The motor effects of some DA autoreceptor agonists and apomorphine in rats with bilateral 6-hydroxydopamine lesions of the median forebrain bundle were ...
j. Neural Transmission 60, 205-223 (1984)
Jouma/of
/g~rll/a'lmnsm/luffon 9 by Springer-Verlag 1984
Postsynaptic Dopamine Agonistic Effects of 3-PPP Enantiomers Revealed by Bilateral 6-Hydroxydopamine Lesions and by Chronic Reserpine Treatment in Rats j . A r n t and J. H y t t e l Department of Pharmacology and Toxicology, H. Lundbeck A/S, Copenhagen, Denmark With 7 Figures Received May 25, 1984; revised August 8, 1984
Summary The motor effects of some DA autoreceptor agonists and apomorphine in rats with bilateral 6-hydroxydopamine lesions of the median forebrain bundle were studied. Whereas (-)-3-PPP, (+)-3-phenethyl-PP and EMD 23448 decreased motility in sham-operated controls, a pronounced hypermotility was induced in 6-OHDA-lesioned rats. 3-PPP enantiomers and apomorphine had similar potency as that found in test models for DA autoreceptor activity in normal rats, e.g. motility inhibition. The DA receptor involvement in the effect of (-)-3-PPP was confirmed by neuroleptic antagonism. (-)-3-PPP and EMD 23448 had similar intrinsic activity as apomorphine, whereas (+)-3-phenethyl-PP and (+)-3-PPP had lower maximal effect. However, the DA autoreceptor agonists differed from apomorphine: The development of postsynaptic supersensitivity to these drugs appeared 4-7 days after the lesion compared to 1-2 days for apomorphine and (+)-3-PPP. Furthermore, no active oral stereotypy was induced by the autoreceptor selective compounds in contrast to the effect observed after apomorphine and (+)-3-PPP. In a separate experiment using circling behaviour in unilaterally 6-OHDA-lesioned rats the different time-course of appearance of supersensitivity to (-)-3-PPP, (+)-3-PPP and apomorphine was confirmed. After chronic reserpine treatment a similar postsynaptic supersensitivity to (-)-3-PPP was observed with a development time between 4 and 7 days
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and with a similar intensity as that observed in 6-OHDA-lesioned rats. In contrast, after chronic neuroleptic treatment for 12 days, (-)-3-PPP was unable to induce hyperactivity 3-7 days after withdrawal. The results indicate that DA autoreceptor agonists are able to stimulate postsynaptic DA receptors in conditions without endogenous transmitter supply for at least 4-7 days, but not after chronic receptor blockade in a similar period. This should lead to consideration of DA autoreceptor agonists as potential antiparkinsonian drugs without stimulant effects on normosensitive postsynaptic DA receptors.
Introduction The investigation ofpre- and postsynaptic dopamine (DA) receptor mechanisms has been greatly stimulated by the development of agents proposed as selective autoreceptor agonists. One of the most promising is 3-PPP 3-(3-hydroxyphenyl)N-n-propylpiperidine which has no excitatory effects in rodents (Hjorth et al., 1981; Martin et al., 1981; A r n t et aL, 1983 ii). However, resolution of 3-PPP into its enantiomers indicated that the picture was more complex: Both enantiomers had postsynaptic effects in high doses, the (+)-enantiomer as an agonist and the (-)-enantiomer as an antagonist (Hjorth et al., 1983; Clark et aL, 1983; Arnt, 1983 a; A r n t et al., 1983 b). Furthermore, it has been shown that both enantiomers (and accordingly the racemate) apparently have postsynaptic DA agonistic activity in a special condition: They induce contralateral circling behaviour in unilaterally 6-hydroxy-DA (6-OHDA)-lesioned rats by stimulation of supersensitive denervated DA receptors (Martin et aL, 1981, 1983 a, b; Arnt, 1982; A r n t et aL, 1983 a, b; Pastor et aL, 1983). In circling models, where the effect is induced by stimulation ofnormosensitive DA receptors, i.e. in rats with striatal KC1 lesions or with hemitransection, racemic 3-PPP and the (-)-enantiomer is ineffective (Hjorth et aL, 1981; Martin et al., 1981; A r n t et al., 1983 a, b). Although 3-PPP enantiomers appeared to be full agonists at unilaterally denervated postsynaptic DA receptors, it cannot be ruled out that the circling behaviour is modified by drug actions and adaptational changes in the intact side of the brain (e.g. Costallet aL, 1983). In the present experiments this problem was overcomed by investigation of the effect of drugs with different selectivity as pre-versus postsynaptic DA receptors in rats with bilateral 6-OHDA lesions of the median forebrain bundle. In addition to 3-PPP enantiomers, two putative DA autoreceptor agonists (+)-3-phenethyl-PP (Wikstrom, 1983; A r n t et aL, 1984) and EMD 23448 (Seyfried et aL, 1982)were included.
