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Haloperidol induces Fos expression in the globus pallidus and substantia nigra of cynomolgus monkeys
Brain Research 835 Ž1999. 154–161 www.elsevier.comrlocaterbres
Research report
Haloperidol induces Fos expression in the globus pallidus and substantia nigra of cynomolgus monkeys David Wirtshafter a
a, )
, Karen E. Asin
b
Department Psychology, M r C 285, The UniÕersity of Illinois at Chicago, 1007 W. Harrison St. Chicago, IL 60607-7137, USA b Department Toxicology, Abbott Laboratories, D468, Bldg. AP13A, Abbott Park, IL 60604, USA Accepted 27 April 1999
Abstract Systemic injections of the dopamine antagonist haloperidol Ž0.1–2.5 mgrkg. induced a dose dependent increase in Fos-like immunoreactivity ŽFLI. in the internal segment of the globus pallidus ŽGPi. and in the substantia nigra ŽSN. of cynomolgus monkeys. These findings are consistent with models of basal ganglia organization which predict that blockade of dopamine receptors should result in a disinhibition of cells in these structures. In the GPi, labeling was most pronounced along the ventral, lateral and medial borders of the nucleus and none of the pallidal cells expressing FLI were immunopositive for choline acetyltransferase. In the SN, immunoreactive nuclei were concentrated in the pars reticulata and the majority of labeled nigral neurons did not display tyrosine hydroxylase-like immunoreactivity. A small number of cells displaying FLI were also observed in the external pallidal segment, but no labeling was seen in the subthalamic nucleus. These findings indicate that blockade of dopamine receptors induces a characteristic pattern of Fos expression in the primate brain which strongly resembles that previously reported in rodents. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Immediate-early genes; c-fos; Subthalamic nucleus; Neuroleptic; Dopamine; Primate
1. Introduction The internal segment of the globus pallidus ŽGPi, entopeduncular nucleus ŽEPN. of rodents. and the pars reticulata of the substantia nigra ŽSNpr. are the major output nuclei of the basal ganglia and have been proposed to inhibit motor processing by means of GABAergic projections to the midbrain tegmentum, the intermediate and deep layers of the superior colliculus and several thalamic nuclei w9,19,39,44x. It has been suggested by a number of workers that interference with dopaminergic transmission increases the activity of cells within the GPi and SNpr, resulting in an inhibition of thalamic and brainstem motor structures and a consequent reduction in motor output w1,2,20,36x. Increases in the activity of GPi neurons have been directly demonstrated in monkeys treated with the dopamine depleting neurotoxin MPTP w20,36x and a similar effect may occur in human Parkinsonian patients w8x. Interference with dopamine transmission has also been reported to increase the firing rates of SNpr cells w8x,
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although this result has not always been obtained w3,38,53x. Furthermore, the parkinsonian symptoms induced by neuroleptic treatment or dopamine depletion can be attenuated by lesions of the GPirEPN or the SNpr or by injections of inhibitory compounds into these structures w7,12,48,52,62x. Several recent studies have examined the effects of disrupting dopaminergic transmission on the expression of the proto-oncoprotein Fos within output structures of the basal ganglia. We were able to demonstrate, for example, that injections of the dopamine antagonists haloperidol and metoclopramide in rats produce a modest but clear expression of Fos-like immunoreactivity ŽFLI. within both the SNpr and the EPN w59x, and similar results have recently been reported in mice w42x. Fos expression within the SNpr and EPN regions can also be induced by injections of the dopamine depleting drug reserpine w11,21x, and this effect in the EPN can be blocked by pretreatment with the D 2-like dopamine agonist quinpirole w21x. Increased Fos expression within the SNpr of rats has also been reported following acute lesions of the ascending dopamine fibers at the level of the lateral hypothalamus w56x. Since Fos expression often appears to be correlated with elevations in the firing rate of neurons w14,18,47x, these results are compatible with the theory that reductions in dopamine
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transmission result in increases in the activity of some SNpr and EPN cells. Interest in the basal ganglia reflects in large part the clinical importance of these nuclei. To date, the effects of dopamine depletion or blockade on Fos expression in basal ganglia output structures have been examined only in rodents. Given the central role that alterations in the activity of the GPi and SNpr play in current theories of the pathophysiology of Parkinson’s disease and other basal ganglia disorders, it is important to determine whether similar effects also occur in primates. Although rodents have frequently provided useful models for the study of the basal ganglia, extrapolation from results obtained in these animals to humans and other primates obviously requires great caution since a number of differences in the anatomy and neurochemistry of the basal ganglia have been described between members of these two orders Že.g., Refs. w5,43,61x.. In the current experiments we therefore examined whether systemic injections of the dopamine antagonist haloperidol are able to induce FLI in the GP and SNpr of cynomolgus monkeys. Some of the current results have been presented in abstract form w57x. 2. Methods and materials 2.1. Subjects Subjects were nine adult cynomolgus monkeys Ž Macacca fascicularis. obtained from Charles Rivers ŽHouston, TX.. Subjects had previously been used in pharmacological studies involving nondopaminergic agents, but had not been given any drugs for at least a month prior to the current study. Animals were individually housed. 2.2. Drug administration and perfusion Subjects received intramuscular injections of haloperidol ŽHaldol w injectable formulation. at doses of either 0.1 mgrkg Ž N s 2., 0.5 mgrkg Ž N s 3. or 2.5 mgrkg Ž N s 2.. Two additional animals were injected with distilled water. Three hours following these injections subjects were anesthetized with ketamine Ž10 mgrkg, i.m.., followed by 50 mgrkg of Nembutal w ŽAbbott Laboratories. given through the saphenous vein. The thorax was then opened and the descending aorta clamped; animals were then perfused transcardially with normal saline Ž500 ml. followed by a 10% solution of formalin prepared in phosphate buffer Ž1500 ml.. Brains were removed from the skulls, blocked into several pieces and post-fixed for 2 h after which time they were transferred to a solution of 20% sucrose in phosphate buffered saline ŽPBS. where they were stored for two days at 48C. 2.3. Immunocytochemistry Thirty-micrometer cryostat sections were taken through the extent of the globus pallidus and the substantia nigra
