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Denervation-like postsynaptic supersensitivity to dopamine agonists induced by microinjection of colchicine into the substantia nigra pars compacta
Brain Research, 367 (1986) 1-7 Elsevier
1
BRE 11480
Research Reports
Denervation-Like Postsynaptic Supersensitivity to Dopamine Agonists Induced by Microinjection of Colchicine into the S_u_bstantjaNigra Pars Compacta KATSUO KAMATA1, TSUTOMU KAMEYAMA1, SHIGERU OKUYAMA2, SANAE HASHIMOTO2 and HIRONAKA AIHARA2
1Department of Chemical Pharmacology, Facultyof Pharmaceutical Sciences, Meijo University, Nagoya and 2Research Center, Taisho Pharmaceuticals, Saitama (Japan) (Accepted June 25th, 1985)
Key words: circling behavior - - colchicine - - 6-hydroxydopamine - - electrolytic lesion - - apomorphine - - methamphetamine
Circling behavior induced by dopamine (DA) agonists following microinjection of colchicine or 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNC) or electrolytic lesions of the SNC was investigated. Methamphetamine produced a contralateral circling behavior 3, 7 and 14 days following injection of colchicine into the SNC. Apomorphine produced an ipsilateral circling behavior followed by a contralateral rotation 3 and 7 days after, the infusion of colchicine, whereas only an ipsilateral circling behavior was produced by apomorphine on day 14. 6-OHDA lesions of the SNC produced a contralateral circling behavior to apomorphine and an ipsilateral circling to methamphetamine, whereas both apomorphine and methamphetamine induced ipsilateral circling behaviors in rats with electrolytic lesions of the SNC. The possible mechanisms of action of colchicine are discussed in relation to the known effects of colchicine on axoplasmic transport.
INTRODUCTION Ungerstedt demonstrated that the degeneration of the nigrostriatal dopamine ( D A ) system by microinjection of 6 - O H D A produced a postsynaptic supersensitivity to D A agonists in the caudate neurons 33,37,38. Since then, there have been a number of reports concerned with the development of supersensitivity to D A in the caudate nucleus following various procedures, e.g. surgical or chemical denervation of nigrostriatal D A neurons, or sustained attenuation or blockade of dopaminergic transmission14,15,21,24,34,39. Development of supersensitivity may be caused by the loss of the transmitter, the blockade of neurotransmission and/or by removing the influence of some neurofactor, e.g. trophic factor, which might be released independently of, or in conjunction with, the transmitter. Denervation-like supersensitivity
can be caused by the block of axoplasmic transport in the peripheral organs. W h e n colchicine, a drug which has been shown to suppress axoplasmic transport, is directly applied to a nerve, axoplasmic transport is blocked causing a denervation-like supersensitivity of skeletal1,13,18.31 and smooth muscle16,17, 30. The purpose of the present study, therefore, was to determine whether denervation-like supersensitivity can be induced by the block of axoplasmic transport in the central nervous system. To measure the development of supersensitivity, we employed the circling behavior model which has been well established. The circling behavior induced by D A agonists in rats with unilateral 6 - O H D A lesions of nigrostriatal D A neurons has been used extensively to study drug effects on central dopaminergic mechanisms. The resulting dopaminergic imbalance is manifested as circling behavior when a D A agonist is administered to the lesioned animals, i.e., the contralateral
Correspondence: K. Kamata, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468, Japan.
circling induced by apomorphine was considered to be due to the stimulation of supersensitive receptors on the lesioned side of the striatum 37,3s. Following the injection of a suitable amount of colchicine into the SNC, the effect of apomorphine (a direct agonist) or methamphetamine (an indirect agonist) on circling behavior was examined. MATERIALS AND METHODS
Animals and surgical procedures All experiments were carried out on male rats of the Wistar strain weighing 200-300 g. The animals were housed in an air-conditioned room at 22 _+ 1 °C with a 12 h light-dark schedule (light on 7.00, off 19.00 h). The surgical procedure for lesioning of the SNC has been described previously22. Briefly, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), placed in a stereotaxic apparatus and subsequently subjected to the surgical operation as follows. Following exposure of the skull, a burr hole was drilled over the left SNC (approximately 2.8 mm anterior to lambda, 2.0 mm lateral to the midline). A stainless-steel cannula attached to a syringe containing 8/~g//~l 6 - O H D A and 0.5 ktg//A ascorbic acid or 5 or 20/~g//~l colchicine in sterile saline was lowered 7.3 m m ventral to the dura. The cannula was kept in position for 5 min before a 1/A volume of the solution of 6 - O H D A or cotchicine was injected at a rate of 0.5 ~g/min. The cannula remained in place for an additional 5 min and then was slowly withdrawn from the brain. The wound area was closed and the animal was injected intramuscularly with 10,000 units penicillin. For the electrolytic lesions, stainless-steel monopolar electrodes (outer diameter 0.7 mm) insulated except at the tip, were lowered into the SNC and lesions were made by passing current (2 mA, for 15 s); the stereotaxic frame served as the inert electrode.
Measurement of the drug-induced circling behavior Rats were placed in individual testing cages (35 cm x 25 cm x 18 cm) and left undisturbed for adaptation to the cage for 1 h. Circling behavior was directly observed for 2 h and a 360 ° turn was considered as one circling count. The number of complete turns and the direction of circling was recorded for 5 rain at 15, 30, 60, 90 and 120 min following the administration of
drugs. Apomorphine HCI and methamphetamine HC1 were administered intraperitoneally as a salt. Circling responses to methamphetamine (2.0 mg/kg) or apomorphine (1.0 mg/kg) were determined 3-14 days following the surgical operations.
