European Journal of Pharmacology, 35 (1976) 85--92

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© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

APORPHINES 16 ACTION OF APORPHINE ALKALOIDS ON LOCOMOTOR ACTIVITY IN RATS WITH 6-HYDROXYDOPAMINE LESIONS OF THE NUCLEUS ACCUMBENS PETER H. KELLY*, RICHARD J. MILLER** and JOHN L. NEUMEYER***

*Department of Experimental Psychology, Downing Street, Cambridge CB2 3EB, and **M.R.C. Neurochemical Pharmacology Unit, Department of Pharmacology, Medical School, Hills Tioad, C~mbridge CB2 2QD, United Kingdom, and ***Department of Medicinal Chemistry and Pharmacology, Northeastern University, Boston, Massachusetts 02115, U.S.A. Received 16 May 1975, revised MS received 22 August 1975, accepted 28 August 1975

P.H. KELLY, R.J. MILLER and J.L. NEUMEYER, Action of aporphine alkaloids on locomotor activity in rats with 6-hydroxydopamine lesions of the nucleus accumbens, European J. Pharmacol. 35 (1976) 85--92. (--)-Apomorphine and (+)-N-n-propylnorapomorphine ((+)-NPA) produce stereotypy but not locomotor activity in normal rats. In rats with selective bilateral lesions of dopamine terminals in the nucleus accumbens induced by microinjection of 6-hydroxydopamine both compounds produced a marked stimulation of locomotor activity. (+)-NPA was considerably more potent than (--)-apomorphine. The maximal intensity of stimulation produced by the two drugs was, however, similar. The locomotor stimulant effects of (--)-apomorphine were inhibited by (+)-bulbocapnine (20 mg/kg) or pimozide (0.5 mg/kg). (+)-N-n-Propylnorapocodeine also produced a long-lasting stimulation of locomotor activity. (+)-Aporphine, (+)-isoapomorphine, (--)-l,2-dihydroxyaporphine, and (--)-nuciferine were all inactive in stimulating locomotor activity. Aporphines Apomorphine Locomotor stimulation

Nucleus accumbens Dopamine receptor

1. Introduction It has been shown that a degeneration of the nigro-striatal dopaminergic system is associated with the various forms of Parkinson's disease (Klawans, 1973). This observation has suggested certain animal models of Parkinsonism in which this pathway may be lesioned by various means either bilaterally or unilaterally (Ungerstedt and Arbuthnott, 1970; Creese and Iversen, 1974). The functional significance of the other major dopaminergic system in the central nervous system, the mesolimbic dopaminergic system, has not been as fully investigated. It has been shown recently that bilateral injection of dopamine into the nucleus accumbens of the rat brain causes a marked stimulation of locomotor activity (Pijnenburg and Van Rossum,

Denervation supersensitivity Dopamine antagonists

1973). In addition peripheral administration of the dopamine agonist apomorphine to rats with selective 6-hydroxydopamine (6-OH-DA) induced lesions of the dopamine terminals in the nucleus accumbens produces a marked stimulation of l o c o m o t o r activity (Kelly et al., 1975). This has been explained by an action of apomorphine on supersensitive dopamine receptors in the nucleus accumbens following degeneration of the presynaptic terminals. Such l o c o m o t o r stimulation is n o t observed following peripheral administration of apomorphine in normal animals or in animals with 6-OH-DAinduced lesions of the striatum (Kelly et al., 1975). The bilateral accumbens lesioned animals therefore provide a convenient model for examining the in vivo effects of drugs with potential actions at CNS dopamine receptors.

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In the present report we have used this model to investigate the in vivo effects of some aporphine analogues in order to compare these with their in vitro effects on a dopamine-sensitive adenylate cyclase described in a previous paper (Miller et at., 1975). 2. Materials and methods 2.1. Drugs The following compounds were used. (-+)Aporphine hydrobromide, (--)-apomorphine hydrochloride, (+-)-isoapomorphine hydrobromide were gifts from R. Pinder, Chemical Defence Establishment, Porton Down, England. (+)-Bulbocapnine hydrochloride was given by Dr. S. Nahorski, Department of Pharmacology, Sheffield University, England. (--)-l,2-Dihydroxyaporphine hydroiodide, (-+)-N-n-propylnorapomorphine hydroiodide ((-+)-NPA), (+-)-N-n-propylnorapocodeine hydroiodide, (--)-nuciferine base were synthesized in the laboratory of Professor Neumeyer (Neumeyer et al., 1973a,b). Drugs were dissolved in 0.9% saline, except nuciferine which was dissolved in saline acidified with HCI. All injections were intraperitoneal (i.p.). 2.2. Animals Male albino Sprague--Dawley rats weighing 250--350 g at the time of surgery were housed in groups of 10--12 with food and water ad libitum.

