LIBRARY Mlchlgan State University PLACE IN RETURN BOX to remove this c heokout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE LT T j; * 1 — ~71 — a %T% 1 MSU to An Afflnnettve Action/Equal Opportunity Institution cmmt CONNECTIONS OF THE NUCLEUS TEGMENTI PEDUNCULOPONTINUS. A COMBINED LIGHT AND ELECTRON MICROSCOPIC STUDY IN THE RAT. BY Bryan Michael-Paul Spann A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1991 ABSTRACT CONNECTIONS OF THE NUCLEUS TEGMENTI PEDUNCULOPONTINUS. A COMBINED LIGHT AND ELECTRON MICROSCOPIC STUDY IN THE RAT. BY BRYAN MICHAEL-PAUL SPANN The present study was focused on furthering our understanding of the connections of the PPN in the rat. Previous studies have indicated that ascending projections to the basal ganglia and descending projections to the spinal cord originate from the same region of the nucleus tegmenti pedunculopontinus (PPN). Therefore some PPN neurons may have axons which divide into ascending and descending collaterals. Single and double-labeling experiments were conducted to determine the distribution of the cells of origin of these projections and to test for the presence of collateralization. The results indicated that the majority of both ascending and descending fibers arise from separate populations of.PPN neurons which are intermingled throughout the nucleus. Numerous studies have indicated that the substantia nigra pars reticulata (SNR) projects to the PPN. Therefore the distribution and mode of termination of the nigropedunculopontine jprojection. were studied. using “the anterograde tracer Phaseolus vulgaris-leucoagglutinin. The results demonstrated that while the nigral fibers terminated in both subdivisions of the PPN, the subnucleus compactus (PPNc) and. dissipatus (PPNd), the bulk of the fibers terminated in the PPNd. The nigral fibers appeared directed towards specific neurons and neuropil regions. The majority of the synaptic terminals were seen in contact with medium size dendrites. In light of the previous observations, the nigral input may be preferentially distributed to particular subpopulation(s) of PPN projection neurons. Using ChAT immunocytochemistry, the final study examined the distribution of cholinergic neurons within the PPN, particularly within the PPNd which receives the bulk of the nigral input, and analyzed the synaptic arrangement of both cholinergic and non-cholinergic PPNd neurons. The results indicated that numerous cholinergic neurons are present in both subnuclei. Both cholinergic and non-cholinergic PPNd neurons are contacted by two morphologically different types of boutons, some of which may be of nigral origin. Thus the present observations suggest, indirectly, that nigral afferents to the PPN may terminate on both cholinergic and non-cholinergic PPN neurons. ACKNOWLEDGMENT I would like to express my sincere appreciation and thanks to my advisor, Dr. Irena Grofova, for her constant advice, support, encouragement, and sometimes great tolerance with me, during my entire graduate training. I also wish to thank the members of my graduate committee, Drs. Gerry Gebber, David Kaufman, Sharleen Sakai, Duke Tanaka, and Charles Tweedle for their advice, support, and invaluable time. I also wish to express my appreciation and gratitude to the students of the Medical Scientist Training Program and the Department of Anatomy for their support and encouragement during this time. I also owe thanks to numerous individuals whose constant encouragement and friendship supported me through this endeavor. Finally, I wish to acknowledge the College of Osteopathic Medicine for the financial support given to me as a member of the Medical Scientist Training Program. iv TABLE OF CONTENTS List of Figures ............................................ vi Abbreviations ............................................ viii Introduction ................................................ 1 Chapter I Origin of ascending and spinal pathways from the nucleus tegmenti pedunculopontinus in the rat ...7 Introduction .................................... 7 Material and Methods ........................... 11 Results ........................................ 15 Discussion ..................................... 24 Summary and Conclusions ........................ 37 Figures ........................................ 39 Chapter II Light and electron microscopic studies on the nigropedunculopontine projection in the rat ....54 Introduction ................................... 54 Material and Methods ........................... 58 Results ........................................ 62 Discussion ..................................... 67 Figures ........................................ 80 Chapter III Ultrastructural study on the cholinergic and non- cholinergic structures of the nucleus tegmenti pedunculopontinus in the rat ................... 96 Introduction ................................... 96 Material and Methods .......................... 101 Results ....................................... 107 Discussion .................................... 117 Figures ....................................... 136 Concluding Remarks ........................................ 156 Bibliography .............................................. 159 CHAPTER Figure Figure Figure Figure Figure Figure Figure CHAPTER Figure Figure Figure Figure Figure Figure LIST OF FIGURES Location and Subdivisions of the PPN ........ 40-41 Distribution of Ascending Projecting Neurons of the Pontomesencephalic Tegmentum ............ 43 Distribution of Descending Projecting Neurons of the Pontomesencephalic Tegmentum ............ 45 Drawings of HRP Deposits in the Forebrain and Midbrain ................................... 47 Photomicrographs of Single- and Double—labeled PPN cells ....................... 49 Bar Histograms Comparing the Shapes and Sizes of HRP-labeled PPN cells projecting to the Forebrain and/or Spinal Cord ................... 51 Distribution of Single and Double-labeled Cells in the PPN ............................... 53 Drawings of PHA-L Deposits in the PPN .......... 81 Brightfield Photomicrographs of PHA-L Deposits in the PPN ............................ 83 Distribution of Labeled Fibers in the PPN in Case PHA-L #15 .............................. 85 Distribution of Labeled Fibers in the PPN ...... 87 Brightfield Photomicrographs of PHA-L .Immunoreacted Sections Showing the Termination of Nigral Fibers in the PPNd ................... 89 Light and Electron Micrographs of Osmium Postfixed and Plastic Embedded PHA-L Labeled Material ....................................... 91 vi CHAPTER II (CONTINUED) Figure 7: Electron Micrographs Illustrating PHA-L Labeled Nigral Boutons in the PPNd ............. 93 Figure 8: Distribution of Contact Sites of Nigral Terminals ...................................... 95 CHAPTER III Figure 1: Synaptic Relationships of PPN Cells ........... 137 Figure 2: Electron Micrographs Illustrating the Major Terminal Types in the PPN ..................... 139 Figure 3: Distribution of Cholinergic Cells in the PPN..141 Figure 4: Brightfield Photomicrographs of ChAT+ Cells in the PPN .................................... 143 Figure 5: Montage of Electron Micrographs of a Cholinergic PPN Cell .......................... 145 Figure 6: Montage of Electron Micrographs of a Non-cholinergic PPN Cell ...................... 147 Figure 7: Electron Micrographs of Cholinergic Elements and Their Synaptic Relationships in the PPNd..149 Figure 8: Cholinergic vs. Non-cholinergic Cells. A Size Comparison ............................. 