Biochemical methods and histology At the end of the series of behavioral experiments the D A contents in the striatum were determined by means of high-performance liquid chromatography with electrochemical detection according to the method of Nabeshima et al.2-L The striatum was quickly removed from rats and homogenated in 4 ml of butanol containing 0.05 M E D T A , 0.025 N HC1 and 6 ~g/ml D H B A (internal standard) for the D A assay. At the end of the behavioral experiments, the animals were sacrificed under deep pentobarbital-Na anesthesia and perfused with 10% formalin. Frozen 50/,m thick sections of the whole brain were cut using a freezing microtome (MA-101, Komatsu). The location of each lesion site was verified. Only the rats in which the lesion target was confirmed histologically were included in the analysis of the behavioral data. RESULTS
Circling behavior induced by DA agonist following 6-OHDA lesions As shown in Fig. 1, rats injected with 6 - O H D A
A
B
50
0
Time ofhlr injecllo~
( min )
15 3 0
120
6O
go
",','
.S E
0
0
I~ 3 0
60
90
Time after injection
120
-5C
( rnm )
Fig. 1. Circling behavior induced by apomorphine (A) and methamphetamine (B) in rats with unilateral 6-OHDA lesions of the SNC. Apomorphine or methamphetamine was given 3 (El), 7 (O) and 14 (rl) days following the lesions. Contralateral circling is indicated by positive numbers (upward direction) and ipsilateral circling is indicated by negative numbers (downward direction).
into the SNC rotated away from the side of the lesion after 1.0 mg/kg of apomorphine (contralateral circling; indicated by positive numbers and an upward direction) and towards the side of the lesion after 2.0 mg/kg of methamphetamine (ipsilateral circling; indicated by negative numbers and a downward direction). Both apomorphine and methamphetamine produced maximal circling behavior 7 days after the 6-OHDA lesions. The characteristic response to both drugs was tight, 'nose-to-tail' turning. While apomorphine induced rotational behavior that lasted for 60 min, methamphetamine-induced circling behavior lasted for nearly 120 min, as shown in Fig. 1. The striatal DA content on the injected side was significantly reduced to 91 + 9% of that on the intact side.
Circling behavior induced by DA agonists following electrolytic lesions Both apomorphine (1.0 mg/kg) and methamphetamine (2.0 mg/kg) produced an ipsilateral circling behavior, although maximal methamphetamine's effect was weaker than that of apomorphine (Fig. 2). The intensity of circling behavior induced by apomorphine was reached 7 days after the lesion. Histological analysis of the animals with electrolytic lesions in the substantia nigra revealed that despite some variability among animals, the lesion was confined primarily to the pars compacta. The pars reticulata and dorsally located midbrain reticular formation also was damaged in some animals. Striatal DA levels on the lesioned side were reduced to 93 + 11% of that on the intact side.
Circling behavior induced by DA agonists following colchicine infusion into the SNC Unilateral injection of colchicine (20/~g/animal) into the SNC produced animals that showed a slight occasional bias in their posture toward the injected side and became aggressive 3-7 days after the treatment, but 14 days later the rats recovered to normal behavior. Apomorhine (1.0 mg/kg) produced an ipsilateral circling at 15 min, followed by a contralateral rotation at 30-120 min 3 days afteran infusion of colchicine into the SNC. Apomorphine-induced contralateral circling behavior lasted over 120 min as shown in Fig. 3A. This apomorphine-induced long-lasting contralateral circling changed to ipsilateral circling behavior 14 days after colchicine infusion. The infu-
A
B Time after in)ection 15 30
60
90
( rain )
Twne after injection
lEO
15 30
60
90
( rain ) 120
.c E "~ -25
-25
-50
-5(:
Fig. 2. Circling behavior induced by apomorphine (A) and methamphetamine (B) in rats with unilateral electrolytic lesions of the SNC. Apomorphine or methamphetamine was given 3 (O), 7 (O) and 14 ([2) days following the lesions. Ipsilateral circlingis indicatedby negativenumbers (downward direction). sion of relatively low doses of colchicine (5/~g/animal) into the SNC produced a weaker but same directional circling to apomorphine as with the larger dose of colchicine. Methamphetamine (2.0 mg/kg) produced a marked contralateral circling behavior 3 and 7 days following colchicine infusion characterized by 'noseto-tail' turning (Fig. 4A). On day 14, methamphetamine produced a weaker contralateral circling behavior. In this case, turning occurred in circles that were larger than those observed on the earlier test days. The weaker but same directional circling behaviors to methamphetamine were observed at 3, 7 and 14 days after an infusion of the low colchicine dose (5 pg/animal) as shown in Fig. 4B. After treatment with colchicine (20 pg/animal), the DA content in the sttiatum ipsilateral to the injection site was 44 + 5% of that on the intact side. DISCUSSION In the present study, 6-OHDA lesions of the SNC produced contralateral circling behavior to apomorphine and ipsilateral circling to methamphetamine, whereas both apomorphine and methamphetamine induced ipsilateral circling behavior in rats with an electrolytic lesion of the SNC. These results are in good agreement with other investigationsS,9, 23,37,38,39,40. Contralateral circling behavior induced by apomorphine in rats with 6-OHDA lesion may be due to the development of postsynaptic supersensitivityl In fact, it has been reported that lesions induced
