Proc. Nail. Acad. Sci. USAVol. 85, pp. 3235-3239, May 1988Neurobiology

Receptor binding sites for substance P, but not substance K orneuromedin K, are expressed in high concentrations by arterioles,venules, and lymph nodules in surgical specimens obtainedfrom patients with ulcerative colitis and Crohn disease

(tachykinins/inflammatory bowel disease/autoradiography/inflammation)

CHRISTOPHER R. MANTYHab, TROY S. GATESa, ROBERT P. ZIMMERMANa, MARK L. WELTONac,EDWARD P. PASSARO, JR.ac, STEVEN R. VIGNAad, JOHN E. MAGGIOe, LAWRENCE KRUGERO,AND PATRICK W. MANTYHaghi'Center for Ulcer Research and Education and 'Department of Surgery, Veterans Administration Wadsworth Medical Center, Los Angeles, CA 90073;Departments of 'Anatomy, hMedicine, and gBrain Research Institute, University of California, Los Angeles, CA 90024; and 'Department ofBiological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Communicated by Jared M. Diamond, December 23, 1987

ABSTRACT Several lines of evidence indicate that tachy-kinin neuropeptides [substance P (SP), substance K (SK), andneuromedin K (NK)] play a role in regulating the inflamma-tory and immune responses. To test this hypothesis in a humaninflammatory disease, quantitative receptor autoradiographywas used to examine possible abnormalities in tachykininbinding sites in surgical specimens from patients with inflam-matory bowel disease. Surgical specimens of colon were ob-tained from patients with ulcerative colitis (n = 4) and Crohndisease (n = 4). Normal tissue was obtained from uninvolvedareas of extensive resections for carcinoma (n = 6). In allcases, specimens were obtained <5 min after removal tominimize influences associated with degradation artifacts andwere processed for quantitative receptor autoradiography byusing '2I-labeled Bolton-Hunter conjugates of NK, SK, andSP. In the normal colon a low concentration of SP receptorbinding sites is expressed by submucosal arterioles and venulesand a moderate concentration is expressed by the external cir-cular muscle, whereas SK receptor binding sites are expressedin low concentrations by the external circular and longitudinalmuscle. In contrast, specific NK binding sites were not ob-served in any area of the human colon. In colon tissue obtainedfrom ulcerative colitis and Crohn disease patients, however,very high concentrations of SP receptor binding sites areexpressed by arterioles and venules located in the submucosa,muscularis mucosa, external circular muscle, external longi-tudinal muscle, and serosa. In addition, very high concentra-tions of SP receptor binding sites are expressed within thegerminal center of lymph nodules, whereas the concentrationsof SP and SK binding sites expressed by the external musclelayers are not altered significantly. These results demonstratethat receptor binding sites for SP, but not SK or NK, areectopically expressed in high concentrations (1000 -2000 timesnormal) by cells involved in mediating inflammatory andimmune responses. These data suggest that SP may be in-volved in the pathophysiology of inflammatory bowel diseaseand might provide some insight into the interaction betweenthe nervous system and the regulation of inflammation and theimmune response in human inflammatory disease.

identified within a subpopulation of these sensory neurons(1). The most extensively characterized of these sensoryneuropeptides is substance P (SP), a member of the mam-malian tachykinin family (Fig. 1), which also includes SKand NK. Though it is clear that SP and SK are expressed bysensory neurons (2) this remains uncertain for NK andderives from difficulty in generating highly specific antibod-ies for each of the tachykinins since they all share the highlyantigenic carboxyl terminus -Phe-Xaa-Gly-Leu-Met-NH2,where Xaa is an aromatic or branched aliphatic amino acid.

Several of these sensory neuropeptides, most notably SP,have been associated with neurons specifically implicated inthe conduction of nociceptive information. Thus, intrathecalinjection of SP produces biting and scratching behavior[consistent with a role for SP as a peptide neurotransmitterassociated with primary afferent nociceptors (3)]; SP releasein the spinal cord is inhibited by opiate analgesics (4);depletion of SP by capsaicin [a neurotoxin that is relativelyselective for unmyelinated sensory neurons (5), includingthose containing SP] is associated with a loss of specificnociceptive response; and release of SP in the spinal dorsalhorn in response to normally innocuous stimuli is enhancedin polyarthritic rats (6).