151 Figure 9: Terminal Types Contacting Cholinergic PPN Dendrites ................................. 153 Figure 10: Distribution of Boutons in the PPNd ........... 155 vii CG CNF CP DPB EP fr GP HYP IC ic LC LG LH 11 LPB ABBREVIATIONS caudal central grey cuneiform nucleus cerebral peduncle dorsal dorsal parabrachial nucleus entopeduncular nucleus fornix fasciculus retroflexus globus pallidus hypothalamus inferior colliculus internal capsule locus coeruleus lateral geniculate nucleus lateral hypothalamic area lateral lemniscus lateral parabrachial nucleus nucleus of the mesencephalic tract of the trigeminal nerve mesencephalic trigeminal tract medial habenular nucleus medial lemniscus viii MT 0t Pf PL PN PNO PPN PPNC PPNd Rmes RN RPO RR SC scp sm SN SNC SNR SO medial mammillary nucleus motor trigeminal nucleus medial parabrachial nucleus mesencephalic reticular formation medial terminal nucleus of the accessory optic tract optic tract parafascicular thalamic nucleus paralemniscal nucleus pontine nucleus pontine reticular nucleus, oral portion pedunculopontine nucleus pedunculopontine nucleus, subnucleus compactus pedunculopontine nucleus, subnucleus dissipatus rostral mesencephalic reticular formation red nucleus pontine reticular nucleus, oral portion retrorubral nucleus retrorubral field superior colliculus superior cerebellar peduncle stria medullaris of the thalamus substantia nigra substantia nigra, pars compacta substantia nigra, pars reticulata superior olive ix SPTg STH TH 21 3N subpeduncular tegmental nucleus subthalamic nucleus thalamus ventral ventral nucleus of the lateral lemniscus ventromedial hypothalamic nucleus ventral parabrachial nucleus ventral tegmental area zona incerta oculomotor nerve INTRODUCT ION The nucleus tegmenti pedunculopontinus (PPN) has been defined by Olszewski and Baxter ('54) in the human as a nucleus which occupies the lateral pontomesencephalic tegmentum and extends from the caudal border of the red nucleus to the parabrachial nuclei. On the basis of cellular density, the authors distinguished two subdivisions of the PPN, the subnucleus compactus (PPNc) and the subnucleus dissipatus (PPNd). While the nucleus and its subdivisions are clearly discernible in other primates, they are far less distinct in non-primates. However, in all species the PPN contains a prominent component of cholinergic neurons which comprise the Cholinergic sector (Ch) 5 of Mesulam and co- workers (‘83, '84, '89). It was not until the advent of modern neuroanatomical tracing techniques that the extensive interaction between the PPN and the forebrain as well as brainstem structures and the spinal cord was established. While a large portion of the ascending projections is directed towards the thalamus (Jackson and crossman, '81, '83; Moon-Edley and Graybiel, '83; Sugimoto and Hattori, '84; Sofroniew et al., '85; Isaacson and Tanaka, '86; Woolf and Butcher, '86; Hallanger et al., '87; Jones et al., '87; Pare et al., '88; Steriade et al., '88; Fitzpatrick et al., '90; Semba et al., '90), the PPN is also reciprocally connected with the hypothalamus and the basal forebrain (Jackson and Crossman, '81; Moon—Edley and Graybiel, '83; Parent and DeBellefeuille, '83; Swanson et al., '84; Mogenson et al., '85; Woolf and Butcher, '86; Jones et al., '87; Hallanger and Wainer, '88; Jones and Cuello, '89; Sakai et al., '90). Furthermore, both cholinergic and non-cholinergic neurons appear to project to these forebrain targets of the PPN ( Sofroniew et al., '85; Isaacson and Tanaka, '86; Woolf and Butcher, '86; Pare et al., '88; Steriade et al., '88; Jones and Cuello, '89; Fitzpatrick et al., '90; Sakai et al., '90; Semba et al., '90). The ascending projections of the PPN appear to have important roles in sleep and wakefulness. The cholinergic input to the thalamus is postulated to be an important component of the ascending reticular activating system or ARAS (Mesulam et al., '83, '89; Woolf and Butcher, '86; Hallanger et al., '87; Pare et al., '88; Steriade and Llinas, '88; Steriade et al., '88). The ARAS is involved in the regulation of thalamocortical transmission which controls EEG desynchronization. In addition, both the hypothalamus and the basal forebrain also take part in the regulation of EEG desynchronization and thus may be influenced by PPN efferents (Woolf and Butcher, '86; Hallanger and Wainer, '88; Jones and Cuello, '89; Sakai et al., '90). Furthermore, cholinergic projections from the PPN to the lateral geniculate nucleus of thalamus have been implicated in the regulation of 3 paradoxical or rapid eye movement (REM) sleep (De Lima and Singer, '87; Hallanger et al., '87; Pare et al., '88; Steriade et al., '88; Mesulam et al., '89; Fitzpatrick et al., '90). In addition to their roles in EEG desynchronization and REM sleep, the PPN neurons appear to participate in various aspects of motor control. Numerous anatomical studies have documented projections from the PPN to the basal ganglia (De Vito et al., '80; Nomura et a1, '80; Saper and Lowey, '82; Parent et al., '83; Jackson and Crossman, '83; Moon-Edley and Graybiel, '83; Sugimoto and Hattori, '84; Woolf and Butcher, '86; Beninato and Spencer, '87; Clarke et al., '87; Rye et al., '87; Scarnati et al., '87a; Lee et al., '88; Gould et al., '89; ). Several investigators have reported that these projections are partially cholinergic (Woolf and Butcher, '86; Beninato and Spencer, '87; Clarke et al., '87; Gould et al., '89) and supply an excitatory input.to the basal ganglia (Gonya-Magee and Anderson, '83; Hammond et al., '83; Scarnati et al., '84, '87b). Furthermore, the PPN receives converging input from several basal ganglia nuclei (globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata) (Nauta and Mehler, '66; Kim et al., '76; Carter and Fibiger, '78; Nauta and Cole, '78; Nauta, '79; Rinvik et al., '79 Beckstead et al., '79; Larsen and McBride, '79; McBride and Larsen, '80; Carpenter et al., '81; Jackson and Crossman, '81, '83; van der Kooy and Carter, '81; Arbuthnott and Wright, '82; De Vito and Anderson, '82; Beckstead and Frankfurter, '82; Gerfen et al., '82; Moon- Edley and Graybiel, '83; Parent, '86; Schneider, '86; Rye et al., '87; Kita and Kitai, '87; Moriizumi et al., '88; Nakamura et al., '89; Smith et al., '90) and these afferents may have a modulating influence on the excitatory feedback loops. Recent studies have demonstrated that the PPN gives rise to descending projections to the pontomedullary reticular formation (Jackson and Crossman, '83; Moon—Edley and Graybiel, '83; Garcia-Rill, '86; Mitani, et al., '90; Rye et al., '88; Woolf and Butcher, '89; Jones, '90; Semba et al., '90; Yasui et al., '90; 'Grofova et al., '91) and to the spinal cord (Jackson and Crossman, '83; Swanson et al., '84; Jones and Yang, '85; Jones et a1, '86; Goldsmith and van der Kooy, '88; Rye et al., '88; Woolf and Butcher, '89). In the rat and cat, the PPN represents the central component of the mesencephalic locomotor region (n: MLR (Garcia-Rill, '86; Garcia-Rill et al., '87). These locomotor effects may be mediated either indirectly through reticulospinal pathways (Garcia-Rill and Skinner, ‘87a, '87b) or directly via spinal connections (Goldsmith and van der Kooy, '88; Rye et al., '88). While both cholinergic and non-cholinergic neurons take part in the PPN projection to the reticular formation, only non-cholinergic neurons project to the spinal cord (Goldsmith and van der Kooy, '88; Rye et al., '88; Woolf and Butcher, '89). In addition, the cholinergic projections to the pontomedullary reticular formation