A
B
25
25
0
I
'7
-25
-25
120
Time after injection (~n)
-50 Time offer injection ( rain ] Fig. 3. Circlingbehavior induced by apomorphine in rats injected with 20#g of colchicine(A) and 5 gg of the drug (B). Apomorphine was given 3 (O), 7 (O) and 14 ([]) days following the treatment. Contralateral circling is indicated by positive numbers (upward direction) and ipsilateral circlingis indicated by negative numbers (downwarddirection). by 6-OHDA in the nigrostriatal pathway increase the number of postsynaptic DA receptors. Such lesions not only produce a significant increase in the binding of DA in the striatum u, but also enhance the inhibitory effects of apomorphine on striatal neurons 33. In addition, these biochemical and electrophysiological changes are accompanied by a dramatic potentiation of the behavioral response to apomorphine 19. On the other hand, ipsilateral circling behavior was produced by the administration of methamphetamine in rats with 6-OHDA lesions of the SNC. The behavioral effects of methamphetamine or amphetamine appear to depend on DA releaseS,Z8,41. It is likely, therefore, that ipsilateral circling induced by methamphetamine may be caused by the release of DA in the intact but not lesioned side of the striatum, thus resulting in circling behavior towards the lesioned side. On the other hand, both apomorphine and methamphetamine produced an ipsilateral circling behavior in rats with unilateral electrolytic lesions of the SNC. In the present study, although reduction of DA
content in the striatum ipsilateral to the electrolytic lesions of the SNC was sufficient (93% reduction) to induce postsynaptic supersensitivity, apomorphine produced rotation towards the lesion side. With regard to this observation, it has been suggested by Watanabe and Watanabe that in addition to the deA
B
~' 2~
0
25
-~;
' ' 15 3O
' 60
90
Time a f t e r iniecfio~
120 [ rn~n ]
0
15 30
60
90
T i m e a f t e r inlect~on
t20 ( mi n )
Fig. 4. Circling behavior induced by m e t h a m p h e t a m i n e in rats
injected with 20/~g of colchi¢ine (A) and 5/~g of the drug (B) into the SNC. Methamphetamine was given 3 (O), 7 (O) and 14 ([2) days following the treatment. Contralateral circling is indicatedby positivenumbers (upward direction).
generation of the nigrostriatal dopaminergic pathway, electrolytic lesions may induce a dysfunction of postsynaptic factors (i.e. DA receptors on efferent neurons) which may be responsible for the difference in the direction of apomorphine-induced rotation in these rats and in rats with 6-OHDA lesions 40. Furthermore, it has been reported that the stereotypy produced by apomorphine is enhanced in rats with bilateral 6-OHDA lesions 38, whereas bilateral electrolytic lesions of the SNC resulted in a drastic reduction of apomorphine-induced stereotyped behavior 10. It is likely, therefore, that electrolytic lesions of the SNC produce a hyposensitivity of postsynaptic DA receptors and this is the reason why apomorphine produced ipsilateral circling behavior in rats with unilateral electrolytic lesions of the SNC. Methamphetamine produced contralateral circling behavior in rats injected with colchicine into the SNC. It is well known that methamphetamine or amphetamine increases the release and blocks the reuptake of newly synthesized DA at the nerve terminals 5-7,28,41. In the present study, DA content in the striatum ipsilateral to the injection side was reduced to 44% of that on the intact side. It is likely, therefore, that methamphetamine can release the DA in the striatum on both sides. However, animals rotated away from the injection side, indicating that postsynaptic DA receptors in the ipsilateral striatum appear to be supersensitive to the released DA. Because DA content in the striatum was reduced by colchicine injection into the SNC (by about 56%), it could be argued that the colchicine effect is actually due to a 'partial' denervation. However, several investigations negate this possibility. With regard to the relationship between the development of supersensitivity and DA levels, it has been suggested that behavioral supersensitivity to apomorphine is not evident until more than 75% of forebrain DA is depleted 36 and that, when the depletions are greater than 75%, there is a high correlation between the extent of DA loss and the degree of behavioral supersensitivity32,36. It is more likely, therefore, that colchicine might produce a variety of changes in postsynaptic DA receptors by interrupting the influence of some neurotrophic factor, e.g., trophic factor via an inhibitory action of the fast axonal transport system as it is known to do in the peripheral adrenergic nerve fibers 3,4,12 and in the central nervous system2,20,35,42.
