Proc. Nati. Acad. Sci. USAVol. 86, pp. 9020-9024, November 1989Neurobiology

Dopamine uptake sites in the striatum are distributed differentiallyin striosome and matrix compartments

(nigrostriatal/ligand binding/mazindol/l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/cocaine)

ANN M. GRAYBIEL* AND ROSARIO MORATALLADepartment of Brain and Cognitive Sciences, E25-618, Massachusetts Institute of Technology, 45 Carleton Street, Cambridge, MA 02139

Contributed by Ann M. Graybiel, August 16, 1989

ABSTRACT A major mechanism of neurotransmitter in-activation at catecholaminergic synapses is reuptake ofreleasedtransmitter at high-affinity uptake sites on presynaptic termi-nals. We have analyzed the anatomical distribution of site-selective ligand binding for dopamine uptake sites in thestriatum of rat, cat, and monkey. We report here that desi-pramine-sensitive [31H]mazindol binding sites have highly het-erogeneous distributions in the dorsal and the ventral striatum.In the caudate nucleus of cat and monkey, [3Hlmazindolbinding observes striosomal ordering, being reduced in strio-somes and heightened in the extrastriosomal matrix. Some localheterogeneity appears in the ventral caudoputamen of the rat.Different subdivisions of the nucleus accumbens also havedifferent binding levels. These rmfdings suggest that somefunctional effects of psychoactive drugs, such as cocaine, thatbind to the dopamine-uptake complex could be related to thedistribution of these specific uptake sites. The rindings alsoraise the possibility that these distributions could result inselective neuronal vulnerability to neurotoxins, such as 1-methyl4-phenylpyridine (MPP+), that depend on the dopam-ine-uptake complex for entry into neurons.

A number of powerful psychoactive drugs, including cocaineand amphetamine, bind to sites on the presynaptic uptakecomplex that constitutes a major means for neurotransmitterinactivation at catecholaminergic synapses (1-3). Catechola-mine-uptake sites are also potential conduits for the entry ofneurotoxins and neurotoxin precursors into catecholaminer-gic synapses. This route is ofspecial interest in relation to thedrug-induced parkinsonian syndrome elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (4-6).The toxic oxidative metabolite of MPTP, 1-methyl-4-phenylpyridine (MPP+), is thought to enter catecholamine-containing neurons by means of the uptake transport mech-anism (7, 8). Blockade of dopaminergic and noradrenergicuptake sites with specific blockers prevents the toxic effectsof MPTP administration on dopaminergic and noradrenergicneurons (9-12). The precise distribution of high-affinity up-take sites within the catecholaminergic systems of the brainis thus important not only in relation to the normal physiologyof these pathways but also in relation to the selective effectsof drugs and environmental toxins on catecholaminergicfunction.

In the experiments reported here we used in vitro ligand-binding autoradiography with the radiolabeled catecholamineuptake blocker [3H]mazindol in the presence of desipramine(13, 14) to study the detailed distribution of high-affinitydopamine uptake sites in the striatum. The results suggestthat in cat and monkey there are notable differences in theamounts of [3H]mazindol binding in the two main tissuecompartments of the dorsal striatum, the striosomes and the

extrastriosomal matrix (15); and they suggest that differentsubdivisions of the ventral striatum in rat, cat, and monkeyare also characterized by different levels of binding. Thesedifferential anatomical distributions may help to account forsome of the functional and toxicological selectivities of drugsacting on the mesostriatal system.

MATERIALS AND METHODS

Brains were removed from one cat, two rats, (one Sprague-Dawley; one Wistar), one squirrel monkey, and one cyno-molgus monkey after induction of terminal anaesthesia withsodium pentobarbital (Nembutal). Blocks containing the stri-atum were frozen in pulverized dry ice and were cut trans-versely at 20 gm on a cryostat immediately or after briefstorage at -70'C. Sections cut at -12'C and thaw-mountedonto "subbed" slides were dried at 40C in a dessicator undervacuum, stored at -20'C for at least 2 weeks, and thenincubated according to the protocols of O'Dell and Marshall(14) and Javitch et al. (13). For autoradiographic studies,sections were preincubated in 50 mM Tris-HCl (pH 7.9)containing 120 mM NaCI and 5 mM KCI at 40C (5 min), thenincubated in 50 mM Tris HCl (pH 7.9) containing 300 mMNaCl, 5 mM KCl, either 2 nM or 15 nM [3H]mazindol (15Ci/mmol; 1 Ci = 37 GBq; DuPont/NEN), and 0.3 ,Mdesipramine (40 min, 4°C), washed in fresh buffer (two 3-minwashes, 4°C), and briefly rinsed in distilled water (10 sec,4°C). Sections were dried under a cool airstream and appliedto 3H-sensitive film (Hyperfilm, Amersham) for 3 weeks at-20°C. Control sections were prepared in parallel by additionof 1 ,uM unlabeled mazindol or 30 ,uM benztropine to theincubation medium. Controls were applied to each film alongwith tritium standards ([3H]Micro-scales; Amersham). Afterautoradiographic exposure, selected sections were stainedfor acetylcholinesterase (AChE) or (for squirrel monkey)butyrylcholinesterase by slightly modified Geneser-Jensenand Blackstad protocols (15, 16) to permit detection ofstriosomes. Densitometry was carried out with a BIOCOM200 computer system (Les Ulis, France). A standard sam-pling zone was superimposed under cursor control on videoimages of [3Hlmazindol-poor zones and on adjacent [3H]maz-indol-rich tissue identified in films of sections through thecat's caudate nucleus. Relative grey-level values were cal-culated for all regions sampled, and values were converted tonCi/mg of tissue (equivalent weight) with BIOCOM softwareusing the [3H]Micro-scales. Mean levels of [3H]mazindolbinding were calculated for summed regions inside andoutside the [3H]mazindol-labeled compartments, and valueswere compared by Student's t test (2-tailed).