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and processed for Fos-like immunoreactivity ŽFLI. using methods we have described in detail elsewhere w59,60x. The primary antibody in these studies was a sheep anti-Fos serum obtained from Cambridge Research Biochemicals ŽWilmington, DE; OA-11-823. which was used at a dilution of 2000 = in PBS containing 0.2% Triton-X100, 0.05% sodium azide and 2% blocking serum ŽPBSTA.. Except as noted below, the secondary antibody was a biotinylated rabbit anti-sheep serum ŽVector Laboratories, Burlingame, CA. used at a dilution of 200 = . Antibodies were detected using a Vectastain Elite ABC kit employing nickel intensified diaminobenzadine ŽDAB. as the chromogen. No staining was seen in control sections in which the primary antibody was either omitted or preadsorbed with the antigenic fragment of the Fos protein ŽCambridge Research Biochemicals, OP-11-3210, 4 mgrml.. Some sections were processed by a sequential double labeling technique which allowed for the simultaneous visualization of FLI and either choline acetyltransferaseŽChAT. or tyrosine hydroxylase- like immunoreactivity. In these sections, a biotinylated donkey anti-sheep antibody preadsorbed with rabbit IgGs ŽJackson Immuno Research, West Grove, PA; 500 = . was used as the secondary antibody for the detection of Fos. After processing for FLI, using intensified DAB, was complete, the sections were thoroughly rinsed in PBS and then incubated for 48 h at 48C in either a rabbit anti ChAT serum ŽChemicon, Temecula, CA; AB143; 2000 = . or a rabbit anti-tyrosine hydroxylase serum ŽEugene Tech International, Allendale, NJ, TE 101; 6000 = .. Both antibodies were prepared in PBSTA containing 2% normal donkey serum. A biotinylated donkey anti-rabbit antibody preadsorbed with sheep IgGs ŽJackson, 500 = . was used as the secondary antibody and detection was accomplished using a Vectastain Elite ABC kit employing unintensified DAB as the chromogen.
3. Results Very few immunoreactive neurons could be seen within the basal ganglia of monkeys injected with water; in contrast, clear staining was apparent within the globus pallidus and substantia nigra of animals injected with haloperidol. Fig. 1. displays a camera lucida drawing of the SN and adjacent structures from a representative subject injected with 0.5 mgrkg of haloperidol. Cells expressing FLI were scattered throughout the SNpr, except for its most lateral regions. The numbers of cells in the SNpr displaying FLI were counted, with the aid of a camera lucida, in one section per animal, at a level similar to that of Fig. 1 in a region extending 1 mm lateral from the medial border of the SN. The results of this analysis are shown in Fig. 2 where it can be seen that haloperidol induced a dose dependent increase in the number of immunoreactive nigral cells Ž F Ž3,5. s 10.3; p - .02.. Further analysis by
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Fig. 1. A camera lucida drawing showing the distribution of Fos immunoreactive cells in the substantia nigra and adjacent regions of a monkey treated with 0.5 mgrkg of haloperidol. The approximate border of the pars compacta is indicated based on examination of adjacent Nissl stained sections. The tendency of immunoreactive cells to be confined to the medial portion of the SNpr was less pronounced in this subject than in many of the others we examined. Abbreviations: CPs cerebral peduncle; OT s optic tract; STNssubthalamic nucleus.
means of polynomial contrasts indicated the presence of a significant first order Žlinear. relation between drug dose and Fos expression Ž F Ž1,5. s 30.4; p - .01.. Although labeling was largely restricted to the pars reticulata, small clusters of immunoreactive nuclei could be observed in some sections within the medial portion of the pars compacta. These nuclei were typically smaller, more lightly stained, and more closely packed than were those seen in the SNpr ŽFig. 3.. Immunoreactive nuclei were also pre-
Fig. 3. Photomicrographs showing Fos immunoreactive nuclei in the medial region of the internal segment of the globus pallidus ŽA., the pars reticulata of the substantia nigra ŽB. and the pars compacta of the substantia nigra ŽC.. Scale bar s100 mm.
Fig. 2. Mean numbers of Fos immunoreactive cells found in the medial substantia nigra pars reticulata for monkeys receiving various doses of haloperidol. ‘Error bars’ represent the observed range of scores.
sent in the ventral tegmental area of both vehicle and haloperidol treated monkeys, although they appeared to be more frequent in drug treated animals. Examination of material processed for both FLI and tyrosine hydroxylase-like immunoreactivity indicated that the great majority of Fos immunoreactive cells seen in the SN did not stain for tyrosine hydroxylase. A small clump of double labeled cells could occasionally be seen, however, within the medial portion of the pars compacta ŽFig. 4a.. Double labeled neurons were relatively common in the ventral tegmental area ŽFig. 4b.. Treatment with either of the two higher doses of haloperidol induced clear FLI within the GPi ŽFigs. 3 and 5.. The general appearance of staining in this region was
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Fig. 4. Photomicrographs of sections processed for the simultaneous detection of Fos like immunoreactivity and tyrosine hydroxylase activity. Panel ŽA. displays a cluster of double labeled cells ŽU . in the pars compacta of the substantia nigra whereas panel ŽB. shows a double labeled neuron in the VTA. Scale bar s 100 mm in panel ŽA. and 45 mm in panel ŽB..
