OO28-3908/92$5.00 + 0.00

Neuropharmncology Vol. 31, No. 3, pp. 293-298, 1992

PergamonPressplc

Printedin GraatBritain

INHIBITION OF ENKEPHALIN METABOLISM AND ACTIVATION OF MU- OR DELTA-OPIOID RECEPTORS ELICIT OPPOSITE EFFECTS ON REWARD AND MOTILITY IN THE VENTRAL MESENCEPHALON CH. HEIDBREDER,’ M. GEWISS,~S. LALLEMAND,’B. P. ROQU& and PH. DE WITTE’ ’ Laboratoire de Psychobiologie, Universite de Louvain, B-l 348 Louvain-la-Neuve, Belgium and ‘Laboratoire de Biochimie Organique, Universid Rend Descartes, F-Paris, France (Accepted 20 September 1991)

Summary-The coexistence of endogenous opioid systems and dopaminergic neurones in the midbrain tegmental area suggests functional interactions between dopamine and enlcephalins. Nevertheless, the identification of the specific opioid receptors associated with modulation of @mental dopamine activity and its behavioural concomitants on motility and reward is far from clear, considering the mixed nature of the ligands usually employed. In this way, kelatorphan, a potent inhibitor of enkephalinases and selective agonists for mu- and delta-opioid receptor subtypes @AGO and DSTBULET, respectively) were infused directly into the ventral @mental area of the rat to study the role of endogenous enkephalins and opioid receptors in regulating spontaneous motor activity and intracranial self-stimulation behaviour. A greater increase in the rate of intracranial self-stimulation behaviour was found after activation of mu-opioid receptors in the ventral tegmental area, as compared to activation of delta-opioid receptors, whereas enhancement of endogenous enkephalins by inhibiting their metabolism through kelatorphan, reduced the rate of intracranial self-stimulation behaviour. On the contrary, spontaneous motor activity was reduced by the delta-opioid receptor agonist, whereas kelatorphan increased the movements of the animal. Taken together, these results show that inhibition of the metabolism of enkephalins in the ventral tegmental area decreased positive reinforcement from the lateral hypothalamic medial forebrain bundle and increased spontaneous movements. On the contrary, activation of both mu- or delta-opioid receptors in the ventral tegmental area significantly increased self-stimulation and decreased spontaneous motor activity, supporting the view that different mechanisms underlie the behavioural effects, resulting from enhancement of endogenous enkephalins and from activation of specific opioid receptors in the ventral mesencephalon. Key words-VTA,

opioids, kelatorphan, reward, motility.

There is a great deal of evidence for a modulatory action of opioids in the ventral tegmental area (VTAA,, dopamine region). Enkephalinergic fibres and perilcarya are present in this structure (Johnson, Sar and Stumpf, 1980; Khachaturian, Lewis and Watson, 1983) whereas mu- and delta-opioid receptors have been reported in moderate and low density, respectively (Moskowitz and Goodman, 1984, Dilts and Kalivas, 1988). More specifically, administration of opioids into the ventral tegmental area was found to activate dopamine (DA) cells (Kelley, Stinus and Iversen, 1980) and to increase DA-related, accumbens-dependent behaviour (Wise and Bozarth, 1982; Kelley and Stinus, 1984). Indeed, this activation may mediate the motor-stimulant effect of morphine (Joyce and Iversen, 1979) and the reinforcing value of this drug of abuse (Wise and Bozarth, 198 1). Furthermore, those areas of the brain which support selfstimulation reward (ICSS) and contain DA fibres, have been reported to display moderate-to-high levels of opioid binding sites (Herkenham and Pert, 1980; Moskowitz and Goodman, 1984) and concentrations of enkephalin (Finley, Maderdrut and Petrusz, 1981; 293

