Phosphoinositide Second Messenger System Is Enriched in Striosomes

The Journal of Neuroscience, August 1993, 13(E): 3300-3308

Phosphoinositide Second Messenger System Is Enriched in Striosomes: lmmunohistochemical Demonstration of lnositol 1,4,5=Trisphosphate Receptors and Phospholipase C ,8 and y in Primate Basal Ganglia

Majid Fotuhi,’ Ted M. Dawson,1s2 Alan H. Sharp,’ Lee J. Martin,3 Ann M. Graybiel,4 and Solomon H. Snyder’ ‘Departments of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry, *Department of Neurology, and 3Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and 4Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

The neurochemical organization of the basal ganglia has rons embedded in the medullary layer between the putamen been studied extensively with respect to neurotransmitters, and the globus pallidus. neuropeptides, and their receptors. The chemoarchitecture These observations demonstrate that the phosphoinosi- of the striatum has been found particularly striking, because tide second messenger system is selectively enhanced in it distinguishes many substances by their relative distribu- neuronal subsystems of the basal ganglia, including strio- tions within the striosome and matrix compartments of the somes, and suggest that signaling by phosphoinositide path- striatum. Very little is yet known about the differential dis- ways elicits discrete effects on input-output processing by tribution of second messenger systems in the basal ganglia, the basal ganglia. however, and no information is available about whether the [Key words: phosphoinositide, phospholipase C, striatum, distribution of second messenger systems is related to the striosome, primate] prominent neurochemical compartmentalization of the stria- turn. We have examined the distribution of the phosphoinosi- An important organizational principle of the basal ganglia in- tide second messenger system in the primate basal ganglia volves differentiation of the neostriatum into compartments and substantia nigra, as detected with polyclonal antisera referred to as striosomes and matrix (Graybiel and Ragsdale, against the inositol 1,4,5-trisphosphate receptor (IP,R), and 1978). Neurons in striosomesand matrix differ in their ontogeny monoclonal antisera against phospholipase C @ (PLC@) and as well as in their afferent and efferent connections. Neurons in phospholipase C y (PLOy). striosomes mature earlier than those in the matrix, and the In the striatum, immunostaining for each of the three pro- striosomal system receives denseinputs from prefrontal orbi- teins was present predominantly in medium-sized neuronal toinsular parts of the cortex, whereasthe matrix input predom- perikarya and in the neuropil. Circumscribed zones of en- inantly originates in other regions including sensorimotor cor- hanced IP,R, PLC& and PLCr immunoreactivity appeared in tical areas (Fishell and van der Kooy, 1982; Gerfen, 1984; a background of generally weaker staining, and these zones Ragsdaleand Graybiel, 1988, 1990;Flaherty and Graybiel, 1991). corresponded to striosomes as identified by calbindin D,,, Striosomal neuronsappear to receive their dopamine-contain- and substance P immunostaining in adjacent sections. Thus, ing input mainly from densocellularislands in ventral parts of the richest representation of the phosphoinositide system the substantia nigra pars compacta (SNc) and to project mainly in the primate striatum appears to be in striosomes. back to medial nigral regions. Neurons of the striatal matrix, In the substantia nigra pars compacta, neurons and neu- by contrast, receive their dopamine-containing inputs mainly ropil were immunopositive, but in the substantia nigra pars from other neurons of the nigral complex and project mainly reticulata and in each segment of the globus pallidus, im- to the external (GPe) and internal (GPi) segmentsof the globus munostaining was mainly confined to the neuropil. Perikaryal pallidus (GP) and to the substantia nigra pars reticulata (SNr) PCL-r immunoreactivity in the absence of detectable PLC/3 (Graybiel et al., 1979; Gerfen, 1984; Donoghueand Herkenham, or IP,R immunolabeling was found in the magnocellular neu- 1986; Gerfen et al., 1987; Jimtnez-Castellanos and Graybiel, 1987, 1989; GimCnez-Amaya and Graybiel, 199 1). Striosomesand matrix differ also in their content of neuro- Received Apr. 21, 1992; revised Nov. 19, 1992; accepted Jan. 12, 1993. transmitters and receptors. For instance, striosomesare selec- This work was supported by USPHS Grant MH-18501, Research Scientist tively enriched in limbic-associatedmembrane protein (LAMP) Award DA-00074 to S.H.S., Javits Award NS-25529 to A.M.G., and a gift from (Chesseletet al., 1991), whereas the matrix is enriched in cal- Bristol-Myers-Squibb. T.M.D. was supported by a Pfizer postdoctoral fellowship and grants from the Dana Foundation, the French Foundation for Alzheimer’s bindin D,,,, AChE, ChAT, somatostatin, and tyrosine hydrox- Research, and the American Academy of Neurology. M.F. was supported in part ylase(TH). Opiate and dopamine D,-like receptorsare enriched by Fonds pour Formation de Chercheurs et L’Aide rl La Recherche (FCAR) of Quebec, Canada. We thank Dr. Sue Goo Rhee for a generous gift of antisera to in striosomes,whereas dopamine D,-like and some muscarinic phospholipase C @ and y. choline& receptors predominate in the matrix (Pert et al., Correspondence should be addressed to Dr. Snyder, Departments of Neuro- 1976; Herkenham and Pert, 1981; Graybiel et al., 1986; Joyce science, Pharmacology and Molecular Sciences, and Psychiatry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. et al., 1986; Besson et al., 1988; for a review, see Graybiel, Copyright 0 1993 Society for Neuroscience 0270-6474/93/l 33300-09%05.00/O 1990). There are also regional variations in these striosomel The Journal of Neuroscience, August 1993, 13(E) 3301 matrix distributions for individual transmitter-related markers perature. After three washes of 10 min each in TBS, the blots were (Graybiel and Ragsdale, 1978; Besson et al., 1990; Martin et developed using 4-chloro- 1-naphthol as the substrate. al., 1991) and species differences as well. Immunohistochemistry. All steps were carried out in TBS and immunostaining for each antiserum was performed under the following This differential distribution of multiple neurotransmitter- conditions. Serial transverse sections were perrneabilized in 0.4% T&on related elements in striosome and matrix compartments sug- X- 100 for 30 min and pretreated for an additional 30 min in 4% normal gests functional specificity, but it is difficult to relate them to goat serum (NGS) or, for sections to be incubated in goat IP,R anti- specific extrapyramidal functions (Albin et al., 1989). Neuro- serum, in normal rabbit serum (NRS) or, for sections to bc incubated in PLC& PLCr, or calbindin D,,,, in normal horse serum (NHS) con- transmitters, however, act through only a few second messenger taining 0.1% Triton X-100. They were then incubated overnight in systems, with the CAMP and phosphoinositide (PI) systems pre- primary