very similar to that observed in the SNpr ŽFig. 3.. Labeling was restricted to the rostral portions of the GPi in all animals and, even at these levels, was not evenly distributed ŽFig. 5.. A narrow band of immunoreactive neurons was typically observed within and just medial to the internal medullary lamina of the GP and labeling continued from this region along the ventral and, at far rostral levels, the dorsal border of the GPi. At the rostral tip of the GPi little labeling was seen in the internal portions of this structure, and immunoreactive cells appeared to form an annulus outlining the borders of the nucleus. At slightly
more caudal levels, however, labeled cells filled much of the medial half of the GPi ŽFig. 5.. Labeling in the region of the internal medullary lamina was usually separated from that along the medial border of the pallidum by a region in the lateral half of the GPi which was virtually devoid of immunoreactive nuclei. Scattered labeling was also seen in GPe and very heavy labeling was apparent in the caudate nucleus and putamen at the level of the GP. Tissue from pallidal region of two monkeys, one injected with the high and one with the middle dose of haloperidol, was processed for the simultaneous demon-
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Fig. 5. A camera lucida drawing showing the distribution of Fos like immunoreactivity in the globus pallidus of a monkey injected with 0.5 mgrkg of haloperidol. Large numbers of labeled cells were also present within the putamen, but are not shown in the diagram. Abbreviations: GPesexternal segment of the globus pallidus; GPis internal segment of the globus pallidus; PUT s putamen.
stration of FLI and choline acetyltransferase-like immunoreactivity ŽChAT.. Examination of these sections indicated that Fos immunoreactive cells in the ventral portion of the GPi were located immediately dorsal to the cholinergic neurons of the nucleus basalis complex Žthe Ch4id sector of Mesulam et al. w34x. and virtually no overlap in the distributions of the two cell types was present. Scattered ChAT positive cells were also observed in the internal medullary lamina of the pallidum; although these neurons were partially intermingled with those expressing FLI, no double labeled cells could be detected. In marked contrast to the labeling seen in other basal ganglia structures, no immunoreactive cells were observed in the subthalamic nucleus in any of the subjects.
4. Discussion The current study demonstrates that administration of the dopamine antagonist haloperidol induces a dose dependent enhancement of FLI within basal ganglia output structures in the primate. Since Fos expression often appears to be correlated with neuronal activation w14,18,47x, these results are consistent with the theory that blockade of dopamine receptors leads to a stimulation of some neurons in the GPi and SNpr w1,2,20,36x. Although some minor differences are apparent, the overall pattern of staining seen here is very similar to that previously observed in rats w59x, suggesting that the mechanisms controlling neuroleptic induced Fos expression are basically similar in rodents and primates. We have shown previously that amphetamine also induces similar patterns of Fos expression in the striatum of rats and of primates w4x.
Given the binding profile of haloperidol, it is likely that the present findings reflect primarily a blockade of D 2 like dopamine receptors. This view is supported by observations that the selective D 1 antagonist SCH-23390 has little effect on nigral Fos expression in rats, whereas a clear response is seen following injections of metoclopramide, which binds to D 2 , but not D 1 , receptors w59x. A role for D 2 like receptors is also supported by the finding that quinpirole is able to antagonize reserpine induced nigral Fos expression in rats w21x. Since substantial data indicate that D 2 receptors are concentrated on striatal neurons projecting to the external segment of the globus pallidus w22,23,31x, the effects observed here in the SNpr and GPi may have been mediated ‘indirectly’ through alterations in the activity of GPe neurons. In support of this suggestion, we have observed in other studies that microinjection of muscimol into the globus pallidus of rats induces an extremely intense expression of FLI in the SNpr and EPN w58x. In monkeys injected with haloperidol, Fos expression was pronounced in the medial region of the rostral GPi, but labeling was rarely seen in the central and lateral portions of this structure, except for a band of immunoreactive neurons found along the external and ventral borders of the nucleus. Little or no labeling was seen in any portion of the nucleus at more caudal levels. These results demonstrate that the primate GPi cannot be viewed as a homogenous structure in terms of its response to neuroleptics. Moderate numbers of cholinergic neurons have been shown to be present along the lateral and ventral borders of the primate GPi w15,34,50x, but our current failure to observe FLI in these cells suggests that they are not major contributors to the pattern of Fos expression. It is tempting to speculate that the mediolateral differences in Fos expression observed here might be related to the subdivision of the GPi into medial and lateral components, separated by the ‘incomplete medullary lamina’, which is present in the brains of some primates, including man w35x. Clinical and experimental studies have indicated that chronic dopamine depletion is associated with increases in the firing rate of some GPi cells w8,20,36x and it is interesting, in view of the above suggestion, that studies in Parkinsonian patients have suggested that firing rates are higher in the medial than in the lateral segment of the GPi w33x. Recent brain stimulation studies in Parkinsonian patients have also led to the suggestion of a functional differentiation of the medial and lateral segments of the GPi w30x. Haloperidol-induced FLI was also unevenly distributed in the SN. Labeling was maximal in the medial portion of the pars reticulata and only a small number of labeled cells could be seen within the pars compacta. Some of the cells in the medial pars compacta were also immunoreactive for tyrosine hydroxylase and were, therefore, presumably dopaminergic, but these neurons represented only a small proportion of the total population of labeled cells. It is interesting that double labeled nigral cells were typically