Khachaturian et al., 1983). Given the relevance ascribed to DA in the medial forebrain bundle, involving self-stimulation behaviour and the coexistence of endogenous opioid systems and A,,, dopamine neurones, it is not surprising that administration of opioids into the ventral tegmental area increases the rate at which animals respond for intracranial self-stimulation and lower the minimum stimulus current for responding in an intracranial self-stimulation paradigm (Broekkamp, Van den Boggard, Heijnen, Rops, Cools and Van Rossum, 1976; Broekkamp, Phillips and Cools, 1979). The discovery of specific opioid receptors in brain has led to the idea that opioid drugs may interact with one or more of the receptor subtypes and that the differential behavioural profiles of opioids may depend upon the relative potencies of these drugs at each of them (Zukin and Zukin, 1981). In this way, the aim of the present study was to assess the role of selective ligands for the mu- and delta-opioid receptors in regulating spontaneous motor activity and intracranial self-stimulation behaviour, by injecting the mu-opioid receptor agonist

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DAGD (Tyr-D.Ala-Gly-(Me)Phe-Gly-al), the deltaopioid receptor agonist DSTBULET (Tyr-D&r(otBu)-Gly-Phe-Leu-Thr) and (N-(3(R)-[(hydroxy amino)carbonyl] - 2 - benzyl - 1 - oxopropyl) - L- alanine) (ICELATORPHAN), a potent inhibitor of enkephalin-degrading enzymes, in the ventral mesencephalon. METHODS

Subjects and surgical procedures

Male rats of the Wistar strain, weighing 30&350 g at the time of surgery, were housed in individual cages throughout the experiment. Food and water were available ad libitum. Each rat was anaesthetized with 0.350 ml of drope ridol 2.5 mg/ml, fentanyl 0.05 mg/ml (Janssen Pharmaceutica), followed by 0.250ml of ketalar (Parke Davis). Intracranial self-stimulation behaviour was obtained through bilateral monopolar nickelchrome electrodes (0.25 mm; 4), insulated except for the cross-section of the tip (Amphenol). Electrodes were implanted stereotaxically, according to the following coordinates (Paxinos and Watson, 1982): 3.5 mm posterior (P) to bregma; 1.2 mm lateral (L) to the midsagittal line, and 8.3 mm below (V) the surface of the dura mater (lateral posterior hypothalamus). The stereotaxic incisor bar was set at 3.0 mm ventral to the interaural line. The indifferent electrode was placed 2.Omm anterior (A) to bregma. One guide cannula was also implanted into the ventral tegmental area. A 24gauge hypodermic stainless steel tube was placed using the following coordinates: 6.3 mm (P); 0.5 mm(L) and 7.9 mm(V). A 31-gauge hypodermic stainless steel tube served for injections and was 1.0 mm longer than the chronically-implanted guide cannula, which was kept clear with wire stylets. The animals were allowed 1 wk for postoperative recovery and 1 wk for training. Behavioural testing procedure Self-stimulation behaviour. Testing for selfstimulation was conducted in plexiglass boxes (40 x 30 x 2Ocm). A lever (0.5 x 4 x 3cm) was placed 2cm above the floor of the test chamber. Depression of this lever delivered electrical stimuli of 0.1 msec pulse width, delivered at 200 Hz for a 200 msec train duration, through flexible leads connected to the chronically-implanted electrodes. A range of intensities (50-200 PA) was evaluated for the response rate they would sustain. Ultimately, each rat was assigned a stimulation intensity that sustained a mean rate of approx 200 self-stimulation responses per period of 5 min. The animals were allowed to self-stimulate for sessions varying from 60 to 120 min. The number of bar-pressings was automatically recorded every 5 min. After 1 wk of daily self-regulation of stimulation of the brain, after which the performance of self-stimulation remained stable, the rats were

