Compartmental origins of striatal efferent projections in the cat

Neuroscience Vol. 32, No. 2, pp. 297-321, Printed

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1989

0306-4522/89 $3.00 + 0.00 Pergamon Press plc 0 1989 IBRO

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COMPARTMENTAL ORIGINS OF STRIATAL EFFERENT PROJECTIONS IN THE CAT J. JI&NEZ-CASTELLANOS* and A. M. GRAYBIELt$ *Universidad de Sevilla, Facultad de Medicina, Departamento de Ciencias Morfokjgicas, Avda. Sgnchez Pizjuan, Sevilla, Spain tMassachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Cambridge, MA 02139, U.S.A. Abstract-Injections of the retrograde tracer, wheat germ agglutinated-horseradish peroxidase were placed in the substantia nigra, in adjoining dopamine-containing cell groups A8 and AlO, and in the internal and external parts of the pallidal complex of 20 cats in order to identify the compartmental origins of striatal efferent projections to the pallidum and midbrain. Patterns of retrograde cell-labeling in the caudate nucleus were analysed with respect to its striosomal architecture as detected in sections stained for acetylcholinesterase. Where possible, a similar compartmental analysis of cell-labeling in the putamen was also carried out. In 15 cats anterograde labeling in the striatum was studied in the sections stained with wheat germ agglutinated-horseradish peroxidase or in autoradiographically treated sections from cases in which [3SS]methionine was mixed with the wheat germ agglutinated-horseradish peroxidase in the injection solution. Predominant labeling of projection neurons lying in striosomes (usually with some labeling of dorsomedial matrix neurons) occurred in a subset of the cases of nigral injection, including all cases (n = 9) in which the injection sites were centered in the densocellular zone of the substantia nigra pars compacta [JimCnez-Castellanos J. and Graybiel A. M. (1987) Neuroscience 23, 223-242.1 Dense labeling of neurons in the extrastriosomal matrix, with at most sparse labeling of striosomal neurons, occurred in all cases of pallidal injection (n = 8) and in two cases of nigral injection in which the injection sites were lateral and anterior to the densocellular zone. Mixed labeling of striosomal and matrical neurons occurred in a third group of cases in which the injection sites were lateral to the densocellular zone but close to it. In a single case with an injection site situated in the pars lateralis of the substantia nigra, there was preferential labeling of striosomal neurons in the caudal caudate nucleus but widespread labeling of neurons in both striosomes and matrix in the putamen. A second type of compartmental ordering of projection neurons was found in the extrastriosomal matrix of the striatum. In cases of pallidal and nigral injection, there were gaps in cell labeling that did not match striosomes precisely, and often clusters of labeled cells appeared that did not correspond to acetylcholinesterase-poor striosomes but, instead, to patches of matrix. Especially prominent were clusters beside striosomes. There was a topographic ordering of striatal projection neurons both in the striosomes and in the extrastriosomal matrix according to their dorsoventral and latitudinal positions. Intranigral injections of [35S]methionine mixed with wheat germ agglutinated-horseradish peroxidase, and nigral cases in which anterograde and retrograde labeling with wheat germ agglutinated-horseradish peroxidase were differentiable, demonstrated patterns of conjoint anterograde and retrograde labeling of corresponding striosomes or of corresponding extrastriosomal matrix. These findings suggest the presence of reciprocal links between the striatum and the substantia nigra pars compacta, or at least links between the striatum and parts of the pars compacta and pars reticulata lying very close to one another. By contrast, injections centered in cell group A8 elicited very little retrograde labeling in either the dorsal or the ventral striatum, and although injections placed in the ventral tegmental area led to extensive retrograde labeling of parts of the ventral striatum, there was little retrograde labeling of the dorsal striatum. Thus the projections from cell groups A8 and A10 to the dorsal striatum may only be weakly reciprocated, if at all. We conclude the following: (1) Striosomal ordering is followed by each major category of striatal efferent connection, but non-striosomal ordering of striatal projection neurons also occurs. (2) The extrastriosomal matrix of the striatum is specialized for conveying information to the two segments of the globus pallidus and the lateral (probably reticular) part of the substantia nigra. (3) Within the matrix, neurons projecting to the pallidum and to the substantia nigra are subject to a form of non-striosomal but striosome-like compartmentalization. (4) The striosomal system of the striatum is specialized for transmitting information from the striatum to the substantia nigra, though a small striosomal output to the pallidum is not excluded by these findings. (5) Cell group A9 may participate in reciprocating striatonigral-nigrostriatal loops, but the projections from cell groups A8 and A10 to the dorsal striatum may lack strong direct return connections. Two characteristics point to the striatum as crucial in establishing the organizational structure of basal ganglia circuitry. Firstly, the striatum is the main receiving station of the basal ganglia. It receives inputs from all areas of the neocortex,‘5,73”4.83,84 from the midline-intralaminar systems of the thalamus,48355,75,77.79 and from the amygdala,74~77~8’and it is

fTo whom correspondence

should be addressed. PB, phosphate buffer; SP, substance P; TH, thyrosine hydroxylase; TMB, tetramethyl benzidine; VTA, ventral tegmental area; WGA-HRP, wheat germ agglutinin-horseradish peroxidase.

_.dbbreuiutions: AChE, acetylcholinesterase;

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the main target of ascending monoamine-containing fiber systems arising in the midbrain.‘,36.38Though the striatum is not the only structure in the basal ganglia receiving such extrinsic afferents, it is the only one with such a breadth of input connections. Secondly, the striatum is the source of massive fiber projections to the globus pallidus’4~67~70*86~s7 and the substantia nigra.4’.86,87.89 As these are the two dominant efferent structures of the basal ganglia, projecting to the thalamus46~53,59~60~67 and to midbrain sites,36,67 the general plan of organization of the extended basal ganglia circuitry appears to be a convergent one in which a broad range of inputs influences narrow output channels by way of dopamine-modulated striatal circuits. Within this general framework there is a compartmental organization of striatal tissue that serves to sort its afferents into different modular units.27.30The compartmentalization is comprehensive. Nearly every afferent system projecting to the striatum has fields of termination that appear in histological sections either as a set of dispersed patches or as one or more larger fields interrupted by lacunae. All striatal afferent systems observe striosomal ordering in that their fibers tend to terminate within or outside the limits of histochemically identifiable striosomes (see Refs 27, 30, 73, 76, 77). Though there is also evidence that individual striatal afferent fibers may have synaptic specializations along a large part of their intrastriatal course (much like parallel fibers of the cerebellar cortex, see Refs 19, ZO), the striatum is thus not simply a region for convergence but a region for dispersal and resorting of inputs.2R Moreover, afferents to the striatum are clustered not only according to whether they project to striosomes or matrix. Certain afferent systems, including those arising in sensory cortex and sensory association cortex, and in certain parts of the amygdala and thalamus, project in a patchy fashion to the extrastriosomal matrix.62,63,76,77 An afferent-fiber “patch” in the striatum thus is not necessarily indicative of a striosomal “patch”; the extrastriosomal matrix has an afferent-mosaic of its own, though this is not readily distinguishable in the histologic preparations used to detect striosomes. The functional impact of such compartmental sorting of afferents clearly depends on the way in which the interneuronal connections of the striatum are organized and the way in which its output pathways originate. If the afferent-fiber compartments were not respected by these connections, a blurring across anatomical input-domains would occur. There is now considerable evidence that for the striosomal compartments, both interneurons and projection neurons respect compartmental boundaries. The processes of sthatal interneurons containing GABA, somatostatin, and acetylchohne are all more densely distributed in the matrix of the striatum than in striosomes.’ ‘,‘2.22,3’,34.72*82 The separation of interneuronal networks of the acetylcholinesterase (AChE)-poor