have been implicated 5 as inducing motor atonia during REM sleep (Woolf and Butcher, '89; Jones, '90; Semba et al., '90). Lesions of the PPN in the rat have been associated with impaired motor functions (Kilpatrick and Starr, '81), and clinical studies have shown an association between PPN cell loss in humans and movement disorders related to progressive supranuclear palsy (Zweig et al., '87; Hirsch et al., '87) and Parkinson's disease (Hirsch et al., '87; Jellinger, '88; Zweig et al., '89). Although significant advances have been made in our understanding of the PPN since the first experimental study 25 years ago (Nauta and Mehler, '66), new knowledge from a variety of disciplines continues to open avenues for further research. At present it is clear that the PPN contains a heterogeneous population of projection neurons with widespread ascending and descending connections which are involved in a variety of motor as well as non-motor functions. The general sources of afferent input to the PPN have also been identified. However, it is still largely unknown whether the different afferent inputs are evenly distributed. to all PPN neurons irrespective of their transmitter content or projection site(s). Furthermore, it is still unclear whether there exist intrinsic interactions among the chemically different subpopulation of the PPN neurons and among the PPN neurons projecting to different targets. Such questions have guided our research efforts which focused mainly on the relationship of the basal ganglia to the PPN. The experiments have been carried out in the rat 6 which has been the most commonly used experimental animal in the previous investigations on the structure and functions of the PPN. The major objective of the first study was to determine whether the PPN neurons projecting to the basal ganglia and those projecting to the spinal cord were contained in the same portion of the nucleus, and whether they represent two different populations or a single population of neurons with collateralized axons. In the second study, the distribution and mode of termination of the nigro-pedunculopontine fibers has been analyzed both light and electron microscopically. The third study explored the synaptic organization and the presence of cholinergic elements in the region of the PPN receiving nigral input. CHAPTER. I The Origin of Ascending and Spinal Pathways from the Nucleus Tegmenti Pedunculopontinus in the Rat INTRODUCTION The nucleus tegmenti pedunculopontinus (PPN) has been defined in the human brainstem by Olszewski and Baxter ('54) as a nucleus of "unknown connections" which occupies the ventrolateral part of the caudal mesencephalic tegmentum lateral to the superior cerebellar peduncle. Furthermore, on the basis of the cell densities, the authors distinguished two subdivisions, the subnucleus compactus (PPNC) and the subnucleus dissipatus (PPNd). The nucleus remained obscure until it was discovered that it receives converging input from the basal ganglia and associated nuclei (Nauta and Mehler, '66; Kim et al., '76; Carter and Fibiger, '78; Nauta and Cole, '78; Nauta, '79; Rinvik et al., '79; Beckstead et al., '79; Larsen and McBride, '79; McBride and Larsen, '80; Jackson and Crossman, '81, '83; van der Kooy and Carter, '81; De Vito and Anderson, '82; Beckstead and Frankfurter, '82; Gerfen et al., '82; Parent and DeBellefeuille, '82, '83; Moon-Edley and Graybiel, '83; Gonya—Magee and Anderson, '83; Noda and Oka, '84). These findings suggested that the PPN may be involved in relaying information from the basal ganglia to the motor centers of the medulla and spinal cord. 8 However, further studies have revealed that the majority of the PPN efferent projections stream rostrally toward the basal ganglia and associated nuclei (De Vito et al., '80; Nomura et al., '80; Saper and Loewy, '82; Parent et al., '83a; Jackson and Crossman, '83; Moon-Edley and Graybiel, '83; Gonya-Magee and Anderson, '83; Lutze and Rafols, '84; Sugimoto and Hattori, '84; Woolf and Butcher, '86; Beninato and Spencer, '87; Clarke et al., '87; Rye et al., '87; Scarnati et al., '87a) thereby establishing feedback loops which provide an excitatory, modulating influence on these nuclei (Gonya-Magee and Anderson, '83; Hammond et al., '83; Scarnati et al., '84). Recent studies on the anatomy and functions of the pontomesencephalic tegmentum have provided convincing evidence that in the rat and cat, the PPN represents the central component of the mesencephalic locomotor region (MLR) (Garcia-Rill, '86). Furthermore, it has been repeatedly demonstrated that the PPN gives rise to descending projections to the medullary reticular formation (Jackson and Crossman, '83; Moon-Edley and Graybiel, '83; Garcia-Rill, '86; Rye et al., '88) and to the spinal cord (Jackson and Crossman, '83; Lutze and Rafols, '84; Swanson et al., '84; Jones and Yang, '85; Jones et al., '86; Goldsmith and van der Kooy, '88; Rye et al., '88). These findings substantiate early suggestions that, via connections with the PPN, the basal ganglia may control some aspects of locomotion circumventing the thalamus and cerebral cortex. In addition, 9 since .the PPN is reciprocally connected with various hypothalamic nuclei and limbic regions of the forebrain (Jackson and Crossman, '81; Moon-Edley and Graybiel, '83; Parent and DeBellefeuille, '83; Swanson et al., '84), it has been proposed that the two descending PPN pathways may play an important role in mediating locomotor responses associated with various behaviors subserved by the hypothalamic and limbic regions (Swanson et al., '84). The interest in the connectivity and functions of the PPN has been further intensified by the results of immunohistochemical studies which have revealed that the pontomesencephalic region including the PPN contains a group of cholinergic neurons designated as the ChS sector by Mesulam et al. ('83). These cholinergic neurons appear to contribute to the descending PPN projections to the medullary reticular formation (Garcia—Rill and Skinner, '87; Goldsmith and van der Kooy, '88; Rye et al., '88) and to the ascending projections to the basal ganglia, hypothalamus and limbic regions (Sugimoto and Hattori, '84; Woolf and Butcher, '86; Beninato and Spencer, '87; Clarke et al., '87). However, they also project heavily to the thalamus (Sugimoto and Hattori, '84; Isaacson and Tanaka, '86; Woolf and Butcher, '86; Rye et al., '87) and provide widespread cholinergic innervation of functionally different groups of thalamic nuclei (Hallanger et al., '87). Thus it seems that the PPN functions are more diversified in that in addition to the locomotor function, the PPN may also be implicated in the 10 control of thalamocortical transmission and cortical activity (Hallanger et al., '87; Rye et al., '87). The present study has focused on the relationships of the PPN to the basal ganglia and to the spinal cord. In particular, it analyzes the distribution patterns and morphological features of the two jpopulations of PPN projection neurons, and examines the possibility that some PPN axons may collateralize into long ascending and descending branches which would innervate both the basal ganglia and spinal cord. A preliminary account of this work has been published (Spann and Grofova, '84). 