Dopaminergic mediation of reward produced by direct injection of enkephalin into the ventral tegmental area of the rat

Life Sciences, Vol. 33, pp. 2505-2511 Printed in the U.S.A.

Pergamon Pres

DOPAMINERGIC M E D I A T I O N OF REWARD PRODUCED BY D I R E C T INJECTION OF ENKEPHALIN

INTO THE VENTRAL TE(~ENTAL AREA OF THE RAT

Anthony G. Phillips,

Fredric G. LePiane and Hans C. Fibiger

Department of Psychology and Department University of Vancouver,

Division of Neurological of Psychiatry British Columbia Canada V6T IW5

Sciences

(Received in final form October 3, 1983) Summary A conditioned place preference paradigm was employed to demonstrate the rewarding property of unilateral injections of 200 ng (D-ala2)-met5-enkephalinamide into the ventral tegmental area of the rat brain. This effect was attenuated in a dose-related manner by systemic injections of the dopamine receptor blocker haloperidol. In addition, selective lesions of the ascending dopamine (DA) pathways ipsilateral to the injection site blocked the rewarding effect when DA levels were reduced by more than 90%. Similar lesions in the contralateral hemisphere had no influence on this behavior. These data suggest that forebrain DA pathways can mediate some of the rewarding properties of opioid drugs. Opioid drugs and the recently discovered opioid peptides have rewarding properties (1,2) that may be related to drug-induced euphoria and the attendant abuse of these compounds by man. Currently, three separate lines of evidence suggest that the affective consequences of opioid administration may be determined to an important degree by an action of these compounds on neurons within the ventral tegmental area (VTA). In the first place, intracerebral injections of morphine or enkephalin into the VTA enhance intracranial self-stimulation in the lateral hypothalamus (3). Furthermore, rats will self-administer morphine directly into the VTA (4) but not at sites in the periventricular gray matter, lateral hypothalamus, nucleus accumbens or caudate nucleus (5). A third approach has employed the conditioned place preference paradigm (6) to demonstrate the rewarding effects of intracerebral mi,croinjections of morphine (5,7) or (D-AIa2) met5-enkephalinamide [D-AIa] (8) into the VTA. The conditioned place preference paradigm is well suited to screening for the possible pharmacological blockade of drug reward as the critical tests are conducted when the animals are in a drug-free state. Recently, for example, two independent studies have used this paradigm to demonstrate that the rewarding property of peripheral injections of heroin is attenuated by the dopamine receptor antagonists haloperidol (9) and pimozide (I0). The VTA contains some of the cell bodies of the mesolimbi~ dopamine projection (ii) and anatomical data suggest an interaction between dopamine and enkephalin containing neurons in this region of the midbrain (12). The present study was

0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

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designed to test the hypothesis that the rewarding effects of opioids may be dependent to an important degree on an enkephalinergic-dopaminergic link in the VTA. The conditioned place preference procedure was used to demonstrate the rewarding effect of unilateral injections of D-AIa into the VTA and this effect was subsequently blocked by either pretreatment with a peripheral injection of the DA receptor antagonist haloperidol (13) or by previous damage to ascending dopaminergic axons ipsilateral to the sites of the D-AIa injections. Method Subjects: Male Wistar rats weighing 300-500 g were anaethetized with Nembutal (50 mg/kg) and stainless steel guide cannulae (23 ga) were implanted unilaterally under stereotaxic control into an area 1.0 nun dorsal to the VTA. Injection needles (30 ga) projected 0.5 mm beyond the tip of the guide cannulae. The following co-ordinates were taken from stereotaxic zero, with head level: anterior +2.1 nra, lateral 0.9 mm, dorsal/ventral -2.6 mm. All cannulae placements were confirmed histologically and only those subjects with accurate placements were included in the statistical analyses. Apparatus: All behavioral testing was conducted in two similar test chambers; each consisting of two large end compartments (34x24 cm), separated by guillotine doors from a smaller middle compartment (Iix25 cm) which served as a choice point in a subsequent test session. To facilitate the association between drug effects and test environment, one end compartment had black walls and a mesh floor while the other had white walls and a parallel grid floor. The testing chambers were housed in a quiet room illuminated by fluorescent overhead lighting. Procedure: On the first 3 days of behavioral testing, each animal was allowed to explore all compartments of the chamber for 15 rain. Time spent by each animal in each of the 2 end compartments was measured to the nearest second and this provided a measure of preference between the 2 compartments. In Experiment I animals were assigned to one of 4 experimental groups, distinguished by the dose of haloperidol (0.025, 0.05, 0.I0 mg/kg) or vehicle administered by intraperitoneal (ip) injection 45 rain prior to each daily intracerebral microinjection of D-AIa. A dose response analysis of the rewarding effect of D-AIa injections into VTA has indicated that effective doses range between 100-250 ng (8). Therefore a dose of 200 ng/0.5 111 was injected 5 min prior to behavioural testing. Each animal was placed into the non-preferred compartment of a shuttlebox and confined there by a guillotine door for 30 rain. This conditioning procedure was repeated for 3 days. On the fourth day, each animal received a sham microinjection prior to being allowed to move freely between both end compartments for 15 min. In theory, reward produced by D-AIa should establish salient environmental stimuli as conditioned reinforcers through the process of classical conditioning (14). Therefore any primary rewarding effect of D-AIa would be confirmed by a significant shift in preference toward the compartment associated with enkephalin microinjections. Experiment 2 involved a more direct test of the critical involvement of ascending DA pathways in mediating the rewarding effects of opioid drugs in general and intracerebral injections of enkephalin in particular. Rats were prepared with unilateral cannulae aimed at the VTA as described above. At the same time, while under anaesthesia, 6-hydroxydopamine HBr (6-OHDA) (4~g base / 2~I) was slowly infused (7-10 rain) into the vicinity of the ascending DA

