3&5 WW“WWW"WWWWHHWWIHH THESII IBIRAR IES NIH II 8923 This is to certify that the thesis entitled Projections from the Pontomesencephalic Tegmentum to the Cranial Nerve Nuclei in the Rat presented by Sheila Keane has been accepted towards fulfillment of the requirements for Mastefis—degree in Anatomy WWW: 0” Wm Major piéfessér Irena Grofova, PhD Date 5/18/90 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution ‘ LXBMRY Michigan State University L _,l *— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE —7 MSU Is An Affirmative Action/Equal Opportunity Institution c:\c|rc\d¢odtnpm3-p.' PROJECTIONS FROM THE PONTOMESENCEPHALIC TEGMENTUM TO THE CRANIAL NERVE NUCLEI IN THE RAT BY Sheila Keane A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 1990 we 51:7 ABSTRACT PROJECTIONS FROM THE PONTOMESENCEPHALIC TEGMENTUM TO THE CRANIAL NERVE NUCLEI IN THE RAT BY Sheila Keane The purpose of this study was to determine whether the nucleus tegmenti pedunculopontinus (PPN) links the output nuclei of the basal ganglia to the cranial motor nuclei. Unilateral injections of an anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) were placed in the PPN and the distribution of labeled fibers in the pontomedullary cranial nerve nuclei was charted. Results of these experiments demonstrated distinct crossed and uncrossed projections from the PPN to the facial nucleus, but only modest bilateral projections to the hypoglossal, ambiguus, dorsal motor vagus, and solitary nuclei. Since the PPN-facial projection exhibited two different distribution patterns in the facial nucleus, additional experiments utilizing retrograde transport of lectin-conjugated horseradish peroxidase (HRP-WGA) from the lateral or medial portions of the facial nucleus were carried out. ‘Ehese latter experiments did not substantiate the existence of PPN-facial projection and demonstrated that facial afferents actually originated from several structures surrounding the PPN. ACKNOWLEDGMENTS I would like to express my sincere appreciation and thanks to my advisor, Dr. Irena Grofova, whose support, advice, encouragement, and occasional sharp prodding greatly facilitated the completion of this research project. I also appreciate the assistance of my graduate committee members: Drs. Jack Johnson, Gerry Gebber, Duke Tanaka, and Charlie Tweedle. I also wish to express my deep appreciation of and gratitude for the technical advise and assistance of our laboratory technician, Ms. Kathy Bruce. Thanks also go to my fellow graduate students for their support and encouragement. iii TABLE OF CONTENTS List of Tables ............ . ........................... v List of Figures ........ . ..... . ........................ vi Abbreviations ............. ... ......................... vii Introduction .......................................... 1 Material and Methods .................................. 4 Results ................................... ............ 8 PHA-L Experiments .............. .................... 10 Projections to the Brainstem ..... ................ 13 Projections to the Facial Nucleus ................ 17 Projections to Medullary Cranial Nerve Nuclei .... 17 HRP Experiments ....... . .......................... 22 Lateral Facial Injections ................... 27 Medial Facial Injections .................... 38 Summary of Results ..... .......................... 47 Discussion ....... ...... .... ........................... 49 PPN Projections to the Facial Nucleus ............ 49 PPN Projections to Medullary Cranial Nerve Nuclei 51 Mesopontine Efferents to the Facial Nucleus ...... 53 Descending PPN Efferents . ........................ 57 Functional Considerations ........................ 59 Bibliography ...................... ....... ...... ....... 63 iv LIST OF TABLES Table 1: Retrogradely Labeled Cells Following Lateral Facial HRP-WGA Injections ............ ......... 33 Table 2: Retrogradely Labeled Cells Following Medial Facial HRP-WGA Injections ..... ..... .. ......... 39 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure l: 2: 3: 10: 11: LIST OF FIGURES PHA-L Injection Sites . ....................... Bright-field Photomicrographs of PHA-L Deposits in the PPN .................. ........ Distribution of Labeled Fibers in the Facial Nucleus in Case PHA-L #28 .. ...... ..... Bright-field Photomicrographs Illustrating the Two Distribution Patterns of PHA-L Labeled Fibers in the Facial Nucleus ................. Distribution of Labeled Fibers in the Facial Nucleus in Case PHA-L #25 ............. Photomicrographs Showing PHA-L Labeled Fibers in the Medullary Cranial Nerve Nuclei ........ Distribution of Labeled Fibers in the Medullary Cranial Nerve Nuclei ........ ....... Lateral Facial HRP-WGA Injection Sites ....... Photomicrographs Showing Center of Injection Sites in Cases CN7 #17 and CN7 #13 ........... Distribution of HRP Labeled Cells in case CN7 #17 ooooooooooooooooooooooo oooooo oo Photomicrographs of HRP Labeled Cells in the Ipsilateral Parabrachial Region in Case CN7 #17 ................. ............ Medial Facial HRP-WGA Injection Sites ....... Photomicrographs of HRP Labeled Cells in the Contralateral Retrorubral Region incaseCN7 #13 00000000009000.0000. oooooooooo Distribution of HRP Labeled Cells in case CN7 #13 ......OOOOOOOOOOOOOOOOOO ....... Circuit Diagram - Orienting Reflex .......... vi 12 16 19 21 24 26 29 31 35 37 41 43 46 6O ABBREVIATIONS Am AP CG Cnf CP g7 GiA IC icp IO IRt KF LC 11 LPB LPGi LRt LSO LVe Me5 mes ml M05 MPB MVe PCRt PN PPNc PPNd Pr5 PY Rmes RPC RPO RRF rs SC scp SNc SNr SO Sol ambiguus nucleus area postrema central gray cuneiform nucleus cerebral peduncle genu facial nerve gigantocellular reticular nucleus, pars alpha inferior colliculus inferior cerebellar peduncle inferior olive intermediate reticular nucleus Kolliker-Fuse nucleus locus coeruleus lateral lemniscus lateral parabrachial nucleus lateral paragigantocellular reticular nucleus lateral reticular nucleus lateral superior olive lateral vestibular nucleus mesencephalic trigeminal nucleus mesencephalic trigeminal tract medial lemniscus motor trigeminal nucleus medial parabrachial nucleus medial vestibular nucleus parvicellular reticular nucleus pontine nucleus pedunculopontine tegmental nucleus, pars compactus pedunculopontine tegmental nucleus, pars dissipatus principal sensory trigeminal nucleus pyramidal tract mesencephalic reticular nucleus red nucleus reticularis pontis caudalis nucleus reticularis pontis oralis nucleus retrorubral nucleus retrorubral field rubrospinal tract superior colliculus superior cerebellar peduncle substantia nigra, pars compacta substantia nigra, pars reticulata superior olive nucleus of the solitary tract vii ABBREVIATIONS (continued) sol sp5 Sp50 SPTg SpVe Tz tz VLL vsc xscp 3 4 6 5n 7 7n 10 12 solitary tract spinal trigeminal tract spinal trigeminal nucleus, pars oralis subpeduncular tegmental nucleus spinal vestibular nucleus nucleus of the trapezoid body trapezoid body ventral nucleus of the lateral lemniscus ventral spinocerebellar tract decussation of superior cerebellar peduncle oculomotor nucleus trochlear nucleus abducens nucleus trigeminal nerve facial nucleus facial nerve dorsal motor nucleus of vagus hypoglossal nucleus viii INTRODUCTION The nucleus tegmenti pedunculopontinus (PPN) was first described in the human caudal mesencephalic tegmentum as being "bounded