antiserum (see above for dilutions) containing 2% NGS (or 2% dominating, so what seems complex at the neurotransmitter NRS for IP,R or 2% NHS for PLCP, PLCr, or calbindin D,,,), 0.1% level may be systematically ordered at the second messenger Triton, and 0.02% sodium azide at 4°C. level. PI markers, such as the inositol 1,4,5-trisphosphate (IP,) After three washes of 10 min each in TBS, sections were incubated with biotinylated secondary anti-goat or anti-rabbit antibodies (1:200) receptor (IP,R) and protein kinase C (PKC), and markers of the (ABC elite kits of Vector Laboratories, Burlingame, CA) in 0.1% Triton CAMP system, such as 3H-forskolin binding, are differentially X-100 at room temperature for 1 hr. Sections were washed 3 x 10 min distributed in the brain with a number of complementary lo- in TBS and incubated in an avidin-biotin-peroxidase complex (1:50) calizations (Worley et al., 1986a,b, 1987, 1989). In the extra- for 45 min. Finally, they were washed (3 x 10 min) and developed in the presence of 0.02% hydrogen peroxide with diaminobenzidine as the pyramidal system, PI markers occur in high densities in the chromogen. Sections were rinsed thoroughly and mounted on subbed corpus striatum and the SNr. The neuroanatomical distributions glass slides, dehydrated, and coverslipped with Permount. Striosomes of phospholipase C (PLC) (Gerfen et al., 1988) PKC (Huang et were identified as zones with few neurons expressing calbindin D,,, or al., 1988; Saito et al., 1988; Yoshihara et al., 1991) and IP,R enkephalin, or by enrichment in substance P-like immunoreactivity. (Nakanishi et al., 1991; Sharp et al., in press) have been de- Controls for immunocytochemical staining for the IP,R antibody were prepared by preadsorbing with excess (loo-fold) IP,R protein. scribed in the rat brain, but these messenger distributions have Control sections for antisera to PLCP, PLCr, calbindin D,,,, TH, sub- not been mapped in primate, in which the striatum and substance P, and met-enkephalin were prepared by incubating sections in stantia nigra are highly differentiated as compared to these regions the appropriate pre-immune serum, respectively. in rat. In particular, the relationship of the PI system to the The density of immunostaining for IP,R, PLC& and PLCy was arbitrarily assigned value based on visual inspection of sections from all striosome and matrix systems has not been characterized. In immunostaining experiments, with the highest densities being assigned the present study we analyzed the distribution of immunoreac- a value of 5 and the lowest densities a value of 0. tivity for IP,R and PLCP and PLCr in the primate basal ganglia and substantia nigra. We report that, in addition to a broad distribution of these PI markers in the basal ganglia, there is a Results selective enrichment of PI markers in striosomes of the striatum. Characterization of immunoreactivityfor IP,R, PLCp, and PLCr. For immunohistochemical localization of IP,R, we employed two antisera, raised in rabbit and in goat, against IP,R protein Methods purified from rat cerebellum (Supattapone et al., 1988; Sharp Tissue preparation. Six rhesus monkeys (Macaca mulatta), two cyno- et al., in press). Both of these antisera have been shown to react molgus monkeys (Macaca.fascicuZaris), and two squirrel monkeys (Sui- selectively and with considerable potency with IP,R protein in miri sciureus) were perfused transcardially, under deep anesthesia, ‘with 0.1 M phosphate-buffered saline (PBS), pH 7.4, followed by 4% para- a range of vertebrate species including salamander, rat, guinea formaldehyde (PF) in 0.1 M PBS with or without 5% sucrose. The brains pig, monkeys, and humans (Peng et al., 199 1). PLCP and PLCr were removed immediately, blocked in coronal planes, postfixed over- were detected with murine monoclonal antibodies prepared nizht in 4% PF in 0.1 M PBS. and crvonrotected in 20% elvcerol. 0.1 M against the two forms of PLC purified from bovine brain (Ryu PBS for 24-48 hr. Blocks containing ihe basal ganglia ind substantia et al., 1987). These antibodies have been shown to bind selec- nigra were frozen and cut on a sliding microtome at 40 wrn into 50 mM T&-buffered saline (TBS; pH 7.4). Serial floating sections were pro- tively to PLC/3 and PLCr in rat brain (Gerfen et al., 1988). cessed for immunohistochemistry. In Western blots of rhesus monkey forebrain extracts, the goat Antisera Affinity-purified polyclonal IP,R antisera were produced in antiserum to IP,R stained one band at about 260 kDa, corre- rabbits and in a goat immunized with purified IP,R, as described else- sponding to the known molecular weight of IP,R monomers where (Supattapone et al., 1988; Peng et al., 1991; Sharp et al., in press). Mouse monoclonal antibodies for PLCP and PLCy were gifts from Dr. (Supattapone et al., 1988) (Fig. 1). Similar staining with the S. G. Rhee, and those for calbindin D,,,, for TH, and for substance P rabbit antiserum marked an identical 260 kDa band (data not were purchased from Sigma, Boehringer Mannheim, and Sera Lab, re- shown). The monomers for PLCp and PLCr are about 145-150 spectively. Antiserum against met-enkephalin was kindly contributed kDa (Ryu et al., 1987). Western blot analysis of the same mon- by Dr. R. P. Elde or purchased from Incstar. For both Western blot key forebrain extracts with monoclonal antibodies against these analysis and immunohistochemistry, the following dilutions of antisera were used: IP,R, 1:500; PLC@ and PLCr, 1:200; calbindin D,,,, 1: 1000; two enzymes showed single prominent bands at about 145-150 TH, 1:200; substance P, 1: 1000; met-enkephalin, 1:2000. kDa. Western blotting. To test the selectivity of the IP,R, PLC& and PLCr To establish the validity of the IP,R immunohistochemical antisera in the monkey brain, Western blot analysis was carried out. staining, we incubated some sections with IP,R antiserum Briefly, samples of frozen monkey forebrain were homogenized, and their protein content was assayed using Pierce BCA reagent.-Equal quan- preadsorbed with IP,R protein. The preadsorption eliminated tities of protein were subjected to SDS-PAGE using 7.5% polyacryl- immunostaining almost completely (Fig. 2B). Immunostaining amide gels, and the separated proteins were transferred to Immobilon-P of rat brain with the monoclonal antibodies to PLCP and PLCr membranes. The blots were pretreated with 3% bovine serum albumin employed here has been shown to be eliminated by preadsorp- (BSA) in TBS for 2 hr before an overnight incubation at 4°C with IP,R, tion with the corresponding proteins (Gerfen et al., 1988). We PLC& and PLC-y antisera (dilutions: 1:500, 1:200, 1:200, respectively) with 3% BSA in TBS. The blots were then washed three times for 20 observed no immunoreactivity for PLCP and PLCy control in min each with 3% BSA in TBS, and were incubated with horseradish sections incubated in preimmune mouse serum (Fig. 2D,F). peroxidase-linked secondary antibodies ( 1: 1500) for 1 hr at room tem- IP,R, PLCp, and PLCy distributions in the striatum. Im- 3302 Fotuhi et al. l Phosphoinositide Markers in Primate Basal Ganglia