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concentrated in small ‘clumps’, an observation which may be related to other reports in primates of clustering of pars compacta neurons with similar connections or staining properties w26,40x. The Fos expression observed in these neurons may be related to the ability of acute neuroleptic treatment to increase the firing rates of dopaminergic cellsw10x. In the rat, we were unable to detect Fos expression in dopaminergic nigral neurons w59x, although, in these animals, as in the macaques used here, double labeled cells were fairly common within the VTA. In macaques, as in rodents w59x, blockade of dopamine receptors by haloperidol induces detectable expression of the Fos protein in only a small proportion of cells present in either the GPi and SNpr. The inconsistent nature of this response suggests that haloperidol may not affect the pattern of synaptic input to all GPi and SNpr cells in a uniform fashion and that, as a result, the types of alterations necessary to induce Fos expression may only be produced in a subpopulation of nigral and pallidal cells. Although many models have assumed that basal ganglia output cells respond to alterations in dopamine transmission in a homogenous fashion, single unit studies have demonstrated that the responses of these cells are often surprisingly variable w37,38,55x, perhaps as a result of local circuit interactions w13,37x. It is also possible that there may be variability in the extent to which different cells in the GPi and SNpr are able to express the Fos protein in response to these alterations. Although interference with dopaminergic transmission has been reported to increase unit activity in the subthalamic nucleus w24,36,53x, haloperidol did not induce detectable Fos expression in this structure in the current studies. Similar results have been reported in rats where subthalamic Fos expression cannot be induced by treatment with either neuroleptics or reserpine w11,59x. These failures do not reflect a general inability of STN cells to synthesize the Fos protein, since clear Fos expression can be induced in this structure by a variety of treatments in both rats and primates w16,27,41,45,54,58x. It is possible that the time course of gene expression in the STN may be different from that of the other structures studied here with the effect that the post treatment time we examined may not have been optimal for demonstrating Fos induction in the STN. It is also possible that the inability of haloperidol to induce FLI in the STN may be related to the magnitude of the effects produced by this drug or to the specific molecular mechanisms through which its effects in the STN are produced. Consistent with other reports w29x, these results further emphasize that an absence of immediate early gene expression cannot be taken as evidence of an absence of neuronal excitation. In agreement with studies in rats w46,51x, haloperidol treatment induced Fos expression in a modest number of cells in the GPe. These results are consistent with electrophysiological data indicating that GPe cells show heterogeneous responses to dopamine blockade w28x, rather than
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uniformly slowing their rates as might be predicted by simplified models of basal ganglia organization. The excitation of GPe cells produced by dopamine blockade could be mediated through a number of mechanisms including activation of STN neurons projecting to the GP w17,32x or blockade of dopamine receptors located within the pallidum itself w6x. It seems likely that the Fos expression observed in the GPe reflects neuronal excitation, but further studies will be necessary to address this issue. Many workers have suggested that immediate-early genes, including c-fos, may play an important role in neuronal plasticity w25,49x. The ability of neuroleptics to induce FLI in the pallidum and nigra, therefore, raises the possibility that these drugs might induce long-term changes in the functioning of cells within these regions. Chronic interference with dopaminergic transmission may, of course, lead to tardive dyskenisia as well as to alterations in responsiveness to dopamine agonists. Although most workers have tended to view these conditions as reflections of abnormalities in the functioning of the striatum itself, the current results suggest that alterations in cells within the GPi and SNpr may play a role as well. Acknowledgements DW was supported, in part, by NIH NS33992. References w1x R.L. Albin, A.B. Young, J.B. Penny, The functional anatomy of basal ganglia disorders, TINS 12 Ž1989. 366–375. w2x G.E. Alexander, M.D. Crutcher, Functional architecture of basal ganglia circuits: neural substrates of parallel processing, TINS 13 Ž1989. 266–271. w3x G. Arbuthnott, N. MacLeod, A. Ryman, The effects of dopamine loss on the output from basal ganglia in the rat, J. Physiology 358 Ž1984. 43. w4x K.E. Asin, D. Wirtshafter, A. Nikkel, Amphetamine induces Fos-like immunoreactivity in the striatum of primates, Brain Res. 719 Ž1996. 138–142. w5x B.D. Bennett, J.P. Bolam, Localization of calcium binding proteins in the neostriatum, in: G. Percheron, J.S. McKenzie, J. Feger ŽEds.., The Basal Ganglia IV, Plenum, New York, 1994, pp. 21–34. w6x M.L. Bouthenet, M.P. Martres, N. Sales, J.C. Schwartz, A detailed mapping of dopamine D-2 receptors in rat central nervous system by autoradiography with w125Ixiodosulpride, Neuroscience 20 Ž1987. 117–155. w7x J.M. Brotchie, I.J. Mitchell, M.A. Sambrook, A.R. Crossman, Alleviation of parkinsonism by antagonism of excitatory amino acid transmission in the medial segment of the globus pallidus in rat and primate, Mov. Disord. 6 Ž1997. 133–138. w8x P. Burbaud, C. Gross, A. Benazzouz, M. Coussemacq, B. Bioulac, Reduction of apomorphine-induced rotational behavior by subthalamic lesion in 6-OHDA lesioned rats is associated with a normalization of firing rate and discharge pattern of pars reticulata neurons, Exp. Brain Res. 105 Ž1995. 48–58. w9x G. Chevalier, J.M. Deniau, Disinhibition as a basic process in the expression of striatal functions, TINS 13 Ž1990. 277–285. w10x L.A. Chiodo, Dopamine-containing neurons in the mammalian central nervous system: electrophysiology and pharmacology, Neurosci. Biobehav. Rev. 12 Ž1988. 49–91.