Of particular interest in this connection is the observation that the 30% decrease of striatal DA content and 72 to 81% loss of [3H]protein in the striatum was observed following an infusion of colchicine into the SNC 20. Methamphetamine-induced contralateral circling recovered 14 days following an infusion of colchicine, indicating that the block of axonal transport by colchicine is a reversible process. On day 14, methamphetamine-induced circling was characterized by large circles compared to the tight circling that was observed 3 and 7 days following colchicine infusion. Electrical stimulation of the ventral tegmental area causes rats to turn in larger circles with little noticeable postural asymmetry29. However, it is unclear at present whether this qualitative difference in circling behavior is the result of postsynaptic supersensitivity to the released DA in the nucleus accumbens and the striatum separately or whether it is the result of the simultaneous development of supersensitivity in the nucleus accumbens and the striatum in one case and the development of supersensitivity in only the nucleus accumbens in the other case. Apomorphine produced an ipsilateral followed by a contralateral circling behavior on days 3 and 7, and induced only ipsilateral circling on day 14 in rats injected with colchicine into the SNC. The contralateral circling behavior may be due to the development of postsynaptic supersensitivity through a colchicineinduced reversible depression in neural transmission 26,27caused by a disruption of the structural organization of the microtubules that are involved in the fast transport of neurosecretory materials (transmitter, neurotrophic substance, etc.) to the axon3,4,12. On the other hand, apomorphine produced an ipsilateral circling on days 7 and 14 in rats injected with colchicine. It is therefore tempting to speculate that the continuous spontaneous release of DA coupled with a blockade of axoplasmic transport that may occur in the striatum ipsilateral to the injection side, results in the postsynaptic subsensitivity of striatal neurons which could account for the ipsilateral circling behavior to apomorphine. However, this is not the case, because on day 3 contralateral circling behavior induced by apomorphine may be due to the development of postsynaptic supersensitivity. Another possible explanation is that in addition to the transient disruption of the nigrostriatal dopaminergic
n e u r o n s , a colchicine i n j e c t i o n into the SNC produc-
ipsilateral circling b e h a v i o r on days 7 and 14 in rats
es functional d a m a g e of an efferent p a t h w a y that is
injected with colchicine. Thus, further investigations
required for the c o n t r a l a t e r a l circling. This also is not the case, however, because m e t h a m p h e t a m i n e pro-
of this issue are required.
duces an ipsilateral circling in rats with electrolytic le-
ACKNOWLEDGEMENTS
sions of the SNC (afferent a n d efferent fibers m a y be d a m a g e d by e l e c t r o c o a g u l a t i o n ) , b u t m e t h a m p h e t a -
The authors wish to t h a n k Dr. G . V . R e b e c of Indiana University, B l o o m i n g t o n , IN, U . S . A . for help-
mine i n d u c e d only c o n t r a l a t e r a l circling on days 3, 7
ful suggestions d u r i n g the writing of this paper. W e
and 14 in rats t r e a t e d with colchicine. U n f o r t u n a t e l y ,
also are grateful to Mr. M. H i r a m a t s u for his technic-
it is u n c l e a r at p r e s e n t why a p o m o r p h i n e p r o d u c e d
al assistance.
REFERENCES
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1 Albuquerque, E.X., Warnick, J.F., Tasse, J.R. and Sansone, F.M, Effects of vinblastine and colchicine on neuronal regulation of the fast and slow skeletal muscle of the rat, Exp. Neurol., 37 (1972) 607-634. 2 Avrith, D., Haas, H.L. and Mogenson, G.J., Behavioral and electrophysiological changes following microinjections of colchicine into the substantia nigra, Neuroscience, 4 (1979) 227-234. 3 Banks, P., Mayor, D., Mitchell, M. and Tomlinson, D., Studies on the translocation of noradrenaline-containing vesicles in postgangtionic sympathetic neurons in vitro. Inhibition of movement by colchicine and vinblastine and evidence for the involvement of axonal microtubules, J. Physiol. (London), 216 (1971) 625-639. 4 Banks, P. and Till, R., A correlation between the effects of anti-mitotic drugs on microtubule assembly in vitro and the inhibition of axonal transport in noradrenergic neurons, J. Physiol. (London), 252 (1975) 283-294. 5 Besson, M.J., Cheramy, A., Feltz, P. and Glowinski, J., Dopamine: spontaneous and drug-induced released from the caudate nucleus in the cat, Brain Research, 32 (1971) 407-424. 6 Besson, M.J., Cheramy, A. and Glowinski, J., Effect of some psychotropic drugs on dopamine synthesis in the rat striatum, J. Pharmacol. Exp. Ther., 177 (1971) 196-205. 7 Carlsson, A., Fuxe, K., Hamberger, B. and Lindqvist, M., Biochemical and histochemical studies on the effects of imipramine-like drugs and (+)-amphetamine on central and peripheral catecholamine neurons, Acta Physiol. Scand., 67 (1966) 481-497. 8 Christie, J.E. and Crow, T.J., Turning behavior as an index of action of amphetamine and ephedrine on central dopamine-containing neurons, Br. J. Pharmacol., 43 (1971) 658-667. 9 Costal, B., Marsden, C.D., Naytor, R.J. and Pycock, C.J., The relationship between striatal and mesolimbic DA dysfunction and the nature of circling responses following 6-OHDA and electrolytic lesions of the ascending DA system of rat brain, Brain Research, 118 (1976) 87-113. 10 Costall, B., Naylor, R.J. and Olley, J., The substantia nigra and stereotyped behavior, Eur. J. Pharmacol., 18 (1972) 95-106. 11 Creese, I., Butt, D.R. and Snyder, S.H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. 12 Dahlstrom, A., Axoplasmic transport (with particular re-
25 Nabeshima, T., Hiramatsu, M., Noma, S., Ukai, M., Amano, M. and Kameyama, T., Determination of methionine-enkephalin, norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) in brain by high-pressure liquid chromatography with electrochemical detector, Res. Commun. Chem. Pathol. Pharmacol., 35 (1982) 421-442. 26 Perisic, M. and Cuenod, M., Synaptic transmission depressed by colchicine blockade of axoplasmic flow, Science, 175 (1972) 1140-1142. 27 Pilar, G. and Landmesser, L., Axotomy mimicked by localized colchicine application, Science, 177 (1972) 1116-1118. 28 Rech, R.H., Borys, H.K. and Moore, K.E., Alterations in behavior and brain catecholamine levels in rats treated with a-methyltyrosine, J. Pharmacol. Exp. Ther., 152 (1966) 412-419. 29 Roffman, M., Bernard, P.S., Dawson, K.M., Sobiski, R.E. and Saelens, J.K., The effects of haloperidol and clozapine on circling induced by electrical stimulation of the substantia nigra and the ventromedial tegmentum, Neuropharmacology, 17 (1978) 943-946. 30 Saito, A., Kasuya, Y. and Goto, K., The role of morphological and functional changes in adrenergic nerves for the development of colchicine-induced supersensitivity in the rat vas deferens, J. Pharmacol. Exp. Ther., 217 (1981) 516-523. 31 Schalow, G. and Schmidt, H., Effect of length and temperature on the induction of action potentials in denervated slow muscle fibers of the frog, Pflager's Arch. Eur. J. Physiol., 372 (1977) 17-22. 32 Schoenfeld, R. and Uretsky, N,., Altered response to apomorphine in 6-hydroxydopamine-treated rats, Eur. J. Pharmacol., 19 (1972) 115-118. 33 Schultz, W. and Ungerstedt, U., Striatal cell supersensitivity to apomorphine in dopamine-lesioned rats correlated to behaviour, Neuropharmacology, 17 (1978) 349-353.