Table 1. Distribution of immunoreactivity for IP,R, PLC& and PLCr in perikarya and neuropil of the primate basal ganglia

IP,R PLCj3 PLCy Peri- Neuro- Peri- Nemo- Peri- Neuro- katya pi1 karya pi1 karya pi1 Striatum 2-4 4 5 5 3 3 GPe and GPi 0 4 0 4 0 4 SNc 4 2 4 2 4 2 SNr 0 4 0 4 2 4 Lateral medullary lamina 0 0 0 0 2 0 Data show a distinct pattern of distribution of IP,R, PLC@, and PLCy immunoreactivity in the primate basal ganglia. In striatum and SNc, IP,R, PLC& and PLCy am present in both perikarya and neuropil. With the exception of the presence of PLCy immunoreactivity in cell bodies of SNr, the immunoreactivity for these three proteins is limited primarily to the neuropil of striatal target nuclei, GPe, GPi, and SNr. Also, PLCr, but not PLC@ or IP,R, immunoreactivity occurs in magnocellular neurons of the intralaminar layer between striatum and GP (see Fig. 8). Throughout the basal ganglia, PLCfi immunoreactivity is denser, in any given area, than IP,R and PLCy, while PLCy immunoreactivity is weaker, though more widespread, than IP,R and PLCj3. In this table, based on visual inspection of all immunostaining experiments considered together, the highest and lowest densities of stainings are arbitrarily assigned values of 5 and 0, respectively.