assigned to 1 of the experimental groups and the injection procedure started. Motility recording. Motility was recorded by an apparatus based on the principle of inertia. The forces generated by the movements of the animal were transported to a plate, supported by steel balls with a minimal mechanical resistance to lower the threshold of detection of body movements of the animal (for detailed technical specifications, see Weyers and Annys, 1982). The animals were implanted with a guide cannula into the ventral tegmental area, according to the procedure described above. After 1 wk of postoperative recovery, the injection procedure started. Drugs and injection procedure Self-stimulation behaviour. The drugs DSTBULET (Tyr-o.Ser(otBu)-Gly-Phe-Leu-Thr) and kelatorphan (N-(3(R)-[(hydroxyamino)carbonyl]-2-benzyll-oxopropyl)-L-alanine) were gratefully provided by Professor B. P. Roques; DAGO (Tyr-D.Ala-Gly (Me)Phe-Gly-al) was purchased from Bachem (H2535-A22041). For intraccrebral injection, the compounds, were dissolved in 0.15 M saline. In preparation for injcctions into the ventral tegmental area, drug or vehicle solutions were administered in a constant volume of 0.5 ~1, over a period of 50sec using the injection needle attached to a microsyringe (Hamilton 7001 N) by polyethylene tubing (Dow Corning). After the injections, the injector cannulae were left in place for an additional 30 set to allow the infused solution to infiltrate the surrounding tissue. After replacement of the occlusion stylets, the rats were immediately subjected to behavioural testing. Six groups of 6 rats, presenting stable self-stimulatory behaviour, were used for behavioural testing: saline solution (0.15 M), kelatorphan (35 nmol), kelatorphan (68 nmol), DSTBULET (1 mnol), DSTBULET (16nmol) and DAGG (1.3 nmol). Motility recording. The animals were injected according to the above-mentioned procedure and immediately placed on the supporting plate of the motility recorder. The differential kinetics of locomotor activity indicated the time-lapse of the effect of the treatment. Five groups of 20 rats were used for the detection of motility. In each group 10 rats always received saline in the ventral tegmental area, whereas the other 10 rats concomitantly underwent infusion of the drug into the ventral tegmental area i.e. kelatorphan (35 nmol), kelatorphan (68 nmol), DSTBULET (1 mnol), DSTBULET (11 nmol) and DAGO (1.3 nmol). Histology

Upon completion of the experiments, the subjects were sacrificed and the brains removed and placed in 10% formalin in saline for 10 days. Then the brains were frozen and cut in the coronal plane at 50pm

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consecutive sections, which were stained with Nissl and W~lck~Heiden~in techniques. Histological confirmation of the placement of the electrodes and cannulae was obtained by examining these stained coronal sections of the brains.

Figure 1 shows the number of bar-pressings, as a function of time (periods of 5 min), under the various drug-treatment conditions. In the pre-injection test, the mean self-stimulation rates on treatment with vehicle (saline 0.15 M), kelatorphan (35 nmol), kelatorphan (68 nmol), DSTBULET (1 nmol), DSTBULET (16 nmol) and DAGO (1.3 nmol) were 196.0, 170.4, 162.4, 149.4, 194.5 and 159.7 bar-pressings per Smin, respectively. For the purposes of display, post-injection values are expressed as a percentage of the pre-inj~tion baseline of selfstimulation. As shown in Fig. l(A), baseline rates of

Analysis of data Results from self-stimulation experiments were analyzed using the two-tailed Wilcoxon matchedpairs signed-ranks T-test @-e-treatment and posttreatment pairs of scores were compared for each subject). Data from motility recording were analyzed using analyses of variance (ANOVA) for repeated measures (general linear models procedure). ICS8 rat0 (S of pro-lnlrotbn toat) 200

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Fig. 1. Rate of intracranial self-stimulation behaviour (ICSS) after infusion of vehicle solution (saline 0.15 M), kelatophan (35 nmol) and DSTBULET (1 nmol) (A); vehicle solution (saline 0.15 M), kelatorphan (68 nmol), DSTBULET (16 nmol) and DAGO (1.3 nmol) (B) into the ventral tegmental area. Bar-pressing rates after each treatment are expressed as a percentage of rates obtained in a preinjeetion test, performed during the same session. Repeated measures (5 min periods) were taken on 6 rats for each treatment. *P < 0.05.

