Substance P and substance P receptor histochemistry in human neurodegenerative diseases

174

Regulator), Pepttdes, 46 (1993) 174-185 © 1993 Elsevier Science Pubhshers B V All rights reserved 0167-0115/93/$0600

REGPEP 01403

Substance P and substance P receptor histochemistry in human neurodegenerative diseases N.W. Kowall a'b, B.J. Quigley Jr. a, J.E. Krause c, F. Lu a, B.E.

Kosofsky ~ and

R.J.

Ferrante b

Neurology Servtce and bDepartment of Neuropathology, Massachusetts General Hospttal and Harvard Medtcal School, Boston, MA (USA), CDepartment of Anatomy and Neurobtologv, Washmgton Umverslty School of Medtcme, St Louts, MO (USA) K e y words." Substance P; Immunocytochemistry; Receptor, In situ hybridization; Human bram, AlzheImer's

disease; Huntington's disease

Summary Substance P immunoreactlvlty is localized in discrete subsets of neurons in the human cerebral cortex and basal ganglia. In the normal human cerebral cortex, a subset of asplny local circuit neurons in deep cortical layers and the cortical subplate contain preprotachyklnm m R N A and substance P lmmunoreactive These neurons, which contain N A D P H dlaphorase (NO synthase) activity, are strikingly depleted in Alzhelmer's disease - in contrast to other local circuit neurons - suggesting that they may be an early target of the degenerative process. In the human basal ganglia, substance P immunoreactlvity and m R N A are localized in a subset of spiny strIatal neurons that project to the internal segment of the globus pallldus These neurons are enriched In D1 dopamlne receptors and dynorphin, and are calblndln and D A R P 32 lmmunoreactlve. A separate subset of aspiny striatal local circuit neurons also contain substance P immunoreactivtty Fiber and terminal staining IS prominent In the matrix compartment of the ventromedlal strlatum and persists dorsally as a rim outlining patches that contain lesser amounts of immunoreactivity Intense fiber and terrmnal staining is found In the pars retlculata of the substantia nlgra In Huntington's disease, substance P is depleted in the striatum in parallel with the dorsoventral gradient of neuronal loss. Terminal staining is progressively depleted in the pallidum and substantla nlgra in tandem with strlatal atrophy Substance P receptor lmmunoreactlvlty, defined with two polyclonal antlsera raised against synthetic peptides derived from the substance P receptor sequence, intensely labels a subset of large neurons in the nucleus basahs and strlatum identical to neurons labeled with choline acetyltransferase and nerve growth factor receptor antibodies (although stnatal cholinergic neurons do not contain nerve growth factor receptor Immunoreactlvlty In the human). These chohnergic neurons resist degeneration in Huntington's disease but are sensitive to degeneration in Alzhelmer's disease. Less intensely labeled neurons include pyramidal neurons in the hlppocampal CA2 field, nonpyramidal neurons In CA1-4, pyramtdal and nonpyramidal neurons in deep neocortlcal Correspondence to N W_ Kowall, Neurology Service, Massachusetts General Hospital, Boston, MA 02114, USA

175

layers and m the cortical subplate. Substance P receptor immunoreactivity is not well defined in the human globus palhdus or substantla nigra.

Introduction

Substance P distribution in postmortem human brain

Substance P, the first bioactive peptide derived from neural tissue, is the archetypical representative of the tachykinm family of peptides It consists of 11 amino acids with a characteristic carboxy-terminal sequence homologous to other members of the family. Tachyklnins are encoded by a pa.tr of very simdar genes that probably arose from a common ancestor by duplication [ 1 ]. Individual actwe peptldes are derived by differential processing of mRNA and protein precursors [2]. Substance P was among the first peptldes anatomically studied in brain with immunocytochemical methods [3]. Its distribution has now been defined in several mammalian species (see for example Refs. 4-7). The effects of substance P are mediated by a specific receptor protein which has been cloned and characterized as a member of the G protein coupled family of transmembrane receptors [ 1,8]. The distribution of substance P receptor mRNA has been studied in rat brain by in situ hybridization [9,10] In the forebraan, message is localized to large striatal cholinergic neurons. More recently, Krause and colleagues [ 11 ] have reported a more widespread dismbutlon of substance P receptor using sensitive lmmunoperoxldase methods with specific substance P receptor antibodies. In the followmg report we summarize the anatomical distribution of substance P immunoreactivlty and mRNA in the human forebrain and upper brainstem in the context of what is known about human neuroanatomy and discuss specific alterations found in two neurodegenerative diseases Alzheimer's disease and Huntington's disease Prehminary immunocytochemical studies of human brain using polyclonal antibodies directed against synthetic peptides derived from the substance P receptor are also presented.