GRAYBIEL

striosomes and AChE-rich matrix is not complete, however, because crossing-over of processes from one histochemically-distinguished compartment to the other is known to occur.9”~22,3’,82 Thus within the striatum, interactions between the striosomes and matrix compartments almost certainly must occur in a highly selective manner. Striosomal ordering has also been demonstrated for certain striatal projection neurons. As was first demonstrated in the cat,37,50neurons projecting to the entopeduncular nucleus (the apparent equivalent of the internal pallidal segment of the primate) originate primarily in the extrastriosomal matrix.37,50 Those projecting to the substantia nigra are divided into striosomal and extrastriosomal categories. For the rat, Gerfen and associates 2223have presented evidence that striosomal (“patch”) neurons project to the pars compacta whereas matrix neurons project to the pars reticulata. The preferential linkage of “patch’ neurons with the pars compacta has been questioned in the experiments of Fishell and van der Koo~,‘*.*~ who reported that the terminal fiber-distributions of “patch” and matrix neurons do not differ postnatally. For the striatonigral system of the cat, weHlpresented evidence that the lateral substantia nigra (excepting the pars lateralis) is the target of extrastriosomal matrix projections, and that striosomal neurons project to the medial part of the substantia nigra. The medial position of the striosomal target was compatible with, but not proof of, a terminus in the substantia nigra’s pars compacta. This evidence, however, does not adequately address the question of how striatal output neurons and interneurons are ordered. Hints of heterogeneity in the distribution of intrinsic striatal cells have been noted for the somatostatin-containing interneurons of the matrix8’ and for striatal projection neurons26.56 (also, Glowinski et al., personal communication), and are also under study in the monkey.26 Secondly, as first reported in the cat by Beckstead and Cruz,4 different sets of striatal neurons project to the two segments of the globus pallidus and to the substantia nigra, so that another level of ordering must exist2’j It is not clear how this other ordering relates to the macroscopic compartments. Thirdly, the original findings on the compartmentalized efferent organization of the cat’s striatum3’ were made before it was appreciated that the dopamine-containing cell groups projecting to the striatum are themselves grouped according to whether they project to stfiosomes or to extrastriosomal matrix.‘6.22.23,M’5’.s’ Accordingly, possible selective affiliation of certain mesostriatal pathways with particular striatomesencephalic pathways could not be evaluated. In the experiments reported here and briefly elsewhere,s” we specifically addressed these issues by making injections of retrograde tracer into each division of the cat’s pallidum and into parts of the nigral complex including not only the pars compacta, pars reticulata and pars lateralis but also the affiliated dopamine-

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similar levels of the mesencephalon from standard series of TH-immunostained sections. Following initial analysis, the retrograde labeling in the striatum and in some cases anterograde labeling as well, was EXPERIMENTAL PROCEDURES analysed in relation to the locations of striosomes detected Deposits of retrograde tracer were placed unilaterally or in serially adjoining sections stained for AChE. An outline bilaterallv in 20 adult cats (Thorsen Breedina Labs, West- drawing was made of the AChE-stained section, the posiboro, MA) (30 hemispheres) deeply anesthetized with tions of AChE-poor striosomes were drawn in, and the sodium pentobarbital (Nembutal) (50mg/kg) and held resulting chart was then used as a form on which to indicate cell labeling in the striosomes and extrastriosomal matrix. in a stereotaxic device. In 14 animals (24 hemispheres) Other patterning in the labeled projection systems was either horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP, 20% aqueous solution) alone entered as necessary. Closely spaced chartings were made (eight hemispheres), or in combination with [3sS]methionine through the striatum in every case. In addition, photographs (2OOpCi/yl) (14 hemispheres) was deposited either in the were made of the labeling patterns present in selected sections. Patterns of anterograde (WGA-HRP and autointernal pallidal segment (entopeduncular nucleus, six cases) radiographic) labeling in these cases have been reported or in the external pallidal segment of the globus pallidus separatelys2 but anterograde labeling in a few of these cases segment (two cases). In all but one of the entopeduncular injections a contralateral approach to the nucleus was fol- was studied again in relation to concomitant retrograde lowed in order to avoid the possibility of contamination of labeling in the same sections. the striatum or its efferent fibers. In all cases the tracer RESULTS solution was ejected from a glass micropipette (internal tip diameter 25-30 pm) in small increments using an air-pulse Four main patterns of retrograde labeling were delivery system. Volumes were c. 15-6Onl total for each evident in the caudate nucleus following injections injection. Survival times after surgery ranged from 2-3 days. Brains were fixed by transcardial perfusion with the placed in the midbrain: (1) predominant labeling of animals under deep barbiturate (Nembutal) anesthesia. neurons in striosomes; (2) predominant and relatively Perfusion was begun with 500 ml of saline, continued with homogeneous labeling of neurons in the extra800 ml 0.5% paraformaldehyde, 1% glutaraldehyde and striosomal matrix with sparse labeling of striosomal 2.5% sucrose in 0.1 M phosphate buffer (PB) and concluded with 800ml of a second stronger fixative made with 0.5% neurons; (3) dense labeling of both of these histoparaformaldehyde, 4% glutaraldehyde and 5% sucrose in chemically distinguished compartments; and (4) PB. Brains were postfixed for 2 h in the stronger fixative, strong labeling of the matrix with either an imprecise then briefly washed in 15% sucrose in PB, and finally kept overnight in 20% sucrose in PB. The brains were cut in the avoidance of striosomal compartments or a frankly heterogeneous distribution of the labeled ma&al transverse plane at 40 pm on a freezing microtome. Sections through the injection sites and striatum were processed for neurons. These patterns were not always present WGA-HRP histochemistrv and, dependina on the case, for through all anteroposterior levels of single cases, autoradiography. In all cases sections through the striatum and the patterns of distribution were not absolute: and midbrain were stained for AChE. Sections immunosome labeled neurons appeared in the extrastriosomal stained for tyrosine hydroxylase (TH), available in other cases,52 were used as reference guides to estimate the matrix even in instances of prominent striosomal locations of the injection sites in the midbrain relative to neuron labeling, and in cases of dense labeling of TH-positive cells and processes in the dopamine-containing neurons of the extrastriosomal matrix, some strioA8-A9AlO cell groups.

containing cell groups A8 (the retrorubral nucleus) and A10 [the ventral tegmental area (VTA) of Tsai].

Histochemical techniques and autoradiography

Both the tetramethyl benzidine (TMB) method of Mesulam@ to demonstrate HRP-reaction product, and the AChE method of Geneser-Jensen and Blackstad” were followed according to modifications reported elsewhere.52 For the radiolabeled cases, other sections were processed for autoradiography13”’ as previously descr&d5* Analysis

Sections were studied with bright-field and dark-field illumination, and for the WGA-HRP material, crossed polarizers were placed in the light path to produce an intensified image of the TMB reaction product.49 Sections were charted onto drawings made with the aid of an overhead projector. Estimates of the size of the effective injection sites were recorded in terms of a central zone of densest labeling and a surrounding less densely labeled region. In the cases of nigral injection, serially adjoining AChE-stained sections through the midbrain were used to compare the location of the injection sites with the AChE-poor zone of the pars compacta of the substantia nigra, which corresponds to the TH-positive densocellular zone of the A9 dopamine-containing cell group. We have previously suggested that this zone projects preferentially to striosomes.r2 In each case of tracer injection into the substantia nigra and paranigral cell groups, an indirect comparison was made between the locations of the injection sites and the A8-A9-Al0 cell complex by reference to sections through

somal neurons were labeled as well. As the following analysis demonstrates, however, the occurrence of each of the four predominant patterns appeared referable in an orderly way to the main loci of the injection sites. Predominant labeling of striosomal neurons

Injections in the densocellular zone. Pronounced retrograde labeling of striosomal neurons was found in each of the nine cases in which injection site centers were located in the densocellular zone of the substantia nigra pars compacta. An example of the selectivity of labeling in one such case (2L) is shown in Fig. 1. Many neurons in the striosomes of the caudate nucleus are heavily labeled, whereas relatively fewer neurons in the matrix are labeled. In this, as in other cases, there were large regions of the extrastriosomal matrix in which hardly any labeled neurons were present despite the large size of the injection site (Figs 2 and 11). In the medial part of the caudate nucleus, however, especially dorsally, fields of scattered retrogradely labeled neurons were present in the matrix, as they nearly always were when there was strong labeling of dorsal striosomes (see also Fig. 3).