with each other (Fig. 2). These zones of enriched immunostaining were most reliably identified with the PLQ3 antiserum, with which immunostaining was most robust (Figs. 2, 4). Immuno- staining for IP,R, PLC& and PLCy was also inhomogeneousin the putamen and ventral striatum, although the zones of heightened immunoreactivity were lessdiscrete than those observed in the caudate nucleus. To determine whether the zones enriched in these PI im- munomarkers were aligned with striosomesin the caudate nucleus,we compared their locations with those of calbindin-poor zones (rhesus monkeys), substance P-enriched zones (rhesus monkeys), or enkephalin-poor zones (cynomolgus monkey, squirrel monkeys; data not shown) known to correspond to striosomes(Graybiel and Chesselet,1984; Gerfen et al., 1985; Figure 1. Characterization of immunoreactivity for IP,R, PLC& and PLCr by Western blot analysis of rhesus monkey forebrain extracts. Martin et al., 1991). As demonstrated in Figures 3 and 4 (and Antisera against IP,R, PLC& and PLQ recognize single bands with data not shown),there wasclose spatial correspondencebetween approximate molecular weights of 260 and 150 kDa, respectively, cor- the striosomesand the regionsexpressing enhanced IP,R, PLCP responding to molecular weights of IP,R and PLC proteins. (Figs. 3,4), and PLCy (data not shown) immunoreactivity. This was true both in the more medial parts of the caudate nucleus munostaining for IP,R, PLC& and PLCy appeared in the cau- and in the middle and lateral parts of the nucleus. For some of date nucleus, putamen, and ventral striatum in each of the mon- the zones, the IP,R immunostaining seemedmore intense in key brains. Immunostaining with each of the antisera appeared the peripheral parts of the striosomes. Some of the dorsal PI- in both the neuropil and cell bodies (Figs. 3, 4; Table 1). The enriched zones were bordered by thin rims of reduced staining abundance and appearance of the IP,R-, PLCp-, and PLCy- (Fig. 2). These variations were not always noticeable, but the immunoreactive cells suggestedthat theseproteins were mainly stronger neuropil stainingof the striosomescompared to nearby in medium-sized neurons of the striatum. Immunostaining for matrix was always pronounced in well-stained sections. PLCP tended to be stronger than that for PLCr (Figs. 2, 3) as IP,R, PLCP, and PLCr immunostaining appeared in nu- noted also by Gerfen et al. (1988) in the rat. The ventral striatum merous neuronal cell bodies of medium size (Figs. 4C, 5), in- expressed higher levels of immunoreactivity for each of the cluding large numbers of medium-sized spiny neurons. The proteins than did the dorsal striatum (data not shown). In some number of immunostained cell bodies appeared similar in the monkeys, the immunostaining within the caudate nucleustend- striosomeand matrix compartments with the IP,R, PLCp (Figs. ed to be highest medially (data not shown). 3,4), and PLCr (data not shown) antisera. The relative enrich- In the caudate nucleus especially, immunostaining with all ment of IP,R, PLCP, and PLCy staining within the striosomes three antisera was distinctly inhomogeneous(Figs. 2-4). Vari- seemedprimarily associatedwith staining of the neuropil. With ably shapedpatches of elevated stainingwere distributed through this generalenhancement of the staining intensity, we could not rostra1 and dorsal parts of the nucleus. Comparisons among determine whether, in addition, the immunostaining of indi- nearly adjacent sections showed that the zones of heightened vidual perikarya was also enhanced. immunostaining for IP,R, PLC& and PLCy were spatially aligned IP,R, PLCP, and PLCy immunostaining in the substantia The Journal of Neuroscience, August 1993, f3(8) 3303

Figure 2. Specificityof immunostainingwith IP,R, PLC& and PLCr antisera.A, C, and E illustratesimilar patterns of immunostainingwith IP,R (A), PLCfi (C), and PLCy (E) antiserain near-serialsections through the caudatenucleus (Sr) of a macaquemonkey. Note patchy zonesof densestaining (examples at Arrows). Immunostainingis absentin near-adjacentcontrol sectionstreated with IP,R antiserumpreabsorhcd with IP,R protein (PA, B) or with normalmouse serum (NMS; 0, F). Arrowheads mark bloodvessels used as landmarks.LK lateral ventricle. The medialaspect of the caudateis by the lateralventricle and the dorsalaspect of the caudateis on the right sideof the figure.Scale bar, 200 pm. nigru and GP. In the substantia nigra, IP,R immunoreactivity Earlier immunohistochemicallocalization of PLCp and PLCr was densein the neuropil of both SNc and SNr (Fig. 6). In SNc, in the rat’s substantia nigra demonstrated staining for both en- weak IP,R-like immunoreactivity wasalso visible in many peri- zymes in neural perikarya in SNc and in fibers in SNr. Some karya (Fig. 7). The distribution and sizes of the IP,R-positive neurons in SNr were stained for PLCy but not PLC@ (Gerfen cells were similar to those of neurons immunostained for TH et al., 1988). In the primate substantia nigra, large numbers of (Fig. 7). These observations suggestthat IP,R may occur in all neuronsin SNc were immunopositive for PLCP (Figs. 5-7, Ta- or most all of the dopamine-producing neuronsof the SNc, as ble l), but staining for PLCy wasonly slightly above background well as in nigra neuropil, especially in SNr (Table 1). (data not shown). In SNr, immunostaining for both enzymes 3304 Fotuhi et at. l Phosphoinositide Markers in Primate Basal Ganglia