The distribution of substance P in human brain generally resembles that of lower mammals but specific differences are often noted in relation to the unique structure of the human brain. The discussion will initially consider the anatomy of the basal ganglia and the distribution of substance P lmmunoreactivity within specific neurons and projection systems Pathological abnormalities associated with Huntington's disease will be presented The normal anatomy of substance P lmmunoreactlve neurons In human cortex and hlppocampus will be discussed followed by a summary of specific alterations associated with AlzheImer's disease

Anatomy of the normal human striatum The patch matrix orgamzation of human stnatum The generally accepted contemporary view of strlatal organization designates two compartments based on histochemical and hodologlcal critena: the patch, or strlosome, and the surrounding matrix This paradigm is mainly based on observations in the rat and cat. In our studies of human striatum we have been impressed by the complexity of substance P and met-enkephalin staining patterns, both considered to be classical markers for stnatal patches Serial sections of normal human stnatum at the level of the nucleus accumbens were stained for acetylcholinesterase, N A D P H diaphorase, tyrosine hydroxylase, calbindin D28K, enkephahn, substance P, cholecystokinin, transforming growth factor alpha, and mlcrotubule associated protein-2 (MAP-2). Substance P, enkephalln, transforming growth factor alpha, and cholecystolonin all showed similar patterns with a core of low immunoreactivity surrounded by a rim of

176 intense staining. Dorsally these patches were smaller and were often cut tangentially resulting in a small immunoreactwe rim without a core. A superimposed gradient of matrix staining was maximal ventrally where it merged with the lmmunoreactive rim of the patches from which it could not be distinguished. Calbindln staining is uniform in the matrix without a dorsoventral gradient. Patches of low calbindln staining correspond exactly with the unstained cores of substance P, enkephalin, transforming growth factor alpha, and cholecystolonln As previously reported, the areas of low acetylchohnesterase, N A D P H diaphorase, MAP-2 and tyrosine hydroxylase correspond. These patches, however, are larger than the calbindin patches and correspond exactly to the core plus immunoreactlve rim of substance P, enkephalln, transforming growth factor alpha, and cholecystokinin. The core region of patches did not stain positively with any method. Our results show that striatal organization in the human is more complex than previously reported A dyadic patch-matrix schema does not adequately define the histochemlcal heterogeneity of the human striatum. Neuronal classes wlth the striatum Neuronal populations of the strlatum can be characterized with respect to their cytoarchitectonic features and the neurochermcal compounds contmned within them. One basis for classification is the relative presence or absence of dendritic sprees Striatal neurons are referred to as spiny and aspiny neurons, as identified in Golga impregnations [ 12]. Biochemical charactenstics further distinguish these neuronal subtypes. Enzyme- and immunohlstochemical methods demonstrate that striatal neurons contain various neurotransmltter substances, neuropeptides, and related enzymes which are specific to particular neuronal subsets. Aspiny striatal neurons contain somatostatln-neuropeptide Y-NADPH dlaphorase [13] acetylcholinesterase [14,15] chromogranin A [16], choline acetyltransferase [17], vasoactive intestinal polypeptide [18] and cholecystoklnln [19] while gamma-amlnobutyric acid [20], substance P

[6], enkephahns [6], dynorphln [21], transforming growth factor alpha [22] and calbindm D28k [23] are among those substances contained within spiny neurons. We have recently performed in situ hybridization histochemical studies of substance P mRNA in postmortem human strlatum using a riboprobe labeled with 35S and we have confirmed the localization of substance P in medium size striatal neurons as initially reported by Chesselet and coworkers [24]. Pathological anatomy of the strtatum in Huntmgton's dtsease Huntington's disease (HD) is an autosomal dominant disorder that leaves a very characteristic pathological signature on the affected brain The striatum is usually most severely affected with atrophy, neuronal loss and gliosls. There is no characteristic cellular inclusion but there are pathological clues to the pathogenesls disorder. The first clue was that a peptide neurotransmitter, somatostatln, was surpnsingly enriched in the atrophic striatum suggesting that there may be differential susceptibility to neurodegeneratlon [ 25 ]. When immunocytochemlcal method s were used to visualize somatostatin neurons in the human striatum these neurons were indeed found to be relatively preserved [26]. Substance P, one of the first neuropeptides to be measured in human brain, is markedly depleted in the basal ganglia in H D [27]. These changes have been attributed to a loss of stnatal spiny projection neurons containing substance P but substance P is present in both spray and aspiny striatal neurons in a 70"30 ratio [28]. The dendrites of spiny neurons develop recurved endings and altered dendritic spines in H D [29] Degeneration and regeneration both occur. There are no qualitative differences between those aspiny neurons impregr~ated m control and H D striatum Medium-sized aspiny substance P neurons persist in H D strlatum [30] Recent findings suggest that proliferative changes precede degenerative changes in H D [31 ]. Prohferative changes, found primarily in moderate grades

