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An immunohistochemical study of the substance P neuronal system in the primate brain : basal ganglia… Beach, Thomas Gerald 1984

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AN IMMUNOHISTOCHEMICAL STUDY OF THE SUBSTANCE P NEURONAL SYSTEM IN THE PRIMATE BRAIN: BASAL GANGLIA AND NEOCORTEX By Thomas Gerald Beach B . S c . U n i v e r s i t y of V i c t o r i a 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n of Neurosc ience , Department of P s y c h i a t r y ) We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA February 1984 © Thomas Gera ld Beach 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) 11 ABSTRACT Using immunohistochemical methods I have studied the distribution of substance P fibers, terminals and perikarya throughout the basal ganglia and neocortex of baboons and at selected levels of the human brain. Immunoreactivity in the substantia nigra pars reticulata, internal segment of the globus pal 1idus and ventral pallidum was dense and of a characteristic, "woolly fiber" morphology. The caudate nucleus and putamen contained sharply circumscribed patches of dense immunoreactivity superimposed on a moderately stained background. The external division of the globus pal 1idus displayed very l i t t l e immunoreactivity. Two morphological types of immunoreactive cell bodies were present in the caudate nucleus, putamen and nucleus accumbens, and were clustered within the dense patches. The distribution of immunoreactive perikarya within the striatum differed from that reported for rats, as stained neurons were distributed evenly throughout the rostro-caudal extent rather than being concentrated in the rostral portions. This work represents the first detailed examination of the distribution of substance P-containing neuronal structures in the primate basal ganglia. It confirms recent published reports which have shown that the striatum is not a homogeneous structure, as it has long been regarded, but is subdivided into zones with differing neuroanatomical connections and neurotransmitter content. The neocortex was not studied as intensively as the basal ganglia, but it was conclusively established that substance P-immunoreactive neurons exist throughout the neocortex. TABLE OF CONTENTS ABSTRACT i LIST OF FIGURES i ACKNOWLEDGEMENT INTRODUCTION pages 1-PART 1: The Basal Ganglia pages 11-Introduction pages 11-Experimental Procedures pages 13-Results pages 16-Figures pages 23-Discussion pages 51-PART 2: Cerebral Cortex pages 57-Results and Discussion pages 58-Figures pages 60-CONCLUSION pages 65-References and Bibliography pages 72-i v LIST OF FIGURES Fig. 1. General patterns of staining in the baboon basal gangl i a 23 Fig. 2-4. Map of cell bodies and nerve terminals in the baboon basal ganglia 26-28 Fig. 5. Baboon basal ganglia rostral to the anterior commi ssure 30 Fig. 6. Baboon basal ganglia at the level of the lenticular nucleus 32 F ig . 7. Caudal putamen and substantia nigra in the baboon 34 Fig . 8a. Patches of intense staining in the baboon putamen 36 F ig . 8b. Detail of substantia nigra in the baboon 36 Fig. 9. Woolly fibers in baboon substantia nigra and globus pal l i dus 38 Fig . 10. Woolly fiber relationship to cel l body in baboon pallidum 40 F ig . 11. Detail of str iatal cel l bodies in the baboon 42 Fig . 12. Detail of cell bodies in baboon striatum and ventral striatum 44 Fig . 13. Human basal ganglia at the level of the lenticular nucleus 46 Fig . 14. Woolly fibers in human basal ganglia 48 F ig . 15. Detail of cel l bodies in human striatum 50 F ig . 16. Representative density of cell bodies in baboon neocortex 62 F ig . 17. Detail of cell bodies in baboon neocortex 64 V ACKNOWLEDGEMENT The author would l ike to thank the Pathology Department of the Health Sciences Centre Hospital at the University of Br i t ish Columbia for their assistance in obtaining the human brains, Dr. J .A. Wada of the Kinsmen Laboratory of Neurological Research for donating the baboon brains, Dr. Keij i Satoh of the same laboratory for providing assistance with technical procedures and data analysis, and Joane and Laura Suzuki for preparing the diagrams. Special thanks to Dr. Edith McGeer for her encouraging advice and guidance. This work was supported by the MRC of Canada. 1 INTRODUCTION Our understanding of central nervous system function has repeatedly been advanced by the development of new neuro-histological techniques. Carmine and Nissl staining methods, developed in 1858 and 1885, respectively, demonstrated that the brain, l ike other tissues, is populated with c e l l s . The Golgi technique (1883) revealed that there are two basic types of brain c e l l s , neurons and g l i a , both of which possess f ine, intr icately branching processes (Haymaker and Sch i l le r , 1970). From these early studies came the "neuron theory" which postulates that the brain "hardware" consists of millions of interconnected yet independent units (neurons). This is s t i l l a basic premise in neuroscience. The latter half of this century has seen the development of several staining techniques which can be used to determine the pattern of neuronal connectivity. The Nauta technique (1950) was the f i r s t of these, but now autoradiographic, florescent tracer and horseradish peroxidase techniques are also available to the neuroanatomist (Heimer and Robards, 1981). These methods are responsible for an exponential increase of tract -tracing studies over the last twenty years and as a result we now have a rough "wiring diagram" of brain pathways. Neurotransmitters, the chemical messengers acting at synapses between neurons, have been intensively studied because of their strategic function and because their susceptibil ity to pharmacological manipulation offers an opportunity for therapeutic intervention in CNS disorders. Histological stains specific for various neurotransmitters have been 2 produced over the last thirty years and are important because they allow us to see where within our wiring diagram a given neurotransmitter acts. In effect we can "color-code" our wiring diagram by identifying neurotransmitters used by each pathway. By comparing the color-coded wiring diagrams of normal and diseased brains, we can identify areas of neurotransmitter deficiency and suggest rational drug treatments. The treatment of Parkinson's disease and psychotic disorders has already been improved through this approach. This thesis presents new information regarding the distribution and connections of neurons containing substance P, a peptide neurotransmitter. An immunohistochemical staining method was used, and the brains of baboons and humans were studied. 3 Substance P. The peptides are the newest class of neurotransmitters, and the most rapidly expanding one (Hokfelt et a l , 1980). Because they can be easily investigated with newly developed immunochemical techniques, they represent one of the most exciting frontiers of brain research. Substance P was the f i r s t peptide to be investigated and is probably the most wel1-characterized in terms of distr ibut ion, release, and biological properties (Pernow, 1983). The story of substance P begins more than f i f t y years ago. In 1930, von Euler and Gaddum, while investigating the effects of acetylcholine upon gut moti l i ty , noted that atropine was only part ia l ly effective in preventing intestinal movements (von Euler, 1981). Further experiments revealed that some gut extracts stimulated movements of the isolated jejeunum, even in the presence of atropine at sufficient concentration to anull the effects of added acetylcholine. Using this property as a bioassay, they isolated and concentrated the active material in the form of a stable, dry powder which they f i r s t called "P" , then "preparation P" and f i n a l l y , "Substance P". Von Euler and Gaddum f i r s t referred to i t as "P" simply because i t was a powder and they knew very l i t t l e else about i t . The final name has stuck even though von Euler tr ied to discourage i ts use. Fortuitously, substance P turned out to be a peptide, making the name seemingly rat ional . For almost 40 years this powder was not chemically ident i f ied , although i t was known to be a peptide and considerable work was done in defining i ts pharmacological properties. It was defined during this time 4 as a pept ide which caused a drop i n blood p r e s s u r e , con t rac ted i s o l a t e d gut specimens, and whose e f f e c t was not antagonized by a t r o p i n e . I t was known to be present i n high c o n c e n t r a t i o n in stomach and b r a i n . In 1970-71 Leeman and her co -workers at Brandise U n i v e r s i t y were i n v o l v e d in an attempt to i s o l a t e a c o r t i c o t r o p i n n r e l e a s i n g f a c t o r from bovine hypothalamic e x t r a c t s . When the var ious e x t r a c t s from one p r e p a r a t i v e column were screened f o r b i o l o g i c a l a c t i v i t y , Leeman n o t i c e d tha t one f r a c t i o n s t i m u l a t e d s a l i v a r y s e c r e t i o n when i n j e c t e d i n t o a n a e s t h e t i z e d r a t s . This s i a l o g o g i c e f f e c t was not i n h i b i t e d by a t r o p i n e and proved to be a s imple q u a n t i t a t i v e assay adaptable to the i s o l a t i o n and p u r i f i c a t i o n of the p e p t i d e . Th is group prepared the pure m a t e r i a l and