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Anatomical and histochemical studies of the globus pallidus and related basal ganglia nuclei Staines, William Alan 1983

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ANATOMICAL AND HISTOCHEMICAL STUDIES OF THE GLOBUS PALLIDUS AND RELATED BASAL,GANGLIA NUCLEI by William Alan Staines B.Sc.(Hons.) University of Alberta 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (I n t e r d i s c i p l i n a r y Studies) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1983 ©William Alan Staines, 1983 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 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 available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis 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 P^AwAv^, b><rs~.p~ o\ t^ax^roVoe^cA ^>O<=^<JL%. The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 requirements for an advanced degree at the University DE-6 (3/81) i i ABSTRACT The anatomical organization of the connections of the major components of the basal ganglia was investigated in d e t a i l . A sensitive procedure for the simultaneous study of afferents and efferents was carried out on the striatum (CP), globus pa l l i d u s (GP), and substantia nigra (SN). Previously well characterized connections of the CP were confirmed, additional evidence for a projection to the CP from the ventromedial nucleus of the thalamus was obtained and a topographically organized projection to the CP from the GP was discovered. A similar study of the SN revealed a n i g r a l projection to the i p s i l a t e r a l l a t e r a l dorsal nucleus of the thalamus and n i g r a l input from the cont r a l a t e r a l posterior l a t e r a l hypothalamus. The projection of the GP to the SN was found to be linked topographically to the s t r i a t o n i g r a l and p a l l i d o s t r i a t a l pathways. A study of the connections of the GP confirmed a massive projection from the CP and provided further evidence of a reciprocal connection. In addition, p a l l i d a l innervations of the entopeduncular nucleus and r e t i c u l a r nucleus of the thalamus were indicated. Because of the potential importance of a p a l l i d o s t r i a t a l projection and the s i g n i f i c a n t number of technical d i f f i c u l t i e s associated with i t s demonstration, additional experiments were carri e d out to confirm the presence of t h i s pathway and to determine i t s anatomical rel a t i o n s h i p to other basal ganglia connections. Retrograde l a b e l l i n g of p a l l i d o s t r i a t a l neurons, studied with electron microscopy and in combination with lesions of the striatum, confirmed that p a l l i d a l neurons project either to or through the striatum. Evidence for possibly two groups of p a l l i d a l neurons that project to the CP was obtained, and i t was observed that both of these c e l l groups were congruent with the s t r i a t o p a l l i d a l terminal f i e l d s . Comparisons of the d i s t r i b u t i o n of c e l l s retrogradely l a b e l l e d after tracer injections into the cortex and CP in combination with histochemistry for acetylcholinesterase demonstrated that the population of p a l l i d a l neurons projecting to the CP was d i s t i n c t from that of p e r i p a l l i d a l cholinergic neurons which may project through the striatum to the cortex. Double retrograde fluorescent tracing experiments indicated that p a l l i d a l neurons which project to the CP also have c o l l a t e r a l projections to the substantia nigra and perhaps to the subthalamic nucleus. The application of a new technique for studying ,efferent projections allowed the confirmation and morphological description of the projection of the globus p a l l i d u s to the striatum. The c h a r a c t e r i s t i c morphology of t h i s projection was shared by p a l l i d a l efferents which project to the entopeduncular nucleus, the r e t i c u l a r nucleus of the thalamus, the subthalamic nucleus and the substantia nigra. The fine morphological d e t a i l afforded by this method of anterograde tracing was u t i l i z e d in combination with a histochemical protocol to show that p a l l i d o s t r i a t a l terminals end in part on somatostatin-containing neurons in the CP. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v L I S T OF FIGURES v L I S T OF ABBREVIATIONS USED IN FIGURES x i i ACKNOWLEDGMENTS xv GENERAL INTRODUCTION 1 STATEMENT OF THE PROBLEMS TO BE EXAMINED 11 EXPERIMENT 1: EXAMINATION OF THE EFFERENT AND AFFERENT 13 CONNECTIONS OF THE STRIATUM EXPERIMENT 2: EXAMINATION OF THE EFFERENT AND AFFERENT 52 CONNECTIONS OF THE SUBSTANTIA NIGRA EXPERIMENT 3: EXAMINATION OF THE EFFERENT AND AFFERENT 100 CONNECTIONS OF THE GLOBUS PALLIDUS EXPERIMENT 4: LIGHT AND ELECTRON MICROSCOPIC DEMONSTRATION 130 OF A PROJECTION FROM THE GLOBUS PALLIDUS TO THE STRIATUM EXPERIMENT 5: COMPARISON OF THE CORTICAL AND STRIATAL 149 PROJECTIONS OF THE GLOBUS PALLIDUS AND PERI PALLIDAL AREAS EXPERIMENT 6: DEMONSTRATION OF COLLATERAL PROJECTIONS OF 170 THE NEURONS OF THE GLOBUS PALLIDUS TO THE STRIATUM AND THE SUBSTANTIA NIGRA EXPERIMENT 7: MORPHOLOGICAL DESCRIPTION OF THE EFFERENTS 181 OF THE GLOBUS PALLIDUS EXPERIMENT 8: INNERVATION OF STRIATAL SOMATOSTATIN- 223 CONTAINING NEURONS BY AFFERENTS FROM THE GLOBUS PALLIDUS GENERAL DISCUSSION 231 REFERENCES 269 LIST OF FIGURES Figure 1. Line drawings of the l a b e l l i n g resultant from an injection into the striatum in case CP-1 . Figure 2. Line drawings i l l u s t r a t i n g other s t r i a t a l WGA-HRP injection s i t e s . Figure 3. WGA-HRP l a b e l l i n g of s t r i a t a l connections with the cortex and globus p a l l i d u s . Figure 4 . Retrograde and anterograde WGA-HRP la b e l l i n g of the s t r i a t a l connections with the globus p a l l i d u s . Figure 5. WGA-HRP l a b e l l i n g of s t r i a t a l connections with the amygdala and entopeduncular nucleus. Figure 6. Anterograde and retrograde WGA-HRP la b e l l i n g of s t r i a t a l connections with the subthalamic nucleus and substantia nigra. Figure 7. Retrograde WGA-HRP l a b e l l i n g of s t r i a t a l afferents in the thalamus, the dorsal raphe and the locus coeruleus. Figure 8. Schematic diagram summarizing the connections of the striatum. Figure 9 . Line drawings of the l a b e l l i n g resultant from an inject i o n of WGA-HRP into the substantia nigra in case SN -1. vi Figure 1 0 . Line drawings i l l u s t r a t i n g other n i g r a l WGA-HRP inj e c t i o n s . Figure 11. Photomicrographs of a nigral WGA-HRP injection and the anterograde and retrograde l a b e l l i n g in the pedunculopontine nucleus. Figure 12. Retrograde and anterograde l a b e l l i n g of a crossed hypothalamic projection to the substantia nigra. Figure 13. WGA-HRP l a b e l l i n g of c e l l s in the substantia nigra which project to the thalamus. Figure 14. Anterograde WGA-HRP l a b e l l i n g of- .the projection of the substantia nigra to the thalamus. Figure 15. Anterograde WGA-HRP l a b e l l i n g of the projection of the substantia nigra to the superior c o l l i c u l u s . Figure 16. Retrograde WGA-HRP l a b e l l i n g of the c e l l s in the substantia nigra which project to the superior c o l l i c u l u s . Figure 17. Retrograde WGA-HRP l a b e l l i n g of the c e l l s of the subthalamic nucleus which project to the substantia nigra. Figure 18. Retrograde and anterograde l a b e l l i n g of n i g r a l connections with' the striatum and globus p a l l i d u s . V I 1 Figure 19. Retrograde and anterograde l a b e l l i n g of the ni g r a l connections with the striatum and globus pa l l i d u s in the special case SN-6. Figure 20. Diagram of the topographical rela t i o n s h i p of connections between the striatum, globus p a l l i d u s and substantia nigra. Figure 21. Schematic diagram summarizing the connections of the substantia nigra. Figure 22. Photomicrograph of a WGA-HRP inj e c t i o n s i t e in the globus p a l l i d u s . Figure 23. Line drawings d e p i c i t i n g the l a b e l l i n g resultant from a WGA-HRP inject i o n into the globus pal l i d u s in case GP-1. Figure 24. Anterograde l a b e l l i n g of the cortex and striatum after WGA-HRP injections into the globus p a l l i d u s . Figure 25. Comparison of the WGA-HRP l a b e l l i n g of s t r i a t a l c e l l bodies and neuropil after injections into the globus pa l l i d u s and substantia nigra. Figure 26. Anterograde WGA-HRP l a b e l l i n g of p a l l i d a l projections to the thalamus and entopeduncular nucleus. Figure 27. Anterograde and retrograde WGA-HRP l a b e l l i n g of p a l l i d a l connections with the subthalamic nucleus. V i l l Figure 28. Anterograde and retrograde WGA-HRP la b e l l i n g of p a l l i d a l connections with the substantia nigra. Figure 29. Anterograde and retrograde WGA-HRP la b e l l i n g of p a l l i d a l connections with the cortex, thalamus and dorsal raphe nucleus. Figure 30. Schematic diagram summarizing the connections of the globus p a l l i d u s . Figure 31. Anterograde and retrograde WGA-HRP la b e l l i n g of the connections between the striatum and globus p a l l i d u s . Figure 32. Line drawings depicting the l a b e l l i n g resultant from s t r i a t a l injections of WGA-HRP with and without kainic acid. Figure 33. Photomicrographs of the l a b e l l i n g in the globus p a l l i d u s , entopeduncular nucleus and substantia nigra resultant from s t r i a t a l injections, of WGA-HRP with and without kainic acid. Figure 34. Line drawings d e t a i l i n g the topography of the s t r i a t o p a l l i d a l and p a l l i d o s t r i a t a l projections. Figure 35. Line drawings d e t a i l i n g the dis t r i b u t i o n s of True blue l a b e l l e d p a l l i d o s t r i a t a l neurons and neurons stained intensely for acetylcholinesterase. ix Figure 36. Photomicrographs of True blue l a b e l l e d p a l l i d o s t r i a t a l neurons and neurons stained intensely for acetylcholinesterase. Figure 37. Photomontage of True blue l a b e l l e d p a l l i d o s t r i a t a l neurons. Figure 38. Neurons in and around the globus pal l i d u s which project to the cortex. Figure 39. Line drawings depicting the d i s t r i b u t i o n of cholinesterase-positive and cholinesterase-negative neurons in and around the globus p a l l i d u s which project to the cortex. Figure 40. Photomicrographs of cholinesterase-positive and cholinesterase-negative neurons in and around the globus pa l l i d u s which project to the cortex. Figure 41. Line drawings depicting the d i s t r i b u t i o n of single and double la b e l l e d p a l l i d a l neurons projecting to the striatum and substantia nigra. Figure 42. Photomicrographs of single and double la b e l l e d p a l l i d a l neurons projecting to the striatum and substantia nigra. Figure 43. D i s t r i b u t i o n of p a l l i d a l and n i g r a l neurons l a b e l l e d by the injection of fluorescent tracers in two locations within the striatum. Figure 44. A PhA-L injec t i o n s i t e in the globus p a l l i d u s . 187 Figure 45. PhA-L l a b e l l i n g of the p a l l i d o s t r i a t a l projection. 190 Figure 46. PhA-L l a b e l l i n g of the p a l l i d o or p e r i p a l l i d o c o r t i c a l projection. 193 Figure 47. PhA-L l a b e l l i n g of the pallidosubthalamic and pallidothalamic projection. 197 Figure 48. PhA-L l a b e l l i n g of the pallidoentopeduncular projection. 199 Figure 50. Diagrammatic comparison of the morphology of p a l l i d o n i g r a l fibers in the pars r e t i c u l a t a and pars compacta. 204 Figure 51. PhA-L la b e l l e d p a l l i d o n i g r a l f i b e r s in the pars r e t i c u l a t a . 206 Figure 52. Apparent axosomatic contacts made by PhA-L la b e l l e d p a l l i d a l efferents. 208 Figure 53. The morphology of PhA-L la b e l l e d s t r i a t a l efferents. 211 Figure 54. Diagrammatic comparison of fiber morphology of PhA-L la b e l l e d subcortical p a l l i d a l efferents. 218 Figure 55. Diagrammatic comparison of fiber morphology of c o r t i c a l PhA-L l a b e l l e d efferents. 220 Figure 56. PhA-L l a b e l l i n g of the p a l l i d o s t r i a t a l projection in combination with s t r i a t a l NADPH dependent diaphorase histochemistry. Figure 57. Drawings of s t r i a t a l neurons stained for NADPH dependent diaphorase a c t i v i t y and their association with PhA-L labelled p a l l i d o s t r i a t a l f i b e r s . Figure 58. Schematic diagram of the functional organization of the connections between some basal ganglia nuclei. Figure 59. Schematic diagram of proposed motor and limbic c i r c u i t s of the basal ganglia. Figure 60. Schematic diagram of the functional organization of the basal ganglion. x i i LIST OF ABBREVIATIONS USED IN FIGURES ab, basolateral nucleus of the amygdala ac , nucleus accumbens AC, anterior commissure BC, brachium conjunctivum ce, central nucleus of the amygdala cem, centromedial nucleus of the thalamus eg, central gray area c l , c e n t r o l a t e r a l nucleus of the thalamus cp, striatum (caudate-putamen) CP, cerebral peduncle ctx, cortex dmt, dorsal midbrain tegmentum dr, dorsal raphe nucleus ep, entopeduncular nucleus F, fornix FM, forceps minor FR, fasciculus retroflexus 9, nucleus gelatinosus 9Pr globus pallidus h, habenular complex ic , i n f e r i o r c o l l i c u l u s i c , internal capsule ICB, bundles of internal capsule fibers xi i i ip, interpeduncular nucleus Id, laterodorsal nucleus of the thalamus l h , l a t e r a l habenula lhp, posterior l a t e r a l hypothalamus LV, l a t e r a l v e n t r i c l e md, mediodorsal nucleus of the thalamus MFB, medial forebrain bundle ML, medial lemniscus MT, mammillothalamic tract na, nucleus accumbens nsp, non-specific thalamic afferents ntp, nucleus tegmenti pedunculopontis pb, peribrachial region pc, paracentral nucleus of the thalamus pf, parafascicular nucleus pfc, prefrontal cortex ppn, nucleus tegmenti pedunculopontis r, red nucleus r r , retrorubral area r t , r e t i c u l a r nucleus of the thalamus sc, superior c o l l i c u l u s snc, substantia nigra pars compacta snr, substantia nigra pars r e t i c u l a t a sp, s p e c i f i c thalamic afferents sum, supramammillary nucleus xiv sut, subthalamic nucleus v l , ventrolateral nucleus of the thalamus vm, ventromedial nucleus of the thalamus vp, ventral pallidum vta, ventral tegmental area of Tsai z i , zona incerta X V ACKNOWLEDGEMENT I would l i k e to thank my thesis advisor, Dr. H.C. Fibiger, for his invaluable support and always constructive c r i t i c i s m , both at the bench and elsewhere. He has taught me how to unflounder myself rather than await rescue. I also thank the other members of my committee for their input into this thesis and my graduate career in general. Dr. E.G. McGeer in par t i c u l a r has been a great help, more than she may be aware, in that I always kept her high standards in mind. For part i c u l a r i n s p i r a t i o n over the years I would l i k e to acknowledge Dr. Garth Bryans, Dr. Jim Nagy and Dr. Hiro Kimura. My other sen sei include Drs. Steven Vincent, Chip Gerfen, K e i j i Satoh and Gordon Arbuthnott from whom I've learned a great deal and with whom I've had a great time. F i n a l l y , I would l i k e to thank S t e l l a Atmadja for keeping l i f e in the lab more or less sane and Lauren Staines who took over from S t e l l a outside the lab. 1 GENERAL INTRODUCTION The two main goals of the study of the neurosciences are to understand the b i o l o g i c a l p r i n c i p l e s and mechanisms underlying normal brain function and to gain an understanding of brain pathology that can be directed toward the more immediate needs of the c l i n i c a l neurosciences. These two ambitions are well met in the study of the basal ganglia. In recent years, much has been learned about the chemistry and physiology of this system and research into the many diseases involving the basal ganglia has benefitted not only the patient but also the researcher. The basal ganglion i s not a circumscribed brain area, but rather a system of i d e n t i f i a b l e brain regions c l o s e l y linked by anatomical and functional considerations. The most readily apparent function carried out by t h i s system i s the control of motor behavior of the type that has come to be termed "extrapyramidal" (DeLong and Georgopoulos, 1981). In essence, these are the coarse background movements upon which more conscious fine motor behavior i s superimposed. Anatomically, the basal ganglia are composed of a number of deep forebrain and bulbar c e l l clusters including the striatum (formed by the caudate nucleus and putamen in humans), globus p a l l i d u s (termed external globus pa l l i d u s in. humans), entopeduncular nucleus (internal globus pa l l i d u s in human), subthalamic nucleus and substantia nigra. Some workers include the nucleus accumbens and olfactory tubercle as well, as anatomical considerations have led them to be considered ventral extensions of the striatum (DeLong and Georgopoulos, 1981). 2 H i s t o r i c a l l y , the i d e n t i f i c a t i o n of this system with motor behavior was inferred from the observation of motor disturbances a r i s i n g from pathological states involving components of the basal ganglia. These include Huntington's chorea, Parkinson's disease, Wilson's disease and hemiballismus (see DeLong and Georgopoulos, 1981). Experimental lesion studies in animals have approximated many of these conditions, allowing corroboration of the anatomical and biochemical presentation of the pathology seen in humans. Our appreciation of the anatomy, biochemistry and function of the basal ganglia, therefore, represents a synthesis of c l i n i c a l and experimental studies. Neurobiological studies usually focus on a single, s p a t i a l l y defined nucleus rather than on a functionally defined system. Often such nuclei represent d i s t i n c t functional units, but modern neuroanatomical techniques have led to a de-emphasis on c l a s s i c a l cytoarchitectural boundaries (Nauta, 1979a). The inputs and outputs of this s p a t i a l l y defined nucleus are investigated to determine what other .brain structures i t is associated with. This information can be obtained by following fiber bundles, the transport of tracer substances in a retrograde or anterograde manner, or from electrophysiological techniques. Once t h i s c i r c u i t r y has been elucidated, biochemical, immunohistochemical or electropharmacological methodologies may be employed to determine the transmitter(s) used by individual connections ( i e . the "biochemical neuroanatomy" of the system). With the data derived by these means the system may be manipulated by the placement of selective lesions or by selective 3 pharmacological perturbation to determine functional p r i n c i p l e s . In some instances, these data also contribute s i g n i f i c a n t l y to our understanding of neurological disease and suggest therapeutic approaches. Discussions of the connections of the basal ganglia most often begin with the c o r t i c a l projection to the caudate nucleus and putamen (CP). The CP receives a divergent input from most c o r t i c a l areas, both motor and sensory, but the majority of fibers from a given area end in c l u s t e r s , indicating a modular type of input (Kemp and Powell, 1971b; Oka, 1980; Royce, 1982). C o r t i c a l areas linked to one another by association fibers give r i s e to overlapping terminal f i e l d s within the striatum (Yeterian and van Hoesen, 1978; Royce, 1982). Most of the c o r t i c a l input to the striatum arises from the cortex on the same side, but there i s also a s i g n i f i c a n t input from the c o n t r a l a t e r a l cortex. C o r t i c a l c e l l s giving r i s e to t h i s projection are located mainly in layer V and in some cases have been shown to have branched axons that continue caudally to the pyramids after innervating the CP via a c o l l a t e r a l (Jinnai and Matsuda, 1979; Donoghue and K i t a i , 1981; Royce, 1982). Biochemical and electrophysiological evidence indicates that glutamate is used as a neurotransmitter by at least a portion of the neurons making up this projection (Divac et a l . , 1977; McGeer et a l . , 1977; Kocsis, et a l . , 1977; Spencer, 1976). In considering the pattern of neuronal a c t i v i t y involved in movement of the extrapyramidal type i t is usual to think in terms of a c o r t i c a l input to the striatum as the f i r s t step. However, 4 i t should be kept in mind that many animals have minimal c o r t i c a l tissue and yet have a well developed striatum. The striatum, therefore, cannot be considered to be a c o r t i c a l l y dependant structure but rather may act in concert with the motor regions of the cortex such that fine c o r t i c a l l y controlled motor behavior i s expressed on a background of s t r i a t a l motor behavior. In this context the c o r t i c a l input to the CP may serve more a coordination role than one of i n i t i a t i o n . The CP receives a well characterized input from the pars compacta of the substantia nigra (SNc) which u t i l i z e s dopamine as a neurotransmitter (see Dray, 1979). The physiological action of dopamine released in the striatum has been variously characterized as excitatory ( K i t a i et a l . , 1976) or inhi b i t o r y (Siggins, 1978) and i t i s possible that i t s action may depend on the state of the postsynaptic neuron at the time i t i s addressed. Anatomically, this input has been shown to be very diffuse and to display a patchy d i s t r i b u t i o n which is correlated with the c o r t i c a l glutamate input and endogenous cholinergic parameters within the CP (Graybeil et a l . , 1981; Lehmann et a l . , 1983). On a behavioral l e v e l the function of this input i s better understood. In Parkinson's disease the loss of DA neurons in the substantia nigra is the main feature and appears to be correlated to the functional abberations (Marsden, 1982). These data indicate a permissive role of s t r i a t a l DA a c t i v i t y in the i n i t i a t i o n of motor behavior. This impression gains support from microinfusion studies in which dir e c t or indire c t DA agonists acting within the striatum increase the le v e l of motor behavior. 5 In rats, infusion s i t e s in the dorsal striatum e l i c i t o r o f a c i a l stereotypies and .those in the ventral striatum (nucleus accumbens) produce an increase in locomotion (Anden and Johnels, 1977). Other s t r i a t a l inputs include those from intralaminar nuclei of the thalamus, a serotonin containing projection from the dorsal raphe nucleus and a noradrenergic innervation originating in the locus coeruleus (see Dray, 1979). Recently, afferents to the striatum from the amygdala, globus p a l l i d u s and nondopaminergic neurons within the pars r e t i c u l a t a of the substantia nigra (SNr) have been described (Dafny et a l . , 1975; Kelley et a l . , 1982; Staines et a l . , 1981; van der Kooy et a l . , 1981b). These la s t two are of p a r t i c u l a r interest in that these regions are in receipt of dense termination from s t r i a t a l neurons and have been suggested to be homologous structures on the basis of c e l l morphology and development (Nauta, 1979b). Lesions of the striatum i t s e l f are the consistant pathological finding in Huntington's chorea (see Dray, 1979; DeLong and Georgopoulus, 1981). The motor disturbances c h a r a c t e r i s t i c of this disorder take the form of a hypermobility with uncontrolled twitches and grimaces. These symptoms respond to treatments which antagonise the permissive influence of the n i g r o s t r i a t a l projection by blockade of DA action. Fiber sparing lesions of the CP of experimental animals mimic the biochemical changes seen in Huntington's chorea (McGeer and McGeer, 1976; Coyle and Schwarcz, 1976). The efferents of the striatum project to only three 6 structures; the globus p a l l i d u s , entopeduncular nucleus and substantia nigra (see Dray, 1979). Over 65% of s t r i a t a l neurons have been estimated to project to the substantia nigra alone (Bolam et a l . , 1981a). As i t can be inferred from lesion studies that not a l l s t r i a t a l projections are c o l l a t e r a l s of those fibers innervating the SN, this figure is undoubtedly an underestimate of the t o t a l percentage of c e l l s which are projection neurons (Staines et a l . , 1980a). Biochemical data suggest that each of the s i t e s of s t r i a t a l termination receive both gamma-aminobutyic acid (GABA)- and substance P-containing projections (Staines et a l . , 1980a) and there are electrophysiological data for dual excitatory and inhibitory inputs (Kanazawa and Yoshida, 1980; Collingridge and Davies, 1982; Mogenson et a l . , 1983). In most animals the globus p a l l i d u s is immediately adjacent to the CP and i s innervated by s t r i a t a l f i bers containing GABA, substance P and enkephalin (Nagy et a l . , 1978a; Cuello and Paxinos, 1978; Staines et a l . , 1980a). The globus p a l l i d u s also receives a DA input from the SNc (Lindvall and Bjorklund, 1979), a serotonergic projection from the dorsal raphe nucleus (Parent et a l . , 1981b; Pasik et a l . , 1981) and a projection from the subthalamic nucleus (Nauta and Cole, 1978; van der Kooy and Hattori, 1980a). The seeming s i m p l i c i t y of the afferents of the GP may be due to the fact that this question has not yet been addressed in a dir e c t manner. The vast majority of efferents from t h i s nucleus project to the subthalamic nucleus (Nauta, 1979a; McBride and Larsen, 1980; Carpenter et a l . , 1981; van der Kooy et a l . , 1981). Attempts to determine the transmitter used by this projection have as yet provided no clear answer (Fonnum et a l . , 1978; Rouzaire-Dubois et a l . , 1980; van der Kooy et a l . , 1981a). The GP may also have projections to the r e t i c u l a r nucleus of the thalamus , substantia nigra, striatum and a minor projection to the mediodorsal nucleus of the thalamus (Carter and Fibiger, 1978; Nauta, 1979a; McBride and Larsen, 1980; Staines et a l . , 1981 ) . The globus pallidus i s involved in the neuropathology in Wilson's disease which i s characterized by tremour, r i g i d i t y , hypertonicity and spasmotic contractions (Owen, 1981). However, animal studies in which u n i l a t e r a l or b i l a t e r a l lesions of the globus p a l l i d u s are made reveal, on the whole, no long term disturbance in motor behavior (see DeLong and Georgopoulos, 1981), and i t may be that the symptomology of Wilson's disease i s related to the degeneration of the putamen which i s more prevalent than that seen to occur in the GP. The features of the disease on the other hand correspond better to the picture of ni g r a l degeneration than that normally associated with caudate-putamen damage (Marsden, 1982). The c l i n i c a l pharmacology sheds l i t t l e l i g h t on t h i s as both the dopamine precursor, 1-dihydroxyphenylalanine (L-DOPA), and the dopamine antagonist, haloperidol, have been claimed to improve the symptoms of Wilson's disease - L-DOPA the "extrapyramidal symptoms" (presumably r e f e r r i n g to the tremour and r i g i d i t y ) and haloperidol the involuntary movements (Owen, 1981). The entopeduncular nucleus receives terminals from the 8 striatum and the nucleus tegmenti pedunculopontis and projects back to the l a t t e r nucleus (van der Kooy and Carter, 1981; Moon-Edley and Graybiel, 1980; Larsen and McBride, 1979). It also innervates the motor d i v i s i o n s of the thalamus, intralaminar c e l l groups of the thalamus, and the l a t e r a l habenular nucleus (Kim et a l . , 1976; Larsen and McBride, 1979; Nauta, 1979a; DeVito and Anderson, 1982). The EP receives a dense innervation from the subthalamic nucleus (SUT) (Carpenter et a l . , 1981). Therefore, although the two d i v i s i o n s of the globus p a l l i d u s (GP and EP) have r e l a t i v e l y minor direct connections (see below), the GP can influence the EP via this relay. As already mentioned, the SUT receives a massive input from the GP and projects to the GP and EP. V i r t u a l l y every c e l l in the SUT appears to project to the EP and each of these seems to also give r i s e to a c o l l a t e r a l innervating the SNr (Deniau et a l . , 1978a; van der Kooy and Hattori, 1980a). An excitatory input to the SUT a r i s i n g from both motor and premotor co r t i c e s has been observed (Hartmann-von Monakow et a l . , 1978; Romansky et a l . , 1979; K i t a i and Deniau, 1981). The substantia nigra receives afferents from each of the other main components of the basal ganglia; the CP, GP and SUT (Gerfen et a l . , 1982). Aside from the ascending dopaminergic projection a r i s i n g from the pars compacta, the pars r e t i c u l a t a of the SN projects to motor d i v i s i o n s of the thalamus, the superior c o l l i c u l u s , the midbrain r e t i c u l a r formation and has reciprocal connections with the nucleus tegmenti pedunculopontis. The projections to the tectum and thalamus have been determined to 9 arise partly as c o l l a t e r a l s of the same neurons (Bentivoglio et a l . , 1979) and to use GABA as a neurotransmitter (Vincent et a l . , 1978a; DiChiara et a l . , 1979a). Much more insight into the function of a nucleus may be obtained by the examination of i t s outputs than i t s inputs. The clearest indication of the function of the striatum has emerged from the study of the electrophysiological response c h a r a c t e r i s t i c s of the EP and SNr (see DeLong and Georgopoulos, 1981). The majority of neurons in these l o c i show a correlation between f i r i n g rate and muscular a c t i v i t y . Of the more interesting findings a r i s i n g from these c o r r e l a t i v e studies are the observations that c e l l f i r i n g within the internal globus pal l i d u s of the monkey ( i e . EP) does not always precede the onset of movement, indicating that the basal ganglia are not necessarily involved in the i n i t i a t i o n of movement, and that correlations were obtained between neuronal a c t i v i t y and fine, d i s t a l movements in addition to more gross, slow movements. These data are at odds with more c l a s s i c a l impressions of basal ganglia function a r i s i n g from c l i n i c a l and lesion studies. Similar attempts to correlate a c t i v i t y of neurons within the external segment of the globus p a l l i d u s ( i e . GP) with motor a c t i v i t y have yielded l i t t l e or no positive data. The same is true of decades of study on the effects of lesions of the GP. Perhaps as a result of th i s and the observation that the GP appears only to interconnect other basal ganglia components, this nucleus has not been subjected to the extensive research that other parts of the basal ganglia have received. The present 10 studies attempt to arrive at a more precise understanding of the anatomy of the basal ganglia, with a mind to biochemical heterogeneity underlying t h i s anatomy, and w i l l emphasize the anatomy of the external globus p a l l i d u s . 11 STATEMENT OF THE PROBLEMS EXAMINED The g r o u n d w o r k was l a i d f o r a more d e t a i l e d s t u d y o f t h e a n a t o m i c a l c i r c u i t r y o f t h e b a s a l g a n g l i a n u c l e i by a r e e x a m i n a t i o n o f t h e c o n n e c t i o n s o f t h e s t r i a t u m , g l o b u s p a l l i d u s a n d s u b s t a n t i a n i g r a . The u n d e r s t a n d i n g o f t h e anatomy o f many b r a i n s y s t e m s h a s been e x p a n d i n g due t o t h e i n c r e a s e d s e n s i t i v i t y o f r e c e n t l y d e v e l o p e d t e c h n i q u e s . The u s e o f wheat germ a g g l u t i n i n c o n j u g a t e d t o h o r s e r a d i s h p e r o x i d a s e (WGA-HRP) a s a n e u r o a n a t o m i c a l t r a c e r a l l o w s f o r s m a l l i n j e c t i o n s t o be made a n d b o t h e f f e r e n t s a n d a f f e r e n t s o f t h e i n j e c t e d a r e a t o be v i s u a l i z e d w i t h g r e a t s e n s i t i v i t y . T h e s e a d v a n t a g e s were e m p l o y e d t o s t u d y t h e i n p u t s a n d o u t p u t s o f t h e t h r e e m a i n c o m p o n e n t s o f t h e b a s a l g a n g l i a . F i n d i n g s f r o m E x p e r i m e n t s 1 and 3, s u g g e s t i n g a p a l l i d o s t r i a t a l p r o j e c t i o n , were e x a m i n e d i n g r e a t e r d e t a i l i n E x p e r i m e n t 4, t o c o n t r o l f o r t h e p o s s i b i l i t y o f a x o s o m a t i c o r t r a n s y n a p t i c a r t i f a c t s i n t h e o b s e r v a t i o n o f t h e a p p a r e n t r e t r o g r a d e l a b e l l i n g o f p a l l i d a l n e u r o n s . The t o p o g r a p h y , o f t h i s p r o j e c t i o n was s t u d i e d w i t h i n j e c t i o n s o f WGA-HRP i n t o t h e CP, i n some i n s t a n c e s i n c o n j u n c t i o n w i t h t h e p e r i k a r y a l n e u r o t o x i n , k a i n i c a c i d , t o b l o c k t h e a n t e r o g r a d e l a b e l l i n g o f t h e GP. The u l t r a s t r u c t u r a l l o c a l i z a t i o n o f t h e p e r o x i d a s e a c t i v i t y w i t h i n p a l l i d a l n e u r o n s l a b e l l e d by s t r i a t a l t r a c e r i n j e c t i o n s was a l s o e x ami n e d . I n E x p e r i m e n t 5 , f u r t h e r c o n t r o l s t u d i e s a d d r e s s e d t h e p o s s i b i l i t y t h a t t h e a p p a r e n t l a b e l l i n g o f a p a l l i d o s t r i a t a l p r o j e c t i o n was i n f a c t due t o l a b e l l i n g o f p e r i p a l l i d a l n e u r o n s 1 2 projecting through the striatum to the cortex. A comparison was made of the d i s t r i b u t i o n of neurons projecting to the striatum and cortex in conjunction with histochemistry for acetylcholinesterase under conditions which d i f f e r e n t i a t e d neurons with c o r t i c a l projections from the t y p i c a l p a l l i d a l neurons. Topographical data from Experiments 1 and 3 had suggested a possible c o l l a t e r a l i z a t i o n of p a l l i d a l projections to the CP and SN. This p o s s i b i l i t y was evaluated by double retrograde fluorescent transport studies in Experiment 6 . In Experiment 7 an attempt was made to provide further evidence for a p a l l i d o s t r i a t a l projection by the application of an anterograde transport technique capable of demonstrating axon terminals at the l i g h t microscopic l e v e l . This was achieved by the in j e c t i o n of a novel l e c t i n into the globus p a l l i d u s and then v i s u a l i z i n g i t s transport using immunohistochemistry. F i n a l l y , Experiment 8 was conducted to determine i f synaptic contact could be observed between terminals of the p a l l i d o s t r i a t a l projection and chemically i d e n t i f i e d c e l l types within the striatum. The anterograde tracing technique used in Experiment 7 was combined with a histochemical procedure for NADPH-dependent diaphorase a c t i v i t y , a r e l i a b l e marker for somatostatin-containing neurons in the striatum. 1 3 EXPERIMENT 1: EXAMINATION OF THE EFFERENT AND AFFERENT CONNECTIONS OF THE STRIATUM INTRODUCTION The striatum i s the most well-studied component of the basal ganglia. It i s in receipt of the majority of the input to the basal ganglia from other brain areas and stands as the f i r s t link in the chain of successive nuclei which make up this system (Nauta, 1979a; McGeer et a l . , 1978). The striatum has been the subject of a great deal of neuroanatomical research aimed at demonstrating both i t s efferent and afferent connections (see Graybiel and Ragsdale, 1979) but the majority of these reports have dealt with observations on single connections. To date, there has been no systematic study mapping the d i s t r i b u t i o n and r e l a t i v e densities of the efferents and afferents of the striatum of the rat. Furthermore, previous studies have used less sensitive neuroanatomical techniques than are now available (Mesulam, 1978; Gonatas et a l . , 1979; Staines et a l . , 1980b) and have not had the advantage of simultaneous examinations of both input and output elements. In the present experiment, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was u t i l i z e d to demonstrate the projections of and inputs to the striatum in the rat. Confirmations of previous findings were obtained and expanded upon, and, in addition, an input to the striatum from the globus pallidus was demonstrated for the f i r s t time. 14 METHODS Male Wistar rats, anesthetized with pentobarbital, were given 50 to 200 nl pressure injections of a solution of 1 to 5% WGA-HRP in isotonic saline via a 5 ul Hamilton syringe mounted on a stereotaxic instrument. WGA-HRP was synthesized as described previously (Staines et a l . , 1980b). The s t r i a t a l coordinates were chosen to correspond to 8.9/1.0/2.0 (anterior/dorsal/medial) in the stereotaxic atlas of the rat brain by Konig and Klippel (1963). Solution was infused over a ten minute period and the cannula was l e f t in place for a further five minutes to l i m i t d i f f u s i o n up the cannula t r a c t . After 24-48 hours, rats were reanesthetized and perfused t r a n s c a r d i a l l y , after clamping the descending aorta, with an isotonic saline solution containing 0.25% procaine and 0.001% heparin. This was followed by perfusion with a f i x a t i v e containing 1% paraformaldehyde and 1% glutaraldehyde in 100 mM sodium phosphate buffer (PB), pH 7.4 (250 mis over 30-45 minutes) and f i n a l l y , an ice cold solution of 10% sucrose in 100 mM PB (200 mis over 30 minutes). Brains were removed and stored for at least four hours in the l a t t e r solution. Sections were cut on a freezing microtome at a thickness of 50 um and mounted on chrome-alum coated s l i d e s from 50 mM PB. Slide mounted sections were reacted for peroxidase using tetramethylbenzidine (TMB) and hydrogen peroxide as substrates under standard conditions (Mesulam, 1978). In b r i e f , s l i d e s were rinsed three times in d i s t i l l e d water and then transferred to a reaction medium containing 100 mg sodium n i t r o f e r r i c y a n i d e and 5 mg TMB (added as a 2% solution in 15 absolute ethanol) per 100 ml of sodium acetate buffer (50 mM, pH 3.3). After presoaking for 5 to 10 min, the reaction was begun by the addition of 250 ul of 3% hydrogen peroxide and allowed to proceed for 20 minutes in t o t a l darkness. The reaction medium was usually replaced at least once during t h i s period. After a number of post-reaction rinses in the reaction buffer and a brief rinse in d i s t i l l e d water, s l i d e s were a i r dried, dehydrated in 100% ethanol for 10-20 seconds, cleared in xylene and coverslipped with permount. Slides were routinely examined with dark f i e l d illumination on a Zeiss Universal Research microscope. In a l l cases, the c r i t e r i o n used to determine a WGA-HRP-labelled terminal f i e l d was the presence of labelled fibers to and within an area in which a moderately dense, punctate d i s t r i b u t i o n of reaction product was evident. The density, d i s t r i b u t i o n , and orientation of the reaction product appeared to allow d i f f e r e n t i a l i d e n t i f i c a t i o n of terminal f i e l d s and areas containing projection f i b e r s , but i t i s acknowledged that this was a subjective evaluation. RESULTS The results of a series of injections w i l l be reported through comparisons of one large i n j e c t i o n (200 n l , 5% WGA-HRP; CP-1; F i g . 