Str~atopall~dal and striatonigral connections

Fig. 2. The dist~bution of the estimated centers of injection sites in the substantia nigra are shown in overlay diagrams for cases with injections giving rise to predominant labeling of striosomes (left side) and cases with injections eliciting preferential labeling of matrical neurons (right side). Injection site centers were first located in TMB-stained sections, and then mapped onto drawings of adjoining AChE-stained sections so that the relation of the deposits to the densocellular zone and other landmarks could be judged. Composite overlay drawings of all cases were then mapped onto standard outlines of AChE-stained sections from a single case. The densocellular zone is shown in black. Note that injections leading to labeling of striosomes were centered in or immediately beside the densocellular zone. By contrast, the injection sites eliciting matrix labeling were centered lateral and anterior to the densoceflular zone. See also Fig. 1I, in which full extent of injection sites are shown. Cases illustrated in other figures are indicated by superscripts: (Pl, P3, P4, P7)--photographs in Figs i, 3,4 and 7; (CS, CI)---charts in Figs 5 and 8. AS, ceil group A8; VTA, ventral tegmental area; SNI, substantia nigra pars lateralis; SNc(dz), substantia nigra pars compacta’s densocellular zone; SNr, substantia nigra pars reticulata; ML, medial lemniscus; RN, red nucleus. Scale bar = 1 mm.

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Fig. 3. ~hoto~raPbs of seriaily adjacent transverse sections through the striatum in case 4L, in which a large WGA-HRP deposit was placed in the densocellular zone of the substantia nigra pars compacta. See Figs 2 and 1I for injection site. (A) Dark-field photograph of a TMB-stained section illustrating patches oflabeted cells and processes in the striatum. (B) Bright-field photograph ofthesame section showing mom clearly the clusters of labeled cells. (C) Serially adjoining AChE-stained section. Note corresponding Jocations of the patches of labeling in (A) and (B) and the AChE-poor striosomes in (Cl. Asterisks mark one such set in correspondence. Scale bar =i 1mm.

StriatopaIIidal and striatonigral connections

Fig. 4. (A) Diagrammatic chartings ofselectedtransverse sections through the striatmn in case IX in which a small &posit of a kxture of WGA-HRP and j3%Jmethionine was placed in the densocellular zone of the substantia nigra pars compacta. The diagrams show the contours of AChE-poor striosames. HRPlabeled striatonigral c&s visible in sections adjacent to those charted are depicted by iarge dots, and a~torad~og~phi~~iy-doled nigrostriatal fibers visible in adjoining sections are represented by small dots. Note that although fiber labeling of striosomes is more extensively distributed than fell labeiing, there is a high degree of correspondence between anterograde and retrograde labeling of striosomes. Note also that the dorsomedial matrix contains labeled cell bodies but not anterograde labeling. (B-D) Photographs of the centers of the WGA-HRP and [35S~eth~o~ne injection sites in serially adjacent sections, respectively processed for TMB Mstochemistry (B) and autoradiography with Cresyi Violet counterstain (C), and of a third serially adjacent section (D) processed for AChE histochemistry to demonstrate the distinctive low AC!& activity which characterizes the densooellular zone of the substantia nigra pars compacta.5’ Injection site invades lateral densocellular zone, see also Fig. 2. Scale bar = 1 mm.

be interpreted unequivocaily as anterogradely transported material, &en that the processes of retrogradely labeled neurons could have been extensively tabeled. We therefore ~~~~ltan~~s~y injected WGAHRP and [3SS]methionine into the nigral complex in 14 hemispheres. The findings strongly supported the impression of reciprocity of the connections in that

there was extensive overlap of pat&es of anterogradely transported radiolabel with clusters of retrogradely labeled ceil bodies. The evidence for case 17L, together with photographs of the injection sites seen with each tracer, is presented in Fig. 4. The injection was caudally situated in the substantia nigra’s pars

compacta, and affected the lateral part of the denso-

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cellular zone. The HRP deposit was one of the three smallest obtained in the entire case material. The distribution of striosomes with anterograde labeling was more extensive than the distribution of strio-

GRAYBIEL

somes with retrograde labeling. It is not clear whether the difference reflected a disparate spread of the tracers away from the pipette tip, because it was not possible to define accurately the outer limits of the

A

Case 11R

B

Case 11L

c Case 13

D

Case 3R

Fig. 5. Schematic drawings of selected transverse sections through the striatum in four cases identified by their case numbers. The cases illustrate examples of regionally restricted striatal cell-labeling (black dots); the location of retrograde labeling depended on the location of the centers of WGA-HRP injection sites relative to the densocellular zone (see Fig. 2 for injection site centers and Pig. 11 for estimates of entire extent of injection sites). (A) Preferential cell labeling of dorsal striosomes in case I LR in which the tracer deposit in the pars compacta of the substantia n&a was ventral. (B) Cell-labeling mainly in ventral striosomes in case 1IL, following a dorsally situated tracer deposit at the dorsal limit of the densocellular zone of the pars compacta. (C) Case 13, with an injection centered in the lateral part of the densocellular zone that gave rise to preferential cell-labeling in laterally situated striosomes. (D) Cell-labeling in caudal and ventrolateral striosomes following an injection site centered in the pars lateralis of the substantia nigra in case 3R.

Striatopallidal and striatonigral connections effective injection sites for either of the two tracers. The relatively restricted fields of labeling observed in the caudate nucleus with both tracers, however, suggest that the anterograde and retrograde connectivities match for at least a part of the striosomal system. Evidence fir topography. All nine nigral deposits centered in the densocellular zone appeared to exceed the boundaries of this nigral zone judging from the spread of TMB reaction product. Nevertheless, there were systematic shifts in the distribution of labeled striosomes in the caudate nucleus depending on the locations of the centers of the injection sites relative to the densocellular zone. These orderly shifts are illustrated in Fig. 5. Deposits with ventrally located centers elicited labeling of dorsal striosomes in the absence of comparable labeling of ventral striosomes. Such ventrally located striosomes were preferentially

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labeled in cases with dorsally situated injection sites. Medial nigral placements were associated with medial labeling of striosomes, and lateral deposits with labeling of laterally (and caudally) situated striosomes. Particularly striking examples of restricted labeling of different parts of the striosomal system appeared in two cases in which an edge of the zone of retrograde labeling cut through striosomal forms visible in the adjoining AChE-stained sections. An example of this partial labeling (from case 13) for a long striosomal form having two swellings connected by a thick neck is shown in Fig. 6. Only the left-hand side of the striosome contains labeled neurons. The border between the labeled and unlabeled parts of the striosome coincides with the edge of the larger field of labeling that occurred in this case (see chartings of the case shown in Fig. 5).