177 (grade 2) of HD, include prominent recurving of distal dendrites, short-segment branching along the length of dendrites, and increased numbers and size of dendritic spines. Degenerative alterations consist of truncated dendritic arbors, focal dendritic-swelling, and marked spine loss and are almost entirely found in severe cases (grades 3 and 4) of HD. The extent to which enkephalin and substance P subsets develop proliferative changes is not known. It IS also not clear if similar dendritic changes affect N A D P H diaphorase aspiny neurons which are spared in H D Transforming growth factor alpha, which localizes with enkephalln in the stnatum, more clearly labels discrete dendritic processes than either enkephalin or substance P. We therefore examined the distribution of transforming growth factor alpha and N A D P H dmphorase in double stained sections of stnatum from 6 H D patients and 5 normal controis. In HD, transforming growth factor alpha neurons were depleted in a dorsoventral gradient, similar to that seen with enkephahn and substance P antisera. The staining mtens~ty of individual neurons, however, was somewhat increased over controls Proliferative dendritic abnormalities and dysomorphic dendriuc recurving affected transforming growth factor alpha but not N A D P H diaphorase neurons Our observations show that dendritic proliferation involves enkephalin neurons that contain a growth factor, transforming growth factor alpha, possibly not present in substance P neurons. If transforming growth factor alpha plays a causal role in the development of dendritic proliferation, spiny substance P neurons should not show evidence of dendritic proliferatlon in HD. Furthermore, transforming growth factor alpha may exacerbate neuronal injury if dendritlc proliferation leads to neuronal degeneration m HD. Pathological anatomy of other subcorttcal structures tn Huntington's disease A cardinal feature of H D is chorea, a disorder characterized by irregularly timed, abrupt and randomly distributed involuntary movements. Both the

neuropathologac findings m H D [32,33] and experimental primate models [34,35] suggest chorea may result from a loss of striatal projections to the external paUidum. The release of the external palhdum from inhibitory stnatal input results in excessive inhibition on the subthalamic nucleus which in turn causes decreased activation of the internal segment of the pallldum and reduced inhibition of the thalamus leading to increased cortical excitation and chorea Consistent with this hypothesis, bicuculline blockade of the GABAergic mput to the external palhdum causes chorea In primates [36]. It has been suggested that there is a differential vulnerability of enkephalin and substance P striatal spiny projection-neurons in H D [32] Under normal conditions, substance P lmmunoreactivity is preferentially located in the internal segment of G P (GP1), while ME immunoreactlvity is primarily within the external segment of the GP (GPe). Both neuropeptides are distributed within the SN. Relner and coworkers have reported that the density of ME immunoreactivlty (ir) in the GPe and substance P-ir In the SN reticulata (SNr) is decreased early on in HD, while the density of substance P-ir In the GP1 and SN compacta (SNc) is decreased late in the disorder. We have been unable to confirm these results [37]. In a preliminary study, the GP from 17 H D patients of all grades ( G 0 - G 4 ) and 14 controls were lmmunocytochemically stained, using antisera against substance P, ME, and transforming growth factor (TGF), an armno acid peptide which extensively colocahzes with ME. There was a gradient loss of substance P-, ME-, and TGF-ir in the GP of H D patients that corresponded to the severity of the disorder, with the most marked reductions occurring in the most severe grades. Within each grade of HD, however, no difference could be ldentLfied.between the density of immunoreactive staining of substance P in the GPi and the density of ME and T G F in the GPe. These findings were supported by densltometnc image analysis in which the density of the stainlng in each H D grade was measured as a percentage of the normal controls. These percentages were nearly

