Chemical heterogeneity of the striosomal compartment in the human striatum

Chemical Heterogeneity of the StriosomalCompartment in the Human Striatum

LUCIA PRENSA,1,2 JOSE MANUEL GIMENEZ-AMAYA,3 AND ANDRE PARENT1*1Laboratoire de Neurobiologie, Centre de Recherche Universite Laval Robert-Giffard,

Beauport, Quebec G1J 2G3, Canada2Departamento de Morfologıa, Facultad de Medicina, Universidad Autonoma de Madrid,

28029 Madrid, Spain3Departamento de Anatomıa, Facultad de Medicina, Universidad de Navarra,

31080 Pamplona, Spain

ABSTRACTThe neurochemical organization of the striosomal compartment in the human striatum

was analyzed by histochemical and immunohistochemical techniques applied to postmortemtissue from normal individuals. The striosomes were delineated by using the followingmarkers: acetylcholinesterase (AChE), enkephalin (ENK), substance P (SP), calbindin-D28k(CB), parvalbumin (PV), calretinin (CR), limbic system-associated membrane protein (LAMP),choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and NADPH-diaphorase. Com-parisons were made between striosomal boundaries, as outlined by each marker applied onadjacent sections, and particular attention was paid to possible variations in the chemicalfeatures of striosomes along the rostrocaudal extent of the striatum. The main findings of thisstudy are as follows: 1) the striosomal compartment is composed of two chemically distinctdomains: a core and a peripheral region; 2) the core is largely devoid of CB and displays a lessintense staining for ENK and LAMP than the peripheral region; 3) although striosomes arelargely devoid of AChE, the activity of this enzyme is slightly higher in the core than in theperipheral region; 4) the core and peripheral regions are weakly stained for PV and intenselystained for SP; 5) ChAT-, CR- and NADPH-diaphorase-positive neurons are preferentiallydistributed in the peripheral region; 6) at rostral striatal levels, striosomes are largely devoidof TH, whereas the inverse is true caudally; and 7) at caudal striatal levels, the peripheralregion of striosomes is intensely stained for CB and ChAT. These results demonstrate that thestriosomes in human display a strikingly complex and heterogeneous chemical architecture.J. Comp. Neurol. 413:603–618, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: human basal ganglia; striosomes; striatal chemospecific compartments;

histochemistry; immunohistochemistry; striatal neurons

In their pioneering study, Graybiel and Ragsdale de-scribed zones poorly stained for the enzyme acetylcholines-terase (AChE) in the striatum of cats, monkeys, andhumans, which they termed striosomes (striatal bodies)(Graybiel and Ragsdale, 1978). Striosomes are also re-ferred to as patches or islands, but the exact correspon-dence between each of these entities remains to be estab-lished (Selemon et al., 1994). The AChE-poor striosomeslie within an AChE-rich matrix, which represents approxi-mately 80% of the total striatal volume (Graybiel andRagsdale, 1978; Graybiel, 1990; Johnston et al., 1990). Thestriosome/matrix subdivision of the striatum is supportedby the distribution of a wide variety of transmitter-relatedsubstances and by the organization of striatal afferent andefferent connections (see Holt et al., 1997 for references).For example, the AChE-poor striosomes are in perfect

register with zones of high enkephalin (ENK)-immunoreac-tivity (Graybiel et al., 1981; Ferrante et al., 1986), whereassubstance P (SP) concentrations appear to vary within thetwo major striatal compartments (Graybiel et al., 1981;Beach and McGeer, 1984; Ferrante et al., 1986; Martin etal., 1991; Jakab et al., 1996; Holt et al., 1997). However,there is as yet not complete agreement as to whether SP is

Grant sponsor: Medical Research Council of Canada; Grant number:MT-5781; Grant sponsor: Killam Program of the Canada Council for theArts; Grant sponsor: Comunidad de Madrid; Grant sponsor: FIS; Grantnumber: 96/0488.

*Correspondence to: Andre Parent, Ph.D., Centre de Recherche Univer-site Laval Robert-Giffard, 2601, de la Canardiere, F-6500, Beauport,Quebec G1J 2G3, Canada. E-mail: [email protected]

Received 1 December 1998; Revised 5 April 1999; Accepted 20 July 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 413:603–618 (1999)

r 1999 WILEY-LISS, INC.

more abundant in the striosomal compartment than in thematrix. The expression of the limbic system-associatedmembrane protein (LAMP) is higher in striosomes than inthe extrastriosomal matrix in the striatum of cats andprimates (Chesselet et al., 1991; Cote et al., 1995).

In regard to calcium-binding proteins, striatal zonesdisplaying a weak calbindin-D28k (CB)-immunoreactivityappear to correspond to patches of high µ-opiate receptordensity and to AChE-poor striosomes (Gerfen et al., 1985;Graybiel, 1990). In humans, certain parvalbumin (PV)-immunoreactive (ir) neurons were exclusively confined tostriosomes (Prensa et al., 1998) and regions of weakPV-immunostaining correspond to ENK-rich striosomes(Waldvogel and Faull, 1993). In non-human primates, thecalretinin (CR)-ir neuropil was found to be intense instriosomes, but these structures contain relatively fewCR-ir perikarya (Parent et al., 1996b).

The immunostaining for tyrosine hydroxylase (TH) andcholine acetyltransferase (ChAT) in the striatum closelyfollows striosomal ordering (Graybiel et al., 1986, 1987;Ferrante and Kowall, 1987; Hirsch et al., 1989; Holt et al.,1996, 1997). Cholinergic perikarya, as well as neurons andneuropil containing the enzyme nicotinamide adeninedinucleotide phosphate-diaphorase (NADPH-d) or nitricoxide synthase (NOS), lie mainly within the extrastrio-somal matrix and are often disposed near compartmentalborders (Graybiel et al., 1981, 1986; Chesselet and Gray-biel, 1986; Kowall et al., 1987; Martone et al., 1994; Holt etal., 1996).

