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The ACTH-(4–9) analogue ORG 2766 facilitates denervation supersensitivity after a unilateral 6-OHDA lesion of the corpus striatum in rats
Neuropeptides(1991) 19,271-279 0 Longman Group UK Ltd 1991
The ACTH-(4-9) Analogue ORG 2766 Facilitates Denervation Supersensitivity After a Unilateral 6-OHDA Lesion of the Corpus Striatum in Rats P. E. VOS, G. J. BLUEMINK,
G. WOLTERINK
and J. M. VAN REE
Department of Pharmacology, Rudolf Magnus Institute, Medical Faculty, University Utrecht, Vondellaan 6,3521 GD Utrecht, The Netherlands (Reprint requests to PEW
Abstract-Direct bilateral 6-OHDA lesioning of the nucleus accumbens causes a temporary reduction in motility, followed by a spontaneous recovery in 3-4 weeks. The ACTH-(4-9) analogue ORG 2766 shortens this period to 1 week. The functional and the peptide-induced facilitation of recovery are accompanied by enhanced motility upon administration of the dopamine agonist apomorphine which may be related to denervation supersensitivity. The present experiments were performed to investigate the interaction between ORG 2766 and denervation supersensitivity in another dopaminergic terminal area i.e. the corpus striatum. After a unilateral 6-OHDA lesion of the right corpus striatum, contralateral rotation was observed upon administration of a high dose of apomorphine 2,3 and 4 weeks after the lesion, indicating supersensitivity of postsynaptic dopaminergic receptor systems. Contralateral rotation upon administration of this dose of apomorphine was observed in ORG 2766 treated animals, already at 1 week after the lesion. Peptide treatment resulted in an enhanced sensitivity for apomorphine, since contralateral rotation was observed in peptide but not in placebo treated, 6-OHDA lesioned animals after a low dose of apomorphine. In conclusion: treatment with ORG 2766 facilitates the development of denervation supersensitivity and enhances sensitivity for apomorphine probably through an increased affinity of dopaminergic receptors for domapine agonists.
Introduction Peptides related to the adrenocorticotrophic hormone (ACTH) e.g., ACTH-(ClO), (x-MSH and the synthetic ACTH-(4-9) analogue ORG 2766 have been shown to facilitate functional recovery of lesions in the peripheral and the Date received 15 February 1991 Date accepted 27 March 1991
central nervous system (l-5). The regeneration of crushed sciatic nerve is accelerated by treatment with ACTH-related peptides including ORG 2766 (2, 3). This peptide has also been shown to influence beneficially cisplatin-induced neuropathy in animals and cancer patients (6, 7). ORG 2766 reduces hyperemotionality and accelerates attenuation of hyperemotionality over repeated testing after septal brain lesions in rats (4). The 271
272
rate of vestibular compensation after unilateral labyrinth lesions is accelerated by ORG 2766 in the frog (8) and the squirrel monkey (9). Also performance deficits in a spatial orientation task induced by fimbria fornix lesions improved after ORG 2766 treatment (10). In rats following parafascicular lesions, chronic treatment with ORG 2766 counteracts the impairment of T-maze leaming (11). Recently it has been shown that destruction of the dopamine system in the nucleus accumbens induced by bilateral injection of the neurotoxic 6-hydroxydopamine (B-OHDA) causes a temporary reduction in motor activity which is followed by a spontaneous functional recovery in approximately 3-4 weeks (12). Treatment with ORG 2766 shortens the recovery period to 1 week (13). Moreover the behavioural response upon administration of apomorphine is enhanced 3-4 weeks after the lesion in spontaneously recovered animals as compared to control animals. This effect is present in ORG 2766 treated animals already 1 week after the lesion. These results suggested that supersensitivity of postsynaptic receptors may underly the spontaneous functional recovery after the lesion and its facilitation by ORG 2766 (12, 13). The present experiments were performed to investigate the interaction between ORG 2766 and development of denervation supersensitivity in more detail. Another dopaminergic innervation area i.e., the corpus striatum was selected for these studies because of the data obtained in the nucleus accumbens were difficult to interpret due to the complexity of the behavioural responses upon challenge with apomorphine i.c. hypo- and hypermotility following low and high doses of the drug respectively.
