6). In contrast, the pu- In rearing tative s-antagonist naltrindole (6 mg/kg, s.c.) failed to attenuate I confined sniffing significantly the catalepsy elicited by 0.5 cLgPLO17 in the ac- GIN gnawing + licking cumbens (Fig. 6). 5 100 -5 Intracaudate PLO1 7 E PLO 17 (0.5 Fg) infused in the dorsal caudate induced only mild % and short-lasting catalepsy and rigidity (not shown). a? 50 Intracerebroventricular (o-Ala-‘)deltorphin II and PLO1 7 Opioids are known to exert biphasic effects after systemic or - intracerebral administration (see introductory remarks). In or- 0 der to evaluate the significance of the effects evoked by the Racloprids Holopsridol SCH 23390 SCH 23390 peptides from the accumbens in terms of their overall central 3 v/Kg 0.2 mg/Kg 0.025 mg/Kg 0.05 mg/Kg effects, we studied the behavioral effects of (D-Ala2)deltorphin Figure 4. Effect of raclopride (3 mg/kg, s.c.), haloperidol (0.2 mg/kg, II and of PLO 17 after intracerebroventricular administration. s.c.), and SCH 23390 (0.025 or 0.050 mg/kg, s.c.) on the behavioral Intracerebroventricular (D-Ala2)deltorphin II (10 Mg; Fig. 7) effectsof (n-AlaZ)deltorphin II injected bilaterally in the accumbens2 elicited within minutes a dose-related motor activation quali- hr after haloneridol and 15min after raclomide or SCH 23390.Controls tatively similar to but less intense than that obtained after intra- were administered saline subcutaneously2 hr or 15 min before (D- Alaz)deltorphinII. Data aremeans of the resultsobtained in five animals accumbens infusion of the same compound. Stereotyped items and are expressedas percentof the values obtained in controls. *, P < (confined sniffing, licking/gnawing) were present also after doses 0.05 as comparedto saline controls. of 5 pg bilaterally. The Journal of Neuroscience, June 1991, 7 f(6) 1569

Table 1. The effect of opioid antagonists and DA antagonists on behavioral effects of i&a-accumbens (r+Ala*)deltorphin II Percenttime/90 min Treatment Number/90 min Confined Gnawing/ @g/k, s.c.1 Locomotion Rearing Grooming sniffing licking (n-Ala2)deltorphin II, 1 rg bilaterally Saline (15 min) 19.2 + 10 62 + 8.3 1.6 + 0.9 15.1 ?z2.0 10.22 k 1.5 Saline ( 120 min) 12.3 + 6 59 t- I 8.1 + 0.5 13.8 k 1.4 11.5 & 1.7 Naloxone, 0.1 67.4 + 8.3 48.2 k 6.3 5.8 zk 0.8 11.2 -t 1.6 9.8 k 1.4 Naloxone, 1 9.6 k 1.2* 5.1 * 0.F 2.8 k 0.3* 1.7 * 0.4* 0.4 f 0.2* SCH 23390,0.05 3.1 * 1.3* 0 0 0 0 SCH 23390,0.025 14 f 1.5* 6.2 + 4.5* 2.2 k 0.6* 0.75 f 0.16* 0.32 + 0.7 Haloperidol, 0.2 61.3 + 8.6NS 53.1 + 9.3NS 5.5 + 0.9NS 12.6 + 1.5NS 10.2 + 1.6NS Raclopride, 3 65.9 k 8.2NS 52.4 I+ 8.2NS 5.2 f 0.7NS 14.5 + l.gNS 13.6 t 1.9NS (nAla*)deltorphin II, 2 pg bilaterally Saline (15 min) 125.6 + 18.6 61.0 f 9.3 9.0 k 0.5 9.0 zk 1.2 16.9 5 1.8 Saline (120 min) 123.9 + 15.3 65.3 + 5.8 8.7 -+:0.7 10.2 + 0.8 16.2 k 2 Naloxone, 0.1 109.4 + 16.7 59.5 ?I 8.5 1.5 f 0.2 9.5 + 1.3 15.7 + 1.8 Naloxone, 1 38.4 + 1.2* 19.2 k 2.0* 3.1 t 0.4* 2.5 + 0.4* 0.5 IL 0.2* Naltrindole, 6 40.2 k 5.3* 27.1 k 3.4* 4.0 + 0.1* 0.6 k 0.2* 0.3 + 0.08* SCH 23390,0.05 6.2 -I l.O* 1.1 f 0.1* 0 0 0 Haloperidol, 0.2 122.5 + 16NS 64.3 + 9.5NS 9.1 + 1.3NS 12.3 I!Z 1.4NS 20.3 + 3.3NS Raclopride, 3 120.8 + lgNS 54.1 + 9.6NS 7.1 Ik l.ONS 11.3 f 1.6NS 17.3 + 2.gNS