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Figure 1 Effect of haloperidol (0.25-0.10 mg/kg ip) on conditioned place preference. Data represent .X+s.e.m. time spent by each group in a shuttlebox compartment before and after pairing with D-AIa (200 ng) injections into the VTA.

pathways at the level of the lateral hypothalamus. An aqueous solution of desipramine HCI (25 mg/kg) was injected ip 30 min prior to surgery to spare noradrenergic axons. In half of the animals the lesion was made ipsilateral to the cannulae placement in the VTA. The remaining animals served as controls and had contralateral lesions. Upon completion of the behavioural tests, the animals were sacrificed and the brains prepared for biochemical and histological analyses. The concentration of DA in the two sides of the forebrain (obtained by making a coronal cut at the level of the decussation of the anterior commissure) was determined spectrofluorometrically for each animal (15). Res ul ts Forty animals were implanted with unilateral cannulae for Experiment I and correct placements were defined as those located within I n~n of the dorsal surface of the interpeduncular nucleus and medial to the substantia nigra. Placements were counterbalanced between hemispheres. Thirty subjects met these criteria and their behavioral data were included in the statistical analyses. A strong conditioned place preference was obtained with the vehicle control group. Prior to D-AIa injections this group spent an average of 155 s e c in the "conditioning" compartment of the shuttlebox. This score had increased to X = 468 sec on tlle post-conditioning test trial (Fig. I). Fig. i also shows that increasing doses of haloperidol produced a progressively greater attenuation of the conditioned place preference. Statistical analyses were performed on the difference scores obtained by subtracting the preconditioning scores for each subject from the post-condltioning scores. Analysis of variance yielded a significant main effect for drug dose (F(3,26) = 3.40, p < 0.03). The groups pretreated with 0.05 and 0.I0 mg/kg of haloperidol both differed significantly from the vehicle control group (p_ < 0.05, Duncan's range test).

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Figure 2 Cannulae placements for subjects with 6-ONDA lesions in the ipsilateral (1)or contralateral hemisphere ( O ) . Misplaced cannulae ( A ) . Brain sections redrawn from Konig and Klippel.

The original subject pool for Experiment 2 consisted of 34 rats, one of which was discarded prior to histology because of a broken injection needle. Cannulae placements for the remaining 33 subjects are shown in Fig. 2 according to lesion location. Four of these animals had misplaced cannulae as indicated and were not included in the statistical analyses. The remaining 29 subjects were categorized further according to the level of DA depletion as revealed by the biochemical analyses. Control values for DA in the unilateral forebrain were X(+s.e.m.) = 2.08 + 0.04 ng/gm. Eight of the 15 animals with ipsilateral lesions had successfuT lesions that depleted forebrain DA levels by more than 95% (~ = 97.48+0.5%) of control values. Seven animals had partial lesions (X = 74.5+5--0%). A similar dichotomy was observed with contralateral lesions. Seven subjects had nearly complete lesions (X = 96.9+0.80%) and the remaining 7 animals had partial lesions (X = 68.7+10.9%).

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33, No. 25, 1983

Dopaminergic

Mediation of Reward by Enkephalin

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Figure 3 Effect of partial (<90%) or complete (>90%) depletion of forebrain DA in the hemisphere contralateral or ipsilateral to D-AIa (200 ng) injections into the VTA. Data represent X+s.e.m. time from pre- and post-conditioning trials.