Materials and Methods Animals and housing conditions
Male Wistar rats obtained from our own live-stock were used. At the moment of operation the rats weighed approximately 160 grams. The animals were exposed to a normal light-dark cycle (lights on 6.00a.m. off 20.00p.m.) in a dimly lighted animal room, in groups of four animals per cage
NEUROPEFTIDES
(40~26~20cm,1Xwxh).TheanimaIshadfree access to food and tap water. One day prior to testing the animals were handled. Induction of the lesion
One hour before operation the rats received an i.p. injection of desipramine (25mg/kg) to prevent destruction of noradrenergic nerve cells. The rats were anaesthetized with Hypnorm@ (O.O7ml/lOO gram bodyweight i.m.) and secured in a stereotaxic apparatus. A stainless steel guide cannula was implanted unilaterally in the brain, the tip ending in the right striatum (coordinates: l.Omm anterior to bregma, 2.5mm lateral of midline, 4.5mm below the surface of the skull at the point of penetration) inserted at an angle of lo”, incissor bar at horizontal zero level. The cannula served to guide the needle of a Hamilton Syringe through which 2 ~1 of 6-OHDA (8 pg/2 ~1, dissolved in saline containing 0.1% ascorbic acid) or vehicle alone was injected over a 2 min period in the right striatum. For each experiment two groups (n = 8-10 per group) were injected with 6-OHDA and two control groups (SHAM operation, n = 8-10 per group) with vehicle. Treatment procedures
From day 1 till day 6 after the operation the animals of one control group and one 6-OHDA lesioned group were treated subcutaneously with ORG 2766 (10 &kg, once daily). The other animals were injected with placebo (saline). Behaviouraf testing
On different times after the lesion (1, 2, 3 or 4 weeks, each week includes a separate experiment) behavioural activities were assessed in a small open field, during 3 consecutive days. The animals were brought to the test room at least 1 h before testing. The small open field consisted of a transparent Plexiglass Tube (diameter 19.5cm, height 3Ocm) placed on a plastic board which was divided into four equal sections. During a 3 min observation period, ipsilateral and contralateral rotations were measured by counting separately, complete 180” ipsilateral and contralateral turns (an ipsilateral rotation = 180” turn towards the lesioned side, a contralateral rotation = 180“ turn towards
THE ACT’H-(4-9) ANALOGUE
ORG 2766FACILITATE.5
DENERVATION
SUPERSENSITIVITY
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IN RATS
7.8
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Fig. 1 Positions of the tip of a cannula in the right corpus striatum obtained from a representative animal. Drawing taken from Pellegrino and Cushman (1967).
the unoperated side). Twenty minutes prior to testing all animals received a S.C. injection with saline on test day 1, 50&kg apomorphine (dissolved in saline) on test day 2 or 1254kg apomorphine (dissolved in saline) on test day 3.
HCl (apomorphine, OPG, Utrecht, The Netherlands). ORG 2766 (Met/O&Glu-His-Phe-D-LysPhe) was obtained from Organon International b.v., Oss, The Netherlands. Statistics
Histological evaluation of lesions
After the third test day the rats were decapitated and the brain was removed and fixated in 4% formaldehyde solution. After sectioning on a freeze microtome 1OOkm thick sections were evaluated with the light microscope for location of the site of injection. Drugs The following drugs were used: desipramine-HCl
(DMI) (CIBA, Arnhem, The Netherlands), 6hydroxydopamine-HBr (6-OHDA) (Sigma Chem. Comp. St Louis, USA) and apomorphinel
Statistical analysis of the behavioural tests, to compare ipsilateral- with contralateral rotation was performed using Student’s paired t-test.
Results Figure 1 shows a representation example of the intrastriatal injection site. The injections were in the dorsolateral part of the nucleus caudatus Lesion-induced lateral rotation
effects on ipsilateral and contra-
Ipsilateral and contralateral
rotation frequencies
274 of the rats after saline administration (test day 1) are shown in Figure 2. No significant differences between ipsilateral and contralateral rotation were found for any group 1,2,3 and 4 weeks after the lesion, except for a contralateral rotation in the ORG 2766 treated lesioned group 3 weeks after the lesion. Low dose of apomorphine Administration of a low dose of apomorphine (test day 2) resulted in a significant contralateral rotation in placebo treated SHAM operated animals 1 week after the lesion (Fig. 3A). No effect was found in SHAM operated animals treated with ORG 2766 (Fig. 3B). In ORG 2766 treated, lesioned animals a tendency to contralateral rotation was observed 1 week after the operation and significant contralateral rotation 2, 3, and 4 weeks after operation (Fig. 3D). This effect was not present in placebo treated, lesioned rats (Fig. 3C). High dose of apomorphine Administration of a high dose of apomorphine (test day 3) did not result in significant ipsilateralor contralateral rotation 1,2,3 and 4 weeks after a SHAM lesion in either placebo or ORG 2766 treated animals (Fig. 4A, B). In placebo treated, 6-OHDA lesioned rats significant contralateral rotation was observed 2, 3 and 4 weeks after the lesion but not 1 week after operation (Fig. 4C). In ORG 2766 treated, 6-OHDA lesioned groups significant contralateral rotation was observed 1, 2,3 and 4 weeks after the lesion (Fig. 4D). Discussion Rats with unilateral 6-OHDA lesions of cellbodies of the nigrostriatal DA-pathway rotate in a direction ipsilateral to the lesion upon administration of amphetamine (14) and in a direction contralateral to the lesion upon administration of the DA precursor GDOPA or direct acting DA agonists (15-17). This model which Ungerstedt (14) called the 6-OHDA rotation model has been used frequently for testing dopamine related pharmacological agents. It is believed that ipsilateral rotation upon administration of amphetamine is the result of activation of normosensitive DA receptors on