It has become increasingly evident in the last decade thata specific class of DRG neurons conveying afferent somato-sensory information from peripheral tissues to the spinalcord is involved in the efferent regulation of the peripheraltissues they innervate. Thus, SP-containing DRG neuronshave been implicated in the afferent central transmission ofnociceptive information and in the efferent regulation ofinflammation and sensitization of joint sensory endings in achronic pain state-e.g., arthritis (7, 8). Support for thisconcept includes observations that the bulk of the SP syn-thesized by DRG neurons is transported to the peripheralterminals rather than to the spinal cord (9); SP is a potentvasodilator in several peripheral tissues (10); terminals ofSP-containing sensory neurons are observed in associationwith blood vessels (11); electrical stimulation of these pe-ripheral nerves at intensities that release SP in peripheraltissues reproduces many of the physiological changes seen in

Neurons with cell bodies located in dorsal root ganglia(DRG) are known to convey specific features of somaticsensory information from peripheral tissues to the centralnervous system. Recently, several neuropeptides have been

Abbreviations: DRG, dorsal root ganglia (ganglion); SP, substanceP; SK, substance K; NK, neuromedin K; IBD, inflammatory boweldisease; H&E, hematoxylin/eosin; GI, gastrointestinal; BH, Bol-ton-Hunter.bPresent address: Medical School, University of Wisconsin-Mad-ison, Madison, WI 53706.dPresent address: Department of Physiology, Duke University Med-ical Center, Durham, NC 27710.'To whom reprint requests should be addressed.

3235

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3236 Neurobiology: Mantyh et al.

Substance P H-Arg-Pro-Lys-Pro-Gln-Gln-PHE-Phe-GL-LEU-MET-NH2Substance K

Neuromedin K

H-His-Lys-Thr-Asp-Ser-PHE-Val-GLY-LEU-MET-NH2

H-Asp-Met-His-Asp-Phe-PHE-Val- L-LEU-MET-NH2

FIG. 1. Structure of mammalian tachykinins. Sequence homologies are noted by underlining, and invariant residues are indicated byuppercase script. Neurokinin a, neurokinin A, and neuromedin L are alternative names for substance K (SK). Neuromedin (8 and neurokininB are alternative names for neuromedin K (NK).

acute inflammation (12), including plasma extravasation (7);and in experimental animals SP and SK are expressed byseveral tissues involved in the inflammatory and immuneresponses (13).What emerges from these observations is the hypothesis

that sensory neurons containing SP and/or other neuropep-tides are involved in conveying nociceptive information tothe spinal cord and in regulating the inflammatory and im-mune responses in the peripheral tissues they innervate.Thus, the same sensory neuropeptide released by a sensoryneuron may signal tissue damage in the spinal cord andparticipate in regulating the inflammation, immune response,and, ultimately, wound healing, in the affected peripheraltissue.To test whether this hypothesis is applicable to human in-

flammatory disease we have examined the changes in tachy-kinin neuropeptide receptor binding sites in chronic inflam-matory bowel disease (IBD) patients, where surgical re-moval of the affected tissue is used to ameliorate the diseasein severe cases. IBD is a generic term that refers to chronicinflammatory diseases of the intestine that are of unknownetiology, principally ulcerative colitis and Crohn disease.Ulcerative colitis is an inflammatory, ulcerating process ofthe colon; Crohn disease is an inflammation of the intestinecharacterized by nodular granulomatous inflammatory le-sions throughout the entire wall that may involve any part ofthe intestine but primarily attacks the distal small intestineand colon (14). The present report is based on an explorationof tachykinin receptor binding sites expressed in IBD andwhether perturbation of this expression is correlated withthese diseases.