34 Schwartz, J.C., Costentin, J., Martres, M.P., Protais, P. and Baudry, M., Modulation of receptor mechanisms in the CNS: hypersensitivityto catecholamines, Neuropharmacology, 17 (1978) 665-685. 35 Sorimachi, M., Changes in noradrenaline and its synthesizing enzymes in the nucleus locus coeruleus after intraventricular administration of colchicine and vinblastine, Exp. Neurol., 59 (1978) 85-98. 36 Thornburg, J.E. and Moore, K.E., Supersensitivity to dopamine agonists following unilateral, 6-hydroxydopamineinduced striatal lesions in mice, J. Pharmacol. Exp. Ther., 192 (1975) 42-49. 37 Ungerstedt, U., Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behaviour, Acta Physiol. Scand., Suppl., 367 (1971) 49-68. 38 Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine system, Acta Physiol. Scand., Suppl., 367 (1971) 69-93. 39 Von Voigtlander, P.F. and Moore, K.E., Turning behaviour of mice with unilateral 6-hydroxydopamine lesions in the striatum: effects of apomorphine, r-dopa, amantadine, amphetamine and other psychomotor stimulants, Neuropharmacology, 12 (1973) 451-462. 40 Watanabe, H. and Watanabe, K., Enhancement of apomorphine-induced rotational behaviour in rats following the combination of 6-hydroxydopamine and electrolytic lesions in the substantia nigra, Jpn. J. Pharmacol., 29 (1979) 93-104. 41 Weisman, A., Koe, B.K. and Tenen, S.S., Antiamphetamine effects following inhibition of tyrosine hydroxylase, J. Pharmacol. Exp. Ther., 151 (1966) 339-352. 42 Willis, G.L. and Smith, G.C., Short-term effects of intrahypothalamic colchicine, Brain Research, 276 (1983) 119-125.
1
BRE 11480
Research Reports
Denervation-Like Postsynaptic Supersensitivity to Dopamine Agonists Induced by Microinjection of Colchicine into the S_u_bstantjaNigra Pars Compacta KATSUO KAMATA1, TSUTOMU KAMEYAMA1, SHIGERU OKUYAMA2, SANAE HASHIMOTO2 and HIRONAKA AIHARA2
1Department of Chemical Pharmacology, Facultyof Pharmaceutical Sciences, Meijo University, Nagoya and 2Research Center, Taisho Pharmaceuticals, Saitama (Japan) (Accepted June 25th, 1985)
Key words: circling behavior - - colchicine - - 6-hydroxydopamine - - electrolytic lesion - - apomorphine - - methamphetamine
Circling behavior induced by dopamine (DA) agonists following microinjection of colchicine or 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNC) or electrolytic lesions of the SNC was investigated. Methamphetamine produced a contralateral circling behavior 3, 7 and 14 days following injection of colchicine into the SNC. Apomorphine produced an ipsilateral circling behavior followed by a contralateral rotation 3 and 7 days after, the infusion of colchicine, whereas only an ipsilateral circling behavior was produced by apomorphine on day 14. 6-OHDA lesions of the SNC produced a contralateral circling behavior to apomorphine and an ipsilateral circling to methamphetamine, whereas both apomorphine and methamphetamine induced ipsilateral circling behaviors in rats with electrolytic lesions of the SNC. The possible mechanisms of action of colchicine are discussed in relation to the known effects of colchicine on axoplasmic transport.
