Brain Research, 513 (1990) 43-59 Elsevier
43
BRES 15326
Afferent connections of the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat Newton S. Canteras, Sara J. Shammah-Lagnado, Bomfim A. Silva and Juarez A. Ricardo Department of Physiology and Biophysics, Institute of Biomedical Sciences of the University of Sao Paulo, Sao Paulo (Brazil)
(Accepted 29 August 1989) Key words: Subthalamic nucleus; Basal ganglia; Motor system; Horseradish peroxidase; Wheatgerm agglutinin; Afferent connection; Rat
A comprehensive characterization of the afferent connections of the subthalamic nucleus of Luys (STN) is a necessary step in the unraveling of extrapyramidal mechanisms. In the present study, the STN afferents in the rat were systematically investigated with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that, besides a massive input from the dorsal pallidum, the STN receives substantial projections from several districts of the cerebral cortex (the medial division of the prefrontal cortex, the first motor and primary somatosensory areas, and the granular insular territory), the ventral pallidum, the parafascicular nucleus of the thalamus and the pedunculopontine tegmental nucleus, as well as a modest innervation from the dorsal raphe nucleus. In spite of the fact that many additional structures were found to contain retrogradely labeled neurons after tracer injections in the STN, no other projection to the latter nucleus could be effectively established in our antero,grade experimental series. INTRODUCTION The subthalamic nucleus of Luys (STN) seems to play an important functional role in somatic motor regulation, as inferred from clinicopathological investigations in humans 6'15 and experimental studies in animals 15-17,34,63. Clinical evidence indicates that ballism, a particularly violent form of dyskinesia, is usually associated with damage of the STN or its circuitry (see ref. 6), a fact that underscores the interest of a precise knowledge of the hodological relationships of the STN for an understanding of the participation of this nucleus in motor mechanisms. The efferent connections of the STN have been thoroughly investigated in different species with both anterograde and retrograde tracing techniques 8,1°,36,53, 59,60,65,84 In line with the involvement of the STN in motor phenomena, these studies have shown that its major projections innervate conspicuously both segments of the globus pallidus and the substantia nigra. Thus, from an anatomical point of view, it appears that the STN is in a strategic position to modulate the main outflow channels of the basal ganglia, an inference corroborated by recent electrophysiological findings (see ref. 39). Some significant species differences in the organization of the STN efferents have also been pointed out (see ref. 59).
In contrast, a comprehensive picture of the STN afferents is still lacking. In fact, most of the available information on this issue derives from reports which have focused on a particular s~urce of input to the STN, such as the cerebral cortex '29'5°'67, the globus pallidus 911,35,45,50-52,54,85.86, the thalamic parafascicular nucleus 79, 81
, or the pedunculopontine tegmental nucleus 27,33,42, TO our knowledge, the sole systematic
50,57,72,75,80,88
attempt to present a general description of the sources of the STN afferent connections that has been published thus far is that by Rinvik et al. 66, who carried out a retrograde study in the monkey and cat, with the horseradish peroxidase (HRP) tracer technique. Their findings suggest the possibility that several other structures, besides the above-mentioned ones, could send projections to the STN; these structures include the central amygdaloid nucleus, the bed nucleus of the stria terminalis, the hypothalamus, the parabrachial complex, and the locus coeruleus. Studies in rats 83,88 have incidentally mentioned the presence of retrograde labeling in the locus coeruleus, the dorsolateral tegmental nucleus, the zona incerta and the thalamic reticular nucleus after fluorescent tracer deposits in the STN. Thus far, these observations have received little attention, and need to be carefully evaluated. In fact, considering that the STN, a small nucleus, is bordered by conspicuous fiber systems coursing in the cerebral peduncle or the field H 2 of Forel,
Correspondence: J.A. Ricardo, Departamento de Fisiologia e Biofisica, Instituto de Ci6ncias Biom6dicas da USP, Cidade Universit~iria, 05508 S~io Paulo, SP, Brazil.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
44 and that the above-mentioned tracers can be taken up by fibers-of-passage23'47'73, it is easy to understand that a considerable degree of uncertainty exists in the interpretation of the reported retrograde data. The precise origin
of the cortical input to the STN is another question that should be considered open: while anterograde anatomical investigations in various species 2"7"2~45"62 indicate that basically rostral cortical areas project to the referred
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45 n u c l e u s , r e c e n t e l e c t r o p h y s i o l o g i c a l e v i d e n c e in t h e rat 68 suggests that m o r e e x t e n s i v e t e r r i t o r i e s o f the c e r e b r a l cortex may influence the STN monosynaptically. In t h e light o f t h e s e c o n s i d e r a t i o n s , w e d e c i d e d , in t h e p r e s e n t study, to c o m b i n e r e t r o g r a d e and a n t e r o g r a d e tracing t e c h n i q u e s in a s y s t e m a t i c a t t e m p t to p r o v i d e a d e t a i l e d a c c o u n t of the S T N afferents in t h e rat, a species in w h i c h m u c h a n a t o m i c a l and physiological i n f o r m a t i o n o n t h e basal ganglia has b e e n o b t a i n e d 19. MATERIALS AND METHODS The present report is based on retrograde and anterograde experimental series carried out on adult albino female rats (weight: 170-200 g), several of which were part of a previous investigation7. All of the surgical procedures were performed under chloral hydrate anesthesia (400 mg/kg, i.p.). The retrograde technique was described in detail elsewhere 7. Briefly stated, microelectrophoretic unilateral deposits of either free HRP (n -- 6) or horseradish peroxidase conjugated with wheatgerm agglutinin (WGA-HRP) (n = 15) were placed stereotaxically in the STN. After a 48 h survival period, the animals were perfused
