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Haloperidol increases and apomorphine decreases striatal dopamine metabolism after destruction of striatal dopamine-sensitive adenylate cyclase by kainic acid
374
Brain Research, 130 (1977} 374- 382 Elsevier/North-Holland BiomedicalPress
Haloperidol increases and apomorphine decreases striatal dopamine metabolism after destruction of striatai dopamine-sensitive adenylate cyclase by kainic acid
GAETANO DI CHIARA, MARIA LUISA PORCEDDU, PIER FRANCO SPAN() and GIAN LUIGI GESSA Institute of Pharmacology, Univer.s'ityof Cagliari, 09100 Cagliari (Italy)
(Accepted March 30th, 1977)
Drugs interacting with dopamine (DA) receptors are known to produce drastic changes in the activity of the nigro-neostriatal DA-neurons. Thus, DA-receptor blockers increase, while DA-receptor stimulants decrease DA metabolism and dopaminergic neuronal firing1,2,4-9. These effects have been postulated to be secondary to the drug-induced blockade or stimulation of striatal post-synaptic DA-receptors and mediated by a negative feedback loop impinging on nigral DA-neurons 8.9 However, the existence of alternative mechanisms has recently been postulated by which these drugs would influence dopaminergic activity and behavior 7,v',-°9. Thus it has been suggested that DA-receptor agonists reduce DA synthesis19 and dopaminergic firing4,5 through a stimulation of pre-synaptic DA-receptors (auto-receptors) 7 located on the membrane of the DA-neurons, More recently, it has been postulated that neuroleptics stimulate dopaminergic firing by blocking nigral DA-receptors, thus relieving DA-neurons from the inhibitory action of DA released from dopaminergic dendrites (self-inhibition)14. Support for this possibility has been provided by the recent discovery ofa DA-sensitive adenylate cyclase within the substantia nigra lv,'?-a,e7 In order to distinguish in vivo between pre- and post-synaptic action of drugs a model is needed which eliminates post-synaptic DA- receptors without destroying dopaminergic neurons. We now report that intra-striatal injection of kainic acid, which produces a degeneration of striatal perikarya but leaves intact dopaminergic terminals 11,21, destroys DA-sensitive adenylate cyclase. With this model we found that haloperidol, a potent and specific neuroleptic, and apomorphine, a DA-receptor agonist, are still able to respectively increase and decrease DA metabolism estimated by measuring the levels of striatat 3,4-dihydroxyphenylaceticacid (DOPAC), the product of the metabolism through monoamine oxidase of released-recaptured DA ')6. Male Sprague-Dawley rats (290-310 g) were anesthetized with Equithesin and placed on a stereotaxic frame (Kopf); kainic acid (Sigma) dissolved in 1/~l of saline was injected in 2 rain in the caudate of one side while that of the other side received
375
Fig. 1. Photomicrographs of a cresyl violet-stained section from a rat injected unilaterally in the striatum with kainic acid (3/tg) 10 days earlier. A and C: control striatum; B and D: kainic-injected striatum. A a n d B 2 . 5 ; C a n d D : 10.
7"
--..21
m~
~v
d
378 TABLE I Effect of intra-striatal administration of kainie acid on basal and DA-stimulated adenylate cy~ lose activio, of striatal homogenates
Rats were injected with 3/tg of kainic acid in the right and with saline in the left striatum and were sacrificed l0 days later. The caudate of each side was homogenized and assayed individually for adenylate cyclase activity. Each value is the mean l: S.E.M. of 5 determinations, run in triplicate. Dopamine concentration
Basal l × 10 6 M 1 × 10 5 M 1 ~: 10 4M *P
Adenylate cyclase activity (pmol eAMP/min/mg prot.) Control side
Lesioned side
210 _-k 15 285 :~ 16" 395 z~ 20* 4203±26"
130 5:12 128 :k 13ns 132 + 15ns 135_i 10n~
0.001; ns, not significantly different from basal values.