3-pPP Enantiomers and Bilateral 6-OHDA Lesions
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Furthermore, the activity o f (-)-3-PPP was also studied in other conditions o f reduced DA function, Le. in chronic reserpinized rats and in rats treated for 12 days with a long-acting neuroleptic.
Materials and Methods
Animals Male Wistar (Mol : Wist) SPF rats weighing 250--300 g were used. They were housed conventionally in Macrolon type III cages in groups of 4 in animal rooms with automatic control of temperature (21+ 1 ~ relative humidity (55 + 5 ~ air exchanges (16 times per hour) and day/night cycle (6 a.m.-6 p.m.). They had free access to a commercial pelleted diet (New Rostock diet-KFK, Aarhus) and tap water. Bilaterally 6-OHDA lesioned and reserpinized rats were orally fed 2-3 times daily with a 30 0/0suspension of Complan artificial diet and further received s.c. injections of isotonic glucose (about 15 ml per day).
Experimental Uni- or bilateral 6-OHDA lesions were made in pentobarbital anaesthetized rats (50-55 mg/kg i.p.) by an injection of a saline solution of 6-OHDA, HC1 (8/lg/4/al/4 minutes, expressed as free base) into the median forebrain bundle rostrally to substantia nigra. Sterotaxic coordinates were A 3.5-4.0 L 1.0-1.2 (Ko'nig and Klippel, 1963). The needle was lowered 8.7 mm below the skull. The solution, containing 0.02 % ascorbic acid was bubbled with Nz and kept ice-cold. Sham-operated rats received the ascorbic acid vehicle only. Rats were used for motility recording and stereotypy observation from 1 to 16 days after bilateral lesioning and were then sacrificed. Usually 2-3 days elapsed between retesting of the same rats. In separate groups of rats saline or reserpine (1.6/amol/kg = 1 mg/kg s.c.) was injected once daily 2 hours before motility testing with (-)-3-PPP (17/amol/kg s.c.) at day 2, 4, 7 and 9. Finally some rats received tefludazine, a long-acting neuroleptic (Arnt, 1982; Bogeso, 1983) once daily (2.6/amol/kg = 1.25 mg/kg p.o.) for 12 days. The response to (-)-3-PPP (17/~mol/kg s.c.) was studied 3 and 7 days after the last injection. Motility was recorded in 15 minute intervals for one hour by placing individual animals in cages (40 x 26 cm) equipped with four horizontal infrared light beams 4 cm above the bottom of the cage. Recording of motility counts required interruption of consecutive light beams in order to decrease the probability to measure stereotypic movements. For drug schedules, see appropriate figures and tables. Along with the motility recordings stereotyped behaviour was scored every 5 minutes according to a 3 point scale: 0: no effect, 1: moderate effect and 2: marked effect. Each stimulation pattern was scored (sniffing, rearing, head movements, licking and biting).
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Circling behaviour in unilaterally lesioned rats was measured by placing the rats in rotometer bowls. The number of complete contra- and ipsilateral turns were recorded in 5 minute intervals for 1 hour.