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transcardially according to procedure II of Rosene and Mesulam (see ref. 47) and the brain sections incubated with tetramethylbenzidine 47 and counterstained with Neutral red. A total of 99 rats were used in the anterograde experiments. In this series, many of the structures in which retrogradely labeled neurons had been consistently found after our STN injections received microelectrophoretic deposits of WGA-HRP: cortical districts (n = 31), the dorsal and ventral pallidum (n = 9 and n = 6, respectively), the striatum (n = 4), the zona incerta and fields of Forel (n = 1), the central amygdaloid nucleus (n = 2), the thalamic parafascicular nucleus (n = 1), the anterior pretectai nucleus (n = 3), the nuclei of the posterior commissure (n = 3), the superior colliculus (n = 3), the mesencephalic reticular formation (n = 3), the red nucleus (n = 3), the dorsal raphe nucleus (n = 3), the pedunculopontine tegmental nucleus (n = 3), the parabrachial complex (n = 3), dorso- and ventrolateral pontine areas (n = 3 and n = 2, respectively), the lateral nucleus of the cerebellum (n = 2), the principal sensory and spinal trigeminal nuclei (n = 5 and n = 3, respectively), and the dorsal column nuclei (n = 6). Cortical, striatal, cerebellar, trigeminal and dorsal column nuclei injections were intended to cover relatively extensive territories, and, therefore, multiple micropipette penetrations, spaced at 0.5-1 mm intervals from one another, were made in these cases. An angulated approach was used to reach the ventral paUidum, the striatum and the central amygdaloid nucleus in order to avoid any ipsilateral leakage of tracer along the pipette track in the dorsal pallidum
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Fig. 1. Chartings of selected frontal sections from case L-9 showing the distribution of the retrograde labeling in the structures indicated by the present anterograde experiments as sources of input to the STN. A photograph of the injection site of this case is seen in Fig. 2A. Each dot represents one labeled neuron. In sections H and I the black area represents the center of the injection site, whereas hatching denotes a region of less dense marker deposition.
46 and/or cerebral cortex. The iontophoretic parameters used in the anterograde studies ranged from 0.05 ILA for 20 s through a micropipette with an internal tip diameter of 9-13/~m to 0.6 eta for 5 min through a micropipette with an internal tip diameter of 27/~m. The experimental procedures adopted in the anterograde series were basically similar to the ones described for the retrograde group. It should be noted, however, that only WGA-HRP was employed for anterograde tracing, since it appears to be superior to free HRP for this purpose 47'82. Moreover, in the anterograde cases the survival time varied from 24 to 48 h. The brain sections were microscopically examined under both bright-field and dark-field illumination for the presence and location of either retrogradely labeled perikarya or anterogradely transported label. In both retrograde and anterograde series, the injection sites and the distribution of transported label were charted onto projection drawings of selected Nissl-stained sections.
present cases (Fig. 1 E - G ) ; the HRP-positive cells were of medium size and many of them appeared very heavily labeled. Some evidence suggesting the existence of a topographic organization in the projection from the dorsal pallidum to the STN could be found in the present material. Thus, injections centered in the rostral portion of the STN (cases L-9, herein illustrated, and L-11) led to a higher concentration of labeled cells in the ventrolateral sector of the dorsal pailidum (Fig. IF); in contrast, when the deposit of tracer involved basically the caudal
TABLE I
Summary of the present anterograde observations RESULTS
Retrograde labeling experiments In 6 animals (4 H R P experiments and 2 W G A - H R P cases) the injections were centered in the STN with only a peripheral involvement of the zona incerta and, to a lesser degree, the crus cerebri. Similar observations were made in all of the 6 rats. Although many structures consistently contained HRP-positive neuronal perikarya in most of these cases, it seems clear that only those whose projections to the STN are also indicated by anterograde findings can be effectively considered as supplying afferents to the STN. Therefore, only the territories indicated by the present companion series of anterograde experiments as sources of input to the STN will be considered in some detail in this section. These territories are districts of the cerebral cortex, the dorsal and ventral pallidum, and the thalamic parafascicular, the dorsal raphe and the p e d u n c u l o p o n t i n e tegmental nuclei. The retrograde labeling pattern observed in these structures is illustrated in Fig. 1, which shows charts of a typical case, L-9, whose injection site can be seen in Figs. 1 and 2A. In the cerebral cortex, the marked perikarya, most of which of pyramidal shape and situated in layer V, were found ipsilaterally. A m o n g cortical districts, the largest n u m b e r s of retrogradely labeled cells appeared in the first motor area (Fig. 1 A - E ) and the medial division of the prefrontal cortex 4°'64, chiefly in the medial precentral area, but also in the dorsal division of the anterior cingulate area (Fig. 1 A - E ) . A still substantial n u m b e r of marked perikarya were seen in the primary somatosensory cortex, mainly in its rostral half and in layer Vb (Fig. 1 C - E , G ) . Finally, a modest contingent of retrogradely labeled n e u r o n s were consistently observed in the granular insular area 12, a general visceral sensory cortex (Fig. 1D,E). The ipsilateral dorsal pallidum 25 contained, by far, the largest n u m b e r of marked perikarya observed in the
The density of the terminal field in the STN is indicated by the following symbols: + + + +, very high; + + +, high; + +, moderate; +, low. The symbol - indicates either absence or very few anterograde labeling in the STN, being noteworthy that structures adjacent to the latter nucleus generally appeared heavily marked. Contralateral projections are indicated in parentheses. The laterality of the projection from the dorsal raphe nucleus was inferred from retrograde data.