the same volume of saline. Coordinates were A 2.2, V 2.8, L 5.0 of Pellegrino and Cushman')4. Ten days post-surgery, rats were sacrificed and the striata of each side dissected out and homogenized in Tris-maleate buffer in order to assay adenylate cyclase activity 16, or frozen in dry ice and kept at - - 2 0 °C until analyzed for DA and D O P A C v'. The brains of three rats were perfused with formaldehyde, cut by cryostat into 40 #m slices and stained by cresyl violet in order to ascertain the extent and topography of the lesion. Haloperidol (Janssen, Beerse) was injected intra-peritoneally while apormophine (hydrocloride, Sandoz, Basle) was administered subcutaneously. Protein was measured by the method of Lowry et al. 2° using bovine serum albumin as a standard. Fig. 1A and B are low power micrographs of rat striatum from the side injected with kainic acid as compared to the contralateral one injected with saline. The kainicinjected striatum appears to be increased in volume and uniformly pale in comparison to the control one. Fig. IC and D are higher magnification micrographs of the kainicinjected and of the control striatum showing marked neuronal loss and slight glyosis in the kainic-injected striatum. Examination of serial cresyl violet-stained sections revealed that the neuronal loss extended through the whole striatum except for a thin subependymal layer in the head of the caudate and for a portion of the tail caudal to level 4.0-3.8 of Petlegrino and Cushman~q Neuronal loss was also extended to about 60°4 of the nucleus accumbens and to the whole bed nucleus of the stria terminalis. In two out of three cases, the globus pallidus also showed some degree of neuronal loss; but the adjacent thalamus and cerebral cortex were relatively unaffected. As soon as the rats awakened from anesthesia they showed continous turning contralateral to the kainic-injected side and intermittent whole body-rockingl This symptomatology lasted for at least 3-4 h. By 24 h post-surgery, rats displayed homolateral torsion of the body and turning which, although diminished, was still present
379 TABLE 1I Effect of haloperidol (H) and apomorphine (A) on DA and DOPAC concentrations in control and kainic-lesioned striatum
Rats were injected with 3/tg of kainic acid in the right striatum and with saline in the left one. After 10 days, rats were given haloperidol or apomorphine and were sacrificed 90 and 45 rain, respectively, after drug administration. Each value is the mean + S.E.M. of the number of determinations indicated in parentheses. Drugs
Dose DA (/Lg/g) (mg/kg) Control
DOPAC (l~g/g) Lesioned
Control
Lesioned
Change of DOPAC~f (/~g/g) Control Lesioned
Saline (30) H (7) H (8) lq (10) A (8) A (7) H t A (10) H t A (10)
-0.30 0.10 0.03 0.5 0.1 0.3 ~0.5 0.3 ~ 0.1
9.52i0.42 9.05%0.65 9.13~0.58 9.35£0.60 9.76~0.65 9.65£0.53 9.33±0.65 9.53~0.60
9.35~0.55 8.87~0.68 9.15~0.53 9.20i0.55 9.63~0.70 9.52~0.63 9.42~0.70 9.25~0.65
1.85~c0.08 6.47t0.30"* 3.76:£0.20** 2.13±0.09 ns 0.92~_0.06'* 1.25i0.07" 4.35~0.25§ 6.15~-0.37§§
3.80~0.18 8.33~0.38"* 8.15:}0.40"* 6.53±0.33** 1.20-a0.06"* 1.37~2:0.08"* 5.52~-0.30§ 7.92£0.42§§
-! 4.62 + 1.91 +0.28 --0.93 --0.60 t 2.50 I 4.30
--4.53 t 4.35 / 2.73 --2.60 --2.43 t 3.72 4.12
* P < 0.01 ; ** P < 0.001 ; ns, not significantly different, with respect to the values of the homolateral striatum of rats administered with saline. P - 0.01, ~.~,not significantly different, with respect to the values of the homolateral striatum or rats administered with haloperidol alone. t with respect to the values of the homolateral striatum of rats administered with saline. l0 days post-surgery. A t this time, a d m i n i s t r a t i o n o f a p o m o r p h i n e (0.1 or 0.5 m g / k g s.c.) stimulated the h o m o l a t e r a l turning while h a l o p e r i d o l (0.3 m g / k g i.p.) reversed the turning into a contralateral one. As shown in T a b l e I, unilateral intrastriataI injection o f 3/zg o f kainic acid ; decrease o f basal adenylate cyclase activity in h o m o g e n a t e s o f kainicresulted in a 40 ',% injected striatum. Moreover, in these homogenates, D A completely failed to stimulate the adenylate cyclase activity even when a d d e d at a c o n c e n t r a t i o n o f 100 # M , which m a x i m a l l y stimulated adenylate cyclase activity of striatal h o m o g e n a t e s f r o m the control side. Similar results were o b t a i n e d with a p o m o r p h i n e . As s h o w n in Table II, unilateral a d m i n i s t r a t i o n o f kainic acid did n o t modify D A concentrations but resulted in a 100 iUooincrease o f D O P A C levels in the lesioned striatum. The p a r e n t e r a l a d m i n i s t r a t i o n o f h a l o p e r i d o l and a p o m o r p h i n e was capable of, respectively, increasing a n d decreasing D O P A C levels b o t h in the kainic-injected and in the control striatum. As Table II shows, 90 rain after a m a x i m a l l y effective dose o f h a l o p e r i d o l (0.3 mg/kg) D O P A C concentrations increased by the same a m o u n t in the kainic-injected and in the control striatum, thus reaching significantly higher levels in the lesioned striatum. A lower dose of h a l o p e r i d o l (0.100 mg/kg), caused a m u c h greater a c c u m u l a t i o n o f D O P A C in the kainic-injected striatum t h a n in the control side a n d the absolute increase o f D O P A C in the kainic-injected side was more t h a n twice that in the control one. Finally, a dose o f h a l o p e r i d o l o f 0.030 mg/kg, failed to p r o d u c e