N = 5; values are means + SEM. Animals were pretreated with subcutaneous saline, naloxone, SCH 23390, or raclopride 15 min before and with saline or haloperidol 120 min before. *, significantly different from saline (P < 0.05); NS, not significantly different from saline (P < 0.05).

Intracerebroventricular (D-Ala*)deltorphin II-induced stereo- the arousing effects of the manipulations involved in the local typy was prevented by subcutaneous doses of naloxone as high injection procedure. as 1.O mg/kg (Fig. 7); lower doses (0.1 mg/kg) were ineffective (not shown). The D, antagonist SCH 23390 (0.05 mg/kg, s.c.) Brain dialysis completely prevented the behavioral stimulation and the stereo- In freely moving rats implanted with dialysis probes in the typies elicited by intracerebroventricular (D-AlaZ)deltorphin II accumbens, (D-AlaZ)deltorphin II, added to the Ringer’s solu- (Fig. 7). Intracerebroventricular PLO 17 (5 wg) elicited a catalepsy tion, stimulated DA release in a concentration-dependent man- syndrome indistinguishable from that elicited by this compound ner (threshold, 5 PM; maximal, 50 PM; maximum increase over after intra-accumbens infusion (Fig. 8). Low doses of systemic basal, + 120%; Fig. 9A). During the application of the highest naloxone (0.1 mg/kg, KC.) prevented intracerebroventricular concentration of (D-Ala2)deltorphin II (50 PM), a syndrome of PLO 17-induced catalepsy (Fig. 8). motor stimulation was observed similar to that evoked by intra- accumbens injection of the same compound. Intra-accumbensand intracaudate saline No changes in DA release and behavior were observed after As shown in Table 2, intra-accumbens or intracaudate saline reverse dialysis of D-Ala2)deltorphin II in the dorsal caudate resulted only in mild, short-lasting motor activation related to (Fig. 9B).

Table 2. The effect of intra-accumbens and intracaudate injection of saline and (D-Ala*)deltorphin II (2 Pf3) Percenttime/90 min Injection site Number/90 min Confined Gnawing/ and treatment Locomotion Rearing Grooming sniffing licking Nucleus accumbens Saline 3 + 1.22 2.2 k 0.66 3.4 z!z1.29 0 0 Dorsal caudate Saline 4.4 5 1.57 2.6 t 0.81 2.8 + 0.66 0 0 Deltorphin (2 pg) 3.0 Ii 1.033 3.83 + 1.22 3.33 k 1.05 0 0 Ventral caudate Saline 4.6 2 0.92 3.4 + 1.07 2.4 k 0.51 0 0 Deltorphin (2 pg) 5 + 1.58 3.8 + 0.86 2.2 k 1.02 0 0 N = 5: values are means f SEM. 1570 Longoni et al. . Dopaminergic Effects of Deltorphin

fZ53 Naltrindols (6 mg/Kg s.c.) I Noloxone (0.1 mg/Kg 6.c.)

Figure 6. Effect of naloxone (0.1 mg/ ki. s.c., 15 min before)and naltrindole (<‘m&g, s.c.)on the catalepsyinduced by bilateral infusion of PLO17 (0.5 fig) in the accumbens.Controls were ad- ministered saline subcutaneously 15 min before PL017. Data are means + SEM of the results obtained in five an- IO 20 30 40 50 60 minutes imals. *, P < 0.05 ascompared to saline controls. Time after intro-accumbens PLO 17 (0.5 pg bilater.)

