Evidence for a role of endogenous opioids in the nigrostriatal system: Influence of naloxone and morphine on nigrostriatal dopaminergic supersensitivity

Brain Research, 270 (1983) 109-117

109

Elsevier

Evidence for a Role of Endogenous Opioids in the Nigrostriatal System: Influence of Naloxone and Morphine on Nigrostriatal Dopaminergic Supersensitivity IRA D. H I R S C H H O R N , DANIEL HITTNER, ELIOT L. G A R D N E R , JOSEPH CUBELLS and M A Y N A R D H. M A K M A N

(I.D.H., D.H. and M.H.M.) Departments of Biochemistry, (M.H.M.) Molecular Pharmacology, (E. L. G. and J. C.) Psychiatry and (E. L. G. and J. C.) N euroscience, Albert Einstein College of Medicine, Bronx, N Y 10461 ( U. S. A .) (Accepted November 30th, 1982)

Key words: rotational behavior - enkephalin - apomorphine - amphetamine - nigrostriatum - supersensitivity

The effects of morphine and naloxone on nigrostriatal function were evaluated by their influence on rotational behavior in rats with unilateral lesions of the substantial nigra. Two different rotational syndromes which result from different lesion placements, were examined. Rats with the contraversive syndrome, when given apomorphine, rotate away from the lesioned side, while rats with the ipsiversive syndrome rotate toward the lesioned side. In both syndromes, rats rotate toward the lesioned side when given amphetamine. Morphine or naloxone, alone, was without effect in either syndrome. Morphine antagonized rotation by either apomorphine or amphetamine in both syndromes. Naloxone stimulated apomorphine-induced rotation in contraversive rats and antagonized amphetamine-induced rotation in ipsiversive rats. These findings support a functional role of endogenous opioids in this dopaminergic system. The effects of morphine and naloxone on apomorphine-induced rotation indicate that opiates act at a postsynaptic site in this system. Finally, the different responses to naloxone and morphine in the two rotational syndromes suggest that an enkephalinergic assymetry may underlie the differences in behavioral responses between these two syndromes. INTRODUCTION

A number of observations suggest that opiates might interact with the dopaminergic nigrostriatal system. A high content of enkephalins42, a high density of opiate binding sites 26,47and enkephalin containing neurons2° are present in the striatum. Lesions of the nigrostriatal pathway which deplete striatal dopamine, also decrease opiate binding in the striatum 38. This suggests that some enkephalinergic neurons synapse on dopamine nerve terminals and, in fact, presynaptic effects of opiates on dopamine release have been reported 28,33,39.The effects of opiates on motor function, such as hypokinesia, catalepsy, and muscle rigidity, resemble those of impaired dopaminergic transmission, and some of these are reversed by dopamine agonists27. A role of endogenous enkephalin in striatal dopaminergic systems is suggested by the effect of chronic haloperidol, a dopamine receptor antagonist, on enkephalin biosynthesis2~. The present 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

experiments investigated the effect of opiates upon nigrostriatal function by utilizing rotational behavior in substantia nigra lesioned rats 3.46. Unilateral substantia nigra lesions cause degeneration of the nigrostriatal dopaminergic neurons and a decrease in striatal dopamine. Drugs such as amphetamine, which release dopamine, produce rotation in the direction toward the side of the lesion (ipsilateral rotation), presumably by releasing dopamine from the intact nigrostriatal neurons. In animals with the lesion placed just medial to substantia nigra pars compacta, as has been the case in most studies, direct-acting dopaminergic drugs, such as apomorphine, produce rotation in the direction away from the lesioned side (contralateral rotation). This contralateral rotation is generally believed to be caused by stimulation of dopamine receptors that have become supersensitive after denervation. This idea is supported by the observation that supersensitivity in the denervated striatum occurs with dopamine stimulated adenylate cy-