~H-DA and 12H-NA Uptake Uptake of 3H-DA ([ring-2,5,6-3H]-dopamine-HCl, New England Nuclear, 5.3 Ci/mmol; 12.5 nM) into brain synaptosomes was estimated as described by Hyttel (1978), except that specific uptake was estimated as the difference between total uptake and uptake in the presence ofl00/aM benztropine (all in triplicate). For whole-striatum samples the synaptosomal pellet was suspended in 55 vol ofbuffer instead ofl0 vol. Uptake was calculated in relation to original wetweight. For brain punches the following procedure was used. The brain was placed with the ventral surface upwards. Three coronal cuts were made approximately 1.5 mm apart, the caudal one 0.5 mm caudal to chiasma opticum (about 7.0, 8.5 and 10.3 mm anterior, K6nig and Klippel, 1963). Three 2 mm circular punches were taken: Nucleus accumbens (A 8.5 to 10.3, L 0.8 to 2.5, V 0.2 to -2.0), striatal tissue dorsal to nucleus accumbens (A 8.5 to 10.3, L 0.8 to 2.5, V 0.2 to 2.4), striatal tissue (A 7.0 to 8.5, L 1.5 to 3.0, V 2.5 to -1.0) (coordinates in parenthesis give the average limits of the punches). The tissue punches were weighed and homogenized in 1 ml of buffer. The final synaptosomal pellet was suspended in 300 vol of buffer. Uptake was calculated in relation to mg protein, determined by the Low~y et al. (1951) method. Uptake of 1-3H-NA(1-[7,8-3H]norepinephrine, Amersham, 1.7Ci/ mmol, 10 nM) into synaptosomes from occipital cortex (which was peeled off) was measured as described by Arnt et al. (1983 a) except that the final synaptosomal pellet was suspended in 25 vol of buffer. Specific uptake was estimated as the difference between total uptake and uptake in the presence of 10/~M talsupram (Hyttel, 1982) (all in triplicate) calculated in relation to mg protein in the samples.
Statistics Comparisons between groups were made using van der Waerden's X-test
(van der Waerden and Nievergelt, 1956). Biochemical comparisons were made by use of two-tailed Student's t-test.
Drugs The following compounds were dissolved in saline: (+)- and (-)-3-PPP, hydrobromide and (+)-3(3-hydroxyphenyl)-N-(2-phenylethyl)piperidine hydrobromide [(+)-3-phenethyl-PP] (synthesized by Dr. K. Bogeso at our Department of Medicinal Chemistry); apomorphine, hydrochloride (Ph. N.; solution containing 0.02 % ascorbic acid); 6-hydroxydopamine, HCI (Labkemi); tefludazine, dihydrochloride (Lundbeck); cis(Z)-flupentixol, dihydrochloride (Lundbeck); SCH 23390, hemimaleate (Schering, U.S.A.).
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EMD 23448, hydrochloride (3-[4-(4-phenyl-1,2, 3, 6-tetrahydropyridyl-[1])butyl)-indole; E. Merck, Darmstadt) was dissolved in 10 % propyleneglycol and diluted with water. Reserpine was used as the commercially available solution (Serpasil, CIBA). Results Bilateral 6 - O H D A Lesions
Upon recovery from the lesion the rats showed pronounced aphagia/adipsia and it was necessary to feed them artificially in order to reduce the severe loss of body weight. The animals remained akinetic and cataleptic throughout the test period which usually was between 2 and 11 days postlesioning. The lesion was verified by measuring the decrease of 3H-DA uptake compared with sham operated controls. In whole-striatum 3H-DA uptake was decreased by 94 %. Furthermore, 3H-DA uptake was decreased in different parts of striatum (central region, anterodorsal region) as well as in nucleus accumbens by 64-79 % in other rats (Table 1). In the latter group of rats, the uptake of 3H-noradrenaline was measured in cortex and was found slightly though not significantly decreased: Control and 6-OHDA lesion values were 4.33 +0.87 and 2.41_ 0.43 pmol/mg protein, respectively (mean + S.E.M., n = 8, 0.05 < P < 0.1) (data not shown). The effectiveness of the lesion was also checked behaviourally by complete loss of the locomotor stimulant effect of d-amphetamine (6.8/amol/kg s.c.) 2 weeks postlesioning: Motility counts during 90 minutes were 63+31 and 1226+129 in 6-OHDA- and shamTable 1. Effect of 6-OHDA (8lag base/41al) injected into median forebrain bundle on 3H-DA uptake in whole striatum or in tissue punches of striatum or nucleus accumbens. The animals (number shown in parentheses) were sacrificed ll days after the lesion. In the whole-striatum experiment the ~ uptake was determined as the total amount of radioactivity only
Sample
Mean + S.E.M. 3H-DA uptake 6-OHDA
(pmoles/mg protein) SHAM
Whole striatum
-
-
Anterior dorsal striatum Central striatum Nucleus accumbens
2.7 • 0.4 (8) 4.0• 3.6+0.3(7)
10.4 + 2.3 (8) 18.8+2.0(8) 10.0+1.7(7)
* P < 0 . 0 2 ; ** P