The behavioral data for these four subgroups are shown in Fig. 3. Successful place conditioning was obtained with both of the contralateral lesions groups and with the ipsilateral group with a partial lesion. No conditioned place preference was observed in the ipsilateral lesion group with nearly complete depletion of forebrain DA ipsilateral to the site of opioid injection. An analysis of the difference scores from subjects in the ipsilateral and contralateral groups with successful lesions revealed a significant difference in the magnitude of conditioned place preference (t14 = 3.60, p < 0.01). These groups did not differ significantly in the magnitude of depletion in forebrain DA levels. Discussion In the present experiment, control rats receiving unilateral injections of D-AIa into the VTA subsequently spent significantly more time in the environment previously associated with the peptide. These data coupled with a previous report that similar injections produced a significantly greater change in place preference than vehicle injections (8), can be interpreted as evidence that the peptide acted as a conditioned reinforcer. Attenuation of opioid-induced conditioned place preference by haloperidol or 6-OHDA lesions does not appear to be an indirect consequence of a deficit in sensory-sensory associative learning. There is considerable evidence suggesting that disruption of brain dopamine systems does not affect this type of learning by the rat (16).

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Dopaminergic systems do, however, play an important role in response initation (17) but this factor cannot account for the present findings as the place preference paradigm does not require the animals to learn or perform specific motor responses. Similarly, the data from the 6-OHDA lesion study do not appear to reflect a reduction in crossing from one compartment to another. The baseline crossing rate for the group with >95% depletion of forebrain D A ipsilateral to the pe_ptide injection had a baseline score of X = 4.3 crosses/15 min as compared to X = 3.0 crosses/15 rain for the contralateral groups with a comparable level of DA depletion. The latter group developed a strong conditioned placed preference but the former group did not. Haloperidol has been used previously in place preference tests to block the rewarding effects of heroin (9) and amphetamine (18), but not cocaine (19). Although the present design of Experiment I did not include a condition in which animals received only haloperidol, other studies have failed to confirm conditioned place aversion with neuroleptics (9,10,18,20). The doses of haloperidol used in these earlier studies ranged from (0.15 - 1.0 mg/kg) and therefore the slight possibility of aversive consequences with lower doses of haloperidol (0.25 - 0. I0 mg/kg) cannot be discounted. An enkephalinergic substrate of reward was first proposed by Belluzi and Stein (2) following the successful demonstration of intraventricular self-administration of leu- and met-enkephalin. The place conditioning paradigm has provided a variety of data in support of this hypothesis. Place conditioning has been obtained with intraventricular injections of D-AIa (21,22) and there have been reports of place conditioning following localized intracerebral injections of morphine into the lateral hypothalamus, nucleus accumbens and periaqueductal gray (23), as well as the VTA (7). To date, the most sensitive area for these effects appears to be the VTA and this conclusion is substantiated by recent studies of intracranial self-administraion of morphine (4,5). Together these studies suggest that neurons in the VTA are an important substrate for the rewarding effects of opiates and enkephalin. Furthermore, the present findings specifically indicate that the dopaminergic elements within the VTA mediate opioid-induced reward, at least when these compounds are applied directly to the VTA. It has been suggested that DA neurons play an important role in mediating the rewarding effects of both opioid and psychomotor stimulant drugs (7). This hypothesis has been challenged recently by several pieces of data. Bilateral 6-OHDA lesions of the dopaminergic axons and terminals in the nucleus accumbens did not attenuate intravenous self-administration of heroin, although similar lesions did block cocaine self-administration (24). Similarly, pretreatment with high doses of the DA antagonist alpha-flupenthixol blocked responding for cocaine but only produced a slight reduction in heroin self-administration (25). One possible explanation for the discrepancy between these data and the present results may be the existence of rmlltiple neural substrates of opioid reward, only one of which is located in the VTA. Systemic administration of opioids would influence many different substrates and would therefore be relatively unaffected by damage to only one sub-system. In contrast, reward produced by direct intracranial activation of a specific substrate would be critically dependent upon the integrity of that single element. It should also be noted that both neuroleptics and 6-OHDA lesions of the nucleus accumbens failed to block conditioned place preference with cocaine (19). These results were attributed to the local anaesthetic effects of cocaine and serve to emphasize that intravenous self-administration and the place preference paradigms may not be measuring identical processes.

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The existence of several types of opioid receptor in the brain is now well established (26) and the question remains as to which of these may mediate the rewarding effects of opioids. Both morphine and the enkephalins have an affinity for the ~ and 6 receptors making them both possible candidates. Leu-e~kephalin appears to have a stronger affinity for the ~ receptor than met-enkephalin and therefore it will be important to compare the potency of these opioid peptides in the conditioned place preference paradigm.

A c k n o w l e d g e m e n t.s

This research was supported by Program grant #23 from the Medical Research Council of Canada to HCF and AGP. The excellent technical assistance of Elaine Chan and Stella Atmadja is acknowledged gratefully. Re ferences i. 2. 3.

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