11 MATERIAL AND METHODS A total of 25 male Sprague-Dawley albino rats (275-350 g) were utilized for this study. Of these, 12 received injections of horseradish peroxidase (HRP) or wheat germ agglutinin-conjugated horseradish peroxidase (HRP/WGA) into the basal ganglia or cervical cord, while 13 received injections of fluorescent dyes Granular Blue (GB) and Diamidino Yellow Dihydrochloride (DYD) into the same targets. Animals were anesthetized with sodium pentobarbital (50-100 mg/kg, i.p.) and atropine sulfate solution (0.7 mg/kg) was administered intramuscularly in order to prevent brain edema. Pressure injections of tracers were made using 5 [.11 Hamilton syringes fitted with 26 gauge bevelled needles and mounted in a holder of a Kopf stereotaxic instrument. Stereotaxic coordinates were derived from the atlas of Paxinos and Watson ('82). Injections into the spinal cord were made under direct visual control following laminectomy. W In ‘the first series of ,’ L k {Vt Figure 3 Figure 4: 86 Distribution of Labeled Fibers in the PPN. Diagrams comparing the distribution of PHA-L labeled fibers in the PPN following injections in either the ventral (lA-C) or dorsal (2A-C) portion of the SNR. Levels A-C correspond to levels B-D in Figure 3. 87 Figure 4 88 Figure 5: Brightfield Photomicrographs of PHA-L Immunoreacted Sections Showing the Termination of Nigral Fibers in the PPNd. A: Arborizations of thin fibers exhibiting numerous varicosities (large open arrows) are concentrated in small areas of the nucleus. Some cells (small open arrows) are located outside of the dense patches of the terminal plexus. Scale bar: 100 um. B: Labeled varicose fibers (open arrows) surround the cell body and dendrites of a medium-sized PPNd neuron. Two adjacent cells (arrows) are devoid of apposing nigral fibers. Scale bar: 20 um. 89 Figure 5 90 Figure 6: Light and Electron Micrographs of Osmium Postfixed and Plastic Embedded PHA-L Labeled Material. A: Low-power photomicrograph showing the PPNd region containing a prominent plexus of PHA—L labeled fibers. The borders of the subnucleus are indicated by the discontinuous line while the solid line outlines the area selected for ultrastructural analysis. Note the large diameter fiber dorsal to the PPNd (arrow). Scale bar: 250 mm. B: Higher magnification of the area outlined by the solid line in A. Arrow indicates PHA-L labeled fiber, arrowhead indicates terminal varicosities. Scale bar: 25 mm. C: Electron micrograph of a labeled, thinly myelinated axon (arrow) adjacent to two nonlabeled axons ( asterisks ). Scale bar: 1.0 mm. 92 Figure 7: Electron Micrographs Illustrating PHA-L Labeled Nigral Boutons in the PPNd. A: Two darkly labeled boutons (Bl and 82) containing prominent centrally placed mitochondria contact the cell body (CB) and proximal dendrite (D) of a PPNd neuron. B: A PHA-L labeled bouton synapsing (open arrow) on a large dendrite (D). C: A medium-sized dendrite (D) is contacted by a large labeled terminal (Bl) and two unlabeled boutons (B2 and B3). Scale bars: 1.0 gm. 93 7 Figure 94 Figure 8: Distribution of Contact Sites of Nigral Terminals. The distribution of labeled terminals was analyzed in electron micrographs from a region indicated in Figure 6A. 95 DISTRIBUTION OF CONTACT SITES OF NIGRAL TERMINALS 50% 4'! N: 63 40%J 38% 30% 30% n 19% 20% “ 13% 10%“ 0% / . SOMA 22* <231* .1. *DENDRITES, width in um Figure 8 CHAPTER. III Ultrastructural study on the cholinergic and non-cholinergic structures of the nucleus tegmenti pedunculopontinus in the rat. INTRODUCTION The brainstem of various primate (Mesulam et al., '84, 89; Smith and Parent, '84; Satoh and Fibiger, '85; Mizukawa et al., '86) species contains a prominent group of cholinergic neurons surrounding the brachium conjunctivum and interspersed among its fibers. These cholinergic neurons are largely situated within a region designated as the nucleus tegmenti pedunculopontinus (PPN) by Olszewski and Baxter ('54) in the human brainstem. While cholinergic neurons are found within both divisions of the PPN, the pars compacta (PPNC) and pars dissipata (PPNd), they appear to peak in density in the PPNc (Mesulam et al., '89). Several studies (Mesulam et al., '84, '89) have also indicated that there is a population of non-cholinergic neurons intercalated with the cholinergic neurons throughout the PPN. This terminology has been applied in the non-primate brainstem to the cells similarly located in the tegmentum surrounding the superior cerebellar peduncle and its decussating fibers in front of the parabrachial nuclei (Armstrong et al., '83; Mesulam et 96 97 al., '83; Beninato and Spencer, '86, '87; Isaacson and Tanaka, '86; Woolf and Butcher, '86; Clarke et al., '87; Jones and Beaudet, '87a; Gould et al., '89; Hall et al., '89; Jones and Cuello, '89; Fitzpatrick et al., '90; Jones, '90; Semba et al., '90). Double-labeling studies have demonstrated that in both primate and non-primate species the cholinergic and non-cholinergic PPN neurons project to the cortex (Vincent et al., '83), thalamus (Sofriniew et al., '85; Isaacson and Tanaka, '86; Woolf and Butcher, '86; Pare et al., '88; Steriade et al., '88; Fitzpatrick et al., '90; Semba et al., '90), superior colliculus (Beninato and Spencer, '86; Woolf and Butcher, '86; Hall et al., '89), basal ganglia (Woolf and Butcher, '86; Beninato and Spencer, '87; Clarke et al., '87; Gould et al., '89), basal forebrain (Woolf and Butcher, '86; Jones and Cuello, '89), hypothalamus (Woolf and Butcher, '86; Sakai et al., '90) and pontine (Semba et al., '90) and medullary (Jones, '90) reticular formation. As a result of these widespread projections, the cells of the PPN have been implicated in a variety of functions. The cholinergic PPN neurons projecting to the thalamus have been considered as important components of the ascending reticular activating system and are also associated with the regulation of paradoxical or REM (rapid eye movement) sleep (Mesulam et al., '83, '89; Woolf and Butcher, '86; De Lima and Singer, '87; Hallanger et al., '87; Pare et al., '88; Steriade et al., '88; Fitzpatrick et al., '90). In addition, 98 the descending cholinergic projections from the PPN to the pontine and medullary reticular formation may play a role in the induction of nwtor atonia during REM sleep (Woolf and Butcher, '89; Jones, '90; Semba et al., '90). However, the cholinergic neurons of the PPN may also be involved in other aspects of motor control through ascending projections to the basal ganglia (Woolf and Butcher, '86; Beninato and Spencer, '87; Clarke et al., '87; Gould et a1., '89), intralaminar nuclei which in turn project to the neostriatum (Isaacson and Tanaka, '86), and the motor relay nuclei of the thalamus (Pare et al., '88; Steriade et al., '88). Furthermore, Garcia-Rill and co-workers ('87) have reported that the cholinergic cells of the PPN are co-localized with the mesencephalic locomotor region (MLR). The sizable population of non—cholinergic neurons intercalated with the cholinergic neuron population throughout the PPN has received very little attention in past experiments. While Steriade and co-workers ('90b) have indicated that both the cholinergic and non-cholinergic neurons of the PPN are responsible for the transfer to the thalamus of brain-stem generated ponto—geniculo-occipital waves, which are closely related to REM, there is little experimental evidence on whether cholinergic and non— cholinergic neurons have similar roles in the other aforementioned functions. Many of the previous studies have focused on examining certain characteristics or functions of only cholinergic neurons and thus de-emphasized the non- 99 cholinergic neurons. The segregation of cholinergic neurons from non-cholinergic neurons has been taken to its most extreme form by Rye and co-workers ('87). The authors have