medially by the superior cerebellar peduncle (scp), laterally by fibers of the medial lemniscus, and dorsally by the nucleus cuneiformis and subcuneiformis (Olszewski & Baxter '54). These authors further identified two subdivisions of the nucleus on the basis of cellular density. The smaller pars compacta "occupies the dorsolateral portion of the caudal half of the nucleus" and the "remainder of the nucleus constitutes the pars dissipata". In subprimate species, the nucleus is less clearly defined. Delineation of the PPN and its subdivisions has been variously made on the basis of cytoarchitectural features (Spann & Grofova '89), cytochemistry (Rye et.al '88, Woolf & Butcher '86), and basal ganglia input (Moon-Edley & Graybiel '83, Nauta & Mehler '66). The functions and connections of the PPN have not been fully established. The PPN has widespread connections with the basal ganglia, thalamus, and limbic structures and has been implicated in a wide variety of functions including motor control, sleep-wake cycles, respiration, locomotion, chewing, and other rhythmic behaviors (Garcia-R111 & Skinner '88) and sensory modulation (Hylden et.al. '85, Katayama et.al. '84, Basbaum & Fields '80, Carstens et.a1. '80). 2 The role of the PPN in the control of movement is well supported by both anatomical and physiological data. Convincing evidence exists that the PPN receives substantial input from.several nuclei of the basal ganglia (Grofova et.al. in press, Spann & Grofova '89, Jackson & Crossman '83, Gerfen et.al. '82, Beckstead et.al. '79, Granata & Kitai '89, Carter & Fibiger '78, Nakamura et.al. '89, Noda & Oka '86, Garcia- Rill et.al. '83, Moon-Edley & Graybiel '83, Larsen & McBride '79, Nauta & Cole '78, Parent & DeBellefeuille '82, Beckstead & Frankfurter '82, Kim et.al. '76) and ascending efferents of the PPN return these basal ganglia projections (Woolf & Butcher '86, Jackson.&iCrossman '83, Saper & Loewy '82, Gerfen et.al. '82, VanDerKooy & Carter '81, Garcia-Rill et.al. '83, Moon-Edley & Graybiel '83, Gonya—Magee & Anderson '83, DeVito & Anderson '82). In addition, physiological experiments have identified a "midbrain locomotor region" in the decorticate cat (Mori et.al. '80) and rat (Garcia-Rill '87) which is located in the pedunculopontine region and includes the PPN (Garcia-Rill '86). Furthermore, lesions of the PPN in the rat have been associated with impaired motor function (Kilpatrick & 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. '85, '87) and Parkinson's disease (Zweig, et.al. '89, Jellinger '88). The pathways by which the PPN affects motor behavior are currently under investigation. Descending PPN efferents 3 include projections to the ventromedial pontomedullary reticular formation (Grofova et.al. in press, Nakamura et.al '89, Mitani et.al. '88, Rye et.al. '88, Moon-Edley & Graybiel '83, Jackson & Crossman '83). This reticular region contains neurons projecting to the ventral horn and cranial motor nuclei (Vertes et.al. '86, Jones & Yang '85, Zemlan et.al. '84, Travers & Norgren '83, Holstege & Kuypers '82, Martin et.al. '81, Peterson '80). In addition, sparse projections from PPN to the spinal cord have been reported in the rat (Spann & Grofova '89, Rye et.al. '88, Goldsmith & VanDerKooy '88). Considering its many connections with the nuclei of the basal ganglia, the PPN is well situated to function as a relay nucleus for descending basal ganglia influence on lower motor structures. While PPN efferents to the reticular formation and spinal cord have been documented by several authors, investigations of PPN projections to the cranial motor nuclei are lacking. The purpose of the present study is to determine the projections from PPN to the pontomedullary cranial motor nuclei. MATERIAL AND METHODS A total of 15 male Sprague-Dawley albino rats weighing 300-350 g were utilized for this study. Eight animals received unilateral injections of Phaseolus vulgaris- leucoagglutinin (PHA-L) in ‘the nucleus tegmenti pedunculopontinus (PPN), while seven received unilateral injections of wheat-germ-agglutinin conjugated horseradish peroxidase (HRP-WGA) in the facial nucleus. Both surgeries and perfusion were performed under deep anesthesia (sodium pentobarbital, 50-100 ‘mg/kg, i.p.), and, atropine sulfate solution (0.7 mg/kg) was administered i.m. prior to the surgery in order to prevent brain edema. Injections of both tracers were made iontophoretically using glass micropipettes with an inside tip diameter of 15-35 pm, and a positive 75 pulsed 5 MA current. The stereotaxic coordinates were derived from the atlas of Paxinos and Watson ('86). PHA-L Experiments Micropipettes filled with a 2.5% solution of PHA-L (Vector Labs) in 10mM Tris buffer (pH 8.0) were inserted vertically through the ipsilateral hemisphere and tectum of the midbrain. Single iontophoretic depositions of PHA-L were made for 30-40 minutes. Following a survival period of 10 to 14 days, deeply anesthetized animals were perfused through the heart with a sodium phosphate buffered saline solution 5 followed by a fixative consisting of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.15M sodium phosphate buffer. The brains were immediately removed and stored overnight at 4°C in fixative. The following day, the brains were divided into a caudal block containing the caudal pons and medulla, and a left and right rostral block including the forebrain, midbrain, and rostral pons. Serial sectioning was done at 30 um on a vibratome in the coronal (caudal block) or sagittal (rostral block) plane. Sections were collected in Tris buffered saline and processed for PHA-L immunohistochemistry using a biotin-avidin (Vector Labs) protocol by Gerfen & Sawchenko ('84). Immuno-reacted sections were mounted onto gelatin-chrom-alum-coated slides, air-dried, dehydrated, and lightly stained with cresyl violet. The sections were examined on a Leitz Orthoplan microscope, using bright-field illumination for the localization of PHA-L injections and the presence of labeled nerve fibers and terminal fields. The localization of the PHA-L deposit in the PPN was charted on a standard map of sagittal sections through the pontomesencephalic region containing the lateral and medial halves of the PPN} The distribution of the labeled fibers and plexuses in the cranial nerve nuclei was documented on projection drawings and photomicrographs of selected sections. HRP-WGA Experiments Single unilateral injections were made in the medial or lateral portions of the facial nucleus using a 2% HRP-WGA solution in Tris buffer delivered iontophoretically for 15 to 25 minutes according to Graybiel & Devor ('74). In order to minimize leakage of HRP-WGA along the needle track, the micropipette remained in situ for’ 10 minutes after the injection, and reversed. polarity' was applied. during its withdrawal. After a 24—48 hour survival period, deeply anesthetized animals were perfused intracardially with a fixative consisting of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.15M sodium phosphate buffer. The brains were blocked into right and left halves, and serial sections were cut sagittally at 30-50 pm on a freezing microtome or vibratome. HRP-WGA histochemistry using the chromogen tetramethyl benzidine (TMB) was performed according to Mesulam ('82) . Some of the sections were additionally stabilized with ammonium.molybdate (Olucha et.al. '85). Reacted