Altogether, these findings reveal that the mammalianstriatum is markedly enriched with a wide variety ofneurochemical markers that are expressed differentiallyin the two major striatal compartments, the striosomesand the matrix. To further our knowledge of the chemicalheterogeneity of the striatum, we have applied immunohis-tochemical and histochemical procedures to postmortemhuman brain materials to study the distribution of some ofthe most abundant striatal chemical markers, namely,AChE, ENK, SP, LAMP, CB, PV, CR, ChAT, TH, andNADPH-d, in the dorsal striatum. The major aim of thisstudy was to examine the chemical organization of thestriosomal compartment in human. We have thus com-pared the pattern of immunostaining of each chemicalmarker as seen on adjacent sections and carefully docu-mented any variations in the staining pattern of strio-somes that may occur along the rostrocaudal extent of thestriatum.

MATERIALS AND METHODS

Subjects

The present observations are based on the analysis ofpostmortem material obtained from 11 normal individualswith no clinical or pathological evidence of neurological orpsychiatric disorders (Table 1). The material was kindlyprovided by Dr. Michel Marois, Service de Pathologie,Hopital Saint-Francois d’Assise, Quebec, and our protocolwas approved by the Laval University Committee onEthics in Research. The brains were sliced unfixed into0.5-cm-thick slabs that were fixed by immersion at 4°Ceither for 2 days in 4% paraformaldehyde or 3 or 4 days inbuffered formalin (3.75% formaldehyde with 1–1.5% metha-nol, pH 7.4). The slabs were then stored at 4°C in 0.1 Mphosphate-buffered saline (PBS, pH 7.4) with 15% sucroseand 0.1% sodium azide. Several adjacent slabs from the

same or different individuals have been used to investigatethe human striatum along its full rostrocaudal extent.

Immunohistochemistry

The slabs were cut with a freezing microtome into50-µm-thick coronal sections that were serially collected incold PBS. Series of adjacent sections were then processedeither immunohistochemically for the visualization of ENK,SP, LAMP, CB, PV, CR, TH, ChAT, or histochemically forAChE and NADPH-d. The immunohistochemical protocolused to visualize either ENK, SP, CB, PV, CR, TH, or ChATwas as follows. After three rinses of 10 minutes each inPBS, the sections were placed for 30 minutes at roomtemperature in hydrogen peroxide (H202; 3%) to eliminateendogenous peroxidase activity. Sections were then prein-cubated during 30 minutes in a solution containing 5% ofeither normal horse serum (for ENK, CB, PV, and TH),normal rabbit serum (for SP and ChAT), or normal goatserum (for CR), and 0.1% Triton X-100. They were thenincubated in a 5% solution containing the appropriatenormal serum, 0.1% Triton X-100, and the primary anti-body. Incubation conditions and sources of primary anti-sera are specified in Table 2. The monoclonal antibodyused to visualized ENK did not distinguish between Met-enkephalin and Leu-enkephalin (Cuello et al., 1984). TheSP antibody was a rat monoclonal antibody that did notcrossreact with other known mammalian brain peptides(Cuello et al., 1979). The CB and PV antibodies werehighly specific mouse monoclonal antibodies (clones 300and 235, respectively; Celio et al., 1988; Celio, 1990). Thepolyclonal anti-CR antiserum was produced in rabbit byimmunization with recombinant human CR and did notcrossreact with CB or other known calcium-binding pro-teins (Schwaller et al., 1993). The TH antibody was amouse monoclonal antibody generated against TH isolatedand purified from rat PC12 cells. This antibody has

TABLE 1. Clinical Data on the Human Cases Used in This Study1

Case SexAge

(years)Postmortem

delay (h) Cause of death Fixation

H-1 M 54 12 Myocardial infarction BFH-2 F 25 20 Closed head injury with basal

and convexity fracturesBF

H-3 M 35 6 Head injuries with lacerationsof the scalp

BF

H-4 M 72 4 Cardiac hypertrophy BFH-5 M 21 10 Drug overdose BFH-6 F 24 8 Stab wound in heart BFH-7 M 25 7 Stab wound in heart PFH-8 M 27 7 Head injury PFH-9 M 20 48 Hypothermia PFH-10 M 46 10 Carbon monoxide asphyxia PFH-11 M 40 24 Suicide by gun shot PF

1BF, buffered formalin; PF, paraformaldehyde.

TABLE 2. Information on the Antibodies Used in This Study1

Antibody SourceAnimalsource Dilution Incubation Revelation

ENK Immunocorp Mouse 1:50 Overnight/4°C DABSP Immunocorp Rat 1:50 Overnight/4°C DABCB Sigma Mouse 1:2,500 Overnight/4°C DABPV Sigma Mouse 1:2,500 Overnight/4°C DABCR SWant Rabbit 1:2,500 Overnight/4°C DABLAMP Dr. P. Levitt Mouse 1:2,000 48 h/4°C DABChAT Chemicon Goat 1:100 72 h/4°C DABTH Incstar Mouse 1:250 48 h/4°C DAB/GO

1ENK, Met- and Leu-enkephalin; SP, substance P; CB, calbindin; PV, parvalbumin; CR,calretinin; TH, tyroxine hydroxylase; LAMP, limbic system associated membraneprotein; ChAT, choline acetyltransferase; DAB, 3,38-diaminobenzidine tetrahydrochlo-ride; GO, glucose oxidase.

604 L. PRENSA ET AL.

recently been used to study the TH immunoreactivity inthe human substantia nigra (Aubert et al., 1997). TheChAT antibody was an affinity-purified polyclonal anti-body raised against goat ChAT (Chemicon, Tamecula, CA;Grosman et al., 1995).

After three rinses of 10 minutes each in PBS, thesections were incubated for 1 hour at room temperature inthe secondary antibodies, which were biotinylated horseIgG (for ENK, CB, PV, and TH), biotinylated rabbit IgG(for SP and ChAT), and biotinylated goat IgG (for CR).After three more rinses in PBS, the sections were reincu-bated for 1 hour at room temperature in 2% avidin-biotincomplex (ABC, Vector Labs, Burlingame, CA), according tothe method of Hsu et al. (1981). Some sections processedfor CB, ENK, or SP were incubated for 1 hour in 2% EliteABC (Vector Labs) to increase the intensity of the staining.The sections were then washed twice in PBS and once in0.05 M Tris buffer (pH 7.6). The bound peroxidase wasrevealed by placing the sections in a medium containing0.05% 3,38-diaminobenzidine tetrahydrochloride (DAB;Sigma) and 0.003% H202 (30%) in 0.05 M Tris buffer (pH7.6) at room temperature. The reaction was stopped afterabout 5 minutes by washing once in 0.05 M Tris buffer (pH7.6) and several times in PBS. Some of the sectionsimmunostained for TH were revealed according to theglucose oxidase method to enhance the immunostainingintensity for fibers and axon terminals (Shu et al., 1988).