380 a significant increase of DOPAC in the control striatum but increased by about 100 o~, the levels of DOPAC in the lesioned striatum. Apomorphine administration (0.5 and 0.1 mg/kg, s.c.) produced a more pronounced decrease of DOPAC in the kainic-injected than in the control side. Finally, as shown in Table II, apomorphine (0. l mg/kg) failed to decrease DOPAC levels both in the kainic-injected and in the control striatum of rats pretreated with haloperidol (0.3 mg/kg). On the other hand, a higher dose of apomorphine (0.5 mg/kg) partly reversed the increase in DOPAC produced by haloperidol (0.3 mg/kg) in the lesioned and in the control striatum. Our results show that the local administration of 3/~g of kainic acid produces a complete loss of DA-sensitive adenylate cyclase in the striatum. Since kainic acid does not seem to damage dopaminergic terminals (as indicated by its failure to decrease DA levels or to modify DA uptake by striatal synaptosomes 11) our results support the concept of a post-synaptic localization of striatal DA-sensitive adenylate cyclasOg, z3. A large body of evidence accumulated in recent years indicates that the DA-sensitive adenylate cyclase is a marker of DA-receptors 3,16. Accordingly, our results can be taken to indicate that intrastriatal kainic acid produces a complete toss of striatal postsynaptic DA-receptors. In agreement with recent studies 2~, our results also indicate that striatal post-synaptic DA-receptors are localized on neuronal perikarya or dendrites and not on axonal afferences, which are not damaged by kainic acid ll,~x. Recently the existence of pre-synaptic DA-receptors localized on striatat DAterminals has been postulated, but their relationship with an adenylate cyctase is unknown l°,~5,~s. Our results indicate that, if these receptors do exist, they are not associated with an adenylate cyclase system. The increase in DA metabolism following degeneration of striatal neurons may result from the loss of neurons conveying inhibitory inputs onto nigral DA-neurons and would be in agreement with studies indicating the existence of a striatal inhibitory influence on the substantia nigra 3°. The complete loss of striatal DA-sensitive adenylate cyclase does not prevent the effects of a DA-receptor blocker, such as haloperidol, and of a DA-receptor agonist, such as apomorphine, on DA metabolism. Since haloperidot and apomorphine were mutually antagonistic, their effects on DOPAC in the kainic-injected striatum should be mediated by an action on DA-receptors. These results, while demonstrating that an action at the level of the post-synaptic DA-receptors is not a prerequisite for the in vivo effects of DA-receptor blockers and antagonists on DA metabolism, indicate the existence of an alternative dopaminergic mechanism exerting an inhibitory influence on DA-neurons. The existence of regulatory mechanisms involving release of DA onto inhibitory DA-receptors different from the post-synaptic ones has recently been postulated in order to explain certain actions of neuroleptics on dopaminergic neurons t4. However, to date no direct evidence has been given that the in vivo effect of neuroleptics on DA metabolism can be mediated by such a mechanism. Our results now appear to provide such evidence ; however, the exact location of this mechanism remains to be established. It might be pre-junctional, being mediated by DA released from DA-terminals onto
381 pre-synaptic DA-receptors regulating tyrosine hydroxylase 1°,15 a n d D A release 13 or located within the substantia nigra and mediated by D A released from dopaminergic dendrites onto DA-receptors localized on dopaminergic n e u r o n s 14 or on connections afferent to the substantia nigra ')8. The lower sensitivity of the intact striatum versus the kainic-injected one to haloperidol and a p o m o r p h i n e is difficult to interpret at the present time. A possible explanation might be that post-synaptic DA-receptor activation by a p o m o r p h i n e in the intact striatum results in stimulation of DA metabolism which tends to counteract an inhibitory effect of the drug. Vice versa, blockade of post-synaptic DA-receptors by haloperidol in the intact striatum activates a n inhibitory input onto the nigra which acts antagonistically to a stimulatory effect of the drug. Thus, post-synaptic DA-receptors would mediate a positive feedback mechanism controlling the activity of D A - n e u r o n s . The above mechanism would not be operative in the kainic-injected striatum due to the loss of post-synaptic DA-receptors. Similar concepts have been expressed by Groves et al. 14 in a different context.