Fig. 6. High magnifiation photomicrographs of striatal fields in serially adjacent transverse sections processed for AChE (A) and TMB (B) histochemistry from case 13, in which a WGA-HRP deposit was centered in the densocellular zone of the substantia nigra pars compacta. (A) Light-field photograph showing an elongated AChE-poor striosome which crosses the full horizontal width of the photograph. (B) Dark-field photograph taken with crossed polarizers placed in the light path. Note retrogradely labeled cells appear only in the left half of the striatal tissue corresponding to the AChE-poor striosome shown in (A). Scale bar = 1 mm.

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These cases point to the presence of a rough topography in the st~osome-to-subst~ntia nigra projection preserving mediolateral dimensions but inverting dorsoventral dimensions. As is also shown in Fig. 5, however, such coordinated displacements of labeling were not as readily identifiable along the anteroposterior dimension of the caudate nucleus. in most cases at least some striosomes were preferentially labeled at levels extending from the anterior pole of the caudate nucleus to the caudal part of the head of the nucleus. Only in two cases was the labeling more restricted: the two cases with the smallest injection sites (16L and 17L), in both of which striosomes were labeled in the caudai and middle part of the head of the caudate nucleus but not in the rostra1 head of the nucleus. Striosomal cell-labeling following injection of the substantia nigra pars lateralis. Near-selective labeling

of striosomal neurons in the caudate nucleus was seen only in one case (3R) in which the center of the injection site did not involve the densocellular zone of the substantia nigra’s pars compacta. The deposit was centered in the pars lateralis of the substantia nigra (Fig. 11). In the caudate nucleus (Fig. 5) ventral and caudal striosomes were labeled as well as small patches that could not be confirmed as striosomes in this caudal zone where, in many cats, AChE is not an optimal striosome marker.35~38+s2 As shown in the charts of Fig. 5, however, there was very extensive and apparently homogeneous labeling of the putamen in this case, so that if the putamen and caudate nucleus are considered together, the labehng does not represent selective labeling of striosomes but a joint pattern in which dorsomedially there is retrograde labeling of striosomes, whereas ventrolaterally there is labeling of matrical neurons as well. Predominant labeling of neurons in the extrastriosomal matrix Injection sites in the substantia nigra. Dense retrograde labeling of matrical neurons with relative sparing of striosomal neurons was the predominant pattern found in two cases of nigral injection. The injection sites were both centered in the lateral part of the substantia nigra, lateral to the densocellular zone but medial to the pars lateralis (cases IR and 9, Figs 2 and 11). Both of the injection sites were very large. A pair of adjacent AChE and TMB-stained sections from case 1R are illustrated in Figs 7A and B.

The zones of sparse cell-labeling that were interspersed in fields of dense retrograde labeling match striosomes quite precisely. Chartings from the second case are shown in Fig. 8. The labeled neurons lie mainly in the anterolateral and caudal parts of the caudate nucleus. Injection sites in the pallidurn. All eight deposits involving the pallidum, whether its external segment (feline “globus pallidus”) or its internal segment (feline “entopeduncular nucleus”), led to preferential labeling of neurons in the matrix of the caudate nucleus. The quite crisp avoidance of striosomes in case 7 is illustrated in Figs 7C and D, in which the deposit was centered in the entopeduncular nucleus, and Figs 7E and F illustrate the preferential matrix labeling in case 5, in which the injection site was centered in the external pallidum. In both cases, enough neurons in the matrix were labeled to give the impression that the striosomes represented sparsely labeled lacunae in otherwise densely labeled striatal tissue. In the four cats with deposits in the entopeduncular nucleus, five hemispheres had medial injection sites and in all of these the labeling in the caudate nucleus was heavier medially and ventrally than laterally and dorsally (see Fig. 8). In the one hemisphere with a lateral placement, the dorsolateral matrix was more strongly labeled than the medial matrix (see Fig. 8). In both cases of lateral pallidal injection, it was also mainly the lateral and dorsal part of the matrix of the caudate nucleus that was labeled together with labeling of the putamen (see Fig. 8). These cases taken together suggest a progression from medial and ventral to lateral and dorsal labeling of matrical neurons in the caudate nucleus with a medial to lateral shift in injection site placement in pallidal tissue extending from the most medial part of the entopeduncular nucleus laterally to the external pallidum (Fig. 11B). As was true for the preferential striosomal labeling in the cases of densoceliular zone injection, the fields of selective matrix labeling seen after pallidal injections were extensive in the anteroposterior dimension. However, the full anterior extent of the head of the caudate nucleus did not contain matrix labeling in any case except one, case 5 (Fig. llB), in which the injection site was very large. The more restricted anteroposterior extent of the matrix labeting in the other pallidal cases suggests that a rostrocaudal as well as a mediolateral topography might be present.

Fig. 7. Three pairs of serially adjacent transverse sections through the striatum demonstrating preferential retrograde cell-labeling in the extrastriosomal matrix following WGA-HRP injections centered in the lateral substantia nigra (A and B, case IR), the internal pallidurn (entopeduncular nucleus) (C and D, case 7) and in the external paIlidum (globus pallidus) (E and F, case 5). TMB-stained sections in (C) and (E) are shown in dark-field photomj~ographs; TMB-stained section in (A) is shown in a hght-field photograph. Corresponding AChE-stained sections shown on the right side of the figure in bright-field photographs (B, D, F) illustrate the distribution of AChE-activity in sections adjoining those in A, C and. E. Note correspondence of the label-poor zones and AChE-poor striosomes (examples indicated by astensks). See also Figs 2. I t and t2 for injection sites. Scale bar = 1 mm.

Striatopallidal

and striatonigral

Fig. 7.

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Mixed labeling of neurons in striosomes and neurons in the extrastriosomal matrix

In three hemispheres with nigral injections, labeling of the matrix predominated in one part of the

A. M. GRAYBIEL

caudate nucleus and labeling of striosomes predominated in another (cases 15, IL, and 2R). The injection sites in these cases were centered rostra1 to the densocellular zone and were all large (Figs 2 and 11). The distribution of labeled cells in one of these cases,

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in Fig. 8. At all anteroposterior levels there is strong labeling of matrical neurons ventrally with gaps visible that could be shown to correspond to AChE-poor striosomes at all but the most caudal levels. Dorsal to this zone of matrix labeling, the pattern switches to one in which most labeled neurons are in striosomes. The striosomal labeling occurs over nearly the full extent of the field of matrix labeling ventral to it, so that the two adjoining fields of labeling, one with striosomal fills and one with “gaps”, appear yoked. This dorsal fill-ventral gap pattern was found for all three nigral cases: in each, heightened labeling of striosomes appeared dorsal to a Freld in which striosomes were visible as interruptions in a field of matrix labeling. The switch from the striosomal to matrical labeling occurred abruptly. In several sections at the t~~sit~on zone, a p~feren~ally labeled striosome was found at the same height in the caudate nucleus as a neighboring striosome distinguished by weaker laheling than that in the su~o~ding matrix. Rarely, a striosomal form identified in the adjoining AChEstained section crossed the border between predomi-

309

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to pr~ominant

labeling

In two other cases of nigral injection (3L and IO), ventral fields of matrical neuronal labeling occurred with some neuronal labeling of dorsally situated striosomes. Thus ventrally, neurons in striosomes as well as in matrix were labeled, so that the pattern was one of nearly uniform labeling, while dorsally the labeling was more selective for striosomes. Taken together, the cases of nigral injection producing mixed labeling of striosomes and matrix suggested a dorsoventral topography both for the striosomal gaps and For the striosomal fills. For the dorsal fill-ventral gap cases, the boundary between ceil labeling in striosomes and in matrix shifted from more dorsal to more ventrat levels in the caudate nucleus with more dorsal placement of the injection sites (see Fig. 2). The most dorsal striosomal gaps occurred in ease IL, the case with the most ventral deposit. In the two cases with the most dorsal deposits the cell-labeling was confined to ventral caudate nucleus and no gaps appeared at all.