sections were :mounted on gelatin-chrom-alum—coated slides and lightly counterstained with neutral red. Sections were analyzed in bright-field illumination for the presence of retrogradely labeled cells in the PPN and surrounding regions. The distribution of labeled cells was documented on projection drawings in representative cases using major'blood vessels and fiber tracts as landmarks. Cell 7 counts for all retrogradely labeled mesopontine nuclei were taken from alternating sections, and the numbers of labeled cells contained in a specific nucleus was pooled from all inspected sections. The borders of the PPN and its subnuclei were identified according to previously established criteria (Spann & Grofova '89). For clarity, the delineation of all other relevant structures was taken fromtthe atlas of Paxinos & Watson ('86). RESULTS Before describing the experimental results, the normal morphology of the pedunculopontine region will be considered briefly. The PPN represents a portion of a continuous cell column surrounding the ascending limb of the superior cerebellar peduncle (scp) at the pontomesencephalic junction. It consists of two subdivisions: 1) the pars compacta (PPNc) composed primarily of larger cholinergic neurons; and 2) the pars dissipata (PPNd) containing a considerable portion of smaller non-cholinergic cells. While the boundaries of the PPN and its subdivisions are well-defined in primates, the outlines of the PPN in carnivores and rodents are less clear. Consequently, there is little consistency in the literature regarding the delineation of this nucleus or its subdivisions in the rat brain. In the present report, we adhere to the definition of PPN based on cytoarchitectural features described in a previous study (Spann & Grofova '89). The rat PPN is located medial to the nuclei of the lateral lemniscus and lateral to the decussation of the scp. It is caudally contiguous with the lateral and medial parabrachial nuclei (LPB and MPB) and is rostrally adjacent to the retrorubral field (RRF) and retrorubral nucleus (RR) as defined by Paxinos & Watson (1986). The PPN borders ventrally on the nucleus reticularis pontis oralis (RPo), and dorsally on the cuneiform (Cnf) and mesencephalic reticular SC ' SC PHA-L # 24 Rmes ' LPB 774?? PHA-L # 25 /( K R ' W; .. __ mes /’< a; PHA'L # 27 @319" \ u m . / \\\ \\\-\‘ PHA-L # 28 §\ SNR \ / / \D \\ u (33$ i"? l/ PHA-L # 31 SNC 1mm \\ I> ’2 ?@ T l 9 / ;{// Figure 1: PHA-L Injection Sites Schematic representation of PHA-L injection sites involving the lateral (A) and medial (B) halves of the PPN in cases in which labeled fibers could be traced to the facial nucleus and to the medullary cranial nerve nuclei 10 (Rmes) nuclei. While both divisions of PPN can be distinguished in the rat, the PPNd comprises the bulk of the nucleus. The PPNd is particularly prominent medially and appears to receive a substantial input from the substantia nigra pars reticulata (Spann & Grofova '88). PHA-L Experiments PHA-L injections were directed toward the PPN region receiving afferents from the substantia nigra (i.e. the medial two thirds of the PPNd). Out of eight cases, five showed excellent anterograde labeling as well as precise localization of the injection in the PPNd. These five cases represent the core material for the present report. The remaining three cases yielded less intense labeling and provided.complementary data. While centered in the medial portions of PPNd, all PHA- L injections involved the entire mediolateral extent of the PPN (Fig. 1). Two zones of different labeling intensity were observed at the injection sites; a central zone in which the DAB reaction product obscured the cytoarchitecture, and a peripheral zone in which single neurons exhibiting Golgi-like labeling could be discerned. In all animals, a variable number of labeled neurons were observed in the surrounding nuclei. However, fiber tracts passing through the injection site were always free of label (Fig.2). The absence of labeling was particularly striking with regard to the scp 11 Figure 2: Bright-field Photomicrographs of PHA-L Deposits in the PPN. A-C: Illustrate the extent of PHA-L injection in case PHA-L #25. The injection extended somewhat beyond the lateralmost border of PPN (C) and the tracer was taken up by a few cells (arrowheads) in the RR. More medially, single labeled cells can be seen in the RRF and in the rostral MPB (arrowheads in B). Unstained fibers of the scp traverse the medial (A) and middle (B) regions of the PPN (arrows). D: The center of PHA—L injection in case PHA-L #28. A few Golgi-like labeled cells are present in the MPB (arrowheads). Scale bar: 0.5mm rips - I V ~ .\ I. an; . .. Figure 2 13 which was inevitably included in the central zone of all PHA- L injections. In case PHA-L #25, there was a dense deposit of PHA-L throughout the entire extent of the PPN. Some cells in the Cnf were also labeled along the course of the needle track. In.addition, a feW'PHArI.labeled cells were observed rostrally in the RR and RRF, ventrally in the RFC, caudally in the MPB, and laterally in the nuclei of the lateral lemniscus (Fig. 2). The inclusion of rostrally adjacent structures in the peripheral zone of PHA-I.uptake'was also observed in case PHA- L #31, where the rostroventral half of PPN was labeled in addition to individual neurons in the surrounding'RR, RRF, and RPo. In cases PHA-L #28, PHA-L #27, and PHA-L #24, injections were also centered in PPN but extended somewhat caudally to include neighboring regions of the MPB and LPB. The PHA-L injection in case PHA-L #28 was the most laterally placed of these, involving the caudoventral PPN and labeling several cells caudal to PPN in the MPB and ventrally in the RFC (Fig. 2). Projections to the Brainstem The bulk of PPN descending projections were distributed within the reticular nuclei of the brainstem. Labeled fibers were seen descending through the pontine reticular nuclei to the ventromedial portions of the nucleus gigantocellularis (GiA and GiV). Although many fibers appeared to cross the l4 midline in the caudal pons and rostral medulla, terminal arborizations were somewhat more numerous ipsilaterally. In the pons, numerous varicose branches of the labeled fibers were seen to terminate around large reticular neurons, especially in the reticularis pontis caudalis (RPc). Projections to the medulla and spinal cord were relatively sparseu The course and.distribution of these projections have been described and illustrated elsewhere (Grofova et.al. in press). In addition to the fibers distributing to the reticular formation, PHA-L labeled fibers were also seen in several cranial nerve nuclei in the pons and medulla. No labeled fibers were found in the motor trigeminal (M05) nucleus. However, two distinct distribution patterns were observed in the facial nucleus. One pattern, present in all cases, consisted. of diffuse labeling in ‘the ipsilateral facial nucleus, while two cases (PHA-L #25 and PHA-L #31) additionally demonstrated a dense contralateral projection to the rostromedial two thirds of the facial nucleus. Projections to the cranial nerve nuclei of the lower brainstem were relatively sparse and were bilaterally distributed. 