The immunohistochemical protocol used to reveal LAMPwas similar to the one described above except that therewas no preincubation and the amount of Triton X-100 wasreduced to 0.005% in the incubation medium containingthe primary antibody and to 0.025% in the solutioncontaining the secondary antibody. The LAMP monoclonalantibody (clone 2G9) was kindly donated by Dr. Pat Levitt,Department of Neurobiology, University of Pittsburgh,and its method of production and specificity have beenreported in detail elsewhere (Levitt, 1984; Zacco et al.,1990).

All immunostained sections were mounted on gelatin-coated slides that were air-dried, rinsed in distilled water,and dehydrated through passages in ascending grades ofalcohol. Sections were cleared in toluene and coverslippedwith Permount. Some sections were treated as aboveexcept that the primary antibody was omitted from theincubation medium. These sections remained virtuallyfree of immunostaining and served as controls.

AChE histochemistry

A modification of Geneser-Jensen and Blackstad’s proce-dure (1971) was used to visualize AChE. Briefly, thesections were washed in distilled water and then incu-bated during a period of time that ranged from 3 to 6 hoursin a solution composed of distilled water to which wereadded sequentially: ethopropazine, acetylthiocholine io-dide, glycine, cupric sulfate, and anhydrous sodium ac-etate. The final pH of the solution was adjusted to 5 by theaddition of some drops of glacial acetic acid. The precipi-tate was visualized by placing the sections for 2–3 minutesin a distilled water solution containing 10% potassiumferricianyde. The reaction was stopped by extensive rins-ing in distilled water, and the sections were mounted,dehydrated, cleared, and coverslipped as above. Controlsections were processed in an identical fashion, except thatacetylthiocholine iodide was omitted from the incubationmedium.

NADPH-diaphorase histochemistry

The presence of the enzyme NADPH-d was revealed byusing a slightly modified version of the histochemicaltetrazolium salt technique described by Scherer-Singler etal. (1983) and Vincent et al. (1983). In brief, free-floatingsections were first rinsed in 0.1 M phosphate buffer (pH7.4) and then incubated in a solution containing 10 ml of0.1 M Tris buffer (pH 8.0) with 0.3% Triton X-100, 8.3 mgb-NADPH (Sigma), and 6.5 mg nitroblue tetrazolium(Sigma). The reaction was made in the dark at 37°C, lastedbetween 20 and 30 minutes, and was terminated byrinsing the sections several times in 0.1 M phosphatebuffer (pH 7.4). Some sections were first treated as abovefor NADPH-d histochemistry and then, after extensivewashing in PBS during 1 or 2 hours, they were processedimmunohistochemically to visualize CB, ENK, or SP.

Data analysis

Photomicrographs were taken at different magnifica-tions from adjacent sections through the entire rostrocau-dal extent of the striatum in all individuals to illustratethe patterns of distribution of the various types of immuno-staining, as well as the morphological characteristics ofsome of the immunostained neurons and fibers. Further-more, the overall distribution of the immunostaining wasillustrated by means of direct prints of adjacent sectionsimmunostained for different neuronal markers. The nega-tive print images were obtained by directly printing immu-nostained sections inserted in a photographic enlarger(Focomat V35, Leitz). Additionally, several adjacent sec-tions processed for CB, CR, and ChAT were drawn using amicroscope equipped with a camera lucida to study thedistribution of the CR- and ChAT-ir neurons in regard tostriosomes. Other drawings were also made from adjacentsections immunostained for CB and TH to depict certainaspects of the distribution of the TH-ir fibers in thestriosomal compartment. The final rendering of the draw-ings was made by using Canvas software (Deneba Sys-tems, Miami, FL).

The present analysis is restricted to the dorsal striatum,which has been subdivided into a rostral and a caudalportion. The term rostral striatum, as used here, refers tothe most anterior regions of the caudate nucleus andputamen up to the level immediately rostral to the ante-rior commissure. The rest of the caudate nucleus and theputamen are included under the term caudal striatum.The ventral striatum, which includes the nucleus accum-bens and the olfactory tubercle, will not be consideredhere.

RESULTS

General patterns of labeling as seenon single sections

Labeling for neuroactive peptides. Along the entirerostrocaudal extent of the striatum, ENK immunoreactiv-ity displays a mosaic-like pattern composed of ENK-richringed areas surrounding ENK-poor centers, as well assmall and uniformly stained zones (Figs. 1C, 5A,D, 7A,D).The immunostaining for SP is more uniform than that ofENK, but some intensely stained SP-ir patches occur alongthe rostrocaudal extent of the striatum (Fig. 4D). Otherpatches completely devoid of both ENK- and SP-ir arefrequently found in the ventral half of the rostral caudatenucleus.

CHEMICAL HETEROGENEITY OF HUMAN STRIATUM 605

Labeling for LAMP protein. This protein is distrib-uted according to a markedly patchy pattern throughoutthe rostrocaudal extent of the human striatum (Fig. 1A,B).LAMP-ir areas are slightly larger and obliquely orientedin the putamen than in the caudate nucleus (Fig. 1A). Asdescribed for ENK, two morphologically different types ofimmunoreactive patches appear intermingled with otherpatches that are smaller and uniformly stained (Fig.1A,B). At the microscopic level the immunostaining forLAMP appears as a multitude of small punctate structureswithin a dense neuropil.

Labeling for calcium-binding proteins. Along therostrocaudal extension of the striatum, CB immunostain-ing is highly heterogeneous and follows a dorsoventralincreasing gradient (Figs. 5C, 7C). This heterogeneity ismainly due to numerous CB-poor areas that are unevenlydistributed in a more intensely stained background (Figs.1D, 5C, 7C). In the dorsal half of the caudate nucleus andputamen, the CB-poor areas are generally small and theirweak CB immunostaining is largely due to the lightneuropil and the paucity of CB-ir perikarya, both elementsbeing weakly stained (Figs. 2C, 3B). In the ventral half ofthe caudate nucleus, however, the CB-poor zones arecompletely devoid of CB-ir elements. Besides the multitu-dinous medium-sized CB-ir striatal neurons lying in a

rather densely stained neuropil that occur throughout thestriatum, a few intensely stained CB-ir neurons, as well asnumerous long, smooth or varicose CB-ir processes occurin the caudal putamen (Fig. 9D).