178

equal In each grade. Immunocytochemical and biochemical studies provide evidence that there is an early and comparable loss of both substance P and ME in H D striatum [37,38]. It is possible that treatment of patients with doparnlne receptor antagonists such as haloperidol affects the pattern of postmortem lmmuno staining We have studied the distribution of DARP-32, a phosphoprotein localized in dopaminoceptlve neurons, in postmortem human brain and find that it intensely labels neurons that project to both segments of the pallidum equally suggesting that is may be localized in both enkephahnerglc and substance P positive neurons. There does not appear to be a consensus of opinion with regard to the extrastrlatal pathologac changes within the substantia nlgra in HD. Early hlstopathologic reports are divided with regard to nerve cell loss [39] The SNr is reported to show fibrillary ghosls with no changes wathin the SNc in the majority of cases A recent analysis of the SN in 4 H D patients, demonstrated a significant reduction in size and number of both pigmented and nonplgmented neurons [40]. Neurochemical analyses of nigral homogenates from H D patients have demonstrated marked reductions in concentrations of substance P, enkephalm and G A B A [41] These findings have been attributed to a loss of strlatal projection neurons that contain these substances. It has been suggested, however, that substance P ~mmunoreacUvlty in SNr is lost early m the disease as compared to that In the SNc [32]. We have examined the SN of 42 H D patients with moderate (n = 9), severe (n = 19), and very severe (n = 14) grades Sections were all at the level of the red nucleus and oculomotor nucleus Counts of pigmented and nonpigmented neurons were made within the SN and respective areas of SNc and SNr were computed using an image analysis system. The total area of SN was significantly reduced in H D ( c o n t r o l = 2 6 + 4 6 m m 2_ H D = 1 6 + 3 . 4 m m 2 ; P< 0.0001) The SNr, however, had a greater area loss than the SNc (control SNr = 10.8 _+3.1 mm 2. H D

S N r = 3 . 5 + 1.1 mm2; P < 0 . 0 0 1 ) (control S N c = 1 5 4 + 2 . 5 m m 2 H D S N c = 1 2 . 6 + 2 . 5 m m 2, p < 0 014) Nonpigrnented neurons associated with the SNr were significantly reduced In number (control = 127 + 22. H D = 44 + 14, P < 0.0001) while pigmented neurons of the SNc were significantly increased in number ( c o n t r o l = 3 8 5 + 6 4 : H D = 435 + 66, P<0.05). The increase in pigmented neurons most likely reflects cell sparing along with moderate neuropil loss of stnatonigral terminal-projections to the SNc The slgntficant reductions of nonpigrnented neurons and neuropil in the SNr in H D may reflect transneuronal degeneration and the loss of matrix afferents, respectively The matrix compartment of the strlatum, which is severely affected in the disorder, projects primarily the SNr [23]. When deprived of their afferent Input, target neurons may die or atrophy. This is consistent with stnatal excitotoxic lesions in rodents in which there is neuronal degeneration in the SNr [42] The administration of musclmol, a specific G A B A agonlst, prevented degeneration In these neurons in this experiment, suggesting that neuronal death may be due to a loss if Inhibitory GABAergic Input with subsequent excessive excitation of SNr neurons_ In normal SN, there is a regional variation in the distribution of substance P and ME, such that the most intense immunoreactivlty of both neuropepUdes is found within the SNr and the pars alpha and beta of the SNc [43]. The nigral patterns of substance P and ME overlap, with the intensity of substance P much greater than ME. We have shown that both substance P and ME lmmunoreactivlties In the SN are severely and equally affected in H D The loss of activity correlates with the severity of the disease These findings have been confirmed, using radioimmunoassay analysis for substance P, m graded cases of H D [44]. Whether or not a better uhderstanding of specific neuronal vulnerability and resistance within H D strlatum might in any way lead to discovering the pathogenesis of this disorder is stiff not known There is no direct ewdence that the neuropathological and neu-

179

Fig 1 Low power overview of mtense substance P receptor ~mmunoreacuwty m large aspmy neurons m human strmtum

Fig. 2 Higher power of substance P receptor lrnmunoreacUve neuron showing extenswe dendntac branches and typical morphology of cholmergac stnatal neurons

180

Fig 3 Overview of substance P receptor Jrnmunoreactive neurons in the nucleus basahs of Mey'nert Note the ~mmunoreactlve fibers

Fig 4 High power view of large cholmerglc neurons in the nucleus basalIs showing substance P receptor lmmunoreactlvaty associated with cell membrane and cytoplasm

181

rochemlcal alterations which have been identified play a role m the etiology of H D . The studies from human postmortem brain tissue, however, have been instrumental in estabhshing an exc~totoxic model of the disease and emphasize the value of such studies Continued examination of the postmortem and experimental alterations may provide clues to the nature of the underlying degenerative process.