In several of the cases showing predominant labeling of neurons in the matrix of the caudate nucleus, the pattern of such labeling was heterogeneous. Uneven ~s~butions of ill-lacing occurred both in cases of injection sites centered in the lateral part of the substantia nigra and also in cases with deposits in the ento~~~cular nucleus. The heterogeneity of cell labeling in these cases took several distinct forms. In some instances the labeled mat&al field resembled a mosaic in which patches of labeled cells could be clearly dis~n~ished in the midst of a larger field in which other labeled neurons were more sparsely distributed. The patchy appearance could not be attributed to the presence of a patchiness of concomitant anterograde labeling because (a) the granular TMB reaction product showed a homogeneous dist~bution clearly visible with polarized optics, and (b) in most of the nigral cases we made simultaneous injections of the anterograde tracer [3sS~ethionine and the radiolabeled nigrostriatal fibers showed a uniform distribution within the labeled striatal field even when patchy cell-labeling appeared in the matrix. It was sometimes quite striking that the clusters of retrogradely labeled neurons lay heside striosomes. Examples of such peristriosomal patches are shown in Fig. 9. A second form of matrical heterogeneity identified occurred in cases in which laeunae free or nearly free of labeled cells, though aligned with AChE-poor striosomes seen in serially adjacent sections, were noticeably larger than the corresponding AChE-poor striosomes. Examples of this pattern are shown in Fig. 9. Evidently, not only the striosomes but also nearby peristriosomal parts of the matrix were excluded from labeling in such cases. Finally, in some cases the matrix Iabeling was non-uniform without being frankly patchy. The nob-unifo~ities did not seem to correspond to detectable striosomes, and there were no sharply de&red clusters of labeled neurons. ~~~~~~~~ant sabering in the ventral striatum injections in the ventral tegmentai area. With depos-

Fig. 8. Schematic chartin~s of selected transverse sections through the striatum in five cases, id~tifi~ by their case numbers, showing preferential ceftiabehng in the extrastriosomal matrix. Retro~ade~~ faheled celfs are represented by black dots. These and other cases suggest the presence of topographic ordering in the striatonigral and st~ato~lIida~ projections. Case 9 shows an aimost uniform labeling of the matrix which includes the fuI1extent of the matrix at caudal levels but which is restricted to the lateral part of the caudate nucleus at rostra1 levels. The WCiA-HELP injection site was centered in the lateral part (pars reticulataf of the substantia nigra. The injection sites in cases 14 and 19R were centered, respectively, in the medial and lateral parts of tire entopeduncular nucleus. Note that the medial injection site in case 14 &cited restricted tabeiing in ventromedial striatat districts of the matrix whereas the injection site centered laterally in the ento~uncusar nucleus (case 19R) gave rise to call-labeling in the dorsal and dorsolateral part of the matrix. Similar but more extensive cell-labeling in the dorsal and lateral matrix was also found in case 5, in which the WGA-HKP injection site was centered in the external segment of the globus pallidus. In case f 5, the WGA-HRP deposit was centered in the medial part of the rostra1 su~t~tia nigra. Note the medial and ventral location of the labehng of neurons in the extrastriosomai matrix and the simultaneous cell-labeling in some dorsally situated striosomes. See Figs 2, Ii and 12 for injection sites.

Fig. 9. Heterogeneity of retrograde celf-labeling in the matrix observed in case 17R, in which the WCA-HRP injection site was in the medial part of the internal pallidurn (enteropeduncular nucleus). (A) Polarized-light photograph of a TM&stained section showing lacunae almost free of labeled cells and patches of strongly labeled neurons. (B) Light-field photograph of adjoining AChE-stained section. Comparison of the sections shows that labeled cells are mostly located in the matrix and that unfabeled “holes” evident in the TMB-stained section (A) are larger than corresponding AChE-poor striosomes shown (example indicated by asterisks). Note also that many of the patches of cell-labeling lie be&& striosomes. Scale bar = I mm. its of HRP placed in the VT.4 (two cases) cell-labeling was restricted to ventral striatal districts (the nucleus accumbens and the olfactory tubercle). The number of iabefed neurons was greater in the case iilustrated in Fig. 10A (XL), which had the more ventrally located injection site (Fig. 1IA). Rostraily, cell-labeling extended dorsally to fill the dorsal AChE-rich zone of the nucleus accumbens. Farther caudally, a mediolateral gradient of labeled cells appeared, including the olfactory tubercie region where clusters of labeled neurons were evident at some levels. In case 8R (Fig, IOB) cell-labeling was restricted at rostra1 Lvets, but at more caudal levels labeled neurons were more numerous, were often clustered together, and were distributed within a broad mediolateral zone stretching from the nucleus accumbens to the lateral part of the olfactory tuber&e. fnjectim sites in cell group A 8. In four cases (see Fig. 11A) a mixture of WGA-IHRP and i3’S]methionine was deposited in ceil group AS. No cell-labeling occurred in the dorsal striatum in any of these cases. In two cases (2OL and ZOR) only isolated labeled &Is

appeared in ventral striatai regions, although prominent anterograde labeling of fibers was detectable in these regions both with WGA-HRP and [?3]meth&nine, and more &@&se labeling was present in the ~xtrastr~osoma1 matrix of the dorsal striatum, In two other cases (19L and 12L) we could not detect any labeled neurons in the striatum. DISCUSSION

The findings reported here demonstrate that there are distinct compartmental arrangements of striatal neurons projecting to different subdivisions of the feline substantia nigra and to the two segments of the pallidurn. This compartmentalization of projection neurons not only serves to distinguish striosomes from matrix, but also deiimits efferent zones within the matrix from one another. A global typography appears to govern the distribution of the projection neurons both in striosomes and in the matrix according to their specific targets within the pallidum and substantia nigra. We conclude that, on the efferent

of

Fig. 10. Schematic drawings selected transverse sections through the striatum in two cases, identified by their case numbers, with WQA-HRP injection rites in the VTA. Lab&d cells are representr?d by black dots. In both cases there were labeled cells in the ventral striatum@ but not in the ventral parts of the caudate nucleus and putamen. Note different patterns of cell labeling in the two cases, and that injection site in case 8R was Gghtfy more dorsal than that of case 8L.

side of the striatum, of projection functional

compartmental

organization

neurons may serve as the basis for specialization within a broad range of

extrapyramidal

circuits.

