Mlcmgfifl 3mm X HERARY University ! L PLACE IN RETURN BOX to remove this checkout from your recocd. To AVOID FINES Mum on or before due due. DATE DUE DATE DUE DATE DUE 3—1 17—“ ll MSU Is An Affinnetlve Action/Equal Opponunlty Institution cmmn‘ DISTRIBUTE PROJECT I 01" 1“ Par DISTRIBUTION AND MORPHOLOGICAL FEATURES OF EFFERENT PROJECTIONS FROM IDENTIFIED SUBDIVISIONAL AREAS OF RAT TRIGEMINAL NUCLEUS INTERPOLARIS BY Mark Steven Cook A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1992 DISTRIBUTE PROJECT. OF The rece trigeminal nu the developme leucoagglutir examination < PHA‘L injectj including 1 Ventrolatera; border “9101 the distribut terllimal ax: Provided. T subdivisiona Posterodeia nuclear 91‘0u nucleus (APT nucleus (RPc cerebellum (: i n dii We: O//_ j/jjg/IX ABSTRACT DISTRIBUTION AND MORPHOLOGICAL FEATURES OF EFFERENT PROJECTIONS FROM IDENTIFIED SUBDIVISIONAL AREAS OF RAT TRIGEMINAL NUCLEUS INTERPOLARIS BY Mark Steven Cook The recent delineation of six distinct subdivisions in trigeminal nucleus interpolaris (Vi) in the rat, along with the development of the anterograde tracer Phaseolus vulgaris- leucoagglutinin (FHA-L) have provided a basis for the re- examination of the efferent projections from Vi. Following FHA-L injections into four subdivisional regions of rat Vi, including the ventrolateral magnocellular (v1Vimc), ventrolateral parvocellular (v1Vipc), dorsomedial (dii) and border regions (brVi), detailed drawings and descriptions of the distribution and morphological characteristics of labeled terminal axonal arborizations in all target nuclei were provided. The results demonstrated that all four Vi subdivisional regions provide input to the ventral posteromedial thalamic, nucleus (VPM), posterior ‘thalamic nuclear group (P0), zona incerta (ZI), anterior pretectal nucleus (APT), superior colliculus (SC), parvocellular red nucleus (RPC), pontine nuclei (Pn), facial motor nucleus (VII), inferior olive (IO), sensory trigeminal complex (SVC), cerebellum (CB) and cervical spinal cord (CSC). Only neurons in dii ‘were found. to jproject to the ‘medial accessory oculonotor nu all four Vi projections 1 were found v four regions and RFC, w? topographica SC. In the projected p' dorsal acce differences the CB, 31 lainly to 1 re‘lions, in 10bu1e and to the deal greatest j Projected . contra late oculomotor nuclei (MAB) in the midbrain. In the diencephalon, all four Vi subdivisional regions gave rise to similar projections to Po and 21, however, topographical differences were found with the projections to VPM. In the midbrain, all four regions of Vi gave rise to similar projections to APT and RFC, while only dii provided input to (MA3) and topographical differences were found with the projections to SC. In the pens and medulla, the four regions of Vi all projected predominantly to medial Pn, dorsal VII, and the dorsal accessory' olivary' nucleus, however, topographical differences were found with the projections to the SVC. In the CB, all four Vi subdivisional regions provided input mainly to the granule cell layer of the orofacial tactile regions, including Crura I and II, paramedian lobule, simple lobule and the uvula, as well as collateralized projections to the deep cerebellar nuclei. Neurons in dii provided the greatest input to CB. In the CSC, all four Vi regions projected to laminae III and IV of the ipsilateral cervical dorsal horn, while only dii and leimc projected to the contralateral dorsal and ventral horns. I would advisor, Dr. and encourag to thank the Grofova, J,] their (Jilidan I also SUpport 0f 1: llY wife, Lir ACKNOWLEDGMENTS I would like to express my appreciation and thanks to my advisor, Dr. William M. Falls, for his leadership, support and encouragement during my graduate training. I also wish to thank the members of my graduate committee, Drs. Irena Grofova, J.I. Johnson, Kathryn Lovell, and Duke Tanaka for their guidance. I also wish to express my deep appreciation for the support of my family and friends. Finally, I want to thank my wife, Linda, for her unrelenting support and patience. iv List of Figur Abbreviation Introduction Chapter I Chapter I I] TABLE OF CONTENTS ListofFigures........ ...... . .................. ..........vi Abbreviations ....................... ...... ..............viii Introduction Chapter I Chapter II Chapter III IOOOOOOOOOOOOOOOOOOOOOOOC0.0.0.00000000000000001 Efferent projections of neurons in four sub- divisional regions of rat trigeminal nucleus interpolaris to the diencephalon and midbrain: [APHA-Lstudy...................................3 Introduction........... ..................... ....3 MaterialsandMethods...........................5 Results.... ..... . ................. ..............8 Discussion.....................................20 Figures........................................47 Bibliography...................................71 Efferent projections of neurons in four sub— divisional regions of rat trigeminal nucleus interpolaris to pontomedullary regions: A FHA-L study..........................................82 Introduction...................................82 HaterialsandMethods..........................84 Results........................................84 Discussion.....................................90 Figures.......................................104 Bibliography..................................118 Efferent projections of neurons in four sub- divisional regions of rat trigeminal nucleus interpolaris to the cerebellum and spinal cord: 4APHA-Lstudy.................. ...... .........125 Introduction.............. ....... .............125 Materials andMethods. . ............ . . . . . . . . . . . 127 Results.......................................128 Discussion....................................138 Figures.......................................160 Bibliography.............. .......... ..........186 '5. 1 I .‘\ on CHAPTERI Figure Figure Figure Figure I"igure Figure Fi‘Jlll'e CHAPTER I Figure 1: Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: Figure 7: Figure 8: Figure 9: Figure 10: Figure 11: Figure 12: CHAPTER II Figure 1: Figure 2: LIST OF FIGURES PHA-L Injection Sites ......................48 Diencephalic Projections from leimc and VIVipc O...OOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOSO Diencephalic Projections from dii and brVi OOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOSZ Midbrain Projections from leimc and VIVipc 0.00...O..0...O...00.000.00.00000000054 Midbrain Projections from dii and brVi ...56 Drawings of Terminal Axonal Arborizations invpu OOIOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOIOOSB Drawings of Terminal Axonal Arborizations in P0 and 21 00.0.0000...0.0.0.000000000000060 Drawings of Terminal Axonal Arborizations inmand SC 0.000....0.0.0.00000000000000062 Drawings of Terminal Axonal Arborizations inRPC andMA3 O0.0..0.0.0.0.00000000000000064 Photomicrographs of Terminal Axonal Arborizations in VPM, Po and 21 ............66 Photomicrographs of Terminal Axonal Arborizations in 21, APT and SC ............68 Photomicrographs of Terminal Axonal Arborizations in SC, RPC and MAB ...........70 PHA-L Injection Sites .....................105 Projections from Vi Subdivisional Regions to Pn OOOOOOOOOOOOOO...0.0.0.00000000000000107 vi Figure 3: Figure 4: Figure 5: DD") 1 Figure Figure Figure Figure Figure CHAPTER III Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10: Figure 11: Figure 12: Figure 13: 7: 1: 2: 9: Pontomedullary Projections from leimc andVIVipc I...000............IOOOOOOOOOOO.109 Pontomedullary Projections from dii and brVi ....... ........... ... ...... ...........111 Drawings of Terminal Axonal Arborizations in Pn andVII ......OOOOOOOOOOOO0.00.00.00.113 Drawings of Terminal Axonal Arborizations in Io and SVC O...........0.00.00.00.000000115 Photomicrographs of Terminal Axonal Arborizations in Pn, VII, IO and SVC ......117 PHA-L Injection Sites .....................161 Cerebellar Projections from dii ..........163 Cerebellar Projections from dii ..........165 Cerebellar Projections from leimc ........167 Cerebellar Projections from leipc ........169 Cerebellar Projections from brVi ..........171 Drawings of Mossy Fibers in CB Granule cell Layer .....OOOOOOOOO00.00.00.000000000173 Drawing of Terminal Axonal Arborizations in DC" 0.0.0.0...O0.0.0....0.0.00.000000000175 Spinal Cord Projections from leipc and VIVimc 0.0.0.0....O......0.0.00.00000000000177 Spinal Cord Projections from brVi and dii 179 Drawings of Terminal Axonal Arborizations in CSC ......OOOOO00......0.0.0.00000000000181 Photomicrographs of Mossy Fibers in CB ....183 Photomicrographs of Terminal Axonal Arborizations in DCN and CSC ..............185 vii brVi CB CE ME mm [KN «Ni duh TWi GCL InG INT Inwh 10 I00 Iopr irvi IS LAT EA 3 ante borc cert cer‘ cer dee dee do: do dc qr ht brVi CB CE CSC DAB DCN chi lei dii GCL InG INT InWh IO IOD IOPr irVi IS ABBREVIATIONS anterior pretectal nucleus border region of Vi cerebellum cervical enlargement cervical spinal cord deep axonal bundle deep cerebellar nuclei dorsal cap of Vi dorsolateral Vi dorsomedial Vi granule cell layer of CB horseradish peroxidase intermediate gray layer superior colliculus intermediate cerebellar nucleus intermediate white layer superior colliculus inferior olive inferior olive dorsal nucleus inferior olive principal nucleus intermediate region Vi injection site lateral cerebellar nucleus medial accessory oculomotor nucleus viii HDH MED 14F PCRt FHA-L PHI. Pn Po RCC RMC RPC SC SIM SVC Vi VII Vlvimc Vlvipc Vm S med mec‘ IDOE me< pa Ph pa MED MF PCRt PHA-L PML Pn Po RCC RF SC SIM SVC Vi VII v1Vimc leipc Vms V0 VPM WGA ZI medullary dorsal horn medial cerebellar nucleus mossy fiber medial lemniscus parvocellular reticular nucleus Phaseolus vulgaris leucoagglutinin paramedian lobule pontine