Substance P in human hippocampus and neocortex We examined the morphology and distribution of substance P elements in normal and AD isocortex and allocortex with a monoclonal anti-substance P antibody to define the morphologic correlates of substance P depletion in AD [45]. Bands of prominent terminal-like staining were found in the dentate gyrus of normal brain. Multipolar substance P immunoreactive neurons were seen in dentate polymorphic layer and CA4 and prominent fiber staining was

present in the CA fields of the hippocampus and adjacent aUocortex. Reactive penkarya, concentrated in deep cortex and infracortical wbate matter, were found in all isocortical regions. Greatest density was in sensory and parietal association cortex; lowest in visual cortex. Fiber density was generally greatest m layers I and II. In Alzhelmer's &sease, staining intensity was reduced in the dentate gyrus. Hilar neurons were unaffected but other CA field neurons were &storted with pruned dendritic trees Isocomcal perikarya and fibers were significantly depleted and distorted in all regions. Globular deposits consisting of &storted neurites or dissolving perikarya were frequently seen. Double staining methods showed that the vast majority of lsocortical, but not hippocampal substance P-like immunoreactive neurons are N A D P H dlaphorase positive. Despite the modest quantitatwe depletion of substance P m Alzheimer's disease cortex as measured by ra&oimmunoassay compared to somatostatln, there is a slgmficant depletion of substance

¢-, "

Fig 5 Lightly substance P receptor immunoreactive pyramidal neurons m CA2 filed of the human hlppocampus

182

P-hke immunoreactive perikarya. This disparity may be due to persistence of afferent projections which make a major contribution to substance P concentrations in cerebral cortex or to the high substance P content of dystrophic fibers in Alzheimer's disease cortex. The antiserum we used likely recognizes other peptides in the tachykinin family which share a common carboxy terminal sequence with substance P We cannot rule out that depletion of substance P immunoreactive neurons reflects the failure of viable neurons to synthesize substance P or, alternatively, to altered posttranslatlonal processing rather than death of immunoreactive cells. Recently, we have studied the dastributton of substance P m R N A in postmortem human cerebral cortex using a specific riboprobe and find a similar distribution in deep cortical layers and infracortIcal white matter. Studies on Alzheimer's disease brain are In progress

Substance P neurons In the dentate gyrus polymorphic layer do not contain N A D P H diaphorase and are resistant to degeneration in AD. In contrast, substance P reactive neurons in other CA fields and the isocortex that are severely depleted in AD are N A D P H dlaphorase reactive. Content of this enzyme may, therefore, define a subset of substance P neurons predisposed to degeneration in AD Recently is has been shown that N A D P H dlaphorase activity is a property of the enzyme N O synthase [46].

Substance P receptor immunocytochemistry human brain

in

Substance P receptor tmmunoreactivity was studied with two polyclonal antisera rinsed against synthetic peptides derived from the second and third extracellular loop of the substance P receptor sequence These antibodies have been characterized

$ 4

J

Fig 6 Dghtly substance P lmmunoreactlve neuron In the cortxcal subplate that morphologically resembles peptiderglc neurons defined with substance P and somatostaUn antlsera