Ii0 clase activity 32 and dopaminergic receptor binding 1l,~8,44. However, when the lesion is placed more laterally, that is, within pars compacta, the animals display a different behavioral syndrome. These animals also rotate ipsilaterally when given amphetamine, but they differ in that they rotate ipsilaterally instead of contralaterally following apomorphinea,15. It is assumed that this ipsilateral rotation is a result of inactivation of the lesioned striatum so that apomorphine can activate only the non-lesioned striatum. Ipsiversive rotators are equivalent to contraversive rotators in the extent of striatal dopamine depletion, enhanced dopamine stimulated adenylate cyclase ~5 and dopaminergic receptor binding44. There is evidence that an enkephalinergic mechanism may be involved in causing the different behavioral effects. In contraversive rats, enkephalin binding in the denervated striatum is decreased, but it is increased in ipsiversive rats ~6. If this enkephalinergic asymmetry is the cause of the different behavioral responses, the opiates or their antagonists should effect the two syndromes differently. In the present experiments we examined and compared the effects of morphine and naloxone on contraversive and ipsiversive rats.

was by a two way analysis of variance. Any rotations in the opposite direction were subtracted from the total. Apomorphine hydrochloride was dissolved in 0.04% ascorbic acid and all other drugs were dissolved in 0.9% saline. Dosages are expressed as the following salts: apomorphine hydrochloride, D-amphetamine sulphate, morphine sulphate, naloxone hydrochloride. Drugs were injected, i.p., in a volume of 1 ml/kg. Following injection of apomorphine or amphetamine, animals were placed immediately in the rotometer. In the drug interaction experiments, morphine or naloxone was injected 10 min prior to apomorphine or amphetamine. RESULTS

Effect of apomorphine and amphetamine Data from saline pretreatment (control) sessions illustrate the characteristics of apomorphine- and amphetamine-induced rotation in rats with the contraversive and ipsiversive rotational syndromes. These data are summarized in Fig. 1. The rats with lesions placed just medial to opomorphine (mg/kg) OA 0.5 1.0 2.0

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MATERIALS A N D METHODS

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Unilateral substantia nigra lesions were produced in male S p r a g u ~ D a w l e y rats (300 g) using standard surgical and stereotaxic techniques. 6-hydroxydopamine hydrobromide (8 ~g in 1 ~1 0.02% ascorbate) was injected over 5 min. The coordinates ~3 for contraversive rats were AP 2.6, L 1.1, DV 2.9, and for ipsiversive rats were AP 2.6, L. 2.15, DV 2.1. Circling was recorded in an automated rotometer and only rats which circled at a rate of at least 4 turns per minute in response to 2.5 m g / k g of apomorphine were used. In numerous previous studies ~5.~6,~8,32,we have found that rats which exhibit rotational behavior under these conditions consistently have almost complete striatal dopaminergic denervation. Rats were tested on Mondays, Wednesdays and Fridays and each drug trial was followed or preceded by a saline trial. Statistical evaluation

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111 the substantia nigra pars compacta exhibited the contraversive rotational syndrome, i.e., they circled away from the lesioned side following the administration of apomorphine as originally reported by Ungerstedt46. In contrast, the rats with lesions placed within the substantia nigra pars compacta exhibited the ipsiversive rotational syndrome as previously reported sJS, i.e., they circled toward the lesioned side following the administration of apomorphine. In animals with either syndrome, amphetamine caused circling toward the lesioned side. Rotation rate, in all cases, varied directly with the dose. In addition, Fig. 1 shows that the rats with the contraversive syndrome are more sensitive to both apomorphine and amphetamine than the rats with the ipsiversive syndrome, i.e., any given dose of either drug produced a higher rate of rotation in the contraversive rats than in the ipsiversive rats. Following apomorphine administration, rats with either the contraversive (Figs. 2A, B and 4A, B, C) or the ipsiversive (Fig. 2C and 4D, E, F) syndromes reached a maximum rate of rotation at 5- 10 min, followed by a gradual decline. Amphetamine, however, produced different changes in response rate with time in the two syndromes. Rats with the ipsiversive syndrome had a maximum rotation rate at 5-15 rain following amphetamine administration, after which it gradually decreased or remained constant (Figs. 3B and 5C, D), a pattern similar to that following apomorphine in either syndrome. In contrast, the rotation rate of contraversive rats gradually increased for 40-45 min before becoming stable or decreasing (Figs. 3A and 5A, B). Since the rotation rate of contraversive rats was increasing as that of ipsiversive rats was decreasing, the differential sensitivity of rats with the two syndromes to amphetamine, as measured by rotation rates, varied with time. Nevertheless, the difference in sensitivity to amphetamine is present throughout the session and only the magnitude of the difference changes with time after amphetamine administration.