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intracranial self-stimulation did not vary significantly following the administration of vehicle solution (saline 0.15 M), kelatorphan (35 nmol) and DSTBULET (1 nmol) into the ventral tefqnental area. At a dose of 16 nmol DSTBULET produced a significant increase in the rate of self-stimulation, from 30 to 35 min after the injection procedure. Similarly, activation of the mu-opioid receptors by DAGO, at a dose of 1.3 nmol, significantly increased the rate of self-stimulation, from 10 min to the end (i.e. 60 min) of the post-injection session. Contrary to activation of the mu- and delta-opioid receptors by DAGO and DSTBULET, respectively, enhancement of endogenous enkephalins by inhibiting the metabolism of enkephalin through kelatorphan, at a dose of

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68 nmol, induced a significant decrease in the rate of self-stimulation, from 4Omin to the end of the post-injection session. These results are shown in Fig. l(B). Motility recording The data de~~bing the dug-indu~d variations of spontaneous motor activity are presented in Fig. 2. Post-injection values are expressed as a percentage of control rats, receiving saline in the ventral tegmental area and tested concomitantly with drug-treated animals. As shown in Fig. 2(A), rats receiving infusion of DSTBULET into the ventral tegmental area, at a dose of 1 nmol, kelatorphan at a dose of 35 run01 or

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Fig. 2. Differential kinetics of spontaneous motor activity, indicating the time-lapse of the effect resulting from infusion of DSTBULET (1 nmol), kelatorphan (35 nmol) and DAGO (1.3 nmol) (A); DSTBULET (I 1 nmol) and kelatorphan (68 mno1) (B) into the ventral tegmental area. The data describing these drug-induced variations are expressed as a percentage of control rats receiving saline (0.15 M) into the ventral tegmental area and tested concomitantly with drug-treated animals. For each treatment, repeated measures (60 min periods) were taken on 20 rats (IO controls vs 10 drug-treated rats). *P -z 0.05.

Effects

of VTA opioid systems on reward and motility

DAGO at a dose of 1.3 mnol, did not significantly vary from their respective controls. Activation of the delta-opioid receptors in the ventral tegmental area by DSTBULET, at a dose of 11 nmol produced a significant decrease in the spontaneous motor activity 4 and 5 hr after the injection procedure. On the contrary, inhibition of the metabolism of enkephalin by kelatorphan, at a dose of 68 nmol, induced a significant increase of motility 4 hr after its administration into the ventral tegmental area. These results are shown in Fig. 2(B). DISCUSSION

The selective mu- and delta-opioid agonists, DAGG and DSTBULET, proved to be extremely potent in self-stimulation experiments and reliably increased the rate of bar-pressings, with a much greater intensity for DAGO rather than for DSTBULET. Indeed, increased rates of self-stimulation have been demonstrated following the injection of morphine, D-Ala-enkephalin (DALA) or [D-Pet&DPet?]enkephalin (DPDPE) into the ventral tegmental area (Broekkamp et al., 1976, 1979; Jenck, Gratton and Wise, 1987) and electrophysiological studies indicate that morphine, injected into the ventral tegmental area, stimulates the activity of dopaminergic neurones (Gys!ing and Wang, 1983; Hommer and Pert, 1983; Matthews and German, 1984). More recently, in uiuo dialysis experiments have reported morphine-induced increases of extracellular concentrations of dopamine in the nucleus accumbens, consistent with increased activity of mesolimbic dopaminergic pathways (Di Chiara and Imperato, 1988a, b), whereas measurements of 3-methoxytyramine in tissue also indicate that opioids increase release of dopamine in mesolimbic and mesocortical terminal areas (Wood and Altar, 1988). Furthermore, stimulation of mu- or delta-opioid receptors, within the ventral tegmental area using either DAGO and DPDPE has been reported to give rise to an appetitive motivational effect, which determines conditioning of specific place preferences (Shippenberg, Bals-Kubik, Spanagel and Herz, 1989). Also, DAGO has been shown to be more potent than the deltaopioid receptor agonist, DPDPE in increasing the metabolism of dopamine in the nucleus accumbens, striatum, septum and prefrontal cortex (Latimer, Duffy and Kalivas, 1987), suggesting that mu-opioid receptors are largely responsible for the activation of dopamine neurones in the ventral tegmental area by morphine and other opioid agonists. This view may also be supported by the fact that mu-opioid receptors predominate in the ventral mesencephalon, whereas delta-opioid receptors are poorly represented in this region of the brain (Zukin and Zukin, 1981). Although endogenous enkephalins display a considerably greater affinity for delta-, rather than for mu-opioid receptors (Paterson, Robson and