concluded that, in the rat, the PPN is composed of only cholinergic neurons which are situated outside of the area that is reciprocally connected. with. the basal ganglia (i.e.midbrain extrapyramidal area or MBA). In addition, they have also suggested that the cholinergic neurons are not involved in either basal ganglia related functions or in the MLR. The segregation of cholinergic neurons and basal ganglia connections has been confirmed by Lee et al., ('88) and a number of authors (Hallanger et al., '87; Hallanger and Wainer, '88; Lee et al., '88; Rye et al., '88) now use the nomenclature of PPN and MBA as defined by Rye and co-workers. Recently, Hall et al., ('89) have indicated there is extensive overlap between the terminal field of nigral fibers and cholinergic neurons of the PPN in the cat. Furthermore, a neurophysiological study on chemically identified neurons in the rat PPN (Kang and Kitai, '90) has suggested that SNR input may be directed to both cholinergic and non-cholinergic cells. Therefore, the definition of the PPN and MBA by Rye and co-workers is becoming increasingly controversial. In a previous study (Spann and Grofova, submitted), we have demonstrated that in the rat the nigropedunculopontine fibers terminate primariLy in the PPNd and suggested that these fibers may be preferentially directed towards a specific subpopulation of PPN neurons. The present study was 100 performed to gain further. insight into the synaptic organization of the PPNd and to establish whether the portion of PPNd receiving nigral input contains cholinergic neurons. Furthermore, particular attention has been paid to the comparison of the fine structure and synaptic organization of cholinergic and non-cholinergic neurons of the PPNd. Preliminary accounts of this work have been previously presented in abstract form (Spann and Grofova, '87, '90). 101 MATERIAL AND METHODS ELECTRON MICROSCOPY The brains of three normal, adult male albino rats were selected from nine brains which were available for the present electron microscopical study. Animals were deeply anesthetized with sodium pentobarbital (80-120 mg/kg, i.p.) and perfused transcardially with 100 mls of sodium phosphate buffered saline solution at 37 °C followed by a fixative consisting of 2% paraformaldehyde and 2% gluteraldehyde (EM grade) in 0.15 M sodium phosphate buffer at pH 7.4 at 4 °C. After perfusion, the brains were immediately removed and kept in the fixative overnight at 4 °C. The brainstem was blocked and cut sagittally on a vibratome into serial sections of 50 (n: 100 um thickness. The sections were thoroughly washed in phosphate buffer and postfixed for 1.0 hrs. in 1.0% osmium tetroxide solution in 0.1 b4 phosphate Ibuffer. Following several washes in distilled H20, the sections were dehydrated in graded acetone solutions and section-embedded in Epon-Araldite between a glass slide and coverslip coated with Liquid Release Agent. Plastic-embedded sections were first inspected in the light microscope and the borders of the PPN were identified according to previously established criteria of Spann and Grofova ('89). The PPN was divided into four lateromedial quarters or levels and one vibratome section was selected from each of the levels for ultrastructural examination. 102 With the 50 um thick sections, the selected region of the PPN for ultrathin sectioning was documented on photomicrographs or line drawings, the coverslip was then removed, and a blank block was cemented on that region under a stereomicroscope. With the 100 pm thick sections, a blank block was first cemented on the PPN and surrounding structures. Semithin sections of 1.0 pm thickness were than taken of this region, stained with P-phenylene-diamine (Hollander and Vaaland, '68) and used to assure correct localization of the PPN region for electron microscopy. Serial ultrathin sections (light-gold interference color) were cut on a Reichart Ultracut E, picked up on Formvar-coated slotted grids and stained with aqueous uranyl acetate and Reynolds lead citrate. The sections were inspected in a JEOL 100 CX electron microscope. For the analysis of appositional relationships of the cell bodies, 41 cells were randomly selected from various levels of the PPN. Only cells where the plane of section had passed through the nucleolus were used. Montages of electron micrographs having a final magnification of 19,800x were made of each cell. The different profiles contacting the somata were color coded and their total length measured using the Bioquant Image Analysis System. Somatic areas of each cell were measured on electron micrographs with a final magnification of 9,900x using the same system. Axonal terminals were categorized according to the subsynaptic specialization, vesicle morphology and organelle content from electron micrographs (final magnification of 103 27,500x) taken at random from the four levels of the PPN. Only terminals with clearly identifiable synaptic contacts were counted. In addition, the length of each terminal (greatest distance between the terminal membranes measured with a line parallel to the synaptic membrane) and postsynaptic target characteristics were recorded. Approximately 3200 um2 of neuropil per level was examined. ChAT IMMUNOCYTOCHEMI STRY Three normal, adult male albino rats were deeply anesthetized with sodium pentobarbital (80-120mg/kg, i.p.) and perfused transcardially with a sodium phosphate buffered saline solution followed by a fixative consisting of 4% paraformaldehyde and 0.1% gluteraldehyde (EM grade) in 0.15M sodium phosphate buffer at pH 7.2. The brains were immediately removed, stored at 4 °C in the fixative for one hour and then transferred to sodium phosphate buffer (pH 7.2) at 4 °C were they remained overnight. Serial 50 mm thick sections were cut on a vibratome in the sagittal plane, collected in Tris buffered saline (TBS) and divided into two series of alternating sections. The first series was processed for light microscopic and the second for electron microscopic ChAT-immunohistochemistry using a modified protocol of Boehringer-Mannheim Biochemicals (BMB). The sections were first rinsed in four changes of TBS (10 mins. each) and then incubated with gentle agitation in a 4 ug/ml 104 dilution of primary monoclonal rat anti-ChAT (BMB) diluted in TBS with 2% Bovine Serum Albumin (BSA), 20% Normal Rabbit Serum (NRS) and 0.1% Triton X-100 for either 48 hrs. at 4 °C or overnight at room temperature. After three 10 min. rinses in TBS, the sections were transferred to a 1:250 dilution of rabbit anti-rat antibody (BMB) with 2% BSA, 20% NRS and 0.1 % Triton X—100 for 60 mins. at room temperature, and after additional rinses, placed for 60 mins. in a 1:250 dilution of rat peroxidase-antiperoxidase (BMB) with 2% BSA, 20% NRS and 0.1% Triton X-100. In order to intensify the reaction, the sections were recycled through the secondary antiserum and peroxidase-antiperoxidase solutions an additional time prior to the DAB reaction. Following several rinses, the sections were placed in a freshly prepared solution containing 100 mg 3, 3' diaminobenzidine or DAB (Sigma), 40 mg NH4Cl, 200 mg beta-D-glucose and 0.4 mg glucose oxidase in 100 ml of 0.15 M Tris buffer for 30-60 mins. After final rinses in Tris buffer, the sections for light microscopy were mounted onto gelatin coated slides, air-dried, dehydrated and either lightly stained with cresyl violet or left unstained. The sections were examined in a Leitz Ortroplan microscope using brightfield illumination. The distribution of labeled cells and fibers