Effect of morphine and naloxone Neither morphine (10 mg/kg) nor naloxone (5 mg/kg), alone, produced circling in contra-

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Fig. 2. The effect of morphine on apomorphine-induced rotation in contraversive (A and B) and ipsiversive (C) rats. Morphine sulphate, 10 mg/kg (O), or saline (O) was injected 10 min prior to apomorphine hydrochloride (2 mg/kg, a and c, 0.5 mg/kg, B) and rotation rate was recorded. Ordinate: average contralateral (A and B) or ipsilateral (C) rotations per minute for 5 rain period. Abcissa: time after apomorphine injection. Each point is the mean of at least two determinations in each of 4 (A, B) or 5 (C) rats. Significant treatment effects: B and C, P < 0.001. Significant time effects: A and C, P < 0.001.

versive or ipsiversive rats. After 5 days of daily morphine injection to render the animals tolerant to morphine's locomotor depressant effects36, morphine still failed to produce consistent rotation. One rat did circle ipsilaterally fol-

112 lowing chronic morphine treatment (1-5 rpm), but this did not occur with the other animals nor was it observed in this same rat upon replication. Neither morphine nor naloxone produced any obvious behavioral effects. In a few experiments in which rotation was recorded prior to the injection of apomorphine or amphetamine, there were no differences in total 1/4 turns, a measure of locomotor activity, between drug and saline pretreatments.

Effect of morphine on apomorphine- and amphetamine-induced rotation The effect of morphine on rotation is shown in Figs. 2 (apomorphine) and 3 (amphetamine) and summarized in Table I. Morphine was strongly inhibitory to apomorphine and amphetamine induced rotation in rats with both the contraversive and ipsiversive syndromes. Only at the higher dose of apomorphine in contraversive rats (2 mg/kg, Fig. 2A) was this effect not observed. The rate of rotation produced by 2 mg/kg of apomorphine in contraversive rats was the highest in these experiments and the fixed dose of morphine was apparently not sufficient to counteract this very large stimulatory effect, yet was sufficient to counteract the lesser stimulation produced by 0.5 mg/kg of apomorphine. In terms of changes of mean rotation rate, as shown in Table I, the magnitude of the inhibition produced by morphine is similar in rats with contraversive and ipsiversive syndromes.

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Effect of naloxone on apomorphine- and amphetamine-induced rotation TABLE I

Effect of morphine on apomorphine- and amphetamine-induced rotational behavior Data are from the experiments represented in Figs. 2 and 3 and are the means ± S.E.M. for the entire 60-min recorded session.

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The effects of naloxone on rotation were different in contraversive and ipsiversive rats. The effect of naloxone on apomorphine-induced rotation is shown in Fig. 4. In the contraversive rats, naloxone increased the apomorphine rotation rate. This stimulatory effect was greatest at 0.5 mg/kg o f a p o m o r p h i n e (Fig. 4B) where rotation rate increased by one-third. At the lower apomorphine dose, 0.1 mg/kg (Fig. 4C), although rotation was of shorter duration and less consistent than at the higher doses, the stimulatory effect ofnaloxone was evident in the first 15

113 greatly reduced for 25 min when the dose of amphetamine was 5 mg/kg (Fig. 5C). There was no effect at 2 mg/kg of amphetamine, but the control rotation rate, at this dose, was only 1-2 turns per minute so a further lowering of the rate would be very difficult to demonstrate.

min. There was no effect ofnaloxone at 2 mg/kg of apomorphine (Fig. 4A), but the reason for this could be that the high rotation rate under control conditions at this dose was already at or close to maximum. It may be noteworthy that naloxone and morphine both had their greatest effects at the same apomorphine dose (0.5 mg/kg) in the contraversive rats, and both had little or no effect at the higher dose of apomorphine (2 mg/kg) in these animals. Naloxone was without effect on apomorphine-induced rotation in the ipsiversive rats (Fig. 4D-F). The effect of naloxone on amphetamine-induced rotation is shown in Fig. 5. Naloxone did not alter amphetamine-induced rotation in contraversive rats (Fig. 5A and 5B). In ipsiversive rats, naloxone was inhibitory. Rotation rate was