15 Figure 3: Distribution of Labeled Fibers in the Facial Nucleus in Case PHA-L #28 Projection drawings of coronal sections through three pontomedullary levels showing the distribution of labeled fibers in the facial nucleus and ventral reticular nuclei in experiment PHA-L #28. labeled varicose fibers are present throughout the ipsilateral facial nucleus with greater concentration caudally. - ~-_ :I’Yier _'_~_. ' ' T ’ l6 ' PHA—L #28 Rostral Middle Caudal Contralateral nnm Ipsilateral Figure 3 17 Projections to the Facial Nucleus Results presented from cases PHA-L #28 and PHA-L #25 are representative of the two patterns of distribution found in the facial nucleus. In case PHA-L #28, labeled varicose fibers were dispersed diffusely throughout the ipsilateral facial nucleus with somewhat greater concentration in its lateral half (Fig; 3). Thick.labeled fibers typically divided into smaller branches exhibiting multiple terminal and preterminal varicosities both in the neuropil and near the somata of lateral facial motoneurons (Fig. 4 A & B). In case PHA-L #25, in addition to the diffuse ipsilateral facial projection which was present in all cases, a dense plexus of anterogradely labeled fibers was observed in the rostromedial two thirds of the contralateral facial nucleus (Fig. 5). Numerous varicose arborizations formed a dense terminal plexus surrounding the medial groups of facial motoneurons (Fig. 4 C & D). This contralateral projection to the medial facial nucleus was also seen in case PHA-L #31, but was not present in other cases. Projections to Medullary Cranial Nerve Nuclei All cases demonstrated a sparse bilateral distribution to the solitary (Sol), dorsal motor vagus (10), and hypoglossal (12) nuclei. In addition, a few thin varicose fibers in the proximity of the large motoneurons of the nucleus 18 Figure 4: Bright-field Photomicrographs Illustrating the Two Distribution Patterns of PHA-L Labeled Fibers in the Facial Nucleus. A&B: C&D: Diffuse ipsilateral projection to the lateral half of the facial nucleus in case PHA-L #28. Figure A illustrates a modest number of labeled fibers (arrows) within the dorsolateral group of facial.motor neurons. 'Fhese fibers are shown at higher magnification in Figure B. Arrows indicate a thick fiber of an even diameter dividing in several thin branches exhibiting multiple varicosities. Contralateral projection to the medial groups of facial motoneurons in case PHA-L #25. A dense terminal plexus of labeled fibers within the ventromedial group of facial motoneurons and LPGi is shown in Figure CL Figure D represents a higher magnification of the region outlined by a rectangle in Figure C, and illustrates numerous preterminal and terminal varicosities surrounding the cell bodies of the medial facial motoneurons (arrowheads) as well as distributing in the neuropil (arrow). Scale bar: 0.1mm ..., 1 ..Im .. 3&5 ...wvmry .fi 1". W.“ 0‘! 9.3.9 a ..I 20 Figure 5: Distribution.of Labeled Fibers in the Facial Nucleus in Case PHA-L #25 Projection drawings of coronal sections through three pontomedullary levels illustrating a dense plexus of labeled varicose fibers in the rostral two thirds of the medial portion of the contralateral facial nucleus in experiment PHA- L #25. 21 PHA—L #25 Ipsilateral Contralateral Figure 5 22 nucleus ambiguus (Am) were occasionally seen bilaterally (Fig. 6A). The distribution of PHA-L labeled fibers to the caudal medullary cranial nerve nuclei are illustrated in Figure 7. Varicose fibers descended bilaterally in the hypoglossal nuclei near midline, subsequently turning and passing to the reticular formation ventrolateral to 12. Similarly, thin varicose fibers were seen coursing from midline along the borders of Sol, branching and terminating in the lateral portions of Sol and 10. While present bilaterally, varicose arborizations were somewhat more numerous ipsilaterally in the lateral regions of Sol and 10, and in the reticular formation ventral to this region (Fig. 6). HRP EXPERIMENTS Results of PHA-L experiments suggested a topographical organization of PPN projections to the facial nucleus, with rostral portions of PPN projecting to the contralateral groups of medial facial neurons, and caudal portions of PPN projecting ipsilaterally primarily to the lateral half of the facial nucleus. To verify this hypothesis, experiments utilizing the retrograde transport of HRP-WGA from medial (N=4) or lateral (N=3) portions of the facial nucleus were carried out. 23 Figure 6: Photomicrographs Showing PHA-L Labeled Fibers in the Medullary Cranial Nerve Nuclei. A: Arrows indicate labeled fibers in the ipsilateral Am in case PHA-L #24. B: Labeled fibers (arrows) in the ipsilateral 12 in case PHA-L #25. C&D: A discrete plexus of fine varicose fibers in the lateral portion of the 801 ipsilateral to the PHA-L injection in case PHA-L #25. Scale bar: 0.1mm ____1 .--?!A. "r_‘.' .’_ 11.. Figure 6 25 Figure 7: Distribution of Labeled Fibers in the Medullary Cranial Nerve Nuclei Projection drawings of coronal sections through the dorsal portion of the lower medulla showing a relatively modest number of varicose fibers in the 12, 10, and Sol in experiment PHA-L #25. 26 PHA—L #25 Rostral Ipsilateral < T > Contralateral Caudal $4 .5. P. ... __ _\_ _ Figure 7 27 Lateral Facial Injections The HRP-WGA injection sites involving the lateral portions of the facial nucleus are shown in Figure 8. Dense deposits of HRP-WGA.were observed in the lateral third.to half of the facial nucleus in all three lateral injections. Despite all precautionary measures, HRP-WGA reaction product was also consistently present along the needle track and in portions of surrounding structures. In case CN7 #17, the dense deposit of HRP-WGA was nearly completely confined to the lateral two thirds of the facial nucleus in its middle antero-posterior extent. A "halo" of diffused reaction product extended ventrally into the trapezoid (tz) and rubrospinal (rs) fiber tracts, and into the reticular formation caudolateral to the facial nucleus (Fig. 9A). No retrogradely labeled cells were observed in the PPN. Labeled neurons were identified primarily in the ipsilateral principal trigeminal (Pr5), medial parabrachial (MPB), and Kolliker-Fuse (KF) nuclei (Fig. 11». Retrogradely labeled cells of the MPB were concentrated rostrally, near the caudal border of the PPN (Fig. 11 C & D). A substantial number of labeled cells in the ipsilateral Pr5 and KF were also present at levels lateral to the PPN (Fig. 11 A & B). Fewer neurons were labeled in the ipsilateral lateral parabrachial and reticularis pontis oralis nuclei. Several labeled neurons were observed in the contralateral red nucleus (RN). In 28 Figure 8: Lateral Facial HRP-WGA Injection Sites Projection drawings of sagittal sections through the center of HRP-WGA injections involving the lateral portion of the facial nucleus in three cases described in the text. The position of dense reaction product around the pipette is indicated by cross-hatching. Hatching indicates a surrounding area of lighter diffusion which may be somewhat overestimated because of use of the sensitive chromogen, TMB. 29 \\ mos . ‘0‘. o. ‘0... 0‘0 \ m °.o\::.;:;:;:o;;:;.,. 7 .0 $55.30.. 