183 extensively and thetr specificity has been confirmed biochemlcally [ 11 ]. P r e a b s o r p t i o n o f antiserum with cognate peptide completely abolishes neuronal staining. The m o s t p r o m i n e n t lmmunoreactivity Is localized to large neurons in the s t n a t u m and b a s a l forebrain (Figs 1-4) In the striatum large lmmunoreactive neurons are distributed throughout the c a u d a t e and p u t a m e n (Fig, 1). In m a n y cases extensive dendritic b r a n c h i n g is visible (Fig. 2). These neurons are acetylchohnesterase positive and cholme acetyltransferase i m m u n o r e a c t w e . In the nucleus b a s a h s large chohnergic neurons are also intensely labeled (Figs. 3, 4). I m m u n o r e a c t l v l t y is a s s o c i a t e d with b o t h the cell m e m b r a n e and c y t o p l a s m (Fig 4) and extends into cellular processes (Fig. 3). The basal forebrain chohnergic neurons are also lmmunoreactive for nerve growth factor r e c e p t o r This suggests an a n a t o m i c a l basis for an interaction between chohnerglc, substance P and nerve growth factor activities These chohnergic n e u r o n s are d a m a g e d in H D but they are not numerically depleted. In experimental excitotoxln lesions they also resist degeneration [47].These neurons, however, are sensitive to degeneration in A l z h e i m e r ' s disease [48] Less intense immunoreactivity is found in other subsets of neurons In the h l p p o c a m p u s pyrarntdal neurons in the C A 2 field are lightly labeled (Fig. 5). This zone is densely innervated by substance P lmmunoreactive fibers [45]. O c c a s i o n a l n o n p y r a m l d a l b a s k e t cells in CA1-4 are also seen In the neocortex the m o s t p r o m i n e n t l y labeled cells are m the cortical subplate (Fig 6). They resemble subplate neurons tdenttfied with somatostatin, n e u r o p e p t i d e Y, M A P - 2 and neurofilament antlsera and N A D P H d l a p h o r a s e hlstochemlstry. D o u b l e staming studies are in progress to further define these relationships. S u b s t a n c e P receptor lmmunoreactivity is not well defined in the h u m a n globus pallidus or substantta nigra This suggests the possibility that other receptors mediate the effects of substance P or that concentratlons o f the r e c e p t o r are below the threshold o f detection in h u m a n bram.

Acknowledgements W e would like to t h a n k K a r e n Harrington, A h d a E v a n s a n d L a w r e n c e C h e r k a s for technical assistance. This study was s u p p o r t e d in part by N I H grants A G 0 5 1 3 4 and N S 25588 (N W . K . , R J F ) and N S 21937 (J E K )

References 1 Yokota, Y, Sasal, Y, Tanaka, K, Fljlwara, T, Tsuchlda, K, Sh/gemoto, R, Kalozuka, A, Ohkubo, H and Nakanlshl, S, Molecular charactenzatlon of a functional cDNA for rat substance P receptor. J Biol Chem 264 (1989) 17649-17652 2 Maggio, J E, Tachykmlns, Annu Rev Neuroscl, 11 (1988) 13-28 3 HOkfelt, T, Kellerth, J O, Nllsson, G. and Pernow, B, Substance P localization in the central nervous system and m some primary sensory neurons, Science, 190 (1975) 889890 4 Cuello, A_C and Kanazawa, I_, The distribution of substance P ~mmunoreactxvefibers m the rat central nervous system, J Comp Neurol, 178 (1978) 129-156 5 Ferrante, R J., Kowall, N.W, Beal, M F_, Richardson, E P Jr, Bird, E_D and Martin, J B_, Topography of enkephalln, substance P, and acetylchohnesterase staining m Huntington's disease stnatum, Neuroscl Lett, 71 (1986) 283-288 6 Izzo, P_N, GraybJel, A M and Bolam, J P_, Characterization of substance P- and [met]enkephalm-lmmunoreactlve neuron s m the caudate nucleus of the cat and ferret by a single section Golgi procedure, Neurosclence, 20 (1987) 577-587 7 Beach, T G and McGeer, E G , The &strtbutlon of substance P m the primate basal gangha An ~mmunohlstochemlcalstudy of baboon and human brain, Neurosclence, 13 (1984) 29-52 8 Hershey, A D and Krause, J E, Molecular characterization of a functional cDNA encoding the rat substance P receptor, Science, 247 (1990) 958-962 9 Elde, R, Schalhng, M, Ceecatelh, S, Nakanlshl, S and HOkfelt, T, Localization of neuropept~de receptor mRNA in rat brain mltml observations using probes for neurotensln and substance P receptors, Neuroscl Lett, 120 (1990) 134-138 10 Geffen, C R, Substance P (neurokmln-l) receptor mRNA is selectivelyexpressed in chollnerglc neurons m the stnatum and basal forebram, Brain Res, 556 (1991) 165-170 11 Veenman, C_L., Medina, L_, Remer, A., Crankshaw, C and Krause, J E, Immunohlstochemlcal localization of SP receptor (SPR) in rat braan Soc Neuroscl Abs, 18 (1992) 1165 12 Graveland, G A, Wflhams, R S and DtFlgha, M A, A Golga