EXPERIMENTAL PROCEDURESRadioligands. The radioligands used in the present study

were '251-labeled tachykinins purified by reverse-phaseHPLC to essentially quantitative specific activity (=2000Ci/mmol; 1 Ci = 37 GBq). The figures are derived fromautoradiograms by using the '251-labeled (mono-iodo) Bol-ton-Hunter (BH) conjugates of SP, SK, and NK. {BHreagent = 3-(p-hydroxy-m-['25I]iodophenyl)propionyl}.BH-SP was a gift from Amersham or was synthesized in ourlaboratory, whereas BH-SK and BH-NK were synthesizedin our laboratory by conventional methods (13).Human Specimens. Specimens were obtained within 5 min

after removal, embedded in Tissue-Tek (Miles), and placedon dry ice to minimize postsurgical degradation artifacts. Inall cases, the diagnosis was independently determined by apathologist to be either ulcerative colitis, Crohn disease, orhistologically normal at the site of resection. The tissue wasthen blocked, placed on a brass microtome chuck, frozen ondry ice, and processed for quantitative autoradiography asdescribed (14). The tissue was serially sectioned (30 um),thaw-mounted onto gelatin-coated microscope slides, andstored at - 20°C in boxes of desiccant for up to 3 months.Receptor Binding Protocols. SP. For SP receptor binding,

slide-mounted tissue sections were brought to room temper-ature and placed in a preincubation medium (19°C for 10 min)consisting of 50 mM Tris HCl (pH 7.4) containing 0.005%(vol/vol) polyethylenimine (Sigma). The slide-mounted sec-

tions were then incubated at 190C for 1 hr in a solution of 50mM Tris HCl (pH 7.4) containing 3 mM MnC12, 200 mg ofbovine serum albumin (Sigma) per liter, 2 mg of chymostatin(Sigma) per liter, 4 mg of leupeptin (Sigma) per liter, 40 mgof bacitracin (Sigma) per liter, and 100 pM radioligand. Thiswas done by placing the slides on a flat surface and coveringthe sections with 1.5 ml of incubation medium. For estimat-ing nonspecific binding, paired serial sections were incu-bated as described above except that 1 AM unlabeled peptidewas added to the incubation solution. Following this incu-bation, the slides were rinsed with four washes of 50 mMTrisHCI (pH 7.4) (4°C, 2 min each) and two washes ofdistilled H20 (40C, 5 sec each) and then quickly dried in thecold room by using a stream of cold air. Sections were thenleft for 3 hr to dry in the cold and stored in desiccant-filledboxes overnight at room temperature.SK and NK. For labeling SK and NK receptor binding

sites, the same protocol was employed except that incuba-tion and wash conditions were slightly altered. The incuba-tion solution for SK was the same as for SP except that thepH was 8.0. The wash conditions were the same as for SPexcept that time was increased to 5 min for each wash step.The incubation solution for NK was the same as for SPexcept that the pH was 8.5. The wash conditions were thesame except the time was increased to 8 min for each step.Polyethylenimine was omitted from the preincubation me-dium for SK and NK and the incubation times were 2 hr ineach case. Nonspecific binding was determined in the pres-ence of 1 ,uM SK or NK, respectively.

Analysis of Autoradiograms. Quantitative autoradiograph-ic analysis of the tachykinin binding sites was performed byplacing the dried, labeled slide-mounted tissue sections inapposition to 3H-sensitive film (Ultrofilm, LKB or Hyper-film, Amersham) along with iodinated brain mash or com-mercially available standards (Amersham). After 1-4 wk, the3H-sensitive film was developed in Kodak D-19 developer,fixed, and washed. In selected sections where a higherdegree of histological resolution of the binding sites wassought, the tissue slices were overlaid with emulsion-coatedcoverslips (15) or treated with paraformaldehyde vapors (16)to fix the radioligand to its binding site; these slide-mountedtissue sections were then processed for standard emulsion-dipped autoradiography. After these autoradiograms weredeveloped, the sections were placed in Carnoy's fixative for3 hr, stained with hemotoxylin/eosin (H&E), and mountedwith Histoclad. Dark-field or bright-field photomicrographswere then taken of the silver grains or counterstainedsections, respectively. By using this approach three comple-mentary images were generated: autoradiograms that wereanalyzed by quantitative densitometry, the autoradiogramsof the emulsion-dipped slides that provided detailed histo-logical resolution of the binding sites, and the counterstainedsection that allowed identification of the cell type expressinga specific binding site. Controls for chemographic artifactswere generated by performing the binding exactly as de-scribed except that the radioligand was omitted from theincubation medium.