Systemic administration of naloxone (1 mg/kg, s.c.) prevented intra-accumbens DADLE is generally preceded by catalepsy, the stimulation of DA output and motor activity evoked by which is likely to be a p-effect (see below). dialysis with 50 PM (D-Ala2)deltorphin II in the accumbens (Fig. The sensitivity of (D-Ala2)deltorphin II effects to blockade by 10). Intra-accumbens dialysis of PLO 17 (50 PM) failed to modify naltrindole (Portoghese et al., 1988) and by 1.O but not by 0.1 the output of DA and of its metabolites (Fig. 1lA) in spite of mg/kg naloxone is consistent with its 6 nature (Yoshimura et it’s ability to induce catalepsy. Intra-caudate dialysis of PLO17 al., 1982). (50 WM) was ineffective on DA output and on behavior (Fig. The motor-stimulant effects of intra-accumbens (D- 11B). Ala2)deltorphin II, like those of DADLE (Pert and Sivit, 1977; Kalivas et al., 1983), were not significantly reduced by halo- Discussion peridol; in contrast, SCH 23390, a specific D, antagonist (Hyttel, (D-Ala2)deltorphin II is a highly specific and potent agonist of 1983), completely prevented the entire behavioral syndrome &opioid receptors (Erspamer et al., 1989). Thus, in binding induced by intra-accumbens (D-Ala*)deltorphin II. If one con- studies, (D-Ala*)deltorphin II has been found to have an affinity siders that haloperidol is a preferential D, antagonist (Seeman, for d-receptors at least 3000 times higher that for p-receptors; 1980), the most parsimonious explanation of the differential in the same experiments, D-Pen, D-Pen was about effect of SCH 23390 and haloperidol on (D-Ala2)deltorphin II 10 times less selective (Erspamer et al., 1989). On isolated or- effects is that they are mediated by DA acting specifically on D, gans, (D-Ala2)deltorphin II showed a potency in eliciting &ac- receptors. In agreement with this, the present study shows that tions at least 10,000 times higher than that required for evoking raclopride, a specific D, antagonist (Ogren et al., 1986), fails to M-actions (Erspamer et al., 1989). PL017, on the other hand, modify the deltorphin syndrome. On the other hand, our study shows quite high selectivity and potency for p-receptors (Blan- also shows that intra-accumbens dialysis of (D-Ala2)deltorphin chard et al., 1987). An additional characteristic of these com- II stimulates DA release in the accumbens. Stimulation of DA pounds is their metabolic stability, which derives from the pres- release by intra-accumbens (D-Ala2)deltorphin II might take place ence of a strategically placed D-amino acid in the peptide chain through presynaptic stimulatory &receptors; it is puzzling, how- (Chang et al., 1976, 1983). ever, that such receptors have been shown in the caudate but The present study demonstrates intense (stereotyped), topo- not in the accumbens (Lubetzki et al., 1982; Petit et al., 1986). graphically discrete (restricted to the accumbens), and receptor- Deltorphin-induced behavioral stimulation appears mediated specific motor stimulation after intracerebral infusion of specifically by D, receptors in the accumbens. Indeed, the motor (D-Ala2)deltorphin II. Sterotypies similar to those elicited by effects of (D-Ala2)deltorphin II are not the only example of a intra-accumbens (D-Ala2)deltorphin II and characterized by mesolimbic DA-mediated response showing preferential block- confined sniffing, licking, and gnawing are typically obtained by ade by the D, antagonist SCH 23390. In fact, high sensitivity stimulation of dopaminergic receptors (Longoni et al., 1987b). to SCH 23390 is also observed for the motor-stimulant effects The motor stimulation previously reported after intra-accum- of systemic morphine (Longoni et al., 1987a; Pollock and Kor- bens infusion of nonspecific opioid peptides such as (D-Ala*-D- netsky, 1989). One explanation of these findings is that in the Leu5)enkephalin (DADLE; Pert and Sivit, 1977; Kalivas et al., mesolimbic DA system the true synaptic receptor for DA is the 1983) was characterized by locomotor stimulation without ste- D, receptor, while D, receptors might be nonsynaptic receptors reotypy and therefore appears mild in comparison with that activated by excess DA released from nerve terminals or by observed in the present study after (D-Ala*)deltorphin II. This exogenous D, agonists. An alternative explanation would be that might be due in part to residual activity of DADLE at p-recep- stimulation of &receptors by (D-Ala2)deltorphin II specifically tors, whose stimulation, by inhibiting motor activity (seebelow), potentiates D,-mediated responses and that stereotypies arise might have prevented the full expression of the behavioral stim- from the combined action of stimulation of D, receptors by ulant effect of b-receptor stimulation. This suggestion is sup- deltorphin-induced release of DA and stimulation of &recep- ported by the fact that the locomotor stimulation induced by tors. The Journal of Neuroscience, June 1991, 1 I(6) 157 1