NEZJROPEPTIDES
the non-lesioned side following DA release from intact nerve terminals (14). Contralateral rotation is the result of stimulation of denervated supersensitive DA receptors on the lesioned side after DA formation from L-DOPA or directly by dopamine agonists (15, 17). During the last decade this model has also been used to study the time course of recovery after such lesions. After 6-OHDA lesioning of the cellbody area of the nigrostriatal DA-pathway and subsequent application of neural grafts in the corpus striatum or treatment with trophic factors, it has been shown by means of functional, biochemical and immunocytochemical measurements that lasting improvement of deficits could be achieved (18-21). After lesioning of the terminal area of the nigrostriatal DA-pathway i.e., the lateral striatum Seigfried and Bures found improvement, within 1 week, on a lateralized reach task in which the ability to retrieve food-pellets with the preferred fore-paw was tested (22). Sabol and co-workers found no improvement on the same task within 3 weeks after the lesion (23). In the present experiments the development of denervation supersensitivity as measured upon challenge with apomorphine was investigated after unilateral 6-OHDA lesioning of the terminal area of the nigrostratial DA-pathway, the corpus striatum. Also the influence of treatment with the neuropeptide ORG 2766 on denervation supersensitivity was investigated. In a pilot study three sites in the corpus striatum were injected with 2~16-OHDA @kg-2~1) and rotation behaviour was measured on several time points (l-12 weeks) after the lesion (data not shown). After elimination of the two lesion sites not significantly contributing to the development of denervation supersensitivity the lesion site described in material and methods was chosen for the present experiments (Fig. 1). A contralateral rotation response upon administration of a high dose of apomorphine was observed 2 weeks after the lesion and was still present 4 weeks after the lesion, in placebo treated, 6-OHDA lesioned animals. In ORG 2766 treated, 6-OHDA lesioned animals a contralateral rotation response was observed already 1 week after the lesion upon administration of a high dose of apomorphine. These results are in accordance with data of earlier experiments from which it was
THE ACM-(4.9)
ANALOGUE
ORG 2766 FACILITATES DENERVATION
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Ffg. 2 Effect of S.C.saline treatment (test day 1) on frequency of ipsilateral and contralateral rotations in rats, 1,2,3, or 4 weeks after a unilateral SHAM or COHDA lesion in the r&ht corpus striatum, pre-treated with placebo or ORG 2766 from day 1 to day 6 after the lesion. Abscissa: time in weeks. Ordinate: rotation frequency. A: SHAM operated, placebo pre-treated. B: SHAM operated, ORG ‘2766pre-treated. C: 6-OHDA lesioned, placebo pre-treated. D: 6-OHDA lesioned, ORG 2766 pre-treated. Each week inch~des a separate experiment. AU animals were treated with saline 20 min prior to testing in a small open held. The frequency of ipsihneral and contralateral rotations were assessed for 3 min. Bach bar represents mean score f SEM per group of rats (n = 8-lQ!group). *Difference between @lateral and contralateral rotations (p -Z 0.05, Student’s paired t-test).
276
NEUROPEPTIDBS
A
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rotation
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Fig. 3 Effect of S.C.apomorphine (50&k& treatment (test day 2)on frequency of ipsilateral and contralateral rotations in rats, 1,2,3 or 4 weeks after unilateral SHAM or 6-OHDA lesion in the right corpus striatum, pre-treated with placebo or ORG 2766 from day 1 to day 6 after the lesion. Abscicca: time in weeks. Ordinate: rotation frequency. A: SHAM operated, placebo pre-treated. B: SHAM operated, ORG 2766 pre-treated. C: 6-OHDA Iesioned, placebo pre-treated. D: 6-OHDA lesioned, ORG 2766 pre-treated. Each week includes a separate experiment. Ah animals were treated with apomorphine 20 min prior to testing in a small open field. The frequency of ipsilateral and contralateral rotations were assessed for 3 min. Each bar represents mean score f SEMper group of rats (n = &lo/group). Difference between ipsilateral and contralateral rotations (Student’s paired t-test): *P < 0.05, #p < 0.01.
THE ACl-li-(4-9) ANALOGUE ORG 2766 FACILITAlES
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Effect of s,c. apomorphine (125 &kg) treatment (test day 3) on frequency of @lateral andcontralateral rotations in rats, 1,2,3 or 4 weeks after unilateral SHAM or 6-OHDA lesion in the right corpus striatum, pre-treated with placebo or ORG 2766
Fig. 4
from day 1 to day 6 after the lesion. Abscicca: time in weeks. Ordinate: rotation frequency. A: SHAM operated, placebo pre-treated. B: SHAM operated, ORG 2766 pre-treated. Fig. 4C: 6-OHDA lesioned, placebo pre-treated. D: COHJIA lesioned, ORG 2766 pre-treated. Each week includes a separate experiment. Ah animals were treated with apomorphine 28 min prior to testing in a small opeo field. The frequency of ipsilateral and contralateral rotations were assessed for 3 mm. Each bar represents mean score + SEM per group of rats (n = 8-IO/group). Difference between ipsilateral and contralateral rotations (Student’s paired t-test): lP -z 0.05, #p < 0.01, + p < 0.001.