Analysis of Data. To estimate quantitatively the density ofradiolabeled tachykinin binding sites, microdensitometrywith 3H-sensitive film was performed (17). In all cases,

Proc. Natl. Acad. Sci. USA 85 (1988)

Proc. Natl. Acad. Sci. USA 85 (1988) 3237

specific binding was obtained by subtracting total bindingfrom nonspecific binding. Nonspecific binding was definedas that binding sustained in the presence of 1 AM unlabeledpeptide.

Statistical Analysis. The results were expressed as mean ±1 SEM and examined for statistical significance by using theStudent's t test for independent samples.

RESULTS AND DISCUSSIONExpression of SP and SK Binding Sites in Normal Tissues.

In the histologically normal colon moderate concentrationsof SP receptors are expressed by the external circularmuscle and by smooth muscle cells comprising the tunicamedia of the large arteries that are just outside of thesubmucosa (Fig. 2a). In addition, a low concentration of SP

FIG. 2. Dark-field autoradiograms showing the distribution of SPbinding sites in transverse sections of colon obtained from themargins of extensive resection for carcinoma (a) and with patientswith Crohn disease (b) and ulcerative colitis (c). In these dark-fieldautoradiograms the 3H-sensitive film was used as the negative;white silver grains represent areas of high concentration of SPbinding sites. Note that whereas in normal colon (a) a moderateconcentration of SP receptor binding sites is expressed by theexternal circular muscle (CM) and the tunica media of a large arteryin the serosa, in Crohn disease (b) and ulcerative colitis (c),arterioles, venules, and lymph nodules express very high levels ofSP receptor binding sites. LM, external longitudinal muscle. (Bar =

1.4 mm.)

binding sites is expressed by arterioles and venules in thesubmucosa. SK receptors are expressed in low concentra-tions by the external circular and longitudinal muscle,whereas specific NK binding sites were not observed in anyarea of the human colon. To control for possible degradationartifacts, one Crohn colon was obtained immediately afterremoval and pieces were frozen at various times to assessthe effects of postsurgical degradation at room temperature.By using this material it was determined that significantdegradation had not occurred within the first 5 min afterremoval, suggesting that postsurgical artifacts should beminimal in this material since all were obtained within thistime period.

Expression of SP and SK Binding Sites in IBD. In surgicalmaterial from Crohn disease patients there was a strikingchange in the tissues expressing SP receptor binding sitescompared to that seen in surgical specimens obtained at themargins of extensive resections for carcinoma "normals"(Figs. 2 and 3). The most notable difference is seen in theexpression of SP receptor binding sites by arterioles, ve-nules, and lymph nodes. In tissue from patients with IBD,arterioles and venules (diameter, 0.1-1.0 mm) in all layers ofthe colon express very high levels of SP receptor bindingsites, whereas in normals, SP receptor binding sites areundetectable in blood vessels, with the exception of theoccasional large arteries just inside the serosa or the arte-rioles and venules in the submucosa (all of which expresslow levels of SP binding sites). Since it is difficult to identifythe total number of arterioles and venules in H&E-stainedsections we have instead compared the total number ofblood vessels expressing SP binding sites in the histologi-cally normal vs. disease specimens in a defined comparablearea (2.6 mm x 3.5 mm) of the tissue sections. Comparisonof the average number of arterioles and venules expressingSP receptor binding sites in samples from six normal andfour Crohn disease patients revealed 6.50 + 2.38 vs. 14.5 ±4.95 in the submucosa, 0 vs. 14.0 ± 1.4 in the externalcircular muscle, and 0 vs. 18.5 + 3.5 in the externallongitudinal muscle.Concomitant with the ectopic expression of SP receptor

binding sites by arterioles and venules is the expression ofSP receptor binding sites by the lymph nodules that borderthe muscularis mucosa. Within each lymph nodule, onlycells associated with the lightly stained (by H&E) germinalcenter expressed a detectable level of SP receptor bindingsites. In a total of 36 lymph nodes found in tissue fromsurgical specimens obtained from patients with Crohn dis-ease, all 36 expressed high concentrations of SP bindingsites. This is in contrast to the 24 lymph nodules we couldlocalize in histologically normal surgical specimens from thesix patients with carcinoma resection in which the expres-sion of SP receptor binding sites was undetectable. In IBDtissues the external circular muscle displays a slightly lowerlevel of SP receptor binding than in normal (Fig. 4), but thisdecrease does not appear to be statistically significant.