in the PPN was documented on both photomicrographs and projection drawings of immuno-reacted sections through the latero-medial extent of the nucleus. The series of sections selected for electron microscopy were processed through all of the aforementioned steps of 105 ChAT immunohistochemistry but the concentration of Triton X- 100 was reduced. to 0.04%. The reacted sections were thoroughly washed in phosphate buffer and then postfixed for 30 mins. in 0.5% osmium tetroxide solution in 0.1 M phosphate buffer. Following several washes in distilled H20, the sections were dehydrated in graded acetone solutions and section-embedded in Epon-Araldite between a glass slide and coverslip coated with Liquid Release Agent. Plastic-embedded sections were first inspected in the light microscope and the distribution of labeled cells and dendrites in the PPN was documented on photomicrographs. A region of the PPNd exhibiting the highest density of labeled cells or dendritic arborizations and previously shown to contain a dense plexus of nigral fibers (Spann and Grofova, submitted) was then identified. The coverslip was removed, a blank block was cemented on the selected region under a stereomicroscope and then trimmed for ultrathin sectioning as described for the normal material. For the analysis of the cell somata, 28 cholinergic and 27 non-cholinergic cells cut through the plane of the nucleolus were selected from the PPNd. All 55 cells were selected from regions of the PPNd containing a ndxture of both cholinergic and non-cholinergic cells. The largest and smallest diameters of each cell soma were measured from montages of electron micrographs having a final magnification of 9000x. Axonal terminals contacting the somata and primary dendrites of (each cell ‘were classified. and. counted in 106 ultrathin sections with a microscope magnification of 10,000x through a 10x viewer. Axonal terminals synapsing on either cholinergic or non- cholinergic dendrites of different sizes in the PPNd were classified and counted from electron micrographs (final magnification of 25,000x) taken at random. 107 RESULTS I. NORMAL ULTRASTRUCTURE OF THE PPN The fOllowing description of tflua fine structure and synaptic organization of the PPN are based on observations from the PPNd and the adjoining portion of the PPNC. Altogether, 41 cells containing the nucleolus and. the surrounding neuropil were analyzed. W The neurons of the rat PPN exhibited soma shapes which ranged from spindle to oval-round. The cross-sectional somatic areas of these neurons ranged between 48 and 270 um2 with a mean of 126.7 umz ( S. D.: 49.8 umz). The perikaryon of the neurons contained rough endoplasmic reticulum, mitochondria, free ribosomes, Golgi apparatus, lysosomes, multivesicular bodies, microtubules and neurofilaments. The nuclear membrane was indented in nearly 90% of the neurons examined. The depth of the invaginations ranged from shallow to deep and was not restricted to any one cell shape or size. Somatic spines or protrusions from the perikaryon of the neurons were only rarely encountered. The surface of the neurons was apposed primarily by myelinated and unmyelinated axons which covered on average 52 and 33% of the somatic membrane. The remaining portion of the somatic membrane was apposed by nerve cell bodies and 108 dendrites, and axon terminals. Particular attention was paid to the number of terminals synapsing on various size somata of PPN neurons (Figure 1). While the overall percentage of the cell membrane covered by axon terminals was relatively small (13%), the range of terminal coverage varied greatly (0-55%). On average, the PPN neurons were contacted by 5 terminals (range: 0-27) per soma. However, the soma of small neurons received fewer terminals than time large neurons. Furthermore, the data suggests that larger neurons (126 um2 or greater in area) may be further subdivided into two groups based on the density of synaptic terminals. One group of the larger neurons was covered by very few terminals (0-9%) while the other group received a substantially greater number of terminals (ls-55%) establishing contact. Ini all neurons examined, the terminals appeared to be randomly distributed along the entire length of the somatic membrane. Of the 41 cells examined, 16 of the neurons had one or more primary dendrites emerging from the cell body. These dendrites did not exhibit any spine-like appendages and nerve terminals seen in contact with these dendrites did not show any particular preference for any specific region of the primary dendrites. Neuronil The neuropil of the PPN consisted of myelinated and unmyelinated axons, glial profiles, dendrites, and axon terminals. The myelinated and unmyelinated axons (Figure 2A) 109 were often organized into Tbundles. The diameters of myelinated axons (myelin sheath included) ranged between 0.4 and 5.0 um. Only a few of the myelinated axons had diameters greater than 3.0 pm. The unmyelinated axons had diameters under 0.6 um. and their cytoplasm contained primarily microtubules. Glial profiles mainly belonged to astrocytes. The majority of dendritic profiles were under 2.0um in diameters. The cytoplasm of dendrites (Figure 2A) contained microtubules, mitochondria, free ribosomes, small sacs of smooth endoplasmic reticulum and vesicular structures. While dendritic profiles with clusters of synaptic vesicles were occasionally noted (Figure 2A), dendrodendritic synapses were not encountered in the present material. Longitudinally cut dendrites were often irregular in shape but did not exhibit the morphological characteristics of dendritic varicosities (Peters et al., '76). Furthermore, the dendrites were generally smooth with only an occasional spine-like process. Ner1e_t.erminals The morphology and distribution of 582 nerve terminals were analyzed in detail. Based on the type of synaptic junction and the shape of the synaptic vesicles, two major types of terminals were identified in the PPN. The Type I terminals (Figure 2A,B) were engaged in asymmetrical synaptic junctions and contained round. or slightly’ ovoid synaptic vesicles. The synaptic cleft measured approximately 25 nm in width. The Type I terminals 110 contained several mitochondria and could be further subdivided into two Subtypes, IA and IB. The Subtype IB terminal (Figure 2A) could be differentiated from the Subtype IA (Figure 2A, B) by the presence of post—junctional dense bodies beneath the postsynaptic density» The Type I terminals ranged in length from 0.4 mm to 3.0 pm with the Subtype IA being slightly larger than the IB. On random sections these type of terminals often represented the ends of unmyelinated axons. The Type II terminals (Figure 2A, C) formed symmetrical synaptic junctions with the synaptic cleft being about 20 nm. They contained clusters of pleomorphic vesicles and numerous mitochondria which were often centrally located. Some of these terminals (Figure 2A) were observed to establish multiple synaptic junctions with 'the same jpostsynaptic target. In addition to the synaptic junctions, the Type II terminals were also attached to the postsynaptic structures by one or more puncta adhaerentia (Figure 2C). The Type II terminals ranged in length from 0.4 to 3.0 pm and were encountered as either en passant or terminal boutons. Occasionally the type I or type II terminals formed a double synapse on two dendritic profiles of various sizes (Figure 2B). However, complex synaptic arrangement were never observed. The Type II terminals were found to represent 56% of the bouton