Explored structures
Telencephalon Cerebral cortex Dorsal division of the anterior cingulate area Medial precentral area First motor Primary somatosensory Granular insular area Retrosplenial (areas 29c and 29d) Primary visual Primary auditory Dorsal pallidum Ventral pallidum Striatum Central amygdaloid nucleus Diencephalon Thalamic parafascicular nucleus Zona incerta and fields of Forel Midbrain and isthmus region Anterior pretectal nucleus Nuclei of the posterior commissure Superior colliculus Mesencephalic reticular formation Red nucleus Dorsal raphe nucleus Pedunculopontine tegmental nucleus Parabrachial complex Dorsolateral pontine area (locus coeruleus, nucleus subcoeruleus and dorsolateral tegmental nucleus) Ventrolateral pontine area (A5 noradrenergic cell group) Pons Principal sensory trigeminal nucleus Cerebellum Nucleus lateralis Medulla oblongata Spinal trigeminal nucleus Dorsal column nuclei
Density of the terminalfield intheSTN
++ ++ +++ ++ + -++++ +++
+++
+ + (+) + + + (+)
47 portion of the STN (case L-30), the retrograde labeling was observed mainly in the dorsal district of the dorsal pallidum (Fig. 4A,B). The ipsilateral ventral paUidum showed a very modest number of marked neurons, which were usually faintly labeled (Fig. 1E). In the thalamic parafascicular nucleus a small number of retrogradely labeled cells with no preferential distribution were observed ipsilaterally; only in case L-9 the marked perikarya were numerous and clustered around
the fasciculus retroflexus (Fig. 1H,I). A moderate number of labeled neurons, with strong ipsilateral predominance, were seen in the dorsal raphe nucleus, chiefly at the level of and immediately caudal to the trochlear nucleus (Fig. 1J,K). Additionally, some marked cells were consistently noticed dorsolaterally to the dorsal raphe nucleus in the ventrolateral sector of the central gray substance, close to the mesencephalic aqueduct (Fig. 1J,K). The location of these cells corresponds
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Fig. 2. Bright-field photomicrographs of injection sites. A: STN (case L-9), HRP. B-P: the various territories which were observed to send projections to the STN in our anterograde series, WGA-HRP. B-D: first motor cortex. E: dorsal division of the anterior cingulate area. F: medial precentral area; G" primary somatosensory cortex. H,I: granular insular area. J-L: dorsal pallidum. M: ventral paUidum. N" thalamic parafascicular nucleus. O: dorsal raphe nucleus. P: pedunculopontine tegmental nucleus. The whole extent of cortical injection sites is represented in Fig. 3. Bar = 1 mm.
48
quite well to that of serotonin-positive neurons recently reported to extend beyond the cytoarchitectonic boundaries of the dorsal raphe nucleus in the ratTM. No
retrograde labeling was seen in the median raphc nucleus. The pedunculopontine tegmental nucleus, an ill-de-
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Fig. 3. Chartings of the anterograde labeling at three different rostrocaudal levels of the STN (rostrai to caudal is represented from left to right in each row) after the WGA-HRP injections represented on the left. The black areas represent the center of the injection sites, whereas hatching denotes regions of less dense marker deposit. Photomicrographs of the labeling observed at the intermediate STN level in some of these cases can be seen in Fig. 4C-G.
50 fined cell group closely associated with the ascending limb of the superior cerebellar peduncle, contained a considerable n u m b e r of m a r k e d perikarya. The labeling was observed bilaterally, the marked neurons on the ipsilateral side being twice as numerous as those on the contralateral side. The HRP-positive cells were found mainly within the cholinergic district of this nucleus, in both its diffuse and densely p o p u l a t e d portions 71, and, to a much lesser degree, in the non-cholinergic territory that has been called 'midbrain extrapyramidal area '71 (Fig. 1J,K). R e t r o g r a d e labeling - - generally m o d e r a t e or even
substantial - - was consistently noticed ipsilaterally in the areas 29c and 29d of the retrosplenial cortex, the areas 17, 18 and 18a of the visual cortex, the striatum, the central amygdaioid and the anterior pretectal nuclei, the deep layers of the superior colliculus, the parvocellular division of the red nucleus, the locus coeruleus, and the nucleus subcoeruleus, contralaterally in the zona incerta and fields of Forel, the large-celled division of the lateral nucleus of the cerebellum, a ventrolateral pontine district that appears to correspond to the A5 noradrenergic cell group, the principal sensory and the spinal trigeminal nuclei, and the dorsal column nuclei, and bilaterally in
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Fig. 4. A: bright-field photomicrograph of the dorsal pallidum of case L-30, in which a deposit of WGA-HRP was placed in the caudal portion of the STN. Note that most of the retrogradely labeled cells are found in the dorsal district of the dorsal pallidum. B: a higher magnification of the inset shown in A. C,E-G: dark-field photomicrographs showing anterograde labeling in the STN (at its intermediate rostrocaudal level) after WGA-HRP injections in the medial precentral area (C), granular insular area (E), dorsal pallidum (F), or ventral pallidum (G). D: bright-field photomicrograph of the anterograde labeling shown in E. Bars in A, 400 t~m; in B,C,E-G, 100/~m; in D, 20 urn.