278 concluded that ORG 2766 accelerated the development of denervation supersensitivity after 6-OHDA lesioning of another DA-ergic terminal area i.e., the nucleus accumbens (12, 13). Upon administration of a low dose of apomorphine no drug response could be detected at any time after the lesion in placebo treated, 6-OHDA lesioned animals. However, in peptide treated, 6-OHDA lesioned animals a significant contralateral rotation response could be elicited 2,3 and 4 weeks after the lesion upon administration of this dose of apomorphine. These results seem in accordance with results obtained after bilateral 6-OHDA lesioning of the nucleus accumbens, in which it was shown that upon administration of a low dose of apomorphine an increased behavioural response could be observed in ORG 2766 treated, 6-OHDA lesioned animals as compared to placebo treated, 6-OHDA lesioned animals (256). However the data from the nucleus accumbens experiments were difficult to interpret, because of the Ushaped curve with respect to the changes in locomotion following apomorphine administration in SHAM operated rats i.e. a decrease and an increase in locomotion after a low and a high dose of apomorphine respectively. The present data clearly reveal an enhanced sensitivity for the dopamine agonist apomorphine in ORG 2766 treated, 6-OHDA lesioned animals as compared to placebo treated, 6-OHDA lesioned animals. From experiments in the nucleus accumbens it has been suggested that this might be due to an increase in the affinity or in the number of dopaminergic receptors in peptide treated animals. The present data favour the idea of an increased affinity, because the peptide treated, 6-OHDA lesioned rats responded on a low dose of apomorphine, in contrast to placebo treated, 6-OHDA lesioned animals, while a high dose of apomorphine induced similar effects in both groups of animals. The absence of behavioural difference between peptide and placebo treated lesioned animals upon administration of a high dose of apomorphine is probably due to a ceiling effect for apomorphine. In conclusion after a unilateral 6-OHDA lesion of the corpus striatum, denervation supersensitivity develops in 2 weeks as evidenced by contralateral rotation upon administration of a high dose
NEUROPEPTIDES
Treatment with ORG 2766 of apomorphine. during the first 6 days after the lesion results in a facilitation of this denervation supersensitivity as evidenced by contralateral rotation already 1 week after the lesion. The peptide-induced facilitation of the denervation supersensitivity is probably the result of an increase in the affinity of the DA-ergic receptor system for DA agonists as evidenced by contralateral rotation upon administration of a low dose of apomorphine 2-4 weeks after the lesion.
Acknowledgements Thiswork was sponsored in part by the Primes Beatrix Fonds, Grant no. 88-2884. References 1. Bijlsma, W., Jennekens, F. G. I., Schotman, P. and Gispen, W. H. (1981). Effects of corticotropin (ACTH) on recovery of sensory-motor function in the rat: structureactivity study. European Joumai of Pharmacology 76: 73-79. 2. Bijlsma, W., Jennekens, F. G. I., Schotman, P. and Gispen, W. H. (1983). Stimulation by ACIH-(4-10) of nerve fiber regeneration following sciatic nerve crush. Muscle & Nerve 6: 104-112. 3. De Koning, P. and Gispen, W. H. (1988). ORG 2766 improves functional and electrophysiological. Peptides 80: 415-422. 4. Isaacson, R. L. and Poplawsky, A. (1983). An ACTH-(49) analogue speeds recovery from septal hyperemotionality in the rat. Behavioral and Neural Biology 39: 52-59. 5. Saint-Come, C. and Strand, F. L. (1985). ACI’I-I-(ClO)/ MSH-(4-10) improves motor unit reorganization during peripheral nerve regeneration in the rat. Peptides 6: 77-83. 6. Gerritsen van der Hoop, R., Vecht, C. J., van der Burg, M. E. L. et al. (1990). Prevention of cisplatin neurotoxicity au ACTH(4-9) anaiogue in patients with ovarian cancer. New England Journal of Medicine 322: 89-94. 7. De Koning, P., Neijt, J. P., Jennekens, F. G. I. and Gispen, W. H. (1988). Org 2766 protects from cisplatiu induced neurotoxicity in rats. Experimental Neurology 97: 746-750. 8. Luneberg, U. and Flohr, H. (1988). Effects of melanocortins on vestibular compensation. In: Pompeiano, 0. and Allurn, J. H. J. (eds): Progress in Brain Research. Elsevier Science Publishers B. V., Vol. 76, p. 421-429. 9. Igarashi, M., Ishikawa, K., Ishii, M. and Schmidt, K. (1985). Effect of ACTH-(410) on equilibrium compensation after unilateral labyrinthectomy in the squirrel monkey. European Journal of Pharmacology 119: 239-242. 10. Spruyt, B. M. (1990). Beneficial effects of the ACTH(C9) analog ORG 2766 in aging and 6mbria fornix lesioned rats.
THEi ACTH-(4-9) ANALOGUE ORG 2766 FACILITATES DENERVATION SUPERSENSITWITY IN RATS
11.
12.
13.
14.
15.
16.
17.