then r e a l i z e d that i t s pharmacolog ica l a c t i o n s and chemical p r o p e r t i e s were i d e n t i c a l w i th those p r e v i o u s l y reported f o r p a r t i a l l y p u r i f i e d substance P (Chang and Leeman, 1970) . The amino a c i d sequence of substance P was determined in 1971 (Chang et a l . ) . I t i s an undecapept ide , and i t s C - t e r m i n a l sequence i s h i g h l y homologous w i th the mol luscan s a l i v a r y gland p e p t i d e , e l e d o i s i n , and w i t h severa l amphibian s k i n p e p t i d e s , the most we l l known of these being physalaemin (Erspamer, 1981) . The C - t e r m i n a l end appears to be r e s p o n s i b l e f o r the fundamental pharmacolog ica l p r o p e r t i e s of these m o l e c u l e s , w h i l e the N - te rmina l sequence, being v a r i a b l e , may account f o r the observed d i f f e r e n c e s i n potency , e f f i c a c y and d u r a t i o n of a c t i o n . Substance P was soon prepared i n s y n t h e t i c form (Tregear et a l , 1971) and made commercia l ly a v a i l a b l e . Th is has r e s u l t e d i n an e x p l o s i o n of p u b l i c a t i o n s on the s u b j e c t , there being only about 25 per year p r i o r to 1970 but more than 250 in 1981 (Pernow, 1983) . Th is has a l s o 5 c l a r i f i e d our u n d e r s t a n d i n g of the f u n c t i o n of t h i s m o l e c u l e . There i s now a g e n e r a l i z e d consensus t h a t s u b s t a n c e P i s a n e u r o t r a n s m i t t e r , h a v i n g met the c r i t e r i a f o r a p p r o p r i a t e l o c a t i o n , b i o s y n t h e s i s , i n a c t i v a t i o n , r e l e a s e , mimicry and antagonism ( N i c o l l et a l , 1980; Pernow, 1 9 8 3 ) . There i s a l s o agreement t h a t s u b s t a n c e P i s t h e n e u r o t r a n s m i t t e r r e l e a s e d i n the d o r s a l horn of the s p i n a l c o r d by p r i m a r y s e n s o r y neurons r e s p o n s i b l e f o r t h e s e n s a t i o n and t r a n s m i s s i o n of p a i n (Henry, 1 9 8 0 ) . However, s u b s t a n c e P i s not l i m i t e d t o s e n s o r y f u n c t i o n s , as i t appears t o p l a y a r o l e i n autonomic and motor systems as w e l l . For i n - d e p t h d i s c u s s i o n s of t h e l i t e r a t u r e on s u b s t a n c e P ( a p p r o x i m a t e l y 2000 p a p e r s ) the r e a d e r i s r e f e r r e d t o t h e f o l l o w i n g r e v i e w s : Skrabanek and P o w e l l (1977) , von E u l e r & Pernow (1977, 1980, 1 9 8 3 ) , and Pernow ( 1 9 8 3 ) . T h i s t h e s i s d e a l s w i t h t h e neuroanatomy o f s u b s t a n c e P - c o n t a i n i n g pathways, so a b r i e f summary of t h e work done i n t h a t f i e l d w i l l now be g i v e n . Comprehensive d e s c r i p t i o n s of the d i s t r i b u t i o n of s u b s t a n c e P w i t h i n t h e CNS are a v a i l a b l e o n l y f o r t h e r a t b r a i n . These are d e r i v e d from radioimmunoassay ( B r o w n s t e i n et a l , 1976) and immunohistochemical approaches ( L j u n g d a h l et a l , 1978; C u e l l o and Kanazawa, 1978; I n a g a k i et a l , 1982; Sakanaka et a l , 1 9 8 2 ) . Substance P appears t o be p r e s e n t i n a l l p a r t s of the CNS, but i t s c o n c e n t r a t i o n v a r i e s w i d e l y from a r e a t o a r e a . The c e r e b e l l u m and c e r e b r a l c o r t e x have very s m a l l c o n c e n t r a t i o n s of the p e p t i d e , w h i l e the b a s a l g a n g l i a , hypothalamus and d o r s a l horn of t h e s p i n a l c o r d are very w e l l endowed. Thus t h e p h y l o g e n e t i c a l l y o l d e r p a r t s of t h e b r a i n seem t o have r e l a t i v e l y h i g h e r c o n c e n t r a t i o n s than t h e more r e c e n t a d d i t i o n s . T h i s i s a l s o r e f l e c t e d by comparison of 6 whole-brain concentrations of substance P in various v e r t e b r a t e s , which decrease in the f o l l o w i n g order: chicken, pigeon, duck, r a t , guinea-pig, r a b b i t , c a t , and human (Pernow, 1983). The more highly d i f f e r e n t i a t e d brains seem to have l e s s substance P per unit of volume. Studies combining l e s i o n techniques with immunohistochemistry have e s t a b l i s h e d several s p e c i f i c substance P pathways w i t h i n the rat brain (Cuello et a l , 1982). Substance P-containing neurons in the caudate-putamen p r o j e c t to the entopeduncular nucleus and s u b s t a n t i a n i g r a . There i s a p r o j e c t i o n from the medial habenula to the l a t e r a l habenula, ventral tegmental area/interpeduncular nucleus and the dorsal raphe, and from the caudal raphe to the spinal t r i g e m i n a l nucleus, ventral horn and dorsal horn of the s p i n a l cord. SP neurons in the medial amygdala innervate the c e n t r a l amygdala and s t r i a t e r m i n a l i s neurons innervate the medial p r e o p t i c hypothalamus. There i s an ascending p r o j e c t i o n from SP neurons in the nucleus 1 a t e r o d o r s a l i s tegmenti of C a s t a l d i (TLD) which innervates the l a t e r a l septal area and the medial f r o n t a l cortex (Sakanaka et a l , 1981, 1983). The nucleus of the t r a c t u s s o l i t a r i u s receives f i b e r s from c e l l bodies located i n the sensory ganglia of the VII, IX and X c r a n i a l nerves (Cuello et a l , 1982). The l o c a l i z a t i o n of substance P in the human brain has been i n v e s t i g a t e d e x t e n s i v e l y by radioimmunoassay (Cooper et a l , 1981) but only s e l e c t e d areas have been described by immunohistochemistry (Pearson,1983; Del F i a c c o et a l , 1983). The RIA studies i n d i c a t e that substance P i s d i s t r i b u t e d i n a pattern s i m i l a r to that seen in the r a t . There are regional d e f i c i t s of substance P in Huntington's disease (Kanazawa et a l , 1977; Gale et a l , 1978; Emson et a l , 1980; Buck et a l , 7 1981), Parkinson's disease (Mauborgne et a l , 1983), Alzheimer's disease (Perry et a l , 1981; Crystal and Davies, 1982), and famil ial dysautonomia (Pearson et a l , 1982). However, in a l l of these, excepting the last , the depletion of substance P is thought to be of minor importance in relation to other pathological features. At present, no therapeutic regimens directed at manipulation of substance P levels have emerged, although this has been considered (Jonsson and Hallman, 1982). There are areas which deserve further exploration by immunohistochemical techniques. The primate brain has not yet been mapped for substance P and the disease states noted above have not been adequately investigated. Even in the rat brain, there are many SP pathways yet to be discovered and we are lacking the fine details of neuronal connectivity in the established pathways. For example, substance P neurons in the striatum project to the substantia nigra but do they synapse with dopaminergic neurons or GABAergic neurons or neurons of other types? This question is highly relevant for Parkinson's disease, since dopaminergic neurons are somewhat selectively depleted in this disorder. The studies presented here were directed at one former gap in our knowledge, the relative lack of information for the primate brain. 8 Immunohistochemical Methodology. The original method of Coons (1958) has been improved upon by many followers, notably Sternberger (1979) and Hsu (1981). Basic to a l l techniques is the production of antibodies which recognize portions of a neurotransmitter molecule or i ts specific synthetic or degradative enzymes. Either monoclonal or polyclonal antibodies may be used. Monoclonal antibodies, especially i f they can be produced continuously by cell culture, offer the advantage of reproducibility of results, since their binding properties do not change from batch to batch (Cuello et a l , 1980). Polyclonal antisera vary in binding properties from animal to animal and even from batch to batch from the same animal (Ljungdahl et a l , 1978). Once a particular batch is used up, exact reproduction of results is no longer possible. In the staining procedure, the antibodies are incubated with thin (5- 50 ym) sections of brain t issue. The antibodies bind to their specific antigens and the excess antibodies are washed away. In the direct florescence technique, this primary antibody is bound to a florescent molecule; viewing the section with light of the exciting wavelength under a l ight microscope reveals the location of the antibody-antigen complexes. In the indirect technique, the florescent molecule is not bound to the primary antibody, but to a secondary antibody which recognizes the primary antibody as i ts antigen. The indirect method results in an amplification of the florescence at the site by increasing the number of florescent molecules in the antibody-antigen complex. Alternatively, an enzyme, horseradish peroxidase, can be bound to primary or secondary antibodies producing an insoluble, colored reaction product (from diaminobenzidine and hydrogen peroxide) at the site of the immune complex. Sternberger improved upon the original method by introducing a third step to the process. In his method, the second antibody, l ike the f i r s t , is unlabel led and a third step involves incubation with a complex of antibodies specif ical ly bound to peroxidase molecules; the peroxidase-anti-peroxidase complex (PAP) binds to the secondary antibody (anti-IgG), already localized in the t issue. This method has at least two advantages over the immunoflorescence techniques. F i r s t , much lower concentrations of antisera can be used (roughly 1/10 to 1/100), because there is much more vis ible product deposited at each primary antibody s i t e . This makes i t more economical than florescent techniques. Second, the slides can be coverslipped with Permount and stored permanently and conveniently at room temperature, whereas florescent slides decay under illumination and must be stored in the freezer with messy glycerine-based mounting media. The PAP method may also be able to localize antigens at lower concentrations than the immunoflorescent techniques can but this is debatable. The ABC method of Hsu et al (1981) takes advantage of the high aff in i ty and specif ic i ty of the avidin-biotin interaction, using i t to attach peroxidase-containing complexes to secondary antibodies conjugated with b iot in . Avidin-biotin immunoperoxidase methods were already in use (Guesdon et a l , 1978; Vincent et a l , 1981); the ABC method improved on these by using large complexes of avidin, biotin and peroxidase in the third step, in a manner analogous to the PAP method, to increase the number of peroxidase molecules aggregated at the reaction s i t e . Although 10 i t has been argued that the ABC method is superior to the PAP method (Hsu et a l , 1981), this has not been convincingly demonstrated. Avidin-biotin techniques were used exclusively in the present work so no comparison with the PAP method is possible here. A comparison of the ABC method with an earl ier avidin-biotin method (Vincent et a l , 1981) was done; the ABC technique seemed to give a lower level of background staining, resulting in a crisper image. An important l imitation of any immunohistochemical method is dictated by the gjture of the antibody-antigen reaction. The antibody recognizes a small sequence of amino acids, perhaps only five or s ix . It is possible that antibodies could recognize the sequence wherever i t is accessible, making i t d i f f i cu l t to say exactly which compounds are being stained in tissue sections. Therefore any staining which is derived from antibodies raised against a particular molecule must indicate this uncertainty. For example in the case of substance P, immunohistochemical staining is usually described as "substance P- l ike immunoreactivity". Elements which might cross-react with substance P antibodies would include longer or shorter peptides that contain the antigenic determinant for that particular antibody, and, part icular ly , closely-related peptides such as physalaemin, which has recently been reported to exist in the mammalian CNS (Erspamer, 1981). 11 PART 1: THE BASAL GANGLIA 12 Recent detailed studies of the localization of certain putative neurotransmitters within the basal ganglia have led to some new insights into the organization of this important nuclear group. The pattern of immunohistochemical staining for substance P and met-enkephalin in the neostriatum (Graybiel et a l , 1981) has added to a growing body of evidence which suggests that this structure is not homogeneous, but is divided into discrete compartments differing in neurotransmitter content and connectivity (Olson et a l , 1972; Goldman and Nauta, 1977; Graybiel et a l , 1979; Haber and Elde, 1982). The distributions of the same two peptides have been used to redefine the borders of the globus pallidus (Haber and Nauta, 1981; Haber and Nauta, 1982). Substance P, in part icular, appears to be a useful marker for the rostral and ventral extension of the globus pal 1idus of rats which has been termed the ventral pallidum. In the primate globus pall idus, substance P has been found to be relatively selectively localized in the internal segment and met-enkephalin in the outer segment (Haber and Elde, 1981); classical studies do not distinguish between the two subdivisions. Both substance P and met-enkephalin immunoreactive structures in the globus pallidus exhibit a remarkable microscopic "woolly fiber" morphology which suggests that nerve terminals containing these peptides completely ensheath receptive pal l idal dendrites (Haber and Nauta, 1981; Haber and Elde, 1982), exerting a powerful influence upon pal l idal neurons. Most of this work has been done in rats or cats. Although there has been some illuminating recent work done on primates, especially on the distribution of met-enkephalin (Haber and Elde, 1981; Haber and Elde, 1982) there has not been a comprehensive study in primates. 13 It was f e l t , therefore, that a detailed immunohistochemical study of substance P in the primate basal ganglia was warranted. EXPERIMENTAL PROCEDURES Subjects. Two baboon brains and four human brains were used in this study. The baboons (Senegalese, Papio papio) were adult males of 10-12 kg body weight. The human brains were obtained through the cooperation of the Department of Pathology at the University of Br i t ish Columbia Health Sciences Centre Hospital in Vancouver, B.C. The subjects were males aged 57, 62, 76 and 91 years of age; a l l had died without signs of central nervous system disorder. Preparation of t issue. The baboons were pretreated with diisopropylfluorophosphate (DFP; 0.2-0.5 mg/kg i.m.) for an unrelated acetylcholinesterase histochemical study. Four to six hours later they were anesthetized with sodium pentobarbital and perfused transcardially or retrogradely through the descending aorta, as well as through the le f t carotid artery. The descending aorta was ligated below the level of cannulation. The vasculature was rinsed with heparinized normal sal ine, followed immediately by a f ixative mixture consisting of 5 l i t res of 10% formalin in 0.04 M phosphate buffer, pH 7.4, with 1% calcium chloride. These solutions were delivered at room temperature over 30-40 minutes. One hour after perfusion the brain was removed from the skull and post-fixed whole in the same fixative for 2-4 hours. At this point the brain was sliced into 10 coronal sections of 8-9 mm thickness, and the 14 post-fixation continued overnight at 4°C. The sl ices were then stored in phosphate-buffered-saline (PBS) with 15% sucrose at 4°C for 1-2 weeks, and sectioned as described below. The human brains were removed from the cranium, within six hours of death, with the c i rc le of Wi l l i s intact . They were perfused via the basilar and/or internal carotid arteries with 1-5 l i t res of PBS, followed by 2.5 l i t res of 4% paraformaldehye in 0.1M phosphate buffer, in some cases with 0.35% glutaraldehyde, at 4°C over 30-40 minutes. The brains were then sliced into 1 cm thick coronal sections and post-fixed at 4°C in the same fixative (without glutaraldehyde) for 16 hours. They were then stored in PBS with 15% sucrose, at 4°C, for up to one month before use. Both human and baboon brains were sectioned coronally on a freezing, sledge-type microtome at 30 y m ; sections were taken from throughout the rostro-caudal extent of the basal ganglia in the case of the baboon brains and from selected levels of the human brains. Substance P antisera. Two antisera were used, one polyclonal and one monoclonal. The polyclonal antiserum was prepared in guinea pigs against synthetic substance P (Sigma), according to the procedure of Vaitukaitis et al (1971). This antiserum has been used in RIA studies (Nagy et a l , 1980) and a previous immunohistochemical study (Vincent et a l , 1981). Its cross-reactivity with the structurally similar peptides, physalaemin and eledoisin, was determined by RIA to be less than one percent (Nagy et a l , 1981). The monoclonal antibody was purchased from Sera Labs, Inc., and has been extensively characterized by both RIA and 15 immunohistochemical techniques (Cuello et a l , 1979; Cuello et a l , 1980). Immunohistochemical procedure. A l l sections were processed free-f loating u t i l i z ing the avidin-biotin-peroxidase complex (ABC) technique (Hsu et a l , 1981). The incubation with monoclonal anti-substance P was performed at a dilution of 1:100 with PBS containing 0.2% Triton X-100. The polyclonal antiserum was diluted 1:10,000 in the same diluent. Both incubations were carried out for 72 hours at 4°C. Control sections were treated with identically prepared solutions to which had been added 200 ng/ml synthetic substance P (Sigma) or, alternatively, the f i r s t incubation was omitted altogether. The following steps were done at room temperature. After washing (3 changes of PBS/0.05% Triton X-100 over 1 hour), the sections treated with monoclonal anti-substance P were incubated for two hours in biotinylated rabbit anti - rat IgG (Vector), while those sections which had been treated with polyclonal antiserum were incubated during the same time period in biotinylated goat anti-guinea-pig IgG (Vector). Both secondary antisera were diluted 1:100 in the same diluent as was used for the primary antibodies. Another washing step was followed by a one hour incubation in an avidin-biotin-peroxidase complex (Vector), diluted in PBS with 0.05% Triton X-100. The sections were then washed with PBS, reacted with 3,3'-diaminobenzidine tetrahydrochloride (0.02% in 0.05 M Tris buffer, pH 7.4, containing 0.006% H 2 0 2 ) , rinsed with PBS and mounted onto slides previously treated with chrome alum gelatin. The next day the mounted sections were dehydrated and coverslipped with Permount. 