1) with numerous smaller injections (CP-2, CP~3, CP-4; Fig. 2) directed at locations within the head of the striatum (that portion r o s t r a l to the decussation of the anterior commissure). The d i s t r i b u t i o n of l a b e l l i n g resultant from the large i n j e c t i o n is depicted in Figure 1. Unless otherwise stated, a l l presentations arise from observations of t h i s case. 16 Figure 1 . Line drawings i l l u s t r a t i n g the WGA-HRP injection s i t e (stippled area) and resultant anterograde and retrograde l a b e l l i n g in case CP - 1 . Labelled perikarya are represented as f i l l e d c i r c l e s . Fibers containing peroxidase reaction product and areas of terminal l a b e l l i n g are drawn in . Abbreviations in t h i s and following figures: ab, basolateral nucleus of the amygdala; AC, anterior commissure; cem, central medial nucleus of the thalamus; c l , ce n t r o l a t e r a l nucleus of the thalamus; cp, striatum; CP, cerebral peduncle; dr, dorsal raphe nucleus; ep, entopeduncular nucleus; gp, globus p a l l i d u s ; IC, internal capsule; pc, paracentral nucleus of the thalamus; pf, parafascicular nucleus; r r , retrorubral area; snc, substantia nigra pars compacta; snr, substantia nigra pars r e t i c u l a t a ; v l , ventrolateral nucleus of the thalamus; vm, ventromedial nucleus of the thalamus. 18 Figure 2. Line drawings i l l u s t r a t i n g the in j e c t i o n s i t e s ( f i l l e d area) in cases CP-2, CP-3 and CP-4. t 19 20 STRIATUM As depicted in Figure 1 the large inj e c t i o n s i t e (CP -1) involved most of the head of the striatum. The injection did not spread ventral or caudal to the anterior commissure and therefore did not label the t a i l of the striatum or accumbens. There was some l a b e l l i n g of the cortex immediately overlying the striatum at the le v e l of the needle t r a c t ; however, most of this tissue had been destroyed by thermal damage during trephanation. The corpus callosum acted as a l a t e r a l boundary to the injection s i t e and medially the injected WGA-HRP did not diff u s e as far as the l a t e r a l v e n t r i c l e , leaving a thin medial s t r i p of striatum free from l a b e l l i n g . At the boundaries of the injection s i t e a disorganized punctate deposit (indicative of a featureless d i s t r i b u t i o n ) rapidly tapered off into clear striatum. A remarkable feature of the inj e c t i o n s i t e was the absence of l o c a l l y l a b e l l e d s t r i a t a l neurons, around i t s perimeter or within the t a i l of the striatum. In case CP-1, only 3 labelled s t r i a t a l neurons could be seen beyond the bounds of the injection s i t e , and a l l of these were within 500 um of the edge of the inject i o n s i t e (Fig. 3 A ) . No neurons were seen in the t a i l of the striatum. The r e l a t i v e lack of retrograde l a b e l l i n g of s t r i a t a l c e l l s was a consistent feature of a l l cases examined (including many not documented in the present report). Heavily la b e l l e d c e l l s were seen within the injection s i t e but their c o d i s t r i b u t i o n with g l i a l elements indicated that they were lab e l l e d by dir e c t uptake of peroxidase by the c e l l body. In cases where WGA-HRP injections were made in caudal regions of the 21 head of the striatum r o s t r a l regions were free of l a b e l l i n g , indicating l i t t l e or no potential for uptake of WGA-HRP into the projection fibers of the anterior c e l l s which passed through the inj e c t i o n s i t e . Again, the la b e l l e d s t r i a t a l neurons which were found beyond the bounds of the injection s i t e were invariably few in number and never more than 500 um from an edge of the inje c t i o n s i t e . There were too few of these neurons for detailed study but most appeared to be large ( > 25 um), triangular or spindle shaped c e l l s . There was also no clear evidence for i n t r a s t r i a t a l anterograde transport of WGA-HRP. A limited amount of f i n e , d i f f u s e reaction product was seen in s t r i a t a l areas beyond the injection s i t e boundaries but thi s was most l i k e l y due to some limited d i f f u s i o n of peroxidase a c t i v i t y or seeding of the peroxidase reaction. In some cases (CP-2 for example) there was clear evidence that no anterograde transport to either the t a i l of the striatum or the l a t e r a l head of the striatum had occurred. There was, however, sparse anterograde l a b e l l i n g in the rostromedial striatum anterior to the inj e c t i o n s i t e . As n i g r o s t r i a t a l fibers are oriented rostrocaudally within the striatum (Tulloch et a l . , 1978), and bear v a r i c o s i t i e s along their length ( D i F i g l i a et a l . , 1978), boutons of these fibers may have taken up WGA-HRP within the injection s i t e and transported i t to more anterior portions of the striatum. Caudal to the s t r i a t a l i n j e c t i o n s i t e , l a b e l l e d f i b e r s were seen to gather into the fiber bundles coursing toward the globus p a l l i d u s . Experience gathered from a large number of WGA-HRP transport experiments in various systems leads to the conclusion 22 that l a b e l l e d projection fibers are prominent when la b e l l e d by anterograde transport but are very seldom seen when labe l l e d retrogradely, and even then only when quite near the c e l l body. Direct evidence that the l a b e l l i n g seen within these fiber bundles in the striatum was s t r i a t o f u g a l i s provided in Experiment 4. CORTEX The d i s t r i b u t i o n of c o r t i c a l c e l l s resulting from the large s t r i a t a l i n j e c t i o n is depicted in Figure 1. Labelled c e l l s were c l e a r l y r e s t r i c t e d to layer V in areas which contained a granular layer (layer IV). L a t e r a l l y located neocortical c e l l s formed a continuum with c e l l s in layer II of the archicortex. A few lab e l l e d c e l l s were seen in layer VI near the inject i o n t r a c t . The prefrontal cortex, around and anterior to the forceps minor, was not examined in case CP-1, but examination of material from other cases indicated that t h i s area gives r i s e to the heaviest c o r t i c a l innervation of the head of the striatum. Fewer and less densely lab e l l e d " c e l l s were found in the cortex contralateral to the injection and their d i s t r i b u t i o n mirrored that in the i p s i l a t e r a l cortex. A l l l a b e l l e d c e l l s appeared to be t y p i c a l medium sized pyramidal c e l l s , with c e l l bodies roughly 14 by 20 um. No large pyramidal c e l l s were seen la b e l l e d . Thick, apical dendrites can be seen in most of the c e l l s in Figure 3B. GLOBUS PALLIDUS At the anterior t i p of the globus pallidus the labe l l e d s t r i a t a l f i b e r bundles merged into the much larger f i b e r bundles 23 Figure 3. (A) Dark f i e l d photomicrograph of the ventral edge of the s t r i a t a l injection s i t e . Note the labelled s t r i a t a l neurons (arrows). Other large bright features are peroxidase l a b e l l e d vascular pericytes or the expression of the endogenous peroxidase a c t i v i t y of red blood c e l l s . (B) Peroxidase-labelled c e l l s in layer V of the cortex i p s i l a t e r a l to the s t r i a t a l i n j e c t i o n s i t e . (C) Anterograde l a b e l l i n g of the anterior globus p a l l i d u s . Note the l a b e l l i n g of fiber bundles (arrows) at thi s anterior l e v e l , although the l a b e l l i n g i s predominantly within the neuropil. (D) The d i s t r i b u t i o n of anterograde l a b e l l i n g at a more caudal l e v e l of the globus p a l l i d u s than that shown in C. Note the l a b e l l i n g of the neuropil and the absence of l a b e l l i n g of the fiber bundles. The whole of the f i e l d of this photograph i s within the globus p a l l i d u s . Bar = 100 um. 24 25 that traverse the globus p a l l i d u s , but a heavy, punctate f i e l d of reaction product was seen within the p a l l i d a l neuropil as well (Fig.1, F i g . 3C), indicating termination of the s t r i a t o p a l l i d a l projection. At more caudal levels of the globus pa l l i d u s (Fig. 3D), the majority of the peroxidase reaction product within the globus p a l l i d u s was found within the neuropil and the l a b e l l i n g within p a l l i d a l fiber bundles was scant. This observation leads to the conclusion that the l a b e l l e d s t r i a t o f u g a l f i b e r s , including those which continue on to more caudal structures, innervate the globus pa l l i d u s rather than just passing through i t . The globus pa l l i d u s also contained lab e l l e d c e l l bodies. A few of these, outside the zones of s t r i a t o p a l l i d a l termination, were c l e a r l y v i s i b l e under dark f i e l d conditions but the majority could be seen only as small dark areas within the la b e l l e d terminal f i e l d (Fig. 4A and 4B). Those c e l l s that were c l e a r l y v i s i b l e were triangular or spindle shaped and measured approximately 25 um along their long axes (Fig. 4C). In one section thrcjugh the globus pallidus in case CP-1, 58 neurons were counted, and t h i s figure i s probably an underestimate due to the d i f f i c u l t y in id e n t i f y i n g neurons within the stained neuropil. Neurons were very occasionally found embedded in the fibers of the internal capsule near the caudal medial pallidum. These appeared to be somewhat larger and to stain more intensely than the c e l l s within the globus pallidus proper (Fig. 4D). Findings similar to those described above were seen in every case of WGA-HRP inject i o n into the striatum. Both the area of 26 Figure 4. Dark f i e l d (A) and l i g h t f i e l d (B) photomicrographs of the l a b e l l e d terminal f i e l d and c e l l bodies (arrows) within the globus p a l l i d u s . (C) Higher magnification of the c e l l in the terminal free area of (A) and (B). The c e l l shown in (D) was located medial to the globus p a l l i d u s , within the fibers of the internal capsule. Bar = 50 um. 27 28 the globus pa l l i d u s displaying terminal l a b e l l i n g and the number of retrogradely la b e l l e d p a l l i d a l neurons was found to be proportional to the size of the s t r i a t a l i n j e c t i o n . While smaller s t r i a t a l injections led to the l a b e l l i n g of fewer p a l l i d a l neurons (eg. a maximum of 35 c e l l s per section in the GP of CP-4), the intensity with which individual c e l l s were labelled did not appear to be affected, arguing against a diffuse projection of individual p a l l i d a l c e l l s over wide areas of the striatum. In the pallidum of CP-2 both the anterograde and retrograde l a b e l l i n g took up a much more medial d i s t r i b u t i o n . In contrast, in CP-3 the globus pa l l i d u s was predominantly l a b e l l e d in i t s l a t e r a l extreme, indicating an ordered, mediolateral topography. Neither CP-2 or CP-4 showed l a b e l l i n g of c e l l s within the internal capsule. AMYGDALA Each case examined revealed weakly staining c e l l bodies in the basolateral nucleus of the amygdala (Fig. 1, F i g . 5A). The intensity of l a b e l l i n g did not decrease in the smaller injections but lab e l l e d c e l l s were far fewer in number. No anterograde l a b e l l i n g was seen within the amygdala. ENTOPEDUNCULAR NUCLEUS Caudal to the globus p a l l i d u s , l a b e l l e d f i b e r s were apparent within the internal capsule. These were far more numerous than those observed in fiber bundles within the globus p a l l i d u s . As depicted in Figures 1 and 5B, the entopeduncular nucleus was heavily l a b e l l e d with peroxidase stained terminals, but retrogradely la b e l l e d c e l l bodies were not seen. This was a 29 Figure 5. The arrows in (A) point out the weakly-labelled c e l l bodies found within the basolateral nucleus of the amygdala. (B) Darkfield photomicrograph of the anterograde l a b e l l i n g of the neuropil of the entopeduncular nucleus. Note the v i r t u a l absence of l a b e l l i n g within the fiber bundles at t h i s l e v e l . Bar = 100 um. 30 31 consistent feature of a l l cases. The smaller injections led to a more diffuse staining of the entopeduncular nucleus. As with the globus p a l l i d u s , l a b e l l i n g within fiber bundles was far less prominent at the le v e l of the entopeduncular nucleus than r o s t r a l or caudal to i t . SUBTHALAMIC NUCLEUS Caudal to the entopeduncular nucleus, l a b e l l e d fibers could c l e a r l y be seen in the medial t i p of the cerebral peduncle In the subthalamic nucleus, which l i e s d i r e c t l y dorsal to thi s fiber bundle, a small number of very f a i n t l y l a b e l l e d c e l l bodies were seen in case CP-1. These numbered only 4-6 per section and were r e s t r i c t e d to the main body of this nucleus (Fig. 6A). Of the other cases, only CP-2 had la b e l l e d c e l l s in the subthalamic nucleus and these were s i m i l a r l y very f a i n t l y l a b e l l e d and few in number. There was no evidence of anterograde transport to this nucleus from the striatum. Two sets of labe l l e d axons were seen at caudal levels of the subthalamic nucleus (Figure 1). One of these, known from other work to be the s t r i a t a l efferents (see Experiment 4), remained within the medial t i p of the cerebral peduncle to the l e v e l of the substantia nigra. The other proceeded dorsomedially into the l a t e r a l hypothalamus (Fig. 6A) where they ran u n t i l the l e v e l of the substantia nigra. At this l e v e l they curved v e n t r o l a t e r a l l y into the substantia nigra pars compacta. These l a t t e r fibers were l i k e l y the proximal axons of the retrogradely la b e l l e d n i g r o s t r i a t a l neurons which run for a short distance in the medial forebrain bundle, and are close enough to the c e l l body to 32 contain detectable levels of peroxidase. SUBSTANTIA NIGRA C e l l bodies and proximal dendrites of neurons in the pars compacta and ventral tegmental area were heavily l a b e l l e d with peroxidase, as were terminals in the ventral half of the pars r e t i c u l a t a . This terminal f i e l d was d i s t r i b u t e d evenly and was without apparent feature. However, with smaller injections (CP-4) the terminal f i e l d in the caudal pars r e t i c u l a t a s p l i t into two t i e r s (Fig. 6D). These two d i s t i n c t f i e l d s of terminal l a b e l l i n g were separated by a band of unreactive neuropil. Both of the l a b e l l e d f i e l d s were confined to the pars r e t i c u l a t a . It was of note that no anterograde l a b e l l i n g was seen in the pars compacta and that at most leve l s there was a very clear separation between the retrogradely la b e l l e d pars compacta c e l l s and the terminal f i e l d in the pars r e t i c u l a t a (Fig. 6B). At more caudal l e v e l s , a few retrogradely l a b e l l e d c e l l s were found within the terminal f i e l d in the pars r e t i c u l a t a (Fig. 6C). At the caudal end of the substantia nigra there was no evidence of l a b e l l e d projection f i b e r s , but a c e l l group continuous with caudal pars compacta was seen to curve d o r s o l a t e r a l l y into the retrorubral pons. In case CP-3, lab e l l e d pars compacta c e l l s were found in the extreme r o s t r a l part of the substantia nigra. The l a b e l l e d terminal f i e l d in the pars r e t i c u l a t a , however, started at a much more caudal l e v e l . In sections in which the s t r i a t o n i g r a l terminal f i e l d was most prominant, only a few very f a i n t l y - l a b e l l e d c e l l s were found in the pars compacta, although 33 Figure 6. (A) Retrogradely l a b e l l e d c e l l bodies within the subthalamic nucleus (arrow). Labelled fibers can be see within the medial forebrain bundle (upper l e f t ) and the bright granular appearance of the cerebral peduncle i s due to l a b e l l e d fibers within this bundle. (B) Photomontage of retrogradely la b e l l e d c e l l bodies within the pars compacta and the terminal f i e l d within the pars r e t i c u l a t a of the substantia nigra. A few labelled dendrites of pars compacta c e l l s can be seen descending into the pars r e t i c u l a t a (arrows). (C) L i g h t f i e l d photomicrograph of l a b e l l e d c e l l bodies in the pars compacta (upper f i f t h ) and pars r e t i c u l a t a of the substantia nigra. The s t r i a t o n i g r a l terminal f i e l d can be seen in the lower t h i r d of the photograph. Note the presence of retrogradely la b e l l e d c e l l bodies within the pars r e t i c u l a t a . (D) Micrograph from the caudal part of the substantia nigra showing that at t h i s l e v e l the l a b e l l e d terminals form two separate f i e l d s within the pars r e t i c u l a t a . The arrows point out a narrow band of densely-labelled terminals lying adjacent to the cerebral peduncle. Bar = 100 um. 34 35 the occasional c e l l in the pars r e t i c u l a t a was heavily l a b e l l e d . Thus, t h i s case exhibited a negative correlation between the d i s t r i b u t i o n of labelled s t r i a t o n i g r a l terminals and labe l l e d pars compacta c e l l s . In a l l cases examined an occasional (one or two per animal) labelled c e l l was seen in the con t r a l a t e r a l pars compacta of the substantia nigra. THALAMUS As depicted in Figure 1, a number of thalamic nuclei contained retrogradely l a b e l l e d c e l l bodies. These included a l l of the intralaminar c e l l groups (central medial, central l a t e r a l , and paracentral; after Jones and Leavitt, 1975) and the parafascicular nucleus. A few c e l l s were observed in the anterior and medial parts of the ventrolateral nucleus and the ventromedial nucleus contained a large number of la b e l l e d neurons. C e l l s in the ventral thalamus were ovoid (20 um in diameter), those in the intralaminar regions were d i s t i n c t l y spindle-shaped ( 10 um by 25 um) and l a b e l l e d parafasicular neurons were polymorphous (20 um maximum diameter) with two to four thick primary dendrites (Fig. 7A,7B and 7C). Labelling of neurons in the parafascicular and intralaminar nuclei was seen in a l l of the cases examined and smaller inje c t i o n s i t e s resulted in a less intense l a b e l l i n g of neurons rather than markedly decreasing the number of c e l l s l a b e l l e d . The reverse was true of l a b e l l i n g of c e l l s in the ventral thalamus, where far fewer c e l l s were seen in cases with small s t r i a t a l i n j e c t i o n s i t e s . These data suggest that intralaminar/parafasicular neurons provide a rather diffuse 36 F i g u r e 7 . Retrogradely l a b e l l e d c e l l s w i t h i n the (A) i n t r a l a m i n a r nucleus of the thalamus, (B) ventromedial nucleus of the thalamus, (C) p a r a f a s c i c u l a r nucleus, (D) d o r s a l raphe and (E) l o c u s c o e r u l e u s . Bar = 100 um. 37 38 innervation of the striatum but that the projections from the ventromedial thalamus is more di s c r e t e . Some f a i l u r e s to label ventromedial neurons were encountered in cases where l a b e l l i n g of other c e l l groups was weak and survival times were shorter. This l i k e l y r e f l e c t s a r e l a t i v e l y small number of terminals per fiber on the axons of this projection. Diffusion of the injection s i t e into the cortex was not a prerequisite for the l a b e l l i n g of c e l l s in the ventromedial nucleus and i t i s doubtful that these c e l l s were la b e l l e d by uptake into f i b e r s of passage (see Discussion). The only marked topographical constrast noted was within the parafascicular nucleus. An abundance of c e l l s medial to the fasciculus retroflexus was noted in the parafascicular nucleus of CP-2 in contrast" to the v i r t u a l absence of la b e l l e d c e l l s in this region in case CP-3, in which l a b e l l e d c e l l s were l a t e r a l to the fasciculus retroflexus. A mediolateral topography of the projection of this nucleus to the striatum i s therefore indicated. OTHER AREAS A small number of neurons was seen in the dorsal raphe nucleus in each case examined (Fig. 1, F i g . 7D), and in cases where the locus coeruleus had been sectioned three or four lab e l l e d neurons were found in t o t a l (Fig. 7E). DISCUSSION Careful consideration must be given to potential procedural or technical p i t f a l l s that could influence the interpretation of these r e s u l t s . This i s p a r t i c u l a r l y true as the use of WGA-HRP for anterograde and retrograde tracing studies i s a r e l a t i v e l y 3 9 new procedure. Although only a modification of the HRP technique which has been in use for some time, there are theoretic a l reasons for expecting some of the problems that are associated with the use of HRP to be less in the present procedure and others to be worse. Although HRP (Nagy et a l . , 1978; Herkenham and Nauta, 1977) and WGA-HRP i t s e l f (Gerfen et a l . , 1982) have demonstrated a capacity for retrograde l a b e l l i n g of c e l l s or anterograde l a b e l l i n g of terminal f i e l d s (Walberg et a l . , 1980) whose axons merely project through an inject i o n s i t e , t h i s phenomenon does not appear to have occurred with the pressure injections employed in t h i s study. Evidence of thi s i s provided by the lack of generalized l a b e l l i n g expected i f afferents to or efferents from the cortex which pass through the head of the striatum had taken up peroxidase. If WGA-HRP had been taken up by projection f i b e r s passing through the striatum, retrogradely l a b e l l e d neurons would have appeared in the mediodorsal nucleus of the thalamus (Jones and Leavitt, 1975; Keefer et a l . , 1980), extensive retrograde l a b e l l i n g in the ventral anterior and ventrolateral thalamic . nuclei would have occurred (Jones and Leavitt, 1975) and neurons in layer VI of widespread areas of the cortex would have been la b e l l e d . Widespread anterograde l a b e l l i n g of the thalamus and cortex would have been expected as well, but none of these were observed in the present experiment. Furthermore, s t r i a t a l c e l l s anterior to injections into the striatum were not la b e l l e d . In some systems, transport markers have been shown to be transported transneuronally, resulting in the p o s s i b i l i t y of 40 apparent retrograde l a b e l l i n g of neurons which are innervated by l a b e l l e d terminals ( T r i l l e r and Korn, 1981; Itayama and van Hoesen, 1982). Anterogradely transported marker may also pass from la b e l l e d terminals into unlabelled terminals, in which case a heavily l a b e l l e d terminal f i e l d may act l i k e a second injection s i t e (CR. Gerfen, personal communication). Transneuronal transport, however, has only been reported to occur after inje c t i o n of very large amounts of material and after survival times in excess of four or fiv e days, much longer than the maximum two day survival time used in t h i s study. Although i t is conceivable that p a l l i d a l and subthalamic c e l l s may have been lab e l l e d by some transneuronal process, the absence of any anterograde l a b e l l i n g in the subthalamus, which receives a dense innervation from the globus p a l l i d u s , argues that t h i s phenomenon did not occur to any appreciable extent. STRIATUM A s t r i k i n g feature of the l a b e l l i n g within the striatum i t s e l f i s the marked paucity of c e l l l a b e l l i n g outside the i n j e c t i o n s i t e . Coordination of a c t i v i t y in widespread areas of the striatum must, therefore, either be unnecessary to the physiological function of t h i s nucleus or occur through polysynaptic mechanisms. The few large c e l l s seen outside the bounds of the injections s i t e f i t the description of the cholinergic s t r i a t a l interneuron (Fibiger, 1982). Local stimulation of s t r i a t a l tissue produces excitatory potentials up to a distance of 0.5 to 1.0 um which have been characterized as cholinergic (Misgeld et a l . , 1982). Other l o c a l e f f e c t s are very 41 rarely seen, indicating that other s t r i a t a l c e l l types have much shorter i n t r a s t r i a t a l connections. STRIATAL EFFERENTS The observation of anterograde peroxidase l a b e l l i n g in the present work i s in complete accord with a number of previous studies (Kemp, 1970; Tulloch et a l . , 1978; Nagy et a l . , 1978a). The globus p a l l i d u s , entopeduncular nucleus and substantia nigra are s t i l l recognized as the only areas in receipt of the output of the striatum. It is of interest to review the anatomical picture presented here in terms of neurochemical findings after lesions of the corresponding s t r i a t a l region. Large lesions of the head of the striatum reduce substance P, enkephalin (to a small extent) and the GABA sythesizing enzyme, glutamic acid decarboxylase (GAD), in the globus pa l l i d u s but reduce only substance P in the entopeduncular nucleus and substantia nigra (Brownstein et a l . , 1977; Jessel et a l . , 1978; Staines et a l . , 1980a). Although lesions of the head of the striatum do not decrease GAD levels in the entopeduncular nucleus and substantia nigra, lesions of the main body and t a i l of the striatum are e f f e c t i v e . This biochemical evidence suggests that substance P could be contained in c o l l a t e r a l s innervating a l l three structures but that GABA-containing c o l l a t e r a l s would be limi t e d to the innervations of the substantia nigra and entopeduncular nucleus. This i s consistent with the anatomical observations presented here. Comparisons with the available biochemical l i t e r a t u r e indicate that the l a b e l l e d terminals seen in case CP-1 represent GABA, substance P and 42 enkephalin-containing terminals in the globus p a l l i d u s , but represent only substance P-containing terminals in the entopeduncular nucleus and substantia nigra. For the most part, the GABAergic innervation of the l a t t e r two structures arises from areas of the striatum caudal to the inject i o n s i t e in case CP-1 . Firm evidence for c o l l a t e r a l i z a t i o n requires electrophysiological or alternate anatomical techniques, but the present data, s p e c i f i c a l l y the observation that s t r i a t a l efferents l e f t the fiber bundles to ramify within the neuropil of the globus p a l l i d u s and entopeduncular nucleus, suggest that there is a cascade of efferents from the head of the CP such that the majority of innervation of each successively caudal structure is via a c o l l a t e r a l . Fox and Raffols (1975) made a similar proposal on the basis of their observations of Golgi material. Some electrophysiological data have been presented upholding the idea of c o l l a t e r a l i z a t i o n but disagree with the biochemical data in asserting that the transmitter involved i s GABA (Yoshida et a l . , 1972). As nearly 70% of s t r i a t a l neurons can be shown to project to the substantia nigra (Bolam et a l . , 1981a), the li k e l i h o o d of c o l l a t e r a l i z a t i o n of these fibers to the globus pal l i d u s and entopeduncular nucleus is high. Recently a dynorphin-containing projection from the striatum to the substantia nigra has been demonstrated (Vincent et a l . , 1982c). Indications are that t h i s opioid peptide has a similar d i s t r i b u t i o n within the basal ganglia to that of substance P, and may occur within the same fibers (Vincent et a l . , 1982b). 4 3 S t r i a t a l c e l l bodies, demonstrated by immunohistochemical means to contain these substances or, in the case of" GABA, the synthetic enzyme, are a l l of the medium spiny class (Ribak et a l . , 1979; Pickel et a l . , 1980; Ljungdahl et a l . , 1978; Somogyi et a l . , 1982). Indeed, only medium spiny neurons appear to innervate the globus pallidus (Woolf and Butcher, 1981) and very few c e l l s not of t h i s class project to the entopeduncular nucleus or substantia nigra (Parent et a l . , 1980; Bolam et a l . , 1981b). Direct demonstration of the innervation of the globus pa l l i d u s by chemically characterized neurons has been obtained in the case of enkephalin (Brann and Emson, 1980;. Del Fiacco, 1982). STRIATAL AFFERENTS Extensive treatment has been given previously to the observation of s t r i a t a l afferents from the cortex (Hedreen, 1977; Yeterian and van Hoesen, 1978; Fallon and Ziegler, 1979; Oka, 1980; Donoghue and K i t a i , 1981; Royce, 1982) amygdala (Royce, 1978; Kelly et a l . , 1982), intralaminar and parafascicular nuclei (Jones and Leavitt, 1975; van der Kooy, 1979; Veening et a l . , 1980), dorsal raphe (van der Kooy, 1979; Loughlin and Fallon, 1982) and locus coeruleus (Mason and Fibiger, 1979). The evidence obtained for a very sparse projection of the subthalamic nucleus to the striatum agrees with the results of an autoradiographic study in the monkey (Nauta and Cole, 1978) in which a sparse innervation of the putamen was observed. Al t e r n a t i v e l y , peroxidase a c t i v i t y may have been taken up from a marginal intrusion of the injected WGA-HRP into the globus p a l l i d u s , although this was not apparent. Neurons of the 44 subthalamic nucleus recently have been shown to innervate the globus pa l l i d u s in addition to having c o l l a t e r a l projections to the entopeduncular nucleus and substantia nigra (Denaiu et a l . , 1978a; van der Kooy and Hattori, 1980a). From the results of the present study, and in agreement with others (van der Kooy and Hattori, 1980a; Ricardo, 1980) i t must be concluded that the subthalamic nucleus probably has, at most, a minor influence on the a c t i v i t y of c e l l s in the head of the striatum, although this projection was at least as s i g n i f i c a n t as that seen from the amygdala. It remains to be determined i f the subthalamic innervation of the t a i l of the striatum, which corresponds more close l y to the putamen, i s more s i g n i f i c a n t . A l t e r n a t i v e l y , the apparent sparse innervation of the striatum by the subthalamic nucleus may r e f l e c t the presence of the occasional neuron of the globus p a l l i d u s within the anatomical boundaries of the striatum. A few examples of a second type of s t r i a t o n i g r a l neuron have been found which are morphologically very similar to the p a l l i d o n i g r a l neuron (Bolam et a l . , 1981b). S i m i l a r l y , a small number of s t r i a t a l neurons of similar size show very dense innervation by enkephalin-containing terminals, a feature not general to the striatum but commonly seen in the globus pallidus (Somogyi et a l . , 1982). There i s l i t t l e evidence in the l i t e r a t u r e supporting a projection of the globus p a l l i d u s to the head of the striatum. In fact, many studies on s t r i a t a l afferents or p a l l i d a l efferents have been c a r r i e d out which might have been expected to have made this observation previously . Among factors which may have 45 contributed to this f a i l u r e are lack of s e n s i t i v i t y of previous methods, d i f f u s i o n of the inje c t i o n s i t e to the pallidum and the presence of terminal l a b e l l i n g within t h i s structure. Golgi and autoradiographic studies of the globus pa l l i d u s have shown axons projecting to the striatum (Nauta, 1979a; Iwahori and Mizuno, 1981), but i t was not clear i f these observations merely re f l e c t e d the projection of fibers from p e r i p a l l i d a l c e l l s bound for the cortex (Divac, 1975; Lehmann et a l . , 1980; Parent et a l . , 1981c). The i n t e r s t i t i a l c e l l s l a b e l l e d medial to. the globus pallidus may have belonged to t h i s population. The projection of the globus pallidus to the striatum i s examined in d e t a i l in a number of subsequent experiments (Expts. 4-8). The observation of l a b e l l e d c e l l s in the ventromedial nucleus of the thalamus confirms the autoradiographic observations of Herkenham (1979) who found a sparse d i s t r i b u t i o n of s i l v e r grains in s t r i a t a l neuropil after injections of t r i t i a t e d amino acids into the ventromedial nucleus. Additional evidence has come from a recent retrograde transport study (Veening et a l . , 1980), but s i g n i f i c a n t negative data exist as well (van der Kooy, 1979). For reasons presented above i t i s not thought that this represents l a b e l l i n g by uptake into fibers of passage. The ventromedial nucleus receives innervation from the substantia nigra (Clavier et a l . , 1976; Experiment 2), the deep cerebellar nuclei (Herkenham, 1979; Sugimoto et a l . , 1981) and possibly the entopeduncular nucleus (Carter and Fibiger, 1978). It projects heavily to layer I of most of the neocortex and to layers III and V of the motor and premotor cortices (Herkenham, 46 1979) . Terminal f i e l d s in the deeper layers correspond remarkably to the d i s t r i b u t i o n of c o r t i c a l c e l l s found to project to the striatum (Fig. 1). A number of considerations indicate that the ventromedial thalamic nucleus in the rat i s a heterogenous nucleus composed of c e l l s with s p e c i f i c and nonspecific c o r t i c a l innervations (see Carter, and Fib i g e r , 1978; Herkenham, 1979). Corroboration of th i s i s supplied by the observation that the ventromedial nucleus in the cat projects solely to layer I (Glenn et a l . , 1982). It is not yet apparent which of these two c e l l types were lab e l l e d a f t e r s t r i a t a l injections in the present material. If i t were the nonspecific c e l l type, the sparse innervation of the striatum inferred here would stand in contrast to the reportedly dense innervation of the cortex. The reverse i s true of the projection pattern of the intralaminar-parafascicular complex which has a dense innervation of the striatum but a more sparse c o r t i c a l projection (Jones and Lea v i t t , 1975). A few c e l l s were also seen in the ventrolateral thalamus. It seems l i k e l y thatt these neurons were outlying members of the ventromedial population, but others have found large numbers of c e l l s in t h i s area after s t r i a t a l HRP injections which produced much less l a b e l l i n g in the ventromedial nucleus (Veening et a l . , 1980) . As both of these thalamic regions act as relays for the output of information from the basal ganglia, the p o s s i b i l i t y that one or both of them project to the striatum deserves more detailed consideration. The present data f i t in well with the impressive l i t e r a t u r e 47 on the s t r i a t a l projections of the dopaminergic c e l l groups in and around the substantia nigra (Lindvall and Bjorklund, 1974; Carter and Fibiger, 1977; Faull and Mehler, 1978; van der Maelin et a l . , 1978; Beckstead et a l . , 1979; Veening et a l . , 1980). It has been shown that v i r t u a l l y a l l c e l l s projecting to the striatum from th i s region, including those found within the pars r e t i c u l a t a , are dopaminergic (van der Kooy et a l . , 1981b). There are, however, a few nondopaminergic pars r e t i c u l a t a neurons which project to the striatum and have c o l l a t e r a l s to other regions in receipt of nondopaminergic n i g r a l innervation (Deniau et a l . , 1978b; Steindler and Deniau, 1980). The present investigation indicates that there are problems associated with the concept of a d i r e c t s t r i a t o n i g r a l - n i g r o s t r i a t a l feedback loop (see Dray, 1979). Neurons within the pars compacta send descending dendrites into the pars r e t i c u l a t a , largely by running along vascular elements (Schiebel and Tomiyasu, 1980). In fact, Golgi studies in the monkey indicate that most of the dendrites of these c e l l s d i s t r i b u t e within the pars r e t i c u l a t a , as far v e n t r a l l y as the cerebral peduncle (Schwyn and Fox, 1974). It i s obvious from Figures 6B and 6C that, although the labelled compacta neurons could receive s t r i a t a l afferents, most would do so only on the d i s t a l parts of their dendrites. A more proximal input would be expected to arise from more ve n t r a l l y located neurons than were lab e l l e d in case CP-1 (Tulloch et a l . , 1978; Nauta et a l . , 1978). This suggests at best a rather vague re c i p r o c i t y of connections between the substantia nigra and striatum. Even more s t r i k i n g i s the observation that in some 48 Figure 8. Schematic diagram summarizing the findings of thi s study and others mentioned in the discussion. The arrows are schematic and not meant to represent c o l l a t e r a l s or monosynaptic connections. 