Abstracts of the 13th annual meeting of the European Neuroscience Association. Supplement No. 3: 73. Nyakas, C., Veldhuis, H. and De Wied, D. (1985). Beneficial effect of chronic treatment with ORG 2766 and a-MSH on impaired reversal learning of rats with bilateral lesions of the parafascicular area. Brain Research Bulletin 15: 257-265. Wolterink G., Van Zanten, E., Kamsteeg, H., Radhakishun, F. S. and Van Ree, J. M. (1990). Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. I. Behavioural and biochemical studies. Brain Research 507: 92-100. Wolterink, G., Van Zanten, E., Kamsteeg, H., Radhakishun, F. S. and Van Ree, J. M. (1990). Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats II. Facilitation by the AClH-(C 9) analog ORG 2766. Brain Research 507: 101-108. Ungerstedt, U. and Arbuthnott, G. (1970). Quantitive recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigrostriatal dopamine system. Brain Research 24: 485-493. Herrera-Marschitz, M. and Ungerstedt, U. (1984). Evidence that apomorphine and pergolide induce rotation in rats by different actions on Dl and D2 receptor sites. European Journal of Pharmacology’98: 165-176. Ungerstedt, U., Ljungberg, T. and Schulz, W. (1978). Dopamine receptor mechanisms: Behavioural and electrophysiological studies. Advances in Biomedical Sciences. 1: 313-321. Ungerstedt, U. (1971). Postsynaptic supersensitivity after
18.
19.
20.
21.
22.
23.
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6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acta Physiologica Scandinavica supplement. 367 III: 69-93. Dunnett, S. B., Whishaw, I. Q., Rogers, D. C. and Jones, G. (1987). Dopamine-rich gratis ameliorate whole body motor asymmetry and sensory neglect but not independent limb use in rats with 6hydroxydopamine lesions. Brain Research 415: 63-78. Iversen, S. D. and DuMett, S. B. (1990). Functional organization of striatum as studied with neural grafts. Neuropsychologia 28: 601-626. Herman, J. P., Lupp, A., Abrous, M., Le Moal, M., Hertting, G. and Jackisch, R. (1988). Intrastriatal dopamine@ grafts restore inhibitory control over striatal cholinergic neurons. Experimental Brain Research 73: 236-248. Dunnett, S. B., Bjorklund, A., Schmidt, R. H., Stenevi, U. and Iversen, S. D. (1983). Behavioural recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell suspensions. Acta Physiologica Scandinavica Supplement 522: 29-38. Siegfried, B. and Bures, J. (1980). Handedness in rats: Blockade of reaching by unilateral 6-OHDA injections into substantia nigra and caudate nucleus. Physiology and Psychology. 8(3): 360-368. Sabol, K.E., Neill, D. A. W. S., Church, W. and Justice, J. B. (1985). Dopamine depletion in a striatal subregion disrupts performance of a skilled motor task in the rat. Brain Research 335: 33-43.
The ACTH-(4-9) Analogue ORG 2766 Facilitates Denervation Supersensitivity After a Unilateral 6-OHDA Lesion of the Corpus Striatum in Rats P. E. VOS, G. J. BLUEMINK,
G. WOLTERINK
and J. M. VAN REE
Department of Pharmacology, Rudolf Magnus Institute, Medical Faculty, University Utrecht, Vondellaan 6,3521 GD Utrecht, The Netherlands (Reprint requests to PEW
Abstract-Direct bilateral 6-OHDA lesioning of the nucleus accumbens causes a temporary reduction in motility, followed by a spontaneous recovery in 3-4 weeks. The ACTH-(4-9) analogue ORG 2766 shortens this period to 1 week. The functional and the peptide-induced facilitation of recovery are accompanied by enhanced motility upon administration of the dopamine agonist apomorphine which may be related to denervation supersensitivity. The present experiments were performed to investigate the interaction between ORG 2766 and denervation supersensitivity in another dopaminergic terminal area i.e. the corpus striatum. After a unilateral 6-OHDA lesion of the right corpus striatum, contralateral rotation was observed upon administration of a high dose of apomorphine 2,3 and 4 weeks after the lesion, indicating supersensitivity of postsynaptic dopaminergic receptor systems. Contralateral rotation upon administration of this dose of apomorphine was observed in ORG 2766 treated animals, already at 1 week after the lesion. Peptide treatment resulted in an enhanced sensitivity for apomorphine, since contralateral rotation was observed in peptide but not in placebo treated, 6-OHDA lesioned animals after a low dose of apomorphine. In conclusion: treatment with ORG 2766 facilitates the development of denervation supersensitivity and enhances sensitivity for apomorphine probably through an increased affinity of dopaminergic receptors for domapine agonists.