1 And6n, N.-E., Butcher, S. G., Corrodi, H., Fuxe, K. and Ungerstedt, U., Receptor activity and turnover of dopamine and noradrenaline after neuroleptics, Europ. J. Pharmacol., I1 (1970) 303 314. 2 Anddn, N.-E., Rubenson, A., Fuxe, K. and H6kfelt, T., Evidence for dopamine receptor stimulation by apomorphine, J. Pharm. Pharmacol., 19 (1967) 627-629. 3 Brown, J. H. and Makman, M., Stimulation by dopamine of adeny[ate cyclase in retinal homogenates and of adenosine-3': 5'-cyclic monophosphate formation in intact retina, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 539-543. 4 Bunney, B. S. and Aghajanian, G. K., D-Amphetamine-induced inhibition of central dopaminergic neurons: mediation by a striato-nigral feedback pathway, Science, 192 (1976) 391-393. 5 Bunney, B. S., Aghajanian, G. K. and Roth, R. H., Comparison of effects of L-DOPA, amphetamine and apomorphine on firing rate of rat dopaminergic neurones, Nature New Biol., 245 (1973) 123-125. 6 Bunney, B. S., Waiters, J. R., Roth, R. H. and Aghajanian, G. K., Dopaminergic neurones: effect of antipsychotic drugs and amphetamine on single cell activity, J. Pharmacol. exp. Ther., 185 (1973) 560-571. 7 Carlsson, A., Receptor-mediated control of dopamine metabolism. In E. Usdin and W. E. Bunney, Jr. (Eds.), Pre- and Post~Tnaptic Receptors, Marcel Dekker, New York, 1975. 8 Carlsson, A., Kehr, W., Lindqvist, M., Magnusson, T. and Atack, C. V., Regulation of monoamine metabolism in the central nervous system, Pharmacol. Rev., 24 (1972) 371 384. 9 Carlsson, A. and Lindqvist, M., Effect of chlorpromazine or haloperidol on formation of 3methoxytyramine and normetanepbrine in mouse brain, Acta pharmacol. (Kbh.), 20 (1963) 140 144. 10 Christiansen, J. and Squires, R. F., Antagonistic effects of apomorphine and haloperidol on rat striatal synaptosomal tyrosine hydroxylase, J, Pharm. Pharmacol., 26 (1974) 367 369. 1I Coyle, J. T. and Schwarcz, R., Lesion of striatal neurones with kainic acid provides a model foJ Huntington's chorea, Nature (Lond.), 263 (1976) 244-246. 12 Di Chiara, G., Porceddu, M. L., Vargiu, L, Argiolas, A. and Gessa G. L., Evidence for dopamine receptors in the mouse brain mediating sedation, Nature (Lond.), 264 (1976) 564 567. 13 Farnebo, L.-O. and Hamberger, B., Drug-induced changes in the release of :~H-monoaminesfrom field stimulated rat brain slices, Acta physiol, stand., Suppl. 371 (1971) 35 44. 14 Groves, P. M., Wilson, C. J., Young, S. J. and Rebec, G. V., Self-inhibition by dopaminergic neurons, Science, 190 (1975) 522-529. 15 Iversen, L. L., Rogawski, M. A. and Miller, R. J., Comparison of the effects of neuroleptic drugs on pre- and postsynaptic dopaminergic mechanisms in the rat striatum, Molec. Pharmacol., 12 (1975) 251-262.