The PV immunostaining follows a ventrodorsal androstrocaudal increasing gradients. Numerous PV-poor ar-eas are scattered throughout the striatum (Figs. 1E, 4F),

Fig. 2. A: Direct ‘‘negative’’ print of a section through the rostralstriatum stained for AChE. Numerous AChE-poor striosomes arescattered throughout the dorsoventral sector of the caudate nucleus.The arrowhead points to the AChE-poor striosome in the head of thecaudate nucleus that is illustrated at a higher magnification in B. Thesame striosome, as seen on adjacent sections stained for CB andNADPH-d/ENK, is depicted in C and D. B: The AChE-poor striosomeis composed of a weakly stained central core (asterisk) surrounded byan unstained periphery. Note that the core of the striosome is lessintensely stained than the matrix. C: The core (asterisk) shows a weakCB-immunostaining, whereas in the peripheral region the CB immu-noreactivity is similar to that displayed by the matrix. D: The core ofthe striosome (asterisk) displays ENK immunostaining that is weakerthan that of the peripheral region. Some NADPH-d-positive neuronslying in the matrix are visible in the upper part of the photomicro-graph (arrows), but none of these neurons occur within the striosomalboundary. Scale bars 5 2.5 mm in A; 250 µm in B (also valid for Cand D).

Fig. 1. Direct ‘‘negative’’ prints of four adjacent sections stained forthe visualization of immunoreactivity for LAMP (A, B), ENK (C), CB(D), and PV (E). The sections are displayed in a rostrocaudal order.A: LAMP is expressed in both ringed patches (with LAMP-poorcenters; see arrows in A and B) and also in small and uniformly stainedareas. The LAMP-ir patch pointed by the arrow in A is shown at highermagnification in B and with various staining in C, B, and E. C: Notethat LAMP-ir areas are in register with ENK-ir striosomes and that

the immunostaining for both LAMP and ENK is intense in theperipheral ringed area that surrounds the poorly stained central core.D: The CB-negative area corresponds only to the LAMP/ENK-poorcore. In the peripheral ring, CB-immunostaining is slightly weakerthan that in the surrounding matrix. E: The PV immunoreactivity isuniformly weak in both the central core and the peripheral region ofthe striosome. Scale bars 5 1.8 mm in A; 1.05 mm in B (also valid forC–E).


Figure 2

but they are particularly obvious in the caudal putamen(Fig. 4F). In the caudate nucleus, the PV-poor areas resideprincipally in the dorsal sector (Fig. 1E) and are not ascrisp as in other portions of the striatum. PV is present in asmall population of striatal neurons, which are distributedin both the PV-poor areas and in the more intenselystained regions (Fig. 4F). In contrast to PV, small areasdisplaying a dense CR-ir neuropil occur in the ventral halfof both caudate nucleus and putamen (Fig. 3D). CR is alsopresent in a large population of medium- and large-sizedneurons rather uniformly distributed throughout the stria-tum.

Labeling for cholinergic markers. The human stria-tum displays intense staining for both AChE (Fig. 2A,B)and ChAT (Figs. 5B,E, 6A,B). Regional variations in theintensity of the AchE- and/or ChAT-positive neurons andneuropil in the caudate nucleus result in a mosaic patternof lightly stained zones embedded in a more intenselystained matrix (Figs. 2A,B, 5B,E, 6A). Most striatal cholin-ergic neurons have a large, round or multipolar cell bodyfrom which emerge numerous dendrites (Fig. 6B). Theseneurons are rather uniformly scattered throughout bothcaudate nucleus and putamen (Fig. 6A,B).

Labeling for NADPH-diaphorase. Numerous me-dium-sized aspiny neurons scattered throughout the hu-

man striatum display intense histochemical staining forNADPH-d (Figs. 3C, 4B,D). The striatal neuropil is alsostained for NADPH-d, but striatal areas that are largelydevoid of both NADPH-d-positive cell bodies and neuropiloccur in both caudate nucleus (Fig. 2D) and putamen (Fig.3C). Overall, the NADPH-d-positive neurons are morenumerous and intensely stained in the lateral and ventralregions of the putamen than elsewhere in the striatum.

Labeling for TH. TH immunoreactivity is heteroge-neously distributed throughout the human striatum (Fig.7B). Numerous small TH-poor zones embedded within anintensely stained TH-ir background occur at rostral stria-tal levels (Fig. 7B,E). In the caudal putamen, however,certain areas exhibit a denser TH-ir neuropil than thesurrounding tissue (Fig. 8B). The rostral striatum and theventromedial sector of both caudal caudate nucleus andputamen harbor a dense field of very thin and smoothTH-ir fibers and isolated varicosities reminiscent of termi-nal boutons among which are scattered some short andthick varicose fibers (Fig. 8D). By contrast, the dorsaltwo-thirds of the striatum exhibits long, smooth or slightlyvaricose TH-ir fibers lying in a background that is largelydevoid of small and isolated terminal bouton-like struc-tures (Fig. 8C).

Fig. 3. Photomicrographs illustrating a striosome located in therostral putamen, as seen on adjacent sections immunostained for ENK(A), CB (B), NADPH-d (C), and CR (D). A, B: The core of the striosomeis less intensely stained for both ENK and CB than the peripheralregion and the CB immunostaining of the peripheral region is asintense as that of the matrix. However, certain unstained myelinated

fascicles (arrows in B) delimit the periphery of the striosome from thesurrounding matrix. C: Numerous NADPH-d-positive neurons andneuropil in the area surrounding the striosome. D: Numerous CR-irneurons and neuropil occur within the striosomal boundary. Note thatthe density of the CR-ir neuropil is higher inside the striosome thanoutside. Scale bar 5 250 µm in A (also valid for B–D).