Introduction Peptides related to the adrenocorticotrophic hormone (ACTH) e.g., ACTH-(ClO), (x-MSH and the synthetic ACTH-(4-9) analogue ORG 2766 have been shown to facilitate functional recovery of lesions in the peripheral and the Date received 15 February 1991 Date accepted 27 March 1991
central nervous system (l-5). The regeneration of crushed sciatic nerve is accelerated by treatment with ACTH-related peptides including ORG 2766 (2, 3). This peptide has also been shown to influence beneficially cisplatin-induced neuropathy in animals and cancer patients (6, 7). ORG 2766 reduces hyperemotionality and accelerates attenuation of hyperemotionality over repeated testing after septal brain lesions in rats (4). The 271
272
rate of vestibular compensation after unilateral labyrinth lesions is accelerated by ORG 2766 in the frog (8) and the squirrel monkey (9). Also performance deficits in a spatial orientation task induced by fimbria fornix lesions improved after ORG 2766 treatment (10). In rats following parafascicular lesions, chronic treatment with ORG 2766 counteracts the impairment of T-maze leaming (11). Recently it has been shown that destruction of the dopamine system in the nucleus accumbens induced by bilateral injection of the neurotoxic 6-hydroxydopamine (B-OHDA) causes a temporary reduction in motor activity which is followed by a spontaneous functional recovery in approximately 3-4 weeks (12). Treatment with ORG 2766 shortens the recovery period to 1 week (13). Moreover the behavioural response upon administration of apomorphine is enhanced 3-4 weeks after the lesion in spontaneously recovered animals as compared to control animals. This effect is present in ORG 2766 treated animals already 1 week after the lesion. These results suggested that supersensitivity of postsynaptic receptors may underly the spontaneous functional recovery after the lesion and its facilitation by ORG 2766 (12, 13). The present experiments were performed to investigate the interaction between ORG 2766 and development of denervation supersensitivity in more detail. Another dopaminergic innervation area i.e., the corpus striatum was selected for these studies because of the data obtained in the nucleus accumbens were difficult to interpret due to the complexity of the behavioural responses upon challenge with apomorphine i.c. hypo- and hypermotility following low and high doses of the drug respectively.
Materials and Methods Animals and housing conditions
Male Wistar rats obtained from our own live-stock were used. At the moment of operation the rats weighed approximately 160 grams. The animals were exposed to a normal light-dark cycle (lights on 6.00a.m. off 20.00p.m.) in a dimly lighted animal room, in groups of four animals per cage
NEUROPEFTIDES
(40~26~20cm,1Xwxh).TheanimaIshadfree access to food and tap water. One day prior to testing the animals were handled. Induction of the lesion
One hour before operation the rats received an i.p. injection of desipramine (25mg/kg) to prevent destruction of noradrenergic nerve cells. The rats were anaesthetized with Hypnorm@ (O.O7ml/lOO gram bodyweight i.m.) and secured in a stereotaxic apparatus. A stainless steel guide cannula was implanted unilaterally in the brain, the tip ending in the right striatum (coordinates: l.Omm anterior to bregma, 2.5mm lateral of midline, 4.5mm below the surface of the skull at the point of penetration) inserted at an angle of lo”, incissor bar at horizontal zero level. The cannula served to guide the needle of a Hamilton Syringe through which 2 ~1 of 6-OHDA (8 pg/2 ~1, dissolved in saline containing 0.1% ascorbic acid) or vehicle alone was injected over a 2 min period in the right striatum. For each experiment two groups (n = 8-10 per group) were injected with 6-OHDA and two control groups (SHAM operation, n = 8-10 per group) with vehicle. Treatment procedures
From day 1 till day 6 after the operation the animals of one control group and one 6-OHDA lesioned group were treated subcutaneously with ORG 2766 (10 &kg, once daily). The other animals were injected with placebo (saline). Behaviouraf testing
On different times after the lesion (1, 2, 3 or 4 weeks, each week includes a separate experiment) behavioural activities were assessed in a small open field, during 3 consecutive days. The animals were brought to the test room at least 1 h before testing. The small open field consisted of a transparent Plexiglass Tube (diameter 19.5cm, height 3Ocm) placed on a plastic board which was divided into four equal sections. During a 3 min observation period, ipsilateral and contralateral rotations were measured by counting separately, complete 180” ipsilateral and contralateral turns (an ipsilateral rotation = 180” turn towards the lesioned side, a contralateral rotation = 180“ turn towards
THE ACT’H-(4-9) ANALOGUE
ORG 2766FACILITATE.5
DENERVATION
SUPERSENSITIVITY
273
IN RATS
7.8
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Fig. 1 Positions of the tip of a cannula in the right corpus striatum obtained from a representative animal. Drawing taken from Pellegrino and Cushman (1967).
the unoperated side). Twenty minutes prior to testing all animals received a S.C. injection with saline on test day 1, 50&kg apomorphine (dissolved in saline) on test day 2 or 1254kg apomorphine (dissolved in saline) on test day 3.
HCl (apomorphine, OPG, Utrecht, The Netherlands). ORG 2766 (Met/O&Glu-His-Phe-D-LysPhe) was obtained from Organon International b.v., Oss, The Netherlands. Statistics
Histological evaluation of lesions
After the third test day the rats were decapitated and the brain was removed and fixated in 4% formaldehyde solution. After sectioning on a freeze microtome 1OOkm thick sections were evaluated with the light microscope for location of the site of injection. Drugs The following drugs were used: desipramine-HCl
(DMI) (CIBA, Arnhem, The Netherlands), 6hydroxydopamine-HBr (6-OHDA) (Sigma Chem. Comp. St Louis, USA) and apomorphinel
Statistical analysis of the behavioural tests, to compare ipsilateral- with contralateral rotation was performed using Student’s paired t-test.