Figure 3. Near-adjacent coronal sections through the head of the caudate nucleus in rhesus monkey demonstrate the striosomal enrichment of IP,R. A and B, Striosomal and matrix compartments were identified by the distributions of calbindin (A) and substance P (B) immunoreactivities. Arrows point to two striosomes. C, IP,R immunoreactiviity is relatively enriched in striosomes (arrows) as compared to the surrounding matrix. D. Both striosomal and matrix compartments contain numerous IP,R-immunoreactive neuronal cell bodies in apparently equivalent abundances within the two compartments. The relative enrichment of IP,R is associated with diffuse staining within the neuropil of striosomes (asterisk). Scale bars: A-C, 583 pm; D, 70 grn. was abundant in the neuropil, but evidence for weak cellular matrix strongly reinforcesthe idea that thesecompartments are immunostaining was only seenwith the PLC& distinct functional entities in the striatum. In the GP, staining for IP,R, PLC& and PLCr was present The general cellular patterns of immunostaining for PLC& in both segmentsand largely confined to the neuropil (Fig. 8, PLCr, and IP,R in the primate basalganglia appear similar to Table 1).Although immunostainingwithin GP appearedsimilar those in the rat; that is, these proteins are mainly within me- with the three antisera, the fiber lamina separating GP from dium-sized neurons. They also are in accord with the distri- striatum showeddifferent staining for PLCP, PLCr, and IP,R butions of mRNAs for theseproteins in the rat brain (Ross et (Fig. 8, Table 1). Very little staining in cells or neuropil was al., 1989; C. A. Ross et al., unpublished observations). PLCS apparent in sectionsstained for IP,R or PLCS (Fig. 7A,B). By and IP,R tend to be strongly represented,whereas PLCy is contrast, the fibrous layer contained abundant PLC+mmu- detectable in lesseramounts (Gerfen et al., 1988; Ross et al., nopositive large neurons and neuropil. There were no PLCy- 1989). The main difference between monkey and rat in the positive cellsdetectable in the fiber layer betweenGPe and GPi. distributions of these PI cycle markers lies in the striosomal compartmentalization of theseproteins in the monkey striatum. Discussion No suchcompartmentalization hasbeen detected in the rat brain The major finding of this study lies in the differential striosome- (Gerfen et al., 1988; Sharp et al., in press;M. Fotuhi, T. M. matrix localizations of markers of the PI system, IP,R and two Dawson, and S. H. Snyder, unpublishedobservations). A similar isoformsof PLC. Thesethree proteins, asjudged by immunohis- sharpeningof neurochemicalcompartments from rat to monkey tochemistry, occur in both striosomesand matrix, but are more (and to human) has been reported previously for many striatal strongly expressedin striosomesthan in most parts of the ma- transmitters and receptors (a notable exception being p-opiate trix. The differencesin stainingintensities mainly seemto reflect receptors as detected by ligand binding) and may reflect the differencesin the neuropil, which may reflect an enrichment of increasedspecialization of subgroupsof striatal neurons with theseproteins in subsetsof dendritic spinesand/or presynaptic respect to their connections and/or functions (Graybiel and axonal terminals. This compartmental difference in the repre- Ragsdale,1983; Graybiel, 1990; Martin et al., 1991). sentationof a major secondmessenger system in striosomesand Regional variations in a number of other neurotransmitter- The Journal of Neuroscience, August 1993, 13(E) 3305

Figure 4. Evidence that striosomes in rhesus monkey caudate nucleus are enriched in PLC@. A and B, The heightened PLC@ immunostaining of striosomes is illustrated in near-adjacent sections stained for calbindin D,,, (A) and PLCj3 (B). Arrowheads identify examples of corresponding striosomes. C. High-magnification photomicrograph showing that in striosomal and matrix compartments, most medium-sized neurons are PLCB- positive examples (arrowheads), whereas the neuropil within striosomes (asterisk) has more intense PLCj3 immunoreactivity than the surrounding matrix. Scale bars: A and B, 563 pm; C, 40 Mm.