16 Analysis and presentation of results. From approximately 150 sections of the baboon brains, nine, stained with the monoclonal antibody, were chosen as representative of the staining seen in the areas of interest. These slides were placed in a photographic enlarger and projected directly onto 8 x 10 i n . photographic paper (Figs. 5-8). Stained areas were considered as specif ical ly marking the presence of substance P i f they were present in the sections stained with the substance P antisera but not in the sections treated as controls. Tracing paper was used to copy the outline of the section and the outlines of prominently stained areas from the photographic print. Further assessment of staining intensity and character, including the location of cel l bodies, was done under the microscope and the location of observed features marked free hand onto the tracing. The sections used for mapping were later rehydrated and counterstained with cresyl violet to reveal the position of nuclear groups in relation to the immunohistochemical staining. Reference was also made to nearby sections stained with cresyl v io let , and to sections stained with the Kluver-Barrera method. Two atlases of the baboon brain were consulted (Davis and Huffman, 1968; Riche et a l , 1968) for aid in the identif ication of brain nuclei and fiber t racts . RESULTS Baboon Brain. The following description of staining patterns and morphological features was derived primarily from studies of the sections treated with monoclonal anti-substance P. The sections treated with 17 polyclonal anti-substance P displayed similar distributions of substance P- l ike immunoreactive structures but, since the staining was much less intense, these sections were not as thoroughly studied. A l l photomicrographs are of sections treated with the monoclonal antibody, with the exception of Figure 15C, which depicts a section of human brain treated with the polyclonal antibody. The putamen, caudate nucleus, globus pallidus and substantia nigra a l l contained a relatively high level of substance P immunoreactivity and stood out against background areas. Microscopic examination revealed that the staining, where i t could be resolved into discrete structures, resembled neuronal elements morphologically. Therefore, i t is assumed that the macroscopic appearance of the staining was due to the staining of neuronal cel l bodies, dendrites and especially, axons and terminals. In general, three types of staining could be distinguished. In the caudate nucleus and putamen there existed a diffuse but specific background staining, upon which was imposed varying numbers of punctate elements and beaded axons (Figs. 1A,B), as well as c e l l s . In the substantia nigra pars compacta the diffuse staining was lacking, there being only punctate elements and beaded axons (Fig. IC). The globus pallidus pars interna, and areas extending ventrally and rostrally from i t into the anterior olfactory area, were found to contain a third type of staining, which consisted predominantly of tangled networks of punctate elements and beaded axons arranged in pairs of parallel lines 2-8 ym apart (Figs. IE,F). As circular profiles were also conspicuous (Figs. 1D,E), i t is possible that cyl indrical dendrites, covered with an immunoreactive coating, are the basis of this staining pattern. These 18 profi les w i l l be termed "woolly f ibers" , following the description by Haber and Nauta (1983) of a similar phenomenon in the homologous areas of the rat brain. The substantia nigra pars reticulata also displayed this type of staining in the lateral parts (Fig. ID). The staining in the medial zones of the reticulata was so dense that morphology was obscured, but i t appeared that the staining might be made up of massed circular profiles of woolly fibers cut in cross-section (travell ing in a parasagittal plane). Caudate, Putamen and Nucleus Accumbens. The caudate nucleus and putamen exhibited a two-tone staining pattern, with intensely stained patches standing out against a uniform background of moderate staining intensity (Figs. 2A-4B, 5A-7A). These patches possess sharp boundaries with the surrounding areas. Although oval, round or squarish shapes were present, long elongated patches with complex curves were most common. The cross-sectional dimensions of the patches were generally 0.3-1 mm in width and, in the case of the elongate patches, 3-7 mm in length. The elongate patches tended to be oriented in a semi-transverse plane rostral ly , and closer to a sagittal plane caudally. The peripheral boundaries of the putamen, especially la tera l l y , appeared to be rimmed by intensely staining patches. Many of the patches were themselves rimmed with bands of even greater intensity (Fig. 8A). The accumbens nucleus (Fig. 5A) and the part of the putamen which extends ventrally below the outlines of the classical lenticular nucleus towards the t a i l of the caudate nucleus (Fig. 6B) did not display these discrete patches of high staining i intensity. 19 Cell bodies immunoreactive for substance P were seen throughout the caudate and putamen (Figs. 2A-4C). They were sl ightly more numerous in the caudal half of these nuclei . In many cases the neurons were obviously concentrated in the densely stained patches (Fig. 11A). There were occasional immunoreactive neurons in the nucleus accumbens (Fig. 2A) The str iatal neurons were round, oval or triangular in shape and 15-20 y m in diameter (Figs. 11B,C,D). A few st r iata l neurons approached 30 um in size and were polymorphic (Fig. 12A). Presumptive substance P cell bodies were also seen ventral to the anterior commissure, in the area which has been variously termed substantia innominata or substriatal grey (Figs. 2C;3A), and also in the anterior olfactory area, amongst the islands of Calleja (Figs. 2A,B). These neurons were of similar size and shape as the str iatal neurons (Figs. 12B,C) and may represent ventrally displaced st r iata l tissue (see discussion). Substantia Nigra. There was a t ightly woven network of woolly fibers in the substantia nigra pars reticulata (Fig.ID); these were very similar to the woolly fibers seen in the globus pallidus pars interna and in the ventral pallidum (Figs. 1E,F). In the substantia nigra pars compacta, the immunoreactivity was predominantly in the form of punctate elements and beaded axons, with occcasional woolly f ibers. Woolly fibers in the pars reticulata were often aggregated into bundles (Fig.9A), particularly in the lateral regions; the medial areas were stained so densely that i t was d i f f i c u l t to discern morphological de ta i l . Neurons of the pars compacta, seen after counterstaining with cresyl 20 v io let , invaded the intensely stained pars reticulata in tongue-like formations (Fig. 8B) . Immunoreactivity around pars compacta cel l bodies varied from moderate to dense. Immunoreactivity around pars reticulata cel l bodies was usually dense. The substantia nigra did not contain substance P immunoreactive neuronal perikarya. Globus Pallidus and Ventral Pal l idal Areas. The interior of the globus pallidus pars externa was devoid of immunoreactivity except for occasional traversing woolly f ibers . These tended to coalesce into loose networks at the peripheral borders of the external segment with the result being an outline of fa i r l y intense staining around the unstained core of this division (Figs. 6A ,B) . This was especially noticeable near the lateral medullary lamina (Fig. 6A) and at the dorsal and ventral borders of the external segment. The medial border of the external segment was outlined s imi lar ly , but this border tended to merge with the tight networks of woolly fibers seen in the internal segment. The loose woolly fiber networks of the ventral border of the external segment merged rostral ly , beneath the anterior commissure, with those of the ventral pallidum (Fig. 5B) . The globus pallidus pars interna was occupied throughout by a tight network of woolly fiber profiles (Figs. 6A ,B) . The lateral border of this division was stained more intensely than the main body; at higher magnification i t was noticed that the woolly fibers here were composed of beaded axons with large varicosities (Fig. 9B), giving this area a grainy appearance compared to the softer look of the main body, where the individual fibers and varicosities were of smaller size (Fig. IE). The concept of a ventral pallidum has evolved in recent years to describe what appears to be a rostral and ventral extension of the globus pallidus of rats into the area previously termed the substantia innominata, and, further rostral ly , into the olfactory tubercle (Ten Donkelaar and Dederen, 1979; Switzer et a l , 1982). As the pattern and character of substance P immunoreactivity seen in this study closely resembles that which has been used to define the ventral pallidum in the rat (Haber and Nauta, 1981; Haber and Nauta, 1983) the term ventral pallidum also seems appropriate here to describe the woolly fiber networks ventral to the anterior commissure (Figs. 2C;5B), ventral and medial to the nucleus accumbens (Figs. 2B;5A), and within the anterior olfactory area (Figs. 2A;5A). Substance P immunoreactive perikarya were not present in the globus pallidus or ventral pallidum. Woolly Fibers. It has been hypothesized that the woolly fibers may consist of unstained dendrites covered with immunoreactive