184

13

14

15

16

17

18

19

20

21

22

23

24

25

study of the human neostnatum Neurons and afferent fibers, J Comp Neurol, 234 (1985) 317-333 Kowall, N W, Ferrante, R J , Beal, M F , Richardson, E P_ Jr, Sofreniew, M , Cuello, A C and Martin, J B, Neuropeptide Y, somatostatm, and NADPH dlaphorase in human stnatum' a combmed immunocytochemical and enzyme histochemlcal study, Neuroscience, 20 (1987) 817 Ferrante, R J , Kowall, N W , Hersh, L B, Bruce, G and Richardson, E P Jr, ColocalIzatlon of chohne acetyltransferase and acetylchohnesterase neurons m Huntington's disease stnatum, Soc Neurosci Abs_, 13 (1987) 214 Ferrante, R J , Beal, M F , Kowall, N W , Richardson, E P Jr and Martin, J B, Sparing of acetylchohnesterase-contammg stnatal neurons in Huntangton's disease, Brmn Res, 411 (1987) 162-166 Ward, R P , Harnngton, K and Kowall, N W , Chromogranin m lmmunoreactlve neurons resist degenerataon In Huntmgton's disease J Neuropathol Exp_ Neurol, 9 (1990) 346 Ferrante, R J , Kowall, N W , Hersh, L B, Bruce, G and Richardson, E P , Jr, Co-localization of chohneacetyltransferase- and acetylchohnesterase-contammg neurons In Huntlngton's disease, 13 (1987) 1030 TherIault, E , Marshall, P E_ and Landis, D M D , Morphology of neurons contmmng VIP-hke immunoreactivity, J , Comp Neurol, 256 (1987) 1 Takagl, H , Mlzuta. H , Matsuda, T, Inagaki, S, Tatelshl, K and Hamaoka, T , The occurrence of cholecystokmm-hke lmmunoreactlve neurons in the rat neostriatum light and electron microscopic analysis, Brain Res_, 309 (1984) 346 Rlbak, C E , Vaughn. J E and Roberts, E, The GABA neurons and their axon terminals In the rat corpus stnatum as demonstrated by GAD immunocytochemxstry, J Comp Neurol, 187 (1979) 261-284 Besson, M J , GraybIel, A M and Qumn, B, Co-expression of neuropeptides in the cat's stnatum an lmmunohIstochemical study of substance P, dynorphm and enkephahn, Neuroscience 39 (1990) 33-58 Harnngton, K . M , Lee, J_M., Ferrante, R J and Kowall, N W, Transforming growth factor alpha is depleted in the substantla nlgra but persists m serotonergic and catecholammergac mldbram neurons in Huntington's disease, J Neuropathol_ Exp Neurol, 49 (1990) 345 Gerfen, C R , Bmmbndge, K G and Miller, J J , The neostrmtal mosaic compartmental distribution of calcium-binding protein and parvalbumm in the basal ganglia of the rat and monkey Proc Natl Acad Sci USA, 82 (1985)8780-8784 Chesselet, M F and Affolter, H U , Preprotachykinln messenger RNA detected by m sItu hybn&zatlon in stnatal neurons of the human brain, Brain Res, 410 (1987) 83-88 Aronm, N , Cooper, P E , Lorenz, L_Z, Bird, E D , Sagar, S M , Leeman, S E and Martin, J B, Somatostatm is in-