related substancesin the striatum have been observed previ- receptors. More than 20 different cell membrane receptors are ously in many species(see, e.g., Graybiel and Ragsdale,1983; known to be linked to the PI system(Chuang, 1989).Many such Bessonet al., 1988; Chesseletet al., 1991; Martin et al., 1991). neurotransmitter and neuropeptide receptors including sub- In some monkeys we also noted regional variations, in that the stanceP are abundant in the striatum and have compartmen- medial and ventral parts of the striatum showed substantially talized and regionally graded distributions (Graybiel, 1990). In greaterintensities ofimmunostaining for IP,R, PLC& and PLQ fact, consideringthe variety of PI-linked receptors in the stria- than the lateral parts. turn, it is of particular interest that PI markersare not uniformly What might be the functional significance of the selective distributed. Their differential representation suggeststhat the distribution of the PI system in the striatum? The largestneu- actions of a number of receptor types may converge on the same ronal input to the striatum is composedof fibers from the ce- secondmessenger system. rebral cortex, which releaseglutamate or a closely related amino Thesecompartmental patterns of distribution of PI markers acid. Glutamate can interact with metabotropic receptorslinked may also be significant in relation to the distribution of other to the PI system and with ionotropic receptors of NMDA and secondmessenger systems. In the rat, complementary localiza- AMPA/kainate subtypes(see Miller, 1991, for a review). Con- tions in the brain have been noted for adenylyl cyclase moni- ceivably, the greater density of immunoreactivity for PI markers tored by 3H-forskolin binding, for PKC by 3H-phorbol ester in striosomesreflects an association with postsynaptic meta- binding, and for IP,R by 3H-IP, binding (Worley et al., 1986a). botropic glutamate receptorsor other G-protein-coupled recep- Recently, we have localized adenylyl cyclase catalytic activity tors. Interestingly, a difference in ionotropic glutamate receptor by a novel histochemical stain, and have observed selectively representation in striosomesand matrix has recently been re- high concentrations in the matrix (M. Fotuhi, T. M. Dawson, ported on the basis of ligand binding autoradiography (Dure et A. Verma, and S. H. Snyder, unpublishedobservations). Thus, al., 1992). Autoradiographic localization of the metabotropic the CAMP and PI systemsmay both be differentially distributed glutamatergic receptorsis not readily performed by direct ligand acrossthe striosome-matrix system of the striatum, and their binding, and the molecular cloning of this receptor has only distributions may in part be complementary. recently been published (Houamed et al., 1991; Masu et al., The expressionof PI-associatedproteins by neuronsand pro- 199l), so the striosome-matrix disposition of its corresponding cessesin the striatum and substantia nigra may in part reflect mRNA or immunoreactivity remains unknown. The striatal interconnections between these regions. Neurons in striosomes regions with heightened immunoreactivity for the PI system are thought to project mainly to SNc, or at least to the medial could alsocoincide with specializationsin cortical or other regions part of the substantia nigra. The axon terminals of striosomal projecting especially strongly to the striosomes. cells may contribute to the limited amount of neuropil staining The differential representationof PI markersin the striosomes for IP,R, PLCp, and PLCr present in SNc. Neurons of the clearly could also reflect the distribution of nonglutamatergic matrix project preferentially to GP and SNr, and it is likely that 3306 Fotuhi et al. - Phosphoinositide Markers in Primate Basal Ganglia

Figure 5. Cellular distribution of immunoreactivity for IP,R, PLCp, and PLCr (A, B, and C, respectively) appear abundantly in medium- sized striatal neurons. The immunostaining with each antiserum is also present in the neuropil. PLC@ staining (B) is substantially more intense than that of IP,R or PLQ. Scale bar, 20 pm. the PI marker-rich neurons of the matrix are associated with the IP,R-, PLCP-, and PLCy-positive terminals fields in these regions. Finally, the immunostained neurons in SNc are likely to be dopamine-containing cells, as their appearance is similar to that of TH-immunostained cells in adjacent sections. Ter- minals from these neurons presumably could contribute to the neuropil in the striatum that is immunopositive for IP,R, PLC& and PLQ. In most of the regions we examined, the relative patterns of staining for IP,R, PLCp, and PLCr were quite similar. The one Figure 6. IP,R and PLCB immunostaining in substantia nigra. In SNc, striking exception was in the fiber layer between the putamen IP,R and PLCD are present in virtually all neurons, and these have and external pallidum, where substantial staining for PLCy ap- distributions similar to those of TH-positive neurons in a nearby section (C). However, the characteristic ventrally extending TH-positive den- peared in magnocellular neurons, but where there was only neg- drites of SNc neurons lack appreciable staining in sections treated with ligible staining for PLCp or IP,R. This difference was particu- IP,R and PLC@antisera (4 and B, respectively). C, SNc; R, SNr. Scale larly notable, because the intensity of PLC@ staining exceeded bar, 100 pm. The Journal of Neuroscience, August 1993. f3(8) 3307

Figure 7. Cellular distribution of IP,R and PLCfi immunostaining in SNc. PLCfi immunoreactivity (B) appears denser than that of IP,R (A) but weaker than that of TH staining (C) in sections treated by similar protocols. Scale bar, 100 pm.

that of PLCy in almost all regions of the basal ganglia proper. The discrepancy highlights the heterogeneity within the extrapyramidal PI second messenger system. Previous immunohistochemical studies in the rat have displayed marked regional Figure 8. Evidence that PLCy immunostaining does occur in the ab- differences in the distribution of subtypes of PKC (Huang et al., sence of detectable IP3R and PLQ3 immunostaining. Magnocellular neurons in the medullary lamina (M) separating the putamen (97’) from 1988; Yoshihara et al., 1991) and some differences between GP express PLCr immunoreactivity (C), but lack IP,R (A) or PLCj3 patterns of PLCB and PLCr immunostaining (Gerfen et al., (B) immunoreactivity. Also note that striatal labeling for all three ep- 1988). Subtypes of IP,R derived from alternative splicing (Dan- itopes is enriched in both perikarya and neuropil, whereas staining in off et al., 199 1; Nakagawa et al., 1991) or from distinct genes GP is limited to the neuropil. Scale bar, 200 pm. (Sudhof et al., 1991; Ross et al., 1992) are not equally represented in all brain regions. It will be interesting to learn whether References different subtypes of IP,R also are differentially distributed in Albin RL, Young AB, Penney JB (1989) The functional anatomy of the basal ganglia and allied nuclei. If so, some of the selective basal ganglia disorders. Trends Neurosci 12:366-375. staining patterns we have observed could be related to such Besson MJ, Graybiel AM, Natsuk M (1988) SCH 23390 ligand binding differences in receptor subtype expression. to D, dopamine receptors in the basal ganglia of the cat and primate: 3308 Fotuhi et al. - Phosphoinositide Markers in Primate Basal Ganglia