Fig. 4. Photomicrographs illustrating three different striosomes inthe putamen, as seen on adjacent sections immunostained for CB andNADPH-D/ENK (A,B), CB and NADPH-d/SP (C,D), and CB and PV(E,F). Blood vessels indicated by arrows serve as landmarks. A: Theintensity of the CB-immunostaining in the peripheral striosomalregion is slightly more intense than that of the surrounding matrix.B: Some NADPH-d-positive neurons located in the ENK-ir peripheryas well as in the less intensely stained core. C: This CB-poor centeredstriosome is surrounded by pale, unmyelinated fiber fascicles (small

arrows). D: The same striosome is uniformly stained for SP. NADPH-d-positive neurons are scattered throughout the matrix and occur alsowithin the striosome itself. E: The pale septa that surround thestriosome are particularly visible in this section immunostained forCB (small arrows). F: The PV-ir neuropil is uniformly weak in both thecore and the periphery of this striosome. Some densely stained PV-irperikarya are scattered within the both the core and the periphery ofthe striosomes. Scale bars 5 250 µm in A (also valid for B, E, and F);250 µm in C (also valid for D).


Comparative studies of the labeling seenon series of adjacent sections

Comparisons of the immunostaining for LAMP andENK, as seen on adjacent sections, demonstrate that thetwo markers are in complete register (Fig. 1B,C). Theyboth label typical structures composed of a LAMP/ENK-rich ring that surrounds a LAMP/ENK-poor center, whichare intermingled with small and uniformly stained LAMP/ENK-ir zones (Fig. 1B,C). On adjacent sections immuno-stained for CB, only the LAMP/ENK-poor centers of thetypical ring-figures match the CB-poor areas (Figs. 1B–D,2C,D, 3A,B, 4A,B). The small and uniformly stainedLAMP/ENK-ir zones that appear intermingled with theringed striosomes on singled sections increase their sizeand become typical ring-figures when they are observedthroughout several serially adjacent sections.

Although the core of striosomes is always largely free ofCB, the peripheral region shows CB immunostainingwhose intensity can be slightly weaker than, similar to, orstronger than that of the surrounding matrix (Figs. 1D,

2C, 3B, 4A, 6C). In the caudal putamen this peripheralzone often displays CB immunoreactivity that is moreintense than that of the extrastriosomal matrix (Figs. 4A,6C, Table 3). The peripheral region of striosomes is fre-quently surrounded by thin, CB-negative striatal areasthat correspond to myelinated fascicles closely surround-ing the striosomes (Figs. 3A,B, 4C,E). These unstainedencircling fibers are particularly obvious at the rostralputamen level, where many of them are in continuity withmyelinated fiber bundles that merge with the white mat-ter laterally or with the internal capsule dorsomedially.

The core and the peripheral region of striosomes alsodiffer in the intensity of AChE staining, the core express-ing a moderate staining that stands out from the un-stained peripheral region (Fig. 2A,B). The intensity of thecore immunostaining is nevertheless weaker than that ofthe extrastriosomal matrix (Fig. 2B). In sections immuno-stained for ChAT, both the core and peripheral region ofstriosomes located in the caudate nucleus display a uni-formly weak staining (Fig. 5B,E). Occasionally, some strio-

Fig. 5. The upper part of the figure shows direct ‘‘negative’’ printsof three adjacent sections through the rostral striatum immuno-stained for ENK (A), ChAT (B), and CB (C). Note that the ChAT-poorzones are in perfect register with both ENK-rich and CB-poor strio-somes (see arrows). The lower part of the figure shows photomicro-graphs of the striosome pointed out by the arrows above, as seen on

sections stained for ENK (D) and ChAT (E), respectively. Note thestrong ENK and weak ChAT immunostaining of the striosome (aster-isks) compared to the matrix. Some cholinergic neurons can be seen inthe matrix and near the striosomal border (arrows). Scale bars 5 3.08mm in A (also valid for B and C); 250 µm in D (also valid for E).


somes located in the dorsal sector of the caudate nucleusshow a ChAT-positive central core. Striosomes in therostral striatum are either completely devoid of ChAT-irperikarya or contain a few ChAT-ir cells confined to theirperipheral region (Fig. 5E). At caudal levels, however,numerous ChAT-ir neurons occur within the peripheralregion of striosomes (Fig. 6C, Table 3). These intrastrio-somal cholinergic neurons are preferentially located at theborder between the peripheral region and the core, as wellas at the frontier between the striosomes and the surround-ing matrix (Fig. 6C). At both rostral and caudal levels, thestriosomal core is largely free of ChAT-ir neurons (Figs.5E, 6C).

The striosomal compartment displays a rather complexSP-immunostaining pattern in human. Striatal zones moreintensely stained for SP than the surrounding matrixcorrespond to striosomes, as identified on adjacent sectionsstained for various other striosomal markers. The SPimmunostaining encompasses both the core and the periph-eral region of striosomes (Fig. 4D). However, there are also

some striosomes that do not show intense SP-immunoreac-tivity. In respect to PV, both the core and the periphery ofstriosomes display a weak immunostaining for this calcium-binding protein (Figs. 1E, 4F). The PV-ir perikarya thatoccur within striosomal boundaries are uniformly scat-tered in both striosomal regions (Fig. 4F).

In the ventral half of the striatum the CR-ir neuropiltends to be denser in the striosomes than in the extrastrio-somal matrix (Fig. 3D). The numerous medium-sizedCR-ir neurons are largely absent from striosomes, whereasthe less abundant large-sized CR-ir neurons are frequentlyfound in the peripheral region, at the same borderlines asthose occupied by ChAT-ir neurons (Fig. 6C).

Some striosomes are totally devoid of NADPH-d stain-ing (Figs. 2D, 3C), whereas others harbor NADPH-d-positive cell bodies (Fig. 4B,D). These intrastriosomalNADPH-d-positive neurons are distributed in both thecore and the peripheral region (Fig. 4B,D). Clusters ofapproximately six to eight NADPH-d-stained cell bodieswith long processes are often encountered close to strio-

Fig. 6. A: Direct ‘‘negative’’ print of one section through the caudalportion of the human striatum immunostained for ChAT. Note lightlystained zones embedded in a more densely stained matrix in thecaudate nucleus. The cholinergic neurons appear as bright dotsscattered throughout the caudate nucleus and the putamen. B: High-power view of ChAT-ir neurons in the caudal putamen. C: Distributionof ChAT-ir neurons with respect to the core and the peripheral regionof striosomes. The photomicrograph illustrates one striosome locatedin the caudal putamen, as seen in CB-immunostained sections. Note

the characteristic intensely stained peripheral region that surroundsa CB-negative central core. Each black dot represents the exactlocation of one ChAT-ir neuron, as visualized on an adjacent section.The CB-negative core is completely free of cholinergic neurons, butmany such neurons occur in the peripheral region of the striosome,particularly at the border between the two striosomal regions. OtherChAT-ir neurons occur along the frontier between striosome andsurrounding matrix. Scale bars 5 2.5 mm in A; 200 µm in B; 250µm in C.


somal boundaries and, in many instances, these clustersclosely surround striosomes (Fig. 3C).