0 / / rs Affiwo. :.;.;.;.;.;.;.. H. , o o .o.o‘:.o.o.o.o.o / 5 n / we .a O %‘0. mt CN7 #14 VL VSC / 1mm W ‘ “(7/ l /5‘ .5/ PCRt / . CN7 #17 Figure 8 30 Figure 9: Photomicrographs Showing Center of Injection Sites in Cases CN7 #17 and CN7 #13 Bright-field photomicrographs illustrate the center of HRP-WGA injection sites in two representative cases CN7 #17 (A) and CN7 #13 (B) . The distributions of retrogradely labeled cells in the pontomesencephalic tegmentum of these two cases are shown in Figures 10 and 14 respectively. Scale bar: 0.5mm 31 a uuzmflh Lmuua 32 addition, a few scattered retrogradely labeled cells were found in the ipsilateral nucleus reticularis pontis caudalis (RPc) and contralaterally in the deep layers of the superior colliculus (SC). Results are summarized in Table 1. Other injections of HRP—WGA in the lateral half of the facial nucleus exhibited larger diffusion of the reaction product into the surrounding tissue. In animal CN7 #16, the injection was centered at the caudolateral edge of the facial nucleus and exhibited diffuse HRP-WGA reaction product with Golgi-like labeling of neurons in the caudolateral two thirds of the facial nucleus as well as the parvicellular reticular nucleus (PCRt) dorsolateral to the facial nucleus. In case CN7 #14, the injection was centered caudal to the facial nucleus and included only the caudal half of the lateral portions of the facial nucleus. HRP-WGA reaction product in animal CN7 #14 was seen as far caudally as the rostral Am and extended dorsally into the reticular formation as well. In these additional cases, the PPN exhibited only a few retrogradely labeled neurons. The larger and more caudally placed injection in case CN7 #14 resulted in ten labeled PPN neurons ipsilaterally, while five PPN neurons were labeled contralaterally. Case CN7 #16 demonstrated only one labeled cell in the ipsilateral PPN. Confirming the results of case CN7 #17, other lateral facial experiments also resulted in heavy HRP labeling ipsilaterally in several nuclei caudal to the PPN (including the MPB, KF, and Pr5) while the core of the 33 .flmaos: Amnmv Hmcflammwuu Hmmflocfium can .Ame mwsmiumxflaaom .Ammqv acwnomuncumm annoyed .3sz Hafinomunwumm H369: on» 939593 zmm new: uwumooa ..aoaosc acum>wm a“ haamnmumaflmmfl uoamnma one mcousoc .um>msom .Hwflucmumnsmcfl ma 2mm may no mafiamnwa mumumouumm A.mmmm£ucouwm ca mum “mans: Hmnwumamuucoo may Eoum mucsoo Hamuv .mCOAuoom guanu couowe om mcflumcuouam Eoum Umucsoo mums mamaosc defies“ may mo mcofiuuoa Hmuwpca may CH mcofluommcfl «UBIQmm mafisoaaom Hoaosc oflamnmmocmmmEoucom may :a mHHwo cmaonma maouwuoonumm fin wanna o we a .mmem a omma .m>:mOH .oqnsmmm no mzm H .mos a Amamv u “owe me on mm Lady «ma AVHC ma Ame son Ame 0H was .mmemm .m>:me ucoa .oansmw "H m>sm H "o o "H “Hat 0 Loy a on 0 Any m on a on mm on a one magma .ooa .mma .ome no AHHV o on ma on mm Ame mad on mm “my and Loy 0 has 0mm m "H nonuo zm 0mm man mm: as mum 2mm #520 wme mcofluomncH «wzimmm deflomm Hmumumq mcfl3oHHom maamo Umamhmq Samccuoouumm "u wanna 34 Figure 10: Distribution of HRP Labeled Cells in Case CN7 #17 Projection drawing of lateromedially arranged sagittal sections through the pontomesencephalic region showing the distribution of HRP labeled cells in the contralateral (A-C) and ipsilateral (D-F) nuclei surrounding the PPN in case CN7 #17. Sections illustrated in A and D are lateral to the PPN. Each dot represents one labeled cell. 35 Figure 10 Fig. 36 11: Photomicrographs of HRP Labeled Cells in the Ipsilateral Parabrachial Region in Case CN7 #17. A&B: C&D: Show the position (A) and morphology (B) of retrogradely labeled neurons in the pontomesencephalic tegmentum lateral to the PPN. Labeled neurons in the ipsilateral KF (open arrows in B) exhibit round somata located ventral to the superior cerebellar peduncle while the smaller fusiform cells of the Pr5 (small arrows in B) lie more ventrally, immediately caudal to the lateral lemniscus. In addition, several larger multipolar neurons within the Pr5 (large arrowheads) also contain HRP granules. Show HRP labeled cells in a more medial region of the pontomesencephalic tegmentum. Most of these cells lie well within the confines of the MPB (small arrows in D). Two neurons in a border zone between the MPB and PPN are indicated by open arrows. A few labeled cells are intermingled with the fibers of the scp (large arrows). Scale bar: 0.1mm 37 38 PPN nucleus was remarkably free of labeled cells. Medial Facial Injections In four animals, HRP-WGA injections were centered in the medial half of the facial nucleus (Fig. 12). In case CN7 #13, dense HRP-WGA reaction product was almost totally confined to the medial half of the facial nucleus. However, slight encroachment of the underlying fibers of the trapezoid body and faint HRP-WGA reaction product was also evident in a small region of the GiA medial to the facial nucleus (Fig. 9B). Results are presented in Table 2. Similar to the observations following HRP-WGA injections in the lateral portions of the facial nucleus, there were no labeled cells well within the confines of the PPN. In case CN7 #13, only one PPN’ cell, located. at 'the rostralmost boundary of the contralateral PPNd, was labeled (Fig. 13). Immediately rostral to the PPN, the contralateral R was heavily labeled. This projection was exclusively contralateral. Labeled. RR. cells 'were located caudally, abutting the rostral pole of PPN and often occupying the border region between these two nuclei. In addition, a more moderate number of labeled neurons were observed in the ipsilateral MPB in case CN7 #13 (Fig. 14). Similar to the pattern observed following lateral HRP-WGA injections, retrogradely labeled.MPB cells were found primarily along the rostral and dorsal borders of MPB in a zone bordering 39 .HmHosc HmmHv HmHnomHnmumm HmumumH new .Hmmv mmsmuumxHHHox .Hmmzv Hwflnowunmuwm HMHUmE .Amumv HmcHEmmHHu Hmmflocflum on» CH maacumumaflmmfl Umaonma mHHwo mu Hams mm Ammv mstosc kuhsuouumu Honoumamupcoo one :H mHHmo pmamnma mo mumnesc mmHmH may muoz A.mmmmnucmuwm :H mum HmHosc Hmuwuwacuucoo on» Eoum mucsoo HHmUv om wcflpccumuam Eoum cmpcsoo muo3 msmHosc HmHomu on» yo mcoHuHom HchmE on» :H mcoflpomncfi .mcofluomm xoflnu COHUHE dozimmm mcflsoHHom HmHosc oHHmcmmocwmeoucom map :a mHHmo umHoan hamuwumouuom "a canes oo H .zm H no 00 H "H Hoe o Hoe o Hoe m Hoe o Hoe o HHHV o e* mac: Hoe o Hoe o on o Hoe o Hov o AHV o ma wo H ”o m>sm m .omm m .Zm s .mwz m "H Hoe a Hoe H Hoe mm Hov oH Loy H AHNV o HHH 0mm m .zm m .mmm NH .mmsm he no 2m m .mmam s .oo 0H .omm sH "H Hoe m Lav m Hoe mm Ame HH “He 0 HHmHv o HH¢ umnuo mmH as mas mum 2mm mm * szo Ommo m:0HuommcH auzummm HmHomm Hcflpoz mcHsoHHom mHHmU cmHmnmq Samumumouumm " CHQGB 40 Figure 12: Medial Facial HRP—WGA Injection Sites Projection drawing of sagittal sections through the center of HRP-WGA injections involving the medial half of the facial nucleus in four representative cases described in the results. See legend to Figure 8 for further explanation. 