Results Figure 1 shows a representation example of the intrastriatal injection site. The injections were in the dorsolateral part of the nucleus caudatus Lesion-induced lateral rotation
effects on ipsilateral and contra-
Ipsilateral and contralateral
rotation frequencies
274 of the rats after saline administration (test day 1) are shown in Figure 2. No significant differences between ipsilateral and contralateral rotation were found for any group 1,2,3 and 4 weeks after the lesion, except for a contralateral rotation in the ORG 2766 treated lesioned group 3 weeks after the lesion. Low dose of apomorphine Administration of a low dose of apomorphine (test day 2) resulted in a significant contralateral rotation in placebo treated SHAM operated animals 1 week after the lesion (Fig. 3A). No effect was found in SHAM operated animals treated with ORG 2766 (Fig. 3B). In ORG 2766 treated, lesioned animals a tendency to contralateral rotation was observed 1 week after the operation and significant contralateral rotation 2, 3, and 4 weeks after operation (Fig. 3D). This effect was not present in placebo treated, lesioned rats (Fig. 3C). High dose of apomorphine Administration of a high dose of apomorphine (test day 3) did not result in significant ipsilateralor contralateral rotation 1,2,3 and 4 weeks after a SHAM lesion in either placebo or ORG 2766 treated animals (Fig. 4A, B). In placebo treated, 6-OHDA lesioned rats significant contralateral rotation was observed 2, 3 and 4 weeks after the lesion but not 1 week after operation (Fig. 4C). In ORG 2766 treated, 6-OHDA lesioned groups significant contralateral rotation was observed 1, 2,3 and 4 weeks after the lesion (Fig. 4D). Discussion Rats with unilateral 6-OHDA lesions of cellbodies of the nigrostriatal DA-pathway rotate in a direction ipsilateral to the lesion upon administration of amphetamine (14) and in a direction contralateral to the lesion upon administration of the DA precursor GDOPA or direct acting DA agonists (15-17). This model which Ungerstedt (14) called the 6-OHDA rotation model has been used frequently for testing dopamine related pharmacological agents. It is believed that ipsilateral rotation upon administration of amphetamine is the result of activation of normosensitive DA receptors on
NEZJROPEPTIDES
the non-lesioned side following DA release from intact nerve terminals (14). Contralateral rotation is the result of stimulation of denervated supersensitive DA receptors on the lesioned side after DA formation from L-DOPA or directly by dopamine agonists (15, 17). During the last decade this model has also been used to study the time course of recovery after such lesions. After 6-OHDA lesioning of the cellbody area of the nigrostriatal DA-pathway and subsequent application of neural grafts in the corpus striatum or treatment with trophic factors, it has been shown by means of functional, biochemical and immunocytochemical measurements that lasting improvement of deficits could be achieved (18-21). After lesioning of the terminal area of the nigrostriatal DA-pathway i.e., the lateral striatum Seigfried and Bures found improvement, within 1 week, on a lateralized reach task in which the ability to retrieve food-pellets with the preferred fore-paw was tested (22). Sabol and co-workers found no improvement on the same task within 3 weeks after the lesion (23). In the present experiments the development of denervation supersensitivity as measured upon challenge with apomorphine was investigated after unilateral 6-OHDA lesioning of the terminal area of the nigrostratial DA-pathway, the corpus striatum. Also the influence of treatment with the neuropeptide ORG 2766 on denervation supersensitivity was investigated. In a pilot study three sites in the corpus striatum were injected with 2~16-OHDA @kg-2~1) and rotation behaviour was measured on several time points (l-12 weeks) after the lesion (data not shown). After elimination of the two lesion sites not significantly contributing to the development of denervation supersensitivity the lesion site described in material and methods was chosen for the present experiments (Fig. 1). A contralateral rotation response upon administration of a high dose of apomorphine was observed 2 weeks after the lesion and was still present 4 weeks after the lesion, in placebo treated, 6-OHDA lesioned animals. In ORG 2766 treated, 6-OHDA lesioned animals a contralateral rotation response was observed already 1 week after the lesion upon administration of a high dose of apomorphine. These results are in accordance with data of earlier experiments from which it was
THE ACM-(4.9)
ANALOGUE
ORG 2766 FACILITATES DENERVATION
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SUPERSENSITIVITY IN RATS
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Ffg. 2 Effect of S.C.saline treatment (test day 1) on frequency of ipsilateral and contralateral rotations in rats, 1,2,3, or 4 weeks after a unilateral SHAM or COHDA lesion in the r&ht corpus striatum, pre-treated with placebo or ORG 2766 from day 1 to day 6 after the lesion. Abscissa: time in weeks. Ordinate: rotation frequency. A: SHAM operated, placebo pre-treated. B: SHAM operated, ORG ‘2766pre-treated. C: 6-OHDA lesioned, placebo pre-treated. D: 6-OHDA lesioned, ORG 2766 pre-treated. Each week inch~des a separate experiment. AU animals were treated with saline 20 min prior to testing in a small open held. The frequency of ipsihneral and contralateral rotations were assessed for 3 min. Bach bar represents mean score f SEM per group of rats (n = 8-lQ!group). *Difference between @lateral and contralateral rotations (p -Z 0.05, Student’s paired t-test).