In the rostral striatum and in the most ventral sector ofboth caudal caudate nucleus and putamen, striosomes areless intensely stained for TH than the extrastriosomalmatrix (Fig. 7B,E). At these levels, TH-immunostainingwithin striosomes is uniformly weak in both the core andthe peripheral region (Fig. 7E). Occasionally, however,some striosomes show a core more intensely stained thanthe periphery. In the caudal portion of the striatum,striosomes located in the dorsal two-thirds of the putamenexhibit a higher density of TH-ir neuropil than the sur-rounding matrix (Fig. 8A,B, Table 3). Furthermore, theTH-ir neuropil is denser in the core than in the peripheralregion of striosomes (Fig. 8B).

Additionally, some long and thick TH-ir fibers occur inthe striosomal core. These fibers can be seen crossingstriosomal boundaries and coursing within the extrastrio-somal matrix (Figs. 8B,E, 9B). Numerous thick and vari-cose CB-ir fibers can also be visualized in the CB-poor core

of striosomes at caudal striatal levels (Fig. 9A,C). TheseCB-ir fibers enter the matrix by running through theperiphery of the striosomes (Fig. 9C).

Fig. 8. A: Schematic representation of a section through the caudalstriatum indicating the location of a striosome in the putamen takenfrom a section immunostained for TH. B: The typical dense THimmunoreactivity in the core of the striosome is better appreciated inthis higher power view of this striosome (see inset in A). Note that theTH-ir neuropil is denser in the striosome than in the extrastriosomalmatrix and even more so in the core compared to the periphery. Somelong TH-ir fibers traverse the periphery of the striosome and course forlong distances in the matrix. C: Photomicrograph illustrating some ofthe thick and varicose TH-ir fibers that occur in the dorsal sector of thecaudal putamen (see inset C in A). D: Rather thick varicose TH-ir fiberin the ventral third of the caudal putamen coursing through a denseneuropil composed of very thin TH-ir varicose fibers and isolated axonalvaricosities (see inset D in A). E: Example of TH-ir fibers that cross theperiphery of the striosomes and course for rather long distances in thematrix. AC, anterior commissure; CD, caudate nucleus; GPe, externalsegment of the globus pallidus; GPi, internal segment of the globuspallidus; PUT, putamen. Scale bar 5 50 µm in C (also valid for D and E).

Fig. 7. The upper part of the figure shows direct ‘‘negative’’ printsof three adjacent sections through the rostral striatum stained forENK (A), TH (B), and CB (C). The pattern of TH-immunostaining ishighly heterogeneous in the caudate nucleus and putamen. In bothnuclei the TH-poor striosomes are in perfect register with areas of highENK- and poor CB-immunoreactivity (see arrows). The lower part of

the figure shows higher power views of the striosome pointed out byarrows above, as seen in ENK (D) and TH (E) stained adjacentsections. Note the strong ENK and weak TH immunostaining of thestriosome (asterisks) compared to the matrix. Scale bars 5 3.1 mm in A(also valid for B and C); 250 µm in D (also valid for E).


Figure 8


DISCUSSION

The results of the present study indicate that thechemical heterogeneity of the human striatum is morecomplex than the simple subdivision into striosome/matrixcompartments. Our detailed analysis of the distribution ofa wide variety of neurochemical markers reveals that thestriosomal compartment is in itself heterogeneous, beingcomposed of at least two distinct compartments: a core anda peripheral region. Our findings also show significantchanges in the chemical features of striosomes along therostrocaudal axis of the striatum.

Our data are largely in agreement with the results ofprevious studies in human and non-human primates,which have reported a complex distribution of certainchemical markers within striosomes (Martin et al., 1991;

Holt et al., 1997). In colchicine-treated monkeys, for ex-ample, some ENK-ir patches in the striatum were found tocontain a large number of ENK-ir neurons and terminals,whereas others harbored few ENK-ir neurons in their centralpart but many ENK-ir cell bodies and terminals at theirperiphery (Martin et al., 1991). In human caudate nucleus andputamen, certain ENK- and SP-ir ringed striosomes, whichcontain a poorly immunoreactive center, were seen to beintermingled with other small and densely stained immu-noreactive patches, as first demonstrated by Graybiel(1984) and recently confirmed by Holt et al. (1997).

Our study demonstrates that other striatal chemicalmarkers, such as LAMP and AChE, are heterogeneouslydistributed in striosomes. In the case of LAMP, our datareveal that the distribution of this protein closely follows

Fig. 9. A,B: Photomicrographs of a typical striosome lying in thecaudal putamen, as seen in adjacent sections stained for CB and TH.Arrows point to blood vessels as landmarks. Note the presence of fibersimmunostained for either CB or TH (small arrows). Some of thesefibers can be followed as far as the matrix. C: High power view of onestriosome in the caudal putamen as seen in CB-stained section. Note

the CB-ir fibers (arrows) that appear to emerge from the core of thestriosome and course through the periphery of the striosome en routeto the matrix. D: High-power view of some intensely stained CB-irneurons (arrows) and numerous long CB-ir processed in the lateralsector of the caudal putamen. Scale bars 5 250 µm in A (also valid forB); 200 µm in C; 100 µm in D.