49 50 animals the l a b e l l i n g of neurons in the pars compacta and terminals within the pars r e t i c u l a t a occurred at very d i f f e r e n t rostrocaudal planes. The l a b e l l e d c e l l s found within the pars r e t i c u l a t a may represent a unique population of n i g r o s t r i a t a l neurons. Their c o d i s t r i b u t i o n with the la b e l l e d terminal f i e l d , even after very small injections, was s i g n i f i c a n t . Hattori et a l . (1975) estimated that only 3.5 % of s t r i a t o n i g r a l terminals end on dopaminergic neurons. A more recent report confirms an innervation but does not supply quantitative data and i s clouded by an almost certain l a b e l l i n g of the nigr a l afferents descending from the globus pallidus as well. (Wassef et a l . , 1981). An exceedingly sparse crossed n i g r o s t r i a t a l projection has been mentioned previously (Loughlin and Fallon., 1982). The importance of this pathway appears to arise from i t s inst r u c t i v e value to neuroanatomists on the question of s i g n i f i c a n c e . SUMMARY The demonstration of a p a l l i d o s t r i a t a l pathway suggests that the striatum may be r e c i p r o c a l l y connected with the globus pal l i d u s but topographical considerations cast doubt on the concept that the s t r i a t o n i g r a l and n i g r o s t r i a t a l projections constitute a similar reciprocal system. The only other nucleus receiving s t r i a t a l outputs, the entopeduncular nucleus, does not project back to the head of the striatum. The striatum receives a massive c o r t i c a l input from the fron t a l cortex and a less massive input from other c o r t i c a l areas including the a r c h i c o r t i c a l entorhinal cortex and the p a l e o c o r t i c a l amygdala. The other major inputs to the striatum 51 a r i s e f r o m t h e d o p a m i n e r g i c n e u r o n s o f t h e s u b s t a n t i a n i g r a a n d t h e i n t r a l a m i n a r / p a r a f a s c i c u l a r n e u r o n s o f t h e t h a l a m u s . C o m p a r a t i v e l y m i n o r i n p u t s t o t h e s t r i a t u m a r i s e f r o m t h e l o c u s c o e r u l e u s , d o r s a l r a p h e , s u b t h a l a m i c n u c l e u s , v e n t r o l a t e r a l t h a l a m u s a n d v e n t r o m e d i a l t h a l a m u s . T h e s e l a t t e r t h r e e r e g i o n s e a c h r e c e i v e i n n e r v a t i o n f r o m n u c l e i t o w h i c h t h e s t r i a t u m p r o j e c t s . T h e s e f i n d i n g s a r e r e p r e s e n t e d d i a g r a m a t i c a l l y i n F i g u r e 8. 52 EXPERIMENT 2: EXAMINATION OF THE EFFERENT AND AFFERENT CONNECTIONS OF THE SUBSTANTIA NIGRA INTRODUCTION The substantia nigra is a major recipient of efferents of the striatum (Grofova and Rinvik, 1970; Hattori et a l . , 1975; Tulloch et a l . , 1978; Experiment 1). It also receives input from other basal ganglia nuclei such as the globus p a l l i d u s and subthalamic nucleus and afferents from outside t h i s system including those from the p e r i b r a c h i a l area, dorsal raphe, parafasicular nucleus, hypothalamus and prefrontal cortex (Kanazawa et a l . , 1976, Bunney and Aghajanian, 1976; Nauta and Domesick, 1978). It in turn projects to the medial prefrontal cortex, striatum, ventromedial, mediodorsal and parafascicular thalamic nuclei, superior c o l l i c u l u s and peribrachial region (Beckstead et a l . , 1979). In the present study, the connections of the substantia nigra were reexamined using anterograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) (Staines et a l . , 1980b). A marked topographical rel a t i o n s h i p was observed for both s t r i a t a l and p a l l i d a l connections with the substantia nigra. B i l a t e r a l innervation of thalamic nuclei and the superior c o l l i c u l u s is demonstrated and a t o t a l l y crossed input from the posterior l a t e r a l hypothalamus i s described for the f i r s t time. In addition, a marked difference was seen in both retrograde and anterograde l a b e l l i n g from dopaminergic and nondopaminergic regions of the substantia nigra. Portions of the results in t h i s report have been published 53 (Gerfen et a l . , 1982) . METHODS Male albino rats, anesthetised with pentobarbital, received 50-100 nl pressure injections of a 1 to 2.5% WGA-HRP solution into the substantia nigra. Injections were made using a 1 ul Hamilton syringe with a 31 gauge cannula. In a few of the nigral injections the solution also contained kainic acid at a concentration of 10 mM. After a 24 hr survival period the animals were reanesthetised and fixed by transcardial perfusion with 1.0% formaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature, followed by a transcardial rinse with buffered ice cold sucrose. The brains were removed and stored in this l a t t e r solution u n t i l sectioned. Slide-mounted 50 um sections were reacted for peroxidase a c t i v i t y using tetramethylbenzidine as substrate as described in Experiment 1. In some cases, f r e e - f l o a t i n g sections were reacted for peroxidase using diaminobenzidine as substrate (Graham and Karnovsky, 1966) and then counterstained for cre s y l v i o l e t to allow morphometric analyses.. In these instances sections were incubated at room temperature in 50 mM Tris-HCl buffer (pH 7.4) containing 0.025% diaminobenzidine (DAB) for 10 minutes. The reaction was started by the addition of 3% hydrogen peroxide to a f i n a l concentration of 0.0075% and allowed to proceed for 20 to 30 minutes. Sections were rinsed in T r i s buffer and mounted onto subbed s l i d e s . After a i r drying, sections were counterstained with c r e s y l v i o l e t , dehydrated through a graded series of alcohol solutions, cleared in xylene and coverslipped with Permount. 54 Control injections of WGA-HRP were made along the angled approach to the substantia nigra and into the regions r o s t r a l , caudal and dorsal to i t . Additional WGA-HRP injections were made into the parafascicular thalamic nucleus, the l a t e r a l dorsal thalamic nucleus, and the peribrac h i a l area. Iontophoretic WGA-HRP injections into the posterior l a t e r a l hypothalamus were made through a micropipette (20 um t i p diameter) f i l l e d with a 1% WGA-HRP solution in 0.9% saline using a 2 uA positive current for 5 minutes. RESULTS Most of the results of n i g r a l injections presented below were obtained from the case i l l u s t r a t e d in Figure 9 (SN-1), in which the inj e c t i o n s i t e was predominantly l o c a l i z e d to the pars r e t i c u l a t a . Seven other cases with injections into various regions and subdivisions of the substantia nigra were examined in d e t a i l and the important differences are noted in the appropriate sections. Some of these other inje c t i o n s i t e s are depicted in Figure 10. NIGRAL INJECTIONS Anterograde and retrograde l a b e l l i n g of neuronal elements resu l t i n g from an injection of WGA-HRP confined to the substantia nigra are shown in Figure 9. The injection was centered in the pars r e t i c u l a t a of the substantia nigra and led to dense l a b e l l i n g of neurons, presumably by direct somal or dendritic uptake, around the injection center in both the pars r e t i c u l a t a and pars compacta (Fig. 11A and 11B). These neurons are marked with tria n g l e s in Figure 9. That the inject i o n did not spread 55 Figure 9 . Line drawings depicting the WGA-HRP pressure in j e c t i o n into the substantia nigra and the resultant anterograde and retrograde transport of peroxidase in case SN - 1 . Labelled perikarya are represented by f i l l e d c i r c l e s , except those in the immediate v i c i n i t y of the inje c t i o n s i t e which are represented by f i l l e d t r i a n g l e s . Labelled projection fibers and terminal f i e l d s are drawn i n . Abbreviations in t h i s and subsequent figures: AC, anterior commissure; BC, brachium conjunctivum; ce, central nucleus of the amygdala; cem, centromedial nucleus of the thalamus; eg, central grey area; c l , cen t r o l a t e r a l nucleus of the thalamus; cp, striatum; CP, cerebral peduncle; dr, dorsal raphe nucleus; F, fornix; FM, forceps minor; FR, fasciculus retroflexus; g, nucleus gelatinosus; gp, globus p a l l i d u s ; i c , i n f e r i o r c o l l i c u l u s ; IC, internal capsule; ip, interpeduncular nucleus; l d , laterodorsal nucleus of the thalamus; l h , l a t e r a l habenula; lhp, posterior l a t e r a l hypothalamic area; md, mediodorsal nucleus of the thalamus; MFB, medial forebrain bundle; ML, medial lemniscus; MT, mammillothalamic t r a c t ; pc, paracentral nucleus of the thalamus; ppn, nucleus tegmenti pedunculopontis; r, red nucleus; sc, superior c o l l i c u l u s ; snc, substantia nigra pars compacta; snr, substantia nigra pars r e t i c u l a t a ; sum, supramammillary nucleus; sut, subthalamic nucleus; vm, ventromedial nucleus of the thalamus; vta, ventral tegmental area; z i , zona incerta. 56 5 7 Figure 10. Line drawings depicting the maximum extent of the i n j e c t i o n s i t e s (as v i s u a l i z e d with tetramethylbenzidine) in cases SN-2 to SN-6. Results of these injections are referred to in the text. SN 4 59 into the medially adjacent ventral tegmental area can be inferred by the absence of dire c t l a b e l l i n g of these c e l l s and by the absence of anterograde transport to the nucleus accumbens known to receive a massive input from that area (Beckstead et a l . , 1979). In the case depicted there was ne g l i g i b l e d i f f u s i o n of WGA-HRP along the injec t i o n t r a c t . There was no evidence that the tracer had spread into the cerebral peduncle ventral to the pars r e t i c u l a t a . RETROGRADE LABELLING The d i s t r i b u t i o n of retrogradely la b e l l e d neurons, mapped in Figure 9 as large dots, confirms previous descriptions of the sources of ni g r a l afferents in the rat (Hattori et a l . , 1975; Bunney and Aghajanian, 1976; Kanazawa et a l . , 1976; van der Kooy and Hattori, 1980a). These include i p s i l a t e r a l inputs from the prefrontal cortex, striatum, globus p a l l i d u s , subthalamic nucleus, dorsal raphe and minor i p s i l a t e r a l inputs from the hypothalamus, central nucleus of the amygdala, and l a t e r a l habenula. In addition, three other regions contain retrogradely l a b e l l e d neurons. These were the con t r a l a t e r a l posterior l a t e r a l hypothalamic region, the i p s i l a t e r a l parafascicular nucleus of the thalamus and the i p s i l a t e r a l and contralateral peribrachial region, including the pedunculopontine nucleus (Saper and Loewy, 1982). In contrast with an e a r l i e r report (Grofova, 1975) no retrograde l a b e l l i n g of the entopeduncular nucleus was observed. ANTEROGRADE LABELLING Terminal l a b e l l i n g was i d e n t i f i e d in the following brain regions: 1) the i p s i l a t e r a l striatum; 2) the i p s i l a t e r a l globus 60 p a l l i d u s ; 3) t h a l a m i c n u c l e i i n c l u d i n g t h e l a t e r a l d o r s a l , p a r a l a m e l l a r m e d i o d o r s a l , a n d v e n t r o m e d i a l n u c l e i , b i l a t e r a l l y a n d t h e i p s i l a t e r a l p a r a f a s i c u l a r n u c l e u s ; 4) t h e s u b t h a l a m i c n u c l e u s ; 5) a d i s c r e t e a r e a o f t h e most v e n t r a l a nd r o s t r a l p o r t i o n o f t h e m i d b r a i n c e n t r a l g r e y ; 6 ) t h e i p s i l a t e r a l d o r s a l m i d b r a i n tegmentum; 7) t h e i p s i l a t e r a l a n d c o n t r a l a t e r a l s u p e r i o r c o l l i c u l u s ; 8) t h e c e n t r a l g r e y r e g i o n t h r o u g h t h e m i d b r a i n and p o n s , b i l a t e r a l l y ; a n d 9) t h e i p s i l a t e r a l p e r i b r a c h i a l r e g i o n . T h e s e r e s u l t s a r e i n c l u s i v e o f p r e v i o u s r e p o r t s on n i g r a l p r o j e c t i o n s ( B e c k s t e a d e t a l . , 1 9 7 9 ) . NIGRAL CONNECTIONS WITH THE PERIBRACHIAL REGION WGA-HRP i n j e c t i o n s i n t o t h e s u b s t a n t i a n i g r a l a b e l l e d n e u r o n s i n t h e p e r i b r a c h i a l r e g i o n , b i l a t e r a l l y . W h i l e i t i s n o t p o s s i b l e t o i d e n t i f y a n t e r o g r a d e l a b e l l i n g w i t h c e r t a i n t y when r e t r o g r a d e l a b e l l i n g i s p r e s e n t i n t h e same a r e a , t h e r e a p p e a r e d t o be t e r m i n a l l a b e l l i n g o f t h e i p s i l a t e r a l b u t n o t c o n t r a l a t e r a l p e r i b r a c h i a l r e g i o n ( F i g . 11C and 1 1 D ) . I t was n o t e d t h a t i n i n j e c t i o n s w i t h more e x t e n s i v e i n v o l v e m e n t o f t h e p a r s c o m p a c t a , p a r s l a t e r a l i s a n d r e t r o r u b r a l a r e a ( S N - 2 and SN - 6 ) t h e r e was f a r more r e t r o g r a d e l a b e l l i n g o f n e u r o n s i n t h e p e r i b r a c h i a l r e g i o n . I n c a s e s JsN-3 and SN-5 v e r y few c e l l s were l a b e l l e d . F u r t h e r m o r e , w h i l e c e l l s l a b e l l e d i n c a s e SN-1 were f o u n d i n t h e r e g i o n n e a r t h e d e c u s s a t i o n o f t h e b r a c h i u m c o n j u n c t i v u m , t h o s e i n SN - 2 a n d SN - 6 were s e e n d o r s a l and v e n t r a l t o t h i s f i b e r t r a c t a t p l a n e s a s c a u d a l a s t h e m o t o r n u c l e u s o f t h e t r i g e m i n a l . As d e p i c t e d i n f i g u r e 9, a n t e r o g r a d e l a b e l l i n g o f t h e w h o l e o f t h i s r e g i o n was a p p a r e n t i n t h o s e c a s e s w i t h i n v o l v e m e n t o f p a r s 61 Figure 11. L i g h t f i e l d (A) and d a r k f i e l d (B) photomicrographs of the inje c t i o n s i t e in the substantia nigra in case SN-1. (C) Peroxidase l a b e l l e d perikarya (arrows) in the co n t r a l a t e r a l and (D) l a b e l l e d c e l l bodies and terminals in the i p s i l a t e r a l pedunculopontine nucleus in case SN-1. (E) Peroxidase l a b e l l i n g of neurons in peribra c h i a l region of case SN-2 contr a l a t e r a l and (F) i p s i l a t e r a l to the i n j e c t i o n . Note that there appears to be two types of l a b e l l e d c e l l s on the i p s i l a t e r a l side , a small c e l l type (arrows) and a larger c e l l type (asterisk), but only the smaller c e l l type on the co n t r a l a t e r a l side. Bar = 200 um in (B) and (C), bar = 50 um in (E) and (F). 62 63 r e t i c u l a t a in the injection s i t e . In case SN-6, in which the injection was r e s t r i c t e d to the pars compacta, only l a b e l l e d c e l l bodies were seen. Evidence was obtained that two c e l l types in the peribrachial region project to the substantia nigra. The majority of labe l l e d c e l l s were medium sized, spindle-shaped neurons (10 by 18 um) but larger, multipolar c e l l s (13 by 25 um) were seen as well (Fig. 11F). Although not examined in great d e t a i l , i t seemed that only the smaller type of c e l l was labe l l e d in the co n t r a l a t e r a l nucleus (Fig. 11E). Injections of WGA-HRP into the peribrachial region, resulted in the l a b e l l i n g of neurons in the i p s i l a t e r a l but not contrala t e r a l pars r e t i c u l a t a . Additionally, these injections resulted in an apparent b i l a t e r a l l a b e l l i n g of terminals in the substantia nigra and subthalamic nucleus, as well as retrograde l a b e l l i n g of neurons in the entopeduncular nucleus and hypothalamus. NIGRAL AFFERENTS FROM THE CONTRALATERAL HYPOTHALAMUS Peroxidase labelled neurons located in the contralateral posterior l a t e r a l hypothalamus were confined to a discrete area just l a t e r a l to the mammillothalamic tract as i t descends into the mammillary nuclei (Fig. 9F, G; F i g . 12A). As many as 14 labell e d neurons were seen per 50 um section. The largest n i g r a l WGA-HRP injections l a b e l l e d a t o t a l of 120 of these neurons. Only WGA-HRP injections into the r o s t r a l half of the substantia nigra l a b e l l e d these c e l l s . To investigate the p o s s i b i l i t y that c o n t r a l a t e r a l posterior hypothalamic neurons were la b e l l e d by 64 uptake into fibres of passage running through the substantia nigra or by uptake of tracer that spread beyond the substantia nigra (although this was not apparent), control injections were made into a number of extranigral midbrain areas. A comparison of the d i s t r i b u t i o n of l a b e l l e d neurons in the hypothalamus after control injections with that seen after n i g r a l injections showed (1) n i g r a l injections l a b e l l e d c e l l s occupying a region containing very few (3 or 4) neurons la b e l l e d by extranigral injections, and (2) while most of the hypothalamic neurons labe l l e d a f t e r control injections were i p s i l a t e r a l to the i n j e c t i o n , n i g r a l injections l a b e l l e d only contralateral neurons in the posterior hypothalmus (although i p s i l a t e r a l hypothalamic neurons were labe l l e d in a much more r o s t r a l region; see Discussion). An i n j e c t i o n of WGA-HRP into the region of the posterior l a t e r a l hypothalamus in which lab e l l e d c e l l s were found gave r i s e to anterograde l a b e l l i n g within the c o n t r a l a t e r a l substantia nigra (Fig. 12B and 12C). The fibers crossed in the supramammillary decussation, progressed l a t e r a l l y and caudally into the substantia nigra pars compacta, and then coursed v e n t r o l a t e r a l l y from the pars compacta into the pars r e t i c u l a t a . Occasionally a labelled f i b e r in the pars compacta was observed with a c o l l a t e r a l that branched into the pars r e t i c u l a t a , where i t appeared to terminate. This pattern of transport was most evident in the r o s t r a l parts of the substantia nigra and became progressively less apparent in more caudal sections. Other l a b e l l i n g contralateral to the i n j e c t i o n appeared in the form of 65 Figure 12. (A) Peroxidase l a b e l l e d perikarya in the posterior l a t e r a l hypothalamus after a WGA-HRP inject i o n into the contral a t e r a l substantia nigra (see F i g . 9G). (B) A single peroxidase l a b e l l e d fiber in the substantia nigra pars compacta with a c o l l a t e r a l descending into the substantia nigra pars r e t i c u l a t a after an iontophoretic WGA-HRP inject i o n s i t e into the contralateral posterior l a t e r a l hypothalamus. (C) A larger f i e l d showing labelled fibers in the anterior substantia nigra pars r e t i c u l a t a . No labe l l e d perikarya were found in the substantia nigra pars r e t i c u l a t a . C a l i b r a t i o n bar =200um. 66 67 l a b e l l e d f i b e r s , terminals, and c e l l bodies in the dorsal midbrain tegmentum, and la b e l l e d terminals and c e l l bodies in both the peribrachial area and in the laterodorsal tegmental nucleus. I p s i l a t e r a l to the hypothalamic i n j e c t i o n , labelled neurons were observed in the ventral tegmental area and the pars compacta. In addition, the pars r e t i c u l a t a contained a moderately dense d i s t r i b u t i o n of punctate reaction product throughout i t s rostrocaudal extent. These results may indicate connections between these areas and the l a t e r a l hypothalamic injec t i o n area. It i s noted that the neurons located in this region lay d i r e c t l y in the path of the dopaminergic projection f i b e r s in the medial forebrain bundle (Fig. 13E) and may receive innervation by boutons en passant. The l a b e l l i n g in the i p s i l a t e r a l pars r e t i c u l a t a i s not thought to have arisen from the posterior l a t e r a l hypothalamic neurons and may have been due to l a b e l l i n g of subthalamonigral f i b e r s . NIGROTHALAMIC CONNECTIONS Parafascicular nucleus Neurons in the i p s i l a t e r a l parafascicular thalamic nucleus were retrogradely l a b e l l e d after n i g r a l WGA-HRP injections. These injections also resulted in an apparent terminal l a b e l l i n g within the same region. Injections into the parafascicular nucleus i t s e l f resulted in retrograde l a b e l l i n g of over 100 neurons in the i p s i l a t e r a l pars r e t i c u l a t a of the substantia nigra (Fig. 13C and 13D). Only rarely were neurons la b e l l e d in the contra l a t e r a l pars r e t i c u l a t a 6 8 Figure 13. (A) An iontophoretic WGA-HRP injection into the laterodorsal nucleus of the thalamus. Some l a b e l l i n g of the fimbria can be seen as well. (B) Peroxidase l a b e l l i n g of perikarya (arrows) in the substantia nigra pars r e t i c u l a t a i p s i l a t e r a l to the injection shown in (A). (C) An iontophoretic WGA-HRP injection into the parafascicular nucleus and (D) the resultant l a b e l l i n g of perikarya in the i p s i l a t e r a l substantia nigra pars r e t i c u l a t a . (E) Peroxidase l a b e l l i n g in the caudal diencephalon after a WGA-HRP injection into the substantia nigra. The anterograde transport of peroxidase to the parafascicular nucleus i s apparent in this section. Note that the posterior l a t e r a l hypothalamus is d i r e c t l y in the path of ascending medial forebrain bundle, which can be seen due to the presence of peroxidase transported in the anterograde di rect ion. 69 70 after parafasicular injections. Additionally, there was evidence of terminal l a b e l l i n g in the i p s i l a t e r a l pars r e t i c u l a t a , striatum, subthalamus and interpeduncular nucleus. Labelled c e l l bodies were seen b i l a t e r a l l y in the entopeduncular nucleus, hypothalamus, superior c o l l i c u l u s and peribrachial area and u n i l a t e r a l l y in the l a t e r a l habenula and laterodorsal tegmental nucleus. Previous studies have i d e n t i f i e d a l l of these connections except for those implied by the l a b e l l i n g seen in the l a t e r a l habenula and interpeduncular nucleus (Ahlenius, 1978). As the t i p of the injection cannula was located in the core of the fasciculus retroflexus, peroxidase l a b e l l i n g of these l a t t e r two areas probably represents transport of tracer by damaged fibe r s of passage. Ventromedial thalamic nucleus Substantia nigra WGA-HRP injections resulted in dense terminal and f i b e r l a b e l l i n g in the i p s i l a t e r a l ventromedial thalamic nucleus (Fig. 14). Additionally, there was evidence of a more sparse d i s t r i b u t i o n of terminal l a b e l l i n g in the contr a l a t e r a l ventromedial nucleus, p a r t i c u l a r l y in i t s most dorsal aspect. Evidence was obtained for a topographical d i s t r i b u t i o n of terminals to the ventromedial nucleus. In very ventrally placed n i g r a l injections the anterograde l a b e l l i n g of th i s nucleus appeared confined to r o s t r a l regions and in the l a t e r a l n i g r a l i n j e c t i o n (SN-2) terminal l a b e l l i n g was r e s t r i c t e d to the dorsolateral half of the the ventromedial nucleus, leaving that portion nearest the mammillothalamic tract free of reaction product. After an injection confined to the pars compacta and 71 Figure 14. Anterograde peroxidase l a b e l l i n g in the thalamus at three coronal planes after a WGA-HRP inj e c t i o n into the substantia nigra in case SN-1. Labelling of the laterodorsal and ventromedial nuclei and b i l a t e r a l l a b e l l i n g of the paralamellar portion of the mediodorsal nucleus i s apparent in (A). The white appearance of the s t r i a medularis i s an a r t i f a c t of dark f i e l d miscroscopy. Note the b i l a t e r a l component to the terminal f i e l d in the ventromedial thalamus in (B) and (C). 72 73 retrorubral areas of the substantia nigra, the ventromedial nucleus was v i r t u a l l y unlabelled. Mediodorsal thalamic nucleus There was d i s t i n c t i v e l a b e l l i n g of terminals in the paralamellar region of the mediodorsal thalamic nucleus, b i l a t e r a l l y , after n i g r a l WGA-HRP injections (Fig. 14). Fibers appeared to enter the mediodorsal nucleus through the intralaminar nuclei, however, i t was not possible to determine whether l a b e l l i n g within the intralaminar region arose from la b e l l e d projection fibers alone or whether terminals were also l a b e l l e d . Injections into the mediodorsal nucleus led to la b e l l i n g of over 400 c e l l bodies in the pars r e t i c u l a t a of the substantia nigra. Some of the other areas found to project to this nucleus were the contral a t e r a l pars r e t i c u l a t a of the substantia nigra, superior c o l l i c u l u s , peribrachial area, magnocellular nuclei of the basal forebrain and the contr a l a t e r a l posterior l a t e r a l hypothalamus. Neurons in thi s l a t t e r region had an id e n t i c a l d i s t r i b u t i o n to those l a b e l l e d c o n t r a l a t e r a l to injections into the substantia nigra. Lateral dorsal thalamic nucleus After n i g r a l WGA-HRP injections, l a b e l l e d fibers were observed exiting in a laterodorsal d i r e c t i o n from the intralaminar nuclei. These fibers continued into the l a t e r a l dorsal thalamic nucleus in which a sparse terminal-like d i s t r i b u t i o n of label was apparent. (Fig. 14). A similar pattern, although less d i s t i n c t , was seen contr a l a t e r a l to the inj e c t i o n . Injections of WGA-HRP centered in the laterodorsal 74 nucleus spread into most of the l a t e r a l t i e r of thalamic nuclei but did not appear to spread more than a minimal amount into the mediodorsal or ventromedial n u c l e i . Such injections resulted in retrograde l a b e l l i n g of c e l l s in the most caudal medial aspect of the pars r e t i c u l a t a of the substantia nigra (Fig. 13A and 13B). NIGROTECTAL CONNECTIONS A d i s t i n c t i v e p u f f - l i k e d i s t r i b u t i o n of la b e l l e d terminals along the entire mediolateral extent of the stratum album intermediale of the superior c o l l i c u l u s was observed i p s i l a t e r a l to n i g r a l WGA-HRP injections (Fig. 15). Additionally, numerous lab e l l e d fibers were observed in the superior c o l l i c u l a r decussation and there was marked terminal l a b e l l i n g in the contrala t e r a l superior c o l l i c u l u s . In contrast to the di s t r i b u t i o n of terminals on the i p s i l a t e r a l side, that in the contr a l a t e r a l superior c o l l i c u l u s was r e s t r i c t e d primarily to the most ventrolateral aspect of the stratum griseum. Iontophoretic applications of WGA-HRP were made into either the r o s t r a l or caudal half of the superior c o l l i c u l u s . Both injections deposited tracer into the intermediate and deep layers of the l a t e r a l half of the tectum and into the most dorsal aspect of the midbrain r e t i c u l a r formation. Each in j e c t i o n resulted in d i s t i n c t l y d i f f e r e n t patterns of retrograde l a b e l l i n g in the substantia nigra (Fig. 16). The caudal inje c t i o n l a b e l l e d a large number of i p s i l a t e r a l pars r e t i c u l a t a neurons located in the r o s t r a l half of that nucleus and confined primarily to a ventral area just above the cerebral peduncle. Only two neurons were la b e l l e d in the con t r a l a t e r a l nucleus. The r o s t r a l 75 Figure 15. (A) Anterograde transport to the anterior superior c o l l i c u l u s and dorsal midbrain tegmentum in case SN-1 shown in re l a t i o n to the inject i o n s i t e into the substantia nigra. (B) A higher magnification of the anterograde l a b e l l i n g within the i p s i l a t e r a l and con t r a l a t e r a l superior c o l l i c u l u s . E s s e n t i a l l y the same pattern of l a b e l l i n g i s seen at more caudal levels of the superior c o l l i c u l u s as well (C). 76 77 Figure 16. A WGA-HRP injection s i t e (A) into the ventrolateral aspect of the anterior superior c o l l i c u l u s and the resultant l a b e l l i n g of perikarya in the substantia nigra pars r e t i c u l a t a i p s i l a t e r a l (B) and cont r a l a t e r a l (C) to the in j e c t i o n . (D) A WGA-HRP inject i o n s i t e into the ventrolateral aspect of the caudal superior c o l l i c u l u s and (E) the perikaryal l a b e l l i n g i t produced within the i p s i l a t e r a l substantia nigra. There was no s i g n i f i c a n t l a b e l l i n g of c e l l s contralateral to the injection shown in (D) . 78 o • m > UJ . \ - V . < 1 < 1 Q 79 injection l a b e l l e d an even greater number of i p s i l a t e r a l n i g r a l neurons than did the caudal i n j e c t i o n . Again these neurons were located in the r o s t r a l half of the nucleus; however, in thi s case, lab e l l e d perikarya were dispersed in the dorsal three fourths of the pars r e t i c u l a t a . The con t r a l a t e r a l pars r e t i c u l a t a contained in excess of 200 labelled perikarya, fewer than on the i p s i l a t e r a l side, primarily confined to r o s t r a l and dorsal regions. The possible inclusion of the midbrain r e t i c u l a r formation in the injec t i o n area precludes a s t r i c t i d e n t i f i c a t i o n of a l l neurons labelled after these injections as exclusively part of the nigrotectal pathway. However, since there was no evidence of crossed fibers terminating in the midbrain r e t i c u l a r formation after n i g r a l WGA-HRP injections, the l a b e l l i n g of contral a t e r a l neurons in the pars r e t i c u l a t a after the injections into the superior c o l l i c u l u s is presumed to r e f l e c t a crossed nigrotectal connection. SUBTHALAMIC NUCLEUS Retrograde l a b e l l i n g of neurons in the subthalamic nucleus was a prominent feature of a l l n i g r a l injections with involvement of the pars r e t i c u l a t a (Fig. 17). In case SN-6, where the injection was confined to the pars compacta, no la b e l l e d c e l l bodies were found within the subthalamic nucleus. Although heavily l a b e l l e d fibers were observed coursing dorsally and medially to the subthalamic nucleus, no apparent innervation of this structure could be discerned. D i s s i m i l a r i t i e s were seen between the d i s t r i b u t i o n s of la b e l l e d neurons in the subthalamic nuclei after the v i r t u a l l y complementary nigra l injections, SN-2 80 and SN-4. The caudolateral injection l a b e l l e d mainly r o s t r a l l y located neurons whereas, after the rostromedial i n j e c t i o n , l a b e l l e d c e l l s were confined predominantly to caudal regions. At the l e v e l of greatest c e l l l a b e l l i n g in each case, positive neurons were seen throughout the whole mediolateral extent. STRIATUM AND GLOBUS PALLIDUS As depicted in Figure 9, n i g r a l injections gave r i s e to retrograde l a b e l l i n g of neurons within both the striatum and globus p a l l i d u s . A marked topographical rela t i o n s h i p with the nigra was noted for both. In case SN-1 most l a b e l l e d s t r i a t a l and p a l l i d a l neurons were found in the core region of these structures. Caudolateral n i g r a l injections (SN-2) gave r i s e to lab e l l e d c e l l s along the l a t e r a l borders of both nuclei, more prominantly at caudal l e v e l s , and, with rostromedial placements, the reverse was true (SN-3; Figs.l8A and 18B). An injec t i o n into the ventral extreme of the pars r e t i c u l a t a l a b e l l e d neurons in the most dorsal parts of the striatum and globus pa l l i d u s (SN-4). An injec t i o n into the pars compacta, apparently devoid of s i g n i f i c a n t involvement of the pars r e t i c u l a t a (SN-6), v i r t u a l l y f a i l e d to label neurons in the striatum (only 10 or 20 were seen in t o t a l ; F i g . 