Abbreviations: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine; AChE, acetylcholinesterase.*To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 86 (1989) 9021

RESULTSDense [3H]mazindol binding appeared in the striatum in allbrains under normal incubation conditions and binding wasabsent in control sections. At mid-anterior levels through thecat's striatum, binding in the caudate nucleus was highlypatterned, with circumscribed zones of relatively reducedbinding punctuating otherwise densely labeled fields (Fig.1A). Most of the [3H]mazindol-poor zones could be identifiedas striosomes when the autoradiographic images were com-pared with AChE staining patterns developed from the samesections: the shapes and positions of the zones of low[3H]mazindol binding matched those ofthe AChE-poor zones(Fig. 1). Even in single films, the degree of patterning of[3H]mazindol was not equivalent at all anteroposterior anddorsoventral levels of the striatum. Binding tended to bemore homogeneous caudally and dorsolaterally in the cau-date nucleus, as did the AChE staining, and little patterningwas visible in the putamen. Occasionally, regions of rela-tively heightened binding were evident. Correspondents forthese were not identified in AChE stains.

Pockets of reduced [3H]mazindol binding were also ob-served in the caudate nucleus of the squirrel monkey (Fig. 2)and cynomolgus monkey (Fig. 3), but the autoradiographywas not as crisp as in the cat and not every AChE-poorstriosome could be matched to a [3H]mazindol-poor zone.Even so, it was possible in both primate species to confirmcorrespondence of many [3H]mazindol-poor zones with stri-osomes identified histochemically in the same sections afterautoradiography. Patterns of binding were quite diffuse in theputamen. No clear patchiness in [3H]mazindol binding wasevident in the central and dorsal regions of rat's caudoputa-men, but ventrally in the caudoputamen some zones of lowbinding did appear.

Densitometry performed for the cat's caudate nucleus(Table 1) indicated that the mean binding in the [3H]mazindol-poor zones was 51.23% of the mean binding in adjacentmatrix tissue (n = 63 sampled zones in four sections).There were marked differences in the intensity of binding

in different parts of the nucleus accumbens. As shown in Fig.4 for the cat, binding was dense rostrolaterally but was veryweak caudally and medially. Comparable variations were

FIG. 2. [3H]Mazindol binding in striatum of squirrel monkey.Zones ofreduced binding in the caudate nucleus (matching histochem-ically identified striosomes, data not shown) were mainly found in theventral half of the nucleus (example at asterisk). Slight heterogeneityis also evident in binding in putamen (see asterisk), and subdivision-specific variations in binding are present in the nucleus accumbens-ventral striatum. From case SQDAR-4. Photograph was printeddirectly from autoradiographic film. CN, caudate nucleus; P. puta-men; IC, internal capsule; NA, nucleus accumbens. (Bar = 1 mm.)

present in the monkeys, and in the rat the "shell" subdivisionof the nucleus accumbens (17) had much reduced bindingcompared to that of the core subdivision.

FIG. 1. [3H]Mazindol binding(A) and AChE activity (B) in serialtransverse sections through thestriatum of an adult cat. Photo-graph in A was directly printedfrom film; photograph in B is neg-ative-image print. Prominentpatches of reduced (3H]mazindolbinding appear in the central partof the caudate nucleus. These cor-respond to the AChE-poor strio-somes visible in B (examples atasterisks). From case CDAR-12.CN, caudate nucleus; P. putamen;IC, internal capsule; AC, anteriorcommissure; NA, nucleus accum-

*,,.^bens. (Bar = 1 mm.)