30 A

20

10

0 30 El locomotion I confined snlfflng 63 t-sating CSZ gnawing + licking

; 20 0) Y % k Naloxone "E I *,. 2 10

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30 0 locomotion I conflnsd sniffing 6Y rearing lxxl gnawing + licking

20 -1 00

10 50 Figure 7. Behavioral effects of the in- tracerebroventricularadministration of SCH 23390 (D-Ala2)deltorphin II and its interaction * with naloxone (1 mg/kg, s.c.) and with ifi SCH 23390 (0.05 mg/kg, s.c.) given 15 ** *** **** ** * *** ** 0 min before. Data are means ? SEM of 10 20 30 40 50 60 70 80 90 minutes the results obtained in five animals. *, P < 0.05 as compared to deltorphin lime after i.c.v. (D-Ala 2, -Deltorphin II (10 pg) alone.

The results obtained with intra-accumbens (D-Ala2)deltorphin diated by D, receptors. Studies are in progress in order to con- II suggest a revision of the concept that opioids exert DA-de- firm this suggestion. pendent behavioral stimulation from the ventral tegmentum Intra-accumbens or intracerebroventricular infusion of the and DA-independent stimulation from the nucleus accumbens k- PLO 17 resulted in a behavioral syndrome quite (Kalivas et al., 1983). This conclusion was based on the differ- opposite to that evoked by (D-Ala2)deltorphin II. Thus, PLO17 ential sensitivity to haloperidol of the effects of intrategmental evoked a syndrome of motor inhibition and catalepsy sensitive as compared to intra-accumbens DADLE. By analogy with (D- to low doses of naloxone (0.1 mg/kg, s.c.) but not to naltrindole; Ala2)deltorphin II, we now suggest that the stimulant effects of this syndrome was reminiscent of the catalepsy elicited by neu- intra-accumbens DADLE are also dopaminergic but are me- roleptics (DA receptor blockers) rather than of the lead-pipe 1572 Longoni et al. l Dopaminergic Effects of Deltorphin

Figure 8. Catalepsy after intracere- broventricular administration of PLO 17 (5 pg) and the effect of naloxone (0.1 mg/kg, s.c.) given 15 min before. Data are means f SEM of the results ob- tained in five animals. *, P < 0.05 as 10 20 30 40 50 60 minutes compared to saline controls. Time after i.c.v. PLO 17 (5 pg)

A

Deltorphin II 50 PM 300- 1 Accumbens 0-O DA A-A DOPAC O--o HVA

0 60 120 180 240 300 B Deltorphin II 50 H Dorsal Caudate

Figure 9. Effect of reverse dialysis of (D-Ala*)deltorphin II (50 PM in the ac- cumbens and in the dorsal caudate) on the output of DA, DOPAC, and HVA in dialysates. Basal values (expressed in pmol/sample) in the nucleus accum- bens and in the dorsal caudate, respec- tively, were as follows: DA, 0.23 _t 0.03, 0.45 + 0.06; DOPAC, 24.6 k 3, 45 f 5; HVA, 17 + 1.5,3&g + 3.2. Data are means + SEM of the results obtained o-i I I1 I I I I I I I, I I, I, in five animals expressed as percent of 0 60 120 180 240 300 basal values. *, P < 0.05 as compared to basal values. time (min.) The Journal of Neuroscience, June 1991, 1 l(6) 1573

Accumbens 150, Nalaxone 1 mg/kg SC.