boutons (Ljungdahl et a l , 1978; Haber and Nauta, 1983), but this has not been conclusively established. Therefore, a close examination of this phenomenon was undertaken. In most cases the immunoreactive structures covering the presumptive dendrites are not easily resolvable due to their small size and dense packing, but i t is sometimes possible to see woolly fibers composed of well-defined axons with large varicosities travel l ing in parallel (Fig. 9B), suggesting that a l l woolly fibers are similarly composed. When sections counterstained with cresyl violet were examined, i t was found 22 that the woolly f ibers, stained brown by the diaminobenzidine reaction product, sometimes merged with neuronal cel l bodies stained blue with cresyl v io let , resulting in what appears to be a completed image of a neuron with dendritic tree (Fig. 10). Such unions were observed in the globus pallidus pars externa, pars interna, ventral pallidum, and in lateral parts of the substantia nigra pars ret iculata. Human Brains. Sections were taken from human brains at the level of the decussation of the anterior commissure, at the level of fu l l development of the lenticular nucleus, and through the midbrain, including the substantia nigra. The pattern of substance P immunoreactivity at these levels resembled that seen at similar levels in the baboon brain. In part icular, there was intense staining ventral to the anterior commissure in the substantia innominata (ventral pallidum), preferential staining of the internal segment of the globus pallidus as compared to the external segment (Fig. 13) and intense staining of the substantia nigra pars ret iculata. The character of staining seen in the ventral pallidum (Fig. 14A), globus pallidus (Fig. 14B) and substantia nigra pars reticulata (Fig. 14C) was of the woolly fiber type. In the substantia nigra pars ret iculata, the woolly fibers often ran together in large bundles (Fig. 14C). Substance P immunoreactive cel l bodies were present in the caudate nucleus and putamen; these were round, oval or triangular in shape, and from 15-20 ym in size (Figs. 15A-D). The human brain did not display prominent st r iata l patches (Fig. 13); possible reasons for this wi l l be presented in the discussion. 23 F ig . 1. Immunoperoxidase photomicrographs of 30 vm frozen sections of baboon brain: putamen, in a matrix region of moderate immunoreactivity (A) ; putamen, in a densely immunoreactive "patch" (B); substantia nigra pars compacta (C); substantia nigra pars reticulata (D); globus pallidus pars interna (E); and ventral pallidum (F) after incubation with monoclonal antibodies to substance P. The character of staining was of three types: evenly stained or amorphous, with superimposed punctae and varicose fibers of small calibre (A,B); varicose fibers and punctae of larger cal ibre, without an amorphous background (C); and networks of profiles resembling cylinders with an immunoreactive coating, hereafter termed "woolly fibers" (D-F). The density of the f i r s t two types of staining varied from sparse or absent (not shown) to moderate (A), dense (B) , and very dense (not shown) and the networks of the third type were categorized as loose (not shown) or tight (D-F), referring to the size of the mesh. Scale bar (A) = 10 pm for A-F. 3 25 F ig . 2-4. Diagrams of the distr ibut ion, morphological character and density of substance P immunoreactive fibers and terminals and of the distribution and numbers of substance P immunoreactive perikarya at representative coronal planes of the basal ganglia of the baboon. Density scales indicate the relative density of fibers and terminals and are assigned values from moderate (M), to dense (D) and very dense (VD) or, where the character of staining was that of interlacing networks of woolly f ibers , loose (L) or tight (T). Other abbreviations are as below. Calibration bar = 3 mm. Abbreviations used in figures 2-15. Acc Nucleus accumbens IP Nucleus interpeduncularis AO Area olfactoria 1ml Lamina medullaris lateral is CA Commissura anterior MM Nucleus mammillaris medial is Cd Nucleus caudatus n l l l Nervus oculomotorius CI Capsula interna NR Nucleus ruber CO Chiasma opticum Put Putamen CrC Crus cerebri SI Substantia innominata CP Commissura posterior SL Nucleus septalis lateral is FMT Fasciculus mammi11othalamicus SNc Substantia nigra pars compacta GL Corpus geniculatum lateral is SNr Substantia nigra pars reticula GP Globus pall idus, pars externa St Nucleus subthalamicus GPi Globus pall idus, pars interna Th Thalamus HP Hypothalamus TO Tractus opticus IC Insula Calleja VP Ventral pallidum ICm Insula Calleja magna ZI Zona incerta 22 29 Figs. 5-8. Projection prints of intensely stained substance P immuno-reactive areas within 30 frozen coronal sections of baboon brain. Areas of intense staining appear white. F ig . 5. Basal ganglia at the level of the insula Calleja magna (ICm;A) and the level of the decussation of the anterior commissure (CA;B). Substance P immunoreactivity is prominent in patches in the putamen (Put) and caudate nucleus (Cd) (A and B), in the nucleus accumbens (Acc;A), around the islands of Calleja (IC;A) and in the area olfactoria (A0;A), around the margins of the external segment of the globus pallidus (6Pe;B) and in the ventral pallidum (VP;B). Calibration bar (A) = 3 mm for both A and B. 30 31 F ig . 6. Basal ganglia at the level of the lenticular nucleus (A and B). Patches of intense substance P immunoreactivity are seen in the putamen (Put;A,B) and caudate nucleus (Cd;A,B). The internal segment of the globus pallidus (GPi;A,B) is also intensely stained. A thin strip of tissue just medial to the lateral medullary lamina (1ml;B) displays some immunoreactivity as does an area within the substantia innominata (SI;B). Scale bar (A) = 3 mm for both A and B. 33 Fig . 7. The caudal most portions of the putamen (Put;A) contain patches of intense immunoreactivity, as do the t a i l and body of the caudate nucleus (Cd;A). The substantia nigra pars reticulata (SNr;A and B) is especially prominent, while the pars compacta (SNc) displays a lesser degree of immunoreactivity (A and B). Scale bar (A) = 3 mm for both A and B. 35 F ig . 8 . Patches of intense immunoreactivity (A) within the caudate nucleus (Cd) and putamen (Put) are rimmed by bands of even greater intensity (arrowheads). Scale bar (A) = 2 mm and serves for both A and B. In (B), the substantia nigra pars reticulata (SNr) appears as an entangled meshwork. The pars compacta (SNc; borders delimited by dashed lines) extends as tongue-like intrusions into the pars ret iculata. 37 Fig . 9. Photomicrograph of the substantia nigra pars reticulata (A) after immunoperoxidase staining for substance P. Individual woolly f iber profi les appear as two dense (dark) lines running in para l le l . These profi les tend to aggregate and run together in intertwined bundles. Scale bar = 50 u m . A photomicrograph of the globus pallidus pars interna (B), after immunoperoxidase staining for substance P, also shows the woolly fiber prof i les . In this case, i t is possible to see that the two parallel lines are made up of structures resembling beaded axons running in para l le l . Calibration bar = 10 y m . 3 8 39 F ig . 10. Photomicrograph of the globus pallidus pars externa after immunoperoxidase staining for substance P, and counterstaining with cresyl v io let . A Nissl-stained neuronal cel l soma (S) appears to form a union with immunostained woolly fiber prof i les . The dark parallel l ines may be formed by deposition of immunoperoxidase reaction product upon the outside of unstained dendrites (d) extending from the neuronal soma, g, g l ia l nuclei ; BV, blood vessel. Calibration bar = 30 vm. v 41 F ig . 11. Immunoperoxidase photomicrographs of baboon putamen after incubation with monoclonal antibodies to substance P. Immunoreactive perikarya in the striatum (A) were often observed to be clustered with densely immunoreactive patches (dp) in preference to moderately immunoreactive matrix (m) areas. Typical str iatal substance P immunoreactive neurons (B,C,D) were medium-sized with oval, round or triangular cell bodies. Calibration bar (B) = 20 vm for B,C,D. 43 F ig . 12. Immunoperoxidase photomicrographs of baboon putamen and forebrain areas. Occasional large, multipolar substance P immunoreactive neurons were observed in the putamen (A), in addition to the more common medium-sized type. Immunoreactive perikarya were also observed in ventral st r iatal areas, ventral to the anterior commissure at the level of i ts decussation (B) and (C) amongst the islands of Calleja (IC; large arrows point to immunoreactive neurons, the other neurons have been stained with cresyl v io le t ) . Pal l idal elements, represented by woolly fiber profiles (small arrowheads), were also seen in the v ic inity of the islands of Cal le ja , which were themselves conspicuously free of immunoreactivity. Scale bar (A) = 30 um for A and B. 45 F ig . 13. Projection print of the human basal ganglia after immunoperoxi-dase staining for substance P. Areas of positive immunoreactivity appear white. The internal segment of the globus pallidus (GPi) is intensely immunoreactive. Other areas with prominent immunoreactivity are a thin str ip of tissue just medial to the lateral medullary lamina (1ml) and the other margins of the external pal l idal segment (GPe). The caudate nucleus (Cd) is moderately stained, and the putamen (Put) is sparsely stained. Both caudate and putamen display patches resembling those seen in the baboon nuclei , but the patches are much less prominent in the human t issue. Scale bar = 5 mm. 47 Fig . 