delineation of striosomal compartments and pallidal and nigral sub- mesostriatal innervation of striosomes and extrastriosomal matrix. division. Neuroscience 26: 10 l-l 19. Neuroscience 23~223-242. Besson MJ, Graybiel AM, Quinn B (1990) Coexpression of neuro- JimCnez-Castellanos J, Graybiel AM (1989) Compartmental origins peptides in the cat’s striatum: an immunohistochemical study of sub- of striatal efferent projections in the cat. Neurosci&ce 32:297-321. stance P, dynorphin B and enkephalin. Neuroscience 39:33-X Jovce JN. Sann DW. Marshall JF (1986) Human striatal donamine Chesselet M-F, Gonzales C, Levitt P (199 1) Heterogeneous distri- ieceptois ai; organized in compakmen&. Proc Nat1 Acad Sci USA bution of the limbic system-associated membrane protein in the cau- 83:8002-8006. date nucleus and substantia nigra of the cat. Neuroscience 40:725- Martin LJ, Hadfield MC, Dellovade TC, Price DL (199 1) Striatal 733. mosaic in primates: patterns of neuropeptide immunoreactivity dif- Chuang D-M (1989) Neurotransmitter receptors and phosphoinositide ferentiate the ventral striatum from dorsal striatum. Neuroscience 43: turnover. Annu Rev Pharmacol Toxic01 29:7 l-l 10. 397-417. Danoff SK, Ferris CD, Donath C, Fischer G, Munemitsu S, Ullrich A, Masu M, Tanabe Y, Tsuchida K, Shigemoto R, Nakanishi S (1991) Snyder SH, Ross CA (199 1) Inositol 1,4$trisphosphate receptors: Sequence and expression of a metabotropic glutamate receptor. Na- distinct neuronal and non-neuronal forms derived by alternative ture 349:760-765. splicing differ in phosphorylation. Proc Nat1 Acad Sci USA 88:295 l- Miller JM (199 1) Metabotropic excitatory amino acid receptors reveal 2955. their true colors. Trends Pharmacol Sci 12:365-367. Donoghue JP, Herkenham M (1986) Neostriatal projections from in- Nakagawa T, Okano H, Furuichi T, Aruga J, Mikoshiba K (199 1) The dividual cortical fields conform to histochemically distinct striatal subtypes of the mouse inositol 1,4,5&sphosphate receptor are ex- compartments in the rat. Brain Res 365:397-403. pressed in tissue-specific and developmentally specific manner. Proc Dure LS IV, Penny JB, Young AB (1992) Compartmentalization of Nat1 Acad Sci USk 88:6244-6248. - _ - excitatory amino acid receptors in human striatum. Proc Nat1 Acad Nakanishi S. Maeda N. Mikoshiba K (1991) Immunohistochemical Sci USA 89:7688-7692. localization of an inositol 1,4,5-triphdsphak receptor, P,,,, in neural Fishell G, van der Kooy D (1987) Pattern formation in the striatum: tissue: studies in developing and adult mouse brain. J Neurosci 11: developmental changes in the distribution of striatonigral neurons. J 2075-2086. Neurosci 7:1969-1978. Peng Y-W, Sharp AH, Snyder SH, Yau K-W (1991) Localization of Flaherty AW, Graybiel AM (199 1) Corticostriatal transformations in the inositol 1,4,5-trisphosphate receptor in synaptic terminals in the the primate somatosensory system. Projections from physiologically vertebrate retina. Ne&on6:525-53 i. - mapped body-part representations. J Neurophysiol 66: 1249-l 263. Pert CB, Kuhar MJ, Snyder SH (1976) Opiate receptor: autoradio- Gerfen CR (1984) The neostriatal mosaic: compartmentalization of graphic localization in rat brain. Proc Nat1 Acad Sci USA 73:3729- corticostriatal input and striatonigral output systems. Nature 311: 13733. 46 1464. Ragsdale CW, Graybiel AM (1988) Fibers from the basolateral nucleus Gerfen CR, Baimbridge KG, Miller JJ (1985) The neostriatal mosaic: of the amygdala selectively innervate striosomes in the caudate nu- compartmental distribution of calcium binding protein and parval- cleus of the cat. J Comp Neurol 269:506-522. bumin in the basal ganglia of the rat and monkey. Proc Nat1 Acad Ragsdale CW, Graybiel AM (1990) A simple ordering of neocortical Sci USA 8238780-8784. areas established by the compartmental organization of their striatal Gerfen CR, Baimbridge KG, Thibault J (1987) The neostriatal mosaic: projections. Proc Nat1 Acad Sci USA 87:6 196-6 199. III. Biochemical and developmental dissociation of patch-matrix me- Ross CA, MacCumber MW, Glatt CE, Snyder