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dopamine system activation on substantia nigra pars reticulata output neurons: variable single-unit responses in normal rats and inhibition in 6-hydroxydopamine-lesioned rats, J. Neurosci. 4 Ž1984. 2369– 2375. M. Weiser, H. Baker, T.C. Wessel, T.H. Joh, Axotomy-induced differential gene induction in neurons of the locus coeruleus and substantia nigra, Mol. Brain Res. 17 Ž1993. 319–327. D. Wirtshafter, Haloperidol and clozapine induce Fos-like immunoreactivity in the primate basal ganglia, Soc. Neurosci. Abstr. 22 Ž1996. 1087. D. Wirtshafter, Fos expression in the basal ganglia following pharmacological inactivation of the globus pallidus, Soc. Neurosci. Abstr. 23 Ž1997. 467. D. Wirtshafter, K.E. Asin, Dopamine antagonists induce Fos-likeimmunoreactivity in the substantia nigra and entopeduncular nucleus of the rat, Brain Res. 670 Ž1995. 205–214. D. Wirtshafter, K.E. Asin, M.R. Pitzer, Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula, Brain Res. 633 Ž1994. 21–26. J. Yelnik, G. Percheron, C. Francois, A. Garnier, Cholinergic neurons of the rat and primate striatum are morphologically different. in: G.W. Arbuthnott, P.C. Emson ŽEds.., Progress in Brain Research. Elsevier, New York, 1993, pp. 25–34. B. Zadow, W.J. Schmidt, Lesions of the entopeduncular nucleus and the subthalamic nucleus reduce dopamine receptor antagonist-induced catalepsy in the rat, Behav. Brain Res. 62 Ž1994. 71–79.
Research report
Haloperidol induces Fos expression in the globus pallidus and substantia nigra of cynomolgus monkeys David Wirtshafter a
a, )
, Karen E. Asin
b
Department Psychology, M r C 285, The UniÕersity of Illinois at Chicago, 1007 W. Harrison St. Chicago, IL 60607-7137, USA b Department Toxicology, Abbott Laboratories, D468, Bldg. AP13A, Abbott Park, IL 60604, USA Accepted 27 April 1999
Abstract Systemic injections of the dopamine antagonist haloperidol Ž0.1–2.5 mgrkg. induced a dose dependent increase in Fos-like immunoreactivity ŽFLI. in the internal segment of the globus pallidus ŽGPi. and in the substantia nigra ŽSN. of cynomolgus monkeys. These findings are consistent with models of basal ganglia organization which predict that blockade of dopamine receptors should result in a disinhibition of cells in these structures. In the GPi, labeling was most pronounced along the ventral, lateral and medial borders of the nucleus and none of the pallidal cells expressing FLI were immunopositive for choline acetyltransferase. In the SN, immunoreactive nuclei were concentrated in the pars reticulata and the majority of labeled nigral neurons did not display tyrosine hydroxylase-like immunoreactivity. A small number of cells displaying FLI were also observed in the external pallidal segment, but no labeling was seen in the subthalamic nucleus. These findings indicate that blockade of dopamine receptors induces a characteristic pattern of Fos expression in the primate brain which strongly resembles that previously reported in rodents. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Immediate-early genes; c-fos; Subthalamic nucleus; Neuroleptic; Dopamine; Primate
1. Introduction The internal segment of the globus pallidus ŽGPi, entopeduncular nucleus ŽEPN. of rodents. and the pars reticulata of the substantia nigra ŽSNpr. are the major output nuclei of the basal ganglia and have been proposed to inhibit motor processing by means of GABAergic projections to the midbrain tegmentum, the intermediate and deep layers of the superior colliculus and several thalamic nuclei w9,19,39,44x. It has been suggested by a number of workers that interference with dopaminergic transmission increases the activity of cells within the GPi and SNpr, resulting in an inhibition of thalamic and brainstem motor structures and a consequent reduction in motor output w1,2,20,36x. Increases in the activity of GPi neurons have been directly demonstrated in monkeys treated with the dopamine depleting neurotoxin MPTP w20,36x and a similar effect may occur in human Parkinsonian patients w8x. Interference with dopamine transmission has also been reported to increase the firing rates of SNpr cells w8x,
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although this result has not always been obtained w3,38,53x. Furthermore, the parkinsonian symptoms induced by neuroleptic treatment or dopamine depletion can be attenuated by lesions of the GPirEPN or the SNpr or by injections of inhibitory compounds into these structures w7,12,48,52,62x. Several recent studies have examined the effects of disrupting dopaminergic transmission on the expression of the proto-oncoprotein Fos within output structures of the basal ganglia. We were able to demonstrate, for example, that injections of the dopamine antagonists haloperidol and metoclopramide in rats produce a modest but clear expression of Fos-like immunoreactivity ŽFLI. within both the SNpr and the EPN w59x, and similar results have recently been reported in mice w42x. Fos expression within the SNpr and EPN regions can also be induced by injections of the dopamine depleting drug reserpine w11,21x, and this effect in the EPN can be blocked by pretreatment with the D 2-like dopamine agonist quinpirole w21x. Increased Fos expression within the SNpr of rats has also been reported following acute lesions of the ascending dopamine fibers at the level of the lateral hypothalamus w56x. Since Fos expression often appears to be correlated with elevations in the firing rate of neurons w14,18,47x, these results are compatible with the theory that reductions in dopamine
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 5 5 0 - 4