14. Photomicrographs of human basal ganglia after immunoperoxidase staining for substance P. Dark structures are positively immunoreactive. Woolly fiber profiles of immunoreactivity were seen ventral to the anterior commissure in the substantia innominata ( A ) . Similar profiles were seen in the globus pallidus pars interna ( B ) , around the margins of the globus pallidus pars externa (not shown) and in the substantia nigra pars reticulata (C). In the latter area, the prof i les , presumably dendrites with an immunoreactive coating, tended to run together in bundles. Scale bar (A) = 20 yin and serves for A and B. The scale bar in (C) = 20 vm. 49 Fig . 15. Photomicrographs of human putamen after immunoperoxidase staining for substance P. Immunoreactive perikarya were a l l medium sized with oval, round or triangular cell bodies (A-D). Scale bar (A) = 20 y m and serves for A-D. 51 DISCUSSION Comparison of the Baboon with Other Species. Substance P immunoreactive f ibers , nerve terminals and cel l bodies are distributed throughout the basal ganglia of the baboon in a pattern which is somewhat similar to that which has been reported for rats (Cuello and Kanazawa, 1978; Ljungdahl et a l , 1978). Notable differences include the non-homogeneous starkly patchy nature of staining in the striatum of baboons and the occurrence of immunoreactive perikarya throughout the rostro-caudal extent of the baboon striatum which is in contrast to their predominantly rostral location in the rat striatum (Ljungdahl et a l , 1978). Substance P immunoreactive patches have been reported in the caudate nucleus of the cat by Graybiel and colleagues (1981); the cat putamen did not consistently display the patches, however, and the patches in the caudate were not defined cr isp ly . The clearly defined patches seen throughout the baboon striatum support the conjecture by Graybiel that technical shortcomings may be responsible for the lack of definition of the patches in the cat nuclei . The observation here that the st r iata l patches have a circumferential band of even greater staining intensity adds a further level of complexity to this phenomenon. As in the rat, substance P immunoreactive cel l bodies were present in the caudate nucleus and putamen but, while in the rat there appears to be a paucity of cell bodies caudal to the level of the anterior commissure (Ljungdahl et a l , 1978), the baboon nuclei contained large numbers of immunoreactive neurons at a l l levels, with even their caudal-most portions possessing substantial numbers of presumptive 52 substance P neurons. The substance P-immunoreactive perikarya appeared to be heavily concentrated within the dense patches of immunoreactivity; this suggests that the patches may represent the relatively concentrated axonal arborizations and terminal f ields of substance P neurons located within them. The visualization of large numbers of substance P immunoreactive perikarya without prior treatment of the experimental animal with co lch i -cine is unusual in i t se l f since this is a prerequisite for their demonstration in adult rats (Cuello and Kanazawa, 1978; Ljungdahl et a l , 1978; Inagaki et a l , 1982). Application of our staining protocol to sections of rat brain did not result in immunoreactive perikarya unless the animals were pretreated with colchicine (unpublished observations); pretreatment with DFP had no effect, so our use of DFP pretreatment in the baboon does not seem responsible for this result. A possible explanation is that substance P-containing perikarya of baboons may contain relatively higher concentrations of the peptide than those of the rat. • The location of immunoreactive perikarya ventral to the anterior commissure in the substantia innominata and amongst the islands of Calleja in the anterior olfactory area are described here because there is considerable evidence that st r iata l tissue may extend ventrally via "bridges" of cel ls through the substantia innominata to the olfactory tubercle (Heimer and Wilson, 1975; Heimer, 1978). These neurons, therefore, may l i e in ventral extensions of the striatum. Substance P-immunoreactive neurons have been observed in these areas in rats (Ljungdahl et a l , 1978; Bolam et a l , 1983) and have been suggested to 53 represent ventral s t r iata l elements in that species (Fallon et a l , 1983). As in the rat (Ljungdahl et a l , 1978; Bolam et a l , 1983), there appeared to be two types of substance P-immunoreactive neurons in the striatum: medium sized round, oval or triangular neurons and occasional medium to large polymorphic neurons. Intense staining of the primate internal pal l idal segment, in high contrast to the relative lack of staining in the external segment, has been reported previously by Haber and Elde (1981). As described by those authors, and by Haber and Nauta (1983) in a later paper, this is reflected in rats by intense staining of the entopeduncular nucleus and relatively sparse staining of the globus pallidus proper, these being the respective homologues of the primate nuclei . The Nature and Distribution of Substance P Immunoreactive Woolly  Fibers. The unique morphology of substance P immunoreactivity in pal l idal areas and in the substantia nigra pars reticulata appears to be due to a coating of stained nerve terminals upon large dendrites, outlining their contours and appearing as parallel lines when cut longitudinally, c ircles when cut in cross-section. This morphology was f i r s t reported by Ljungdahl et a l . (1978) in the globus pallidus of the rat. These authors also f i r s t conjectured that i t was due to an outlining of large dendrites. Recently Haber and Nauta (1981, 1983) have furthered the description of the phenomenon in rats and Haber and Elde (1981) have described i ts occurrence in monkeys with both enkephalin and substance P immunohistochemical staining. Pearson (personal comm.) has observed identical features in the human substantia nigra. 54 The distribution of this type of substance P immunoreactive morphol-ogy, termed "woolly fiber networks" by Haber and Nauta (1983), appears to be confined, according to these authors, to the substantia nigra pars reticulata , entopeduncular nucleus and ventral pallidum in rats. The woolly fiber networks have been proposed by the same authors to be a relatively specific marker of ventral pal l idal areas in that species. Substance P immunoreactive woolly fiber networks have also been observed in the internal pal l idal segment in the monkey (Haber and Elde, 1981). The distribution of woolly fiber networks in both baboon and human brain, as described here, conforms to that found in rats and monkeys. The l imitation of the phenomenon to substantia nigra pars reticulata and pal l idal areas suggests that the s imi lar i t ies between these areas, well known from ultrastructural studies (Fox et a l , 1974), continue when putative neurotransmitter content and disposition are compared. The proposal put forth by Nauta (1979) that the two areas might better be considered as a single nucleus, spl i t in two by the internal capsule, is attracti ve. The woolly fibers form unions with Nissl-stained cel l bodies in the baboon ventral pallidum, globus pallidus internal and external segment, and in the pars reticulata of the substantia nigra. These neurons are of medium to large s ize, with abundant Nissl substance and prominent nucleoli and resemble in s ize, frequency and distribution GAD and GABA-T-containing neurons in these areas in the rat (Vincent et a l , 1982; Oertel et a l , 1982; Nagai et a l , 1983). It is possible that GABA-containing neurons are the common post-synaptic elements in the woolly-fiber networks. 55 Human Brains. As the human brains were not sectioned systematically throughout the extent of the basal ganglia, a fu l l comparison of the staining pattern with that of the baboon or of other species is not possible. However, enough sections were taken to verify that woolly f iber networks immunoreactive for substance P exist in the substantia innominata beneath the anterior commissure (ventral pallidum), in the internal segment of the globus pall idus, at the peripheral borders of the external pal l idal segment and in the substantia nigra pars ret iculata. The caudate and putamen exhibited patches which were stained relatively more intensely than the matrix, although this was not nearly as str iking as in the baboon. Immunoreactive cel l bodies were abundant in the caudate nucleus and putamen and resembled the medium-sized neurons seen N in the baboon. Multipolar neurons were not seen, possibly because not enough sections were examined; these neurons were scarce in the baboon st r i atum. Post-mortem delay prior to f ixat ion, although limited to six hours or less in this study, may result in a loss of immunohistochemical staining intensity, perhaps explaining the faded nature of the str iatal patches of the human brains. Radioimmunoassay studies (Gale et a l , 1978; Cooper et a l , 1981) have found substance P to be remarkably stable in human brains, with no drop in immunoreactivity up to 66 hours after i n i t i a l sampling, i f the brains are refrigerated. However, the