382 16 Kebabian, J. W., Petzold, G. L. and Greengard, P., Doparnine-sensitive adenytate cyclase in caudate nucleus of rat brain, and its similarity to the 'doparnine receptor', Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2145-2149. 17 Kebabian, J. W. and Saavedra, J. M., Doparnine-sensitive adenylate cyclase occurs in a region of substantia nigra containing doparninergic dendrites, Science, 193 (1976) 683-685. 18 Kehr, W., Carlsson, A., Lindqvist, M., Magnusson, T. and Atack, C., Evidence of a receptormediated feedback control of striatal tyrosine hydroxylase activity, J~ Pharm. Pharmacol.~ 24 (1972) 744-747. 19 Laduron, P., Verwirnp, M., Janssen, P. 1:7.M. and Leysen, J., Subcellular localization of doparninesensitive adenylate cyclase in rat brain striaturn, Life Sci., 18 (1976) 433-440. 20 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 21 McGeer, E. G. and McGeer, P. L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutarnic and kainic acids, Nature (Lond.), 263 (1976) 517--519. 22 McGeer, E. G., McGeer, P. L., Grewaal, D. S. and Singh, V. K., Striatal cholinergic interneurons and their relation to dopaminergic nerve endings, J. Pharmaeol., 6 (1975) 143-152. 23 Mishra, R. K., Garner, E. L , Katzman, R. and Makrnan, M. H., Enhancement ot' doparninestimulated adenylate cyclase activity in rat caudate after lesions in substantia nigra : evidence for denervation supersensitivity, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 3883-3887. 24 Pellegrino, L. J. and Cushman, A. J., A Stereotaxic Atlas o f the Rat Brain, Meredith, New York, 1971. 25 Phillipson, O. T. and Horn, A. S., Substantia nigra of the rat contains a dopamine sensitive adenylate cyclase, Nature (Lond.), 261 (1976) 418~420. 26 Roth, R. H., Murrin, L. C. and Waiters, J. R., Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid, Ettrop. J~ PharmacoL. 36 (1976) 163-171. 27 Spano, P. F., Di Chiara, G., Tonon, G. and Trabucchi, M., A dopamine-sensitive adenylate-cyclase in rat substantia nigra, J. Neurochem., 27 (1976) 1565-1568. 28 Spano, P. F., Trabucchi, M. and Di Chiara, G., Localization of nigral DA-sensitive adenylatecyclase on neurons originating from the corpus striaturn, Science (1977) in press. 29 Str6mborn, U., Catecholamine receptor agonists. Effects on motor activity and rate of tyrosine hydroxylation in mouse brain, Naunyn-Schmiedeberg,'s Arch. exp. Path. Pharmak.~ 292 (1976) 167-176. 30 Yoshida, M. and Precht, W., Monosynaptic inhibition of neurons of the substantia nigra by caudate-nigral fibers, Brain Research, 32 (1971) 225-228.