PAt. K E L L Y ET AL.

of ascorbic acid. 2 pl of this solution was injected into each nucleus accumbens at the rate of 1 pl/min. There is evidence that 6-OHDA, as used here, destroys catecholamine -containing neurones mainly by a specific action. First, desipramine pretreatment completely protects the destruction of noradrenergic neurones innervating the forebrain which this lesion normally produces (Iversen and Kelly, 1975). Second, the 6-OH-DA lesions of the NAS do n o t reduce the concentration of choline acetyltransferase in the NAS (Kelly, unpubl, data). 2.4. Behavioural testing L o c o m o t o r activity was measured in a bank of wire cages (25 X 40 X 70 cm) each fitted with two horizontal photocell beams along the long axis. The cages were housed in a temperature controlled (22°C) uniformly illuminated room. Interruption of the photocell beams activated electro-mechanical counters in a separate room. Rats were habituated to the cages for 30 min immediately before drug injection. Recordings of photocell beam interruptions were taken every 10 min after drug injection. The locomotor response to apomorphine (1 mg/kg and 0.1 mg/kg) was determined 10 days and 12 days post lesion. On the basis of these responses the rats were assigned to matched groups of 5--6 rats. The responses of the animals to the various drugs were determined between 14 and 28 days post operation.

2.3. Methods

2.5. Biochemical methods

Rats were anaestheti;:ed with Equithesin (3.5 ml/kg) and positioned in a stereotaxic apparatus. Bilateral injections of 6-hydroxydopamine were made into the nucleus accumbens septi (NAS) through a 30 gauge stainless steel cannula, using stereotaxic coordinates 3.4; 1.7; 7.2, according to the atlas of Pellegrino and Cushman (1967). 6-Itydroxydopamine (6-OtI-DA) hydrobromide (4 mg base/ml) was dissolved in 0.9% saline containing I mg/ml

At the end of the series of experiments (60--90 days post-lesion) 9 lesioned animals and 9 unoperated animals were selected at random and decapitated. The brains were rapidly removed and placed on ice. The nuclei accumbens were dissected as previously described (Horn et at., 1974) and assayed for dopamine by a radioenzymatic technique (Cuello et at., 1973). Dopamine levels of the lesioned rats were 26 + 6% of control.

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APORPHINES AND LOCOMOTOR ACTIVITY

2.6. Statistical methods Comparisons of total locomotor activity during sessions following drug or placebo injection were made using Student's t-test.

3. Results

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(--)-Apomorphine does r~ot produce a marked increase in locomotor activity when administered peripherally to normal rats (Kelly et al., 1975). When administered to animals with bilateral destruction of the dopaminergic innervation of the nucleus accumbens produced by

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6-OH-dopamine, (--)-apomorphine produced a marked stimulation of locomotor activity. Apomorphine produced significantly more activity than saline in doses of 0.5 mg/kg (p < 0.05) and (1.0 mg/kgp < 0.005) (fig. 1). With a higher (5 mg/kg) dose of (--)-apomorphine the duration but not the intensity of the locomotor activity increased further. With this dose the locomotor stimulation during the early phase was suppressed by the emergence of intense stereotyped behaviour. The action of the putative dopamine receptor antagonists (+)-bulbocapnine and pimozide on the locomotor stimulation produced by (--)-apomorphine was also investigated. Both of these drugs have been shown previously to inhibit the stimulating effect of dopamine on striatal adenylate cyclase (Miller et al., 1975). Both of these agents were effective in inhibiting the stimulation of locomotor activity produced by (--)-apomorphine (figs. 2 and 3). Bulbocapnine (20 mg/kg) significantly reduced the locomotor stimulation produced by 0.5 mg/kg of apomorphine (p < 0.05) (fig. 2b), although a lower dose (15 mg/kg) of bulbocapnine did not significantly reduce the effect of 1.0 mg/kg of apomorphine (fig. 2a). eimozide (0.5 and 5.0 mg/kg) administered 2 hr before apomorphine (1.0 mg/kg) abolished the locomotor response (fig. 3; p < 0.05 and p < 0.025 respectively, compared to apomorphine alone).