interval between death and removal of the brain from the cranium (4-6 hours in this study) may result in an i n i t i a l depletion of immunoreactivity. Substance P is not stable in aqueous solution at room temperature, as i t tends to hydrolyze spontaneously (technical note, Beckman). In the 56 post-mortem, pre-perfusion time period, the temperature of the brain slowly drops from 37°C to 20°C. There is good reason to suspect some loss of substance P during this period. Supporting this conjecture is the fact that radioimmunoassay values of brain peptides in various animal species are generally higher than those reported for humans (Cooper et a l , 1981). The staining of the human brain was, with the exception of the faded st r iata l patches, surprisingly similar in intensity to the staining of baboon brain, suggesting that post-mortem degradation should be anticipated, but should not unduly hamper, future immunohistochemical investigations of the distribution of substance P in the human brain. PART 2: CEREBRAL CORTEX 58 Immunohistochemical studies have indicated that several neuro-peptides are present in neurons of the cerebral cortex (Emson and Lindval l , 1979). The substance P content of cortex (Brownstein et a l , 1976; Gale et a l , 1978; Cooper et a l , 1981) has been localized to varicose fibers (Hokfelt et a l , 1976; Ljungdahl et a l , 1978) and has been attributed to ascending afferents originating from the brainstem (Paxinos et a l , 1978; Sakanaka et a l , 1983) since cortical substance P neurons have been observed only rarely (Ljungdahl et a l , 1978; Inagaki et a l , 1982). This author wishes to report here on the existence of a substantial population of substance P- l ike immunoreactive neurons within the cerebral cortex of baboons. These neurons could contribute to the substance P fiber networks of the cortex. Two adult male baboons (Papio papio, 10-12 kg body weight) were used (see Part 1, Basal Ganglia, for details of fixation and immunohisto-chemical methods: the same baboons and methods were used in this study). Sections (30 ym) were taken on a freezing microtome from frontal , par ieta l , temporal and occipital lobes and from cortex of cingulate gyrus and insula. Substance P- l ike immunoreactive perikarya were seen in a l l areas of cortex examined when the monoclonal antibody was employed. (Sections treated with the polyclonal antibody contained only occasional, very faintly stained perikarya. Therefore the remainder of this section wi l l deal solely with findings from sections treated with the monoclonal antibody.) There were small numbers of very intensely stained neurons, as well as much larger numbers stained with lesser degrees of intensity. Figure 1 shows two intensely-stained neurons against a background of 59 cresyl violet-stained neurons. There were no stained cortical perikarya in the sections treated with substance P-adsorbed antisera. In a l l cortical areas, immunoreactive neurons occurred in layers III-VI but were most common in layers V and VI. Positively stained neurons were most abundant in the anterior cingulate gyrus and in the fusiform gyrus of the temporal lobe and least numerous in the occipital lobe. A count of intensely-stained neurons in the anterior cingulate gyrus, frontal lobe, temporal lobe and insular cortex gave an average of approximately one intensely-stained neuron per circumferential millimeter of cortex, measured along the grey matter-white matter interface (F ig . l ) . Many more neurons were faintly stained; these were approximately ten times more numerous. The immunoreactive neurons were of many shapes. Figure 2 depicts the wide range of morphologies observed. Bipolar neurons (Fig.2e) were the most numerous type, representing almost 20% of the intensely-stained neurons. Most of these had dendrites oriented vert ical ly in the cortical columns but a horizontal orientation was also common. Multipolar neurons, resembling the one shown in figure 2a, were common, accounting for approximately 10% of the t o t a l . A few neurons of the unipolar type (Fig.2d) and the pyramidal type (Fig.2c) were seen. Besides these identif iable types, there were many multipolar neurons of widely varying form (Fig. 2b, f ) . The sizes of the immunoreactive neurons varied from 7 um to 42 ym, measured along their longest axes. Two-thirds of the measured neurons were 15-20 ym, however, and this appeared to be the predominant size in a l l areas. It is evident that substance P neurons, or neurons containing a 60 substance which is immunologically s imilar , exist in the cerebral cortex of baboons. The existence of cortical substance P neurons has not been widely recognized in the past, perhaps because existing reports (Ljungdahl et a l , 1978; Inagaki et a l , 1982) are few in number and lacking in de ta i l . This i s , therefore, the f i r s t detailed description of the phenomenon. This finding leads to speculation upon related topics. It is possible that cortical substance P neurons could give rise to at least part of the cortical SP-LI fiber networks described previously, with brainstem neurons also contributing. The decreases in substance P content of cortex reported in Alzheimer's disease (Perry et a l , 1981; Crystal et a l , 1982) might be due to loss of cortical substance P neurons. A further interesting aspect of this study was the demonstration of considerable numbers of substance P- l ike immunoreactive neurons without colchicine pretreatment, both in the cerebral cortex and in the basal ganglia. Colchicine-induced disruption of axonal transport is usually required to demonstrate substance P-containing perikarya by immunohistochemical techniques (Ljungdahl et a l , 1978; Cuello and Kanazawa, 1978; Inagaki et a l , 1982). It is possible that DFP, an acetylcholinesterase inhibi tor , caused an elevation of perikaryal substance P levels , since acetylcholinesterase is capable of hydrolyzing substance P (Chubb et a l , 1980). However, preliminary experiments with DFP-treated rats did not produce similar results. The baboon, in comparison to animals investigated previously, may simply have high levels of perikaryal substance P. 61 Figure 1. Two intensely-stained substance P- l ike immunoreactive (SP-LI) neurons (arrows and insets) in the cortex of anterior temporal lobe. The section has been counterstained with cresyl v io let . This f ie ld is representative of the average density of intensely-stained SP-LI neurons. 63 Figure 2. An i l lust rat ion of the wide range of somatic shapes displaying substance P- l ike immunoreactivity. Dot-l ike background structures are SP-LI nerve terminals. See text for further discussion. CONCLUSION 65 The primary objective of this work was to chart the location of substance P-containing neurons and terminals in the baboon basal ganglia. This has been accomplished; we have therefore added some details to what is known about substance P. In the process, however, we have found that the pattern of staining demonstrated levels of organization within these nuclei which are not readily apparent with classical staining techniques. Our quest for detailed knowledge has led us to a vantage point from which we can view the basal ganglia with a new perspective. The Striatum as a Compartmentalized Structure. The striatum appears to be a homogeneous structure in Nissl-stained sections, but in the last 15 years, various investigators have shown that i t has at least two sharply differing regions. Afferent fibers and groups of efferent neurons tend to be concentrated into "islands", "compartments", or "clusters", surrounded by the remainder of the tissue ("matrix"). Various neuro-transmitter markers show a similar arrangement. The baboon striatum displays precisely this type of organization when stained for substance P. Graybiel et al (1981) have previously reported a "mosaic distr ibution" for substance P in the caudate nucleus of the cat, but stated that the zones formed by l ight ly and intensely staining regions were not sharply defined, in contrast to the clearly dist inct compartmentation seen with other markers. They conjectured that their staining methods might have been inadequate. This appears to have been a 66 c o r r r e c t assumption as the " i s l a n d s " or "patches" of substance P - r i c h areas found in t h i s work are very d i s c r e t e and suggest that substance P may be added to the l i s t of n e u r o t r a n s m i t t e r markers compartmental ized w i t h i n the s t r i a t u m . I t i s worth not ing that substance P immunoreact iv i t y i s a l s o poor ly o rganized i n the rat s t r i a t u m . Could i t be p o s s i b l e tha t a l l p rev ious s t u d i e s on the rat were t e c h n i c a l l y inadequate? Or i s i t p o s s i b l e t h a t the o r g a n i z a t i o n of substance P - c o n t a i n i n g t e r m i n a l s becomes p r o g r e s s i v e l y more r e f i n e d as one ascends the e v o l u t i o n a r y s c a l e , e x p l a i n i n g why the p a t t e r n becomes more obvious as one looks from rat t o cat to baboon. Going aga ins t the l a t t e r hypothes is i s the e x i s t e n c e of a f u l l y developed compar tmenta l i za t ion of rat s t r i a t u m when s t a i n e d f o r other parameters , such as o p i a t e r e c e p t o r s , t h a l a m i c a f f e r e n t s and a c e t y c h o l i n e s t e r a s e (Herkenham and P e r t , 1981) . I t i s t e n t