19). Far fewer c e l l s were seen in the globus pa l l i d u s of SN-6 than in other case, and these were di s t r i b u t e d widely throughout the nucleus. Except for t h i s l a s t case, the density of labe l l e d p a l l i d a l neurons was very high in those regions containing them, leading to the impression that most p a l l i d a l neurons project to the substantia nigra. The p a l l i d a l c e l l s were generally triangular or spindle shaped and ranged from 81 Figure 17. Labelled c e l l bodies in the subthalamic nucleus in case SN-2. Note the absence of anterograde l a b e l l i n g of t h i s nucleus. Note also that the medial aspect of the nucleus (to-the l e f t of the f i e l d ) i s well l a b e l l e d despite the fact that the inje c t i o n s i t e placement in t h i s case was very l a t e r a l . Figure 18. Labelled neurons in the medial striatum (A) and globus pa l l i d u s (B) after the n i g r a l injection in case SN-3. This animal was coinjected with kainic acid which has abolished the anterograde l a b e l l i n g in contrast to that seen in the dorsal globus pa l l i d u s of case SN-5 in which a l i g h t anterograde l a b e l l i n g of globus p a l l i d u s was seen as well as the retrograde l a b e l l i n g of c e l l bodies (C). Bar = 100 um. 82 83 Figure 19. Labelling in the striatum (A) and globus pal l i d u s (B) after a n i g r a l i n j e c t i o n r e s t r i c t e d to the pars compacta (case SN-6). The large bright features seen within the striatum are arte f a c t u a l , no l a b e l l e d c e l l s were seen in thi s f i e l d , although many labe l l e d fibers can be seen within the s t r i a t a l neuropil. Three l a b e l l e d c e l l s are shown by arrowheads within the globus p a l l i d u s . Labelled fibers can be seen within this structure as well. Bar = 100 um. 84 85 19 um to 28 um in diameter (mean = 23.7 um, sem = 1.0 um, n 20). • This was s i g n i f i c a n t l y greater than that of l a b e l l e d s t r i a t a l c e l l s which ranged from 12 um to 21 um (mean = 15.6 um, sem = 0.6 um, n = 20). In addition, p a l l i d a l c e l l s were more intensely l a b e l l e d with reaction product (Fig. 18). Anterograde l a b e l l i n g of both the striatum and globus pallidus was noted in a l l cases except those in which kainic acid had been coinjected with the WGA-HRP (Figs. 18A and 18B). The heavy f i e l d of punctate reaction product, c h a r a c t e r i s t i c of lab e l l e d terminal f i e l d s , was less apparent in the globus p a l l i d u s . Rather, the anterograde l a b e l l i n g took the form of heavily l a b e l l e d single f i b e r s . It was noted that a pars compacta injec t i o n (SN-5) produced prominent anterograde l a b e l l i n g throughout the striatum and globus p a l l i d u s (Fig. 19). DISCUSSION The present data demonstrate that the substantia nigra in the rat receives afferents from the peribrachial area b i l a t e r a l l y and projects efferents to the i p s i l a t e r a l p e r i b r a c h i a l region. Complementary anterograde and retrograde data were obtained after both n i g r a l and peribr a c h i a l i n j e c t i o n s . This semireciprocal system has been reported previously in the cat (Moon-Edley and Graybiel, 1980; Nomura et a l . , 1980) and this report confirms the previous demonstration of an efferent projection to the i p s i l a t e r a l p eribrachial region in the rat (Beckstead et a l . , 1979). The correspondence with observations in the cat suggest that the d i s t r i b u t i o n of l a b e l l i n g seen within the peribrachial region represents l a b e l l i n g of the nucleus tegmenti 86 pedunculopontis pars compacta described in other species (Kim et a l . , 1976; Larsen and McBride, 1979; Nauta, 1979a). Results from the present study indicating that the projections of t h i s nucleus end predominantly in the substantia nigra pars compacta are in agreement with a study of the projections of the pedunculopontine tegmental nucleus in the rat (Saper and Loewy, 1982). The d i s t r i b u t i o n of c e l l s giving rise to t h i s projection i s similar to that of a diffuse group of neurons which stain intensely for acetylcholinesterase in the rat (Fibiger, 1982) and for choline acetyltransferase in the cat (Kimura et a l . , 1981). A cholinergic input to the dopaminergic c e l l s in the substantia nigra is suggested from biochemical and neuropharmacological studies (Lichtensteigler et a l . , 1982; James and Massey, 1978) and lesion studies have previously ruled out a forebrain source for such an innervation (McGeer et a l . , 1971). However, some electrophysiological studies have found neurons of the pars compacta to be insensitive to iontophoretically applied acetylcholine, and instead, point to the marked excitatory effect on pars r e t i c u l a t a neurons as in d i c a t i v e of the relevant post-synaptic elements (Collingridge and Davies, 1981; Pinnock and Dray, 1982). It has been suggested that central cholinergic neurons are generally large c e l l s (Fibiger, 1982) and therefore would more l i k e l y represent the less numerous c e l l type found in the pedunculopontine nucleus which appear to have a u n i l a t e r a l input to the substantia nigra. It i s interesting to note that, although the pedunculopontine nucleus projects predominantly . to the pars 87 compacta, i t receives afferents from the pars r e t i c u l a t a of the substantia nigra. A few c e l l s in the pars compacta have been noted to project to. this area by other authors (Jackson and Crossman, 1981b) but this may be attributed to the presence of some nondopaminergic c e l l s within the pars compacta (see Beckstead et a l . , 1979). The peribrachial region i s p a r t i c u l a r l y well connected with other components of the extrapyramidal system. It receives afferents from the i p s i l a t e r a l and possibly contralateral entopeduncular nucleus (Nauta, 1979a; Moon-Edley and Graybiel, 1980; Larsen and McBride, 1979) via c o l l a t e r a l s of the entopedunculothalamic pathway which arises from the "motor" portion of this nucleus (van der Kooy and Carter, 1981). A reciprocal relationship with the subthalamic nucleus has been suggested (Jackson and Crossman, 1981a). Additional inputs include those from the amygdala, hypothalamus and ventrocaudal regions of the striatum and globus pa l l i d u s (Jackson and Crossman, 1981b). As w i l l be discussed in Experiment 3 this l a t t e r projection probably r e f l e c t s afferents from nucleus basalis magnocellularis. Efferent projections from the pedunculopontine nucleus have been described to a l l of the areas from which i t receives afferents (Tohyama et a l . , 1978; Moon-Edley and Graybiel, 1980; Saper and Loewy, 1982). Some of the widespread connections presently attributed to t h i s nucleus may, in fact, arise from the nucleus parabrachialis, the c e l l bodies of which surround the brachium conjunctivum and correspond almost exactly to the d i s t r i b u t i o n of terminal l a b e l l i n g shown in 88 Figure 9G. This nucleus i s a relay for second order gustatory and v i s c e r a l afferents and has been shown to have afferents to the insular cortex, which others have attributed to the pedunculopontine nucleus (Shiply and Sanders, 1982), and projections to the amygdala and ventromedial nucleus of the thalamus (Voshart and van der Kooy, 1981). The present report indicates for the f i r s t time a t o t a l l y crossed input to the nigra from a discrete region of the posterior l a t e r a l hypothalamus. Additional significance is attached to this finding in l i g h t of the observation that these same hypothalamic neurons appear to project to areas of the co n t r a l a t e r a l mediodorsal thalamus which are in receipt of n i g r a l terminals. Previous studies have described n i g r a l afferents from the i p s i l a t e r a l l a t e r a l hypothalamus (Bunney and Aghajanian, 1976; Nauta and Domesick, 1978). In our material, when WGA-HRP injections were centered in the pars r e t i c u l a t a with spread to but not dorsal to the pars compacta, only two or three neurons were retrogradely lab e l l e d in the i p s i l a t e r a l l a t e r a l hypothalamus, at levels considerably more r o s t r a l than the co n t r a l a t e r a l hypothalamic neurons lab e l l e d after pars r e t i c u l a t a injections . More dorsally placed injections, including the area just dorsal to the pars compacta lab e l l e d a considerably larger number of these i p s i l a t e r a l anterior hypothalamic neurons. Previous studies have demonstrated the existence of n i g r a l input to the i p s i l a t e r a l parafascicular and ventromedial thalamic nuclei, and to the i p s i l a t e r a l and contralateral paralamellar portion of the mediodorsal thalamic nucleus (Faull and Carmen, 89 1968; Carpenter and Peter, 1972; Carpenter et a l . , 1976; Clavier et a l . , 1976; F a u l l and Mehler, 1978; Herkenham, 1979; Beckstead et a l . , 1979; Bentivoglio et a l . , 1979). The present data confirm and extend these reports with the demonstration of a minor n i g r a l afferent connection from the i p s i l a t e r a l parafasicular nucleus and a crossed n i g r a l input to the ventromedial thalamic nucleus. P r i t z e l and Huston (1980) have reported that u n i l a t e r a l n i g r a l lesions induce the contra l a t e r a l substantia nigra to send fibers to the ventromedial nucleus i p s i l a t e r a l to the les i o n . However, the present findings and those of Herkenham (1979), who reported an occasional la b e l l e d neuron in the cont r a l a t e r a l pars r e t i c u l a t a after HRP injections into the ventromedial nucleus, point to an existing crossed input to the ventromedial thalamus from the nigra in the unlesioned animal. Thus the report of an induction of this crossed system after u n i l a t e r a l n i g r a l lesions may in fact have been a demonstration of the expansion of an already existing projection. P a r t i c u l a r l y problematical among the present findings was the observation of terminal l a b e l l i n g in the laterodorsal thalamic nucleus after substantia nigra WGA-HRP inje c t i o n s . This n i g r a l connection has not been described previously. The laterodorsal thalamic nucleus has been reported to receive afferents from the pretectal nuclei, subicular and presubicular cortex, dorsal mesencephalic nucleus, and subcuneiform nucleus (Ryska and Heger, 1979; Robertson et a l . , 1980). The l a t t e r three areas are in close proximity to the ni g r a l WGA-HRP injection s i t e . In the present study, neurons in each of these 90 areas were labelled after laterodorsal nucleus WGA-HRP injections. Although such"injections also l a b e l l e d c e l l s in the pars r e t i c u l a t a , the p o s s i b i l i t y of minor d i f f u s i o n of the thalamic i n j e c t i o n s i t e into the adjacent mediodorsal nucleus precludes a d e f i n i t i v e i d e n t i f i c a t i o n of a n i g r a l efferent system to the laterodorsal nucleus. The connections of the thalamic nuclei that are in receipt of n i g r a l input include ascending projections to the s t r i a t a l and c o r t i c a l regions that in turn provide descending input to the substantia nigra (Akert et a l . , 1979; Gerfen and Clavier, 1979; Herkenham, 1979; Krettek and Price, 1979; Beckstead, 1979a; Bentivoglio et a l . , 1981; Experiment 1), the superior c o l l i c u l u s and p e r i b r a c h i a l areas that receive n i g r a l input. While s t r i a t a l afferents o r i g i n a t i n g in the mediodorsal and ventromedial thalamic nuclei may be considered l i g h t , the parafascicular nucleus provides a massive input to the striatum. This thalamic nucleus i s p a r t i c u l a r l y well connected with regions involved in motor function and, as well as projecting to the cortex and striatum, the parafascicular nucleus projects to the i n f e r i o r o l i v e (Walberg, 1981), thereby allowing i t to influence both the basal ganglia and cerebellar motor systems. Using electrophysiological techniques, Deniau et a l . (1977) demonstrated the existence of a crossed nigrotectal projection in the rat. Anatomical techniques have been used to demonstrate b i l a t e r a l innervation of the superior c o l l i c u l u s by efferents from the substantia nigra in the cat and monkey (Hopkins and Niessen, 1976; Rinvik et a l . , 1976; Beckstead et a l . , 1981). 91 However, previous HRP injections into the rat superior c o l l i c u l u s have been reported to result in the l a b e l l i n g of only a few (1 or 2) neurons in the cont r a l a t e r a l substantia nigra. These differences in d i s t r i b u t i o n have prompted some authors to suggest species differences in the basic function of the basal ganglia (Beckstead et a l . , 1981). The present data, using complementary WGA-HRP injections into the superior c o l l i c u l u s and substantia nigra indicate that the nigr o t e c t a l projection i s , in fact, b i l a t e r a l in the rat. The con t r a l a t e r a l n i g r a l projection to the superior c o l l i c u l u s appears to be less extensive, in terms of both i t s d i s t r i b u t i o n and the number of neurons contributing to the pathway, than i t s i p s i l a t e r a l .counterpart. However, the present l a b e l l i n g of over 200 neurons in 'the contralateral pars r e t i c u l a t a a f t e r c o l l i c u l a r WGA-HRP injections suggest that t h i s crossed pathway i s much more extensive than previously suspected. The d i s t r i b u t i o n of the crossed system, primarily into the l a t e r a l and ventral aspect of the r o s t r a l tectum, probably accounts for the f a i l u r e of previous investigations, using more s u p e r f i c i a l and caudal injections, to label t h i s pathway as extensively as was the case in the present study. Of further note is the suggestion of a topographic organization to the nigrotectal pathway; i.e. more dorsally located pars r e t i c u l a t a neurons projecting to the ro s t a l superior c o l l i c u l u s and more ventrally located nig r a l neurons projecting to the caudal superior c o l l i c u l u s . For the most part previous studies have i d e n t i f i e d only ventral n i g r a l neurons as contributing to the nigrotectal pathway (Faull and Mehler, 1978; Beckstead et a l . , 92 1981), consistent with the fact that these studies employed injections of retrograde tracer into the caudal superior c o l l i c u l u s . Some reports have parcelled the pars r e t i c u l a t a into separate regions which project to the superior c o l l i c u l u s or thalamus (Faull and Mehler, 1978), but recent double retrograde tracing and electrophysiological studies have shown that a large number of nigr a l neurons project to both areas (Bentivoglio et a l . , 1979; Anderson and Yoshida, 1980). In l i g h t of the present data i t is l i k e l y that most, i f not a l l n i g r a l neurons which innervate the thalamus send a c o l l a t e r a l to the tectum as well. A similar c o l l a t e r a l i z a t i o n has recently been proposed between the n i g r o c o l l i c u l a r and nigropedunculopontine pathways in the monkey (Beckstead and Frankfurter, 1982). This conclusion was arrived at by indirect means however, and following the same l i n e of reasoning would force the authors to conclude from their data that in the monkey there i s no c o l l a t e r a l i z a t i o n to the tectum and thalamus. Rather than point to species difference, t h i s i s l i k e l y a commentary on the l i m i t a t i o n s of the protocol. A point that has received l i t t l e attention in the past i s the remarkable s i m i l a r i t y between the projections of the pars r e t i c u l a t a and those of cerebellar efferents ascending in the brachium conjunctivum (said to exclude those a r i s i n g from the f a s t i g i a l nucleus; F a u l l and Carman, 1978). These include the superior c o l l i c u l u s , dorsal midbrain tegmentum, parafascicular nucleus and ventromedial thalamus. The thalamic f i e l d innervated by the f a s t i g i a l nucleus i t s e l f i s v i r t u a l l y confined to the 93 ventromedial nucleus (Haroian et a l . , 1981), and, therefore, completely overlaps with that portion of the nigrothalamic projection. These overlapping inputs, together with the divergent inputs from the parafascicular nucleus, may allow for the coordination of basal ganglia and cerebellar influences on motor control. The recognition that the subthalamic nucleus projects to the substantia nigra has been r e l a t i v e l y recent (Kanazawa et a l . , 1976). Although i t might be inferred from these authors' work that the subthalamic nucleus projects primarily to the pars compacta, in the present study the projection was determined to be predominantly to the pars r e t i c u l a t a . This p a r t i t i o n i n g agrees with a number of autoradiographic studies (Nauta and Cole, 1978; Carpenter et a l . , 1981). The subthalamic nucleus also projects to the nucleus pedunculopontinus, the globus pa l l i d u s and the entopeduncular nucleus (Nauta and Cole, 1978; Larsen and Suttin, 1978; Perkins and Stone, 1980; Jackson and Crossman, 1981). A l l of i t s connections to other basal ganglia nuclei are via c o l l a t e r a l s from the same neurons (van der Kooy and Hattori, 1980a; Deniau, 1978a). Although not apparent from the present material there is convincing histochemical and biochemical evidence for a dopaminergic innervation of the subthalamic nucleus (Brown et a l . , 1979; Meibach and Katzman, 1979). Its major input arises from the globus p a l l i d u s (see Experiment 3) and i t also receives afferents from the nucleus tegmenti pedunculopontis, a somatotopically organized input from the motor cortex and separate input from the prefrontal cortex 94 Figure 2 0 . Diagrammatic representation of the topographical relationship between s t r i a t o n i g r a l p a l l i d o n i g r a l and p a l l i d o s t r i a t a l projections. C o l l a t e r a l i z a t i o n i s not implied. c p 96 (Hartmann-von Monakow et a l . , 1978). A topographical projection of the striatum to the substantia nigra has been demonstrated previously by both autoradiographic and HRP techniques (Tulloch et a l . , 1978). The present data confirm t h i s observation and expand i t to include a similar topography for the p a l l i d o n i g r a l projection. In fact, the two d i s t r i b u t i o n s seem to be related to the p a l l i d o s t r i a t a l topography presented in Experiment 1. P a l l i d a l regions projecting to a r e s t r i c t e d region of the striatum also appear to project to the area of the substantia nigra to which that region of the striatum projects (see Figure 20). SUMMARY The substantia nigra pars r e t i c u l a t a receives a major topographically organized projection from the striatum and a topographically related projection from the globus p a l l i d u s . The pars compacta of the substantia nigra projects back to both of these structures, but anatomical evidence for any but a very low resolution feedback loop is lacking. Other major inputs to the substantia nigra include those from the subthalamic nucleus, prefrontal cortex and dorsal raphe. Moderate to l i g h t n i g r a l innervation arises from the central nucleus of the amygdala, peribrachial region, l a t e r a l habenula, parafascicular nucleus, l a t e r a l hypothalamus and an anatomically d i s t i n c t region of the cont r a l a t e r a l hypothalamus. The efferent connections of the substantia nigra are s i m i l a r l y quite divergent, with major innervation of the ventromedial and mediodorsal nuclei of the thalamus, the dorsal 97 F i g u r e 2 1 . Diagrammatic summary of the f i n d i n g s . 98 99 midbrain tegmentum, peribr a c h i a l region and superior c o l l i c u l u s a r i s i n g from nondopaminergic pars r e t i c u l a t a neurons and a major projection to the striatum a r i s i n g from the dopaminergic pars compacta neurons. Other nondopaminergic n i g r a l projections include those to the parafasicular and laterodorsal nuclei of the thalamus. Minor dopaminergic innervations of the globus p a l l i d u s and subthalamic nuclei have been described. These observations are summarized diagramatically in Figure 21 . TOO EXPERIMENT 3: EXAMINATION OF THE EFFERENT AND AFFERENT CONNECTIONS OF THE GLOBUS PALLIDUS INTRODUCTION Although the connections of the internal segment of the globus pallidus (entopeduncular nucleus in the rat) have been the subject of a great deal of anatomical research (see van der Kooy and Carter, 1981) there have been r e l a t i v e l y few studies conducted to examine the efferent and afferent connections of the external segment (globus pallidus in the r a t ) . Some authors have concluded that this nucleus projects fibers solely to the subthalamic nucleus (Kim et a l . , 1976); others state that i t innervates both the subthalamus and substantia nigra (Hattori et a l . , 1975; Carter and Fibiger, 1978) and s t i l l others describe possible additional connections to the pons, the neocortex and the r e t i c u l a r nucleus of the thalamus (Nauta, 1979a; McBride and Larsen, 1980). The d i f f i c u l t i e s represented by this lack of consensus l i e largely in the lack of s e n s i t i v i t y of past techniques in revealing diffuse projections and in the i n a b i l i t y to r e s t r i c t the injection s i t e to the nucleus of interest (McBride and Larsen, 1980; DeVito et a l . , 1980). Furthermore, to date there has been no systematic study of the afferents to the globus pallidus in the rat, although i t is known from anterograde studies to receive fibers from the subthalamic nucleus, substantia nigra, dorsal raphe and a massive innervation from the striatum. The present study reports the d i s t r i b u t i o n of the efferents and afferents of the globus pa l l i d u s revealed using injections of 101 WGA-HRP into this nucleus. Particular attention was paid to an examination of ascending efferents to the striatum predicted from the results of Experiment 1 . METHODS Rats were anesthetized with pentobarbital and the sk u l l trephaned for either a v e r t i c a l or angled approach to the globus p a l l i d u s . WGA-HRP was injected s t e r e o t a x i c a l l y through a 5 ul syringe. Injections of 0.1 ul of 2% WGA-HRP were made over a 5 minute period and the cannula l e f t in place for a further 5 minutes. After a survival time of from 24 to 48 hrs, animals were reanesthetized and fixed by transcardial perfusion as described in Experiment 1. Tetramethylbenzidene was used as a peroxidase substrate for the reaction of s l i d e mounted or fre e - f l o a t i n g sections as described in Experiment 1. In some cases, f r e e - f l o a t i n g sections were reacted for peroxidase with diaminobenzidine as substrate (Graham and Karnovsky, 1966), as detailed in Experiment 2. RESULTS Four cases involving d i f f e r e n t p a l l i d a l injections were examined and related to the inject i o n locus. The majority of the results presented pertain to the large p a l l i d a l injection (Case GP-1) depicted in Figure 22. The WGA-HRP deposition in the cortex in thi s animal was negligible and the majority of the la b e l l i n g along the cannula tract was due to an accumulation of erythrocytes. The d i s t r i b u t i o n of l a b e l l i n g seen in thi s case i s depicted in Figure 23. A second case (GP-2) occupied the same coordinates but was much smaller. In case GP-3 a large injection 102 Figure 22. Photomicrograph of a counterstained section showing WGA-HRP inject i o n into the globus p a l l i d u s in case GP-1. Peroxidase reaction using diaminobenzidine substrate. 103 1 0 4 Figure 23. Line drawings depicting the anterograde and retrograde l a b e l l i n g in case GP-1. Retrogradely lab e l l e d c e l l s are denoted by f i l l e d c i r c l e s . Anterogradely lab e l l e d f i b e r s and terminals are drawn i n . Abbreviations used in th i s and subsequent figures; AC, anterior commissure; BC, brachium conjuntivum; cp, striatum; CP, cerebral peduncle; dr, dorsal raphe nucleus; ep, entopeduncular nucleus; gp, globus p a l l i d u s ; F, fornix; FM, forceps minor; FR, fasciculus retroflexus; IC, internal capsule; h, habenular complex; md, mediodorsal nucleus of the thalamus; ML, medial lemniscus; MT, mammillothalamic t r a c t ; ntp, nucleus tegmenti pedunculopontis; pf, parafascicular nucleus; r r , retrorubral area; r t , r e t i c u l a r nucleus of the thalamus; snc, substantia nigra pars compacta; snr, substantia nigra pars r e t i c u l a t a ; sut, subthalamic nucleus; v l , ventrolateral nucleus of the thalamus; vm, ventromedial nucleus of the thalamus. 105 106 s i t e spread throughout the caudal globus p a l l i d u s , internal capsule and anterior extreme of the entopeduncular nucleus and the injection s i t e of GP-4 involved the caudal globus p a l l i d u s , i n t e r n a l capsule, entopeduncular nucleus and part of the r e t i c u l a r nucleus of the thalamus. EFFERENT PROJECTIONS The results of the anterograde transport are largely in agreement with previous work (Carter and Fibiger, 1978). By far the heaviest l a b e l l i n g was seen in the subthalamic nucleus. The substantia nigra and entopeduncular nucleus showed moderate anterograde l a b e l l i n g , and l i g h t l a b e l l i n g of the r e t i c u l a r and mediodorsal nuclei of the thalamus were seen. A l l cases gave ri s e to retrograde l a b e l l i n g of c e l l s in the striatum, within which moderate levels of fine extraperikaryal reaction product were also seen, indicative of a sparse terminal f i e l d l a b e l l e d by anterograde transport (Fig. 23). In cases GP-1 and GP-2 a f i e l d of faint terminal l a b e l l i n g was seen throughout layers III to V of the dorsal cortex anterior to the le v e l of the globus p a l l i d u s (Fig. 24A). This feature was far more prominent when the p a l l i d a l injection s i t e s were centered caudally and medially in the nucleus (GP-3 and GP-4). In these cases, fibers could be seen very c l e a r l y throughout large areas of the neocortex. C o r t i c a l fibers were observed running along the dorsal surface of the corpus callosum in layer V i , radiating out through the c o r t i c a l layers at right angles to the c o r t i c a l surface and branching out p a r a l l e l to the c o r t i c a l surface in a l l c o r t i c a l layers (Fig. 24B). In animals showing 107 Figure 24. (A) Sparse anterograde l a b e l l i n g in the cortex of GP-1. A l l of the larger bright features are red blood c e l l s . (B) Anterograde l a b e l l i n g in the cortex in case GP-3. Note that in t h i s case heavily la b e l l e d fibers can be seen. The f i e l d depicted shows layers VI (at bottom) through I I I . (C) Heavily l a b e l l e d fibers can be seen running through the striatum in case CP-3. Some anterograde and retrograde l a b e l l i n g of striatum can be seen as well, but this f i e l d does not portray the main s i t e of s t r i a t o p a l l i d a l interconnections. Bar = 100 um. 108 109 heavy and d i s t i n c t c o r t i c a l l a b e l l i n g , heavily l a b e l l e d fibers were seen projecting through the striatum (Fig. 24C) and traversing the corpus callosum to branch within the cortex. The ch a r a c t e r i s t i c s of labelled fibers found in the cortex in these rats corresponds closely to a recent description given to the c o r t i c a l projection of the nucleus basalis magnocellularis (Fibiger, 1982). In case GP-1 the head of the striatum contained a f i e l d of terminal l a b e l l i n g in and around the area that contained retrogradely la b e l l e d c e l l bodies (Fig. 25). No heavily l a b e l l e d fibers were seen traversing the striatum in th i s case; such features appeared only in animals with dense l a b e l l i n g of the afferents to the cortex. The reaction product within the s t r i a t a l neuropil in case GP-1 was much more dense than that found in experiments in which anterograde transport to the CP had been blocked by the coinjection of kainic acid with the WGA-HRP (Fig. 25). In case GP-1, neither retrograde nor anterograde l a b e l l i n g was seen in the t a i l of the striatum, but both were seen in th i s area in cases GP-3 and GP-4. Figure 26A depicts the l a b e l l i n g seen in the r e t i c u l a r nucleus in case GP-1. This was a consistant feature of a l l cases examined. The l i g h t l a b e l l i n g followed the contours of th i s nucleus throughout i t s rostrocaudal extent. Wide areas of the ventrolateral thalamus also contained a punctate reaction product somewhat above background l e v e l s , possibly indicating a very sparse innervation of this region (Fig. 23 and 26A). A very r e s t r i c t e d f i e l d of moderately intense terminal l a b e l l i n g was no Figure 25. (A) Light f i e l d photomicrograph of the striatum in case GP-1. Retrogradely l a b e l l e d neurons were found within f i e l d s of diffuse neuropil staining thought to represent peroxidase a c t i v i t y transported into p a l l i d o s t r i a t a l f i b e r s and terminals. A similar d i s t r i b u t i o n of l a b e l l i n g within the striatum i s found after injections into the substantia nigra (B) but note that after blocking anterograde transport by coinjection of kainic acid into the substantia nigra the diffuse l a b e l l i n g of the neuropil i s greatly reduced (C). Bar = 20 um. 112 Figure 26. (A) Anterograde l a b e l l i n g of the r e t i c u l a r nucleus of the thalamus in case GP-1. The dark region at the far right i s the internal capsule, and the ventrolateral thalamus occupies the l e f t t h i r d of the f i e l d . (B) Anterograde l a b e l l i n g in the mediodorsal nucleus of the thalamus in case GP-1. Note the r e s t r i c t e d terminal f i e l d and i t s r e t i c u l a r appearance. The l a t e r a l habenula can be seen at the top of the f i e l d . (C) Anterograde l a b e l l i n g in the entopeduncular nucleus in case GP-1. Some of the positiv e f i b e r s in this f i e l d are within internal capsule fiber bundles but many are within the neuropil. The r e t i c u l a r appearance of the l a b e l l i n g within this nucleus contrasts markedly to that seen after s t r i a t a l i n j e c t i o n s . The arrows denote heavy l a b e l l i n g outlining neurons in the entopeduncular nucleus; these c e l l s are not retrogradely l a b e l l e d but rather appear to receive a dense innervation around the region of their c e l l body. Bar = 100 um. 1 1 3 114 seen within the mediodorsal nucleus of the thalamus (Fig. 26B). A remarkable feature of the l a b e l l i n g in t h i s region was i t s irregular or r e t i c u l a r appearance rather than the diffuse f i e l d seen after the l a b e l l i n g of s t r i a t a l efferents (see Experiment 1). A similar pattern of terminal l a b e l l i n g was seen in the other cases except that GP-4 showed dense termination in the medial part of the ventrolateral nucleus and in the l a t e r a l habenular nucleus and the zona incerta, undoubtedly r e f l e c t i n g the involvement of the entopeduncular nucleus in the injection s i t e (Carter and Fibiger, 1978). In two cases (GP-1 and GP-2), the WGA-HRP inject i o n s i t e was confined to the globus p a l l i d u s with no spread to the entopeduncular nucleus. In both of these cases, anterograde l a b e l l i n g was apparent within the neuropil of the entopeduncular nucleus which did not appear to derive merely from lab e l l e d projection fibers passing through t h i s nucleus. This putative terminal f i e l d had a very irregular appearance, and in some instances, seemed to outline the c e l l bodies of neurons in the entopeduncular nucleus (Fig. 26B). By far the heaviest terminal f i e l d in case GP-1 was seen in the subthalamic nucleus (Fig. 27). A few retrogradely la b e l l e d c e l l bodies could be seen within t h i s nucleus as well (Fig. 27B). In case GP-4 terminal l a b e l l i n g was far less dense while the number of c e l l bodies l a b e l l e d was much greater (Fig. 27C). A f a i n t , d i f f u s e terminal f i e l d was seen in the parafascicular nucleus in a l l cases (Fig. 29A). This f i e l d corresponded with the d i s t r i b u t i o n of labe l l e d c e l l bodies that 115 Figure 27. (A) Anterograde l a b e l l i n g of the subthalamic nucleus and i t s intrusion into the cerebral peduncle. (B) Higher power bright f i e l d photomicrograph of the subthalamic nucleus in case GP-1 showing the presence of retrogradely l a b e l l e d c e l l bodies (arrows). (C) Bright f i e l d photomicrograph of the subthalamic nucleus in case GP-4. Note that in th i s case the anterograde l a b e l l i n g of the nucleus is much more sparse. Bar = 50 um. 1 1 6 1 1 7 Figure 28. (A) Substantia nigra of GP-1. Note the presence of l a b e l l e d c e l l bodies in pars compacta and anterograde l a b e l l i n g in pars compacta and pars r e t i c u l a t a . (B) At a more caudal l e v e l in the same case, a l l of the anterograde l a b e l l i n g i s seen in pars r e t i c u l a t a . (C) The appearance of the anterograde l a b e l l i n g in the pars r e t i c u l a t a in case GP-3 in d a r k f i e l d and ( D ) ' l i g h t f i e l d after counterstaining with c r e s y l v i o l e t . The features suggestive of dense termination around c e l l bodies are denoted by arrows in (C) and by asterisks in the corresponding f i e l d in (D). Bar = 100 um. 119 were sometimes found in t h i s region (see below). The terminal f i e l d in t h i s region appeared to be a caudal continuum of the l a b e l l i n g seen in the mediodorsal nucleus of the thalamus but i t did not share the same irregular appearance. Within the substantia nigra the predominant terminal l a b e l l i n g was found in the pars r e t i c u l a t a . Some anterograde l a b e l l i n g of the pars compacta was seen at r o s t r a l levels (Fig. 28A) but more caudally, the l a b e l l i n g within t h i s d i v i s i o n was almost' exclusively within c e l l bodies (Fig. 28B). The terminal l a b e l l i n g in the substantia nigra was very d i f f e r e n t from that seen after s t r i a t a l injections (see Expt. 1), but similar to the l a b e l l i n g of the entopeduncular nucleus. It seemed that l a b e l l e d elements had d i s t i n c t targets within the neuropil of the pars r e t i c u l a t a . The l a b e l l i n g was very r e t i c u l a r and uneven, some lab e l l e d elements seemed to form bundles and others were concentrated around perikarya and proximal dendrites of unlabelled neurons within the pars r e t i c u l a t a (Fig. 28C and 28D) and pars l a t e r a l i s . Although d i f f i c u l t to ascertain, there did not appear to be a similar concentration of lab e l l e d f i b e r s around the retrogradely la b e l l e d neurons. Anterograde l a b e l l i n g caudal to the substantia nigra was found only in case GP-4 in which a moderately dense terminal f i e l d was seen in the region of the brachium conjunctivum on the side i p s i l a t e r a l to the i n j e c t i o n . AFFERENT PROJECTIONS A few f a i n t l y staining neurons were found in the prefrontal cortex after a l l p a l l i d a l injections (Fig. 29B). No c o r t i c a l 120 Figure 29. (A) Very f a i n t l y l a b e l l e d c o r t i c a l c e l l s (arrows) in case GP-2. (B) Anterograde l a b e l l i n g of the striatum (right) and globus p a l l i d u s ( l e f t ) after a WGA-HRP injecti o n into the frontal cortex. Heavily l a b e l l e d fiber bundles (presumably corticofugal) were seen within the globus p a l l i d u s but are outside the f i e l d of this photomicrograph. (C) Retrogradely labelled c e l l in the ventromedial nucleus of the thalamus in case GP-1. (D) Weakly l a b e l l e d c e l l s (arrows) and fibers (asterisk) in the parafasicular nucleus in case GP-1. (E) Retrograde l a b e l l i n g of c e l l bodies in the dorsal raphe nucleus of case GP-1. Bar = 50 um. 121 122 c e l l s were found caudal to the r o s t r a l t i p of the striatum. WGA-HRP injections into the prefrontal cortex led to a dense and diffuse l a b e l l i n g of the striatum but also revealed a l i g h t l a b e l l i n g of the globus pa l l i d u s (Fig. 29C). Many neurons in the striatum were l a b e l l e d . A few l i g h t l y stained c e l l bodies were seen in the ventrolateral, ventromedial, parafascicular and intralaminar nuclei of the thalamus (Fig. 23D to 23F and F i g . 29) of case GP-1. Comparison with other cases confirmed the observations in the ventral thalamus but not those found in the intralaminar group. A few la b e l l e d neurons were found in the caudal extreme of the entopeduncular nucleus but i t was f e l t that these c e l l s may have been part of the r o s t r a l subthalamic nucleus in which lab e l l e d neurons were numerous. Within the substantia nigra l a b e l l e d c e l l bodies were found mainly in the pars compacta. At more caudal levels c e l l s were seen within the pars r e t i c u l a t a and pars l a t e r a l i s as well. In the caudal extreme of th i s nucleus, c e l l s formed a continuum with la b e l l e d c e l l s in the retrorubral area (Fig. 23H). This d i s t r i b u t i o n was similar in a l l cases. The dorsal raphe nucleus contained a large number of la b e l l e d c e l l bodies (Fig. 23H, F i g . 29D) . The region around the brachium conjunctivum was examined c a r e f u l l y in a l l cases but no la b e l l e d c e l l s could be found in cases GP-1 or GP-2. One neuron was located in th i s region in case GP-3 and in GP-4 d i s t i n c t groups of la b e l l e d c e l l s were found both i p s i l a t e r a l and contralateral to the in j e c t i o n corresponding to the nucleus tegmenti pedunculopontis. 1 2 3 DISCUSSION The greater morphological d e t a i l provided by the use of anterograde peroxidase compared with the autoradiographic technique (due to the l o c a l i z a t i o n of p r e c i p i t a t e within the tissue rather than in an overlying emulsion) proved to be of great value in the present study. The d i s t r i b u t i o n of p a l l i d a l efferents depicted in Figure 23 i s in good agreement with the results obtained by Carter and Fibiger (1978). However, whereas they were unable to conclude from their data that the globus pal l i d u s innervates the r e t i c u l a r nucleus of the thalamus or the entopeduncular nucleus, the present data indicate both connections. The l a b e l l i n g of these structures was beyond the d i f f u s i o n boundaries of the i n j e c t i o n s i t e and d i f f e r e d markedly in appearance from that which would be attributed to d i f f u s i o n of tracer. Previous autoradiographic (Hattori et a l . , 1975) and retrograde HRP studies (DeVito et a l . , 1980) have given indications of a p a l l i d a l projection to the entopeduncular nucleus (or i t s counterpart in the primate). The previous l i t e r a t u r e on a projection to the r e t i c u l a r nucleus of the thalamus i s controversial (Hattori et a l . , 1975, McBride and Larsen, 1980). The r e t i c u l a r nucleus of the thalamus has not been previously thought to be related to the basal ganglia, but i t does receive c o r t i c a l input from the same region which projects to the substantia nigra (Gerfen and Clavier, 1979; Reep and Winans, 1982) and receives c o l l a t e r a l s of the thalamostriatal projection o r i g i n a t i n g from the intralaminar c e l l groups (Nguyen-Legros et a l . , 1982). 1 2 4 A very sparse anterograde l a b e l l i n g of parts of the ventral thalamus and mediodorsal thalamus has been 'detected previously (Carter and Fibiger, 1978) but was not regarded as p a l l i d a l in o r i g i n . In agreement with these studies, injections of retrograde fluorescent tracers into the thalamus f a i l e d to label neurons in the GP of the cat (Parent and de B e l i e f e u i l i e , 1982) or the rat (van der Kooy and Carter, 1981). On the other hand, Herkenham (1979) did observe neurons in the ventral globus pa l l i d u s after HRP injections into the ventromedial nucleus of the thalamus and raised the p o s s i b i l i t y that neurons of the nucleus basalis magnocellularis (see Fibiger, 1982) rather than p a l l i d a l neurons may project to the thalamus. Similar conclusions have been reached regarding the innervation of the mediodorsal nucleus of the thalamus (Sapawi and Divak, 1978; Gerfen et a l . , 1982). However, i t has been suggested that the term nucleus basalis magnocellularis be reserved for neurons staining intensely for acetylcholinesterase (Lehmann et a l . , 1980), and those neurons within the globus pallidus that project to the mediodorsal nucleus of the thalamus do not appear to contain high lev e l s of t h i s enzyme (Hardy et a l . , 1976). As most of the c e l l s in t h i s region which project to the thalamus are l o c a l i z e d ventral to the globus pa l l i d u s (Carter and Fibiger, 1978, Gerfen et a l . , 1982), i t seems l i k e l y that the neurons found within the boundaries of the globus p a l l i d u s represent outlying members of a second (noncholinergic) population of basal forebrain neurons. Controversy also surrounds the innervation of the substantia 125 nigra by the globus p a l l i d u s . Early reports of a projection to the substantia nigra indicated that the majority of the innervation was confined to the pars compacta (Hattori et a l . , 1975). More recent work has suggested that the globus p a l l i d u s innervates the pars r e t i c u l a t a as well (Carter and Fibiger, 1978) or exclusively (Grofova, 1975; McBride and Larsen 1980). The present data are consistent with a major innervation of the pars r e t i c u l a t a and, furthermore, indicate an innervation of the soma and proximal dendrites of neurons within this area. This is in agreement with morphological studies showing a large number of axosomatic terminals of p a l l i d a l o r i g i n in the substantia nigra (Hattori et a l . , 1975). The observation of presumed anterograde l a b e l l i n g in * the striatum in and around the regions containing retrogradely la b e l l e d neurons lends support to evidence for a p a l l i d a l projection to the caudate-putamen in the rat (see Experiment 1). Previously, similar observations were attributed to d i f f u s i o n of tracer or the l a b e l l i n g of the c o r t i c a l projection a r i s i n g from neurons of the nucleus basalis magnocellularis, some of which occupy p a l l i d a l or p e r i p a l l i d a l regions (Carter and Fibiger, 1978; Nauta, 1979a; McBride and Larsen, 1980). This phenomenon was shown to occur here with caudal p a l l i d a l injections of tracer. However, the anterograde l a b e l l i n g of the cortex in case GP-1 was n e g l i g i b l e compared to that seen within the striatum. The present data therefore add support to the proposal that a p a l l i d o s t r i a t a l projection exists and indicates that i t may have a reciprocal topographical relationship to the s t r i a t o p a l l i d a l 126 projection. Conclusive evidence for a p a l l i d o s t r i a t a l projection awaits the demonstration of l a b e l l e d terminals on p a l l i d o s t r i a t a l f i bers (see below). It has been claimed on the basis of retrograde transport studies that the globus p a l l i d u s , l i k e the entopeduncular nucleus, projects to the nucleus tegmenti pedunculopontis (Jackson and Crossman, 1981b). Like others (Carter and Fibiger, 1978; Nauta, 1979a; McBride and Larsen, 1980), the present experiments did not provide any indication of t h i s . The d i s t r i b u t i o n of neurons portrayed by Jackson and Crossman (1981b) is very similar to that of the histochemical designation of the nucleus basalis magnocellularis (Fibiger, 1982). A caudally directed projection from t h i s c e l l group to the hindbrain has been indicated (Divak, 1975), and i t may be these neurons, rather than those of the globus p a l l i d u s , that project to the nucleus tegmenti pedunculopontis or adjacent regions. There i s now l i t t l e doubt of a dopaminergic input, a l b e i t l i g h t , to the globus p a l l i d u s from the substantia nigra (Lindvall and Bjorklund, 1979; DeVito et a l . , 1980). P a l l i d a l inputs from the subthalamic nucleus, dorsal raphe, and striatum also have a large l i t e r a t u r e (Nauta and Cole, 1978; van der Kooy and Hattori, 1980a; Ricardo, 1980; Carpenter et a l . , 1981; Nagy et a l . , 1978a; DeVito et a l , 1980; Brann and Emson, 1980; Parent et a l . , 1981b; Pasik et a l . , 1981). However, no previous report of a c o r t i c a l projection to the globus p a l l i d u s has appeared. Diagrammatic representations of the data in a recent paper (Reep and Winans, 1982) appear to provide support for a minor input to the globus 127 Figure 30. Schematic diagram summarizing the efferent and afferent connections of the globus p a l l i d u s . 