nuclei posterior thalamic nucleus rostral cervical cord receptive field magnocellular red nucleus red nucleus parvocellular red nucleus superior colliculus simple lobule sensory trigeminal complex trigeminal nucleus interpolaris facial motor nucleus ventrolateral magnocellular Vi ventrolateral parvocellular Vi trigeminal main sensory nucleus trigeminal nucleus oralis ventral posteromedial thalamic nucleus wheat germ agglutinin zona incerta ix Trigem; of the sc intonation relaying’ it nidbra in , ; researchers Vi 0f the r eqarding g Specit ic airbcrizat 1. character 1 infomat 10 Project ion dif f erenc e provided,i Of 13er ac INTRODUCTION Trigeminal nucleus interpolaris (Vi), one of the nuclei of the sensory trigeminal complex, receives tactile information from orofacial regions and modifies it before relaying it to several target nuclei in the diencephalon, midbrain, pons, medulla, cerebellum and spinal cord. Many researchers have investigated the projections from neurons in Vi of the rat, however, few have provided information regarding subdivisional differences with these projections, specific distribution patterns of , terminal axonal arborizations in target nuclei, or the morphological characteristics of the terminal arbors. The lack of information. pertaining to these aspects of Vi efferent projections was due to insufficient data on Vi subdivisional differences and the utilization of neuronal tracers which provided inadequate labeling of terminal axonal arborizations of projection neurons. The recent delineation of six Vi subdivisions has served as a basis, in the present study, for the reexamination of projections from. Vi utilizing the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA- L). The present study describes the projections from neurons in four subdivisional regions of Vi in the rat, including rial ventrolateral parvocellular (brVi) area: distribution axonal arbor nucleus (VPH incerta (le colliculus accessory o. facial Noto tl‘igeminal c cOrd (CSC) . 2 ventrolateral magnocellular (v1Vimc), ventrolateral parvocellular (v1Vipc), dorsomedial (dii) and border region (brVi) areas. This study also describes the specific distribution and morphological characteristics of terminal axonal arborizations in the ventral posteromedial thalamic nucleus (VPM), posterior thalamic nuclear complex (Po), zona incerta (ZI) , anterior pretectal nucleus (APT), superior colliculus (SC), parvocellular red nucleus (RPC), medial accessory oculomotor nucleus (MA3) , pontine nuclei (Pn) , facial motor nucleus (VII), inferior olive (IO), sensory trigeminal complex (SVC), cerebellum (CB) and cervical spinal cord (CSC). Efferent Pro of Ra Di Trigemi information orofacial r sensory inf before bei no Unclei alo CHAPTER I Efferent Projections of Neurons in Four Subdivisional Regions of Rat Trigeminal Nucleus Interpolaris to the Diencephalon and Midbrain: A PHA-L Study INTRODUCTION Trigeminal nucleus interpolaris (Vi) receives sensory information from primary trigeminal neurons innervating orofacial receptive fields. Within Vi, this orofacial sensory information undergoes processing and modification before being relayed by projection neurons to several target nuclei along the neuraxis (39,40,49,51). Utilizing anatomical axonal tracing techniques andmelectrophysiological recordings, neurons in rat Vi have been shown to emit axons which terminate in various nuclei of the thalamus and midbrain. These nuclei include ventral.posteromedial thalamic nucleus (17,21,22,28,29,35,49,67,81,84,87,118,102), zona incerta (29,84,96), posterior thalamic nuclei (21,22,84) and superior colliculus (1,17,34,45,61,91,92,99,102,106). Efferent ‘Vi projections to ‘well-known thalamic and midbrain.areas have been described.without.much.consideration for their subdivisional origin within the nucleus. This is to be expected, since only recently have six separate and distinct subdivisions of rat Vi been delineated based on 4 differences in their overall cyto- and myeloarchitecture, as well as connectional criteria (85). The six Vi subdivisions include ventrolateral magnocellular (v1Vimc), ventrolateral parvocellular (v1Vipc), border region (brVi), dorsolateral (lei), dorsal cap (chi) and intermediate region (irVi). In addition, previous studies have provided relatively little information regarding the morphological characteristics of the terminal axonal arborizations of Vi projection neurons. The present study was conducted toldetermine the distribution of efferent axons from neurons in four distinct Vi subdivisional regions to target nuclei along the neuraxis, as well as to describe the morphological characteristics of the terminal axonal arborizations of these neurons. The four subdivisional regions of Vi from.which these projections are described include v1Vimc, v1Vipc, brVi and dorsomedial Vi (dii). The subdivsional region, dii, includes lei, chi and irVi. Iontophoretic injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) , permit one to make injections into Vi subdivisional regions and visualize the distribution and morphological characteristics of the axons and terminal arborizations of Vi neurons in the various target nuclei. The morphological details of Vi axons and their terminal arborizations are necessary if one is to begin to understand the effects of Vi inputs on the activity of neurons in target nuclei in the thalamus and midbrain. All anz? for postOp guidelines. grams) usec Pentobarbit al>13ill'atus. Opened and (tip diame‘ PHA'L (rec: at 2Suki/mi according atlas or P Phelan anc‘ ihj acted a MATERIALS AND METHODS All animals were housed, prepared for surgery and cared for postoperatively according to federally prescribed guidelines. The eight adult male Sprague-Dawley rats (250-300 grams) used in this study were anesthetized with sodium pentobarbital (35-40 mg/kg) and placed in a stereotaxic apparatus. The cranium overlying the cerebellar cortex was opened and the dura mater reflected. A glass micropipette (tip diameter approximately 20 pm), filled with 1.0 pl of PHA-L (reconstituted in 10 mM phosphate buffer at a pH of 8.0 at 25mg/m1), was lowered into a Vi subdivision region according to precalculated stereotaxic coordinates from the atlas of Paxinos and Watson (83) and the previous studies of Phelan and Falls (85,87). The FHA-L was iontophoretically injected at 5.0 microamperes of alternating positive current for approximately 25-35 minutes. Following the removal of the micropipette, Gelfoam was placed over the cranial opening and the overlying skin was closed with wound clips. After 10-12 days of survival, each rat was transcardially perfused under heavy anesthesia (sodium pentobarbital), with a fixative solution (500 ml) containing 4% paraformaldehyde in a 0.1 M acetate buffer (pH 6.5) followed immediately by another fixative solution (500 ml) containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M borate buffer (PH stored in a Twenty-four sectioned a1 transverse collected i adjusted to processed fr Gerfen and : conducted a' three rinS¢ sections in normal Rain: by a 5 m: incubation Anti“)! t. agitatiOn' antibody Bich ) 6 buffer (pH 9.5) . The brain was immediately removed and stored in a fresh solution of the latter fixative at 4°C. Twenty-four hours later, the thalamus and midbrain were sectioned at 30-50 pm on an Oxford Vibratome in either the transverse or horizontal plane. Tissue sections were collected in a 0.05 M Tris-buffered 0.15 M saline (TBS), adjusted to a‘pH of 7.6 at room.temperature , and immediately processed for PHA-ereaction.product by'a modification of the Gerfen and Sawchenko method (37). All subsequent steps were conducted at room temperature unless otherwise noted. After three rinses in TBS with 0.2% Triton X100 (TBS-TXIOO), sections were incubated for 30 minutes in TBS-TX100 with normal Rabbit Serum (Vector Labs. Inc.).. This was followed by’ a. 5 minute rinse in. TBS-TXIOO and then a 48 hour incubation in primary antibody, consisting of a 1:2,000 Goat Antibody to PHA-L (Vector Labs. Inc.) in TBS-TX100, with agitation, at 5°C. After the incubation in the primary antibody, sections were rinsed 3 times in TBS-TXIOO (5 min. each) and then incubated for 90 minutes in secondary antibody, which consisted of Rabbit antibody Goat (Vector Labs. Inc.) in TBS-TX100. Sections were then rinsed (3 times at 5 min. each) with TBS-TX100 followed by a 1-2 hour incubation in a solution of Avidin and Biotinylated horseradish peroxidase (Vector Labs. Inc.) in'TBS-TX100. The sections were then rinsed 4 times (5 min. each) with TBS- TX100. The final step consisted of exposing the sections to 7 a solution of diaminobenzidine (DAB) and glucose oxidase at a ratio of 10:1. The reaction was terminated with 4 rinses of TBS-TX100. Sections were then placed on gelatin-coated glass slides, stained.with cresyl violet and coverslipped for light microscopic analysis. All sections were viewed with a Leitz Laborlux 12 microscope fitted with a drawing tube. Low-power drawings of the location and distribution of V1 efferent axons to thalamic and midbrain regions were made by using a 2.5x objective lens at a magnification of 31x. Morphologically distinct types of Vi terminal axonal arborizations in each of the described target nuclei were visually assessed by observing each area at high power light microscopy, using a 100x oil immersion . lens, and several terminal arborizations, representative of each. type identified, were drawn using a drawing tube at a magnification of 1,250X. .Although most.of the representative terminal arborizations drawn were from single tissue sections, some terminal arbors were reconstructed from adjacent sections. The distinction of the various types of Vi terminal arborizations in this study were based on axonal diameter, degree of branching, as