41 SCD/ / MVB " 22 Me5 \._i: m 1“" '— RPo 7n " 5 31%;. l c3339 \ Mo / / N2 80 Wfi‘éf/é-P‘fl. fl; / ,1 PCRt I ”226;- CN7 #11 CN7 #13 1mm Figure 12 ~¢ ‘ “n #1 n! - - 42 Figure 13: Photomicrographs of HRP Labeled Cells in the Contralateral Retrorubral Region in Case CN7 #13. A&B: The location (A) and morphology (B) of retrogradely labeled cells in the contralateral RRF. Most of these cells lie well within the caudal half of the RRF (B). An arrow (B) indicates one labeled cell in the border zone between the PPNd (**) and the RRF; The PPNc is indicated by a single asterisk. C&D: Show HRP reactive neurons in a more lateral region of the pontomesencephalic tegmentum. Labeled cells (arrows in D) are observed along the caudal border of the RR. Scale bar: 0.1mm 44 the caudal PPN. The other mesopontine nuclei exhibiting HRP— labeled.neurons in.case CN7 #13 included, in decreasing order: the bilateral Rmes (more contralaterally); bilateral RPo (more ipsilaterally); contralateral RRF; ipsilateral Pr5; ipsilateral CG (in clusters along the ventral border); bilateral KF; bilateral RN; and the ipsilateral LPB. In the other three animals with injections in medial portions of the facial nucleus, moderately dense HRP-WGA reaction product was observed in neighboring regions of the adjacent reticular nuclei. GiA was labeled medial to the facial nucleus in cases CN7 #11, CN7 #4, and CN7 #9, while the reticular formation caudodorsal to the facial nucleus was labeled only in cases CN7 #11, and CN7 #4. Results from these animals confirmed those of animal CN7 #13. There were essentially no labeled cells in the PPN following HRP-WGA injections into medial portions of the facial nucleus. Diffusely labeled neurons were observed in nuclei surrounding (but not including) the ipsilateral PPN, particularly the MPB and Pr5. Distinct labeling in the contralateral RR was present in all medial cases, even in one case (CN7 #9) with an extremely small injection site. 45 Figure 14: Distribution of HRP Labeled Cells in Case CN7 #13 Projections drawings of sagittal sections illustrating the distribution.of HRP labeled cells in the contralateral (A- C) and ipsilateral (D-F) pedunculopontine region following HRP-WGA injection involving the medial part of the facial nucleus in case CN7 #13. Each dot represents one labeled cell. 46 O p. . a R O I , ..O/ I s N I P D. . s It udsolll 9 “sun m 2.. Cr. R ’ od% 0.. o N O. 8 1mm Figure 14 47 Summapy of Results In summary, results of unilateral FHA-L injections centered in the PPN showed anterograde labeling primarily in the ventromedial pontomedullary reticular nuclei, but also in several nuclei of the cranial nerves. Labeled fibers were most dense in the facial nucleus, and relatively sparse in the caudal cranial nerve nuclei (12, Sol, 10, and Am). PmmrL projections to the facial nucleus exhibited two distinct patterns: 1) a diffuse ipsilateral projection present in all cases, and 2) a punctate contralateral projection to the rostromedial two thirds of the facial nucleus in two cases. The HRP-WGA experiments which were designed to clarify the organization of this PPN-facial pathway failed to confirm the existence of such projections. Retrograde labeling of cells located well within the PPN was not observed after injections of HRP-WGA.in either the lateral or medial portions of the facial nucleus. The exception was case CN7 #14, in which HRP-WGA was also deposited in the reticular formation caudal to the facial nucleus. Similarly, retrograde labeling in the PPN (136 cells ipsilaterally, 54 contralaterally) was observed in one additional case (CN7 #18) in which the core area of the HRP-WGA injection included the entire extent of the facial nucleus and substantial portions of the surrounding reticular nuclei. In contrast to the absence of labeled cells in PPN, retrograde. experiments showed labeling' of several nuclei 48 surrounding the PPN. HRP-WGA injections into the medial facial nucleus resulted in distinct labeling of cells in the contralateral RR. Furthermore, both medial and lateral facial injections resulted in a primarily ipsilateral pattern of HRP- labeled neurons in the MPB, KF, LPB, and Pr5. These nuclei were labeled most distinctly in lateral facial injections. DISCUSSION PPN Projections to the Facial Nucleps The anterograde and retrograde labeling experiments yielded contradictory results. While PHA-L injections in the PPN clearly demonstrated two distribution patterns of labeled fibers in the facial nucleus, the HRP-WGA experiments failed to retrogradely label cells located in the core of the PPN. Particularly significant. was. a complete absence of retrogradely labeled cells in the medial two thirds of the PPNd which appear to receive the bulk of nigral afferents (Spann & Grofova '88). It may be argued that the negative results obtained from the HRP experiments were due to technical failure. However, this seems unlikely since retrogradely labeled cells were consistently present in the areas surrounding the PPN. Furthermore, the PPN cells did exhibit distinct labeling in one of the experimental animals in whidh the HRP-WGA injection involved mostly the ventral part of the reticular formation adjacent to the medial aspect of the facial nucleus. Taken together, these observations indicate that the PPN does not give rise to the facial projections demonstrated in the experiments utilizing anterograde transport of PHA-L. Although there exist occasional reports (Cliffer & Giesler '88, Schofield '89) that the PHA-L can be taken up by fibers passing through the injection site, most authors agree with the original 49 50 observations of Gerfen & Sawchenko ('84) that PHA-L does not appear to be taken up and transported effectively by fibers of passage. Our own observations fully support the latter notion. The PPN is traversed by several prominent fiber systems including the superior cerebellar peduncle and the central tegmental tract.which were unavoidably involved.in.all PHA-L injections. However, there was no evidence of PHA-L uptake and transport by these fiber systems. In fact, the fiber bundles of the scp traversing the center of PHA-L deposits were clearly discernable by the absence of reaction product (Fig.2), and no labeled fibers could be traced to the red nucleus. Thus labeling of fibers "en passage" does not provide a reasonable explanation. of the results of the anterograde tracing experiments. Most likely, the labeled fibers in the facial nucleus originated from scattered PHA-L labeled cells located within the nuclei surrounding the lateral aspect of the PPN. This conclusion is substantiated by careful comparisons between the distributions of HRP labeled cells following medial and/or lateral facial injections, and the distribution of single PHA- I.labeled.cells surrounding the:dense center of PHA-I.deposit. The HRP-WGA injections into the medial groups of facial motor neurons resulted in labeling of a discrete group of cells in the contralateral RR and lateral RRF. Correspondingly, single Golgi-like labeled cells were seen.in these regions in the two PHA-L experiments (#25 and #31) which demonstrated a 51 contralateral projection to the medial portion of the facial nucleus. 