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transmission result in increases in the activity of some SNpr and EPN cells. Interest in the basal ganglia reflects in large part the clinical importance of these nuclei. To date, the effects of dopamine depletion or blockade on Fos expression in basal ganglia output structures have been examined only in rodents. Given the central role that alterations in the activity of the GPi and SNpr play in current theories of the pathophysiology of Parkinson’s disease and other basal ganglia disorders, it is important to determine whether similar effects also occur in primates. Although rodents have frequently provided useful models for the study of the basal ganglia, extrapolation from results obtained in these animals to humans and other primates obviously requires great caution since a number of differences in the anatomy and neurochemistry of the basal ganglia have been described between members of these two orders Že.g., Refs. w5,43,61x.. In the current experiments we therefore examined whether systemic injections of the dopamine antagonist haloperidol are able to induce FLI in the GP and SNpr of cynomolgus monkeys. Some of the current results have been presented in abstract form w57x. 2. Methods and materials 2.1. Subjects Subjects were nine adult cynomolgus monkeys Ž Macacca fascicularis. obtained from Charles Rivers ŽHouston, TX.. Subjects had previously been used in pharmacological studies involving nondopaminergic agents, but had not been given any drugs for at least a month prior to the current study. Animals were individually housed. 2.2. Drug administration and perfusion Subjects received intramuscular injections of haloperidol ŽHaldol w injectable formulation. at doses of either 0.1 mgrkg Ž N s 2., 0.5 mgrkg Ž N s 3. or 2.5 mgrkg Ž N s 2.. Two additional animals were injected with distilled water. Three hours following these injections subjects were anesthetized with ketamine Ž10 mgrkg, i.m.., followed by 50 mgrkg of Nembutal w ŽAbbott Laboratories. given through the saphenous vein. The thorax was then opened and the descending aorta clamped; animals were then perfused transcardially with normal saline Ž500 ml. followed by a 10% solution of formalin prepared in phosphate buffer Ž1500 ml.. Brains were removed from the skulls, blocked into several pieces and post-fixed for 2 h after which time they were transferred to a solution of 20% sucrose in phosphate buffered saline ŽPBS. where they were stored for two days at 48C. 2.3. Immunocytochemistry Thirty-micrometer cryostat sections were taken through the extent of the globus pallidus and the substantia nigra
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and processed for Fos-like immunoreactivity ŽFLI. using methods we have described in detail elsewhere w59,60x. The primary antibody in these studies was a sheep anti-Fos serum obtained from Cambridge Research Biochemicals ŽWilmington, DE; OA-11-823. which was used at a dilution of 2000 = in PBS containing 0.2% Triton-X100, 0.05% sodium azide and 2% blocking serum ŽPBSTA.. Except as noted below, the secondary antibody was a biotinylated rabbit anti-sheep serum ŽVector Laboratories, Burlingame, CA. used at a dilution of 200 = . Antibodies were detected using a Vectastain Elite ABC kit employing nickel intensified diaminobenzadine ŽDAB. as the chromogen. No staining was seen in control sections in which the primary antibody was either omitted or preadsorbed with the antigenic fragment of the Fos protein ŽCambridge Research Biochemicals, OP-11-3210, 4 mgrml.. Some sections were processed by a sequential double labeling technique which allowed for the simultaneous visualization of FLI and either choline acetyltransferaseŽChAT. or tyrosine hydroxylase- like immunoreactivity. In these sections, a biotinylated donkey anti-sheep antibody preadsorbed with rabbit IgGs ŽJackson Immuno Research, West Grove, PA; 500 = . was used as the secondary antibody for the detection of Fos. After processing for FLI, using intensified DAB, was complete, the sections were thoroughly rinsed in PBS and then incubated for 48 h at 48C in either a rabbit anti ChAT serum ŽChemicon, Temecula, CA; AB143; 2000 = . or a rabbit anti-tyrosine hydroxylase serum ŽEugene Tech International, Allendale, NJ, TE 101; 6000 = .. Both antibodies were prepared in PBSTA containing 2% normal donkey serum. A biotinylated donkey anti-rabbit antibody preadsorbed with sheep IgGs ŽJackson, 500 = . was used as the secondary antibody and detection was accomplished using a Vectastain Elite ABC kit employing unintensified DAB as the chromogen.
3. Results Very few immunoreactive neurons could be seen within the basal ganglia of monkeys injected with water; in contrast, clear staining was apparent within the globus pallidus and substantia nigra of animals injected with haloperidol. Fig. 1. displays a camera lucida drawing of the SN and adjacent structures from a representative subject injected with 0.5 mgrkg of haloperidol. Cells expressing FLI were scattered throughout the SNpr, except for its most lateral regions. The numbers of cells in the SNpr displaying FLI were counted, with the aid of a camera lucida, in one section per animal, at a level similar to that of Fig. 1 in a region extending 1 mm lateral from the medial border of the SN. The results of this analysis are shown in Fig. 2 where it can be seen that haloperidol induced a dose dependent increase in the number of immunoreactive nigral cells Ž F Ž3,5. s 10.3; p - .02.. Further analysis by
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Fig. 1. A camera lucida drawing showing the distribution of Fos immunoreactive cells in the substantia nigra and adjacent regions of a monkey treated with 0.5 mgrkg of haloperidol. The approximate border of the pars compacta is indicated based on examination of adjacent Nissl stained sections. The tendency of immunoreactive cells to be confined to the medial portion of the SNpr was less pronounced in this subject than in many of the others we examined. Abbreviations: CPs cerebral peduncle; OT s optic tract; STNssubthalamic nucleus.
means of polynomial contrasts indicated the presence of a significant first order Žlinear. relation between drug dose and Fos expression Ž F Ž1,5. s 30.4; p - .01.. Although labeling was largely restricted to the pars reticulata, small clusters of immunoreactive nuclei could be observed in some sections within the medial portion of the pars compacta. These nuclei were typically smaller, more lightly stained, and more closely packed than were those seen in the SNpr ŽFig. 3.. Immunoreactive nuclei were also pre-
Fig. 3. Photomicrographs showing Fos immunoreactive nuclei in the medial region of the internal segment of the globus pallidus ŽA., the pars reticulata of the substantia nigra ŽB. and the pars compacta of the substantia nigra ŽC.. Scale bar s100 mm.