51 the nuclei of the posterior commissure, the mesencephalic reticular formation, the parabrachial complex, and the dorsolateral tegmental nucleus. It is important to mention, however, that, in companion experiments, massive deposits of WGA-HRP centered in the abovementioned territories led to few if any anterograde transport in the STN, while surrounding s t r u c t u r e s either the zona incerta/fields of Forel, the hypothalamus or the substantia nigra - - generally appeared heavily marked. Thus, the possible existence of projections from these districts to the STN could not be established by the present observations. A few retrogradely labeled cells were observed ipsilaterally in the lateral and ventrolateral orbital cortical areas, the dorsal and posterior agranular insular cortical subdivisions, the prelimbic cortex, the ventral part of the anterior cingulate cortex, the secondary somatosensory cortical area, the entopeduncular nucleus, the substantia innominata, the ventromedial nucleus of the hypothalamus, the lateral hypothalamic area, the posterior pretectal nucleus, the interstitial nucleus of Cajal and the partes compacta and reticulata of the substantia nigra, and bilaterally in the nucleus of Darkschewitsch and the magno- and parvocellular districts of the pontomedullary reticular formation. The possibility that these structures might represent minor sources of input to the STN was not investigated in our anterograde experimental series.
Anterograde labeling experiments In order to confirm the existence of presumptive sources of input to the STN suggested by our retrograde data, and also in an attempt to study the pathways and terminal fields of these STN afferents, injections of the sensitive WGA-HRP anterograde tracer were made into many cortical and subcortical territories. The results of these anterograde experiments are summarized in Table I. Photomicrographs illustrating the location of representative injection sites in the diverse territories which were observed to send projections to the STN are shown in Fig. 2, and the correspondent chartings of the labeling pattern in the STN can be seen in Fig. 3. Cerebral cortex. The cortical injections were placed in either the first motor area, the medial division of the prefrontal cortex, the primary somatosensory, the granular insular, the temporal, the retrosplenial or the visual areas. None of these tracer deposits involved the underlying white matter. In each of these cases, labeled fibers left the respective injection site, coursed through the striatum, and, via the internal capsule and cerebral peduncle, reached the ipsilateral subthalamic region. It should be kept in mind that the terminal fields that were observed may not reflect the whole distribution of the corticosubthalamic pathways, because, in spite of multi-
ple micropipette penetrations, the tracer deposits failed to cover the explored cytoarchitectonic cortical districts in their totality. Eight rats received injections in the first motor area. In two of these cases the W G A - H R P deposit covered a relatively large anteroposterior extent of the referred cortical district, whereas in the 6 others the injection sites were smaller and located respectively in the rostralmost (n = 3), the rostromedial (n = 2) and the rostrolateral (n = 1) sectors of the primary motor area (Figs. 2B-D and 3). All of these districts innervated the STN substantially, but the pattern of labeling resulting from these injections differed to some extent, suggesting the existence of a coarse topographic arrangement within this corticosubthalamic projection along the anteroposterior dimension of the STN. Thus, the rostral deposits marked essentially the rostral two-thirds of the STN, whereas the more extensive injections led to an additional conspicuous labeling of the lateral half of the caudal third of the STN (Fig. 3). On the other hand, very similar terminal fields were observed in the STN in the rostromedial and rostrolateral cases. No labeling was seen in any of the present first motor cases in a small ventromedial sector of the STN at intermediate levels of the nucleus or in the medial half of the STN at caudal levels (Fig. 3). In 4 animals the injection sites were located in the medial division of the prefrontal cortex. The tracer deposits were either confined almost entirely to the dorsal division of the anterior cingulate area (n = 2) (Fig. 2E) or to the medial precentral cortex (n = 1) (Fig. 2F), or involved extensively both of these cortical districts (n = 1). In each of these cases a relatively substantial dust-like labeling was noticed in the caudal two-thirds of the STN, where the distribution of the labeling appeared to be, to a large extent, complementary to the abovedescribed pattern of the projection from the first motor area (Figs. 3 and 4C). As already reported in a previous publication 7, after multiple WGA-HRP deposits placed along the rostral half of the primary somatosensory cortex (n = 4) (Fig. 2G), a moderate amount of anterograde labeling was observed in the dorsolateral portion of the STN at the midrostrocaudal level of this nucleus (Fig. 3). In the present anterograde series an additional rat received a large injection centered in the caudal half of the primary somatosensory cortex. In this case, very few if any anterograde labeling appeared in the STN, a finding in good agreement with conclusions drawn from the retrograde set of experiments. Injections of WGA-HRP confined almost entirely to the granular insular cortex were obtained in 3 rats (Fig. 2H,I). Ipsilateral labeled fibers left the cerebral peduncle, traversed the lateral portion of the STN, and gave