------lx= ------Deltorphin II 50 p&i II Figure IO. Effect of systemic nalox- one (1 mg/kg, s.c.) on the effect of (D- Ala2)deltorphinII (50 WM) on the output o-o DA of DA and metabolites in the accum- A-A DOPAC bens.Basal values (in pmol/sample)in I-WA the nucleus accumbensand in the dor- o--o II,,> sal caudate, respectively, were as fol- lows: DA. 0.32 f 0.03. 0.45 + 0.05: 0 60 li0 180 ’ 240 I ’ 360 DOPAC, ‘26.1 f 3, 53 ‘k 6; HVA, 18: k 2.0, 37.5 + 3.5. Data are means + time (min.) SEM of the resultsobtained in five rats.

catalepsy elicited by systemic morphine-like opiates (see Di and Koob (1985). In fact, as shown by the results obtained in Chiara et al., 1979, for discussion of this issue). However, apart the present study with PL017, an action of morphine in the from this apparent relationship with DA, local application of accumbens would rather result in catalepsy. The motor-stim- PLO 17 by accumbens dialysis failed to modify DA release, in- ulant effects of systemic morphine are more likely to be due to dicating that the behavioral effects of PLO 17 are not due to a stimulation of p-receptors in the ventral tegmentum area (VTA)- presynaptic interference with DA transmission, but eventually medial substantia nigra (SN), resulting in disinhibition of DA to a modulatory action of p-receptors on the effects of stimu- neurons projecting to the accumbens (Di Chiara and Imperato, lation of postsynaptic DA receptors. In fact, recent results show 1988b; Morelli et al., 1989). that stimulation of p-receptors reduces the stimulatory action It is interesting, therefore, that while both p- and d-opioids of D, receptor activation on CAMP production (Groppetti et al., can stimulate DA transmission in the mesolimbic DA system 1990). and produce behavioral activation, the mechanism of these ef- The observation of clearcut differential effects of b-versus fects is different for the two types of opioid receptors, taking p-receptor stimulation in the accumbens provides strong be- place proximally (i.e., in the SN-VTA region) in the case of havioral and biochemical evidence for separate functions of p- M-opiates and distally (i.e., in limbic terminal DA regions) in and b-receptors in specific brain areas. In the accumbens, a the case of d-opioids. morphological substrate of such differential behavioral effects The present results are in contrast with the notion that oral might be the differential distribution of opioid receptors, as stereotypies arise from the caudate rather than from the accum- demonstrated by receptor autoradiography; this distribution is bens (Creese and Iversen, 1975); indeed, this conclusion derives diffuse for 6- and patchy for b-receptors (Mansour et al., 1988). from studies performed with , whose effects, in The accumbens appears to be a specific site of origin of the contrast with those of (D-Ala’)deltorphin II, are blocked central motor effects of CL-or 6- stimulation, be- not only by D, but also by D, antagonists (Creese and Iversen, cause no motor effects were obtained from dorsal or ventral 1975). The differential sensitivity of amphetamine and (D- caudate administration of PLO 17 or (D-Ala2)deltorphin II. In- Ala*)deltorphin II to D, antagonism indicates that the popula- deed, the lack of responses from the caudate is surprising in tion of DA receptors stimulated by amphetamine is different view of the fact that this area contains high concentrations of from that stimulated following intra-accumbens deltorphin. This p- as well as &receptors and that the presence of &receptors in turn might be the basis for the ability of (D-Ala2)deltorphin stimulating DA release has been shown in this area rather than II, in contrast with amphetamine, to elicit oral stereotypies from in accumbens (Lubetzki et al., 1982). Failure to obtain behav- the accumbens. In this respect, it is notable that (D- ioral responses after intracaudate administration of opioid pep- Ala2)deltorphin II is capable of eliciting D,-dependent oral tides might be due to the fact that the area to be influenced for stereotypies even under strong blockade of D, receptors by ra- obtaining an effect is larger than that which can be reached by clopride or by haloperidol. This would imply that, under &re- diffusion by relatively polar compounds such as the peptides. ceptor stimulation, concurrent D, and D, stimulation (Longoni Alternatively, the responsive area in the caudate is quite small, et al., 1987b) is not essential for the expression of oral stereo- and we simply failed to hit the right spot (Kelley et al., 1988). typies. A reason for this might be that delta-receptor stimulation If indeed, as indicated by the present study, the motor-stim- strongly potentiates the effect of D, receptor stimulation by DA ulant effects of intra-accumbens opioid peptides are mediated released by deltorphin. by &receptors, it is unlikely that the motor-stimulant effects of The observation of the present study that &receptors exert systemic morphine-like opiates, which are mu in nature, are a powerful facilitatory influence on dopaminergic transmission due to a direct action on the accumbens as suggestedby Amalric in the accumbens might have far-reaching consequences. In fact, 1574 Longoni et al. * Dopaminergic Effects of Deltorphin