0n the other hand, retrogradely labeled neurons were most numerous in the ipsilateral MPB, KF, LPB and Pr5 following HRP-WGA injections centered laterally in the facial nucleus, and uptake of PHA-L by isolated cells within these regions was more prominent in cases PHA-L #28, #27 and #24 which demonstrated a diffuse projection terminating predominantly ipsilaterally in the lateral division of the facial nucleus. PPN Projections to Medullary Cranial Nerve Nuclei While retrograde experiments were not carried out to clarify our findings of labeled fibers in the caudal cranial nerve nuclei (12, 10, Sol, and Am) following PHA-L injections centered in PPN, the literature supports the impression that these projections may also originate in nuclei surrounding the PPN. Several authors have shown retrogradely labeled KF, and to a lesser extent MPB and LPB neurons following HRP-WGA injections in several regions of the Sol in the rat (Herbert et.al. '90, Fulwiler & Saper '84, Rye et.al. '88). The largest proportion of these cells have been reported in the KF, with the remaining retrogradely labeled cells surrounding the ventrolateral scp in the MPB and LPB nuclei. Rye and colleagues ('88) have additionally shown that while many cells were retrogradely labeled in the parabrachial nuclei following injections of HRP-WGA in the ventrolateral Sol region, only 52 a few cells in the PPN were so labeled. In the present experiment, PHA-L injections sites did not include cells in the KF but did include several cells of the rostral and middle regions of the LPB and MPB. This may explain the sparse distribution of PHA-L labeled fibers to Sol in our results. Parabrachial projections to the ventrolateral medulla (including Am) and the hypoglossal nucleus have also been reported in the rat (Rye, et.al. '88; Saper & Loewy, '80; Fulwiler & Saper, '84; Herbert, et.al '90) and in the pigeon (Wild, et.al. '90). The ‘Ventrolateral medulla is widely thought to be involved in respiratory control (Ellenberger et.al. '90, Feldman & Grillner '83), and it has been suggested that the ventrolateral portion of the Sol also contributes to the control of respiration (Herbert et.al., '90). Moreover, the muscles controlling the patency of the upper airway are innervated by the hypoglossal (Krammer, et.al. '79; Lewis, et.al. '71; Odutola, '76) and dorsal motor vagus (Lewis, et.al. '70) nuclei and receive parabrachial input (Saper & Loewy '80). On the basis of their connections with these respiratory and oral motor nuclei, it has been proposed that the parabrachial nuclei (particularly the KF) may contribute to the control of respiration (Herbert, et.al. '90) and vocalization (Wild, et.al. '90). While it seems unlikely that the PPN contributes directly to these functions, the present study can not rule out this possibility. 53 Mesopontine Efferents to the Facial Nucleus: 1) Parabrachial Nuclei An ipsilateral projection from the KF, MPB, and LPB to intermediate and lateral portions of the facial nucleus has been reported previously in the rat (Isokawa-Akesson & Komisaruk '87, Hinrichsen & Watson '83, Travers & Norgren '83, Saper & Loewy '80), cat (Fort et.al. '89, Holstege et.al. '86, Takeuchi et.al. '80), and opossum (Panneton & Martin '83). Our findings confirm these results. In many species, the lateral facial subnuclei contain motoneurons innervating the buccolabial musculature (Watson et.al. '82, Komiyama et.al. '84, Dom et.al. '73). Our data implicates the MPB to a greater degree than other parabrachial nuclei in projecting to lateral portions of the ipsilateral facial nucleus, and these results are supported in the literature by autoradiographic studies (Saper & Loewy '80). The MPB has been identified as having gustatory functions (Saper & Lowey '80, Hill '87, Herbert et.al. '90) and, as was previously discussed, the ventrolateral parabrachial region has been associated with respiratory function. It follows that projections from parabrachial nuclei to the lateral facial subnuclei may contribute to the control of oral musculature in respiratory and feeding behaviors. 54 2) Retrorubral Nucleus In addition to parabrachial facial afferents, our data confirm a contralateral projection from RR to medial subdivisions of the facial nucleus which was first reported in the rat by Isokawa-Akesson & Komisaruk ('87). The rat RR is a compact group of small to medium sized cells located rostral to the ventral nucleus of the lateral lemniscus and rostromedial to the fibers of the lateral lemniscus (Paxinos & Watson '86). In other retrograde tracing studies in the rat, the contralateral "midbrain reticular fermation" (Hinrichsen & Watson '83) or the "paralemniscal zone" (Travers & Norgren '83) have been identified as sources of afferents to the medial groups of facial motoneurons. Similar observations have also been reported in the cat (May et.al. '89, Fort et.al. '89, Takeuchi et.al. '79, Henkel & Edwards '78) and opossum (Panneton & Martin '83). Since all these regions are rather vaguely defined, it is possible that the discrepancies are more semantic than factual. The cat "paralemniscal zone" has been. described in coronal sections as a narrow, vertical region consisting of darkly staining“ clusters, of :medium-sized, densely' packed neurons extending from the nuclei of the lateral lemniscus to the caudal pole of the RR (Henkel & Edwards '78). The cat retrorubral nucleus represents a distinct entity which is characterized by the presence of catecholaminergic cells projecting to the striatum (Vandermaelen et.al. '78) and 55 having similar morphological and functional properties as the nigral cells in the pars compacta (Preston et.al. '81). No similar information is available for the rat RR delineated by Paxinos and Watson ('86) . In fact, the cytoarchitectural features of the rat RR resemble more those described for the cat paralemniscal zone. This zone appears to receive projections from the superior colliculus and other structures known to be involved in visual and auditory orienting responses (Henkel '81), and it has been suggested that the connection from.this zone:to the medial facial motoneurons:may play a role in the pinnae orienting response. Although no such function has been previously proposed for the rodent RR, it is interesting that the projection from the rat RR to the contralateral facial nucleus is restricted only to medial facial motoneurons which innervate the pinnae (Watson et.al. '82). Thus, the rodent RR is clearly in a position to affect pinnae movements during the orienting response. It would be of interest to further explore the functional connectivity of the rat RR, particularly with regard to its involvement in the orienting reflexes. 3) Red Nucleus Our experiments confirm a crossed rubro-facial pathway terminating in the lateral and intermediate facial subnuclei as described in the literature (Travers & INorgren '83, Hinrichsen & watson '83, Isokawa-Akessan & Komisaruk '87, 56 Edwards '72, Dom et.al. '73, Panneton & Martin '83). Our data shows this to be a sparse projection. The large number of RN cells labeled in case CN7 #14 is very likely due to uptake of HRP-WGA by damaged fibers of the rubrospinal tract passing caudolateral to the facial nucleus. 4) Principal Sensopy Trigeminal Nucleus Finally, we have demonstrated a rather substantial projection from Pr5 to the lateral and intermediate facial subnuclei. Many neurons located just caudal to the ventral spinocerebellar tract (rostrolateral to M05) were labeled following HRP-WGA injections in both the medial and lateral portions of the facial nucleus. These were more numerous after lateral facial injections. Physiological studies demonstrating disynaptic responses of facial motoneurons to stimulation of the trigeminal nerve seem to support these findings (Tanaka et.al., '71). In contrast to our results, previous investigators have described Pr5 innervation of the facial nucleus to be rather sparse in the rat (Travers & Norgren.'83, Erzurumlu.