well as frequency and size of boutons. PEA-L Inje The . successful two for ear two separa‘ subdivisior for each . rePl’esentat centered in region of c‘ labeled Ce: labeling was “3 devoid . RESULTS PEA-L Injection Sites The data provided for this study were based on successful injections of PHA-L in a total of eight animals, two for each of the four Vi subdivisional regions. Although two separate injections were made into each of the four Vi subdivisional regions, only one representative injection site for each will be used for illustrative purposes. The representative injection site in v1Vimc (Fig. 1A) was centered in the middle of this subdivision with a dark brown region of dense PHA-L reaction product, defined by solidly labeled cell bodies. Surrounding this dense "core" of labeling was a lighter brown spread of reaction product which was devoid of labeled cell bodies. A slight spread of the reaction product extended laterally into brvi, medially into the adjacent parvocellular reticular formation (PCRt) and rostrally into the caudalmost extent of trigeminal nucleus oralis (Vo) , where it was located in the ventrolateral portion of the nucleus. 'The representative injection site in leipc (Fig. 13) was centered in the middle of this subdivision, with a dark brown region of reaction product in the middle and a lighter brown periphery extending laterally into brvi and. medially into PCRt. The representative injection site in dii (Fig. 1C) showed dark brown reaction 9 product in dlvi, dcvi and irvi and a lighter brown spread of the reaction product extending ventrally into the dorsal portion of brvi and dorsal v1Vimc. The representative injection site in brVi (Fig. ID) showed a dark brown reaction product in the caudal two-thirds of the subdivision and a lighter brown spread of reaction product in the lateral-most portion of leipc. Only a few retrogradely labeled cells were located in the medullary dorsal horn (MDH: trigeminal nucleus caudalis) , and no retrogradely labeled axons were found in the sensory root of the trigeminal nerve. Projections to the Thalamus Injections of PHA-L into any of the four Vi subdivisional regions, v1Vimc, v1Vipc, dii and brVi, labeled projection neurons whose axons exited Vi ventromedially and coursed through the parvocellular reticular formation toward the midline. The converging axons then coursed immediately dorsal to the ipsilateral inferior olive, 'decussated, and collected into a loose bundle located dorsal to the medial portion of the pyramidal tract. As they ascended, the fibers shifted dorsally in the rostral pons, coursed rostrally through the midbrain and entered the diencephalon interspersed with fibers of the medial lemniscus (ML). Labeled axons from all Vi subdivisional regions exited ML and coursed either dorsally or ventrally and gave rise to terminal arborizations in the contralateral ventral 10 posteromedial nucleus of the thalamus (VPM) , the medial portion of the posterior nuclei (Po) and the zona incerta (21) (Figs. 2,3). With each injection, the density of terminal axonal arborizations was greatest in 21, less in VPM and least in Po. No labeled terminal axonal arborizations were found ipsilaterally in these target nuclei. Projections to the ventral Posteromedial Thalamic Nucleus In the caudal diencephalon, Vi-VPM parent axons (approximately 1.5 pm in diameter) exited the contralateral ML and traveled dorsally and rostrally in‘VPM where they gave rise to terminal arbors in various portions of the nucleus depending on the Vi subdivisional region of origin. Axons from neurons in v1Vimc, v1Vipc, dii and brVi gave rise to terminal arborizations throughout the rostrocaudal extent of contralateral VPM, with the majority of arbors located in the middle one-third of the nucleus. Aside from these consistencies, topographical differences were found with the distribution of terminal arbors from the various Vi subdivisional regions. Axons from v1Vimc neurons gave rise to terminal arborizations located. dorsomedially in ‘VPM (Fig. 2A-D). Terminal arbors from axons of leipc neurons were found dorsomedially in rostral VPM (Fig. 2E,F) and dorsolaterally in caudal VPM (Fig. ZG,H). The terminal arbors from axons of dii neurons were primarily located ventrolaterally in VPM 11 (Fig. 3B-D) , and those from axons of brVi neurons were primarily found in the dorsal-most portion of VPM (Fig. 3E- H). A small number of terminal arborizations from axons of neurons in each Vi subdivisional region were located dorsomedially in the rostral-most.portion of VPM (Figs. 2A,E; 3A,E). In all cases, the number of terminals at the rostral- and caudal-most extent of VPM were sparse. All of the PHA-L injections into Vi subdivisional regions produced two morphologically distinct types of terminal axonal arborizations in VPM, a thin-VPM type and a thick-VPM type. Both types of terminal arbors were usually found intermingled throughout the receptive regions of VPM and were formed either by collaterals of the parent fiber or at the terminal end of the parent axon. The terminal arbors were often intermingled and formed patches (6A). The thin- VPM type (Figs. 63 3,4; 108) was characterized by a relatively thin, branched terminal strand (approximately 0.5 pm in diameter), with oval to irregularly shaped boutons (approximately from 1.0 to 2.5 pm in diameter). The thick- VPM type (Figs. 68 1,2; 10A) was characterized by a thicker, branched terminal strand (approximately 1.0 pm in diameter). It gave rise to irregularly shaped boutons (approximately from 1.0 to 4.0 pm in diameter). 