Neurobiology: Graybiel and Moratalla

9022 Neurobiology: Graybiel and Moratalla

FIG. 3. Sector of medial caudate nucleusof cynomolgus monkey illustrating corre-spondence (see asterisks) between zones ofreduced [3H]mazindol binding (A) and zonesof reduced AChE-staining (B). Note similar-ity in size and shape of zones in the twoimages. (A) Printed directly from film. (B)Printed in negative image. From case MR-1.Sections are 60 ,um apart. CN, caudate nu-cleus; P, putamen; IC, internal capsule; NA,nucleus accumbens. (Bar = 1 mm.)

DISCUSSIONThese observations have implications of at least two types.First, the findings indicate that pharmacologic treatmentsaffecting the dopamine uptake mechanism may have differ-ential effects on the striosome and matrix subdivisions of thedorsal striatum and on distinct subdivisions of the ventralstriatum. Second, the findings suggest that entry of potentialtoxins into dopamine-containing fibers in the striatum couldbe different for these striatal compartments.For the dorsal striatum the differences in [PH]mazindol

binding were clearest in the caudate nucleus and, amongspecies, were sharpest in the cat and only occasionallydetectable, and then only ventrally, in the rat's caudoputa-men. The lower numbers of uptake sites available for thebinding of [3H]mazindol in striosomes suggest that thereeither are fewer dopamine-containing terminals in this striatalcompartment or are fewer sites per terminal in striosomesthan in the extrastriosomal matrix, or, conceivably, that thesites in the two striatal compartments have different molec-ular conformations although both bind [3H]mazindol. Thereis no direct evidence yet to settle this issue. Both striosomesand matrix are richly innervated by fibers from the midbrain,but different cell groups within the nigral complex innervatethe two striatal compartments to different extents (18-20).Striosomes have lower tyrosine hydroxylase-like immuno-reactivity than the extrastriosomal matrix (21, 22). Thisdistinction, like that for the [3H]mazindol binding reportedhere, could reflect metabolic differences between striosome-directed and matrix-directed fibers rather than differences inthe respective densities of such fibers. Olson et al. (23) haveproposed such metabolic differences on the basis of experi-ments with tyrosine hydroxylase inhibitors.The compartmental selectivity in [3H]mazindol binding in

the caudate nucleus documented here provides additionalevidence for fundamental differences between dopaminergicmechanisms in striosomes and matrix as judged not only byDi-selective ligand binding [heightened in striosomes (24)] and

Table 1. Densitometry on [3H]mazindol binding in the caudatenucleus of the cat

[3H]Mazindol bound, Ci/mg of tissue matrixSection Striosome Matrix ratio, %763 10.60 ± 2.69* (10) 22.71 ± 2.30 (8) 46.7643 13.66 ± 1.98* (8) 22.45 ± 3.06 (5) 60.8715 10.60 ± 2.69* (10) 22.70 ± 2.30 (8) 46.7571 11.85 ± 3.91* (6) 22.67 ± 2.63 (6) 52.2

Total 11.73 ± 3.36 (34) 22.89 ± 3.55 (29) 51.23

Higher section numbers are for more rostral sections. Values for[3H]mazindol binding to striosome and matrix are mean ± SD.Numbers in parenthesis refer to n.*Significantly different from binding in matrix at P < 0.0001.

D2-selective ligand binding [heightened in the matrix (25)] butalso by presynaptic markers. Interestingly, all of these mark-ers display greater compartmental selectivity in the caudatenucleus than in the putamen and are not as clearly compart-mental in distribution in the rat (if they are at all) as they arein carnivores and primates. Amphetamine treatment in the rathas, however, been found to induce patchy anatomical distri-butions of transmitter-related compounds, including (presyn-aptic) tyrosine hydroxylase-immunoreactive patches (26) and(postsynaptic) dynorphin immunoreactive patches (27).

If synpatic release of dopamine is mainly from the newlysynthesized pool of transmitter (28), the difference in numberof uptake sites shown here for striosomes and matrix (about50% maximum detected difference) might not be reflectedfunctionally in these normally slow-firing neurons. Clearly,however, with intense firing and transmitter release or underthe influence of drugs inducing massive transmitter release,differences in the capacity for transmitter clearance by re-uptake could be functionally significant. Uptake-site block-ade by agents such as cocaine could also have markedlydifferent effects on striosomes and matrix. Interpretation ofsuch differences is now constrained by lack of informationabout the distribution of uptake sites on single terminals instriosomes and matrix; but given that the [3H]mazindolbinding site has been directly related to the [3H]cocainebinding site on the dopamine uptake-site complex (2), thepresent results suggest that striosome-matrix differences inuptake-site distribution should be taken into account inaddition to compartmental differences in D1/D2-binding-siteratios in analyzing drug effects on the nigrostriatal system.There are considerable dose-response differences in the