INTRODUCTION Ungerstedt demonstrated that the degeneration of the nigrostriatal dopamine ( D A ) system by microinjection of 6 - O H D A produced a postsynaptic supersensitivity to D A agonists in the caudate neurons 33,37,38. Since then, there have been a number of reports concerned with the development of supersensitivity to D A in the caudate nucleus following various procedures, e.g. surgical or chemical denervation of nigrostriatal D A neurons, or sustained attenuation or blockade of dopaminergic transmission14,15,21,24,34,39. Development of supersensitivity may be caused by the loss of the transmitter, the blockade of neurotransmission and/or by removing the influence of some neurofactor, e.g. trophic factor, which might be released independently of, or in conjunction with, the transmitter. Denervation-like supersensitivity
can be caused by the block of axoplasmic transport in the peripheral organs. W h e n colchicine, a drug which has been shown to suppress axoplasmic transport, is directly applied to a nerve, axoplasmic transport is blocked causing a denervation-like supersensitivity of skeletal1,13,18.31 and smooth muscle16,17, 30. The purpose of the present study, therefore, was to determine whether denervation-like supersensitivity can be induced by the block of axoplasmic transport in the central nervous system. To measure the development of supersensitivity, we employed the circling behavior model which has been well established. The circling behavior induced by D A agonists in rats with unilateral 6 - O H D A lesions of nigrostriatal D A neurons has been used extensively to study drug effects on central dopaminergic mechanisms. The resulting dopaminergic imbalance is manifested as circling behavior when a D A agonist is administered to the lesioned animals, i.e., the contralateral
Correspondence: K. Kamata, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468, Japan.
circling induced by apomorphine was considered to be due to the stimulation of supersensitive receptors on the lesioned side of the striatum 37,3s. Following the injection of a suitable amount of colchicine into the SNC, the effect of apomorphine (a direct agonist) or methamphetamine (an indirect agonist) on circling behavior was examined. MATERIALS AND METHODS
Animals and surgical procedures All experiments were carried out on male rats of the Wistar strain weighing 200-300 g. The animals were housed in an air-conditioned room at 22 _+ 1 °C with a 12 h light-dark schedule (light on 7.00, off 19.00 h). The surgical procedure for lesioning of the SNC has been described previously22. Briefly, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), placed in a stereotaxic apparatus and subsequently subjected to the surgical operation as follows. Following exposure of the skull, a burr hole was drilled over the left SNC (approximately 2.8 mm anterior to lambda, 2.0 mm lateral to the midline). A stainless-steel cannula attached to a syringe containing 8/~g//~l 6 - O H D A and 0.5 ktg//A ascorbic acid or 5 or 20/~g//~l colchicine in sterile saline was lowered 7.3 m m ventral to the dura. The cannula was kept in position for 5 min before a 1/A volume of the solution of 6 - O H D A or cotchicine was injected at a rate of 0.5 ~g/min. The cannula remained in place for an additional 5 min and then was slowly withdrawn from the brain. The wound area was closed and the animal was injected intramuscularly with 10,000 units penicillin. For the electrolytic lesions, stainless-steel monopolar electrodes (outer diameter 0.7 mm) insulated except at the tip, were lowered into the SNC and lesions were made by passing current (2 mA, for 15 s); the stereotaxic frame served as the inert electrode.
Measurement of the drug-induced circling behavior Rats were placed in individual testing cages (35 cm x 25 cm x 18 cm) and left undisturbed for adaptation to the cage for 1 h. Circling behavior was directly observed for 2 h and a 360 ° turn was considered as one circling count. The number of complete turns and the direction of circling was recorded for 5 rain at 15, 30, 60, 90 and 120 min following the administration of
drugs. Apomorphine HCI and methamphetamine HC1 were administered intraperitoneally as a salt. Circling responses to methamphetamine (2.0 mg/kg) or apomorphine (1.0 mg/kg) were determined 3-14 days following the surgical operations.
Biochemical methods and histology At the end of the series of behavioral experiments the D A contents in the striatum were determined by means of high-performance liquid chromatography with electrochemical detection according to the method of Nabeshima et al.2-L The striatum was quickly removed from rats and homogenated in 4 ml of butanol containing 0.05 M E D T A , 0.025 N HC1 and 6 ~g/ml D H B A (internal standard) for the D A assay. At the end of the behavioral experiments, the animals were sacrificed under deep pentobarbital-Na anesthesia and perfused with 10% formalin. Frozen 50/,m thick sections of the whole brain were cut using a freezing microtome (MA-101, Komatsu). The location of each lesion site was verified. Only the rats in which the lesion target was confirmed histologically were included in the analysis of the behavioral data. RESULTS
Circling behavior induced by DA agonist following 6-OHDA lesions As shown in Fig. 1, rats injected with 6 - O H D A
A
B
50
0
Time ofhlr injecllo~
( min )
15 3 0
120
6O
go
",','
.S E
0
0
I~ 3 0
60
90
Time after injection
120
-5C
( rnm )
Fig. 1. Circling behavior induced by apomorphine (A) and methamphetamine (B) in rats with unilateral 6-OHDA lesions of the SNC. Apomorphine or methamphetamine was given 3 (El), 7 (O) and 14 (rl) days following the lesions. Contralateral circling is indicated by positive numbers (upward direction) and ipsilateral circling is indicated by negative numbers (downward direction).
into the SNC rotated away from the side of the lesion after 1.0 mg/kg of apomorphine (contralateral circling; indicated by positive numbers and an upward direction) and towards the side of the lesion after 2.0 mg/kg of methamphetamine (ipsilateral circling; indicated by negative numbers and a downward direction). Both apomorphine and methamphetamine produced maximal circling behavior 7 days after the 6-OHDA lesions. The characteristic response to both drugs was tight, 'nose-to-tail' turning. While apomorphine induced rotational behavior that lasted for 60 min, methamphetamine-induced circling behavior lasted for nearly 120 min, as shown in Fig. 1. The striatal DA content on the injected side was significantly reduced to 91 + 9% of that on the intact side.