population. The Type IA represented 37% and the Type IB accounted for only 7% of the total number of terminals 111 within the PPN. With the exception of the Type 18 terminals which were observed only on dendrites, both Type IA and II were found on somata/primary dendrites as well as on dendritic profiles of various sizes within the neuropil of the PPN. However, there was a significant difference in the distribution of the Type I and Type II terminals. The Type II terminals were concentrated on the somata/primary dendrites which appeared to receive 56% of the total number of the Type II terminals (N=326). Their numbers declined on the medium size dendrites (i.e.1.00-1.99 pm in width), and slightly increased again on small dendritic profiles measuring less than 1 pm in width. On the other hand, 50% of the total number of Type IA terminals (n=215) and 71% of the Type IB terminals (n=41) were found on small dendrites. II. ChAT IMMUNOCYTOCHEMISTRY Microscopy The ChAT positive (ChAT+) neurons could be easily identified on the basis of a homogenous dark brown staining of the soma and dendrites. In the lateral pontomesencephalic tegmentum (Figure 3), cholinergic cells were most numerous in the PPN. A smaller number of cholinergic cells were observed in the ill-defined caudal and dorsal border of the SNR, the retrorubral nucleus/field, the rostral poles of the lateral and. medial parabrachial nuclei, and the subpeduncular 112 tegmental nucleus. The cholinergic neurons were seen in both subnuclei of the PPN (Figs. 3, 4A), however a slightly greater number (58%) were located in the PPNd. The majority of the PPNd cholinergic neurons were seen in the middle portion of the subnucleus (Figure BB,C) and often in close proximity to the fibers of the superior cerebellar peduncle. The ChAT+ PPNd cells had fusiform or polygonal cell bodies and ranged in size from 20 to 60 um along the longest axis. The PPNd contained dendrites forming extensive arborizations among a diffusely organized group of cholinergic cell somata (Figure 4A,B). The fusiform cells were seen to give rise to two primary dendrites which were oriented in a rostrocaudal direction. From the polygonal cell somata, several primary dendrites radiated in all directions. The primary dendrites were relatively straight and divided into secondary dendrites approximately within 100nm of the soma. The secondary and tertiary dendrites branched infrequently and exhibited a moderate degree of undulation along their course. While varicosities were occasionally encountered on the secondary or tertiary dendrites, spines or short appendages were not observed arising from any of the dendrites examined. Dendrites could be traced up to 300 um from the cell somata of the fusiform cells and slightly over 425 pm from the polygonal cell somata. While a moderate number of dendrites were followed beyond the borders of the PPNd, the majority remained confined within the territory of the subnucleus. 113 The second subdivision of the PPN, the PPNc, contained a densely packed group of ChAT+ neurons. The lateral half of the PPNC consisted of a single compact group of cholinergic neurons (Figs. 3A,B and 4A). At more medial levels (Figure BC), the PPNc was traversed by the expanding scp coursing rostrally and separated into a dorsal and ventral cluster of cholinergic cells which diminished in number medially. The cholinergic PPNC cells exhibited similar shapes and sizes as the PPNd cells. However, the polygonal cells were more numerous than the fusiform cells and both cell types appeared to be less elongated than the cells in the PPNd. In addition to the cell bodies, the PPNc contained a dense plexus of ChAT+ dendrites which were interlaced among clusters of cholinergic somata (Figure 4A). In general, these dendrites appeared more undulated and branched sooner than the dendrites of PPNd neurons. They were rarely traced beyond 250 pm from the cell somata but a few could be followed for nearly 400 pm. While a greater number of dendrites appeared to extend beyond the borders of the PPNC when compared to the PPNd, the bulk of the dendrites remained within the PPNC. No varicosities, spines or appendages were encountered on the dendrites of the PPNc. In addition to the ChAT+ cell bodies and dendrites, we have also observed a small number of ChAT+ thin, frequently varicose fibers. The majority of the varicose fibers were located in the medial PPNd. No such fibers were noted within the PPNC. 114 Warm At the electron microscopic level, we have observed ChAT+ cell bodies and dendrites, a few myelinated axons and one nerve terminal in the PPNd. All ChAT+ cells (Figure 5) exhibited a dense, homogenous reaction product throughout the cytoplasm of their somata and dendrites. However, the reaction product was excluded from the interior of organelles, vesicles and the nucleus. The ChAT+ reaction product within axons and dendrites (Figs. 7A and 9) of the neuropil appeared to adhere directly to the surface of the microtubules and formed periodic patches along the arrays of microtubules. Cholinergic profiles were studied in tissue from the middle portion of the PPNd and compared to non- cholinergic cells (Figs. 5,6) contained within the same region. Twenty-eight ChAT+ cells cut through the nucleolus were examined in detail. The size of the somata ranged from 14 x 7 pm to 33 x 16 pm. The endoplasmic reticulum, Golgi apparatus, lysosomes and mitochondria of the perikaryon of ChAT+ neurons could be recognized. The nuclear membrane of both large and small cells often exhibited one or more shallow, randomly—located invaginations (Figure 5). Somatic appendages or spines were only rarely encountered. The somatic membrane of cholinergic neurons (Figure 5) was contacted by only a moderate number of terminals (average: 2/soma; range: 0-7). Five of the twenty-eight neurons 115 examined had one or more primary dendrites emerging from the cell body. The 27 ChAT- neurons ranged in size from 12 x 5 to 40 x 14 pm. The nucleus of the ChAT- neurons exhibited one or more rather prominent, deep indentations which were most evident in the smaller size neurons (Figure 5). Terminals contacting the somata and primary dendrites of the non- cholinergic neurons (Figure 6) were observed with greater frequency than for the cholinergic neurons (average: 6/soma; range: 0-26). The somatic and dendritic membranes of all ChAT- neurons were aspinous. Approximately 45% of the neurons examined had one or more primary dendrites emerging from the cell body. A comparison of cholinergic and non-cholinergic neurons revealed that the sizes of the somata (Figure 8) were roughly similar. However, the non-cholinergic cells tended to be somewhat smaller and more spindle-shaped than the cholinergic ones and received a richer synaptic input. A particularly interesting observation was that nearly 30% of the cholinergic somata were directly apposed to other cholinergic or non-cholinergic cell bodies (Figure 5) or large diameter dendrites. Similar- appositions Ibetween .non-cholinergic profiles were not observed. On the other hand, the ChAT+ and ChAT- dendrites were morphologically quite similar. Both exhibited occasional spine-like processes and were contacted by similar number of terminals. 