Brain Research, 130 (1977} 374- 382 Elsevier/North-Holland BiomedicalPress
Haloperidol increases and apomorphine decreases striatal dopamine metabolism after destruction of striatai dopamine-sensitive adenylate cyclase by kainic acid
GAETANO DI CHIARA, MARIA LUISA PORCEDDU, PIER FRANCO SPAN() and GIAN LUIGI GESSA Institute of Pharmacology, Univer.s'ityof Cagliari, 09100 Cagliari (Italy)
(Accepted March 30th, 1977)
Drugs interacting with dopamine (DA) receptors are known to produce drastic changes in the activity of the nigro-neostriatal DA-neurons. Thus, DA-receptor blockers increase, while DA-receptor stimulants decrease DA metabolism and dopaminergic neuronal firing1,2,4-9. These effects have been postulated to be secondary to the drug-induced blockade or stimulation of striatal post-synaptic DA-receptors and mediated by a negative feedback loop impinging on nigral DA-neurons 8.9 However, the existence of alternative mechanisms has recently been postulated by which these drugs would influence dopaminergic activity and behavior 7,v',-°9. Thus it has been suggested that DA-receptor agonists reduce DA synthesis19 and dopaminergic firing4,5 through a stimulation of pre-synaptic DA-receptors (auto-receptors) 7 located on the membrane of the DA-neurons, More recently, it has been postulated that neuroleptics stimulate dopaminergic firing by blocking nigral DA-receptors, thus relieving DA-neurons from the inhibitory action of DA released from dopaminergic dendrites (self-inhibition)14. Support for this possibility has been provided by the recent discovery ofa DA-sensitive adenylate cyclase within the substantia nigra lv,'?-a,e7 In order to distinguish in vivo between pre- and post-synaptic action of drugs a model is needed which eliminates post-synaptic DA- receptors without destroying dopaminergic neurons. We now report that intra-striatal injection of kainic acid, which produces a degeneration of striatal perikarya but leaves intact dopaminergic terminals 11,21, destroys DA-sensitive adenylate cyclase. With this model we found that haloperidol, a potent and specific neuroleptic, and apomorphine, a DA-receptor agonist, are still able to respectively increase and decrease DA metabolism estimated by measuring the levels of striatat 3,4-dihydroxyphenylaceticacid (DOPAC), the product of the metabolism through monoamine oxidase of released-recaptured DA ')6. Male Sprague-Dawley rats (290-310 g) were anesthetized with Equithesin and placed on a stereotaxic frame (Kopf); kainic acid (Sigma) dissolved in 1/~l of saline was injected in 2 rain in the caudate of one side while that of the other side received
375
Fig. 1. Photomicrographs of a cresyl violet-stained section from a rat injected unilaterally in the striatum with kainic acid (3/tg) 10 days earlier. A and C: control striatum; B and D: kainic-injected striatum. A a n d B 2 . 5 ; C a n d D : 10.
7"
--..21
m~
~v
d
378 TABLE I Effect of intra-striatal administration of kainie acid on basal and DA-stimulated adenylate cy~ lose activio, of striatal homogenates
Rats were injected with 3/tg of kainic acid in the right and with saline in the left striatum and were sacrificed l0 days later. The caudate of each side was homogenized and assayed individually for adenylate cyclase activity. Each value is the mean l: S.E.M. of 5 determinations, run in triplicate. Dopamine concentration
Basal l × 10 6 M 1 × 10 5 M 1 ~: 10 4M *P
Adenylate cyclase activity (pmol eAMP/min/mg prot.) Control side
Lesioned side
210 _-k 15 285 :~ 16" 395 z~ 20* 4203±26"
130 5:12 128 :k 13ns 132 + 15ns 135_i 10n~
0.001; ns, not significantly different from basal values.
the same volume of saline. Coordinates were A 2.2, V 2.8, L 5.0 of Pellegrino and Cushman')4. Ten days post-surgery, rats were sacrificed and the striata of each side dissected out and homogenized in Tris-maleate buffer in order to assay adenylate cyclase activity 16, or frozen in dry ice and kept at - - 2 0 °C until analyzed for DA and D O P A C v'. The brains of three rats were perfused with formaldehyde, cut by cryostat into 40 #m slices and stained by cresyl violet in order to ascertain the extent and topography of the lesion. Haloperidol (Janssen, Beerse) was injected intra-peritoneally while apormophine (hydrocloride, Sandoz, Basle) was administered subcutaneously. Protein was measured by the method of Lowry et al. 2° using bovine serum albumin as a standard. Fig. 1A and B are low power micrographs of rat striatum from the side injected with kainic acid as compared to the contralateral one injected with saline. The kainicinjected striatum appears to be increased in volume