The striatomgrai projection fkmz striosomes. in our initial brief report on the striatonigral pathway in the cat3 we concluded that the striosomal system of the caudate nucIeus projects to the medial part of the subs~ntia nigra (including, apparently, the medial part of its pars ~rn~a~~~ and also to the pars lateralis of the substantia nigra. By contrast, these preliminary findings suggested that the extrast~os~m~~ matrix innervates the lateral part of the substantia nigra, possibly its pars reticulab. The observations reported here confirm most of these results, but also extend them by a~~o~g a more precise ~den~~tio~ of the ~nrn~a~tmenta~~z~ origins of the striatonigraf pathway and the nigrai subdivisions within which it terminates. The most striking conclusion from our analysis of this pathway is that the AChEgoor striosomes of the caudate nucleus pro&et to the AChE-poor densoeelfufar zone of the substantia n&m’s pars compacta or to its immediate vicinity. This conch&on is based on two sorts of evidence. Firstly, it was clear from the cases with large HBP deposits in the substautia nigra that the more medial the centers of

the injection

sites were, the greater the number of striosames labeled and, depending on the size of the deposits, the greater the selectivity of the striosomal tabehng. when the centers of these mjection sites were anatysed in relation to the location of the densocellufar zone, we found that afl injection sites with centers in the densocellular zone produced celllabeling predominantly in striosomes. Secondly, in the cases with very small injection sites in the densocellular zone, there was quite restricted labeling of striosomal neurons except in the do~ome~al quadrant of the eaudate nucleus where more scattered labeling was always found (see below). The techniques we used in this study do not permit crisp definition of the limits of the nigral target of striosomes, We cannot, for example, discount the possibility that the target includes the n&al zone subjacent to the ~s~e~~~ zone. This region is righ in (RI-t-t-)8 - chloro - 2,3,4,5 - tetrahydro - 3 -methyl - 5 - phenyt 1~-3-~n~p~n-?-o~ ~[3H]SCH~3390~ D, dopamine receptor-selective binding sites, and D, sites are thought to he in large part oft striatonigral fibers6~” Dendrites of densoceliular zone neurons probably lie in this region, however; so that even this ~~~~rne~~ of the striosomaf target zone couki still impty a reciprocal striosome-densoceIlular zone-striosome circuit, There was a remarkable similarity between the patterns of retrograde cell-labeling in the caudate nucleus following small injections in the densocellular

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and A. M. GRAYBIEL

Fig. It. The location of all of the injection sites in the n&al complex indicated semi-schematically in drawings of transverse sections through the cat’s mesencephalon. The full size of the darkened WGA-HRP injection sites are indicated; cf. Fig. 2, in which the centers of injection sites are shown. Cases with injections giving rise to predominant labeling of striosomes are drawn on the left side and cases with injections eticiting preferential labeling of neurons in the matrix are represented on she right side. Note that whereas the centers of the injection sites (Fig. 2) clearly distinguish striosome-related and matrix-related cases, the full extent of the WGA-HRP darkened zones do not. The densocellular zone of the pars compacta of the substantia nigra is represented in solid black.

Striatopallidal and striatonigral connections

zone and the distribution of neurons expressing substance P (SP)-like and dynorphin B-like immunoreacIt is in the dorsal part of the caudate nucleus that clusters of SP-positive neurons and dynorphin B-positive neurons are prominent and where they lie in correspondence with tyrosine hydroxylase-positive patches in kittens32,33 and AChE-poor striosomes in cats.3*8.32.33,63 The SP- and dynorphin B-positive neurons are also scattered through the matrix, especially in the dorsomedial part tivity.4*7A32

313

of the caudate nucleus. Thus the SP/dynorphin Bcontaining neurons and the populations of cells retrogradely labeled from the densocellular zone injections are not completely restricted to striosomes, but both populations lie predominantly in striosomes and have parallel extrastriosomal distributions. Aside from the striosomal projection to the region of the densocellular zone, our single case of injection confined to the pars lateralis of the substantia nigra suggests that caudal and lateral striosomes in the

2’

Fig. 12(A).

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J. JIMBNEZ-CASTELLANOS and A. M. GRAYBIEL

B 1

Fig. 12. Semi-schematic drawings of the injection sites placed in (A) the entopeduncular nucleus (internal pallidurn) and in (B) the external pallidum (globus pallidus). All injection sites were drawn according to procedure described in the legend of Figs 2 and 11. The center and the peripheral parts of each injection site are shown by dark and light stippling, respectively. Each case is identified by its own case number. In A, at levels 2 and 3, the injection sites have been drawn in two panels (2 and 2, 3 and 3’) in order to make it easier to identify each site and its center. Cases illustrated in other figures are indicated by superscripts: (P7, P9), photographs in Figs 7 and 9; (C8), charts in Fig. 8; CN, caudate nucleus; P, putamen; Am, amygdaloid complex; GPe, globus pallidus; En, entopeduncular nucleus; OT, optic tract; Fx, fornix; MTT, mamillothalamic tract; AC, anterior commissure; RT, nucleus reticularis of thalamus.

Striatopailidal and striatonigral connections PREFRONTAL & tNSULAR CORTEX, AMYGOALA

SENSORY & MOTOR CORTEX, CINGULATE GYRUS

315

PREMOTOR, SUPPLEMENTARY MOTOR 8 PREFRONTAL CORTEX

Dopamine-containing nigral complex

Fig. 13. Highly schematic summary of findings, placed in context of trans.striatal pathways of the basaf gangha shown in simplified form. The striatum is shown as the stippled region at the upper left. The main striatofugal pathways identified were: (1) from striosomes (light stipple) leading to the medial part of the substantia nigra (SNm), to the region of the densocellular zone (dz) of the substantia nigra par compacta; (2) from the extrastriosomal matrix (heavy stipple) to the two segments of the pallidum; and (3) from the matrix to the lateral part of the substantia nigra, probably the pars reticutata of the substantia nigra (S. Nigra pr). Pathways arising from the matrix were identified in some cases as originating in clusters of projection neurons in the matrix, symbolized by the darkest stipple in the diagram. Inputs from the cerebral are shown together with outputs hemisphere to the striosome and matrix subdivisions’5~62~73~7”75~76~77~78 leading toward the thalamus. A more complete summary of the efferent pathways is given elsewhere.%

caudate nucleus-and both striosomal and matrical neurons in the putamen-project to this subdivision of the cat’s nigral complex. The ~tri~t~n~gral projection from the matrix. We were unable to pinpoint the precise nigral termination zones of the majority of matrical projection neurons. None of the nigral HRP deposits were small enough to involve exclusively either of the two large subdivisions of the substantia nigra outside of the densocellular zone (the diffuse rostrolateral part of the pars compacta and the pars reticulata). Further, though we identified the densocellular zone ~stochemically by its selectively low AChE-activity, we did not, for technical reasons, compare in the same cases the extent of the HRP deposits and the distribution of TH-immunoreactive neurons in the nigral complex. Despite these limitations, the evidence favors a ventrolateral target for the matrical projection. All lateral injections in the substantia nigra gave rise to more abundant cell-ladling in the matrix than more medially located injections. Moreover, in two cases the pattern of striatonigral labeling corresponded to almost pure matrical cell-labeling and in each of these cases the micropipette’s tip was located laterally in the cerebral peduncle ventral to the pars reticulata of the substantia nigra. Presumably, though the injection sites in these cases were large, the nigral region infiltrated was mainly the pars reticulata.

In the rat22.23.24it has been suggested that the “patch” (striosomal) compartment may project to the ventral tier of the pars compacta and to the ventrolateral caudal pars reticulata where dopaminergic neurons are located in the rat. Striatal neurons in the matrix are reported to project to the remainder of the pars reticulata. No reference is made in these papers to the afferent connections of the dorsal tier of the pars compacta of the substantia nigra where (together with cell groups A8 and AlO) the main mesostriatal input to the extrastriosomal matrix is thought to originate in the rat.2s The spatial distribution of dopamine-containing cells in the substantia nigra of the cat is different from that seen in the rat. On the basis of distinct THimmunostaining and differential nigrostriatal connections’* the cat’s substantia nigra pars compacta can be divided into a caudomedially situated densocellular zone (a main source of the nigrostriatal projection to striosomes) and a rostrolateral diffuse zone where TH-immunoreactive cells and fibers are more sparsely distributed. At rostra1 levels of the substantia nigra, the diffuse zone forms a medio1ateralIy oriented wing overlying the substantia nigra pars reticulata. The diffuse zone contains nests of clustered TH-immunoreactive cells and neuropil which may represent rostra1 finger-like extensions of the densoceIlular zone.