& Killackey '79) and opossum (Panneton & Martin '83). The discrepancy with respect to the abundance of facial- projecting Pr5 cells may be due to the difficulties in the delineation of the Pr5 from the KF. In the sagittal plane, the KF and Pr5 nuclei intermix and the exact borders are difficult to distinguish. We defined KF cells as round or 57 pyramidal, darkly staining cells with a distinct nucleolus and a cell diameter exceeding 16pm (Fulwiler & Saper '84). Pr5 cells were smaller and more lightly stained than KF cells (Fukushima & Kerr '79). It is also possible that some of the retrogradely labeled cells in this transitional region represent neurons of the catecholaminergic A7 cell group. A7 cells are dispersed throughout the subcoeruleus, KF, and Pr5 nuclei and are known to project to the facial nucleus in the rat (Grzanna et.al. '87) and cat (Fort, et.al. '89). Cells labeled in the ipsilateral Pr5 may have also resulted from uptake onHRP-WGA.in.reticular regions lateral to the targeted facial nucleus. However, this can not entirely explain our results since several Pr5 cells were also labeled ipsilaterally with injections of HRP-WGA in the medial portions of the facial nucleus which completely avoided this reticular region. Since the Pr5 receives primary facial sensory input, a projection from Pr5 to the lateral subdivisions of the facial nucleus may provide a pathway mediating the tactual guidance of oromotor behavior. Descending PPN Efferents Our data suggests there does not exist a direct projection from the PPN to the cranial nerve nuclei, but descending PPN efferents clearly project to the ventromedial pontomedullary reticular nuclei. The majority of PPN 58 efferents to the reticular formation are concentrated ventromedially in the RFC, GiA, and Giv (Grofova et.al. in press, Nakamura et.al. '89, Mitani et.al. '88, Rye et.al. '88, Jackson & Crossman '83, Moon-Edley & Graybiel '83) , and these nuclei have been shown to project to somatic and autonomic motor columns in the medulla and spinal cord (Vertes et.al. '86, Jones & Yang '85, Travers & Norgren '83, Zemlan et.al. '82, Holstege & Kuypers '82, Martin.et.al. '81, Peterson '80), thus establishing a potential pathway by which PPN may affect motor behavior. The existence of a PPN-reticulo-spinal pathway has been proposed by Garcia-Rill & Skinner ('87) on the basis of electrophysiological experiments. Extracellular recordings of‘singletmedioventral.medullary'neurons.in the.cat showed. short latency' orthodromic .responses following stimulation of the mesencephalic locomotor region (MLR) coexistent with the ability to antidromically activate these same neurons from stimulation of the spinal cord. Since the reticular nuclei receiving input from the PPN project not only to the spinal cord but also to several motor and autonomic nuclei of the cranial nerves, it is possible that the PPN may influence both the spinal and cranial motor systems indirectly, through a relay in the brainstem reticular formation. Clinical syndromes associated with neuronal loss in the PPN in humans seem to support this suggestion since they invariably include disorders related to dysfunctions of the cranial nerves. 59 In progressive supranuclear palsy (PSP) , symptoms include unsteady gait, dysarthric or dysphonic speech, and impaired ocular movements, particularly in the vertical plane (Jellinger '88, Maher & Lees '86). Another syndrome associated with PPN cell loss is Meige syndrome, a rare disorder involving involuntary head turning, blepharospasm, and grimacing movements of the orofacial musculature (Tolosa & Marti '88, Zweig et.al. '88). Functional Considerations The PPN has been implicated in motor control by virtue of its abundant connections with the basal ganglia and spinal cord projecting reticular nuclei, and by its co-localization within the physiologically identified.mesencephalic locomotor region. However, it became increasingly obvious that it would be an oversimplification to consider PPN functions only in terms of locomotion.or motor control in general. In.the light of various lines of evidence, it is tempting to speculate that the PPN may represent.a part.of a complex substrate underlying orienting reflexes. While the precise pathways involved in the orienting reflex are far from clear, several contributing nuclei have been identified (Fig. 15). In particular, the deep layers of the SC are necessary to elicit orienting behaviors (Peterson '80). It is possible that.the orienting reflex, characterized by turning of the head, pinnae and eyes toward a novel 6O ISNrF PPN ,. SC RPc Gi cervical cord CN 7 CN 3, 4, 6 Figure 15: Circuit Diagram - Orienting Reflex Circuit diagram illustrates the connections of some of the structures thought to be involved in the orienting reflex. Abbreviations: intermediate and deep layers of the superior colliculus (SC), substantia nigra pars reticulata (SNr) , pedunculopontine nucleus (PPN), nucleus reticularis pontis caudalis (RPc) , nucleus gigantocellularis (Gi) , cranial nerves (CN) 3, 4, 6, and 7. 61 stimulus, may be executed via deep tectal efferents projecting directly to appropriate motor structures. Intermediate and deep layers of the SC project to the upper cervical segments, innervating axial musculature required for head turning (Huerta & Harting '82), to the cranial motor nuclei involved in extraocular and pinnae movements (Vidal et.al. '88, Keller '79, Graham '77) and to the pontomedullary reticular formation projecting to these cranial motor nuclei (Vertes et.al. '86, Kawamura & Hashikawa '78, Edwards '80, Isokawa—Akesson & Komisaruk '87, Panneton & Martin '83, Takeuchi et.al. '79, Fort et.al. '89). Since the PPN sends descending projections to these same reticular nuclei (i.e. the medioventral RPc, GiA, and Giv) (Grofova et.al. in press, Mitani et.al. '88, Rye et.al. '88, Moon-Edley & Graybiel '83, Jackson & Crossman '83, Zemlan '84) it is well situated to modulate neural activity occurring during the orienting response. On the other hand, the SNr is in a position to influence these two structures (i.e. the SC and PPN) which both project either directly or indirectly to the motor nuclei required for execution of the orienting response. The SNr projects substantially to the ipsilateral deep layers of the SC (Grofova et.al. in press, Williams & Faull '88, Beckstead & Frankfurter '82, Gerfen et.al. '82) as well as extensively to the ipsilateral PPN (Spann & Grofova '88, Noda & Oka '84, Garcia-Rill et.al. '83, Gerfen et.al. '82, Beckstead et.al. 62 '82). Physiological experiments have also confirmed a SNr- PPN-reticular pathway (Kelland & Asdourian '89, Nakamura et.al. '89, Garcia-Rill.& Skinner '87), and monosynaptic input from SNr to tectospinal neurons has been described (Williams & Faull '88). There seem to be considerable interconnections within this circuitry. In this regard, it is of interest to note that the PPN also sends input to the SC (Hall et.al. '89, Woolf & Butcher '86, Beninato & Spencer '86). Thus, nigral efferents may assist in coordinating the influence of the SC and PPN on the medial RPc, GiA, and Giv reticular nuclei. Physiological observations of directionally specific behavioral abnormalities following unilateral lesions of the rat PPN (Kilpatrick & Starr '81) also support a possible role of the PPN in the orienting reflex. Because of the multiplicity of nuclei involved in control of the orienting response, and the complexity of interconnections among them, further detailed studies will be required to elucidate the role of the PPN in the orienting reflex as well as other functions. 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