Circling behavior induced by DA agonists following electrolytic lesions Both apomorphine (1.0 mg/kg) and methamphetamine (2.0 mg/kg) produced an ipsilateral circling behavior, although maximal methamphetamine's effect was weaker than that of apomorphine (Fig. 2). The intensity of circling behavior induced by apomorphine was reached 7 days after the lesion. Histological analysis of the animals with electrolytic lesions in the substantia nigra revealed that despite some variability among animals, the lesion was confined primarily to the pars compacta. The pars reticulata and dorsally located midbrain reticular formation also was damaged in some animals. Striatal DA levels on the lesioned side were reduced to 93 + 11% of that on the intact side.
Circling behavior induced by DA agonists following colchicine infusion into the SNC Unilateral injection of colchicine (20/~g/animal) into the SNC produced animals that showed a slight occasional bias in their posture toward the injected side and became aggressive 3-7 days after the treatment, but 14 days later the rats recovered to normal behavior. Apomorhine (1.0 mg/kg) produced an ipsilateral circling at 15 min, followed by a contralateral rotation at 30-120 min 3 days afteran infusion of colchicine into the SNC. Apomorphine-induced contralateral circling behavior lasted over 120 min as shown in Fig. 3A. This apomorphine-induced long-lasting contralateral circling changed to ipsilateral circling behavior 14 days after colchicine infusion. The infu-
A
B Time after in)ection 15 30
60
90
( rain )
Twne after injection
lEO
15 30
60
90
( rain ) 120
.c E "~ -25
-25
-50
-5(:
Fig. 2. Circling behavior induced by apomorphine (A) and methamphetamine (B) in rats with unilateral electrolytic lesions of the SNC. Apomorphine or methamphetamine was given 3 (O), 7 (O) and 14 ([2) days following the lesions. Ipsilateral circlingis indicatedby negativenumbers (downward direction). sion of relatively low doses of colchicine (5/~g/animal) into the SNC produced a weaker but same directional circling to apomorphine as with the larger dose of colchicine. Methamphetamine (2.0 mg/kg) produced a marked contralateral circling behavior 3 and 7 days following colchicine infusion characterized by 'noseto-tail' turning (Fig. 4A). On day 14, methamphetamine produced a weaker contralateral circling behavior. In this case, turning occurred in circles that were larger than those observed on the earlier test days. The weaker but same directional circling behaviors to methamphetamine were observed at 3, 7 and 14 days after an infusion of the low colchicine dose (5 pg/animal) as shown in Fig. 4B. After treatment with colchicine (20 pg/animal), the DA content in the sttiatum ipsilateral to the injection site was 44 + 5% of that on the intact side. DISCUSSION In the present study, 6-OHDA lesions of the SNC produced contralateral circling behavior to apomorphine and ipsilateral circling to methamphetamine, whereas both apomorphine and methamphetamine induced ipsilateral circling behavior in rats with an electrolytic lesion of the SNC. These results are in good agreement with other investigationsS,9, 23,37,38,39,40. Contralateral circling behavior induced by apomorphine in rats with 6-OHDA lesion may be due to the development of postsynaptic supersensitivityl In fact, it has been reported that lesions induced
A
B
25
25
0
I
'7
-25
-25
120
Time after injection (~n)
-50 Time offer injection ( rain ] Fig. 3. Circlingbehavior induced by apomorphine in rats injected with 20#g of colchicine(A) and 5 gg of the drug (B). Apomorphine was given 3 (O), 7 (O) and 14 ([]) days following the treatment. Contralateral circling is indicated by positive numbers (upward direction) and ipsilateral circlingis indicated by negative numbers (downwarddirection). by 6-OHDA in the nigrostriatal pathway increase the number of postsynaptic DA receptors. Such lesions not only produce a significant increase in the binding of DA in the striatum u, but also enhance the inhibitory effects of apomorphine on striatal neurons 33. In addition, these biochemical and electrophysiological changes are accompanied by a dramatic potentiation of the behavioral response to apomorphine 19. On the other hand, ipsilateral circling behavior was produced by the administration of methamphetamine in rats with 6-OHDA lesions of the SNC. The behavioral effects of methamphetamine or amphetamine appear to depend on DA releaseS,Z8,41. It is likely, therefore, that ipsilateral circling induced by methamphetamine may be caused by the release of DA in the intact but not lesioned side of the striatum, thus resulting in circling behavior towards the lesioned side. On the other hand, both apomorphine and methamphetamine produced an ipsilateral circling behavior in rats with unilateral electrolytic lesions of the SNC. In the present study, although reduction of DA
content in the striatum ipsilateral to the electrolytic lesions of the SNC was sufficient (93% reduction) to induce postsynaptic supersensitivity, apomorphine produced rotation towards the lesion side. With regard to this observation, it has been suggested by Watanabe and Watanabe that in addition to the deA
B
~' 2~
0
25
-~;
' ' 15 3O
' 60
90
Time a f t e r iniecfio~
120 [ rn~n ]
0
15 30
60
90
T i m e a f t e r inlect~on
t20 ( mi n )
Fig. 4. Circling behavior induced by m e t h a m p h e t a m i n e in rats
injected with 20/~g of colchi¢ine (A) and 5/~g of the drug (B) into the SNC. Methamphetamine was given 3 (O), 7 (O) and 14 ([2) days following the treatment. Contralateral circling is indicatedby positivenumbers (upward direction).