316

J. JIM~EZ-CASTELLANOSand A. M. GRAYBIEL

In the series of experiments reported here, only two of the cases of lateral nigral injection had almost exclusive cell-labeling in the extrastriosomal matrix. All other cases of lateral nigral injection showed a pattern of mixed striosomal and matrical cell-labeling. The injection sites in these cases were large, and though their centers were either rostra1 or lateral to the densocellular zone, the peripheral parts of the injection sites might have invaded the striosomerecipient region in or immediately adjacent to the densocellular zone. In addition, they may also have involved the rostra1 finger-like extensions of the densocellular zone. Either factor could have led to labeling of striosomal neurons in these cases, as could involvement of the diffuse zone of the pars compacta. We therefore can suggest only with reservation that the matrical projection to the substantia nigra seems to innervate the pars reticulata, for years the only accepted destination of the striatonigral pathway. Topography. A topographic ordering of projections from both striosomal and matrical neurons to nigral targets can be clearly inferred from our results. Whether an anteroposterior topography also exists is less certain. For the striosomal system, the analysis showed that a direct mediolateral topographic ordering of the projection exists, but that a dorsoventral inversion also occurs. Particularly striking were instances in which only parts of striosomes visible in single sections were filled with labeled cells, with the edge of the striosomal cell-labeling corresponding to the transition between labeled and unlabeled fields in the striatum. The cases with nearly pure matrix cell-labeling and those with mixed striosomal and extrastriosomal cell-labeling also indicated mediolateral and inverted dorsoventral topographic ordering. Two or more independent fields of striatal labeling were never found in the same case. However, in three cases predominant cell labeling in dorsal striosomes of the caudate nucleus coexisted with gaps of labeling corresponding to ventral striosomes in an otherwise fully labeled or partially (ventrally) labeled extrastriosomal matrix. This particular pattern of striatal labeling has also been found for anterogradely labeled striatal afferent fiber systems,76*77,78 including the mesostriatal projection.52 The presence of some topographic ordering of striatonigral fibers is a fact almost universally accepted, but the specific arrangement of the projection is still a matter of dispute and differences among species have been noticed. In the rat, Bunney and Aghajanian” reported a mixed anteroposterior and mediolateral topographical distribution of the striatonigral projection. In the cat, a mediolateral topography of caudatonigral projections was found with degeneration methods, but the terminal zone was reported to be confined to the rostra1 pars reticulata of the substantia nigra.69 With anterograde transport methods Royce and Laine”’ confirmed a mediolateral topography and added that (a) the caudatonigral

PK+ction involves the entire rostrocaudal length (excepting the rostrdmost pole) of the pars reticulata, and that (b) an additional pathway, mostly originating in the dorsolateral part of the caudate nucleus, innervates the substantia nigra’s pars compacta, especially its caudolateral parts. In the macaque monkey, a certain degree of striatonigral topography has been reported in the mediolateral axis but little or none in the anteroposterior and dorsoventral axes4’ It has further been reported in the squirrel monkey” that a territorial dissociation of striatal neurons occurs in which those projecting to the substantia nigra are mainly located in the caudate nucleus whereas those innervating the globus pallidus are located mainly in the putamen. In the rat, Gerfen2’ has suggested that topographically as well as non-topographically ordered striatonigral projections coexist. According to this author, the non-topographic projection originates in the “patch” (striosomal) compartment and is directed to the pars compacta and caudal ventrolateral pars reticulata where dopamine neurons are also located. A topographically ordered projection originating in the striatal matrix is reported to innervate the remaining pars reticulata of the substantia nigra. Our own findings in the cat favor the existence of a topographic ordering of striatonigral projections both from the striosomal compartment and from the matrix compartment.

Ventral striatum

The ventral striatum, including the nucleus accumbens and the olfactory tubercle,44 contained labeled neurons in cases with HRP deposits in the ventral tegmental area (cell group AlO) and in the retrorubral nucleus (cell group AS) but only in a subset of the cases in which HRP was deposited in the substantia nigra. The only cases of nigral injection with labeled cells in the ventral striatum were cases in which the HRP deposits were centered in the medial part of the substantia nigra adjacent to the VTA. This medial part of the substantia nigra contains the diffuse zone of the pars compacta rostrally, whereas caudally the densocellular zone occupies this position. None of the three small injections in the densocellular zone, even the one located most medially, elicited any retrograde labeling in the ventral striatum. Presumably, therefore, it is the medial part of the diffuse zone or the junction between this zone and the paranigral cell groups A10 and A8 that forms the major mesencephalic recipient zone of the ventral striatonigral projection. These findings are in agreement with previous which show that the efferent projections of reports 40,68 the nucleus accumbens in the rat terminate not only in cell groups A10 and A8 but also in the adjoining medial part of the pars compacta of the substantia nigra. Because we observed the ventral striatal labeling with injections involving the matrix-projecting diffuse zone but not with injections involving the

317

Striatopallidal and striatonigral connections striosome-projecting densocellular zone, our evidence in the cat further suggests that the ventral striatum would indirectly influence extrastriosomal matrix by this trans-nigral pathway. It is interesting that the matrix is also favored by the VTA itself, which comprises the other arm of the mesostriatal system.52 We cannot discount an additional projection from the accumbens nucleus to more ventrally located zones in the substantia nigra, including the pars reticulata as reported for the rat.@ Our findings do indicate that in the cat, as in the rat, such a projection would be directed to the medial pars reticulata. Retrograde tracer injections in the VTA elicited a prominent cell-labeling in ventral striatal regions but not in any part of the caudate nucleus or putamen. Surprisingly, we could not detect more than very rare labeled cells either in the dorsal striatum or in the ventral striatum in cases of cell group A8 injection. Previous reports in the rat6* have shown that both cell group Al0 and the rostra1 retrorubral nucleus receive abundant projections from the nucleus accumbens, whereas the caudal retrorubral nucleus receives projections at least from other ventral striatal regions situated lateral and caudal to the nucleus accumbens. In the cat,” the medial nucleus accumbens (which corresponds to the part of the nucleus where we found retrograde labeled cells) has also been found to project both to VTA and to the retrorubral nucleus; and the lateral part of the nucleus accumbens according to these authors projects to the retrorubral nucleus and to the substantia nigra. The retrogradely labeled cells that we found in the ventral striatum after VTA injections were organized in clusters, as are many neurotransmitter-related substance and afferent fibers there (see e.g., Refs 47 and 39). We did not make extensive correlations between the patches of retrogradely labeled cells in the ventral striatum with such other compartmental markers, but it was obvious in the VTA cases that cell-labeling in the striatum was at least partly related to the pattern of AChE staining, for example, in the nucleus accumbens where HRP-labeled cells were mainly distributed in AChE-rich zones. Reciprocity