Fig. 2. Mean numbers of Fos immunoreactive cells found in the medial substantia nigra pars reticulata for monkeys receiving various doses of haloperidol. ‘Error bars’ represent the observed range of scores.
sent in the ventral tegmental area of both vehicle and haloperidol treated monkeys, although they appeared to be more frequent in drug treated animals. Examination of material processed for both FLI and tyrosine hydroxylase-like immunoreactivity indicated that the great majority of Fos immunoreactive cells seen in the SN did not stain for tyrosine hydroxylase. A small clump of double labeled cells could occasionally be seen, however, within the medial portion of the pars compacta ŽFig. 4a.. Double labeled neurons were relatively common in the ventral tegmental area ŽFig. 4b.. Treatment with either of the two higher doses of haloperidol induced clear FLI within the GPi ŽFigs. 3 and 5.. The general appearance of staining in this region was
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Fig. 4. Photomicrographs of sections processed for the simultaneous detection of Fos like immunoreactivity and tyrosine hydroxylase activity. Panel ŽA. displays a cluster of double labeled cells ŽU . in the pars compacta of the substantia nigra whereas panel ŽB. shows a double labeled neuron in the VTA. Scale bar s 100 mm in panel ŽA. and 45 mm in panel ŽB..
very similar to that observed in the SNpr ŽFig. 3.. Labeling was restricted to the rostral portions of the GPi in all animals and, even at these levels, was not evenly distributed ŽFig. 5.. A narrow band of immunoreactive neurons was typically observed within and just medial to the internal medullary lamina of the GP and labeling continued from this region along the ventral and, at far rostral levels, the dorsal border of the GPi. At the rostral tip of the GPi little labeling was seen in the internal portions of this structure, and immunoreactive cells appeared to form an annulus outlining the borders of the nucleus. At slightly
more caudal levels, however, labeled cells filled much of the medial half of the GPi ŽFig. 5.. Labeling in the region of the internal medullary lamina was usually separated from that along the medial border of the pallidum by a region in the lateral half of the GPi which was virtually devoid of immunoreactive nuclei. Scattered labeling was also seen in GPe and very heavy labeling was apparent in the caudate nucleus and putamen at the level of the GP. Tissue from pallidal region of two monkeys, one injected with the high and one with the middle dose of haloperidol, was processed for the simultaneous demon-
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Fig. 5. A camera lucida drawing showing the distribution of Fos like immunoreactivity in the globus pallidus of a monkey injected with 0.5 mgrkg of haloperidol. Large numbers of labeled cells were also present within the putamen, but are not shown in the diagram. Abbreviations: GPesexternal segment of the globus pallidus; GPis internal segment of the globus pallidus; PUT s putamen.
stration of FLI and choline acetyltransferase-like immunoreactivity ŽChAT.. Examination of these sections indicated that Fos immunoreactive cells in the ventral portion of the GPi were located immediately dorsal to the cholinergic neurons of the nucleus basalis complex Žthe Ch4id sector of Mesulam et al. w34x. and virtually no overlap in the distributions of the two cell types was present. Scattered ChAT positive cells were also observed in the internal medullary lamina of the pallidum; although these neurons were partially intermingled with those expressing FLI, no double labeled cells could be detected. In marked contrast to the labeling seen in other basal ganglia structures, no immunoreactive cells were observed in the subthalamic nucleus in any of the subjects.
4. Discussion The current study demonstrates that administration of the dopamine antagonist haloperidol induces a dose dependent enhancement of FLI within basal ganglia output structures in the primate. Since Fos expression often appears to be correlated with neuronal activation w14,18,47x, these results are consistent with the theory that blockade of dopamine receptors leads to a stimulation of some neurons in the GPi and SNpr w1,2,20,36x. Although some minor differences are apparent, the overall pattern of staining seen here is very similar to that previously observed in rats w59x, suggesting that the mechanisms controlling neuroleptic induced Fos expression are basically similar in rodents and primates. We have shown previously that amphetamine also induces similar patterns of Fos expression in the striatum of rats and of primates w4x.
Given the binding profile of haloperidol, it is likely that the present findings reflect primarily a blockade of D 2 like dopamine receptors. This view is supported by observations that the selective D 1 antagonist SCH-23390 has little effect on nigral Fos expression in rats, whereas a clear response is seen following injections of metoclopramide, which binds to D 2 , but not D 1 , receptors w59x. A role for D 2 like receptors is also supported by the finding that quinpirole is able to antagonize reserpine induced nigral Fos expression in rats w21x. Since substantial data indicate that D 2 receptors are concentrated on striatal neurons projecting to the external segment of the globus pallidus w22,23,31x, the effects observed here in the SNpr and GPi may have been mediated ‘indirectly’ through alterations in the activity of GPe neurons. In support of this suggestion, we have observed in other studies that microinjection of muscimol into the globus pallidus of rats induces an extremely intense expression of FLI in the SNpr and EPN w58x. In monkeys injected with haloperidol, Fos expression was pronounced in the medial region of the rostral GPi, but labeling was rarely seen in the central and lateral portions of this structure, except for a band of immunoreactive neurons found along the external and ventral borders of the nucleus. Little or no labeling was seen in any portion of the nucleus at more caudal levels. These results demonstrate that the primate GPi cannot be viewed as a homogenous structure in terms of its response to neuroleptics. Moderate numbers of cholinergic neurons have been shown to be present along the lateral and ventral borders of the primate GPi w15,34,50x, but our current failure to observe FLI in these cells suggests that they are not major contributors to the pattern of Fos expression. It is tempting to speculate that the mediolateral differences in Fos expression observed here might be related to the subdivision of the GPi into medial and lateral components, separated by the ‘incomplete medullary lamina’, which is present in the brains of some primates, including man w35x. Clinical and experimental studies have indicated that chronic dopamine depletion is associated with increases in the firing rate of some GPi cells w8,20,36x and it is interesting, in view of the above suggestion, that studies in Parkinsonian patients have suggested that firing rates are higher in the medial than in the lateral segment of the GPi w33x. Recent brain stimulation studies in Parkinsonian patients have also led to the suggestion of a functional differentiation of the medial and lateral segments of the GPi w30x. Haloperidol-induced FLI was also unevenly distributed in the SN. Labeling was maximal in the medial portion of the pars reticulata and only a small number of labeled cells could be seen within the pars compacta. Some of the cells in the medial pars compacta were also immunoreactive for tyrosine hydroxylase and were, therefore, presumably dopaminergic, but these neurons represented only a small proportion of the total population of labeled cells. It is interesting that double labeled nigral cells were typically