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Kosterhtz, 1983) and although kelatorphan-induced behavioural activation can be blocked by a deltaopioid receptor antagonist, following intraventricular infusion (De Witte, Heidbreder and Roques, 1989), the present results show that enhancement of endogenous enkephalins by inhibiting the metabolism of enkephalin by kelatorphan, produced a decrease in the rate of self-stimulation, contrary to the above-reported data resulting from activation of mu- or delta-opioid receptors. Functional interactions between specific opioid receptor systems and dopamine thus appear to represent only one of the multiple neuromodulatory links within the Alo dopamine region. Indeed, one of the main problems is not only related to the subtype of opioid receptors which mediate the behavioural response but also refers to the question of establishing whether this response is indeed dependent upon dopamine activity and if so, whether or not a subdivision of the ascending dopamine pathways from the ventral midbrain is primarily responsible for the effects of opioids. As regards the motility recording, infusion of DSTBULET into the ventral tegmental area induced a significant decrease of the spontaneous motor activity, whereas kelatorphan significantly increased the movements of the animal. Inhibition of the metabolism of enkephalin in the ventral tegmental area by administration of thiorphan, has been reported to produce a naloxone-reversible increase in spontaneous motor activity and mesolimbic metabolism of dopamine (Kalivas, Duffy, Dilts and Athold, 1988). The failure of DAGO to affect motility in these experiments would suggest that larger doses are necessary to modulate this behaviour as estimated in this model. Other recording methods, however, such as measurement of horizontal motor activity by a photocell apparatus, detected a motor-stimulant effect after injection of DAGO (0.1 nmol) into the ventral tegmental area, whereas an equimolar dose of the delta-receptor agonist (n-Pens-enkephahn (DPEN) was revealed to be ineffective (Kalivas et al., 1988; Latimer et al., 1987). Two main differences may also explain the considerable discrepancies from study to study: it seems likely that the first one lies in the kind of injection (unilateral vs bilateral) whereas the second difference may be related to the duration of the recording method. Indeed, the present data from analysis of motility were obtained after a 5-hr recording period, which contrasts with the paradigms most commonly used in this field. In addition to the anatomical heterogeneity of the ventral tegmental area with its mediolateral, dorsoventral and anteroposterior aspects and the more than likely specific distribution of opioid receptors within the ventral tegmental area, it may be suggested that opioid-induced variations of selfstimulation reward and spontaneous motility could also be explained by the occurrence of multiple opioid

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receptors on neurones in the ventral tegmental area with different terminal areas. In this way, binding of a ligand, selective for a given subtype of opioid receptor, might allosterically change the binding of ligands selective for the associated type of receptor.

neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescenceimmunocytochemistry. Brain Res. 194: 566571. Joyce E. M. and Iversen S. D. (1979) The effects of morphine applied locally to mesencephalic dopamine cell bodies on spontaneous motor activity in the rat. Neurosci.

Acknowledgemen&-Ch. Heidbreder is research associate of the Fonds de Wveloppement Scientifique from the Univer-

Kalivas P. W., Duffy P., Dilts R. and Athold R. (1988) Enkephalin modulation of A,, dopamine neurons: A role in dopamine sensitization. Ann. N.Y. Acad. Sci. 557:

sity of Louvain. This work was supported by grants from FRSM (90-93) and FMRE (89-92). REFERENCES

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