52 rise to small varicosities in a region that encompassed a dorsomedial narrow strip of the STN and the immediately adjacent field H e of Forel and lateral hypothalamus (Figs. 3 and 4D,E). The light anterograde labeling found in this diencephalic region appeared to be somewhat more pronounced in the STN than in the adjacent districts. No conclusive evidence of anterograde transport in the STN was found after extensive W G A - H R P injections involving either the areas 29c and 29d of the retrosplenial cortex, the primary visual or the primary auditory cortices. Dorsal and ventral pallidum. Two animals received large W G A - H R P deposits in the dorsal pallidum (Fig. 2J). Issuing from the injection site, labeled fibers coursed in the internal capsule and cerebral peduncle and led to a very dense terminal field in the STN. Though dust-like anterograde transport filled the whole extent of the STN, a strip of higher density was observed in each of these cases; in the rostral STN, this strip is located in the ventral sector of the nucleus (not shown), but, as we move caudally, it is seen in gradually more dorsal STN districts (Figs. 3 and 4F). It is noteworthy that most of the STN retrogradely labeled cells were also found within this strip*. The analysis of cases with smaller dorsal pailidai injections further reinforced the retrograde evidence, reported in the preceding section, suggestive of the existence of some topographic organization in this pallidosubthalamic projection. Thus, when tracer deposits were confined to the dorsal sector of the dorsal pallidum (n = 1) (Fig. 2K) or were centered in the striatum or internal capsule but encroached on the dorsal portion of the dorsal pallidum (n = 2), the caudal district of the STN appeared more intensely marked than the rostral one (Fig. 3); a reverse pattern of anterograde labeling was observed in 2 cases with injections confined to the ventrolateral sector of the dorsal pallidum (Figs. 2L and 3). Tracer deposits in the ventral pailidum that apparently did not infringe on the dorsal pallidum were obtained in 2 rats. In each of these cases an appreciable W G A - H R P leakage was noticed along the pipette track in the septal region and the bed nucleus of the stria terminalis (Fig. 2M); it will be recalled, however, that none of these structures showed retrogradely marked cells following tracer deposits in the STN. From the injection site, labeled fibers reached the subthalamic region via the medial forebrain bundle and innervated conspicuously
the dorsomedial sector of the caudal two-thirds of the STN (Figs. 3 and 4G), Thalamic parafascicular nucleus. In one rat a large injection was placed in the thalamic parafascicular nucleus (Fig. 2N). Emerging from the injection site, labeled fibers crossed the subthalamic region and led to a relatively dense terminal field in the ipsilateral STN. The labeling was chiefly concentrated in the rostral two-thirds of the STN, but also marked lightly the lateral portion of the caudal STN (Fig. 3). Dorsal raphe nucleus. Injection sites involving bilaterally the dorsal raphe nucleus and the immediately adjacent central gray substance were obtained in 2 cases (Fig. 20). Labeled fibers, ascending in the medial forebrain bundle, were found to innervate a large extent of the STN, the labeling being somewhat preponderant in the dorsomedial sector of the nucleus. Pedunculopontine tegmental nucleus. Three animals received W G A - H R P deposits in the pedunculopontine tegmentai nucleus involving both its cholinergic district and the so-called 'midbrain extrapyramidal area '7J (Fig. 2P). From the injection site, labeled fibers ran in the ventral mesencephalic tegmentum in a region between the medial lemniscus and the substantia nigra and gave rise to a substantial and mainly ipsilateral terminal field throughout the whole extent of the STN (Fig. 3). DISCUSSION The present study represents the first systematic attempt to describe the afferent connections of the STN in a single species, the rat, using combined retrograde and anterograde tracing techniques. Our results suggest that, besides a massive input from the dorsal pallidum, the STN receives substantial projections from several districts of the cerebral cortex, the ventral pallidum, the parafascicular nucleus of the thalamus and the pedunculopontine tegmental nucleus, as well as a modest innervation from the dorsal raphe nucleus. These observations, in general, confirm those of other investigators and, in addition, reveal more details on the precise origin and terminal field of some of the STN afferents. A further point of interest of the present study is represented by some of our negative findings. Thus, retrograde labeling after tracer injections into the STN was observed in many structures (see Results) other than those mentioned above; a similar retrograde labeling had already been reported in the literature 45~66'~3'~ in the
* It should be kept in mind that coexistence of anterograde and retrograde labeling in the same structure seriously hampers any precise quantitative estimate of the density of the respective projections, since under these circumstances it is impossible to tell whether the fine dust observed is located in a dendrite or axon terminal. In our experimental series, such a situation occurred in the STN particularly after WGA-HRP injections in the dorsal and ventral pallidum.
53 case of some of these structures, such as the locus coeruleus, the dorsolateral tegmental nucleus, the parabrachial complex, and the central amygdaloid nucleus. It is noteworthy that the possibility that these territories could be the sources of direct projections to the STN could not be substantiated in the rat by the present anterograde data. Significantly, it was seen in our anterograde cases that WGA-HRP injections into the structures under consideration lead, in general, to heavy labeling of districts (the zona incertaJfields of Forel, the hypothalamus, or the substantia nigra) that are adjacent to the STN. It will be appreciated that, under these circumstances, a slight spread of the retrograde tracer beyond the confines of the small STN could easily result in labeling of neural districts that do not, in fact, give rise to STN afferents. Another conceivable source of some of the false negative retrograde findings (e.g., in the case of the striatum) would be the occurrence of transneuronal retrograde transport of tracer TM. Cerebral cortex