128 129 p a l l i d u s . This c o r t i c a l region also projects to the substantia nigra and the r e t i c u l a r nucleus of the thalamus (Gerfen and Clavier, 1979; Reep and Winans, 1982). Mutually confirmatory anterograde and retrograde demonstrations of this projection were obtained in the present study but both indicated that i t i s minor. Some previous indications of a thalamic projection to the globus p a l l i d u s can be c i t e d in support of the observations presented here (Herkenham, 1979; DeVito et a l . , 1980), but for the present these data should be taken only as encouragement for further study. SUMMARY In summary, the present results confirm a massive projection from the globus pallidus to the subthalamic nucleus and a more modest innervation of the pars r e t i c u l a t a of the substantia nigra. Additional efferents of the globus pallidus include those to the striatum, entopeduncular nucleus and the r e t i c u l a r and parafascicular nuclei of the thalamus. Major inputs to the globus p a l l i d u s arise from the striatum, subthalamic nucleus, dorsal raphe nucleus and substantia nigra. Evidence was obtained for minor inputs to the globus p a l l i d u s from the prefrontal cortex, the parafascicular nucleus and the ventrolateral and ventromedial nuclei of the thalamus. These observations are summarized schematically in Figure 30. 130 EXPERIMENT 4: LIGHT AND ELECTRON MICROSCOPIC DEMONSTRATION OF A PROJECTION FROM THE GLOBUS PALLIDUS TO' THE STRIATUM IN THE RAT INTRODUCTION Evidence obtained from retrograde transport studies on the connections of the striatum, described in Experiment 1, indicated that this nucleus receives a projection from the globus pallidus (GP). This finding is quite s t a r t l i n g both in terms of the implications i t has to present concepts of basal ganglia function and the fact that such a connection has gone previously undetected in a system that has been subjected to such intense neuroanatomical research. Previous studies examining the efferents of the globus pallidus (McBride and Larsen, 1980; Kim et a l . , 1976; Carter and Fibiger, 1978; Carpenter et a l . , 1981) or the afferents of the striatum (Royce, 1978; Oka, 1980; Veening et a l . , 1980; Royce, 1982) have made no mention of this projection. The following experiments were designed to investigate further the p o s s i b i l i t y that the globus p a l l i d u s does indeed project to the striatum, and to gather anatomical data about th i s projection which might serve as a basis for speculation concerning the role of t h i s pathway in the functional organization of the basal ganglia. In the present study, the phenomenon of retrograde l a b e l l i n g of p a l l i d a l neurons aft e r injection of WGA-HRP into the striatum was examined in d e t a i l to (1) confirm that the apparent l a b e l l i n g of these neurons was due to i n t r a c e l l u l a r accumulation of peroxidase, (2) rule out the p o s s i b i l i t y that neurons were 131 l a b e l l e d trans-synaptically, and (3) investigate the relationship between the p a l l i d o s t r i a t a l and s t r i a t o p a l l i d a l pathways. Some of the observations reported in this experiment have been previously published (Staines et a l . , 1981). METHODS Male Wistar rats (Woodlyn Farms, Guelph, Ontario) were anesthetised with pentobarbital and injected s t e r e o t a x i c a l l y with 0.05 to 0.10 ul of 0.25 to 1.0% solutions of WGA-HRP in saline or s t e r i l e saline containing 10 mM kainic acid (KA, Sigma). WGA-HRP was synthesized as described previously (Staines et a l . , 1980b). Four to fourty-eight hours after the injection the animals were anesthetized with pentobarbital and perfused t r a n s c a r d i a l l y with 100 mM sodium phosphate buffer (PB) containing 0.9% NaCl, followed by a 1/2 hr perfusion with glutaraldehyde and formaldehyde (1%:1%) in PB at room temperature. The aldehydes were washed out by perfusion with ice cold 10% sucrose in PB and brains were stored 3 hrs in thi s solution before cutting. Sections 50 um thick were cut on a freezing microtome and processed for HRP by the tetramethylbenzidine (TMB) method (Mesulam 1978; Experiment 1). Alternate sections were counterstained with 1% neutral red (NR). Some unreacted sections at the lev e l of the injection s i t e were stained with c r e s y l v i o l e t for h i s t o l o g i c a l examination. Measurements of the dimensions of lab e l l e d neurons were made using a grid eyepiece to observe sections which had been reacted for peroxidase a c t i v i t y using diaminobenzidine (DAB) as substrate (see Experiment 2) and counterstained with cresyl v i o l e t . 132 For electron microscopy, animals were perfused as above except that the aldehyde mixture was 1% formaldehyde and 2% glutaraldehyde. Sections were cut at 50 um on an Oxford vibratome and alternate sections were reacted for HRP using TMB or DAB. TMB reacted sections were examined to insure that the injecti o n s i t e s were confined to the head of the striatum. DAB reacted sections were treated with 1% osmium tetroxide, dehydrated, and f l a t embedded in Epon as described by Wilson and Groves (1979). Labelled p a l l i d a l neurons were located under the li g h t microscope and dissected out of the Epon-embedded material. The resultant block was mounted, and pale gold sections cut for examination on a P h i l i p s model 201 electron microscope without prior heavy metal counterstaining. RESULTS In the case i l l u s t r a t e d in Figure 31a, both the WGA-HRP and WGA-HRP-KA injections were confined to the head of the striatum. As shown in Figure 31b, in neither i n j e c t i o n did the WGA-HRP diffu s e as far caudally as the GP. At the caudal pole of the WGA-HRP injection s i t e , reaction product could be seen in fi b r e bundles in the ventromedial aspect of the striatum. The appearance of th i s reaction product suggested that i t was lo c a l i z e d to fibers but these fibers d i f f e r e d markedly in appearance from those l a b e l l e d by uptake into damaged axons. Axons la b e l l e d through damage most often appear to be stained heavily in a G o l g i - l i k e manner. The fiber bundles in thi s instance however showed a f a i n t , discontinuous reaction deposit arranged in linear array. Within the GP, reaction product was 133 Figure 31. (a) B i l a t e r a l injection of WGA-HRP (50 n l , 0.25%) into the striatum. The solution injected on the l e f t also contained 10 mM KA (WGA-HRP-KA). Survival time 24 hrs. Reacted with tetramethylbenzidine (TMB), counterstained with neutral red (NR). (b) Section caudal to (a) showing that the injection s i t e s have not spread to the le v e l of the anterior commissure. Reacted with TMB, counterstained with NR. (c) Section from anterior GP i p s i l a t e r a l to the WGA-HRP injecti o n ((a) right) showing retrogradely lab e l l e d c e l l body (arrow) surrounded by anterograde reaction product. Reacted with TMB, uncounterstained. Bar = 50 um. (d) Same section as (c) but showing c e l l s in the GP i p s i l a t e r a l to the WGA-HRP-KA injection ((a) l e f t ) . Note that the anterograde reaction product i s absent. Reacted with TMB, uncounterstained. Bar = 50 um. (e) Epon-embedded section through the GP of a rat receiving an injection of WGA-HRP (100 n l , 1.0%) into the i p s i l a t e r a l striatum. Reacted with diaminobenzidine (DAB). The arrow denotes the area of GP taken for electron microscopy. (f) Electron micrograph of a p a l l i d a l neuron showing multivescicular bodies stained with DAB. S e r i a l sections revealed the nucleus of thi s neuron to be deeply indented. Uncounterstained. Bar = 2 um. 1 34 135 seen in the p a l l i d a l neuropil between the capsular bundles. As shown in Figure 31c, in addition to thi s anterograde label, the anterior GP contained what appeared to be large retrogradely la b e l l e d neurons (mean maximum diameter = 22.1um, sem = 1.1um, n = 10). The electron micrograph (Fig. 31f) taken from the p a l l i d a l section seen in Figure 31e c l e a r l y shows that GP perikarya contain WGA-HRP reaction product within multivesicular bodies, as i s c h a r a c t e r i s t i c for neurons retrogradely labelled in conventional HRP experiments (Somogyi et a l . , 1979). Results from a limited number of electron microscopic p r o f i l e s that were obtained from s e r i a l sections revealed that a l l l a b e l l e d neurons have deeply indented nuclei, sparse axosomatic connections, and are 10 um by 20 to 30 um in size . Coinjection of WGA-HRP or HRP with KA has already been shown to abolish anterograde l a b e l l i n g of the s t r i a t o n i g r a l pathway while sparing the retrograde l a b e l l i n g of n i g r o s t r i a t a l neurons (Staines et a l . , 1980b). Results consistent with this were obtained in the present study. After s t r i a t a l injections of WGA-HRP together wj.th KA, no reaction product was seen in fibre bundles caudal to the inject i o n s i t e or in the neuropil of the GP. However, l a b e l l e d p a l l i d a l c e l l bodies could s t i l l be c l e a r l y seen (Fig. 3ld). This feature was u t i l i z e d to map the di s t r i b u t i o n of p a l l i d a l neurons projecting to the head of the striatum (Fig. 32). The presence of anterograde label often makes i t d i f f i c u l t to v i s u a l i z e retrogradely l a b e l l e d c e l l s . In the case depicted in Figure 32, the coinjection of KA with WGA-HRP abolished 136 Figure 32. Line drawings depicting the d i s t r i b u t i o n of l a b e l l i n g a f t e r s t r i a t a l injections of 100 nl of 2% WGA-HRP with ( l e f t ) and without (right) 10 nM kainic acid in the injection buffer. Labelled neurons are represented by f i l l e d c i r c l e s . Areas of terminal l a b e l l i n g are shown with a fine s t i p p l i n g and projection fibers are drawn i n . Abbreviations in this and subsequent figures: cp, striatum; ep, entopeduncular nucleus; gp, globus p a l l i d u s ; IC, internal capsule ICB, bundles of internal capsule f i b e r s ; snc, substantia nigra pars compacta; snr, substantia nigra pars r e t i c u l a t a . 1 37 138 anterograde transport of tracer from the injection s i t e , as seen by the absence of labelled fibers emanating in a caudal d i r e c t i o n from the inj e c t i o n s i t e and the absence of terminal l a b e l l i n g in the GP, EP or SN (Fig. 33). Sections from the same animal stained for cr e s y l v i o l e t revealed early degenerative changes on the KA injected side that involved an area of striatum only s l i g h t l y larger than the extent of the peroxidase inje c t i o n s i t e . I l l u s t r a t e d in Figure 32 i s the d i s t r i b u t i o n of l a b e l l e d neurons in the presence and absence of labelled s t r i a t o p a l l i d a l terminals. The number and d i s t r i b u t i o n of these c e l l s on either side of the brain is very similar and the s l i g h t differences e a s i l y a t t r i b u t a b l e to differences in the two inj e c t i o n s i t e s . Neurons within the whole rostrocaudal extent of the GP project to the head of the striatum but as shown in Figure 34, there i s an obvious r e s t r i c t i o n to their mediolateral d i s t r i b u t i o n . At r o s t r a l l e v e l s both l a b e l l e d p a l l i d a l neurons and labe l l e d afferent terminals occupy an extreme l a t e r a l position within the GP. At more caudal levels both p o s i t i v e soma and terminals are seen in a much more medial position. Rather than a progression in the medial dir e c t i o n there appears to be discontinuity in the d i s t r i b u t i o n of labelled c e l l s and terminals. From the examination of a large number of cases in both coronal and horizontal section i t seems that two c e l l groups are la b e l l e d within the GP after injections into the head of the striatum. One t i e r of c e l l s extends from the r o s t r a l pole of the GP, through the l a t e r a l GP in r o s t r a l sections and ends in a s l i g h t l y medial position at midpallidal l e v e l s . Another begins at 139 Figure 33. Darkfield photomicrographs of the l a b e l l i n g seen in the GP (A and B), EP (C and D) and SN (E and F) after the coinjection of KA and WGA-HRP (A, C and E) or WGA-HRP by i t s e l f (B, D and F) into the head of the striatum. Note that there i s no terminal l a b e l l i n g in the GP, EP or SN on the kainic acid injected side (A, C and E) although retrogradely l a b e l l e d c e l l s are seen in the GP and SN (A and E). Bar = 100 um. 140 141 Figure 3 4 . Detailed topography of anterograde (stippling) and retrograde l a b e l l i n g ( f i l l e d c i r c l e s ) of the globus pa l l i d u s after a s t r i a t a l injection of 100 nl of 2% WGA-HRP into the head of the striatum. Each c e l l recognized i s plotted out in projection drawings of the section. Abbreviations: AC, anterior commissure; LV, l a t e r a l v e n t r i c l e . 142 143 midpallidal l e v e l s , in a position more medial than the f i r s t c e l l group, and extends as a continuous band of c e l l s into the caudal extreme of the GP. Thus, p a l l i d o s t r i a t a l neurons can be seen to be d i s t r i b u t e d into two t i e r s . The terminal l a b e l l i n g of the s t r i a t o p a l l i d a l pathway shows an i d e n t i c a l d i s t r i b u t i o n . As indicated in Figure 32 and from observations of a large number of other cases, injections into the head of the striatum do not lead to l a b e l l i n g of neurons within the entopeduncular nucleus, although anterograde l a b e l l i n g of the striatoentopeduncular projection pathway and terminal f i e l d are c l e a r l y v i s i b l e . Near the caudal pole of the GP, however, a few neurons are occasionally seen embedded within the internal capsule . These c e l l s , although generally of a similar morphology, d i f f e r e d from neurons found within the confines of the pallidum in that they were larger and often were more heavily l a b e l l e d for peroxidase . It was noted that their presence was correlated to d i f f u s i o n of the injec t i o n s i t e into the cortex overlying the striatum. No neurons were found within t h i s region in f i v e cases where the injec t i o n s i t e did not spread into the cortex. DISCUSSION The results reported here represent a consistent finding so far observed in over twenty animals. Anterograde and retrograde reaction product in the GP can be seen as early as four hours after WGA-HRP injections wholly confined to the striatum. The injections depicted in Figure 31a led to the l a b e l l i n g of roughly 17 cells/50 um section, indicating that t h i s i s not a minor 144 projection. As i s the case for the n i g r o s t r i a t a l pathway, the number of retrogradely l a b e l l e d neurons i s not affected by the coinjection of KA. The inject i o n coordinates used here consistently led to l a b e l l i n g of neurons in the l a t e r a l GP at ro s t r a l levels of this nucleus. At more caudal levels the labell e d neurons took up a position in the medial GP. Anterograde label followed a similar d i s t r i b u t i o n . In fact, l a b e l l e d c e l l s were seldom seen outside areas lab e l l e d by anterograde transport as well. The congruence shown by the two forms of label i s suggestive of a re c i p r o c i t y between s t r i a t o p a l l i d a l and p a l l i d o s t r i a t a l connections. The present results confirm and expand on the demonstration of a projection from the globus p a l l i d u s to the striatum presented in Experiment 1. An important observation added in the present work i s that the l a b e l l i n g of p a l l i d a l c e l l bodies i s independent of the anterograde transport of WGA-HRP to the GP. As is the case with other tracers, WGA-HRP can cross the synaptic c l e f t and be taken up by c e l l bodies and dendrites postsynaptic to heavily l a b e l l e d terminals (Itaya and van Hoesen, 1982). As discussed in Experiment 1 i t i s unlikely that t h i s process occurs to any appreciable extent under the conditions employed here. However, the dendrites of p a l l i d a l neurons are often described as being covered with a layer of synaptic endings (Fox et a l . , 1974; D i F i g l i a et a l . , 1982a) and as most of the terminals of thi s immense input would be expected to contain WGA-HRP i t was f e l t necessary to repeat the observation in the absence of anterograde transport. 145 Used by themselves, each of the approaches employed here to demonstrate the p a l l i d o s t r i a t a l projection could lead to erroneous conclusions. However, the combination of l i g h t and electron microscopic techniques as well as the use of coinjections of kainic acid with WGA-HRP rule out the p o s s i b i l i t i e s that perikarya in the GP appeared to be labelled due to a dense input of anterogradely l a b e l l e d p a l l i d o s t r i a t a l f i bers surrounding unlabelled p a l l i d a l neurons or that l a b e l l i n g of p a l l i d a l perikarya in the GP was due to trans-synaptic transport of WGA-HRP. Although previous investigations of the projections of the GP have f a i l e d to identify t h i s pathway, some support for the present finding has recently been provided by Nauta (1979). He reported that injections of t r i t i a t e d amino acids into the GP of the cat resulted in sparse and . widespread appearance of autoradiographic grains in the caudate nucleus and putamen. However, on the basis of his r e s u l t s , Nauta concluded that i t was not possible to determine whether these grains reflected pathways wtj,ich originated in the GP or whether they represented transport from regions adjacent to the GP. The present results provide clearcut evidence for a p a l l i d a l o r i g i n . Since the o r i g i n a l report of thi s projection (Staines et a l . , 1981) i t has been confirmed by retrograde fluorescent marker transport in the rat (Arbuthnott et a l . , 1982a) and retrograde WGA-HRP transport in the cat (Buchwald et a l . , 1981). Furthermore, Arbuthnott and coworkers (1982b) have reported electrophysiological evidence consistent with a p a l l i d a l 146 projection to the striatum. In a recent Golgi study of the mouse GP, axons of l a t e r a l l y located p a l l i d a l c e l l s were traced into the striatum although their ' termination within this structure could not be ascertained (Iwahori and Mizuno, 1981). It has also been reported that p a l l i d a l lesions lead to the degeneration of s t r i a t a l terminals recognized at the u l t r a s t r u c t u r a l l e v e l (Chung and Hassler, 1982). At present, the precise population of p a l l i d a l neurons that project to the striatum i s not known. One p o s s i b i l i t y i s that this population represents the cholinergic neurons of the nucleus basalis magnocellularis (nBM). These neurons, many of which are found in the ventromedial aspect of the GP (Lehmann et a l . , 1980), may project through the striatum to the cortex (Nauta, 1979a; Staines et a l . , 1980a). This hypothesis i s dealt with (and rejected) in Experiment 5, although i t i s l i k e l y that some of these were seen in the present study and represent those neurons found embedded within the internal capsule fibers (Fig. 32) . The nature of the p a l l i d a l neurons that give r i s e to the p a l l i d o s t r i a t a l projections, therefore, requires further investigation. For example, i t would be of considerable interest to determine i f the same or d i f f e r e n t populations of p a l l i d a l neurons give r i s e to the p a l l i d o s t r i a t a l , pallidosubthalamic, and p a l l i d o n i g r a l projections. The general morphology and c e l l size of the p a l l i d a l neurons lab e l l e d after s t r i a t a l injections are very similar to those l a b e l l e d after n i g r a l injections (see Experiment 2). Two types of c e l l s are seen within the GP of the 147 monkey in Golgi preparations. A large c e l l type (20-50 um) , with a deeply indented nucleus, predominates and i s thought to be the projection neuron. A more scarce, medium-sized c e l l (12 um) has been suggested to be a p a l l i d a l interneuron (Fox et a l . , 1974; D i F i g l i a et a l . , 1982). The projection neuron in the mouse is much smaller (25 um) (Iwahori and Mizuno, 1980) and corresponds closely to those retrogradely la b e l l e d from the striatum. The detailed description of p a l l i d o s t r i a t a l connections given above indicates a discontinuity in both the anterograde and retrograde l a b e l l i n g of the GP. This i s more apparent when s t r i a t a l i n j e c t i o n s i t e s have a limited mediolateral extent. One explanation of th i s feature could be that a toxic reaction developed to the WGA-HRP at the core of the injec t i o n s i t e (along the cannula tract) which inh i b i t e d transport from th i s region. However, a dual topographical representation of s t r i a t a l efferents within the GP has previously been indicated in autoradiographic studies (Wilson and Phelan, 1982) and after i n t r a c e l l u l a r i n j e c t i o n of s t r i a t a l neurons with horseradish peroxidase (Chang et a l . , 1981). It would appear that there may also be a dual topographical representation within the p a l l i d o s t r i a t a l projection as well. Additional significance is attached to this finding in l i g h t of the observation of morphological differences found between l a t e r a l l y located p a l l i d a l c e l l s and those located within the core of the GP (Iwahori and Mizuno, 1980;. Park et a l . , 1982) and the fact that the l a t e r a l zone and core of the GP in the monkey show d i f f e r e n t i a l immunoreactivity for substance P and enkephalin 148 (Haber and Elde, 1981). As mentioned in Experiment 1, two d i s t i n c t f i e l d s of labelled s t r i a t o n i g r a l terminals can often be seen in caudal regions of the substantia nigra as well, suggesting that there may be some degree of duality to the topography of other s t r i a t a l efferents. 149 EXPERIMENT 5: COMPARISON OF CORTICAL AND STRIATAL PROJECTIONS OF THE GLOBUS PALLIDUS AND PERI PALLIDAL AREAS INTRODUCTION It has been demonstrated by retrograde transport studies in a number of species that a rather disperse group of basal forebrain neurons has a d i r e c t projection to the cortex (Kievit and" Kuypers, 1975; Parent et al.,1981c; Reinoso-Suarez et al.,1982). In most species, including the rat, there i s an i n f i l t r a t i o n of some of these neurons into the anatomical boundaries of the globus p a l l i d u s . Although most authors agree that these c e l l s are r e s t r i c t e d to the medial and caudal portions of the globus pallidus (Divak,l975; Lehmann et a l . , 1980; Wenk et al.,1980), in contrast to the d i s t r i b u t i o n of p a l l i d o s t r i a t a l neurons, i t was necessary to c l e a r l y distinguish the p a l l i d o s t r i a t a l projections from those of these basal forebrain c e l l s . This d i s t i n c t i o n would address the objection that retrograde l a b e l l i n g of p a l l i d a l neurons after s t r i a t a l injections results from uptake of tracer by fibers of passage or s t r i a t a l c o l l a t e r a l s of the projection of basal forebrain neurons to the cortex. Indirect evidence from biochemical studies exists which can be interpreted as indicating that these neurons project through the striatum on t h e i r way to the corte*:-'^Staines et a l . , 1980a) and a similar conclusion was arriveid at from data presented in Experiment 3. Four approaches were taken to achieve t h i s d i f f e r e n t i a t i o n . The f i r s t made use of the observation that the majority of the basal forebrain neurons projecting to the cortex stain intensely for acetylcholinesterase (AChE) (Mesulam and van Hoesen,l976; 150 Lehmann et a l . , 1980; Parent et al.,1981c; Ribak and Kramer,1982). The histochemical v i s u a l i z a t i o n of acetylcholinesterase a c t i v i t y may be used as a r e l i a b l e and reproducable marker for forebrain cholinergic neurons in animals which have been pretreated with the i r r e v e r s i b l e acetylcholinesterase i n h i b i t o r diisopropylfluorophosphate (DFP). At short survival times, intense acetylcholinesterase a c t i v i t y i s seen in the c e l l bodies of neurons which have a high rate of de novo synthesis of t h i s enzyme (Lehmann et a l . , 1980, Fibiger, 1982). The d i s t r i b u t i o n of p a l l i d a l neurons l a b e l l e d after s t r i a t a l injections of the fluorescent tracer true blue (Tb)(Kuypers et a l . , 1980) was compared to the d i s t r i b u t i o n of AChE-positive c e l l s in the same sections and the c o l o c a l i z a t i o n of these two markers was evaluated. In the second approach, retrograde transport of fluorescent tracer from the striatum to the globus pallidus was examined in rats which had been previously subjected to c o r t i c a l ablation. Under these conditions projection axons passing through the striatum to the cortex would be expected to degenerate and the striatum would become the most d i s t a l structure, obviating the problem of uptake into f i b e r s of passage. In the t h i r d approach, a d i r e c t comparison was made of the d i s t r i b u t i o n and morphology of neurons retrogradely l a b e l l e d by injections of tracer into the striatum with those seen following c o r t i c a l i n j e c t i o n s . F i n a l l y , the acetylcholinesterase staining of neurons retrogradely l a b e l l e d by c o r t i c a l i n j e c t i o n of the fluorescent tracer nuclear yellow were examined. 151 Taken together, the results of these studies support the proposition that neurons in the globus pallidus proper do in fact project to the striatum in the rat. METHODS Male Wistar rats, anesthetized with pentobarbital, received stereotaxic injections of True Blue (Tb). Pressure injections of 100 to 300 nl of Tb as a 5% suspension in d i s t i l l e d water were made through a 5 ul Hamilton syringe into the striatum. Some rats receiving s t r i a t a l injections had been subjected to c o r t i c a l ablation by suction one week or three months previous to the adminstration of tracer. Three days after Tb i n j e c t i o n , animals were injected intramuscularly with diisopropylfluorophosphate (DFP; 1.5 mg/kg in peanut o i l ) and intraperitoneally with atropine (1.0 mg/kg). After a survival time of four to six hours rats were anesthetized with pentobarbital and perfused t r a n s c a r d i a l l y with normal saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB; pH 7.4). Brains were removed and postfixed in the same f i x a t i v e containing 10% sucrose overnight at 4 C. Sections were cut on a freezing microtome at a thickness of 15 and 30 um. The l a t t e r were mounted immediately onto glass s l i d e s from 50 mM PB, a i r dried and coverslipped with paraf f i n o i l . Sections to be reacted for AChE (the 15 um section) were co l l e c t e d into ice cold PB containing 10% sucrose, rinsed in this solution for 1 hr, and mounted onto subbed sl i d e s from 50 mM PB. They were then rinsed in ice cold acetate buffer for 15 min and incubated at room temperature in Karnovsky's reaction medium for 152 the v i s u a l i z a t i o n of AChE. The progress of the reaction was monitored closely by microscopic examination of the sections and terminated when c e l l s became c l e a r l y v i s i b l e in the striatum and nucleus basalis magnocellularis (Lehmann et a l . , 1980) (generally 20-40 min). Nuclear Yellow (NY: a g i f t from Prof. 0 Dann) was injected into the cortex as a 1% solution and after a survival time of 24 hrs the rats were treated as for Tb. Injections of WGA-HRP into striatum (single injection) or fronta l cortex (double injection) were also made and the animals processed for tetramethylbenzidine v i s u a l i z a t i o n of peroxidase a c t i v i t y as described in preceding sections. RESULTS Scrutiny of sections throughout the brains of animals receiving s t r i a t a l injections of Tb confirmed the observations of s t r i a t a l afferents presented in Experiment 1. Experience with both tracers led to the conclusion that in terms of retrograde transport, Tb is superior to HRP and WGA-HRP both in s e n s i t i v i t y and ease of application. C e l l groups c l e a r l y l a b e l l e d by WGA-HRP were also well labelled by Tb, but those neurons weakly la b e l l e d by small injections of WGA-HRP (such a those in the cortex and amygdala) were strongly la b e l l e d by Tb. The inject i o n s i t e obtained with small injections of Tb was eas i l y controlled and r i v a l l e d in size those obtained with WGA-HRP. NY, on the other hand, produced a larger, more diffuse injection s i t e which made i t the tracer of choice for the c o r t i c a l injections. Another advantage of Tb is i t s diff u s e cytoplasmic l o c a l i z a t i o n in 153 lab e l l e d neurons, allowing description of the c e l l body and proximal dendrites. Evidence was obtained that Tb undergoes anterograde transport but th i s merely had the effect of increasing the background in the GP and never obscured the retrogradely l a b e l l e d c e l l bodies. Flourescent l a b e l l i n g with both Tb and NY was found to be compatible with AChE staining i f the l a t t e r was controlled and not allowed to progress farther than necessary. The high fluorescent background reported by others (Nagai et a l . , 1982a) was not found to be a problem i f sections were examined soon after they were reacted. The d i s t r i b u t i o n s of retrogradely la b e l l e d p a l l i d o s t r i a t a l neurons seen after a Tb injection into the CP and intensely stained AChE-positive c e l l s in the same animal are presented diagrammatically in Figure 35. Each neuron is characterized as AChE-positive ( t r i a n g l e ) , Tb-positive ( c i r c l e ) both (asterisk) and mapped onto a projection drawing of the section. At r o s t r a l p a l l i d a l levels AChE-positive neurons were seen in the striatum and subcommissural regions (labelled ventral pallidum by Lehmann et a l . , 1980) but not within the GP i t s e l f . The reverse was true of the Tb fluorescent neurons which are confined to the GP proper. At midpallidal levels (Fig. 35D) a few AChE-positive c e l l s could be seen within the extreme ventromedial GP, a region where the retrogradely la b e l l e d GP neurons were very scarce. A few AChE-positive neurons were found in the l a t e r a l GP, which contained a large number of fluorescent c e l l s , but no co l o c a l i z a t i o n of the two markers was detected. At caudal levels of the GP, the number of AChE-positive neurons increased markedly 154 F i g u r e 35. D i s t r i b u t i o n o f f l u o r e s c e n t n e u r o n s l a b e l l e d f r o m a s t r i a t a l Tb i n j e c t i o n ( o p e n c i r c l e s ) a n d n e u r o n s s h o w i n g i n t e n s e AChE a c t i v i t y 4 h r s . a f t e r a DFP i n j e c t i o n ( t r i a n g l e s ) a t v a r i o u s l e v e l s t h r o u g h t h e g l o b u s p a l l i d u s . E a c h p o s i t i v e c e l l h a s b e e n p l o t t e d o n p r o j e c t i o n d r a w i n g s o f t h e s e c t i o n . N e u r o n s s h o w i n g b o t h f l u o r e s c e n t l a b e l l i n g a n d AChE a c t i v i t y a r e i n d i c a t e d ( a s t e r i s k ) . A b b r e v i a t i o n s i n t h i s a n d f o l l o w i n g f i g u r e s : A C , a n t e r i o r c o m m i s s u r e ; c p , s t r i a t u m ; g p , g l o b u s p a l l i d u s ; I C , i n t e r n a l c a p s u l e . 155 156 Figure 36. (A) L i g h t f i e l d photomicrograph of an AChE stained neuron in the globus p a l l i d u s . (B) The same f i e l d showing Tb labelled neurons. Note that the Tb labe l l e d neuron at the center of the f i e l d has no v i s i b l e AChE r e a c t i v i t y . (C) L i g h t f i e l d photomicrograph of AChE stained neurons at a more caudal l e v e l of the GP. The two AChE positive c e l l s denoted by asterisks are labelled retrogradely la b e l l e d with Tb as indicated by the arrows in the fluorescence micrograph (D). Bar = 50 um. 158 in the medial and ventral portions of the GP and the population of Tb neurons took up a more medial position in the GP, such that these two populations of neurons p a r t i a l l y overlapped in their d i s t r i b u t i o n s . Within overlapping regions a small number of neurons showed both intense acetylcholinesterase a c t i v i t y and Tb fluorescence (Fig. 35H-K, F i g . 36). Quantitative analysis ( c e l l counts) in unreacted sections indicated that there were more fluorescent c e l l s in these regions than were seen in AChE-reacted material (even after correcting for differences in section thickness). This was no doubt due to the screening of fluorescence in some AChE stained neurons, and was found to result in a 50% underestimate of the number of double labelled c e l l s . This discrepancy was confined to regions containing double l a b e l l e d c e l l s , in a l l other regions comparisons of the two sets of sections were quantitively s i m i l a r . A v i r t u a l l y i d e n t i c a l d i s t r i b u t i o n of fluorescent labelled GP neurons was obtained in an animal which had undergone an extensive ablation of the cortex dorsal and r o s t r a l to the striatum three months prior to the in j e c t i o n of Tb (Fig. 37). Acetylcholinesterase reaction of sections from th i s brain indicated that a large number of AChE-positive c e l l s survived in and around the GP (distributed as Figure 35), but in this material only three double l a b e l l e d neurons could be found. The general d i s t r i b u t i o n of c e l l bodies l a b e l l e d in the p a l l i d a l and p e r i p a l l i d a l regions after injections of WGA-HRP injections into the fron t a l cortex was similar to that seen with NY injections into the same area (refer F i g . 38). A larger 159 Figure 37. Photomontage of the anterior globus p a l l i d u s in an animal injected with Tb into the striatum 3 months after extensive c o r t i c a l ablation. The diffuse pale f i e l d represents a minor anterograde transport of Tb. 160 161 number of c e l l s were seen within the boundaries of the globus pallidus than reported in previous studies in the rat (Divac, 1975). Labelled c e l l bodies were found mainly within the internal capsule and caudomedial globus pallidus but some were found along the l a t e r a l and medial borders as well (Fig. 38A and 38B). In addition, the main body of the globus pallidus contained a few very l i g h t l y l a b e l l e d neurons which appeared to belong to a population of a smaller sized c e l l (Fig. 38C and 38D). Morphometric analysis was not attempted as la b e l l e d c e l l s were polymorphous and highly i s o d e n d r i t i c , but the fact that the qu a l i t a t i v e difference apparent in Figure 38 was correlated with a d i s t r i b u t i o n a l difference was f e l t s i g n i f i c a n t . The smaller, weakly-stained c e l l s were r e s t r i c t e d to the GP and, although few in number, were as common in thi s region as the larger c e l l s . The size, appearance and d i s t r i b u t i o n of heavily labelled c e l l s contrasts dramatically to that already shown for p a l l i d a l neurons retrogradely la b e l l e d from the tracer injection into the s t r i a t um. In rats receiving injection of NY into the cortex, fluorescent neurons were found in the diagonal band, ventral pallidum, ventrocaudal globus p a l l i d u s , and within and ventral to the internal capsule (Fig. 39). The number found in the GP was much less than in the other areas and much fewer than t y p i c a l l y seen in th i s nucleus after a s t r i a t a l injection ( i e . 