116 Only one ChAT+ terminal was encountered in the present study. All of the ChAT+ axons (Figure 7A) were myelinated and had diameters (myelin sheath included) ranging in size from 0.5 to 2.2 gm. Quantitative analysis of nerve terminals (n=421) found in synaptic contacts or close appositions with ChAT+ profiles (33 somata/proximal dendrites and. 84 m O O O O O 0 l!) V C') N v- suomog Aq peJerg HIDUG'I aumqwew % O (D 138 Figure 2: Electron Micrographs Illustrating the Major Terminal Types in the PPN. A: Type 1 terminals (1A and 1B) containing round synapticvesicle and forming asymmetrical synapses (open arrows) with small-size dendrites. The Subtype 18 is differentiated from the Subtype 1A by its post-junctional dense bodies, which appear to be continuous with a portion of the smooth endoplasmic reticulum in this dendrite (arrowheads). A Type 2 terminal containing pleomorphic vesicles forms symmetrical synapses (open arrows) with a cell somata. Note the small cluster of presumed synaptic vesicles (solid arrows) in a medium-size dendrite. B: A Subtype 1A terminal synapsing (open arrow) on a small-size dendrite (d1). Another Subtype 1A terminal is establishing both a definitive synapse (open arrow) on a dendritic spine (solid star) and suggestive synapse on a large-size dendrite (d2). C: A Type 2 terminal containing centrally placed mitochondria forming multiple synapses on a cell body (CB). A second Type 2 terminal is attached to the cell body by a puncta adherens (solid arrow). Scale bars: 1.0 pm 140 Figure 3: Distribution of Cholinergic Cells in the PPN. Projection drawings of sagittal sections of the lateral (A) through medial (D) levels of the pontomesencephalic tegmentum showing the distribution of ChAT positive cells in the PPN and adjacent structures. One dot equals one cell. \/ 142 Figure 4: Brightfield Photomicrographs of ChAT+ Cells in the PPN. A: Low-power photomicrograph of the lateral half of the PPN showing numerous ChAT+ cells in both subnuclei. The PPNc has reached its maximum dimensions and contains densely packed ChAT+ cell somata and dendritic plexuses. Scale bar: 250 um. B: Higher magnification of the medial half of the PPNd showing ChAT+ cells interlaced by extensive dendritic arborizations. Scale bar: 50 pm. 143 Figure 5: 144 Montage of Electron Micrographs of a Cholinergic PPNd Cell. Electron microscopic montage from the PPNd of a large ChAT+ cell in direct apposition with a smaller ChAT- cell. Open arrows indicate terminals forming either definitive or suggestive synapses with the somatic membrane of the two neurons. Scale bar: 3.0 pm. 145 Figure 5 Figure 6: 146 Montage of Electron Micrographs of Non- cholinergic PPNd Cell. Electron microscopic montage from the PPNd of a large ChAT- cell. Note.the striking increase in the number of terminals (Open arrows) contacting the somata in contrast to the ChAT+ cell demonstrated in Figure 5. Scale bar: 3.0um. 147 148 Figure 7: Electron Micrographs of Cholinergic Elements and Their Synaptic Relationships in the PPNd. A: A Subtype 1B terminal simultaneously synapsing onto ChAT+ (d1) and ChAT- (d2) small-size dendrites. B: Spine-like evaginations (arrows) arising from a ChAT- cell body (CB) are postsynaptic to presumed Subtype 1A and Type 2 terminals. C: A portion of a ChAT+ cell body (CB) postsynaptic to a Subtype 1A terminal. Scale bars: 1.0 um. 149 . c. Refund in. r h 93.5.: 150 Figure 8: Cholinergic vs. Non-cholinergic Cells. A Size Comparison. Dot diagram of the sizes of 28 ChAT+ and 27 ChAT- neurons in the central region of the PPNd . 151 mv m ounmnm .53 2.2 5.5.2 ow mm on mm om m. or 0 L1 0 o o o m . o e o e o o o I O O O o o o o o I o m w o O O O O O O O O O O O O O O O o I O O O O C I. O o -- O o 0685.05-52 i- 0 0555620 .. O mjmo 0_0mmz_._0_._0-zoz .m> 0.0mmz_.._0_._0 O ,- LO ,_ O N mm on (er) S!XV JOUIW Figure 9: 152 Terminal Types Contacting Cholinergic PPNd Dendrites. Electron micrographs illustrating the Type 1 (A and B) and Type 2 (C and D) terminals synapsing on various diameter ChAT+ dendrites in the PPNd. Note the subtype 1B (B) post-junctional dense body (arrow) beneath the post-synaptic density is clearly distinguishable from the surrounding reaction product. Scale bar: 1.0 um. 153 m own—mam Figure 10: 154 Distribution of Boutons in the PPNd. The distribution of 421 terminals (Type 1:207; Type 2:214) establishing distinct synapses with 117 ChAT+ and 102 ChAT— profiles were analyzed on electron micrographs containing a mixture of both ChAT+ and ChAT- profiles. 155 DISTRIBUTION OF BOUTONS IN PPNd 80%— TYPE” TYPEI TOTAL ChAT - - ChAT + Figure 10 CONCLUDING REMARKS The present study involved three major projects which were focused on furthering our understanding of the circuits between the basal ganglia and the PPN. The first project examined the distribution of the PPN neurons projecting to the basal ganglia and spinal cord, and established whether some of the cells give rise to collateralized projections. The retrograde transport studies demonstrated that both ascending PPN neurons projecting to the basal ganglia and descending PPN neurons projecting to the spinal cord were distributed throughout the PPNd. An analysis of the labeled ascending and descending projection neurons indicated there was no distinct morphological features to differentiate with certainty one population from the other. The fluorescent dye studies revealed an almost insignificant number of PPN projection neurons had axons branching into an ascending and descending collateral. Therefore, these results indicated that the basal ganglia-projecting cells are neither spatially segregated nor morphologically distinct from the cells projecting to the spinal cord and both represent separate populations of PPN projection neurons. In the following project, the distribution and mode of termination of the nigropedunculopontine projection was studied light and electron microscopically by using the PHA-L technique. While the SNR was observed to project to both subnuclei, the bulk of the nigral fibers terminated in the 156 157 PPNd. Furthermore, the nigral fibers demonstrated a distinct preference for some neurons and islands of neuropil. Electron microscopic examination confirmed that nearly all of the varicosities observed in the light microscope represented either terminal boutons or boutons en passant. The majority of nigral terminals were seen in contact with various-width dendrites. The region containing the nigral plexus has previously been shown to contain both basal ganglia and spinal cord projecting PPNd neurons. However, the patchy distribution of the nigral plexus suggests that the nigral fibers may be related to a specific subpopulation of PPN projection neurons. The final. project focused (N1 the distribution. and synaptic organization of both cholinergic and non-cholinergic neurons in the PPNd. The cholinergic neurons were visualized using ChAT immunocytochemistry. This study demonstrated that cholinergic somata and dendrites were present throughout the subnuclei. Furthermore, some of the cholinergic dendrites in the PPNd were observed to arise from cells in the PPNc. Both cholinergic and non-cholinergic somata and dendrites were contacted by terminals which had been classified as Types I and II in the ultrastructural analysis of the PPN. The Type II terminals were morphologically quite similar to the nigral boutons identified in the previous project. Therefore, it is possible nigral afferents to the PPN may terminate on both cholinergic and non-cholinergic neurons of the PPNd. In light of the previous observations that nigral terminals were 158 also encountered in the PPNC and a portion of the cholinergic dendrites originate from PPNc neurons, it is plausible that the nigral input may modulate cholinergic cells not only in the PPNd, but also in the PPNC. 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