12 Projections to the Posterior Thalamic Nuclear Group Parent axons (approximately 1.0 to 1.5 pm in diameter) which gave rise to terminal arbors in medial Po coursed dorsally from contralateral ML, traversed VPM, and traveled medially before giving rise to terminal arbors in Po. The arbors in Po were widely scattered throughout the rostrocaudal extent of the nucleus, just medial to‘VPM (Figs. 2,3). Although Vi-Po axons traversed VPM receptive zones on their way to P0, it could not be determined whether or not they provided collateralized input to VPM. Only one ‘morphologically'distinct.typerof'terminal axonal arborization was located in medial Po following injections in each of the Vi subdivisional regions (Figs. 7A, 10C). The Po-type of terminal arbor was characterized by a thin, sparsely branched terminal strand (approximately 0.5 pm in diameter) with relatively few, widely spaced boutons en passant measuring approximately from 1.0 to 1.5 pm.in diameter. The Po-type of arbor was unique from the other terminal arbors described in this study. It.had a long filamentous terminal strand, which either came off as a side branch of the parent fiber, or as the termination, and invariably produced a relatively large bouton (approximately 2.0 pm in diameter) at the end of the terminal strand. 13 Projections to Zona Incerta Neurons in v1Vimc, v1Vipc, dii and brVi gave rise to axons (approximately 1.0 to 1.5 pm in diameter) which exited ML ventrally in the contralateral diencephalon, and coursed to the subjacent 21. The labeled axons passed ventrally, through.thea single type of terminal arborization (Fig. 8), which consisted of several thin, branched terminal strands (approximately 0.5 pm in diameter) with numerous ‘widely spaced. boutons en. jpassant and. end. boutons (approximately 1.0 to 2.0 pm in diameter). Terminal arbors in DCN were mainly oriented dorsoventrally. 136 Projections to the Cervical Spinal Cord PHA-L labeled axons gave riSe to terminal arborizations in the cervical spinal cord (CSC). The greatest density of terminal arbors was in the rostral cervical cord (RCC) with the cervical enlargement (CE) receiving considerably fewer terminal arbors. Projections to the CE did not extend caudally past the level of C7. Labeled axons from.neurons in all four Vi subdivisional regions descended directly through MDH, in the deep axonal bundles, to the ipsilateral dorsal horn, where they gave rise to terminal arborizations predominantly in laminae III and IV of the RCC and CE (Figs. 9,10). The density of labeled terminal axonal arborizations decreased from RCC to CE, and the overall density was less following brVi injections than they were from injections into the other subdivisional regions. An occasional labeled terminal arbor was seen in laminae V and VI of the ipsilateral.dorsal.horn following the various injections, and no labeled axon extended into the ipsilateral ventral horn. While v1Vimc and dii injections produced labeling in the contralateral CSC (Figs. 9C,D; 10C,D), v1Vipc and brVi injections did not (Figs. 9A,B; 10A,B). PHA-L injections into v1Vimc and dii labeled axons which coursed medially from the respective injection sites, decussated and descended in the contralateral ventral funiculus. From.the‘RCC ventral funiculus, labeled.axons coursed.dorsally through.the ventral horn, where they gave rise to terminal arbors in lamina IX, 137 and several continued dorsally through the intermediate gray to the dorsal horn where sparse numbers of axons ended in laminae III and IV. Although a substantial number of axons from v1Vimc neurons extended to the CE in the contralateral ventral funiculus, where they gave rise to substantial terminal arbors in laminae IX and laminae III and IV (Fig. 9D), axons from dii did not. The terminal arborizations located in the ipsilateral dorsal horn originated from axons (approximately 1.0 to 1.5 pm in diameter) which descended within the deep axonal bundles and were of one type (Fig.11). They were characterized by thin, branched terminal strands (approximately 0.5 pm in diameter) with multiple en passant and end boutons (approximately 1.0 to 2.0 pm in diameter). The terminal arbors were generally oriented rostrocaudally, often with ascending and descending limbs spanning considerable distances (up to 580 pm). The labeled terminal arborizations located in the contralateral CSC were from axons that were thicker (approximately 2.0 pm in diameter) than those that projected to the ipsilateral dorsal horn, however they were similarly branched and had multiple en passant and end boutons (Figs. 13C,D). 