6 c e l l s per section as apposed to 60 c e l l s per section with s t r i a t a l tracer i n j e c t i o n s ) . In a l l areas the majority of c e l l s were also found to be AChE reactive, but a few fluorescent neurons in both 162 Figure 38. Labelled neurons in globus p a l l i d u s after a front a l c o r t i c a l injection of WGA-HRP. Ce l l s shown in (A) and (B) were on the p a l l i d o s t r i a t a l border and adjacent to the internal capsule respectively. A few smaller, less intensely l a b e l l e d c e l l s were seen in the body of the globus pal l i d u s as well (C and D). Bar = 50 um. 163 164 Figure 39. Line drawings depicting the NY inject i o n s i t e s ( f i e l d s of open c i r c l e s in A and B) and the resultant retrograde fluorescent l a b e l l i n g of neurons in and around the globus p a l l i d u s . Sections were reacted for AChE a c t i v i t y and NY labelled c e l l s showing intense AChE a c t i v i t y are represented by tr i a n g l e s . NY la b e l l e d c e l l s not showing AChE a c t i v i t y are represented by open c i r c l e s . Abbreviations: FM, forceps minor. 165 166 Figure 40. (A) Fluorescent NY labe l l e d neurons in the globus p a l l i d u s after a NY injection into the fron t a l cortex. Fluorescent nuclei appear in negative image (dark on a pale background). Only the two neurons denoted by asterisks were AChE-reactive and are shown by arrows in the negative image l i g h t f i e l d photomicrograph (B). In the caudomedial globus pa l l i d u s AChE positive neurons were far more abundant but not a l l were retrogradely l a b e l l e d (C and D). (E) A positive image of the NY labelled c e l l s within the internal capsule. Bar = 100 um. 167 168 p a l l i d a l and p e r i p a l l i d a l regions did not show s i g n i f i c a n t AChE a c t i v i t y (Fig. 39 and 40). DISCUSSION The neuropathology of Alzheimer's disease is now seen to include a loss of basal forebrain cholinergic neurons which have projections to the neocortex (Nagai et a l . , 1982b; Whitehouse et a l . , 1982; Rosser, 1982; Pearson et a l . , 1983). The d i s t r i b u t i o n of these c e l l s overlaps the boundaries of the globus pallidus (and putamen) and has led to claims of a p a l l i d o c o r t i c a l cholinergic projection (Edstrom and P h i l l i s , 1980; Kelley and Moore,1980). These (magnocellular) c e l l s can, however, be d i f f e r e n t i a t e d from t y p i c a l parvocellular (to use the terminology of Parent et a l . , 1981c) p a l l i d a l neurons by their larger size, intense acetylcholinesterase a c t i v i t y , as revealed by the pharmacohistochemical procedure, and by their projections (Lehmann et a l . , 1979; Parent et a l . , 1981c). The major p a l l i d a l c e l l type stains neither for AChE (Lehmann et a l . , 1980) nor choline acetyltransferase (Kimura et a l . , 1981). The present data c l e a r l y indicate that the majority of the p a l l i d a l neurons found to project to the striatum are parvocellular. There is i n s u f f i c i e n t evidence in this or previous experiments to indicate any s i g n i f i c a n t c o l l a t e r a l i z a t i o n of the magnocellular projection within the striatum on i t s way to the cortex. The t h i r d and fourth sections of the present work confirm observations in the cat that the fro n t a l cortex also receives a non-magnocellular projection from the globus pa l l i d u s and subp a l l i d a l regions (Ribak and Kramer, 1982). Whether or not 169 those seen within the GP are parvocellular neurons (as defined by projections to other basal ganglia nuclei) has yet to be established. It is noted with interest that t h i s second type of neuron i s not seen after injections into other c o r t i c a l areas (Parent et a l . , 1981c), but has so far only been observed after injections into the f r o n t a l cortex. 170 EXPERIMENT 6: COLLATERAL PROJECTIONS OF THE NEURONS OF THE GLOBUS PALLIDUS TO THE STRIATUM AND THE SUBSTANTIA NIGRA INTRODUCTION With the completion of Experiment 5, the retrograde demonstration of a p a l l i d o s t r i a t a l pathway is considered confirmed. The globus pallidus (GP) also has a massive projection to the subthalamic nucleus (SUT) (Carter and Fibiger, 1978; van der Kooy et a l . , 1981a; Nauta, 1979; McBride and Larsen, 1980) and a l i g h t e r projection to the substantia nigra (SN) (Hattori et al.,1975; Kanazawa et al.,1976; Bunney and Aghajanian, 1976, Experiments 2 and 3). The density of neuronal c e l l bodies within the GP is not great and the observations from Experiment 1 and 2 indicate that in some regions at least a majority of neurons within the GP project to either SN or CP. Topographical considerations discussed in Experiment 2 suggest that p a l l i d a l c e l l s projecting to an area of striatum are in close proximity to those projecting to the portion of the substantia nigra which i s innervated by that area of striatum. Taken together, therefore, previous experiments have provided data suggesting that at least some p a l l i d a l neurons innervating SN have c o l l a t e r a l s to the CP. In this experiment, the proposal was tested d i r e c t l y using double retrograde transport of fluorescent tracers (van der Kooy, 1979; Bentivoglio et a l . , 1979; Kuypers et a l . , 1980). This technique was also used to confirm topographical claims made in Experiment 1 concerning the p a l l i d o s t r i a t a l projection. 171 METHODS Male Wistar rats (150 gm) were anaesthetized with pentobarbital and injected s t e r o t a x i c a l l y with 100 to 200 nl of True Blue (Tb; yiel d s blue fluorescent cytoplasm in retrogradely l a b e l l e d neurons) into the CP as described in Experiment 5. One to two days l a t e r , an injection of 50-100 nl Nuclear Yellow (NY; yield s yellow fluorescent nuclei in retrogradely labelled neurons) was made into the substantia nigra, SUT or a separate region of the CP. In cases of s t r i a t a l NY in j e c t i o n , the injecti o n buffer contained 10 mM kainic a c i d . Twelve to twenty-four hours after the second i n j e c t i o n , animals were fixed with paraformaldehyde and sections cut and vi s u a l i z e d for fluorescence as described in Experiment 4. In some cases nigr a l injections of another tracer, primuline ( P r i ; yiel d s golden granules in cytoplasm of retrogradely lab e l l e d neurons) were made in animals also receiving a s t r i a t a l i n j e c t i o n of Tb. Survival time in these cases were three days for both tracers. RESULTS Short survival times are required with the use of NY as at larger intervals i t may diffuse out of retrogradely or anterogradely la b e l l e d neuronal elements and into neighbouring neurons not projecting to the s i t e of i n j e c t i o n . The occurrence of t h i s i s readily apparent by the l a b e l l i n g of g l i a l nuclei in areas containing retrogradely l a b e l l e d neurons (Bentivoglio et a l . , 1980a; Bentivoglio et a l . , 1980b). Results obtained with the use of these tracers provided support for a l l of the retrograde observations contained in Experiments 1 and 2, 172 including s t r i a t a l inputs from ventral thalamus, SUT, and amygdala, although the invariable l a b e l l i n g of the cortex along the cannula tract made i t impossible to determine with assurance that the l a b e l l i n g reflected s t r i a t a l connections. Coinjection with KA was found to be a necessary adjunct to the use of short survival times to prevent l a b e l l i n g of g l i a l nuclei in the globus p a l l i d u s . PALLIDAL COLLATERALS TO THE CP AND SN Confirmation was obtained of the topography of p a l l i d a l projections to CP and SN presented.in Experiments 1 and 2. When the s t r i a t a l i n j e c t i o n s i t e s were located outside of those areas showing retrograde l a b e l l i n g with the ni g r a l tracer, there was l i t t l e overlap in the d i s t r i b u t i o n of the d i f f e r e n t i a l l y l a b e l l e d p a l l i d a l neurons. However, when s t r i a t a l injections of Tb f e l l within areas containing NY labe l l e d c e l l bodies, there was coincidence of Tb and NY l a b e l l e d p a l l i d a l c e l l s and, moreover, a large number of c e l l s were double l a b e l l e d (Fig. 41 and 42). The incidence of double labelled neurons varied with the locations of the injection s i t e s and their size and ranged from 20% to 50% of the smaller population. Although not studied in d e t a i l , the occasional double labelled c e l l was noted in the fron t a l cortex but not in the parafascicular or intralaminar nuclei of the thalamus. PALLIDAL COLLATERALS TO CP AND SUT Injection of Tb into the CP and NY into the SUT gave r i s e to double l a b e l l e d neurons in the GP, but invariably produced retrogrde l a b e l l i n g of some s t r i a t a l neurons as well. This 1 7 3 Figure 41. Line drawings (after Konig and K l i p p e l , 1968) depicting a s t r i a t a l Tb injection s i t e and resultant neuronal l a b e l l i n g (open c i r c l e s ) and a nigr a l NY injection s i t e and i t s resultant retrograde neuronal l a b e l l i n g (open t r i a n g l e ) . Neurons showing both Tb and NY fluorescence are denoted by f i l l e d c i r c l e s . Abbreviation in t h i s and subsequent figures: ac, nucleus accumbens; cp, striatum; gp, globus p a l l i d u s ; snc, substantia nigra pars compacta; snr, substantia nigra pars r e t i c u l a t a . 1 74 175 Figure 42. (A and B) Photomicrographs of the fluorescent l a b e l l i n g of p a l l i d a l neurons after NY inje c t i o n into the substantia nigra and Tb in j e c t i o n into the striatum. (A) A p a l l i d a l neuron near the p a l l i d o s t r i a t a l border is labelled for both NY and Tb (arrow). Note the NY l a b e l l e d s t r i a t a l neurons at the top of the f i e l d . In (B) a double lab e l l e d neuron (arrow) is shown along with a single labelled p a l l i d o n i g r a l c e l l demonstrating the d i f f e r e n t i a l i n t r a c e l l u l a r l a b e l l i n g of the two tracers. Note the absence of g l i a l l a b e l l i n g . (C) P a l l i d a l neurons labelled by a nigra primuline inje c t i o n and a s t r i a t a l True Blue i n j e c t i o n . Some primuline-containing neurons are singly l a b e l l e d but the majority of True Blue neurons are double la b e l l e d (arrows). 1 76 177 Figure 43. Line drawings of the s t r i a t a l i n j e c t i o n s i t e s and resultant retrograde l a b e l l i n g of NY (triangles) and Tb (open c i r c l e s ) in the globus p a l l i d u s and substantia nigra. One double labelled c e l l was i d e n t i f i e d in each of the globus pallidus and substantia nigra and these are indicated by f i l l e d c i r c l e s . 1 78 179 indicates that NY was taken up into fibers of passage and therefore double labelled neurons could have arisen through l a b e l l i n g of p a l l i d o n i g r a l c o l l a t e r a l s . PALLIDAL COLLATERALS TO DIFFERENT AREAS OF STRIATUM As seen in Figure 43, non-contiguous s t r i a t a l injections gave r i s e to the l a b e l l i n g of separate populations of p a l l i d a l neurons. A mediolateral topography i s maintained and only a few (1 or 2) double l a b e l l e d c e l l s were seen. In agreement with van der Kooy (1979), no double la b e l l e d c e l l s were seen in the thalamus and very few (only 3) in the SN. DISCUSSION The present report demonstrates that some p a l l i d a l neurons which innervate the striatum also project c o l l a t e r a l s to the substantia nigra. It was not possible to determine with assurance whether neurons of the GP innervate the striatum and subthalamus via c o l l a t e r a l s as well. Furthermore, individual neurons do not appear to have widespread connections within the striatum. These data suggest that the GP may play a more dire c t role in the o v e r a l l regulation of a c t i v i t y in basal ganglia than previously thought. It i s known to have a dense innervation of the SUT, a nucleus with widespread c o l l a t e r a l innervation of many of the component nuclei of the basal ganglia (Deniau et a l . , 1978a; van der Kooy and Hattori, 1980a; Jackson and Crossman, 1981). The present results indicate that at least some of the projections of the GP are s i m i l a r l y c o l l a t e r a l i z e d . A c t i v i t y in the p a l l i d a l neurons may modify the f i r i n g rate of SN neurons 180 both d i r e c t l y , via the p a l l i d o n i g r a l pathway, and i n d i r e c t l y , through the topographically related s t r i a t o n i g r a l projection (see Fig.. 20). 1 8 1 EXPERIMENT 7: MORPHOLOGICAL CHARACTERIZATION OF THE EFFERENTS OF THE GLOBUS PALLIDUS INTRODUCTION Evidence has been presented in the preceding experiments for a projection from the globus pa l l i d u s to the striatum. Implicit in these demonstrations is termination within the striatum. In the past, conclusive demonstration of termination has usually entailed u l t r a s t r u c t u r a l observation of a pathway marked by means of a lesion (eg. Kemp, 1970). A new technique developed by Gerfen and Sawchenko (1983), which u t i l i z e s a unique l e c t i n as a neuroanatomical tracer, allows the v i s u a l i z a t i o n of projection fibers and their associated terminal elements in their entirety and with morphological "detail r i v a l l i n g the Golgi technique. This procedure was applied in the present study to provide a morphological description of the projections of the globus pal l i d u s to the striatum, thalamus, subthalamic nucleus and substantia nigra. METHODS Male Wistar rats were anesthetized with pentobarbital and secured in a stereotaxic instrument. Glass m i c r o c a p i l l a r i e s , broken back to a t i p diameter of 10 to 20 um, were f i l l e d with a 2.5% solution of Phaseolus Vulgaris - Leucogglutinin (PhA-L, Vector Labs) in 100 mM sodium phosphate buffered saline, 0.9%/pH 7.4 (PBS), and centered within the globus p a l l i d u s using empirically determined coordinates. In one animal an injection was made into the striatum to serve as a control. Lectin was injected by iontophoresis into the striatum or globus pallidus 182 using a current of 5 uAmp pulsed (7 sec. on/7 sec. off) for from 5 to 15 min. After a 5 to 8 day survival time animals were reanesthetized and perfused sequentially with ice cold normal saline, 4% paraformaldehyde in ice cold sodium acetate buffer (100 mM/pH 6.5), and f i n a l l y , 4% paraformaldehyde and 0.05% glutaraldehyde in sodium tetraborate buffer (100 mM/pH 9.5). Brains were postfixed overnight in a cold 10% sucrose solution made up in the basic f i x a t i v e . Sections 30 um in thickness were cut on a freezing microtome and colle c t e d s e r i a l l y in ice cold potassium phosphate buffer, pH 7.6 (KPBS). A series consisted of one section out of every s i x . Sections for immunohistochemistry were transferred through the following : (1) Incubation in rabbit anti-PhA-L antibodies (Vector Labs), at a 1:2000 d i l u t i o n in KPBS containing 1% Triton-X 100 and 2% normal goat serum, for 24 to 48 hrs. at 4 C. (2) Rinse for 15 min in a solution of normal saline buffered with Tris-HCl (50 mM/pH 7.4; TBS) containing 1% Triton X 100 and 2% normal goat serum. (3) Incubation for 45 minutes in biotinylated goat antirabbit antibodies (Vector Labs) at an 1:250 d i l u t i o n with TBS containing 1% Triton-X 100 and 2% normal goat serum. (4) Rinse for 15 min. in TBS. (5) Incubation for 60 min. in avidin-dh/HRP complex (Vector Labs) 1:250 d i l u t i o n (6) Rinse in TBS for 10 min. (7) Incubation for 10 min in 0.5% cobalt chloride in TBS. (8) Rinse in TB for 5 min. (9) Rinse in 100 mM sodium cacodylate buffer (CB), pH 5.1, for 3 min. (10) Incubation in CB containing 0.05% diaminobenzidine (DAB) and 0.04% hydrogen peroxide for 10 min. (11) Rinse in TBS for 10 min. (12) Mount 183 sections from 10 mM PB, dehydrate through graded series of alcohols and c o v e r s l i p . Some series were processed from step 7 for the HRP reaction protocol using DAB as substrate outlined in Experiment 2. Both procedures y i e l d comparable results in terms of enzyme v i s u a l i z a t i o n but d i f f e r in the background staining of the tissue. With the procedure outlined above, nonreactive tissue stains l i g h t l y in the manner of cresyl v i o l e t . To avoid confusion, t h i s reaction w i l l be referred to as CoDAB in figure captions to d i f f e r e n t i a t e i t from the noncounterstaining reaction (DAB). RESULTS The PhA-L injec t i o n s i t e for case Ph-1 (from which most of the following observations were made) is depicted in Figure 44A. Unless otherwise stated i t w i l l be understood that a l l data presented derive from this case. A second p a l l i d a l injection (Ph-2) was much larger and involved part of the striatum as well, and a t h i r d p a l l i d a l i n j e c t i o n (Ph-3) was v i r t u a l l y i d e n t i c a l to that in Ph-1. The area of involvement of the i n j e c t i o n s i t e in case Ph-1 is obviously well confined to the globus p a l l i d u s (GP). Under higher magnification i t took the appearance of a diffuse peroxidase reaction in the p a l l i d a l neuropil and a much more intense peroxidase reaction within the c e l l bodies and dendrites of a limited number of p a l l i d a l neurons which f e l l within the bounds of stained neuropil. Axons could be seen to ramify within the GP at short distances from the injection s i t e , and axons leaving the GP could be followed. In addition, fibers and 1 8 4 terminals were found within other brain nuclei including the striatum, entopeduncular nucleus, subthalamic nucleus, substantia nigra and thalamus. Labelled fibers appeared to be t o t a l l y f i l l e d with reaction product and to be v i s i b l e throughout their extent, including v a r i c o s i t i e s and terminals. Their appearance was v i r t u a l l y i d e n t i c a l to the staining of axons and terminals seen in Golgi material, or after i n t r a c e l l u l a r HRP injections (Chang et a l . , 1981). The only s i m i l a r i t i e s with the anterograde l a b e l l i n g produced by WGA-HRP injections were in terms of d i s t r i b u t i o n and, as the d i s t r i b u t i o n of the efferent projections was v i r t u a l l y i d e n t i c a l to that reported in Experiment 3, the mapping of p a l l i d a l efferents w i l l not be repeated in this exper iment. In case Ph-1, only two retrogradely l a b e l l e d neurons were observed in the striatum even though v i r t u a l l y every s t r i a t a l section obtained was reacted for the presence of PhA-L. These two were very weakly reactive and immunoreactivity appeared limited to the c e l l body. No retrogradely labelled neurons were seen in any of the other brain sections. These observations are in keeping with previous reports that PhA-L, when used under the present conditions, i s almost exclusively an anterograde transport marker (Gerfen and Sawchenko, 1983). The naming of structures as axons, axon v a r i c o s i t i e s and terminals follows the precedent set in the Golgi l i t e r a t u r e . In some cases, l i g h t microscopic observations suggestive of synaptic contact with an i d e n t i f i e d postsynaptic element were made. Rather than repeat modifiers such as "terminal-like" or "suggestive of" i t w i l l be 185 acknowledged at this point that they are not proven e x p l i c i t l y as such. With th i s proviso in mind, the detailed observations made on the efferents of the GP revealed using t h i s technique are reported below. GLOBUS PALLIDUS The PhA-L injection s i t e occupied approximately 10% of the volume of the GP and was situated medially at midlevels of thi s nucleus. The main features seen emanating from the injection s i t e at thi s level were varicose fibers which ramified quite l o c a l l y within the the GP i t s e l f (Fig. 44B). The majority of the GP l a t e r a l to the inject i o n s i t e remained free of labe l l e d structures. In the GP r o s t r a l to the inject i o n s i t e , single, l a b e l l e d projection fibers were commonly observed. These smooth, straight f i b e r s had a predominantly rostrocaudal orientation and were seen both within fiber bundles and running freely within the p a l l i d a l neuropil between the fiber bundles. STRIATUM At the r o s t r a l pole of the GP, fibers were seen to radiate r o s t r a l l y and l a t e r a l l y within s t r i a t a l f i b e r bundles and occasionally in the s t r i a t a l neuropil. More commonly observed within the s t r i a t a l neuropil, however, were randomly oriented, twisting, varicose axons. A f a i r l y dense plexus of the varicose fi b e r s was seen in the medial 1/4 of the striatum r o s t r a l to the GP but similar features could be observed throughout the medial 1/2 of the caudal head of the striatum (Fig. 45A and 45B). They were also apparent throughout the whole of the r o s t r a l half of the head of the striatum (Fig. 45C). V a r i c o s i t i e s were sometimes 186 Figure 44. (A) Low power photomicrograph showing the PhA-L inject i o n s i t e in the GP of case Ph-1. Reacted for CoDAB. (B) Higher magnification showing an immunoreactive varicose axon or dendrite within the GP ventral to the injection s i t e . Bar = 25 um. 1 8 7 188 very large (2 or 3 um by 1 um) and e l l i p t i c a l in shape, but were more commonly only s l i g h t l y e l l i p t i c a l and somewhat smaller (1.5-2.0 um x 1.5 um). Within varicose segments of an axon, the v a r i c o s i t i e s appeared at roughly regular, 4-5 um int e r v a l s . Some individual fibers could be followed in 1 section for several hundred um, through which distance they were seen to give off four to six beaded c o l l a t e r a l s . However, with the section thickness employed, the most commonly observed elements were short segments of beaded f i b e r s . It was not uncommon to observe these in close apposition to s t r i a t a l c e l l bodies (Fig. 45D), but l i t t l e significance should be attached to this finding alone. CORTEX Several smooth PhA-L la b e l l e d projection fibers were found within the fiber bundles in the r o s t r a l pole of the striatum and were seen within the fi b e r s of the forceps minor at s t i l l more anterior l e v e l s . Very rarely, l a b e l l e d fibers could be seen ascending through the corpus callosum overlying the head of the striatum. Examination of the cortex surrounding and anterior to the head of the striatum revealed 8 - 10 segments of stained fibers bearing terminal l i k e v a r i c o s i t i e s . These appeared to be of two types. The f i r s t consisted of fine, straight fibers which radiated perpendicular to the c o r t i c a l laminae (Fig. 46A). They were very sparsely varicose during their passage through c o r t i c a l layers V and III and bore very short c o l l a t e r a l processes ending in terminal end bulbs of 0.5 um in diameter which were i d e n t i c a l to the axonal v a r i c o s i t i e s . One of these fine c a l i b e r fibers was traced as far as layer II. In another part to the cortex of the 189 Figure 45. (A) PhA-L positive f i b e r s in the striatum. Note the v a r i c o s i t i e s d i s t r i b u t e d along the fibers and the extensive branching of that portion in the upper right of the f i e l d . Reacted for CoDAB. (B) Higher magnification of a portion of the f i e l d in (A). (C) PhA-L positive fiber in the r o s t r a l extreme of the striatum in case Ph-2. Reacted for DAB. (D) Segment of la b e l l e d varicose axon lying just ventrolateral to a medium-sized s t r i a t a l neuron in case Ph-1. The c e l l body i s s l i g h t l y darker than the surrounding neuropil and the nucleus is pale. Reacted for CoDAB. Bar = 1 0 um. 190 191 same section, a fiber of similar caliber was seen to bifurcate and end in small boutons within layer I (Fig. 46B). The second, more commonly observed immunoreactive elements were short, randomly oriented, segments of beaded f i b e r s as depicted in Figure 46C. These were judged to be morphologically similar to those described in the striatum but di f f e r e d from the fine caliber f i b e r s seen in the cortex in that the v a r i c o s i t i e s were di s t r i b u t e d along the axon rather than on short branches off the main axon and the v a r i c o s i t i e s were much larger. Segments of beaded fibers were sometimes found within a few hundred microns of the fine c a l i b e r radiating fibers but i n s u f f i c i e n t material was coll e c t e d to determine i f they originated from the radiating f i b e r s . Fragments of beaded fibers were most commonly observed in the agranular cortex, dorsomedial and ventrolateral to the forceps minor. Their d i s t r i b u t i o n was much broader than that of the fine c a l i b e r f i b e r s . In sections anterior to the striatum, the major orientation of these fibers was within the coronal plane of the section, but in more caudal sections (at the le v e l of the r o s t r a l pole of the CP) their orientation was predominantly rostrocaudal. Terminals of th i s fiber type were found predominantly within layer V in the cortex anterior to l e v e l 9650 of Konig and Klippel (1968). Labelled f i b e r s of either type were not detected caudal to th i s plane and were therefore r e s t r i c t e d to the agranular frontal cortex. It should be emphasized that these fibers were not a predominant feature of the l a b e l l i n g seen in any of the cases studied. The t o t a l l a b e l l i n g of cortex appeared to arise from only six or eight 192 Figure 46. Labelling in the cortex in case Ph-1. (A) Projection fiber within layer III of the cortex oriented perpendicular to the c o r t i c a l surface. This fiber was traced through layers V to I I . (B) Fine caliber f i b e r ending in small boutons (arrows) within layer I of the cortex. (C) A segment of beaded axon bearing large v a r i c o s i t i e s found in layer V in apparent axosomatic contact with the c e l l whose nucleus i s denoted by an asterisk. Reacted for CoDAB. Bar = 10 um. 193 194 p r o j e c t i o n f i b e r s . THALAMUS C a u d a l t o t h e P h A - L i n j e c t i o n s i t e i n t h e G P , f i b e r s w e r e s e e n t o t r a v e r s e t h e i n t e r n a l c a p s u l e ( I C ) i n a d o r s o m e d i a l d i r e c t i o n . No f i b e r s p a s s e d l a t e r a l l y i n t o t h e t a i l o f t h e s t r i a t u m . Once t h e f i b e r s h a d c r o s s e d i n t o t h e r e t i c u l a r n u c l e u s o f t h e t h a l a m u s , t h e y b r a n c h e d q u i t e e x t e n s i v e l y a n d d i s p l a y e d s p h e r o i d v a r i c o s i t i e s a t r e g u l a r i n t e r v a l s a l o n g t h e a x o n a l b r a n c h e s ( F i g . 4 7 C ) . T h e s e f i b e r s w e r e m o s t c o m m o n l y o b s e r v e d i n t h e a n t e r i o r r e g i o n s o f t h i s n u c l e u s . I d e n t i c a l i m m u n o r e a c t i v e f i b e r s w e r e s e e n i n more c a u d a l p o r t i o n s o f t h e n u c l e u s a s w e l l , b u t a t t h e s e l e v e l s o c c u p i e d i t s d o r s a l e x t r e m e . The f i b e r s a n d t h e i r v a r i c o s i t i e s c o u l d n o t be d i s t i n g u i s h e d f r o m t h o s e s e e n i n t h e s t r i a t u m . M e d i a l t o t h i s n u c l e u s v e r y r a r e ( t h r e e i n t o t a l ) p r o j e c t i o n f i b e r s w e r e s e e n w i t h i n t h e v e n t r a l t h a l a m i c t i e r . E a c h was q u i t e l o n g a n d s t r a i g h t a n d t h e y a l l p r o j e c t e d i n t h e d i r e c t i o n o f t h e s t r i a m e d u l l a r i s . S e c t i o n s f r o m c a s e P h - 1 w e r e e x a m i n e d i n d e t a i l , b u t n e i t h e r c o n t i n u a t i o n n o r t e r m i n a t i o n c o u l d be f o u n d . I n c a s e P h - 2 , h o w e v e r , a few l a b e l l e d v a r i c o s e f i b e r s w e r e f o u n d i n a r e s t r i c t e d r e g i o n w i t h i n t h e m e d i o d o r s a l n u c l e u s o f t h e t h a l a m u s . No l a b e l l i n g was s e e n w i t h i n t h i s r e g i o n i n c a s e P h - 3 , b u t a few l a b e l l e d v a r i c o s e f i b e r s w e r e s e e n w i t h i n t h e p a r a f a s c i c u l a r n u c l e u s . ENTOPEDUNCULAR NUCLEUS I m m u n o r e a c t i v e p r o j e c t i o n f i b e r s w i t h a p r e d o m i n a n t l y r o s t r o c a u d a l o r i e n t a t i o n w e r e f o l l o w e d w i t h i n t h e i n t e r n a l c a p s u l e t o t h e a n t e r i o r p o r t i o n o f t h e e n t o p e d u n c u l a r n u c l e u s . 195 At t h i s l e v e l , beaded axons appeared within the neuropil inserted within the capsular fiber matrix (Fig. 48). Most often these beaded fibers had a featureless, random orientation, but at times appeared to outline c e l l u l a r p r o f i l e s . Beaded axons were found more frequently within anterior portions of this nucleus. At the le v e l where the inclusions of entopeduncular neuropil into the IC become large,, the fibers were once again only v i s i b l e as projection f i b e r s with a mainly rostrocaudal orientation. SUBTHALAMIC NUCLEUS The subthalamic nucleus received a much more dense innervation than any of the other nuclei examined. Varicose axons were evident throughout the medial part of the nucleus and along the dorsal border of the l a t e r a l area (Fig. 47A). They could also be seen within those segments of the nucleus which protruded into the internal capsule. The morphological description of the beaded axons is as that given for those found in the striatum (Fig. 47B). It appeared that a mediolateral orientation of the beaded segments of fibers predominated within the body of the subthalamic nucleus. It was noted that very few projection fibers were seen within the internal capsule at the le v e l at which innervation of the SUT was most dense, far fewer than observed within this f i b e r bundle either r o s t r a l or caudal to this nucleus. In the caudal SUT, the network of l a b e l l e d varicose f i b e r s was much less dense. Long, single beaded axons were seen running p a r a l l e l to the cerebral peduncle and within the plane of the section while the majority of fibers were found projecting caudally within the peduncle. This d i s t r i b u t i o n was 196 F i g u r e 47. (A) PhA-L l a b e l l i n g i n t h e s u b t h a l a m i c n u c l e u s . The c e r e b r a l p e d u n c l e i s i n t h e l o w e r r i g h t o f t h e f i e l d . (B) H i g h e r m a g n i f i c a t i o n o f t h e l a b e l l i n g i n t h e s u b t h a l a m i c n u c l e u s . R e a c t e d f o r CoDAB. (C) P h o t o m o n t a g e o f a l a b e l l e d v a r i c o s e f i b e r i n t h e r e t i c u l a r n u c l e u s o f t h e t h a l a m u s . R e a c t e d f o r CoDAB. B a r = 100 um. 197 198 Figure 48. Photomontages of PhA-L immunoreactive varicose fibers in the entopeduncular nucleus. Note that some v a r i c o s i t i e s are in close apposition to c e l l bodies (arrows in C). Reacted for CoDAB. Bar = 10 um. 199 200 seen to continue back to the r o s t r a l t i p of the substantia nigra, in agreement with the c y t o l o g i c a l delineation of the SUT provided by van der Kooy and Hattori (1980). SUBSTANTIA NIGRA Projection f i b e r s ran within the medial portion of the internal capsule from the subthalamic nucleus to the substantia nigra. From th i s point to the caudal reaches of the SN, projection fibers followed a path through the pars compacta. Varicose axons as described in the striatum were seen within the pars compacta but were far more common in the pars r e t i c u l a t a (Fig. 49A). This was true throughout the whole rostrocaudal extent of t h i s nucleus. Although the morphology of l a b e l l e d axons and v a r i c o s i t i e s was as described in a l l previous regions except for the cortex, their d i s t r i b u t i o n in the substantia nigra was remarkable. Single varicose axons were r e l a t i v e l y rare and instead several segments of beaded axons would associate in tight plexes. This occurred in i t s simplest form as two varicose segments wound around one another. However, many instances were noted in which four to six f i b e r s would form elaborate arrays, apparently ensheathing neuronal c e l l bodies within the pars r e t i c u l a t a (Figs. 49B, 50A, 51 and 52). Similar, although infrequent and not so marked, examples of t h i s were also found within the pars compacta (Fig. 50B) . It appeared, from the l i g h t counterstaining afforded by the CoDAB reaction that f u l l y 50% of the varicose fibers in the pars r e t i c u l a t a were in intimate contact with c e l l bodies and the proximal portions of their dendrites. In each instance, c r e s y l v i o l e t counterstaining 201 Figure 49. (A) PhA-L immunoreactivity in the substantia nigra. Labelled varicose fibers can be seen throughout much of the pars r e t i c u l a t a (snr) but only one or two fibers with v a r i c o s i t i e s on them can be seen in the pars compacta (snc). The region denoted by the arrow i s shown in higher magnification in (B) and in the drawing in Figure 50A. The area denoted by the asterisk is shown on Figure 50B. Reacted for DAB. Bar = 10 um. 2 0 2 203 Figure 50. Drawings of l a b e l l e d varicose fibers in the pars r e t i c u l a t a (A) and pars compacta (B) of the substantia nigra (refer to Figure 49A). Bar = 10 um. 204 205 Figure 51. Photomontages of selected features of the l a b e l l i n g in the pars 'reticulata of the substantia nigra. Note that there appears to be a neuronal c e l l body at the center of the fiber plexus in (A). This was confirmed by counterstaining with c r e s y l v i o l e t . The same was true of the plexus shown in (B) (refer to Fig.53). (A) reacted for CoDAB, (B) reacted for DAB. Bar = 10 um. 206 A 207 Figure 52. Apparent axosomatic connections in the (A and C) substantia nigra and (E) striatum. The corresponding f i e l d after c r e s y l v i o l e t counterstaining i s shown at the right. Bar = 2 5 um. 2 0 8 209 revealed the presence of a neuronal nucleus within these arrays of l a b e l l e d varicose f i b e r s (Fig.52). In l i g h t of the prevalence of t h i s type of contact within the SN, sections from the striatum were examined after c r e s y l v i o l e t counterstaining and, although individual examples were not as dramatic, apparent axosomatic contacts were common in t h i s nucleus as well (Fig. 52F). Projection f i b e r s , v i r t u a l l y perpendicular to the plane of sectioning, were observed in the caudal extreme of the SN and could be traced through a few sections into the pons, at which point they were l o s t . Available sections back to the l e v e l of the locus coeruleus were scrutinized, but neither fibers nor v a r i c o s i t i e s were observed. STRIATAL INJECTION The iontophoretic inj e c t i o n of PhA-L into the head of the striatum (Ph-4) gave r i s e to a minor c o l l a t e r a l l a b e l l i n g within the striatum, and l a b e l l e d s t r i a t a l projections to the GP, EP and SN (Fig. 53). Both the d i s t r i b u t i o n and morphology of s t r i a t a l projections contrasted dramatically with that seen after injections into the GP. Far fewer labelled f i b e r s were seen within the striatum in case Ph~4 than in any of the cases involving inj e c t i o n of the GP and the boutons associated with these f i b e r s appeared as terminal end-bulbs rather than v a r i c o s i t i e s along the length of the fiber (compare Figs. 45 and 53). These c h a r a c t e r i s t i c s also applied to the l a b e l l i n g seen in the GP, EP and SN in case Ph-4. Furthermore, the l a b e l l e d fibers seen in the SN after the s t r i a t a l i n j e c t i o n of PhA-L appeared to be evenly and randomly di s t r i b u t e d within the pars r e t i c u l a t a . 