276
NEUROPEPTIDBS
A
B 25 -
25 -
weeks
lpsilateral m
rotation
Contralateral
weeks
rotation
Fig. 3 Effect of S.C.apomorphine (50&k& treatment (test day 2)on frequency of ipsilateral and contralateral rotations in rats, 1,2,3 or 4 weeks after unilateral SHAM or 6-OHDA lesion in the right corpus striatum, pre-treated with placebo or ORG 2766 from day 1 to day 6 after the lesion. Abscicca: time in weeks. Ordinate: rotation frequency. A: SHAM operated, placebo pre-treated. B: SHAM operated, ORG 2766 pre-treated. C: 6-OHDA Iesioned, placebo pre-treated. D: 6-OHDA lesioned, ORG 2766 pre-treated. Each week includes a separate experiment. Ah animals were treated with apomorphine 20 min prior to testing in a small open field. The frequency of ipsilateral and contralateral rotations were assessed for 3 min. Each bar represents mean score f SEMper group of rats (n = &lo/group). Difference between ipsilateral and contralateral rotations (Student’s paired t-test): *P < 0.05, #p < 0.01.
THE ACl-li-(4-9) ANALOGUE ORG 2766 FACILITAlES
DENERVATION SUPERSENSITIWIY
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IN RATS
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Effect of s,c. apomorphine (125 &kg) treatment (test day 3) on frequency of @lateral andcontralateral rotations in rats, 1,2,3 or 4 weeks after unilateral SHAM or 6-OHDA lesion in the right corpus striatum, pre-treated with placebo or ORG 2766
Fig. 4
from day 1 to day 6 after the lesion. Abscicca: time in weeks. Ordinate: rotation frequency. A: SHAM operated, placebo pre-treated. B: SHAM operated, ORG 2766 pre-treated. Fig. 4C: 6-OHDA lesioned, placebo pre-treated. D: COHJIA lesioned, ORG 2766 pre-treated. Each week includes a separate experiment. Ah animals were treated with apomorphine 28 min prior to testing in a small opeo field. The frequency of ipsilateral and contralateral rotations were assessed for 3 mm. Each bar represents mean score + SEM per group of rats (n = 8-IO/group). Difference between ipsilateral and contralateral rotations (Student’s paired t-test): lP -z 0.05, #p < 0.01, + p < 0.001.
278 concluded that ORG 2766 accelerated the development of denervation supersensitivity after 6-OHDA lesioning of another DA-ergic terminal area i.e., the nucleus accumbens (12, 13). Upon administration of a low dose of apomorphine no drug response could be detected at any time after the lesion in placebo treated, 6-OHDA lesioned animals. However, in peptide treated, 6-OHDA lesioned animals a significant contralateral rotation response could be elicited 2,3 and 4 weeks after the lesion upon administration of this dose of apomorphine. These results seem in accordance with results obtained after bilateral 6-OHDA lesioning of the nucleus accumbens, in which it was shown that upon administration of a low dose of apomorphine an increased behavioural response could be observed in ORG 2766 treated, 6-OHDA lesioned animals as compared to placebo treated, 6-OHDA lesioned animals (256). However the data from the nucleus accumbens experiments were difficult to interpret, because of the Ushaped curve with respect to the changes in locomotion following apomorphine administration in SHAM operated rats i.e. a decrease and an increase in locomotion after a low and a high dose of apomorphine respectively. The present data clearly reveal an enhanced sensitivity for the dopamine agonist apomorphine in ORG 2766 treated, 6-OHDA lesioned animals as compared to placebo treated, 6-OHDA lesioned animals. From experiments in the nucleus accumbens it has been suggested that this might be due to an increase in the affinity or in the number of dopaminergic receptors in peptide treated animals. The present data favour the idea of an increased affinity, because the peptide treated, 6-OHDA lesioned rats responded on a low dose of apomorphine, in contrast to placebo treated, 6-OHDA lesioned animals, while a high dose of apomorphine induced similar effects in both groups of animals. The absence of behavioural difference between peptide and placebo treated lesioned animals upon administration of a high dose of apomorphine is probably due to a ceiling effect for apomorphine. In conclusion after a unilateral 6-OHDA lesion of the corpus striatum, denervation supersensitivity develops in 2 weeks as evidenced by contralateral rotation upon administration of a high dose
NEUROPEPTIDES
Treatment with ORG 2766 of apomorphine. during the first 6 days after the lesion results in a facilitation of this denervation supersensitivity as evidenced by contralateral rotation already 1 week after the lesion. The peptide-induced facilitation of the denervation supersensitivity is probably the result of an increase in the affinity of the DA-ergic receptor system for DA agonists as evidenced by contralateral rotation upon administration of a low dose of apomorphine 2-4 weeks after the lesion.
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