A

---- I PLO--- 17 zL!&I Accumbens

O-O DA A-A DOPAC 0-n HVA

O! I I I I I I I I I I I I I I ( I 0 60 120 180 240 300

150, ~PLO1750ixl Dorsal Caudate B

Figure 11. Effect of PLO17 (50 PM), = applied by reversedialysis, on the out- : put of DA and metabolites in the ac- n cumbensand in the dorsal caudateas 50 estimated by transcerebraldialysis in 75 freely moving rats. Basal values (in 8? pmol/sample) in the nucleus accum- bensand in the dorsal caudate,respec- tively, wereas follows: DA, 0.35 f 0.04, 0.05 f 0.05; DOPAC, 23.6 k 5, 54 + 0 1 5;HVA, 16-+2,35 f4.Dataaremeans 0 60 120 180 240 300 * SEM of the results obtained in five rats. time (min.)

the mesolimbic dopamine system (which includes as a major itory influence on motor activity (catalepsy), which appears in- terminal area the accumbens) is currently regarded as an inter- dependent from changes in DA release. Therefore, at least for face for the conversion of motivation into action (Mogenson et motor behavior, the actions of p- and &receptor stimulation al., 1980). In particular, stimulation of DA transmission is re- can be clearly separated; this shows that the two receptors can garded as a major factor in the rewarding properties of various be distinguished not only at the recognition site but also at the stimuli, including natural reinforcers (food, sex) and drugs of behavioral level. abuse (Wise and Rompre, 1989); &receptors, on the other hand, Third, the motor stimulant effects of intra-accumbens (D- have been shown to exert DA-dependent rewarding effects Ala2)deltorphin II are specifically mediated by D, receptors. (Shippenberg et al., 1987). Given these premises, an interaction Because these effects appear to be due to a stimulation of DA like the one reported in the present study might provide a pos- release in the accumbens, the results suggest that D, rather than sible neurochemical basis for the postulated role of the limbic D, receptors are the synaptic receptors for DA in the accumbens system in normal and abnormal behavior (Stevens, 1973). or, alternatively, that stimulation of &receptor specifically po- To summarize, a number of conclusions can be drawn from tentiates D, responses. Finally, as shown by the similarity of the present study. First, d-receptors in the accumbens exert a the effects of intracerebroventricular and intra-accumbens (D- powerful stimulant influence on DA transmission consisting of Ala2)deltorphin II and PLO 17, the accumbens is among the most stimulation of DA release and resulting in typical stereotyped influential sites for the motor effects of 6- and p-opioids in the behavior. forebrain, and this role can be clearly distinguished from that Second, p-receptors in the accumbens elicit a powerful inhib- of the caudate. The Journal of Neuroscience, June 1991, 17(6) 1575

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