Corticosubthalamic projections have been shown to exert a tonic 69 and very powerful excitatory38,68 action on STN neurons; thus, the precise identification of the cortical territories that give rise to these projections is a matter of considerable interest. This is a controversial issue in the literature, since, contrary to anatomical studies 2'29'66, Rouzaire-Dubois and Scarnati 68 have suggested, in a recent electrophysiological investigation in the rat, that nearly the entire cortical mantle contributes afferents - - of bilateral origin - - to the STN. According to the present results, cortical projections to the STN are ipsilateral and arise mainly from the first motor area and districts of the medial division of the prefrontal cortex, but also from the primary somatosensory region and, to a much lesser degree, from the granular insular territory; on the other hand, temporal, visual and retrosplenial cortices do not appear to send fibers to the STN. In line with Rouzaire-Dubois and Scarnati's 68 electrophysiological findings in the rat, the present retrograde observations indicate that the cortical projections to the STN originate from layer V; in the cat, part of this system has been reported to represent collaterals of the pyramidal tract 22. It is noteworthy that these two features are exhibited also by the corticostriate pathway x8,46,88, the other conspicuous route that directly conveys cortical impulses to basal ganglia circuitry. The existence of a projection from the first motor area to the STN has been indicated in the rat with autoradiography 2, and in the c a t 45'62 and monkey 29,62,66 with both retrograde and anterograde tracing techniques; it has also been confirmed in the cat by electron microscopy5°'67. In agreement with the autoradiographic
study of Afsharpour 2 in the rat, the present observations indicate that this projection distributes to a large extent of the STN. However, whereas in Afsharpour's chartings (see Fig. 10 in ref. 2) the anterograde label spares the lateral half of the caudal STN, in our material it is the medial half of the caudal STN that appears devoid of labeling. In spite of a pronounced overlap of the STN terminal fields of the projections from different sectors of the primary motor cortex, Afsharpour's 2 data suggest the existence of a coarse topographic organization along the mediolateral dimension of this cortical area, whereas the present findings point to the existence of a topographic organization along the anteroposterior dimension of the first motor area. The above-mentioned discrepancies might perhaps be ascribed to different locations of the injection sites within the rat primary motor cortex. In contrast to the rat, in the monkey 29 this corticosubthalamic projection appears to be confined to the dorsolateral sector of the STN and to present a clear-cut topographic organization. It has been suggested that the projection under consideration could relay to STN information relative to the direction of movement 17. According to the present data, substantial projections to the medial sector of the caudal STN originate from both the medial precentral area and the dorsal division of the anterior cingulate area, districts that seem to correspond in part to the rat's frontal eye field (see e.g. refs. 43, 55 and 64). To our knowledge, only a projection from the medial precentral cortex to the STN had already been reported in autoradiographic studies in the rat2'64; the detailed description of the terminal field of this corticosubthalamic pathway made by Afsharpour 2 is in good agreement with the present anterograde observations. In the monkey, anterograde anatomical experiments 29,31 indicate that regions of the prefrontal cortex, as well as districts of the premotor cortex that include the frontal eye field, send fibers to the STN. Physiological studies 16 have described STN units whose activity was related to eye movements, and a neural link between the frontal eye field and the STN might be involved in the integration of visuo- and skeletomotor processes. In a previous publication 7 we have reported the existence of a direct projection from the primary somatosensory cortex to the STN in the rat. The present anterograde data further reinforce the idea that this pathway originates essentially from the rostral half of the referred cortical district. The existence of a modest cortical projection from the granular insular area to the STN is suggested by the present material. This cortical district appears involved in the regulation of numerous visceral functions 12. Although the presence of an important autonomic center has been recognized in a territory immediately adjacent to the
54 STN (see refs. 77 and 87), there is no evidence suggesting a participation of the latter nucleus itself in this functional domain 77. Interestingly, it was shown in a recent mapping study of the rat motor cortex 55 that part of the motor representation of the lower lip and tongue is located in the rostral insular territory. It seems conceivable, therefore, that the projection under consideration conveys to the STN information that may be relevant to the motor control of buccal and glossal musculature.
Basal ganglia The dorsal pallidum, the rodent homologue of the external segment of the globus pallidus of primates, is classically recognized as the major source of the STN afferents (see e.g. refs. 8-11, 35, 45, 52, 54, 85 and 86), and this has been confirmed once again in the present work. In the rat this pathway has been described with retrograde techniques using fluorescent compounds s3, 86.88 and also with autoradiography 1~'3°. Cytological and hodological evidence indicates that the dorsal pallidum is composed of heterogeneous cell populations 86. In line with the retrograde study of Van der Kooy and Kolb s6 in the rat, the present data suggest that dorsal pallidal efferents to the STN originate essentially from the medium size perikarya, which seem to be noncholinergic 86. It should be emphasized that, according to the present anterograde observations, this projection distributes to the whole extent of the STN, and not just to the medial half of the nucleus as previously reported by Carter and Fibiger 11 in an autoradiographic study in the rat. The short survival times and exposure periods used in their experiments, perhaps, or the location and size of their injection sites, may have led the referred authors to underestimate the extent of the terminal field of this pallidosubthalamic pathway. In the cat, autoradiographic evidence 45, supported by electron microscopic findings 5~, indicates that dorsal pallidal efferents terminate in the whole STN, though more conspicuously in the lateral two-thirds of the nucleus. In the monkey, however, this projection appears to be more restricted 9"35"54. It is noteworthy that the present cases suggest the existence of some topographic organization in this pallidosubthalamic projection in the rat. Thus, it appears from our retrograde and anterograde observations that the dorsal and ventrolateral sectors of the dorsal pallidum project predominantly to the caudal and rostral districts of the STN, respectively. In the cat, Fonnum et al. 2° noticed that a small lesion located in the dorsolateral region of the dorsal pallidum led to a reduction of glutamate decarboxylase in the lateral part of the caudal half of the STN. Autoradiographic data reported by Ricardo 65 indicate that, in the rat, the caudal portion of the STN innervates mainly the dorsal sector of the dorsal