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concentrated in small ‘clumps’, an observation which may be related to other reports in primates of clustering of pars compacta neurons with similar connections or staining properties w26,40x. The Fos expression observed in these neurons may be related to the ability of acute neuroleptic treatment to increase the firing rates of dopaminergic cellsw10x. In the rat, we were unable to detect Fos expression in dopaminergic nigral neurons w59x, although, in these animals, as in the macaques used here, double labeled cells were fairly common within the VTA. In macaques, as in rodents w59x, blockade of dopamine receptors by haloperidol induces detectable expression of the Fos protein in only a small proportion of cells present in either the GPi and SNpr. The inconsistent nature of this response suggests that haloperidol may not affect the pattern of synaptic input to all GPi and SNpr cells in a uniform fashion and that, as a result, the types of alterations necessary to induce Fos expression may only be produced in a subpopulation of nigral and pallidal cells. Although many models have assumed that basal ganglia output cells respond to alterations in dopamine transmission in a homogenous fashion, single unit studies have demonstrated that the responses of these cells are often surprisingly variable w37,38,55x, perhaps as a result of local circuit interactions w13,37x. It is also possible that there may be variability in the extent to which different cells in the GPi and SNpr are able to express the Fos protein in response to these alterations. Although interference with dopaminergic transmission has been reported to increase unit activity in the subthalamic nucleus w24,36,53x, haloperidol did not induce detectable Fos expression in this structure in the current studies. Similar results have been reported in rats where subthalamic Fos expression cannot be induced by treatment with either neuroleptics or reserpine w11,59x. These failures do not reflect a general inability of STN cells to synthesize the Fos protein, since clear Fos expression can be induced in this structure by a variety of treatments in both rats and primates w16,27,41,45,54,58x. It is possible that the time course of gene expression in the STN may be different from that of the other structures studied here with the effect that the post treatment time we examined may not have been optimal for demonstrating Fos induction in the STN. It is also possible that the inability of haloperidol to induce FLI in the STN may be related to the magnitude of the effects produced by this drug or to the specific molecular mechanisms through which its effects in the STN are produced. Consistent with other reports w29x, these results further emphasize that an absence of immediate early gene expression cannot be taken as evidence of an absence of neuronal excitation. In agreement with studies in rats w46,51x, haloperidol treatment induced Fos expression in a modest number of cells in the GPe. These results are consistent with electrophysiological data indicating that GPe cells show heterogeneous responses to dopamine blockade w28x, rather than
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uniformly slowing their rates as might be predicted by simplified models of basal ganglia organization. The excitation of GPe cells produced by dopamine blockade could be mediated through a number of mechanisms including activation of STN neurons projecting to the GP w17,32x or blockade of dopamine receptors located within the pallidum itself w6x. It seems likely that the Fos expression observed in the GPe reflects neuronal excitation, but further studies will be necessary to address this issue. Many workers have suggested that immediate-early genes, including c-fos, may play an important role in neuronal plasticity w25,49x. The ability of neuroleptics to induce FLI in the pallidum and nigra, therefore, raises the possibility that these drugs might induce long-term changes in the functioning of cells within these regions. Chronic interference with dopaminergic transmission may, of course, lead to tardive dyskenisia as well as to alterations in responsiveness to dopamine agonists. Although most workers have tended to view these conditions as reflections of abnormalities in the functioning of the striatum itself, the current results suggest that alterations in cells within the GPi and SNpr may play a role as well. Acknowledgements DW was supported, in part, by NIH NS33992. References w1x R.L. Albin, A.B. Young, J.B. Penny, The functional anatomy of basal ganglia disorders, TINS 12 Ž1989. 366–375. w2x G.E. Alexander, M.D. Crutcher, Functional architecture of basal ganglia circuits: neural substrates of parallel processing, TINS 13 Ž1989. 266–271. w3x G. Arbuthnott, N. MacLeod, A. Ryman, The effects of dopamine loss on the output from basal ganglia in the rat, J. Physiology 358 Ž1984. 43. w4x K.E. Asin, D. Wirtshafter, A. Nikkel, Amphetamine induces Fos-like immunoreactivity in the striatum of primates, Brain Res. 719 Ž1996. 138–142. w5x B.D. Bennett, J.P. Bolam, Localization of calcium binding proteins in the neostriatum, in: G. Percheron, J.S. McKenzie, J. Feger ŽEds.., The Basal Ganglia IV, Plenum, New York, 1994, pp. 21–34. w6x M.L. Bouthenet, M.P. Martres, N. Sales, J.C. Schwartz, A detailed mapping of dopamine D-2 receptors in rat central nervous system by autoradiography with w125Ixiodosulpride, Neuroscience 20 Ž1987. 117–155. w7x J.M. Brotchie, I.J. Mitchell, M.A. Sambrook, A.R. Crossman, Alleviation of parkinsonism by antagonism of excitatory amino acid transmission in the medial segment of the globus pallidus in rat and primate, Mov. Disord. 6 Ž1997. 133–138. w8x P. Burbaud, C. Gross, A. Benazzouz, M. Coussemacq, B. Bioulac, Reduction of apomorphine-induced rotational behavior by subthalamic lesion in 6-OHDA lesioned rats is associated with a normalization of firing rate and discharge pattern of pars reticulata neurons, Exp. Brain Res. 105 Ž1995. 48–58. w9x G. Chevalier, J.M. Deniau, Disinhibition as a basic process in the expression of striatal functions, TINS 13 Ž1990. 277–285. w10x L.A. Chiodo, Dopamine-containing neurons in the mammalian central nervous system: electrophysiology and pharmacology, Neurosci. Biobehav. Rev. 12 Ž1988. 49–91.
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