SH (1989) Brain phos- sostriatal systems. J Neurosci 7:3935-3944. pholipase C isozymes: differential mRNA localizations by in situ Gerfen CR, Choi WC, Suh PG, Rhee SG (1988) Phospholipase C I hybridization. Proc Nat1 Acad Sci USA 86:2923-2927. and II brain isozymes: immunohistochemical localization in neuronal Ross CA, Danoff SK, Schell MJ, Snyder SH, Ullrich A (1992) Novel systems in rat brain. Proc Nat1 Acad Sci USA 85:3208-3212. inositol 1,4,5-trisphosphate receptors: molecular cloning and differ- GimCnez-Amaya JM, Graybiel AM (199 1) Modular organization of ential localization in brain and peripheral tissues. Proc Nat1 Acad Sci projection neurons in the matrix compartment of the primate stria- USA 89:5265-5269. turn. J Neurosci 11:779-79 1. Ryu SH, Cho KS, Lee K-Y, Suh P-G, Rhee SG (1987) Purification Graybiel AM (1990) Neurotransmitters and neuromodulators in the and characterization of two immunologically distinct phosphoinosi- basal ganglia. Trends Neurosci 13:244-254. tide-specific phospholipase C from bovine brain. J Biol Chem 262: Graybiel AM, Chesselet MF (1984) Compartmental distribution of 12511-12518. striatal cell bodies expressing met-enkephalin-like immunoreactivity. Saito N, Kikkawa U, Nishizuka Y, Tanaka C (1988) Distribution of Proc Nat1 Acad Sci GSA 8 i:7980-7984. protein kinase C-like immunoreactive neurons in rat brain. J Neu- Gravbiel AM. Raasdale CW Jr (1978) Histochemicallv distinct com- rosci 8:369-382. p&tments in thi striatum of hbman, monkey and cai demonstrated Sharp AH, Dawson TM, Ross CA, Fotuhi M, Mourey RJ, Snyder SH by acetylcholinesterase staining. Proc Nat1 Acad Sci USA 75:5723- (in press) Inositol 1,4,5-triphosphate receptors: immunohistochem- 5726. ical-localization to discreet &ea, of rat brain. Neuroscience, in press. Graybiel AM, Ragsdale CW Jr (1983) Biochemical anatomy of the Sudhof TC. Newton CL. Archer BT III. Mianerv GA (199 1) Structure striatum. In: Chemical neuroanatomy, pp 427-504. New York: Ra- of a novel InsP, recedtor. EMBO J iO:3199:3206. ~ ’ ven. Supattapone S, Worley PF, Baraban JM, Snyder SH (1988) Solubi- Graybiel AM, Ragsdale CW, Moon Edley S (1979) Compartments in lization, purification and characterization of an inositol trisphosphate the striatum of the cat observed by retrograde cell-labeling. Exp Brain receptor. J Biol Chem 263: 1530-l 534. Res 34:189-195. Worley PF, Baraban JM, DeSouza EB, Snyder SH (1986a) Mapping Graybiel AM, Baughman RW, Eckenstein F (1986) Cholinergic neu- second messenger systems in the brain: differential localizations of ropil of the striatum observes striosomal boundaries. Nature 323: adenvlate cvclase and nrotein kinase C. Proc Nat1 Acad Sci USA 83: 625-627. 4053-4057: Herkenham M, Pert CB (198 1) Mosaic distribution ofopiate receptors, Worley PF, Baraban JM, Snyder SH (1986b) Heterogeneous localiza- parafascicular projections and acetylcholinesterase in rat striatum. tion of protein kinase C in rat brain: autoradiographic analysis of Nature 291:415-418. phorbol ester receptor binding. J Neurosci 6:199-207. Houamed KM, Kuijper JL, Gilbert TL, Haldeman BA, O’Hara PJ, Worley PF, Baraban JM, Colvin JS, Snyder SH (1987) Inositol tris- Mulvihill ER, Almers W, Hagen FS (199 1) Cloning, expression, and phosphate receptor localization in brain: variable stoichiometry with gene structure of a G protein-coupled glutamate receptor from rat protein kinase C. Nature 325: 159-l 6 1. brain. Science 252: 13 18-1321. Worley PF, Baraban JM, Snyder SH (1989) Inositol 1,4,5-trisphos- Huang FL, Yoshida Y, Nakabayashi H, Young WS III, Huang K-P phate receptor binding: autoradiographic localization in rat brain. J (1988) Immunocytochemical localization of protein kinase C iso- Neurosci 9:339-346. zymes in rat brain. J Neurosci 814734-4744. Yoshihara C, Saito N, Taniyama K, Tanaka C (1991) Differential Jiminez-Castellanos J, Graybiel AM (1987) Subdivisions of dopa- localization of four subspecies of protein kinase C in the rat striatum mine-containing AS-A9-A10 complex identified by their differential and substantia nigra. J Neurosci 11:690-700.