138 DISCUSSION This study examined the projections from Vi to the cerebellum and spinal cord based on recently delineated Vi subdivisions (68), using the anterograde PHA-L method (30). This study also provided detailed descriptions of the morphological characteristics of PHA-L labeled MFs in the GCL, as well as the characteristics of the terminal axonal arborizations in the DCN and CSC. For a discussion of differences between the various Vi subdivisions and the benefits of utilizing the anterograde PHA-L method in this study, see the Discussion section of Chapter I. However, it is important to mention here that although it has been shown that PHA-L can be transported retrogradely (74) and anterogradely by fibers of passage (21), these modes of transportation did not appear to affect the results of this study. Only a few retrogradely labeled neuronal somata were located in rostral MDH. Regarding the labeling of fibers of passage, one of the main routes for trigeminospinal axons is through the more caudal trigeminal sensory nuclei (31,40,41,61). So although the potential exists for labeling the fibers of more rostral trigeminal sensory nuclei, which travel through Vi to the spinal cord, this did not appear to be the case in the present study. The projections from Vi to the CSC described in this study, are 139 different from those described from ‘Vo using' the same technique (85). If PHA-L injections into Vi did not label fibers of passage from neurons in V0, there is no reason to believe that fibers of passage from.neurons in Vms would also be labeled. Projections to the GCL of the Cerebellar Cortex Projections from Vi to CB have been extensively studied anatomically and electrophysiologically in the rat (39,46,51,65,84,93,96,97) and.cat (33,86) as well as in other mammalian species (64,80). Vi has been shown to be the main nuclear contributor to the trigeminocerebellar projection (93). Up to 70% of its neurons have been shown to project directly to CB, mainly ipsilaterally (86), with a smaller constituent of cells projecting contralaterally (26,64,65). Many’ previous studies. which investigated 'trigemino- cerebellar projections described their distribution in areas of the cerebellar cortex known to receive orofacial tactile input. These orofacial tactile areas, lobule 9a of the vermis (46,86), PML, Crura.I and II (83,86), and SIM (82,86), contain representations of orofacial regions that are disproportionately large compared to other body parts. The present study adds support to these findings by showing that these orofacial tactile areas are the main CB targets of Vi neurons. The major projection to the vermis from all Vi subdivisional regions was to lobule 9a. Lobule 9a has been 140 shown to contain projection areas from cutaneous receptive fields (RFs) mostly from mystacial vibrissae and the upper lip (83). The primary Vi projections to the hemisphere was to Crura I and II, PML and SIM. These regions also receive tactile information from orofacial regions (82,83). Crus I receives patch-like inputs from the head and upper face (including the crown, ear, eyelids, nose, upper and lower lips, and mystacial vibrissae) , however lacks input from perioral regions. Crus II receives input from perioral structures, as well as from incisors and intraoral skin of the furry buccal pad. PML receives tactile input from the entire body, but primarily from perioral structures. Finally, SIM has been shown to receive input only from regions within and around the face and mouth (including the gingiva, incisors, lips and vibrissae). The neurons in the dorsal portion of Vi (dii), presumably receiving mandibular primary afferent input, are shown by the present study, and supported by other studies (39,65), to be the main contributor of axons to these regions. The MFs from Vi neurons described in the present study, branched within the white matter and GCL, while giving rise to large, irregular swellings only in the GCL. During their course through the white matter, MFs divide several times, giving off as many as 30 collaterals to the GCL as they course towards the distal portion (crown) of the folia (62). Some fibers branch. deep in the white matter to serve 141 different folia, as well as within a particular folium to serve its different parts. This branching of MFs may be, in part, responsible for the fractured pattern of distribution of orofacial RFs in CB (46,82,83), where multiple representations of body parts exist in a particular folium, or in several folia, forming patches sometimes