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As with (--)-apomorphine, (-+)-NPA did not markedly stimulate locomotor activity in normal animals (fig. 4), although it does produce stereotypy (Schoenfeld et al., 1975). In animals with 6-OH-DA lesions of the nucleus accumbens (+-)-NPA stimulated locomotor activity (fig. 4). (-+)-NPA was considerably more potent than (--)-apomorphine (compare fig. 1 and fig. 4). The locomotor stimulant effect of 0.05 mg/kg of (-+)-NPA was not significantly different from that of 1.0 mg/kg of (--)-apomorphine (p > 0.05). (+-)-NPA is therefore approximately 20 times more potent than (--)apomorphine. Increasing doses of (-+)-NPA pro-

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Several of the compounds tested at doses up to 5 mg/kg did not stimulate locomotor activity (p > 0.05 compared to saline) in animals with 6-OH-DA lesions of the nucleus accumbens. These were (+)-aporphine (fig. 5), (+)-isoapomorphine, (--)-l,2-dihydroxyaporphine, (--)-nuciferine in doses up to 5 mg/kg. (+)-N-nPropylnorapocodeine (fig. 5), however, produced a long-lasting stimulation of locomotor activity (p < 0.025 compared to saline). (--)Apocodeine has been shown previously to produce stereotypy in rats (Lal et al., 1972).

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Rats with a 6-OH-DA lesion of the nucleus accumbens show an enhanced locomotor activity response when injected i.p. with the dopamine agonist apomorphine (Kelly et al., 1975). In the present investigation we have used this preparation as an in vivo model for studying the effects of aporphine alkaloids at mesolimbic dopamine receptors. (+)-N-n-propylnorapomorphine stimulated locomotor activity in doses which were ineffective in unoperated rats, and was approximately 20 times more potent than (--)-apomorphine. It is known that both of these compounds are also effective in producing stereotypy in rats (Smelik and Ernst, 1966; Schoenfeld et al., 1975). In agreement with our results, (+)-N-npropylnorapomorhine was more potent than (--)-apomorphine in stimulating stereotypy. This is in contrast to the effects of these

Fig. 2a a n d b. E f f e c t of ( + ) - b u l b o c a p n i n e ( B U L B ) o n s t i m u l a t i o n of l o c o m o t o r activity p r o d u c e d b y (--)a p o m o r p h i n e in rats w i t h bilateral 6-OH-DA lesions of the nucleus accumbens. Bulbocapnine and apomorp h i n e were a d m i n i s t e r e d s i m u l t a n e o u s l y (i.p.). Values are m e a n s o f 6 animals. M e a n + S.E.M.

APORPHINES AND LOCOMOTOR ACTIVITY

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compounds on the dopamine-sensitive adenylate cyclase of the rat striatum where (--)-apomorphine was slightly more potent than (+)-Nn-propylnorapomorphine (Miller et al., 1975). This apparent discrepancy between the relative potencies in vitro and in vivo may be due to a difference in topography of dopamine receptors in the nucleus accumbens and the striatum. However, from previous studies this does not appear to be the case (Horn et al., 1974). An alternative explanation may be that the N-propyl group in (+)-N-n-propylnorapomorphine confers greater lipid-solubility on the molecule thus producing easier access to the CNS. It has also been shown that (+)-N-n-propylnorapoco-

deine is inactive in stimulating striatal adenylate cyclase (Miller et al., 1975). However, this c o m p o u n d was active in producing locomotor stimulation in rats with 6-OH-DA lesions of the nucleus accumbens. Moreover (--)-apocodeine has been shown to produce stereotypy in rats (Lal et al., 1972). From previous structural considerations it seems unlikely that (+)-N-npropylnorapocodeine would be a direct acting dopamine receptor agonist. It is conceivable that (+)-N-n-propylnorapocodeine may be metabolized to (+)-N-n-propylnorapomorphine which may then activate the dopamine receptor. A similar situation has been shown to occur in the case of the antiparkinsonian drug

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ET495 which is metabolized to its active catechol metabolite $584 (Miller and Iversen, 1974). The other aporphine alkaloids tested were inactive in producing a stimulation of locomotor activity. This parallels their lack of activity in stimulating striatal adenylate cyclase. It has been observed that (+}-bulbocapnine is a potent inhibitor of the effects of both dopamine and (--)-apomorphine on striatal adenylate cyclase (Miller et al., 1975). This inhibition is reflected in the present study by the ability of (+)-bulbocapnine to inhibit stimulation of locomotor activity produced by (--)-apomorphine. It has been shown previously that (+)-

bulbocapnine is an effective cataleptic agent which also suggests it has dopamine receptor antagonist properties (Costall and Naylor, 1973}. The neuroleptic agent pimozide has also been previously reported to inhibit striatal dopamine-stimulated adenylate cyclase. It was found to be rather less potent however than would be predicted from its in vivo potency (Miller et al., 1974). It has therefore been proposed that the in vivo activity of pimozide may be related to a predominantly presynaptic action as an inhibitor of dopamine release (Seeman and Lee, 1975}. However the observations reported here that pimozide will inhibit the effects of apomorphine on denervated dop-

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amine receptors argues in favour of some direct action of pimozide on postsynaptic dopamine receptors.