DISCUSSION

The results of the present study add to the already considerable evidence from various kinds of experiments 1,21.27,38that the function of the dopaminergic nigrostriatal system is modulated by opiates. The greatest and most consistent effect was that of morphine inhibiting rotational behavior. This occurred with rotation produced by both apomorphine and amphetamine and in

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Fig. 4. The effect of naloxone on apomorphine-induced rotation in contraversive (A-C) and ipsiversive (D-F) rats. Naloxone hydrochloride, 5 mg/kg (O), or saline (O) was injected 10 min prior to apomorphine hydrochloride (various doses) and rotation rate was recorded. Ordinate: average contralateral (A-C) or ipsilateral (IN-F) rotations per minute for 5 min period. Abcissa: time after apomorphine injection. Each point is the mean of 3 determinations in each of 5 rats (A) or 2 determinations in each of 4 rats (all others). Significant treatment effect: B, P ~ 0.005. Significant time effects: C, P ~ 0.005; D, P ~ 0.001 ; E, P ~ 0.05.

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Fig. 5. The effect of naloxone on amphetamine-induced rotation in contraversive (A and B) and ipsiversive (C and D) rats. Naloxone hydrochloride, 5 mg/kg (O), or saline (©) were injected, 10 min prior to D-amphetamine sulfate (5 mg/kg, A and C; 2 mg/kg, B and D) and rotation rate was recorded. Ordinate: average ipsilateral rotations per minute for five minute period. Abcissa: time after amphetamine injection. Each point is the mean of 2 determinations in each of 5 rats. Significant treatment effect: C, P < 0.05. Significant time effects: A and B, P < 0.001; C, P < 0.05.

rats with either the contraversive or the ipsiversive rotational syndrome. It is unlikely that this is a non-specific depressant effect since the dose of morphine that was used usually has no effect or is even stimulatory to locomotor activity in the rat 5J2J9 and, in the present study, no depression of locomotor activity was observed with morphine alone. Morphine inhibition of apomorphine m and amphetamineg.41-induced rotation has been previously reported for contraversive rats only, and the present results extend it to rats with the ipsiversive rotational syndrome. Morphine has been shown to inhibit the release of striatal dopamine in vitro28,43 and Slater and BlundelW proposed this mechanism for the inhi-

bition of amphetamine-induced rotation. However, this mechanism cannot explain the inhibition of apomorphine-induced rotation because apomorphine, as a direct dopamine receptor agonist, does not require the release of dopamine for its action. In addition, even if apomorphine were to cause the release of dopamine, this would not be possible on the lesioned side because following lesion of nigrostriatal neurons, there is little or no striatal dopamine remaining and apomorphine is believed to produce contraversive rotation by predominantly activating the lesioned striatum. Finally, the nature of the effect of morphine on striatal dopamine neurons is not clear. Not only inhibition, but also no ef-

115 fect33,34 and stimulation 1,7,17,39 have been reported. In addition to the presynaptic nigrostriatal location38, significant numbers of striatal opiate receptors are also present on intrastriatal neurons37 and corticostriatal neurons 6. Morphine probably acts at one of these latter sites to inhibit apomorphine-induced rotation. Inhibition of amphetamine-induced rotation might also occur at the same site, and indeed, there is no particular reason to invoke a different site of action. The inhibition of rotational behavior by morphine suggests that endogenous opiates may have an inhibitory influence on dopaminergic systems in the striatum. If this is the case, a specific opiate antagonist, such as naloxone, by antagonizing the effects of endogenous opiates should have the opposite effect, i.e., stimulation. This result was obtained in the rats with the contraversive syndrome as naloxone increased the apomorphine rotation rate. However, it was not observed with apomorphine-induced rotation in the ipsiversive rats nor with amphetamine-induced rotation in rats with either syndrome. Since enhancement did not occur in these cases, naloxone is apparently not acting as a non-specific stimulant. In all the cases in which naloxone did not stimulate rotation, the intact striatum is thought to be activated to produce ipsiversive rotation, while for the one case in which naloxone did stimulate rotation, the lesioned, supersensitive, striatum is believed to be predominantly activated to produce contraversive rotation. Therefore, it may be concluded that the stimulatory effect of naloxone is observed only when rotation is produced by an action on the lesioned, supersensitive, striatum. Perhaps, under the normal conditions of the intact striatum, the amount of inhibition by endogenous opiates is small so that the net stimulation produced by blocking it is also small, while in the denervated striatum, the enhanced dopaminergic responsivity, in some manner, magnifies the effect of endogenous opiates and naloxone. If endogenous and exogenous opiates inhibit dopaminergic neurons at a postsynaptic (striatal) site instead of a presynaptic (nigrostriatal) site, then morphine and naloxone, by themselves, would not be expected to produce rota-