pallidum. Furthermore, in the present material, after W G A - H R P deposits in the dorsal pallidum, the bulk of retrogradely labeled neurons were found specifically in the STN district that exhibited the densest dust-like anterograde transport. Taken together, these facts suggest that precisely organized feedback loops may exist between the dorsal pallidum and the STN. Recent ultrastructural studies in the rat ss and cat s° have described reciprocal connections between these two nuclei at a cellular level, i.e. axon terminals of dorsal pallidum neurons make synaptic contacts with STN cells that project back to the dorsal pallidum. In contrast, in the monkey 9 reciprocal relationships between these two structures seem to be only partial and not point for point. The projection from the ventral pailidum to the STN indicated by the present data had already been reported in the rat in a retrograde study with fluorescent tracers ~, as well as in anterograde autoradiographic 25 and Phaseolus vulgaris leucoagglutinin (PHA-L) z4 investigations. In good agreement with the findings of Haber et al. 2s, the present observations indicate that the ventral pailidal efferents terminate heavily in the dorsomedial sector of the caudal two-thirds of the STN. It is interesting to note that, according to the present anterograde data, the ventral pallidum and the medial division of the prefrontal cortex, two structures affiliated with the limbic system, innervate the same STN district. The present observations, in line with autoradiographic evidence in the rat 3°'83, do not support the existence of a direct projection from the striatum to the STN in this species; such a projection has been described in anatomical studies in the cat 47°, but not in the monkey 1°'35,59. It may be noted that the STN seems to send fibers to the striatum in the rat 36, cat 4'7° and monkey 53's9, but only in the latter species this projection appears to be a prominent one 59. In the present material, retrogradely labeled neurons were found in the entopeduncular nucleus and in the compacta and reticulata divisions of the substantia nigra after tracer injections into the STN. Considering that the bulk of the entopeduncular outflow courses in the field H 2 of Forel and through the STN, the existence of an entopeduncular input to the STN cannot be stated with certainty from the present observations, as had already been the case in previous anterograde 9'11'41'52'54 and retrograde 45'66'~3 tracing studies. However, the present data, in agreement with other retrograde investigations 45'66's3, indicate that such a projection, if present at all, is a very modest one. On the other hand, recent P H A - L evidence in the rat supports the existence of a substantial input from the pars reticulata of the substantia nigra to the STN37; a projection from the pars compacta of the substantia nigra to the STN is compatible with the presence of dopamin-
55 ergic varicosities in the latter nucleus 56. The STN in turn innervates heavily the entopeduncular nucleus and the reticulata and compacta divisions of the substantia nigra 8'1°'36'53'59'65'84. The intimacy of the STN relationships with dopaminergic mechanisms is further underscored by the fact that STN lesions with kainic acid induce spontaneous ipsiversive turning behavior34; moreover, some patients with hemiballism exhibit clinical improvement after administration of dopaminergic receptor antagonists 6.
Thalamic parafascicular nucleus A projection from the parafascicular nucleus of the thalamus to the STN has already been indicated with anterograde tracer techniques in the rat EL81 as well as in autoradiographic and retrograde HRP experiments in the cat 81, and confirmed by electron microscopic autoradiography in the rat 79. The present observations are in agreement with the detailed description of this pathway provided by Sugimoto el al. 8~. It is interesting to point out that, according to our data, the thalamic parafascicular nucleus and the primary motor cortex, two profusely interconnected structures 3,14,44, give rise to widely overlapping terminal fields in the STN. The intralaminar nucleus under discussion is also reciprocally connected with the striatum and the substantia nigra 14,zl,44, and represents a major target of the entopeduncular nucleus efferents (see e.g. refs. 8, 14 and 44). Taken as a whole, these hodological observations indicate a close participation of the thalamic parafascicular nucleus in somatic motor mechanisms. Interestingly, recent anatomical findings 14"58 suggest that this nucleus may channel influences from the reticular activating system to basal ganglia circuitry.
Dorsal raphe nucleus In line with the presence of serotonin-immunoreactive fibers in the S T N 49'78, retrogradely labeled neurons have been found in the dorsal raphe nucleus after HRP or fluorescent tracer deposits in the STN in the rat 88, cat 57"66 and monkey 66. The sole conclusive anterograde evidence of a dorsal raphe projection to the STN was reported by Peschanski and Besson 61 in a WGA-HRP study in the rat. The present data confirm the existence of such a pathway, and indicate that it consists of a modest contingent of fibers originating from the rostral part of the dorsal raphe nucleus and distributing rather diffusely throughout the STN. A topographic organization of the dorsal raphe efferents was reported in the rat in a recent retrograde study 32 according to which the rostral district of this nucleus projects to the striatum and the substantia nigra, whereas its caudal part sends fibers to the hippocampus. The present retrograde material further
strengthens the notion that the rostral dorsal raphe district is the one closely associated with basal ganglia structures. Considering that the pattern of discharge of dorsal raphe neurons shows profound alterations along the sleep-waking cycle (see e.g. ref. 1), it is conceivable that the projection from this nucleus to the STN may be involved in the fact that ballistic movements are usually absent during sleep 6. It should be pointed out that no retrograde labeling was observed in our material in any of the other raphe nuclei, in spite of a previous suggestion in the literature of a projection from the median raphe nucleus to the STN 6].