TABLE 3. Relative Densities of Stained Elements Between the Two Striosomal Regions, Core and Periphery, and the Matrix Compartment at Rostraland Caudal Levels of the Striatum1

ENK SP LAMP CB PV CR AChE ChAT NADPH-d TH

Rostral striosomesCore 6 1 6 2 2 6 6 2 6 2Periphery 1 1 1 6/1 2 6 2 2 6 2

Caudal striosomesCore 6 1 6 2 2 6 6 2 6 111Periphery 1 1 1 11 2 6 2 1 6 11

Matrix 2 2 2 1 1 1 1 1 1 1

1AChE, acetylcholinesterase; NADPH-d, NADPH-diaphorase. For other abbreviations, see Table 2.


that of ENK, both substances being mainly confined to theperipheral region of striosomes in human. Earlier studieshave shown that LAMP is a specific marker of the strio-somal compartment in cats and squirrel monkeys (Chesse-let et al., 1991; Cote et al., 1995), but no allusion was madeto a possible heterogeneity in regard to the intrastriosomaldistribution of this protein. In fact, the presence of LAMPin the human striatum has never been previously re-ported.

The present study has also revealed that the heterogene-ity of striosomes is obvious even in sections stained forAChE, which was the first marker to be used for theidentification of striosomes (Graybiel and Ragsdale, 1978).In the human, the AChE-poor striosomes correspond toareas containing high levels of µ-opiate receptors andprodynorphin mRNA and low densities of dopamine up-take sites (Hurd and Herkenham, 1995). In our study, theAChE-poor striosomes were found to be composed of aperipheral region largely devoid of AChE surrounding acore showing a moderate AChE staining. In a previousinvestigation of the human striatum, Faull et al. (1989)have described an AChE-negative annular region mark-edly enriched in neurotensin receptors that lay betweenthe AChE-poor striosomes and the matrix. These authorsconsidered this annular region as a third neurochemicalcompartment of the human striatum, distinct from thestriosome/matrix compartments. However, our data indi-cate that the annular region described by Faull et al.(1989) cannot be considered as a distinct striatal compart-ment. Instead, this area appears to correspond to theAChE-poor periphery of striosomes, as described in thepresent study.

One of the major findings of the present study is that CB,which is considered one of the most reliable markers of thematrix (Gerfen et al., 1985), also intensely labels theperipheral region of striosomes. In humans, therefore, theabsence of CB can be taken as indicative of only the core ofstriosomes. In contrast, striatal patches outlined by PV-immunoreactivity, which were found to align with ENK-irstriosomes in humans (Waldvogel and Faull, 1993), corre-spond to entire striosomes, as shown in the present study.Both the core and the periphery of striosomes display asimilarly weak PV immunoreactivity. The fact that theentire striosomes are devoid of PV whereas only the corelacks CB, may explain that certain PV-poor regions werefound to match CB-rich regions in the human striatum(Holt et al., 1997). It is also worth noting that the smalland densely stained zones, which are intermingled withringed striosomes in both ENK- and LAMP-immuno-stained coronal sections, do not represent distinct entities.Instead, when carefully followed on several adjacent sec-tions, these small structures appear to correspond toeither the beginning or the end of striosomes.

Overall, our data regarding the compartmental distribu-tion of ChAT- and TH-ir neuropil are in agreement withthe results of previous studies in humans (Hirsch et al.,1989; Mesulam et al., 1992; Holt et al., 1996, 1997).However, the present investigation has provided the firstdemonstration that TH-poor striosomes contain a centralcore that stains more intensely for the enzyme than itsperipheral region. Furthermore, our study has broughtnew information on the distribution of ChAT-ir perikaryawithin striosomes. Among other things, we have shown 1)that the distribution of ChAT-ir neurons within striosomesobeys the pattern of subdivision of these entities into a core

and a peripheral region, as defined by other chemicalmarkers; and 2) that the number of ChAT-ir cell bodieswithin striosomes is significantly higher at caudal thanrostral levels (Table 3). We also found that the peripheralregion of striosomes contains a large number of bothChAT-ir neurons and large-size CR-ir neurons. This find-ing concurs with the results of our previous colocalizationstudies, which show that about 80% of all cholinergicinterneurons in the human striatum express CR (Parent etal., 1996a; Cicchetti et al., 1998). Interestingly, striatalneurons that display ChAT and/or CR immunoreactivityhave been shown to express the SP receptor subunitneurokinin-1 (NK1) (Aubry et al., 1994; Cicchetti et al.,1996). The peripheral region of striosomes being markedlyenriched in ChAT- and CR-ir neurons may thus correspondto the so-called striocapsular zone, which surrounds theCB-poor striosomes in macaque monkeys (Jakab et al.,1996). This striocapsular zone was characterized by thefact that it harbors numerous NK1-expressing perikaryascattered within a dense SP- and NK1-ir neuropil (Jakabet al., 1996).

The compartmental distribution of NADPH-d has beenreported previously in rats, cats, and humans (Graybiel etal., 1981; Chesselet and Graybiel, 1986; Sandell et al.,1986; Kowall et al., 1987; Kubota and Kawaguchi, 1993;Morton et al., 1993). All these studies agree that NADPH-dactivity is higher in the matrix than in striosomes and thatNADPH-d-positive neurons abound preferentially at theborders of the two major striatal compartments. Ourresults confirm that striosomes in humans are oftencompletely surrounded by clusters of NADPH-d neurons.However, our data indicate that isolated NADPH-d neu-rons also occur within striosomal boundaries in human.Furthermore, these intrastriosomal NADPH-d-positiveneurons, which represent less than 5% of the total striatalNADPH-d neurons in humans (Kowall et al., 1987), werefound to be equally abundant in the core and peripheralregion of striosomes.

Functional considerations

On the basis of the fact that the core and peripheralregion of striosomes display different chemical features,we postulate that these two regions represent two distinctfunctional domains of the striosomal compartment. Theconcept of a dual striosomal organization presupposes thatthe two sectors of striosomes belong, at least in part, to twodistinct anatomical systems. Although anatomical connec-tions cannot be studied directly in human, our immunohis-tochemical data suggest that the two striosomal domainsare indeed hodologically distinct. For example, LAMP is areliable marker of limbic system connections (Levitt, 1984)and striosomes are believe to be the preferential targets oflimbic striatal afferents (Gerfen, 1984; Donoghue andHerkenham, 1986; Graybiel, 1990). Hence, the fact thatLAMP immunostaining is much more intense in theperiphery than in the core of striosomes suggests thatstriatal limbic afferents arborize more profusely in theperipheral zone than in the core of striosomes in human.