Previous studies5@52have demonstrated in the cat that the mesostriatal projections arising in dopaminecontaining cell groups A8, A9 and A10 are compartmentalized in the dorsal striatum, and are predominantly directed either to the striosomes (those originating in the densocellular zone of the pars compacta, and to a lesser degree in the pars lateralis of the substantia nigra) or to the extrastriosomal matrix (those from cell group A8, the rostra1 diffuse zone of the substantia nigra pars compacta, and the VTA in this order). The VTA and A8 also give rise to a prominent projection to the ventral striatal region. A comparison between these mesostriatal systems and the striatonigral projections reported here suggests coordinate patterning of the origins and terminal sites

of at least a subset of the mesostriatomesencephalic control loops. First, the findings suggested, though they did not prove, reciprocity between the densocellular zone of substantia nigra pars compacta and the striosomal system. The fmdings also suggested reciprocal patterns of connectivity between the VTA and the ventral striatum. In both instances, however, labeled mesostriatal fibers ended in a striatal region that was more extensive than the distribution of striatal cells labeled by tracer injections in these mesencephalic subdivisions. The existence of an electrophysiologically-detected monosynaptic reciprocity between the striatonigral and nigrostriatal pathways involving the caudoputamen has been denied in the rat.57 Evidence for striatal and nigral reciprocity at the electron microscopic level has been reported,85 although the nigral neurons which are thought to receive monosynaptic contacts from (ventral) striatal fibers project back to a wider (more dorsal) region of the striatum. Strikingly, in the present experiments, injections in cell group A8 gave rise to only exceptional labeling of cells in the ventral striatum, even though in the very same cases there was anterograde labeling of fiber projections to the extrastriosomal matrix and to the ventral striatum. Striatopallidal

projection

We confirm here previous reports suggesting that the feline striatopallidal projection originates mainly in the extrastriosomal matrix.37~50Taking together all cases with WGA-HRP deposits in the entopeduncular nucleus, cell-labeling in the caudate nucleus included the full dorsoventral extent of the nucleus. However, the two HRP injections which involved most of the external segment of the globus pallidus gave rise to well circumscribed fields of matrical cell-labeling in the dorsolateral or lateral regions of the head of the caudate nucleus. A mediolateral topography of cell-labeling in the caudate nucleus was evident in the striatopallidal projection to both segments of the pallidum, and was especially clear for the cases of medial and lateral HRP injection in the entopeduncular nucleus. The putamen also contained labeled cells in our cases of pallidal injection but a topographical ordering of putaminopallidal projections was not evident. Because the injection sites involved the full dorsoventral extent of the internal and external pallidurn, we could not analyse the striatopallidal relation in this axis. There is general agreement, however, that the striatal projections to both pallidal segments are organized by simple non-inverted topographical relationships in the cat.69.80 Heterogeneous organization the extrastriosomal matrix

of projection

neurons in

A major finding in these experiments was the nonstriosomal heterogeneity of retrograde cell-labeling seen in the matrix following HRP deposits in the

318

J. JI~~Z-CAS~~LANOS and A. M. GRAYB~EL

lateral substantia nigra and in the entopeduncular nucleus. An apparently comparable heterogeneity of distribution of labeling in the matrix has also been observed by the Glowinski groups6 (Kernel et al., personal communication) and has recently been confirmed in the monkey.26*58 The concomitant anterograde nigrostriatal transport of HRP in the cases of nigral injection did not account for such a heterogeneous labeling of the matrix, for the anterograde labeling did not show comparable heterogeneity. This was confirmed also in the cases in which we simultaneousiy injected ~3sSJ-meth~onine and WGA-HRP in the substantia nigra, and found a homogeneous distribution of autoradiographically labeled fibers in the striatal matrix. Peristriosomal patterns. The patterns of celllabeling in the matrix took different forms. In some cases, pockets of sparse cell-ladling in the field of extrastriosomal labeling were centered in the same locations as AChE-poor striosomes, but the cell-poor zones were larger. This suggested that the matrix neurons along the rims of some striosomes did not share certain projection targets with cells deeper in the extrastriosomal matrix. A somewhat complementary pattern of labeling was also found: sometimes clusters of labeled neurons lay directly alongside striosomes. Again, the evidence suggested differences between peristriosomal neurons and neurons lying deeper in the matrix. It is interesting that several transmitter-reiated markers also show specializations at striosomal borders. Striatal somatostatinergic neurons, considered to be interneurons, tend to be located near AChEpoor striosomes, rimming the striosomal borders in the cat.“.‘” This rimming pattern has also been demonstrated for NADP~~iaphorase activity, considered a sensitive somatostatinergic marker in the striatum.82 Staining for AChE activity in the neuropil sometimes shows densification around the striosomal boundaries in the cat and in other species.35,82SP-like immunoimmunoreacreactivity2 and [metlenkephalin-like tivity,29.‘a both characteristic of striatal projection neurons, are particularly dense along the borders of striosomes in the primate striatum. Most recently, neurotensin binding sites have been identified in such peristriosomal positions. ISaThis pattern is not striking in the cat, but the present evidence suggests that some of the projection neurons rimming striosomes may be specialized matrix neurons in the cat as well. Patches in the matrix. In some cases, the heterogeneity in distribution of projection neurons took the form of clustering of labeled cells within otherwise unlabeled fields of the matrix. The finding of such clusters suggests the possibility that there may be separate clusters of striatonigral neurons and of striatopallidal neurons projecting either to the entopeduncular nucleus (internal pallidum) or to the globus pallidus (external pallidum). This possibility would be consistent with the observation, based on fluorescent double-labeling experiments4 that different

cell populations project to the substantia nigra, to the internal pallidum, and to the external pallidum in the cat. No interdigitation of groups of neurons projecting to different sites was reported in these experiments, but in view of the present endings, such a mosaic organization of projection neurons should be carefully tested. In the rat some clustering of striatal projection neurons has been reported,6’ but it has been shown that as many as 40% of the projection neurons in the caudoputam~n send axon collaterals both to the mesencephalon (substantia nigra-VTA complex) and to the external pallidum. Apparently, segregation of neurons projecting to only one of these striatal targets is more highly developed in the cat and monkey.4,26g58One might expect, then, a further elaboration of clustering patterns in the monkey than in the cat, as indeed appears to be the casez6x5’(Gimenez-Amaya and Graybiel, unpublished findings). The observation of clustering of projection neurons could help to clarify the organization of neurochemically distinct striatofugal projection systems specifically innervating the substantia nigra and the two segments of the paliidum.5,29~38”Conceivably, the cells of origin of the tackykinin-containing projections to the pallidum and substantia nigra could be preferentially grouped together in relation to [met]enkephalin-immunoreactive neurons projecting to the external pallidum (and to each other if different tackykinin-containing groups exist). Even if striatal projection neurons are not fully segregated according to their efferent targets, the heterogeneous distributions of these neurons almost certainly must mean that a mosaic organization of projection cells exists in the extrast~osomai matrix. This finding is of great interest in relation to two other sorts of evidence that the matrix has a compartmental organization. Firstly, afferents to the striatum are arranged in modular fashion in the matrix,62.63,76.77 and for some of these afferents the evidence suggests that the modular terminal fields may serve to bring into close proximity fibers representing different subsets of fibers related to a given functional domain. For example, in the cat, corticostriatal fibers originating in somatic sensory areas SI and 3a of the cat have heen found to distribute in striosome-like striatal compartments within the extrastriosomai matrix.62 Secondly, most neurotransmitter-related substances localized to the striatum have distributions that show some heterogeneity in the matrix (see, for example, Refs 6, 11, 30, 31, 35, 54, 65). Though these uneven distributions in the matrix do not have the sharpness of striosomes, they suggest that neurochemically-specified subsystems may exist in the matrix and may have inde~ndent access to striatal projection neurons projecting to different parts of the pallidum and substantia nigra. These findings suggest a general frame of organization in the striatum in which different sets of afferent

Striatopallidal and striatonigral connections fibers can be targeted to individually distinct striatopallidal and striatonigral pathways. The segregation of striosomes and matrix, in this view, would be one part of a comp~hensive compartmental plan estabIishing sets of internal processing units and throughconduction routes for the striatum as a whole.

319

Acknowledgements-This work was funded by the Seaver Institute, the McKnight Foundation, and a Javits award NIH 2 ROl NS 25529-OlAl. We thank H. F. Hall, who is responsible for the photography, D. Major and G. Holm for help with the histology, and L. Connors for help with word processing.

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