Acknowledgements Richard Miller is an M.R.C. Scholar. P.H. Kelly acknowledges his I.C.I. Postdoctoral Fellowship. We would like to thank Drs. L.L. Iversen and S.D. Iversen for help and encouragement during this study.

References Costall, B. and R.J. Naylor, 1973, Is there a relationship between the involvement of extrapyramidal and mesolimbic brain areas with the cataleptic action of nueroleptic agents and their chemical antipsychotic effect, Psychopharmacologia 32, 161. Creese, I. and S.D. Iversen, 1973, Blockade of amphetamine induced motor stimulation and stereotypy in the adult rat following neonatal treatment with 6-OH dopamine, Brain Res. 55, 369. Cuello, A.C., R. Hiley and L.L. Iversen, 1973. Use of catechol- O-methyltransferase for the enzyme radiochemical assay of dopamine, J. Neurochem. 21, 1337. Horn, A.S., A.C. Cuello and R.J. Miller, 1974, Dopamine in the mesolimbic system of the rat brain. Endogenous levels and the effect of drugs on the uptake system and stimulation of adenylate cyclase activity, J. Neurochem. 22, 265 Iversen, S.D. and P.H. Kelly, 1975, The use of 6-hydroxydopamine (6-OH DA) techniques for studying the pathways involved in drug-induced m o t o r behaviours, in: Chemical Tools in Catecholamine Research (Elsevier,North-Holland) (in press). Klawans, H.L. Jr., 1973, The Pharmacology of Extrapyramidal Movement Disorders (S. Karger, Basel). Kelly, P.H., P. Seviour and S.D. Iversen, 1975, Amphetamine and apomorphine responses in the rat following 6 - O H - D A lesions of the nucleus accumbens septi and corpus striatum, Brain IRes.(in press). Lal, S., T.L. Sourkes, K. Missala and G. Belendiuk, 1972, Effects of aporphine and emetine alkaloids

P.H. KELLY ET AL. on central dopaminergic mechanisms, European J. Pharmacol. 20, 71. Miller, R.J. and L.L. Iversen, 1974, Stimulation of a dopamine sensitive adenylate cyclase on homogenates of rat striatum by a metabolite of piribedil (ET495), Naunyn-Schmiedeb. Arch. Pharmacol. 282, 213. Miller, R.J., A.S. Horn and L.L. Iversen, 1974, The action of neuroleptic drugs on dopamine stimulated adenosine-3'5'-monophosphate production in rat striatum and limbic forebrain, Mol. Pharmacol. 10, 759. Miller, R.J., P.H. Kelly and J.L. Neumeyer, 1975, Action of aporphine alkaloids on dopaminergic mechanisms in rat brain, European J. Pharmacol. 35,77. Neumeyer, J.L., B.R. Neustadt, K. Oh, K.K. Weinhardt, C.B. Boyce, F.J. Rosenberg and D.G. Teiger, 1973a, Aporphines 8. Total synthesis and pharmacological evaluation of (+)-apomorphine, (-+)-apocodeine, (+)-N-n-propylnorapomorphine and (+)-N-n-propylnorapocodeine, J. Med. Chem. 16, 1223. Neumeyer, J.L., M. McCarthy, S.P. Battista, F.J. Rosenberg and D.G. Teiger, 1973b. Aporphines 9. Synthesis and pharmacological evaluation of (+)9,10-dihydroxyaporphine, (+)-, (--)-, and (+)-, ],2dihydroxyaporphine and (+)-1,2,9,10-tetrahydroxyaporphine, J. Med. Chem. 16, 1228. Pellegrino, L.J. and A.J. Cushman, 1967, A Stereotaxic Atlas of the Rat Brain (Appleton-CenturyCrofts, New York). Pijnenburg, A.J. and J.M. Van Rossum, 1973, Stimulation of locomotor activity following injection of dopamine into the nucleus accumbens, J. Pharm. Pharmacol. 25, 1003. Schoenfeld, R.I., J.L. Neumeyer, W. Dafeldecker and S. Roffler-Tarlov, 1975. Comparison of structural and stereoisomers of apomorphine on stereotyped sniffing behaviour of the rat, European J. Pharmacol. 30, 63. Seeman, P. and T. Lee, 1975, in: An~ipsychotic Drugs, Pharmacodynamics and Pharmacokinetics, ed. G. Sedvall (Wenner-Gren International Centre Symposia, Pergamon Press, Oxford) (in press). Smelik, P.G. and A.N. Ernst, 1966, Role of nigrostriatal dopaminergic fibres in compulsive gnawing behaviour in rats, Life Sci. 5, 1485. Ungerstedt, U. and G. Arbuthnott, 1970, Quantitiative recording of rational behaviour in rats after 6-OH dopamine lesions of the rat nigro-striatal system, Brain Res. 24,486.