Surgical material from ulcerative colitis patients revealeda pattern and level of SP receptor binding sites similar to thatobserved in the specimens obtained from Crohn diseasepatients. In specimens from ulcerative colitis patients veryhigh levels of SP receptor binding sites are expressed bysmall arterioles and venules located in the muscularis mu-cosa, submucosa, external circular muscle, external longitu-dinal muscle, and serosa. In samples of tissue obtained fromsix carcinoma resection patients and four patients withulcerative colitis, the average number of arterioles andvenules in the histologically normal and ulcerative colitistissue in a 2.6 mm x 3.5 mm sample was 6.50 + 2.3 vs. 13.3± 1.5 in the submucosa, 0 vs. 15.0 ± 6.2 in the externalcircular muscle, and 0 vs 19.0 ± 5.0 in the external longitu-dinal muscle, respectively (Fig. 5).

Neurobiology: Mantyh et al.

3238 Neurobiology: Mantyh et al.

t.,/*$t ,u.c. sN

The germinal center of lymph nodules in tissue obtainedfrom the ulcerative colitis patients also expresses very highlevels of SP receptor binding sites compared to those foundin normals (Fig. 4). Thus, of the 29 lymph nodes histologi-cally localized in H&E-stained sections from the four casesof ulcerative colitis, 28 expressed very high levels of SPbinding sites, whereas 24 nodules localized in normal colonfailed to express detectable levels of SP binding sites.

Involvement of SP in Inflammatory Diseases. Although theetiology of IBD remains unknown, recent research into avariety of inflammatory diseases such as arthritis (18) andasthma (19) suggests that the nervous system may exagger-

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SUBSTANCE Pbinding sites

Lymph SM-bvNodule

CM CM-bv LM-bv

FIG. 3. Higher-power autoradio-grams of transverse sections of colonshown in Fig. 2 demonstrating the lo-calization of SP receptor binding sitesin the histologically normal human co-lon (a and b), Crohn disease patients (cand d), and ulcerative colitis (U.C.) (eand f) patients. (a, c, and e) Bright-field photomicrographs of the humancolon stained with H&E. (b , d, and f)Dark-field photomicrographs obtainedby using 3H-sensitive film that overlaidthis tissue as the negative. In b, d, andf the white silver grains represent highconcentrations of SP binding sites.Note that in the normal tissue a mod-erate concentration of SP binding sitesis expressed by the external circularmuscle (CM), whereas in the Crohndisease and ulcerative colitis tissue SPreceptor binding sites are expressed byarterioles and venules (arrows) and thelightly stained outer rim of the lymphnodule (arrowheads), which corre-sponds to the germinal center. Controlsections were treated identically to thesections shown in b and c except that 1,uM unlabeled peptide was added tothe incubation medium. Unless other-wise noted, control sections were es-sentially blank. SM, submucosa. (Bar= 0.63 mm.)

ate associated immune and inflammatory responses ingenet-ically predisposed individuals. The present findings revealthat in a disease state a specific tachykinin binding site forSP appears to be strategically expressed by specific cells thatare involved in producing two of the salient symptoms ofIBD. These include a hyperactive inflammatory and immuneresponse, often accompanied by abdominal pain.A key question is whether SP receptor binding site expres-

sion by arterioles, venules, and lymph nodules is an etiolog-ical factor in IBD or whether it is simply concomitant withthe inflammation. One observation that suggests it is anetiological factor is that whereas corticosteroids are effica-

SUBSTANCE Kbinding sites

FIG. 4. Histogram showing changes in

LJNormal the location and concentration of SP andSK binding sites in surgical specimens of

Crohn normal colon obtained from carcinomaresection (open bars), Crohn disease

UC (hatched bars), and ulcerative colitis (UC)(dark bars). In this histogram, 100lo spe-cific binding is that concentration of spe-

0.001 < p < 0.05 cific SP binding sites expressed by thesmooth muscle of the normal externalp (0.001 circular muscle (CM). Note that whereasSP receptor binding sites are undetectablein blood vessels (bv) vascularizing theexternal circular muscle (CM) and exter-nal longitudinal muscle (LM) and in thelymph nodules of normal patients, in theCrohn disease and ulcerative colitis pa-tients they reach levels that are the highestobserved in the human gastrointestinal(GI) tract. Distinct binding sites for NKwere not detected in any area of the hu-

CM LM man GI tract. bv, blood vessel (arterioleor venule); SM, submucosa.