26

27

28

29

30

31

32

33

34

35 36

37

38

creased in the basal ganglia in Huntington's disease, Ann_ Neurol, 13 (1983) 519-526 Ferrante, R J , Kowall, N W , Beal, M F , Richardson, E P Jr, Bird, E.D and Martin J B, Selective spanng of a class of stnatal neurons in Huntington's &sease, Science, 230 (1985) 561-563 Gale, J S,BIrd, E D,Spokes, E G,Iversen, L L andJessell, T, Human substance P &stnbutlon in controls and Huntington's chorea, J Neurochem, 30 (1978) 633-644 Bolam, J P , Somogyl, P , Takagl, H , Fodor, I and Smith, A D , LoeallzaUon of substance P-like lmmunoreactlvity m neurons and nerve terrmnals in the neostnatum of the rat a correlated light and electron microscopic study, Neurocytology, 12 (1983) 325-344 Graveland, G A , Wdhams, R S and DlFlglIa, M A , Evidence for degenerative and regenerative changes in neostnatal spray neurons in Huntington's &sease, Science, 227 (1985) 770-773 Ferrante, R J , Kowall, N W , Martin, J B and Richardson E P Jr, Substance P-containing strlatal neurons m Huntington's &sease, J Neuropathol Exp_ Neurol, 46 (1987) 375 Ferrante, R, J_, Kowall, N W and Richardson, E P , Prohferative and degenerative changes in stnatal spmy neurons m Huntington's disease A combined study using the section Golgl method and calbmdm D28k immunocytochemlstry, J NeuroscI, 11 (1991) 3877-3887 Reiner, A , Albin, R L , Anderson, K_D, D'Amato, C J , Penney, J B and Young, A B, Differential loss of strmtal projection neurons in Huntington's disease, Proc Nail Acad ScL USA, 85 (1988) 5733-5737 Albln, R_L, Remer, A , Anderson, K D , Penney, J B and Young, A B, Stnatal and mgral neuron subpopulations in rigid Huntington's disease Implicalaons for the functional anatomy of chorea and ngadity-akmesia, Ann Neurol, 27 (1990) 357-365 Crossman, A_R, Primate models of dyslonesla The experimental approach to the study of basal ganglia-related involuntary movement disorders, Neurosclence, 21 (1987) 1-40 DeLong, M R , Pnmate models of movement disorders of basal ganglia, Trends Neurol, Scl, 13 (1990). Crossman, A R , Mitchell, I J , Sambrook, M A and Jackson, A , Chorea and myoclonus m the monkey induced by gamma-ammobutyric acid antagonism m the lenuform complex, Brain, 111 (1988) 1211-1233 Ferrante, R.J_, Kowall, N W , Harrlngton, K and Richardson, E P , Terminal strlatal substance P and met enkephahn projections in the globus palhdus are equally affected in Huntlngton's disease, J Neuroscl, 16 (1990) Marshall, P E , Landis, D M D_ and Zlanentls, E L , Immunocytochemical studies of substance P and leu-enkephalm in Huntington's disease, Brain Res , 289 (1983) 11-26

185 39 Fomo, L S. and Jose, C., Huntington's chorea a pathologtcal study In A Barbeau T N. Chaseand and G W Paulson (Eds), Huntington's Chorea, Raven Press, New York, 1973, pp 450-470 40 Oyanagl, K , Takeda, S., Takahashl, H., Ohama, E. and Ikuta, F , A quanUtatwe mvestlgat~on of the substantla nlgra m Huntmgton's &sease, Ann Neurol, 26 (1989) 13-19 41 Beal, M F and Martin, J B, Neuropeptldes m neurologacal &sease, Ann Neurol,, 20 (1986) 547-565 42 Sajl, M and Rels, D J , Delayed transneuronal death of substantla mgra neurons prevented by gama-ammobutync acid agomst, Science, 235 (1987) 66-69 43 Fen-ante, R J , Kowall, N.W and Rachardson, E P., Selective loss of lmmunocytochemlcal markers m substantla mgra of Huntington's disease, J Neuropathol Exp Neurol., (1990). 44 Beal, M F , Elhson, D W , Mazurek, M F , Swartz, K_S,

45

46

47

48

Malloy, J R , Bird, E.D and Martm, J_B, A detmled exammation of substance P m pathologacally graded cases of Huntmgton's &sease, J Neurol Scl, 84 (1988) 51-61 Qmgley, B J_ J and Kowall, N_ W , Substance P-hke lmmunoreactwe neurons are depleted m Alzheuner's &sease cerebral cortex, Neurosclence, 41 (1991) 41-60 Hope, B T , Michael, G-J., Kmgge, K M and Vincent, S.R, Neuronal NADPH dlaphorase is a mtnc oxide synthase, Proc Natl Acad Scl USA, 88 (1991) 2811-2814 Beal, M F , Swartz, K J , Ferrante, R J_ and Kowall, N . W , Chronic qumohnlc acid lesions m rats closely resemble Huntmgton's disease, J Neuroscl, 11 (1991) 1649-1659 Whltehouse, P J , Price, D L, Clark, A_W , Coyle, J T_ and Delong, M R , Alzhelmer disease evidence for selectwe loss of chollnerglc neurons in the nucleus basahs, Ann Neurol, 10 (1981) 122-126