The peripheral region of striosomes displays a moreintense ENK-immunoreactivity than the central core. Ifthe density of this immunostaining reflects the number ofENK-ir neurons in human, as has been demonstrated incats and monkeys treated with colchicine (Graybiel andChesselet, 1984; Martin et al., 1991), this finding wouldindicate that the peripheral region of striosomes harbors a


greater population of ENK-ir neurons than the core. ThisENK-rich periphery may thus be an important source ofenkephalinergic striatopallidal fibers, which terminatepreferentially in the external segment of the globus palli-dus. This finding may have some implications in thepathophysiology of certain neurodegenerative diseases,such as Huntington’s chorea. In Huntington’s disease theENK-containing striatal projection to the external seg-ment of the globus pallidus has been shown to degeneratebefore the SP-containing striatofugal fibers projecting tothe internal segment of the globus pallidus and/or thesubstantia nigra pars reticulata (Reiner et al., 1988). Thisearly degeneration of striatopallidal enkephalinergic neu-rons has been associated with the choreiform movementsthat characterize the early stages of this disease (Reiner etal., 1988). Furthermore, Hedreen and Folstein (1995) haverecently demonstrated that striosomes are selectively af-fected at the early stages of Huntington’s disease, and theysuggested that this loss of striosomal neurons mightconcur with the development of choreiform movements. If,as postulated above, the peripheral region of striosomes isindeed the main source of enkephalinergic projectionsfrom striosomes to the globus pallidus, the choreic move-ments that characterize the early stages of Huntington’sdisease could be specifically related to the degeneration ofthis striosomal domain.

The finding that striosomes display a core region whosestaining for AChE is more intense than that of theperipheral region, although less intense than that of thematrix, may also reflect some specific connections. The aretwo major sources of AChE in the striatum: 1) the largecholinergic interneurons, which express high levels ofChAT and AChE (Levey et al., 1983; Eckenstein andSofroniew, 1983); and 2) the axon terminals of the nigro-striatal neurons (Greenfield et al., 1983; Weston andGreenfield, 1986), which can release a significant amountof the enzyme in the striatum (Greenfield et al., 1983). Thefact that the striosomal core is largely devoid of cholinergicinterneurons, as shown in the present study, indicates thatthe AChE staining found in this portion of striosomesoriginates principally from the dopaminergic neurons ofthe substantia nigra pars compacta. In support of such aview is the fact that the core of striosomes stains moreintensely for TH than their periphery. Altogether, thesefindings indicate that the dopaminergic innervation ofstriosomes in the human is largely oriented toward thecore region.

Previous tract-tracing studies in rats, cats, and monkeyshave revealed the existence of subsystems within thedopaminergic nigrostriatal pathway that terminate prefer-entially in striosomes or in the matrix compartment of thestriatum (Moon-Edley and Herkenham, 1983; Gerfen etal., 1987; Jimenez-Castellanos and Graybiel, 1987; Langerand Graybiel, 1989). In cats, the zone of the substantianigra pars compacta projecting more specifically to strio-somes was found to display a less intense AChE stainingthan that projecting to the matrix (Jimenez-Castellanosand Graybiel, 1987). Our data in the human show that theAChE staining in the core of striosomes is less intensethan that in the matrix, but more intense than that in theperipheral zone of striosomes. These findings suggest thatthe innervation of the matrix and striosomes, as well asthat of the two striosomal domains, in the human mayoriginate from nigral cell populations that are distinct

from one another and display different levels of AChEactivity.

In the caudal portion of the putamen, numerous TH- andCB-ir fibers were seen crossing the boundary of strio-somes, and these fibers could be followed for long distanceswithin the matrix. These long fibers could in fact expressboth TH and CB and originate from the dorsal tier of thesubstantia nigra pars compacta, which harbor neuronsthat express both TH and CB and project specifically to thematrix (Gerfen et al., 1987; Lavoie and Parent, 1991).Assuming that these fibers are coursing from striosomes tothe matrix and indeed coexpress TH and CB, it may behypothesized that all nigrostriatal axons course throughthe striosomal network, including those that arise fromthe dorsal tier of the substantia nigra and that target thematrix.

Some striatal interneurons, particularly those contain-ing NADPH-d or expressing ChAT or CR, have been foundto be largely confined to the borders of the two majorstriatal compartments (Kubota and Kawaguchi, 1993;Holt et al., 1996; present study). Because of their preferen-tial localization, NADPH-d- and ChAT-positive interneu-rons are believed to mediate interactions between striatalprojection neurons of both compartments (Graybiel et al.,1986, 1994; Aosaki et al., 1995; Kawaguchi, 1997). In thepresent study, clusters of NADPH-d-containing neurons,as well as more isolated large CR-ir interneurons, wereencountered along the borders between the two striatalcompartments. Neurons immunoreactive for ChAT or CRwere also found to be confined to the peripheral region,abounding particularly at the borders between either thetwo striosomal regions or between the peripheral region ofstriosomes and the extrastriosomal matrix. Their strategicposition at these various border zones indicates that thesetwo populations of chemospecific striatal interneuronsmay act as a functional interface between the two majorstriatal compartments, as well as between the core and theperiphery of single striosomes.

Concluding remarks

The present study has provided a detailed account of thechemical features of the striosomal compartment through-out the entire rostrocaudal extent of the human striatum.This investigation has demonstrated that striosomes arecomposed of two chemically distinct regions, a core and aperiphery, which are likely to play a different role in thefunctional organization of the human striatum. The pre-sent study has also detected certain variations in thechemical features of striosomes that occur along therostrocaudal axis of the striatum and that may concerneither one of the two striosomal sectors or both of thesesubdivisions. Our data indicate that the chemical anatomyof the striosomal compartment is particularly complex inhumans and that this chemical heterogeneity may havesome bearing on the functional organization of this majorcomponent of the basal ganglia.

ACKNOWLEDGMENTS

The authors express their sincere gratitude to CaroleEmond and Lisette Bertrand for their skillful technicalassistance. We also thank Dr. Pat Levitt for his generousgift of LAMP antibodies and Dr. Michel Marois for havingprovided the postmortem material used in the presentstudy. This work was supported by grants MT-5781 from


the Medical Research Council of Canada and by the KillamProgram of the Canada Council for the Arts to A. Parent. L.Prensa was supported by a grant from the Comunidad deMadrid and J. M. Gimenez-Amaya by FIS 96/0488.

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Chemical heterogeneity of the striosomal compartment in the human striatum