210 Figure 53. Anti-PhA-L immune-reactivity after a PhA-L injection into the striatum (case Ph-4). (A) Labelling around the injection s i t e in the striatum. Note the r e l a t i v e scarcity of lab e l l e d f i b e r s around the injection s i t e (arrows). (B) Higher magnification of one of the labe l l e d f i b e r s around the injec t i o n s i t e . Note the marked difference in axonal and terminal (arrows) morphology compared to that seen in the striatum after a p a l l i d a l i n j e c t i o n . (C) Low power photomicrograph of the l a b e l l i n g in the globus pallidus after a s t r i a t a l i n j e c t i o n . (D) Higher magnification of the lab e l l e d fibers and terminals in the globus pallidus after a s t r i a t a l injection of PhA-L. Bar = 25 um. 211 212 There was no evidence of the obvious convergence of terminals around n i g r a l c e l l somata as was seen after injections into the GP. LARGE PALLIDAL INJECTION Observations of material resulting from a large p a l l i d a l i njection which also involved part of the striatum (Ph-2) are of some additional interest. The terminal l a b e l l i n g in the substantia nigra reflected the fact that two morphologically d i s t i n c t inputs had been lab e l l e d (morphological heterogeneity was not an obvious feature after injections of the striatum alone). It was noted that a l l areas receiving p a l l i d a l projections showed a much more profuse l a b e l l i n g , due no doubt to the larger injection s i t e , but the shorter survival time in t h i s case - fiv e days rather than eight - seemed to have led to a less complete l a b e l l i n g of individual elements). DISCUSSION Before discussing the present observations in terms of the projections of the globus p a l l i d u s a number of possible sources of misinterpretation must be examined. Perhaps the most serious source of error would be that a r i s i n g from the uptake and transport of l e c t i n by fibers from other sources passing through the p a l l i d a l i n j e c t i o n s i t e . This would force the admission that terminals in the substantia nigra could in fact have arisen from l a b e l l i n g of the s t r i a t o n i g r a l pathway, and that the features in the striatum could be explained in terms of any number of ascending inputs. The c h a r a c t e r i s t i c s of PhA-L transport have been rigourously 213 investigated in the hippocampus and i t was concluded that t h i s l e c t i n , under the conditions used here, does not give r i s e to s i g n i f i c a n t uptake by fibers of passage (Gerfen and Sawchenko, 1983). Furthermore, the morphological d e t a i l afforded by th i s technique allows one to assess th i s p o s s i b i l i t y through a comparison of axonal and terminal morphologies. Both the d i s t r i b u t i o n pattern and morphology of l a b e l l i n g in the substantia nigra after p a l l i d a l injections d i f f e r e d markedly from those seen after s t r i a t a l i n j e c t i o n s . This shows that, although s t r i a t o n i g r a l fibers pass through the globus p a l l i d u s , they do not take up appreciable amounts of tracer and, furthermore, indicates that is i s unlikely that any of the l a b e l l i n g attributed to projections of the GP arose instead from l a b e l l i n g of fibers that originated outside the injection s i t e . The morphologies of a number of s t r i a t a l afferents have been described in the monkey using the Golgi technique ( D i F i g l i a et a l . , 1978). Examination of the photographs and drawings of s t r i a t a l afferents thought to arise from the cortex, substantia nigra and dorsal raphe nucleus reveals s t r i k i n g differences in morphology with the s t r i a t a l f i b e r s l a b e l l e d after p a l l i d a l injections of PhA-L. Although similar in some respects, the morphology of immunoreactive elements in the striatum also d i f f e r s s i g n i f i c a n t l y from the Golgi charcterization of afferents suggested to arise from the thalamus ( D i F i g l i a et a l . , 1978). The p a l l i d a l afferents d i f f e r from the thalamic afferents in having a much more consistent axonal diameter throughout the length of the f i b e r , a far greater prevalence of long varicose. 214 segments as opposed to short varicose or end-bulb c o l l a t e r a l s and a much more regular shape of v a r i c o s i t i e s . Thus T not only is i t very unlikel y that fibers passing through the GP were labelled , but the morphology of the labe l l e d axons within the CP d i f f e r e d from that of any of i t s other known afferents. Another phenomenon with the potential to confound the interpretation of the present observations would be the retrograde l a b e l l i n g of l o c a l c o l l a t e r a l s of neurons projecting to the injec t i o n s i t e . In agreement with previous work (Gerfen and Sawchenko, 1983), the retrograde l a b e l l i n g of c e l l bodies after the injection in case Ph-1 and Ph-3 was v i r t u a l l y nonexistent. Direct comparison of the morphology of p a l l i d o s t r i a t a l fibers (Fig. 45") with that of l o c a l c o l l a t e r a l s of s t r i a t a l neurons seen after PhA-L injections into the striatum (Fig. 53) or by i n t r a c e l l u l a r HRP injections into s t r i a t a l efferent neurons (Wilson and Groves, 1980; Preston et a l . , 1980) also serves to indicate that the terminal l a b e l l i n g studied did not arise from retrograde l a b e l l i n g of l o c a l c o l l a t e r a l s of s t r i a t a l efferent neurons. It appears, therefore, that this novel tracing technique can provide r e l i a b l e data on the efferent projections of the injected nucleus. Some examples can be found of l o c a l c o l l a t e r a l s of s t r i a t a l neurons which do resemble the p a l l i d o s t r i a t a l fibers (Somogyi et a l . , 1981), but i t would not be anticipated that the morphology of the p a l l i d o s t r i a t a l fibers be t o t a l l y unique. The d i s t r i b u t i o n of terminal l a b e l l i n g found in this experiment i s in t o t a l accord with that implied by the 215 anterograde l a b e l l i n g of p a l l i d a l efferents by WGA-HRP presented in Experiment 3, including a minor efferent projection to the mediodorsal and parafascicular n u c l e i . The fact that l a b e l l i n g of these l a t t e r two nuclei was not a consistant feature after p a l l i d a l injections may have been due to the small number of efferents l a b e l l e d in each case, such that minor connections might be missed in a single animal. In addition, many of the other observations presented in previous sections seem to be borne out by observations made in the present study. As the l i t e r a t u r e in support of the existance of these projections has been c i t e d previously, the present findings w i l l be discussed in terms of the conclusions of the previous experiments. It i s of special note that the examination of c o r t i c a l material provoked a heterogenous description of afferent morphology. A l l of the other nuclei receiving afferents from the globus pallidus showed the same beaded, axon en passant type of synaptic formation (Figs. 54 and 55) The l a b e l l i n g within the cortex can in part be attributed to the fact the injec t i o n s i t e in case Ph-1 undoubtably involved some neurons of the nucleus basalis magnocellularis. As the f i n e - c a l i b e r axon with small v a r i c o s i t i e s and end bulbs were unique to the cortex, i t i s l i k e l y that the observation of a small number of these f i b e r s in case Ph-1 represented c o r t i c a l afferents a r i s i n g from the magnocellular neurons of the nucleus ba s a l i s . The fragments of larger, beaded fibers seen within the cortex were indistinguishable from the p a l l i d a l efferents to other nuclei and could represent the c o r t i c a l terminals of the small number of 216 parvocellular p a l l i d a l neurons which were demonstrated in Experiment 5 to project to the fro n t a l cortex. It i s noted with interest that the morphology of the l o c a l c o l l a t e r a l s of the l a t e r a l p a l l i d a l neuron described by Park et a l . (1982) i s i d e n t i c a l to that which has been seen in the CP, EP, SUT and SN in the present study after p a l l i d a l PhA-L inje c t i o n s . The same i s true of the l o c a l c o l l a t e r a l s i l l u s t r a t e d by D i F i g l i a et a l . (1982a) for p a l l i d a l neurons in the monkey studied by the Golgi technique. In fact, in a l l areas except for the cortex, the l a b e l l e d p a l l i d a l efferents shared the same rather unique axonal morphology (Fig. 54). The remarkable maintenance of morphological homogeneity seen within a l l areas receiving input from the GP supports the conclusion drawn in Experiment 6 on the basis of double retrograde l a b e l l i n g that the p a l l i d o s t r i a t a l and p a l l i d o n i g r a l pathways arise at least partly as c o l l a t e r a l outputs. The present results suggest that t h i s concept might be expanded to include the projections to the subthalamic nucleus and thalamus as well. As described in Experiment 5 there i s evidence that the GP is not a homogeneous nucleus. The inje c t i o n s i t e , depicted in Figure 44, in the more well studied case in this report was situated in the medial part of the GP. This region i s distinguished from the l a t e r a l GP in that (1) i t s neurons do not c o l l a t e r i z e within the GP, (2) i t s neurons have a d i f f e r e n t dendritic morphology and orientation and (3) in the monkey i t demonstrates a much heavier enkephalin immunoreactivity and a much l i g h t e r substance P immunoreactivity (Park et a l . , 1982; 217 Figure 5 4 . Drawings of t y p i c a l labelled fibers and v a r i c o s i t i e s in the striatum (A), r e t i c u l a r nucleus of the thalamus (B), entopeduncular nucleus (C), subthalamic nucleus (D) and substantia. nigra pars r e t i c u l a t a (E). Note the marked s i m i l a r i t i e s in the appearance of lab e l l e d features in these fiv e nuclei. Bar = 1 0 um. 219 Figure 55. Camera lucida drawings of some of the PhA-L lab e l l e d f i b e r s in the cortex of case PhA-1. (A) An example of the f i n e - c a l i b e r , radiating f i b e r . (B) Example of the short segments of beaded fiber found within the prefrontal cortex. Compare thi s l a t t e r type to the beaded fibers in the subthalamic nucleus (C). Bar = 10 um. 221 Haber and Elde, 1981). However, as the d i s t r i b u t i o n of efferents of the GP did not d i f f e r in any s i g n i f i c a n t way between the small p a l l i d a l injection (which l a b e l l e d only the medial GP) and the large p a l l i d a l injection (which l a b e l l e d the medial GP, l a t e r a l GP and the bordering s t r i a t a l tissue) i t would appear that the basis for nonhomogeneity within the GP does not include it's efferent projections. As shown in Experiments 2 and 3, the major p a l l i d a l projection to the SN ends within the pars r e t i c u l a t a rather than compacta and d i f f e r s markedly from the s t r i a t a l input to the pars r e t i c u l a t a in i t s concentration around c e l l bodies and primary dendrites, and in i t s morphology. Previous demonstrations of the projection of the globus p a l l i d u s to the substantia nigra have described i t as l i g h t . While th i s may be true in some contexts, i t i s obvious from Figure 49 that the p a l l i d a l innervation of some pars r e t i c u l a t a neurons i s heavy indeed. The large v a r i c o s i t i e s studding the c e l l soma and primary dendrites are l i k e l y to have a s i g n i f i c a n t functional advantage over more d i s t a l l y located afferent elements. Axosomatic contacts of p a l l i d a l terminals were also suggested by observations made in the CP and EP, but were not as numerous or obvious as those seen in the SN. Neither, however, were those seen in the SUT but recent u l t r a s t r u c t u r a l observations of the p a l l i d a l efferents to th i s nucleus show that in fact, p a l l i d a l terminals do end predominantly on the c e l l bodies and proximal dendrites of neurons in the SUT (Romansky et a l . , 1980). It may be that techniques better able to answer t h i s question w i l l show that 222 proximal innervation of target c e l l s (termination predominantly on the c e l l soma and proximal dendrites) by p a l l i d a l terminals is generalizable to nuclei other than the SN and SUT. The major finding of significance in the present report is the observation of varicose fibers projecting from the globus pallidus to the striatum. This represents the f i r s t clear demonstration of anterograde transport from the GP to the CP and serves as f i n a l confirmation of the existence of a p a l l i d o s t r i a t a l projection in the rat. A recent electron microscopic study reports the morphology of a reputed p a l l i d o s t r i a t a l projection. After electrocoagulation of the external globus pallidus in the monkey, degenerative changes were observed in a myelinated afferent which made broad, asymmetric, axospinous, synaptic contacts in the CP (Chung and Hassler, 1982), characterised as plump axospinous type I I I . Degenerating terminals ranged from 0.5 to 2.0 um in diameter and contained large round v e s i c l e s . The large v a r i c o s i t i e s seen on l a b e l l e d p a l l i d o s t r i a t a l fibers in the present study are c e r t a i n l y within th i s size range but i t is probable that Chung and Hassler were examining the degenerating processes in a whole range of s t r i a t a l afferent populations due to their choice of lesioning technique. Anterograde l a b e l l i n g of t h i s pathway with a tracer or with a fi b e r sparing lesion of the GP i s required before r e l i a b l e u l t r a s t r u c t u r a l conclusions can be reached. 223 EXPERIMENT 8: DEMONSTRATION OF A PALLIDAL INNERVATION OF STRIATAL SOMATOSTATIN CONTAINING NEURONS INTRODUCTION Recent immunohistochemical studies have demonstrated that the neuropeptide somatostatin is contained within a population of s t r i a t a l neurons ( D i F i g l i a and Aronin, 1982; Vincent et a l . , I982d). Somatostatin (SST)-containing neurons found within the striatum can also be r e l i a b l y and s e l e c t i v e l y stained by using a histochemical technique to v i s u a l i z e nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) a c t i v i t y (Vincent et a l . , I982e). Thus, within the striatum there i s a perfect correspondence between perikaryal SST immunoreactivity and NADPH-d a c t i v i t y , indicating that t h i s enzyme can be used to iden t i f y s t r i a t a l SST-containing c e l l s . The histochemical technique i s , in fact, superior to the immunohistochemical method in that i t provides much greater morphological d e t a i l and is more readily amenable to combinations with other histochemical procedures (Vincent et a l . , 1983). In the present report, NADPH-d a c t i v i t y was combined with anterograde l a b e l l i n g of the p a l l i d o s t r i a t a l projection to demonstrate that p a l l i d a l terminals innervate the.proximal dendrites of SST-containing neurons within the CP. METHODS Sections through the s t r i a t a of the cases discussed in Experiment 7 (the small p a l l i d a l injections, PhA-1 and PhA-3, and the large p a l l i d a l i n j e c t i o n , PhA-2) were used in the present study. Therefore, the procedure for injection of PhA-L, fix a t i o n 224 and sectioning are as described in Experiment 7. Sections which had been stored for six days, in ice cold potassium phosphate buffer (100 mM, pH 7.6) containing 0.9% NaCl, after cutting were reacted for NADPH-d a c t i v i t y using a modification of the method of Scherer-Singler et a l . (see Vincent et a l . , I982e). Sections were incubated at 37 C for 30 min. in Tris-HCl buffer (100 mM, pH 8.0) containing 1.0 mM NADP, 0.2 mM ni t r o blue tetrazolium and 15 mM monosodium malate. After a 10 min. rinse in phosphate buffered saline, the sections were reacted for the immunohistochemical demonstration of PhA-L. Dehydration through a graded series of alcohols was found to produce c r y s t a l l i n e a r t i f a c t within the sections so some sections were simply a i r dried and coverslipped with p a r a f f i n o i l . Sections were observed and photographed using a Zeiss universal microscope f i t t e d with a 100X o i l immersion objective and drawings were made with a Wild microscope equipped with a drawing tube. RESULTS The appearance of NADPH-d stained neurons within the striatum was as described previously (Vincent et a l . , I982e; Vincent et a l . , 1983). Dark blue formazan precipitate f i l l e d the perikarya and dendrites of medium sized polymorphic neurons dis t r i b u t e d throughout the striatum (Fig. 56). The s t r i a t a l neuropil also contained numerous beaded fi b e r s stained dark brown for the immunoperoxidase reaction revealing anterogradely transported PhA-L as described in Experiment 7 (Fig. 56). However, the c l a r i t y of the staining was somewhat diminished compared to sections reacted for PhA-L alone. In a l l three 225 Figure 56. Photomicrographs of NADPH-d-stained neurons (blue) in the striatum showing apparent innervation by terminal v a r i c o s i t i e s of p a l l i d o s t r i a t a l fibers l a b e l l e d by the immunohistochemical procedure for anterogradely transported PhA-L (brown). A l l examples are from case PhA-2. The neuron in (A) is shown in higher magnification in (B). Bar = 10 um. 226 227 Figure 57. Camera lucida drawings of NADPH-d-stained neurons and la b e l l e d p a l l i d o s t r i a t a l f i b e r s . The neuron shown in (A) i s from case PhA-1, the other two are from PhA-2. Note in pa r t i c u l a r the correspondence between the orientations of the afferent fiber and one of the dendrites of the neuron in (B). Bar = 10 um. 228 B 229 cases, instances were found in which NADPH-d reactive neurons had beaded fibers stained for PhA-L running along their dendrites (Fig. 56 and 57). In some of these cases, the fibers and the v a r i c o s i t i e s they bore followed the dendrites for some distance and matched i t in focal plane (Fig. 57B), suggestive of a longitudinal axodendritic innervation. In areas containing l a b e l l e d p a l l i d o s t r i a t a l f i b e r s , an apparent apposition to NADPH-d-labelled dendrites was noted for approximately 5% of the terminal v a r i c o s i t i e s . DISCUSSION Although the association of a few v a r i c o s i t i e s with an i d e n t i f i a b l e post-synaptic element is of questionable significance, the results of this experiment showed a congruence between labelled axon and primary dendrite which could hardly be attributed to a random process. Labelled terminal v a r i c o s i t i e s were rare within the f i e l d s from which the examples presented above were taken, and thus, the probability of coincidental observation of apposition was small. As many other immunoreactive fibers and v a r i c o s i t i e s were found which were devoid of i d e n t i f i a b l e association with NADPH-d reactive perikarya or dendrites, i t would seem that only one of the targets of the p a l l i d a l efferents within the striatum is the primary dendrite of the s t r i a t a l somatostatin containing neuron. This neuron is thought to correspond to the aspiny medium sized type III neuron described as an interneuron in the Golgi studies of Dimova et a l . , (1980). In recent ultrastructu.ral studies, the SST-containing c e l l 230 of the striatum has been described as receiving only two or three types of synaptic endings ( D i F i g l i a and Aronin, 1982; Takagi et a l . , 1983). Large synaptic boutons were frequently seen on proximal dendrites and represent an excellent candidate for the p a l l i d o s t r i a t a l terminals. These boutons were seen to form symmetrical synapses and to contain pleomorphic v e s i c l e s , features often associated with GABAergic terminals (Ohara et a l . , 1983). Another, smaller type of bouton was seen to form asymmetrical synaptic contacts with more d i s t a l portions of the dendrites of SST-containing CP neurons and contained small round ve s i c l e s such as those found within terminals originating in the cortex or thalamus (Kemp and Powell, 1971b). Although the observation of innervation of SST-containing s t r i a t a l neurons by p a l l i d o s t r i a t a l terminals bears confirmation by electron microscopic techniques, the present data provide strong evidence for such an association. Similar combinations of the PhA-L tracing technique with retrograde l a b e l l i n g and histochemistry for acetylcholinesterase have already proved successful (Staines and Gerfen, unpublished observations) and these strategies w i l l undoubtedly enhance our understanding of the c i r c u i t r y underlying brain function. 231 GENERAL DISCUSSION As the major thrust of the research presented above has been an investigation of the connections of the globus pallidus within the context of the c i r c u i t r y of the basal ganglia, t h i s discussion w i l l concentrate mainly on the role of the GP in the function of t h i s system. In speculating about this function, a number of primary and secondary assumptions have been made. (i) Neurons of the GP have axon c o l l a t e r a l s which innervate the CP, SUT and SN. In the double retrograde fluorescent tracing experiment, i t was shown that at least a portion of p a l l i d a l neurons innervate the CP and SN via c o l l a t e r a l s (Expt. 6 ) . The extension to include c o l l a t e r a l i z a t i o n to the SUT as well is based on observations made using PHA-L to trace the efferent projections of the GP. With this method, similar terminal morphology was seen in the CP, SUT, and SN. Furthermore, although beaded axons were seen within the subthalamic nucleus, descending projection fibers could not be found at t h i s l e v e l . Projection fibers were c l e a r l y v i s i b l e r o s t r a l and caudal to this nucleus. These observations suggest that the innervation of the substantia nigra arose from a caudal extension of fibers giving off terminals in the SUT. ( i i ) P a l l i d a l endings within the CP, SUT and SN are i n h i b i t o r y . This assertion i s based on the assumption of c o l l a t e r a l i z a t i o n and the observations from electrophysiological studies on the pallidosubthalamic projection. The effects of 232 p a l l i d a l stimulation on the f i r i n g of rate of neurons in the SUT were reported in e a r l i e r work as a mixed inhibitory-excitatory response but more recent studies have shown that this input is purely i n h i b i t o r y (Tsubokawa and Sutin, 1972; Ohye et a l . , 1976; Rouzaire-Dubois et a l . , 1980; Kita et a l . , 1983). ( i i i ) GABA i s (one of) the neurotransmitter(s) used by the efferents of the GP. At present, although there i s no consensus as to the transmitter(s) used by neurons of the GP in any of their connections, the majority of available evidence points to GABA as the major p a l l i d a l neurotransmitter. The data bearing on this point are reviewed b r i e f l y below. In early immunohistochemical work, substance-P positive c e l l bodies were found in the GP (Kanazawa et a l . , 1977), but other studies, employing colchicine to i n h i b i t axonal transport and increase the concentration of the peptide in the perikarya, f a i l e d to confirm this observation (Ljungdahl et a l . , 1978). Other data indicate that the majority of the substance-P immunoreactivity in the GP i s contained within afferents from the CP (Staines et a l . , 1980a; but see Hong et a l . , 1977). Projections from the SNr to the superior c o l l i c u l u s , ventromedial thalamus, dorsal midbrain tegmentum and peribrachial region a l l appear to contain GABA (Vincent et a l . , 1978a; DiChiara et a l . , 1979; Childs and Gale, 1983). S i m i l a r l y , the projection of the EP to the l a t e r a l habenula is also GABAergic (Gottesfeld et a l . , 1977; Nagy et a l . , 1978b; Starr and K i l p a t r i c k , 1981; Vincent et a l . , 1982a) and that to the ventral 233 t i e r of the thalamus may be as well (Uno et a l . , 1978). Frequent references to the remarkable s i m i l a r i t i e s between the GP, EP and SNr (see Nauta, 1979a) suggest that the p o s s i b i l i t y of a GABAergic component to the outputs of the GP deserves consideration. An immunohistochemical study on the d i s t r i b u t i o n of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), in the forebrain contrasted the numerous positive c e l l bodies seen within the CP with the r e l a t i v e r a r i t y of perikaryal staining in the GP (Ribak et a l . , 1979). The few stained c e l l s located within the GP were medium sized (15-20 um) and the more numerous large c e l l s received a heavy innervation of GAD-postive terminals but were themselves unreactive. On the other hand, recent studies from another laboratory, reported in abstract form, state that nearly a l l of the neurons in the GP, EP and SNr show perikaryal staining for GAD (Oertel et a l . , 1982). There are c o n f l i c t i n g biochemical data concerning the p o s s i b i l i t y of a GABAergic projection from the GP to the SUT. Fonnum and his colleagues (1978) found decreased levels of GAD in the SUT of the cat following e l e c t r o l y t i c lesions of the GP. Kainic acid lesions in the rat GP however f a i l e d to s i g n i f i c a n t l y reduce GAD a c t i v i t y in the SUT although they were e f f e c t i v e in blocking the anterograde l a b e l l i n g of the pallidosubthalamic projection (van der Kooy et a l . , 1981a). Nagy and Fibiger (1980) reported that there was no s i g n i f i c a n t decrease in the GAD a c t i v i t y in the SN after kainic acid lesions of the GP and a portion of the surrounding CP. This 234 observation has been confirmed, but with the addendum that at shorter post-lesion survival times a s i g n i f i c a n t decrease in n i g r a l GAD can be seen (van der Kooy et a l . , 1981a). GAD a c t i v i t i e s have been shown to increase in some brain areas after lesions of non-GABAergic inputs (Vincent et a l . , 1978b; Gilad and Reis, 1979), probably as a result of sprouting of GAD-containing fibers within the target locus. It is possible, therefore, that at longer survival times, a similar post-lesion increase in n i g r a l GAD a c t i v i t y due to sprouting may mask a small post-lesion decrease in n i g r a l GAD levels due to a loss of p a l l i d o n i g r a l terminals. It remains to be determinined i f lesions of the GP cause a decrease in the GAD a c t i v i t y of the CP but ' t h i s would be d i f f i c u l t to discern, given the high levels of this enzyme contained within i n t r i n s i c elements of the striatum (McGeer and McGeer, 1976; Nagy et a l . , 1978a) and the comparatively few neurons projecting to i t from the GP. It has been noted that kainic acid lesions of the GP produce s i g n i f i c a n t decreases in the GAD within t h i s nucleus (Nagy and Fibiger, 1980). An almost i d e n t i c a l percentage of the GAD a c t i v i t y in the SN i s lost after n i g r a l kainic acid lesions (Nagy et a l . , 1978c). In the case of the SN, and perhaps the GP as well, these data r e f l e c t the fact that, although most of the a c t i v i t y of t h i s enzyme i s located within afferent terminals, a portion of i t derives from neurons found within that nucleus. The i n h i b i t o r y effect of GP stimulation on the f i r i n g rate of SUT neurons i s mimicked by iontophoretic application of GABA 235 or muscimol (a GABA agonist) and i s antagonized by the GABA antagonists bicucculine and picrotoxin (Rouzaire-Dubois et a l . , 1980). Furthermore, the in h i b i t o r y postsynaptic potentials recorded from neurons in the SUT after p a l l i d a l stimulation result from an increase in chloride ion conductance (Kita et a l . , 1983). The inhi b i t o r y action of GABA i s well known to act via thi s ionic mechanism (see McGeer et a l . , 1978). In a recent series of papers, the histochemical demonstration of GABA-transaminase (GABA-t) a c t i v i t y has shown potential for the i d e n t i f i c a t i o n of GABAergic projections. Lesions of nuclei containing GABAergic projection neurons lead to decreases in the histochemical reaction for GABA-t within the corresponding terminal regions. Thus, lesions of the CP decrease GABA-t in the GP, EP and SN; lesions of the EP decrease GABA-t in the ventral thalamus and the l a t e r a l habenula; and , bearing on the present question, lesions of the GP decrease GABA-t in the SUT (Vincent et a l . , 1981; Vincent et a l . , 1982a). These findigs suggest that p a l l i d a l efferents, l i k e those of the CP and EP, have a GABAergic component. The u l t r a s t r u c t u r a l immunohistochemistry of GABA-t has been examined in the cerebellum and this enzyme has been shown to occur within GABAergic neurons, on the membrane postsynaptic to GABAergic nerve terminals, and within g l i a l elements associated with GABAergic perikarya (Chan-Palay et a l . , 1979). The histochemical findings c i t e d above suggest that GABA-t i s l o c a l i z e d within the terminals of GABAergic projection neurons as well. Using combined immunofluorescence and retrograde fluorescent tracing 236 techniques (see van der Kooy and Sawchenko, 1982) i t has been determined that a l l of the p a l l i d a l neurons projecting to the SN and CP display moderate GABA-t immunoreactivity (Staines and Vincent, unpublished observation). In fact, v i r t u a l l y every p a l l i d a l neuron, and a s i g n i f i c a n t population of p a l l i d a l g l i a l c e l l s stain for GABA-t. Although hardly d e f i n i t i v e , this observation further correlates features of the p a l l i d a l neurons with those of other c e l l s known to be GABAergic, i e . the SNr neuron (Vincent, personal communication). There i s , therefore, evidence both for and against the proposal that GABA is a neurotransmitter used by the efferents of the GP. Under these circumstances one either draws no conclusions or adopts a tentative working hypothesis. The working hypothesis supported by the majority of the available data and employed in the succeeding discussion is that GABA is a major transmitter of the efferents of the GP. (iv) P a l l i d a l terminals predominantly innervate the nondopaminergic neurons of the substantia nigra. Observations of the retrograde and anterograde transport of WGA-HRP (Experiments 2 and 3), studies of p a l l i d a l efferents using PHA-L (Experiment 7) and the l i t e r a t u r e c i t e d within these experiments show that the GP projects predominantly to the pars r e t i c u l a t a of the SN. This does not mean that p a l l i d a l terminals do not innervate the ventral dendrites of pars compacta neurons or the dopaminergic neurons located within the pars r e t i c u l a t a . Although i t i s of importance to distinguish between inputs to dopaminergic and nondopaminergic neurons of the SN this cannot be 237 accomplished simply by noting d i f f e r e n t i a l input to the two zones of the nigra. There is a dopaminergic c e l l type whose c e l l body and dendrites are thought to be confined to the pars compacta, but these neurons project to the allocortex (olfactory bulb and amygdala) rather than the striatum (Fallon et a l . , 1978). The functional implications of the p a l l i d a l input to the substantia nigra d i f f e r r a d i c a l l y for a major termination on dopaminergic SNc as opposed to nondopaminergic SNr neurons. Not only do their neurotransmitters d i f f e r , but these two c e l l types have markedly d i f f e r e n t efferent projections. Those of the nondopaminergic neurons constitute outputs from the basal ganglia, while those of the dopaminergic c e l l s , for the most part, remain i n t r i n s i c to t h i s system. As described in Experiments 3 and 7, fibers from the GP terminate mainly in the SNr, and appear to contact the c e l l bodies of neurons within t h i s region. Although a few dopaminergic c e l l s are found within the SNr, these are confined to the caudal half of the nucleus, a more r e s t r i c t e d d i s t r i b u t i o n than that observed for the apparent axosomatic features seen in the anterograde tracing experiments. Another commonly observed feature of the p a l l i d a l termination in the SNr was that of several fibers running in a bundle. This might be related to the bundling of v e n t r a l l y directed dopaminergic dendrites described within the SNr. However, whereas these dendritic bundles have a predominantly v e r t i c a l orientation in coronal sections (Scheibel and Tomiyasu, 1980), the axonal bundles displayed no such preferred orientation. It seems more l i k e l y that these f i b e r s were engaged in longitudinal 238 axodendritic association with the dendrites of pars r e t i c u l a t a neurons. The conclusion drawn from t h i s work (that the GP predominantly innervates the nondopaminergic neurons of the SNr) is in accord with the findings of Hattori et a l . , (1975) who concluded that although the p a l l i d o n i g r a l projection ends in part on dopaminergic n i g r a l neurons, for the most part i t innervates nondopaminergic neurons. PALLIDAL INNERVATION OF THE SUBSTANTIA NIGRA In order to consider the possible function of the p a l l i d a l innervation of the substantia nigra i t i s f i r s t necessary to examine the present understanding of the s t r i a t o n i g r a l projection. (i) Function of the s t r i a t o n i g r a l projection. The projection of the CP to the SN is neurochemically heterogeneous, with at least three transmitter candidates being carried in d i f f e r e n t descending fibers of t h i s system, i e . GABA, substance P and dynorphin. A l l end predominantly within the pars r e t i c u l a t a region but could presumably innervate the ventral dendrites of the dopaminergic neurons of the pars compacta. Although according to Hattori and coworkers (1975) SNc neurons are not the major nigra l targets of the s t r i a t o n i g r a l terminals, recent studies have provided morphological evidence for some descending s t r i a t o n i g r a l termination onto dopaminergic neurons within the SN (Wassef et a l . , 1981). Neuropharmacological work has indicated that while substance P produces an activation of SNc neurons (Starr, 1978; Cheramy et a l . , 1977; James and Starr, 1979), the neurons of the SNr are 239 much more sensitive to the excitatory effects of iontophoretically applied substance P (Pinnock and Dray 1982). Si m i l a r l y , an inhib i t o r y influence of descending GABA projections onto dopaminergic n i g r o s t r i a t a l neurons has been described (Bunney and Aghajanian, 1978; Guidotti et a l . , 1978; Kelly and Moore, 1978b; but see Cheramy et a l . , 1978). However, the behavioral e f f e c t s of the injec t i o n of GABA agonists or antagonists into the SN are opposite to those which would be predicted from a major dir e c t effect on dopaminergic n i g r o s t r i a t a l neurons (Scheel-Kruger et a l . , 1980) and on this basis the major action of GABA within the SN is believed to be exerted on SNr neurons. In analogy to the iontophoretic studies on the n i g r a l actions of substance P, SNr neurons are twenty times more sensitive to the application of GABA agonists than are SNc neurons (Grace et a l . , 1982). Thus, although responses to substance P and GABA can be e l i c i t e d from both SNc and SNr neurons, SNr neurons are more sen s i t i v e . Taking the anatomical data into account as well, i t appears that the s t r i a t o n i g r a l pathway exerts i t s major influence over the nondopaminergic SNr neurons. A number of observations have led to the recent conclusion that the influence of ni g r a l afferents on the a c t i v i t y of the dopaminergic n i g r o s t r i a t a l neurons may be mediated through a projection of SNr neurons onto SNc c e l l s (Grace et a l . , 1982). These observations include the finding that these two c e l l types show reciprocal alterations in f i r i n g rates in response to noxious stimuli or drug application within the SNr. 240 To summarize to thi s point, the SNr receives dual excitatory and inhibitory inputs from the CP. The s t r i a t a l influences on the SNc are much weaker and may act in part through the SNr. As i t i s the neurons of the pars r e t i c u l a t a which constitute the output elements, effects on the ascending dopaminergic system a r i s i n g from the SNc w i l l be ignored for the present, except to rei t e r a t e that dopaminergic ac t i v a t i o n of the striatum and nucleus accumbens is f a c i l i t a t o r y to movement. A large body of evidence now indicates that i n h i b i t i o n of SNr neurons leads to an increase in motor behavior. Direct e l e c t r i c a l stimulation of the CP can e l i c i t some crude movements in experimental animals (Lee and Slater, 1981) and results in a mixed e x c i t a t i o n - i n h i b i t i o n response in the nondopaminergic projection neurons of the SNr, with i n h i b i t i o n as the predominant response (Deniau et a l 1976; Collingridge and Davies, 1981). Furthermore, i n t r a n i g r a l i n j e c t i o n of ethanolamine o-sulphate (EOS), which i n h i b i t s degradation of GABA, or n i g r a l injections of the GABA agonist muscimol, increase the le v e l of motor behavior in animals and antagonize the cat a l e p t i c effects of dopaminergic antagonists such as haloperidol (Dray et a l . , 1975; Scheel-Kruger, 1977). B i l a t e r a l lesions of the substantia nigra produces chronic stereotyped behavior in animals (Olianas et a l . , 1978), suggestive of a release of these behaviors from an inhibi t o r y n i g r a l control. Rats subjected to asymmetrical dopaminergic stimulation of the striatum show turning behavior away from the more stimulated side, c o n t r a l a t e r a l turning (Ungersted.t, 1971; Yamamoto et a l . , 241 1982). C i r c l i n g resultant from an i n t r a n i g r a l injection of GABA agonists i s also in the contralateral d i r e c t i o n (K i l p a t r i c k et a l . , 1980). U n i l a t e r a l lesion of the SN induces contralateral turning (DiChiara, 1976) and nigral injections of tetanus toxin, which remove nigral neurons from descending inh i b i t o r y and (to a lesser extent) descending excitatory influences, cause animals to turn towards the injected side ( i p s i l a t e r a l turning) (Collingridge and Davies, 1982). That lesions of the SN or the in t r a n i g r a l infusion of inhibitory substances e l i c i t movement and mimic the e f f e c t s of s t r i a t a l stimulation indicates that a c t i v i t y within neurons of the SNr i s inhibitory to