a t i v e l y concluded here tha t d e f i n i t i v e compar tmenta l i za t ion of substance P probably e x i s t s i n rat and cat s t r i a t u m but has not yet been c l e a r l y demonstrated. Pharmacolog ica l man ipu la t ion of substance P c o n c e n t r a t i o n s may be necessary to b r i n g out the p a t t e r n ; h i s t o c h e m i c a l methods f o r dopamine only reveal compar tmenta l i za t ion i n the adu l t rat b r a i n when the animals have been p r e t r e a t e d wi th a t y r o s i n e hydroxy lase i n h i b i t o r (Olson et a l , 1972) . The baboons in t h i s study were p r e t r e a t e d wi th an a c e t y l c h o l i n e s t e r a s e i n h i b i t o r ; t h i s may have r a i s e d substance P t i s s u e c o n c e n t r a t i o n s p r i o r to s a c r i f i c e , s i n c e a c e t y l c h o l i n e s t e r a s e has been reported to be capable of h y d r o l y z i n g substance P (Chubb et a l , 1980) . P r e l i m i n a r y exper imentat ion wi th AChE i n h i b i t o r pretreatment of r a t s was done i n an e f f o r t to r e p l i c a t e the s t a i n i n g p a t t e r n seen in the 67 baboons, but was not s u c c e s s f u l . Only one set of drug dosage/staining c o n d i t i o n s was t e s t e d , however, so t h i s i s an area that needs f u r t h e r study. The f u n c t i o n a l s i g n i f i c a n c e of s t r i a t a l compartmentalization has not yet been determined. In an approach to t h i s question several i n v e s t i g a t o r s have attempted to c o r r e l a t e s t a i n i n g patterns produced by various d i f f e r e n t methods. "Islands" of opiate receptors have been found to correspond to vacancies in the termination of thalamic p a r a f a s c i c u l a r p r o j e c t i o n s and a c e t y l c h o l i n e s t e r a s e - p o o r zones (striosomes) in the rat s t r i a t u m (Herkenham and Pert, 1981); e n k e p h a l i n - r i c h patches r e g i s t e r with AChEase-poor areas in the cat putamen (Graybiel et a l , 1981); AChEase-poor patches mirror dopamine i s l a n d s in f e t a l and neonatal cats (Graybiel et a l , 1981a); p r e f r o n t a l c o r t i c a l a f f e r e n t s to the s t r i a t u m end only in matrix and not in i s l a n d areas (Goldman-Rakic, 1982). Neurotensin-rich areas c o i n c i d e with striosomes, while neurotensin receptors are markedly denser in the AChE-rich matrix (Goedert et a l , 1984). A r e t r o s p e c t i v e a n a l y s i s of N i s s l - s t a i n e d s e c t i o n s in the rhesus monkey caudate nucleus found that i s l a n d s of densely packed neurons were embedded in a matrix of l a r g e r , more l o o s e l y packed neurons (Goldman-Rakic, 1982). A l l methods demonstrate two compartments, one r i c h in the marker of i n t e r e s t , and one poor. It might be ventured that markers grouped together in e i t h e r i s l a n d or matrix zones are l i k e l y to have a f u n c t i o n a l r e l a t i o n s h i p as w e l l ; t h i s appears to be the most e x c i t i n g aspect of the s u b j e c t . It has been suggested that the i s l a n d component i s made up of concentrations of e f f e r e n t neurons (Graybiel et a l , 1979). Since 68 afferents from prefrontal cortex and from the parafascicular thalamus end in matrix areas, i t might be reasonable to guess that interneurons l i e largely in matrix, receiving inputs which they relay to output neurons lying in the islands. The results of this work appear to support such a model. Substance P neurons are believed to be efferent neurons, giving rise to a striatonigral projection. Striatonigral neurons labelled by retrograde transport of horseradish peroxidase are reportedly concentrated into is land- l ike compartments (Graybiel et a l , 1979). Our finding that substance P- l ike immunoreactive neurons are concentrated within the island areas therefore f i t s well into this hypothetical scheme. Further correlative studies of afferents, efferents and neurotransmitter markers wi l l be needed to ful ly evaluate this model. Woolly Fiber Networks as Markers of Homologous Areas In 1979, H.J.W. Nauta proposed a "conceptual reorganization" of the basal ganglia, arguing that the traditional nuclear boundaries might be masking a more basic pattern and thereby hampering our efforts to understand the operation of this system. The subdivision of a mass of grey matter by a tract of white matter should not necessarily endow separate functional identit ies to the grey matter on opposite sides of the t ract . The striatum of rats is a single mass of grey matter so i t is easy to accept the idea that i t represents a single nucleus. The striatum of primates, however, is cleaved by the internal capsule and thus we have given each side a name: caudate and putamen. We now know that the caudate and putamen are best considered as a functional unit, their spatial separation not reflecting functional differences. Nauta suggests that other subdivisions may be equally arbitrary. The substantia nigra may be 69 only "art i factual ly" separated from the caudal globus pallidus by the internal capsule (in whales and porpoises the two are not separated). S imi lar ly , the anterior commissure may be only an inconsequential intrusion into the rostral part of a larger globus pall idus, not a barrier delimiting pallidum from substantia innominata. Since that time Nauta's suggestions have received experimental support. Switzer, H i l l and Heimer demonstrated (in the rat) that the globus pallidus does indeed extend rostrally and ventrally beneath the anterior commissure, claiming a large piece of the substantia innominata (1982). Five separate histochemical stains were used to distinguish pallidum-like areas from the surrounding brain substance. The area beneath the anterior commissure has become known as the ventral pal 1i dum. In spite of the histochemical s imi lar i t ies between dorsal and ventral pallidum demonstrated by Switzer et a l , some differences have emerged. Switzer et al found that immunohistochemical staining for enkephalin "woolly fiber networks" (this term was actually coined later by Haber and Nauta) was uniform throughout the dorsal and ventral globus pal l idus. But Haber and Nauta (1981) found that substance P woolly f iber networks were confined to the ventral pallidum, being scarce or nonexistent in the dorsal pallidum. On the other hand, prominent substance P woolly fiber networks extended beyond the boundaries of the rat globus pallidus into the entopeduncular nucleus, which was devoid of enkephalin woolly f ibers. Ear l ier , Haber and Elde (1981) had reported equivalent results in the primate globus pall idus: the external segment (corresponding to globus pallidus proper of the rat) showed dense 70 enkephalin woolly fiber networks while the internal segment (corresponding to entopeduncular nucleus in the rat) had a relative scarcity of these structures. Our results complement those of Haber and Elde in this issue, for we found that substance P woolly fiber networks were absent from the external segment and very dense in the internal segment and ventral pallidum. Thus, although the ventral pallidum has been shown to be histochemically s imilar , in many ways, to dorsal pallidum, other histochemical methods have shown that there are differences between dorsal pallidum (globus pallidus proper of rats and external segment of primates) on the one hand and ventral pallidum and entopeduncular nucleus on the other hand. It is therefore not possible to say that a l l these areas are ident ical , but this is not what Nauta is suggesting. He only suggests that certain regions regarded as being unique (such as globus pall idus, ventral pallidum, entopeduncular nucleus and substantia nigra pars reticulata) may in fact have certain fundamental structural and functional s imi la r i t ies , upon which local specializations may be superimposed. The basic structure of the above-mentioned areas, as seen under the electron microscope, is very similar (pointed out by Fox) and the light microscopic manifestation of this structure appears to be the woolly fiber networks. The woolly fiber networks are found exclusively in these brain areas. The fact that the woolly fiber networks contain enkephalin in some areas and substance P in others might be regarded as a local specialization on the fundamental pattern. Nauta's suggestion that these areas might a l l perform a similar computational task is s t i l l appealing. 71 Summary. This study has shown that the disposition of substance P immunoreactivity within the primate basal ganglia is fundamentally very similar to that previously reported for the rat. Use of the rat as a model for human basal ganglia function would therefore be reasonable, at least as far as substance P is concerned. In addition, the use of substance P as a marker of organizational patterns within the basal ganglia has demonstrated levels of organization both more complex and more simple than that which classical neuroanatomy teaches. The striatum appears to consist of at least two different regions which have characteristic neurotransmitter content and neuroanatomical connections; classical neuroanatomy regarded the striatum as homogeneous. Substance P perikarya and terminals are much more highly concentrated within the "island" areas than within the "matrix" areas. 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