and uniformly pale in comparison to the control one. Fig. IC and D are higher magnification micrographs of the kainicinjected and of the control striatum showing marked neuronal loss and slight glyosis in the kainic-injected striatum. Examination of serial cresyl violet-stained sections revealed that the neuronal loss extended through the whole striatum except for a thin subependymal layer in the head of the caudate and for a portion of the tail caudal to level 4.0-3.8 of Petlegrino and Cushman~q Neuronal loss was also extended to about 60°4 of the nucleus accumbens and to the whole bed nucleus of the stria terminalis. In two out of three cases, the globus pallidus also showed some degree of neuronal loss; but the adjacent thalamus and cerebral cortex were relatively unaffected. As soon as the rats awakened from anesthesia they showed continous turning contralateral to the kainic-injected side and intermittent whole body-rockingl This symptomatology lasted for at least 3-4 h. By 24 h post-surgery, rats displayed homolateral torsion of the body and turning which, although diminished, was still present
379 TABLE 1I Effect of haloperidol (H) and apomorphine (A) on DA and DOPAC concentrations in control and kainic-lesioned striatum
Rats were injected with 3/tg of kainic acid in the right striatum and with saline in the left one. After 10 days, rats were given haloperidol or apomorphine and were sacrificed 90 and 45 rain, respectively, after drug administration. Each value is the mean + S.E.M. of the number of determinations indicated in parentheses. Drugs
Dose DA (/Lg/g) (mg/kg) Control
DOPAC (l~g/g) Lesioned
Control
Lesioned
Change of DOPAC~f (/~g/g) Control Lesioned
Saline (30) H (7) H (8) lq (10) A (8) A (7) H t A (10) H t A (10)
-0.30 0.10 0.03 0.5 0.1 0.3 ~0.5 0.3 ~ 0.1
9.52i0.42 9.05%0.65 9.13~0.58 9.35£0.60 9.76~0.65 9.65£0.53 9.33±0.65 9.53~0.60
9.35~0.55 8.87~0.68 9.15~0.53 9.20i0.55 9.63~0.70 9.52~0.63 9.42~0.70 9.25~0.65
1.85~c0.08 6.47t0.30"* 3.76:£0.20** 2.13±0.09 ns 0.92~_0.06'* 1.25i0.07" 4.35~0.25§ 6.15~-0.37§§
3.80~0.18 8.33~0.38"* 8.15:}0.40"* 6.53±0.33** 1.20-a0.06"* 1.37~2:0.08"* 5.52~-0.30§ 7.92£0.42§§
-! 4.62 + 1.91 +0.28 --0.93 --0.60 t 2.50 I 4.30
--4.53 t 4.35 / 2.73 --2.60 --2.43 t 3.72 4.12
* P < 0.01 ; ** P < 0.001 ; ns, not significantly different, with respect to the values of the homolateral striatum of rats administered with saline. P - 0.01, ~.~,not significantly different, with respect to the values of the homolateral striatum or rats administered with haloperidol alone. t with respect to the values of the homolateral striatum of rats administered with saline. l0 days post-surgery. A t this time, a d m i n i s t r a t i o n o f a p o m o r p h i n e (0.1 or 0.5 m g / k g s.c.) stimulated the h o m o l a t e r a l turning while h a l o p e r i d o l (0.3 m g / k g i.p.) reversed the turning into a contralateral one. As shown in T a b l e I, unilateral intrastriataI injection o f 3/zg o f kainic acid ; decrease o f basal adenylate cyclase activity in h o m o g e n a t e s o f kainicresulted in a 40 ',% injected striatum. Moreover, in these homogenates, D A completely failed to stimulate the adenylate cyclase activity even when a d d e d at a c o n c e n t r a t i o n o f 100 # M , which m a x i m a l l y stimulated adenylate cyclase activity of striatal h o m o g e n a t e s f r o m the control side. Similar results were o b t a i n e d with a p o m o r p h i n e . As s h o w n in Table II, unilateral a d m i n i s t r a t i o n o f kainic acid did n o t modify D A concentrations but resulted in a 100 iUooincrease o f D O P A C levels in the lesioned striatum. The p a r e n t e r a l a d m i n i s t r a t i o n o f h a l o p e r i d o l and a p o m o r p h i n e was capable of, respectively, increasing a n d decreasing D O P A C levels b o t h in the kainic-injected and in the control striatum. As Table II shows, 90 rain after a m a x i m a l l y effective dose o f h a l o p e r i d o l (0.3 mg/kg) D O P A C concentrations increased by the same a m o u n t in the kainic-injected and in the control striatum, thus reaching significantly higher levels in the lesioned striatum. A lower dose of h a l o p e r i d o l (0.100 mg/kg), caused a m u c h greater a c c u m u l a t i o n o f D O P A C in the kainic-injected striatum t h a n in the control side a n d the absolute increase o f D O P A C in the kainic-injected side was more t h a n twice that in the control one. Finally, a dose o f h a l o p e r i d o l o f 0.030 mg/kg, failed to p r o d u c e