behavioral effects of drugs affecting catecholamine uptakesites, and some of these relate to the different expression ofbehaviors thought to be mediated preferentially by sensory-motor and limbicohypothalamic mechanisms (2, 29, 30). Thecontrasting pre- and postsynaptic character of the dopamineinnervations of striosomes and matrix could be essential to anunderstanding of these drug effects, for striosomes are pref-erentially related to a subset of limbic pathways and projectto the substantia nigra, whereas the extrastriosomal matrix islinked to sensorimotor regions and other limbic pathways andprojects preferentially into pallidothalamic and nigrotectalpathways (for review, see ref. 31).For the dopamine-containing mesolimbic system, there

also were striking regional inhomogeneities in uptake-sitedistribution, and these regional differences were at least asmarked as those for striosomes and matrix in the dorsalstriatum. Marshall (32) has already noted lower levels of[3H]dopamine uptake and [3H]mazindol binding in the ventralthan in the dorsal striatum in the rat, and the present findingsextend his observations by suggesting that different parts ofthe nucleus accumbens have different levels of [3H]mazindolbinding. Selective functional effects of drugs would be ex-

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 9023

IFIG. 4. Transverse sections at rostral (A)

and caudal (B) levels through the ventralstriatum of the cat illustrating the strong[3H]mazindol binding in anterior and lateralparts of the nucleus accumbens and olfac-tory tubercle and the weak binding in caudaland medial parts of the ventral striatum. Thesections were approximately 960 ,um apart.From case CDAR-4. Photographs wereprinted directly from the autoradiographicfilm. NA, nucleus accumbens; Olf T, olfac-tory tubercle; CN, caudate nucleus; IC, in-ternal capsule; AC, anterior commissure.(Bar = 1 mm.)

pected according to these differences as well (see refs. 17 and33). As in the dorsal striatum, there is no obvious relationbetween the degree of [3H]mazindol binding and knownintensities of total innervation by dopamine-containing fi-bers; for example, the dopamine-containing innervation isintense in the medial "shell" of the nucleus (17), where[3H]mazindol binding is relatively weak.The present observations have special significance in sug-

gesting that different subdivisions of the nigrostriatal andmesolimbic systems might have different vulnerabilities toMPTP intoxication. In fact, there is a striking similaritybetween the striosomal patterning of [3H]mazindol bindingshown here for the cat and the pattern of fiber degenerationobserved by Wilson et al. (34) and Turner et al. (35) in dogsacutely exposed to MPITP. At 8-14 days after MPTP injection,these authors found intense degeneration of fibers in theextrastriosomal matrix with relative sparing of fibers in thestriosomes. They also describe the striosomal pattern as beingclearest in the caudate nucleus at rostral and midanterior levelsand illustrate fiber degeneration in the nucleus accumbensmainly in the lateral rather than the medial part of the nucleus.Given the findings presented here, serious consideration

should be given to the possibility that such differentialpatterns of MPTP-induced fiber degeneration might reflectlocal inhomogeneities in uptake-site distribution within thestriatum, just as, in general, the greater vulnerability of moredorsal than more ventral striatal regions might be related togradients of uptake-site distribution (27). The possibility thatlocal variations in uptake-site distribution within the striatumconstrain the level of degeneration induced by acute expo-sure to MPTP is in accord with the finding that intrastriataladministration of MPTP can induce degenerative changes inneurons in the midbrain (36, 37). Such differential vulnera-bility of fiber terminals could account for patterns of partialsparing of midbrain neurons seen with dose schedules pro-ducing submaximal damage to the system (38, 39). To theextent that the MPTP model relates to other forms of par-kinsonism, the compartmental distributions of uptake sitesdescribed here could be important in constraining patterns ofmesencephalic neuronal loss in these disorders as well (40).

It is a pleasure to thank Mr. Glenn Holm, Mrs. Yona Dornay, andMrs. Judy Rankin for their technical support, Dr. Ann Kelley forreading the manuscript, Mr. Henry F. Hall, who is responsible for thephotography, and Dr. William Houlihan of the Sandoz ResearchInstitute for the gift of nonradioactive mazindol (Sanorex). This workwas funded by Grant NS25529-02 (Javits Neuroscience InvestigatorAward) from the National Institutes of Health, the National Alliancefor Research on Schizophrenia and Depression, the Scottish RiteSchizophrenia Research Program, The Seaver Institute, and the

National Parkinson Foundation. R.M. is a recipient of a fellowshipfrom Consejo Superior de Investigaciones Cientificas (Spain).

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