movement and that movement i s correlated with an i n h i b i t i o n of these c e l l s by the descending s t r i a t o n i g r a l projection. A l l of the projections of the SNr, excluding the small population of dopaminergic c e l l s found within t h i s region, have been shown to contain GABA. Stimulation of the SNr produces i n h i b i t i o n in the superior c o l l i c u l u s (Chevalier et a l . , 1981) and the ventromedial nucleus of the thalamus (Yoshida and Omata, 1978). It would be predicted, therefore, that activation of the GABA receptors within these nuclei, simulating stimulation of the neurons of the pars r e t i c u l a t a , should i n h i b i t movement and, indeed, i t has been observed that i n j e c t i o n of the GABA agonist muscimol into the ventromedial nucleus produces catalepsy (DiChiara et a l . , 1979), and the c i r c l i n g produced by u n i l a t e r a l injections i s opposite to that of asymmetrical s t r i a t a l a c t ivation ( K i l p a t r i c k et a l . , 1980). Thus, i t appears that movement is correlated with an 242 in h i b i t i o n of SNr neurons and furthermore, the motor effects of fiber systems descending to the substantia nigra act mainly through the projection neurons of the SNr. The motor effects attributed to a c t i v i t y of these neurons are disrupted by lesions of the regions receiving afferents from the SNr (Mulas et a l . , 1981; Garcia-Munoz et al.,1982), s p e c i f i c a l l y , the superior c o l l i c u l u s and dorsal midbrain tegmentum.. ( i i ) Function of the p a l l i d o n i g r a l projection. In contrast to the substantia nigra, the a c t i v i t y of neurons in the globus pallidus seems to be p o s i t i v e l y correlated with movement. Although the globus p a l l i d u s is analogous to the pars r e t i c u l a t a in i t s receipt of a massive innervation from the striatum, functionally i t appears to be analogous to the CP. Like the CP, a c t i v i t y in the efferents of the GP appears to in h i b i t c e l l s which are themselves inhibitory to movement. Injections of neuroleptic drugs, such as haloperidol, block dopamine receptors and produce a catalepsy in experimental animals which is correlated to some of the extrapyramidal symptoms seen in human patients given these drugs. It .has been suggested that t h i s catalepsy arises from an increased i n h i b i t i o n of the a c t i v i t y of c e l l s in the GP by their inputs from the CP (Costall and Olley, 1971). Although t h i s proposal was based on the e f f e c t s of lesions which undoubtedly disrupted far more than the s t r i a t o p a l l i d a l projection, i t has gained support from more selective experiments. Thus, muscimol injections into the GP potentiate the catalepsy induced by haloperidol (Matsui and Kamioka, 1978; Scheel-Kruger et a l . , 1980) and EOS injections 243 into the GP, acting to i n h i b i t the degradation of GABA within th i s structure, block the increase in locomotor behavior e l i c i t e d by the inject i o n of dopamine agonists into the nucleus accumbens (Pycock and Horton, 1976). This i s consistent with the observation that apomorphine administration increases the f i r i n g rate of GP neurons (Bergstrom et a l . , 1982). In addition, GABA turnover, an indicator of synthesis and release of this neurotransmitter, is seen to increase within the GP in animals treated with neuroleptic drugs (Marco et a l . , 1976). With repeated exposure to dopamine antagonists a tolerance develops to the effects on GABA turnover in the GP which shows a time course similar to the development of tolerance to the cataleptogenic e f f e c t s of the drugs (Marco et a l 1976; Ezrin-Waters and Seeman, 1977). A similar increase in GABA turnover i s seen within the nucleus accumbens which does not show tolerance and may therefore be related to the antipsychotic e f f e c t s of neuroleptics (Marco et a l . , 1976; Costa et a l . , 1978). Effects on motor behavior independent of dopaminergic systems can also be e l i c i t e d by pharmacological manipulations of the GP. Direct inj e c t i o n of EOS into the GP produces an akinesia which i s not reversed by the dopamine agonist amphetamine (Pycock et a l . , 1976) . Opiate drugs can also produce catalepsy and have a concomitant stimulatory effect on the GABA turnover within the GP (Moroni et a l . , 1979). Local inj e c t i o n of stable analogues of enkephalin into the GP, however, produce an increase in coordinated locomotor behavior (Joyce et a l . , 1981) which is 244 reversed by naloxone but not by dopaminergic antagonists. It i s evident, therefore, that the behavioral consequences of pharmacological manipulation of GABA systems within the GP and SN d i f f e r . An increase in GABA receptor stimulation within the SN leads to an increase in motor behaviors and within the GP i t leads to a decrease in motor behaviors. It is of interest to note that the administration of apomorphine, at doses having a locomotor stimulant e f f e c t , has opposite effects on the metabolic a c t i v i t y in the GP and SN (Kozlowski and Marshall, 1982). Note that a motor stimulant effect rather than motor depression would be expected from p a l l i d a l EOS injections i f the GP gave ri s e to a major inhibitory projection to dopaminergic SNc neurons. These findings from pharmacological studies and the pathways which may mediate them are summarized in Figure 58. Lesion studies indicate that s t r i a t a l GABAergic projections to the SN and GP are not c o l l a t e r a l s (Jessel et a l . , 1978; Brownstein et a l . , 1977; Staines et a l . , 1980a). This independence of the s t r i a t a l GABA projections to the GP and SN implies that under certain conditions, the s t r i a t a l and p a l l i d a l projections to the SN could act in concert. Experimental evidence of this i s provided by neuropharmacological manipulations of the GP in animals subjected to stimulation of the striatum. In rats, CP stimulation causes the animal to turn i t s head to the side opposite the s i t e of stimulation. These motor ef f e c t s are not blocked by prior c o r t i c a l ablation, indicating that they do not arise from activation of corticofugal f i b e r s passing near the stimulating electrode and that t h i s motor 245 Figure 58. Schematic diagram showing some of the connections of the basal ganglia l a b e l l e d for the neurotransmitter used, the major postsynaptic response to stimulation (+ or -) and the the overall behavioral consequence of stimulation (increase or decrease in motor behaviors; arrows). C o l l a t e r a l i z a t i o n and monosynaptic associations are not implied by the figure. 246 247 function i s not mediated through a feedback a c t i v a t i o n of c o r t i c a l motor centers. It is observed that injections of muscimol into the GP decrease the speed with which animals perform this movement in response to s t r i a t a l stimulation and that p a l l i d a l injections of the indirect GABA antagonist picrotoxin cause the movement to be performed much more quickly (Lee and Slater, 1981). These data indicate that the GP acts in p a r a l l e l with the striatum in the control of some types of motor behavior. The motor response given in th i s example i s not wholly dependent on the descending projections to the SN as the movement is s t i l l performed, a l b e i t slowly, after lesions of the i p s i l a t e r a l SN. These lesions do, however, abolish the above-mentioned effects that pharmacological manipulation of the GP has on the motor response (Lee and Slater, 1981). As seen in experiments presented above, the nigral innervation originating in the striatum is quantitatively much greater than that a r i s i n g from the GP, but the location of p a l l i d a l terminals on the c e l l soma and proximal dendrites of ni g r a l neurons allows for the p o s s i b i l i t y that the projection from the GP i s of an equal or even greater functional sig n i f i c a n c e . As mentioned in Experiment 7, there was no evidence of a perisomal innervation of SNr neurons by s t r i a t o n i g r a l terminals. In summary, both the CP and GP have an inhib i t o r y input to the SNr. These two descending projections are topographically, and, therefore, somatotopically linked and a c t i v i t y in both i s f a c i l i t a t o r y to movement. As a c t i v i t y in the p a l l i d o n i g r a l 248 projection is largely determined by the input that the GP receives from the CP i t may be thought of as a more highly integrated form of descending i n h i b i t i o n . It would seem l o g i c a l in such a system that the more highly integrated input play a r e l a t i v e l y greater role in the control of the f i r i n g rate of SNr neurons, and the dense concentration of p a l l i d a l terminals on the c e l l soma and proximal dendrites of the neurons in the SNr may in fact r e f l e c t just that. PALLIDAL INNERVATION OF THE STRIATUM As discussed above, a large portion of the GABA within terminals in the CP i s assumed to reside within l o c a l c o l l a t e r a l s of GABA-containing projection neurons (McGeer and McGeer, 1976; Nagy et a l . , 1978a; Ribak et a l . , 1979) and l o c a l c i r c u i t interneurons (Bolam et a l . , 1983). It must be assumed then that i f the p a l l i d o s t r i a t a l pathway i s GABAergic i t must have some ch a r a c t e r i s t i c not shared by the far more numerous GABAergic l o c a l c o l l a t e r a l s in order to exert i t s influence within the s t r i a t a l neuropil. One way th i s could come about i s suggested by the p o s s i b i l i t y that p a l l i d a l terminals end on the c e l l soma and proximal dendrites of neurons in the CP, as discussed in Experiment 7. This would make them el e c t r o p h y s i o l o g i c a l l y more eff i c a c i o u s in c o n t r o l l i n g the f i r i n g of s t r i a t a l neurons than GABAergic l o c a l c o l l a t e r a l s , which end predominantly on the dendritic spines of other GABAergic neurons (Ribak et a l . , 1979). Alt e r n a t i v e l y , GABAergic terminals originating in the GP may innervate c e l l types within the striatum which do not receive input from the l o c a l GABAergic c o l l a t e r a l s . In Experiment 8 249 evidence was presented indicating that p a l l i d a l endings terminate in part on the proximal dendrites of s t r i a t a l somatostatin (SST)-containing neurons. SST-containing neurons have extensive l o c a l projections within the CP ( D i F i g l i a and Aronin, 1982), and i f the GP proved to be the only source of inhibitory input to these c e l l s i t could provide the p a l l i d o s t r i a t a l projection with an e f f e c t i v e means of influencing the f i r i n g rate of other CP c e l l s . Evidence for a reciprocal innervation of topographically related portions of the CP and GP was presented in the WGA-HRP tracing studies in Experiment 4. This c i r c u i t may function as a point to point reciprocal feedback system regulating s t r i a t a l efferent a c t i v i t y , the requirements of which do not seem to be met by the c i r c u i t s connecting the striatum and the substantia nigra. The c o l l a t e r a l i z a t i o n of p a l l i d a l projections to the SN and to the regions of the CP which project to that same area of the SN, indicates that the GP may influence the SN not only by direct innervation but also by modulating the a c t i v i t y of the s t r i a t o n i g r a l projection. Additional access to the SN i s provided by the projection of the GP to the SUT which in turn projects to the SN. PALLIDAL INNERVATION OF THE ENTOPEDUNCULAR NUCLEUS The studies presented above represent the f i r s t clear demonstration of an innervation of the EP by the GP. Previous studies have alluded to such a connection but lacked the anatomical resolution to draw d e f i n i t i v e conclusions (Hattori et 250 a l . , 1975; Carter and Fibiger, 1978). There is very l i t t l e data on the function of the EP on which to base speculation concerning the involvement of an input from the GP. In many developmental and anatomical ways, the EP is similar to the SNr (Nauta, 1979b). Biochemical data indicates that the EP, l i k e the SN, receives a GABAergic input which is dissociable from the input to the GP, and in fact the GABAergic innervation of the EP and SN may be c o l l a t e r a l s of the same neurons (Staines et a l . , 1980a). Like the nondopaminergic projections of the SNr, the EP sends branched projections to the ventrolateral thalamus, intralaminar thalamus and nucleus tegmenti pedunculopontis which are inhibitory and may use GABA (Uno et a l . , 1978). Presumably, these connections of the EP function in a manner similar to those of the SNr. Like the SNr, the EP appears to receive dual descending i n h i b i t i o n from the the CP and GP. The majority of the neurons in the EP project to the l a t e r a l habenula (van der Kooy and Carter, 1981) and this projection has also been determined to use GABA (see above). The l a t e r a l habenula projects to the SNc and the dorsal raphe (Herkenham and Nauta, 1977) and therefore could control both the dopaminergic and serotonergic innervations of the striatum. Although the functional significance of these anatomical findings have yet to be determined, stimulation of the l a t e r a l habenula has been shown to decrease serotonin release within the striatum (Reisine et a l . , 1982). PALLIDAL PROJECTIONS TO THE CORTEX Findings from both retrograde fluorescent transport studies 251 (Experiment 5) and anterograde l a b e l l i n g with PHA-L (Experiment 7) point to a small p a l l i d a l innervation of the prefrontal cortex. This region of cortex gives r i s e to the c o r t i c a l innervation of the SN (Gerfen and Clavier, 1979), and part of the c o r t i c a l innervation of the CP (Experiment 3). It is tempting to speculate that t h i s p a l l i d a l projection contributes to the regulation of these corticofugal pathways. PALLIDAL INNERVATION OF THE THALAMUS The significance of the findings concerning the p a l l i d a l innervation of the r e t i c u l a r and mediodorsal nuclei are d i f f i c u l t to evaluate. The apparent innervation of the mediodorsal nucleus by the GP is l i k e l y to have arisen from neurons more properly c l a s s i f i e d as belonging to the ventral pallidum and w i l l be discussed below. After p a l l i d a l injections of WGA-HRP (Experiment 3), an apparent innervation . of the whole of the r e t i c u l a r nucleus of the thalamus (RTN) was observed. However, a much more r e s t r i c t e d d i s t r i b u t i o n of terminals was found within the RTN when PHA-L was used as an anterograde tracer of p a l l i d a l projections (Experiment 7), and thi s d i s t r i b u t i o n matches very well that seen for cholinergic terminals in the RTN (Kimura et a l . , 1981). Although the morphology of the anterogradely l a b e l l e d elements within t h i s nucleus corresponded to that seen in the CP, SUT and SN and did not match that of the putative cholinergic f i b e r s in the cortex, i t i s possible that neurons of the nucleus basalis magnocellularis rather than those of the GP innervate the RTN. In the absence of retrograde l a b e l l i n g data no d e f i n i t i v e claim can be made for an innervation of the RTN by 252 the parvocellular neurons of the GP. PALLIDAL INNERVATION OF THE SUT The heaviest output of the GP i s i t s projection to the SUT. P a l l i d a l terminals end on the c e l l bodies and proximal dendrites of SUT neurons which project back to the GP (Romansky et a l . , 1980; van der Kooy et a l . , 1981) and send additional axon c o l l a t e r a l s to the EP and SN (Deniau et a l , 1978; van der Kooy and Hattori, 1980a; Kita et a l . , 1983b), and presumably the cortex as well (Jackson and Crossman, 1981). The SUT also receives a very powerful excitatory input from the cortex (Kitai and Deniau, 1981) which i s somatotopically organized (Harmann-von Monakow et a l . , 1978). Although the projection distances of subthalamic neuron axon c o l l a t e r a l s to the GP and SN d i f f e r considerably, the conduction v e l o c i t i e s of descending and ascending fibers are such that impulses a r i s i n g from the subthalamic nucleus arrive at the SN and GP simultaneously (Kita et a l . , 1983b). In a c a r e f u l l y controlled study, stimulation of the SUT was observed to give r i s e to an excitation of neurons in the substantia nigra (Hammond et a l . , 1978). Most of the c e l l s activated were located in the SNr but a few c e l l s in the SNc also responded. Other studies (Yoshida et a l . , 1971; Perkins and Stone, 1980) describe the effects of stimulation of the SUT as producing an inhibitory or a mixed e x c i t a t i o n / i n h i b i t i o n response in the GP. These findings appear i r r e c o n c i l a b l e with the fact that these are c o l l a t e r a l projections. Nauta and Cuenod (1982) have suggested that the SUT projections are GABAergic, based on 253 the retrograde transport of t r i t i a t e d GABA to the SUT from the GP, but the r e l i a b i l i t y of t h i s technique has yet to be determined. Use of an inhibitory transmitter by the projections of the SUT would c e r t a i n l y f i t the c l a s s i c a l impression of the function of the SUT, that of exerting a powerful behaviorally-inhibitory control over several basal ganglia nuclei (see DeLong and Georgopoulos, 1981) but, as discussed below, the observations pointing to an excitatory subthalamic neurotransmitter actually predict the behaviorally-inhibitory role of t h i s nucleus. Hemiballismus, a violent movement disorder, has long been regarded as due to a loss of the i n h i b i t o r y influence of the SUT on the output neurons i t innervates. The pioneering work of Carpenter and others (1950) on the hyperkinesia resulting from the destruction of the SUT has recently been confirmed using the more selective kainic acid lesioning technique (Hammond et a l . , 1980). The consequence of this lesion is an episodic, b a l l i s t i c hyperkinesia of the c o n t r a l a t e r a l extremities. Interepisodic limb function i s normal save for a minor bradykinesia. Similar effects have been observed after the infusion of picrotoxin into the internal GP of primates (EP; Crossmann et a l . , 1980). Paradoxically, the most r e l i a b l e electrophysiological data on the projections of the SUT suggests that i t has excitatory actions, at least in the SN (Hammond et a l . , 1978). As represented in Figure 58, the loss of an excitatory input to the SNr would be expected to result in a movement disorder characterized by increased motor a c t i v i t y because decreasing the 254 f i r i n g rate of these nigr a l c e l l s i s associated with increased movement. In t h i s context then, hyperkinesia i s the natural consequence of a lesion of excitatory projections a r i s i n g from the SUT. The types of movements seen with damage of the SUT d i f f e r from those associated with SNr efferents, however, and i t is l i k e l y , considering the lesion placements which counteract the b a l l i s t i c movements ( i . e . the entopeduncular nucleus and the ventralateral nucleus of the thalamus; see DeLong and Georgeopoulos, 1981), that the c l i n i c a l syndrome i s related more to the SUT innervation of EP than of SNr. The hyperkinesia a r i s i n g from picrotoxin injections into the EP (Crossman et a l . , 1980) cannot be accounted for in this scheme. GLOBUS PALLIDUS The major source of afferents to the GP i s the CP. The dendrites of p a l l i d a l neurons are almost t o t a l l y covered by GABA or enkephalin-containing terminals derived from th i s projection (Ribak et a l . , 1979; Somogyi et a l . , 1982). Electrophysiological data indicate that the GABAergic input innervation of the GP by the CP i s inhibitory (Yoshida and Obata, 1977), as i s the enkephalinergic input (Napier et a l . , 1983). This finding agrees with the observation that i n h i b i t i o n is the major response of GP neurons to CP stimulation. A small (15 %) excitatory component is seen as well (Mogenson et a l . , 1983), perhaps from the substance P and/or enkephalin-containing projection (Staines et a l . , 1980a). In some animal preparations, p a l l i d a l neurons are noted to have a high f i r i n g rate with a periodic pattern (DeLong and Georgopouos, 1981; Park et a l . , 1982). This periodic pattern 255 does not appear to arise from i t s s t r i a t a l input and i t has not yet been determined i f i t is due to one of the other afferents to the GP or whether i t is an i n t r i n s i c property of p a l l i d a l neurons (Park et a l . , 1982). In the putamen of the monkey, a study of the f i r i n g rate of neurons in r e l a t i o n to various movements revealed a somatotopic organization with the hindlimb represented in the dorsal putamen, forelimb representation in the ventrolateral putamen, and representation of the head and neck in the ventromedial putamen (see DeLong and Georgopoulos, 1982). The f i r i n g rates of caudate c e l l s were not well correlated to these d i s t a l movements. A similar somatotopic d i s t r i b u t i o n was found for the f i r i n g of GP neurons associated with these same movements, with the exclusion of the rostrodorsomedial GP which is connected to the caudate nucleus. Considerations from other work would suggest that the caudate nucleus, and presumably the rostrodorsomedial GP, is associated with activation of a x i a l musculature (see Schneider and Lidsky, 1981). These data, derived from correlations between f i r i n g rates and movements, are borne out by the somatotopy suggested by the d i s t r i b u t i o n of the c o r t i c a l input to the CP ( G a r c i a - R i l l et a l . , 1979; Kunzle et a l . , 1975; Tanaka et a l . , 1981 ). The observation that p a l l i d a l f i r i n g rate is correlated to movement in the same topographical manner as are s t r i a t a l neurons points to the active involvement of the GP in movement. Given the largely i n h i b i t o r y nature of the s t r i a t o p a l l i d a l projection, one might predict that p a l l i d a l neurons would be inactive during movement. However, the above data indicate that 256 this is not the case, and suggest instead that those p a l l i d a l neurons relevant to the movement are spared s t r i a t a l i n h i b i t i o n or actually excited by the CP. It should be mentioned that some of the excitatory influences acting on the GP may derive from the innervation i t receives from the SUT, although t h i s i s based on inference from the excitatory effects of the SUT on the SN. Direct evaluation of the effects of SUT stimulation on the a c t i v i t y of p a l l i d a l neurons has suggested that although excitatory effects occur, the predominant effect i s i n h i b i t i o n (Perkins and Stone, 1980); however, a well controlled study may well show the subthalamic projection to the GP to be excitatory. There i s a large but generally inconclusive l i t e r a t u r e on the e f f e c t s o f ' p a l l i d a l lesions on motor behavior in experimental animals. On the whole, these studies have found that such lesions do not produce obvious a l t e r a t i o n s in gross motor behaviors (see DeLong and Georgopoulos, 1981). Some workers have attributed this lack of effect to the a b i l i t y of visual information to allow the animals to compensate for the effects of p a l l i d a l l e s i o n . Experimental strategies eliminating t h i s variable reveal greater motor dysfunction subsequent to lesions of the GP (Hore et a l . , 1977). Another possible reason for these negative findings i s that the majority of studies have used electrocoagulation of the GP, which i s in fact a multiple lesion, destroying not only the GP but also many of the efferents and and afferents of the CP and cortex. It i s widely observed that the effects of lesions of basal ganglia nuclei can be reversed by lesions of'other brain areas associated with t h i s system. Thus, 257 the hemiballistnus resulting from lesions of the SUT may be p a r t i a l l y counteracted by lesions of the EP, ventromedial nucleus of the thalamus, or motor cortex and lesions of the p a l l i d a l complex or ventrolateral thalamus can p a r t i a l l y reverse some of the symptoms of Parkinson's disease (see DeLong and Georgopoulos, 1981) . There is abundant data from experiments using pharmacological manipulation of the GP (discussed above) to suggest that t h i s nucleus is important for normal motor function. GLOBUS PALLIDUS VS. VENTRAL PALLIDUM As mentioned b r i e f l y in the introduction, the nucleus accumbens (NA) is considered one of the components of the basal ganglia. Its s i m i l a r i t i e s to the CP suggest that in some respects i t may be viewed as a ventral extension of t h i s nucleus. An impressive array of histochemical and immunohistochemical data indicate that the GP has an analagous rostromedial extension which has been termed the ventral pallidum (VP) (Switzer et a l . , 1982) . This analogy may be applied to many other components of the basal ganglia as well and gives r i s e to the impression that there are p a r a l l e l motor and limbic basal ganglia systems. The CP d i f f e r s somewhat from the NA in a number of ways, but most notably in that the l a t t e r receives telencephalic inputs from the amygdala and hippocampus as opposed to the neocortex (see Nauta et a l . , 1978), although some input from parts of the fr o n t a l cortex and entorhinal cortex are now recognised (Beckstead, 1979b; Krayniak et a l . , 1981; Reep and Winans, 1982). The NA receives a dense dopaminergic input from the ventral tegmental area (VTA) and retrorubral nucleus (RR) which l i e 258 adjacent to and p a r t i a l l y overlap the dopaminergic SNc neurons innervating the CP (Nauta et a l . , 1978; Carter and Fibiger, 1977). Rather than project to the GP, as i t i s demarcated by most workers, the NA projects to the ventral pallidum, l a t e r a l preoptic area and f i e l d s within the l a t e r a l hypothalamus medial to the entopeduncular nucleus (Nauta et a l . , 1978). The innervation of the GP and VP by the CP and NA, respectively forms a topographical continuum (Mogenson et a l . , 1983) and agrees with the proposal that these systems should be considered p a r a l l e l . Furthermore, in the rat there i s some degree of overlap such that p a l l i d a l neurons cannot be assumed to belong to the ventral pallidum (defined as in receipt of NA afferents) merely by their location ventral to the decussation of the anterior commissure. Like the response of p a l l i d a l neurons to s t r i a t a l stimulation, the response of VP neurons to NA stimulation in predominantly inhib i t o r y (Mogenson et a l . , 1983), and there i s evidence that GABA i s used as a transmitter in both pathways (Nagy et a l . , 1978a; Fonnum and Walaas, 1979). Injections of retrograde tracers into the ventral parts of the CP (but s t i l l not within the NA) results in the l a b e l l i n g of c e l l bodies in the ventral pallidum (unpublished observation), but i t has not as yet been determined i f there is a VP projection to NA. Nauta et a l . (1978) commented on the absence of an efferent pathway from the NA analogous to the striatoentopeduncular projection. Considerations of the efferent projections of the EP in the rat, however, suggest that a population of c e l l s medial to 259 the t i p of the internal capsule and a population of c e l l s ventral to the EP, in the nucleus of the ansa l e n t i c u l a r i s , might be analogous to the EP (Larsen and McBride, 1979; van der Kooy and Carter, 1981; Parent et a l . , 1981a). Drawings of the efferent projections of the NA reveal that both of these regions could receive inputs and, therefore, could represent a p a r a l l e l of the CP innervation of the EP (Nauta et a l . , 1978; Mogenson et a l . , 1983). Furthermore, c e l l s in the l a t e r a l hypothalamus, medial to the t i p of the internal capsule, project to the VTA and RR and t h i s terminal f i e l d precisely overlaps the regions giving r i s e to the dopaminergic innervation of the NA (Nauta and Domesick, 1978). This finding represents a departure from the analogy, as the EP has not been seen to project to the SN. In keeping with the topography of s t r i a t o n i g r a l projections, in which successively more ventral s t r i a t a l areas project to successively more dorsal n i g r a l regions, the NA (as a ventral continuation of the CP) projects heavily to the SNc and upper reaches of the SNr (Nauta et a l . , 1978). The NA projects to a much greater region of the dopaminergic perikarya than i t receives input from. In fact, the efferents of the NA could influence v i r t u a l l y a l l of the dopaminergic c e l l s innervating the striatum (Nauta et a l . , 1978). This observation has c a l l e d into question the concept of the point to point r e c i p r o c i t y in the n i g r o s t r i a t a l feedback loop discussed in Experiment 1. As mentioned above, the efferent projections of the NA appear to p a r a l l e l the system of s t r i a t a l efferents. They deviate however in that the accumbens also projects to the 260 mediodorsal nucleus of the thalamus (Nauta et a l . , 1978). It w i l l be recalled that a noncholinergic projection to the mediodorsal nucleus from the ventral GP and VP was discussed in Experiment 2. Thus, i t appears that the NA and VP share in a thalamic projection which has no analogy in the connections of the CP and GP, but may share a role similar to the projection from the EP to the ventrolateral nucleus of the thalamus. The mediodorsal nucleus projects to prefrontal cortex which then in turn innervates the NA (Jones and Leavitt, 1975; Beckstead, 1979b) and the ventrolateral thalamus projects to the motor and premotor f r o n t a l cortex which form part of the innervation of the CP (Jones and Leavitt, 1975; Kunzle et a l . , 1975; Jones et a l . , 1977). One of the histochemical features c h a r a c t e r i s t i c of the GP, EP and VP is their intense reaction for the presence of iron. This feature is also noted in the ventral thalamus and the mediodorsal nucleus . The presence of iron within the ventral thalamus has been suggested to arise from the entopeduncular efferents to i t , and i t may be that that in the mediodorsal thalamus arises from a projection from VP (Switzer et a l . , 1982). As shown by Lehmann et a l . , (1980), the VP at r o s t r a l levels contains r e l a t i v e l y few neurons c l a s s i f i e d as belonging to the nucleus basalis magnocellularis (nBM), defined by histochemical means. Although both the NA projection to VP and the CP projection to GP could innervate portions of the nBM, neither seem to be s p e c i f i c a l l y targeted on this neuronal population. In primates the nBM forms a more d e f i n i t e nucleus well removed from 261 the terminal f i e l d s of s t r i a t a l efferents and i t is doubtful that these neurons are involved to any great extent with' the c i r c u i t s of the basal ganglia. There appear, therefore, to be p a r a l l e l systems within this expanded concept of the basal ganglia related to both motor and limbic areas, (Fig. 59). The anatomical associations of the motor basal ganglion are compatable with modulation of the a c t i v i t y of s k e l e t a l musculature but those of the limbic basal ganglia may modulate aff e c t and motivation (see Nauta and Domesick, 1978). In the rat, stimulation of the CP results in o r o f a c i a l movements and stimulation of the NA gives r i s e to an increase in locomotor a c t i v i t y (Anden and Johnels, 1977). Somatotopic considerations discussed above however, suggest that representations of the musculature involved in locomotion would reside in the dorsal and l a t e r a l CP and that i f indeed the NA were simply an extension of the ventral parts of the CP i t would be associated with movements of r o s t r a l body parts. Furthermore, the NA does not receive sensorimotor c o r t i c a l input l i k e that projecting to the CP, but instead has inputs a r i s i n g from limbic structures such as the amygdala, hippocampus and prefrontal cortex. From an anatomical viewpoint the NA appears i l l suited to control of the mechanics of locomotion and may instead control the motivational aspects of locomotion. Bearing on t h i s point are experimental data in animals which show that the d i f f e r e n t i a l symptoms of Parkinson's disease, akinesia and r i g i d i t y , produced in t h i s case by reserpine administration, are obviated by the infusion of dopamine agonists into the NA and CP respectively 262 Figure 5 9 . Schematic diagram of some the connections of the motor c i r c u i t and the limbic c i r c u i t of the basal ganglia. C o l l a t e r a l i z a t i o n and monosynaptic connections are not implied. 262 264 (Anden and Johnels, 1977). These data f i t the c o r o l l a r y that dysfunction of the NA would result in a motivational motor d e f i c i t , akinesia, and s t r i a t a l dysfunction would lead to a mechanical motor d e f i c i t , r i g i d i t y . One pathway by which the limbic basal ganglia c i r c u i t s may influence the motor basal ganglia c i r c u i t s i s , as described above, the possible innervation by the NA of v i r t u a l l y a l l of the dopaminergic neurons projecting to the CP. The significance of t h i s pathway to the locomotor effects of NA stimulation could be tested by examining the effect that selective lesion of the s t r i a t a l dopamine input has on locomotion induced by infusion of dopaminergic drugs into the accumbens. Existing psychopharmacological data are consistent with an interaction between these two systems (Kelly, 1975; Kelly and Moore, 1977). Another pathway by which these two c i r c u i t s could interact is via the projection from the EP and l a t e r a l hypothalamus to the l a t e r a l habenula which in turn gives r i s e to the major innervation of the dorsal raphe. The dorsal raphe sends d i f f u s e , c o l l a t e r a l , serotonergic projections to the SN and CP, innervates the NA, and is also one of the major sources of input to the GP (van der Kooy and Hattori, 1980b; Parent et a l . , 1980b; Pasik et a l . , 1981). Thus, projections of the limbic c i r c u i t s of the basal ganglia can p o t e n t i a l l y influence both the serotonergic and dopaminergic inputs to the motor regions of the basal ganglia. Injection of dopamine into the NA increase locomotor a c t i v i t y in the rat (Anden and Johnels, 1977) as do picrotoxin injections into the ventral tegmental area, which presumably 265 reduce i n h i b i t i o n of these dopaminergic c e l l s projecting to the NA (Mogenson et a l . , 1980). It has been proposed that t h i s increase in motor a c t i v i t y occurs through a projection from the NA to the GP (Mogenson et a l . , 1980) as injections of picrotoxin into the GP increase the locomotor a c t i v i t y (an eff e c t most often associated with the NA as opposed to the CP) in otherwise untreated animals, and injections of GABA into the GP antagonize the hyperactivity induced by dopaminergic stimulation of the NA. This proposal however lacks an anatomical basis. The NA does not have a s i g n i f i c a n t projection to the GP (Mogenson et a l . , 1983; Experiment 3). An alternate hypothesis which could account for these findings and s t i l l be compatable with the known anatomical organization would suggest that the reversal of the locomotor ac t i v a t i o n results not from the GABA induced blockade of the NA ef f e c t s on the GP but by blocking the CP effects on the GP which arise from a NA stimulation of the CP through the SNc. SUMMARY The CP has long" been considered the only s i g n i f i c a n t fenestra for entry of afferent inputs to the basal ganglia. It has largely inhibitory projections to the output nuclei of the basal ganglia, the EP and SNr. The somatotopically organized c o r t i c a l input to the SUT would seem to force consideration of th i s nucleus as an additional channel for input into t h i s system. The SUT appears to to have excitatory inputs to the SNr and therefore, because of the c o l l a t e r a l i z a t i o n of the outputs from the SUT, probably to the EP as well. The GP has reciprocal connections with both the CP and the SUT and i t also innervates 266 the-SN and EP. Therefore, i t would appear to be in a position to influence the c i r c u i t that f a c i l i t a t e s motor function (CP efferents) as well as that which i n h i b i t s motor a c t i v i t y (SUT efferents). S p e c i f i c a l l y , the GP may have the unique property of being excited by the c i r c u i t which is ultimately inhibitory and inhibited by the c i r c u i t which i s ultimately f a c i l a t o r y to movement. It also has inhi b i t o r y terminals advantageously positioned on the output neurons of the SNr and EP which would result in motor f a c i l i t a t i o n . These concepts are depicted in Figure 60. 267 Figure 60. Schematic diagram summarizing the major connections of the basal ganglia. Pathways are la b e l l e d (+ or -) according to their o v e r a l l effect on motor behavior ( f a c i l i t a t o r y or i n h i b i t o r y ) . The abbreviations sp and nsp refer to s p e c i f i c and nonspecific thalamocortical projections. 268 V + coarse V^. motor control 269 REFERENCES Anden,N.-E. and Johnels, B. (1977) Effect of l o c a l application of apomorphine to the corpus striatum and to the nucleus accumbens on the reserpine-induced r i g i d i t y in rats. 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