tional behavior because postsynaptic striatal opiate receptors are unchanged following nigrostriatal lesion and, therefore, equivalent in both striata. The inability of naloxone, alone, to produce rotational behavior in the present investigation is consistent with the results of Iwamoto et al.23 However, the effect of morphine, alone, is less clear. Morphine did not produce rotation in the present study, but Iwamoto et al.23 and Pert et al.36 found it to cause ipsiversive rotation (in contraversive rats). The significance of these results, however, is uncertain because in both of these other studies, a special manipulation (tail pinch and chronic morphine treatment, respectively) was required to bring out the effect and the rate of rotation was much lower than that obtained with dopaminergic agents. One of the primary objectives of the present investigation was to obtain information on the possible involvement of enkephalinergic mechanisms in determining the direction of rotation following administration of apomorphine in the contraversive and ipsiversive rotation syndromes. Apomorphine, as a direct dopamine receptor agonist, activates both the intact and lesioned striata, but has a greater effect upon the lesioned striatum as a result of the enhanced dopaminergic sensitivity there. In rats with the contraversive syndrome, this produces rotation in the dir etion away from the lesioned striatum and the highest rotation rates because the receptors producing the response are supersensitive. Ipsiversive rats, in spite of equivalent dopaminergic supersensitivity in the denervated striarum t5.44, rotate toward the lesioned side and at a lower rate. This implies that the intact striatum, with its normally sensitive dopamine receptors is predominantly activated and, for this to occur, the lesioned striatum must be inactivated. It is apparent that another nerve pathway besides the nigrostriatal dopaminergic pathway is involved and that this pathway is damaged in one syndrome, but intact in the other. There are several pathways in the region of the lesion sites which could possibly be damaged with one lesion placement and not the other. For example, the median forebrain bundle passes just medial to the substantia nigra 45 and is in the region of

116 the lesion placement for the contraversive syndrome. The possibility that serotonergic or noradrenergic fibers there may be lesioned in the contraversive syndrome, but not the ipsiversive syndrome, is currently being explored. The differences in striatal enkephalin binding in contraversive and ipsiversive rats ~6is a possible clue to the anatomical basis of the two syndromes and the differences in the effects of morphine and naloxone on rotational behavior in the two syndromes is further evidence that an enkephalinergic mechanism is involved. Since the discovery of the enkephalins as endogenous ligands for opiate receptors 22, evidence has accumulated for a role of enkephalins in a variety of physiological functions including modulation of pain 2.3°, f e e d i n g 29,31,35, m e m o r y 24.25, and reward 4.14.39. Some of the studies concerning reward suggest an influence ofendo-

genous opiates on dopaminergic systems outside the nigrostriatum. The present results provide evidence for a functional role in the nigrostriatum in the expression of rotational behavior. The opiate effects appear to occur at a postsynaptic (striatal) site. In addition, differential damage to these enkephalinergic systems may underlie the different responses to apomorphine in the contraversive and ipsiversive rotational syndromes. ACKNOWLEDGEMENTS

This work was supported by USPHS research Grants NS09649 and DAO1560 to M.H.M and E.L.G.I.D.H. was supported by NIH Training Grant AG00052. We thank Jeffrey Bernstein for excellent technical assistance.

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