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Proc. Natl. Acad. Sci. USA 85 (1988)

Proc. Natl. Acad. Sci. USA 85 (1988) 3239

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cious in ameliorating the symptoms of IBD (14) they do notappear to affect SP receptor binding expression in affectedtissue. Thus, though corticosteroids suppress the inflamma-tion and the immune response, withdrawal of corticosteroidsresults in a return of the symptoms, suggesting that theunderlying pathology is still present. The location or levelsof SP receptor binding expression do not appear to besignificantly different in the two Crohn disease patients whoreceived high doses of corticosteroid (Pregnesone) treatmentas compared to two Crohn disease patients who had notreceived any corticosteroid treatment for >12 months.Another suggestive link between tachykinins and IBD

pathophysiology is that the SP/SK-containing axons that areknown to innervate blood vessels and regulate the inflam-matory response are the same sensory fibers hypothesized totransmit nociceptive input from the GI tract to the spinalcord (11, 13, 20, 21). Capsaicin, the active ingredient in redpeppers, is known to release SP/SK from the central andperipheral ends of these sensory nerves (2, 12, 20, 21) andalso appears to produce a set of responses in the GI tract ofnormal subjects similar to that seen in IBD patients. Thus,ingestion of capsaicin or a similar compound by a subjectaccustomed to bland food may elicit a crampy diarrhea,which although less severe, is similar to the symptomspresented in IBD patients. Capsaicin-sensitive sensorynerve fibers also appear to be involved in other pathophys-iological states such as arthritis (8) and skin disorders, whichare among the common extraintestinal manifestations ofpatients with IBD (14).These data support speculation that in some cases of IBD,

tachykinins and their receptors may be involved in thepathophysiology of the disease. We suggest that the tachy-kinin system is involved in the pathophysiology of IBD andthat this involvement may be relevant to several criticalfeatures: (it) DRG neurons, which are known to innervate thesubmucosal blood vessels in the GI tract and express SP andSK, alter what they synthesize and release in the peripheraltissues during times of stress (such as the herpes simplexvirus), possibly accounting for the increase in the severity ofIBD during stress; (it) in IBD, SP binding sites are expressedin high concentrations by small arterioles and venules thatare known to be involved in the inflammatory response inthe GI tract; (iii) SP receptor binding sites are expressed bycells in gut-associated lymph tissue that is involved inregulating the immune response; (iv) DRG neurons thatrelease SP and SK in the periphery also are thought to beinvolved in conveying nociceptive sensory information to

-1 00 FIG. 5. Histogram showingC changes in the number of bloodW vessels (arterioles and venules)

80 ) and percentage of lymph nod-ules expressing SP binding sitesx a) in surgical specimens of normal. colon obtained from the margins

- 60 ( " of carcinoma resection (open3 O bars), Crohn disease (hatchedc-

bars), and ulcerative colitis (UC)40 C C (dark bars). Values for blood

n vessels are the average numbera- of blood vessels in a 2.6 mm x

E U1 3.5 mm (9.1 mm2) sample ex-- 20 -> pressing SP receptor binding

4- sites, whereas for the lymph01 nodules it is the percentage of

lymph nodules that express SP0 binding sites. For abbreviations,

see Fig. 4.

the spinal cord, which may account for the accompanyingabdominal pain in IBD; and (v) DRG neurons that expressSP and SK have also been hypothesized to be involved in thepathophysiology of arthritis and several skin disorders,which are common extraintestinal manifestations of patientswith IBD. Thus, alterations in tachykinin-containing sensoryneurons and tachykinin receptors may contribute to thepathophysiology of IBD. If this hypothesis is correct, itmight provide a rationale for the development of new ther-apies employing specific tachykinin antagonists to helpcombat what is now an inadequately understood group ofdiseases.

This work was supported by the Southern California ArthritisFoundation, Sloan Foundation, and by Grants 23970, NS-22961, andDCB85-42227 from the National Institute of Neurological and Com-municative Disorders and Stroke.

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