Pedunculopontine tegmental nucleus A projection from the pedunculopontine tegmental nucleus to the STN has already been extensively investigated. In the rat 21'27'33'42'72'75'8°'88 and cat 45'4s'5°'57'66, it has been described with both retrograde and anterograde tracer techniques and confirmed at the ultrastructural level, while in the monkey 1°,66 it has been indicated in retrograde HRP experiments. According to the present findings and previous anterograde reports 33'48'57,8°, the pathway under discussion innervates conspicuously the whole extent of the STN. The precise origin of this projection is still a matter of dispute, despite the several studies addressed specifically to this issue. Thus, in a fluorescent tracer investigation in the rat 7s retrogradely labeled neurons were observed throughout the pedunculopontine tegmental nucleus. On the other hand, in a combined choline-O-acetyltransferase (CHAT) WGAHRP retrograde study in the rat 42, the neurons projecting to the STN appeared to be essentially confined to a non-cholinergic district of that nucleus, embedded in the superior cerebellar peduncle and termed 'midbrain extrapyramidal area '71 in view of its reciprocal connections with basal ganglia structures. Other retrograde experiments in the rat, however, using either [3H]choline8° or a combination of fluorescent tracers associated with ChAT immunohistochemistry ss, have emphasized the cholinergic nature of the projection under discussion. In the present material, most of the retrogradely labeled neurons were found in what appears to correspond to the cholinergic district of the pedunculopontine tegmental nucleus, in both its diffuse and its densely populated parts, and, to a much lesser degree, in the noncholinergic 'midbrain extrapyramidal area'. It should be kept in mind, however, that it is difficult to settle this controversial issue in a retrograde study, since neural districts adjacent to the STN (the zona incerta, hypothalamus and substantia nigra) are also innervated by the pedunculopontine tegmental nucleus 26'42'48,57'80. It is apparent that the question of the origin of the cholinergic terminals known to be present in the STN 56 should be
56 considered open. Physiological studies indicate that the p e d u n c u l o p o n t i n e tegmental nucleus, which appears to be involved in the regulation of l o c o m o t o r behavior 5"13, is capable of modulating the activity of neurons in the STN 27 and o t h e r basal ganglia structures TM. F r o m an anatomical point of view, it may be noted that the p e d u n c u l o p o n t i n e tegmental nucleus seems to project directly both to the STN and to several structures, such as the medial division of the prefrontal cortex 72, the dorsal 33"42'72"88 and ventral 26 pallidum, and the thalamic parafascicular nucleus 14'33"75's°, which in turn project to the STN.
Interspecies differences The organization of basal ganglia circuitry presents m a n y species differences (see e.g. refs. 8, 19, 48, 59, 60, 83, 84), and the available anatomical evidence, however fragmentary, suggests that this may be the case also of the STN afferent connections. Thus, a direct projection from the p r i m a r y s o m a t o s e n s o r y cortex to the STN has been described so far only in the rat (see ref. 7 and present observations), while a striatal input to the STN has been r e p o r t e d in the cat 4'7° but neither in the rat 3°'83 nor in the m o n k e y 1°'35's9. M o r e o v e r , it appears that projections to the STN arising from different cortical districts as well as from the dorsal pallidum give rise to more restricted ABBREVIATIONS IN THE FIGURES AC ac
acd acv
aid aip am av
bst C
CC cg cp CS
DBC dr Fx gld glv gP gri Gv H1 Hz ha
anterior commissure nucleus accumbens septi dorsal division of the anterior cingulate area ventral division of the anterior cingulate area dorsal agranular insular area posterior agranular insular area anteromedial thalamic nucleus anteroventral thalamic nucleus bed nucleus of the stria terminalis central nucleus of the amygdaloid complex crus cerebri central gray substance nucleus caudatus putamen median raphe nucleus decussation of the bracbium conjunctivum dorsal raphe nucleus fornix dorsal lateral geniculate nucleus ventral lateral geniculate nucleus globus pallidus granular insular area ventral tegmental nucleus of Gudden Forel's field H 1 Forel's field H 2 anterior hypothalamic nucleus
REFERENCES 1 Adrien, J. and Lanfumey, L., Ontogenesis of unit activity in the raphe dorsalis of the behaving kitten: its relationship with states of vigilance, Brain Research, 366 (1986) 10-21.
terminal fields and exhibit a more clear-cut topographic organization in the m o n k e y 9"29'35'54 than in non-primate species (refs. 2, 45 and 51; also, present observations). C o m p a r a t i v e cytological studies (see ref. 28), taking into account the relative sizes of the STN and of its dendritic fields, also reinforce the idea that p r o b a b l y only in primates a rather precise spatial organization of the STN afferents becomes possible. Interestingly, recent findings with double-labeling r e t r o g r a d e techniques indicate a similarly higher degree of specificity in the organization of the primate 59'6° STN efferents c o m p a r e d with their counterparts in rodents ~4. Needless to say, much physiological e x p e r i m e n t a t i o n is required before the functional implications of the anatomical observations herein r e p o r t e d can be fully understood, Nonetheless, it is clear that the comprehensive characterization of the STN afferent connections that was the aim of the present work is a necessary step in the unraveling of basal ganglia mechanisms, which seem to be powerfully m o d u l a t e d by th6 STN,
Acknowledgements. This work was supported by grants from FAPESP (86/0237-1 and 87/1749-9), FINEP (43 86 0771 01) and CNPq (303264-84 and 303265-84). The authors are indebted to J.D.S. Vitor for excellent histological assistance. The technical help of N. Teixeira, R.S. Nascimento and R. Vieira is also gratefully acknowledged. hi hp IC icc ML MT MI OT ot P pf pg pl pn po pp prcm pv sc SM Sml SmlI st tv vp ZI IV
lateral hypothalamic region posterior hypothalamic nucleus internal capsule central nucleus of the inferior colliculus medial lemniscus mammillothalamic tract first motor area optic tract olfactory tubercle pyramid nucleus parafascicularis thalami nucleus parabigeminalis prelimbic area pontine nuclei nucleus reticularis pontis oralis pedunculopontine tegmental nucleus medial precentral area nucleus paraventricularis thalami superior colliculus stria medullaris thalami primary somatosensory area secondary somatosensory area subthalamic nucleus of Luys ventrobasal complex ventral pallidum zona incerta trochlear nucleus
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