generation of the nigrostriatal dopaminergic pathway, electrolytic lesions may induce a dysfunction of postsynaptic factors (i.e. DA receptors on efferent neurons) which may be responsible for the difference in the direction of apomorphine-induced rotation in these rats and in rats with 6-OHDA lesions 40. Furthermore, it has been reported that the stereotypy produced by apomorphine is enhanced in rats with bilateral 6-OHDA lesions 38, whereas bilateral electrolytic lesions of the SNC resulted in a drastic reduction of apomorphine-induced stereotyped behavior 10. It is likely, therefore, that electrolytic lesions of the SNC produce a hyposensitivity of postsynaptic DA receptors and this is the reason why apomorphine produced ipsilateral circling behavior in rats with unilateral electrolytic lesions of the SNC. Methamphetamine produced contralateral circling behavior in rats injected with colchicine into the SNC. It is well known that methamphetamine or amphetamine increases the release and blocks the reuptake of newly synthesized DA at the nerve terminals 5-7,28,41. In the present study, DA content in the striatum ipsilateral to the injection side was reduced to 44% of that on the intact side. It is likely, therefore, that methamphetamine can release the DA in the striatum on both sides. However, animals rotated away from the injection side, indicating that postsynaptic DA receptors in the ipsilateral striatum appear to be supersensitive to the released DA. Because DA content in the striatum was reduced by colchicine injection into the SNC (by about 56%), it could be argued that the colchicine effect is actually due to a 'partial' denervation. However, several investigations negate this possibility. With regard to the relationship between the development of supersensitivity and DA levels, it has been suggested that behavioral supersensitivity to apomorphine is not evident until more than 75% of forebrain DA is depleted 36 and that, when the depletions are greater than 75%, there is a high correlation between the extent of DA loss and the degree of behavioral supersensitivity32,36. It is more likely, therefore, that colchicine might produce a variety of changes in postsynaptic DA receptors by interrupting the influence of some neurotrophic factor, e.g., trophic factor via an inhibitory action of the fast axonal transport system as it is known to do in the peripheral adrenergic nerve fibers 3,4,12 and in the central nervous system2,20,35,42.
Of particular interest in this connection is the observation that the 30% decrease of striatal DA content and 72 to 81% loss of [3H]protein in the striatum was observed following an infusion of colchicine into the SNC 20. Methamphetamine-induced contralateral circling recovered 14 days following an infusion of colchicine, indicating that the block of axonal transport by colchicine is a reversible process. On day 14, methamphetamine-induced circling was characterized by large circles compared to the tight circling that was observed 3 and 7 days following colchicine infusion. Electrical stimulation of the ventral tegmental area causes rats to turn in larger circles with little noticeable postural asymmetry29. However, it is unclear at present whether this qualitative difference in circling behavior is the result of postsynaptic supersensitivity to the released DA in the nucleus accumbens and the striatum separately or whether it is the result of the simultaneous development of supersensitivity in the nucleus accumbens and the striatum in one case and the development of supersensitivity in only the nucleus accumbens in the other case. Apomorphine produced an ipsilateral followed by a contralateral circling behavior on days 3 and 7, and induced only ipsilateral circling on day 14 in rats injected with colchicine into the SNC. The contralateral circling behavior may be due to the development of postsynaptic supersensitivity through a colchicineinduced reversible depression in neural transmission 26,27caused by a disruption of the structural organization of the microtubules that are involved in the fast transport of neurosecretory materials (transmitter, neurotrophic substance, etc.) to the axon3,4,12. On the other hand, apomorphine produced an ipsilateral circling on days 7 and 14 in rats injected with colchicine. It is therefore tempting to speculate that the continuous spontaneous release of DA coupled with a blockade of axoplasmic transport that may occur in the striatum ipsilateral to the injection side, results in the postsynaptic subsensitivity of striatal neurons which could account for the ipsilateral circling behavior to apomorphine. However, this is not the case, because on day 3 contralateral circling behavior induced by apomorphine may be due to the development of postsynaptic supersensitivity. Another possible explanation is that in addition to the transient disruption of the nigrostriatal dopaminergic
n e u r o n s , a colchicine i n j e c t i o n into the SNC produc-
ipsilateral circling b e h a v i o r on days 7 and 14 in rats
es functional d a m a g e of an efferent p a t h w a y that is
injected with colchicine. Thus, further investigations
required for the c o n t r a l a t e r a l circling. This also is not the case, however, because m e t h a m p h e t a m i n e pro-
of this issue are required.
duces an ipsilateral circling in rats with electrolytic le-
ACKNOWLEDGEMENTS
sions of the SNC (afferent a n d efferent fibers m a y be d a m a g e d by e l e c t r o c o a g u l a t i o n ) , b u t m e t h a m p h e t a -
The authors wish to t h a n k Dr. G . V . R e b e c of Indiana University, B l o o m i n g t o n , IN, U . S . A . for help-
mine i n d u c e d only c o n t r a l a t e r a l circling on days 3, 7
ful suggestions d u r i n g the writing of this paper. W e
and 14 in rats t r e a t e d with colchicine. U n f o r t u n a t e l y ,
also are grateful to Mr. M. H i r a m a t s u for his technic-
it is u n c l e a r at p r e s e n t why a p o m o r p h i n e p r o d u c e d
al assistance.
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