380 a significant increase of DOPAC in the control striatum but increased by about 100 o~, the levels of DOPAC in the lesioned striatum. Apomorphine administration (0.5 and 0.1 mg/kg, s.c.) produced a more pronounced decrease of DOPAC in the kainic-injected than in the control side. Finally, as shown in Table II, apomorphine (0. l mg/kg) failed to decrease DOPAC levels both in the kainic-injected and in the control striatum of rats pretreated with haloperidol (0.3 mg/kg). On the other hand, a higher dose of apomorphine (0.5 mg/kg) partly reversed the increase in DOPAC produced by haloperidol (0.3 mg/kg) in the lesioned and in the control striatum. Our results show that the local administration of 3/~g of kainic acid produces a complete loss of DA-sensitive adenylate cyclase in the striatum. Since kainic acid does not seem to damage dopaminergic terminals (as indicated by its failure to decrease DA levels or to modify DA uptake by striatal synaptosomes 11) our results support the concept of a post-synaptic localization of striatal DA-sensitive adenylate cyclasOg, z3. A large body of evidence accumulated in recent years indicates that the DA-sensitive adenylate cyclase is a marker of DA-receptors 3,16. Accordingly, our results can be taken to indicate that intrastriatal kainic acid produces a complete toss of striatal postsynaptic DA-receptors. In agreement with recent studies 2~, our results also indicate that striatal post-synaptic DA-receptors are localized on neuronal perikarya or dendrites and not on axonal afferences, which are not damaged by kainic acid ll,~x. Recently the existence of pre-synaptic DA-receptors localized on striatat DAterminals has been postulated, but their relationship with an adenylate cyctase is unknown l°,~5,~s. Our results indicate that, if these receptors do exist, they are not associated with an adenylate cyclase system. The increase in DA metabolism following degeneration of striatal neurons may result from the loss of neurons conveying inhibitory inputs onto nigral DA-neurons and would be in agreement with studies indicating the existence of a striatal inhibitory influence on the substantia nigra 3°. The complete loss of striatal DA-sensitive adenylate cyclase does not prevent the effects of a DA-receptor blocker, such as haloperidol, and of a DA-receptor agonist, such as apomorphine, on DA metabolism. Since haloperidot and apomorphine were mutually antagonistic, their effects on DOPAC in the kainic-injected striatum should be mediated by an action on DA-receptors. These results, while demonstrating that an action at the level of the post-synaptic DA-receptors is not a prerequisite for the in vivo effects of DA-receptor blockers and antagonists on DA metabolism, indicate the existence of an alternative dopaminergic mechanism exerting an inhibitory influence on DA-neurons. The existence of regulatory mechanisms involving release of DA onto inhibitory DA-receptors different from the post-synaptic ones has recently been postulated in order to explain certain actions of neuroleptics on dopaminergic neurons t4. However, to date no direct evidence has been given that the in vivo effect of neuroleptics on DA metabolism can be mediated by such a mechanism. Our results now appear to provide such evidence ; however, the exact location of this mechanism remains to be established. It might be pre-junctional, being mediated by DA released from DA-terminals onto
381 pre-synaptic DA-receptors regulating tyrosine hydroxylase 1°,15 a n d D A release 13 or located within the substantia nigra and mediated by D A released from dopaminergic dendrites onto DA-receptors localized on dopaminergic n e u r o n s 14 or on connections afferent to the substantia nigra ')8. The lower sensitivity of the intact striatum versus the kainic-injected one to haloperidol and a p o m o r p h i n e is difficult to interpret at the present time. A possible explanation might be that post-synaptic DA-receptor activation by a p o m o r p h i n e in the intact striatum results in stimulation of DA metabolism which tends to counteract an inhibitory effect of the drug. Vice versa, blockade of post-synaptic DA-receptors by haloperidol in the intact striatum activates a n inhibitory input onto the nigra which acts antagonistically to a stimulatory effect of the drug. Thus, post-synaptic DA-receptors would mediate a positive feedback mechanism controlling the activity of D A - n e u r o n s . The above mechanism would not be operative in the kainic-injected striatum due to the loss of post-synaptic DA-receptors. Similar concepts have been expressed by Groves et al. 14 in a different context.
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