referred.to as a "mosaic”. For example, vibrissae and/or dorsal head regions are represented on five different folia (Crus Ia,b and c, Crus 11a, and PML), and the perioral tissues on three folia (Crus 11a and b, and PML). .Also, within a particular folium, a specific body structure may project to more than one patch, often separated from each other by patch projections from other body regions. One previous study in the rat ( 96) provides additional evidence that trigeminocerebellar MF branching is related to this somatotopical organization of the GCL. Although this pattern of MP distribution may not appear indicative of a well-organized system, microelectrode mapping of these tactile areas have revealed that they are highly organized, but multiple, representations (83). Recordings from orofacial regions to the respective patches in CB, have shown latencies of 3-5 msec. A similar study recorded latencies of 2-4 msec., in lobule 9a of the uvula and crura I and II, when the snout was stimulated (6). These latency times are short enough to allow only a single relay in the medulla, most likely in part, in Vi. The RFs for a particular patch can be recorded in a 142 vertical column in the GCL. This may be due to the morphology of the MFs, as described in the present study as well as in other examinations (62,81), which illustrate that the large irregular swellings of a particular MF, or groups of MFs, are distributed throughout the deep, middle and superficial portions of the GCL. Although three morphologically distinct types of MFs have been described (62), only two have been identified in the present study, a simple and complex type. The present study shows that dii provides the most robust input from Vi to all of the CB receptive areas. Although previous studies have shown that this appears to be true for projections to PML (65) and Crura I and II (39) in the rat, this is not supported for projections to PML in the tree shrew (64) . A more extensive study of subdivisional origin of Vi-CB neurons in the rat revealed that they were distributed throughout the rostrocaudal extent of Vi (69). Vi has also been shown to provide substantial input to major pre-cerebellar nuclei which are known to project to the cerebellum, including the pontine nuclei (Pn) (23,56) and inferior olive (IO) (23,58). Another Vi target which has been shown to provide input to the cerebellum is the superior colliculus ( 22) . The neurons in Pn provide a major source of MFs to most regions of CB cortex and DCN, and it is generally accepted that all cells in Pn project predominantly to CB contralaterally. The areas of Pn which receive which Vi 143 input have been shown to project to orofacial portions of CB including SIM, PML, and Crura I and II (57). The neurons in IO are thought to be the sole source of climbing fibers to the CB cortex in the rat (4,24), and it is generally accepted that all neurons in IO send. their axons to CB. The trigemino- receptive regions of IO have also been shown to provide climbing fiber input to some of the same areas that receive direct Vi MF input, including Crura I and II, PML and SIM. The superior colliculus (SC), although not generally considered a major pre-cerebellar nucleus (66) , is another region which receives direct Vi input (22) and also provides input to tactile areas of the CB hemispheres in rat (47). Using micromapping techniques, stimulation of the tactile- responsive intermediate layers of SC evoke responses in tactile regions of contralateral GCL of CB. Responses to SC stimulation were found in Crura I and II, SIM and PML, but not in the uvula (47). The peripheral receptive fields of interconnected SC and GCL loci consisted exclusively of facial structures, especially from the vibrissae, crown, bridge of nose and eyelids. This pathway, along with an additional pathway from SC to IO and subsequently to the CB (3,38), appears to provide a significant alternative pathway for information from V1 to reach CB. Thus, Vi not only has the ability to influence CB activity by means of direct projections, but also through indirect routes through Pn, IO and SC. This indicates that 144 a major role of Vi is to convey orofacial tactile information to the cerebellum by direct and indirect paths, and has the ability to significantly effect the activity in CB through various MF and climbing fiber endings. This is important because the control of movement and.posture by CB is achieved by adjusting the degree of contraction of skeletal muscles, which requires that CB be provided with a continuous flow of information about the events in the periphery during the course of movement. It appears that direct Vi input to CB, as well as indirect projections through Pn, IO and SC, provides this kind of continuous flow of information. Projections to the Deep Cerebellar Nuclei In addition to describing MF distribution in the cerebellar cortex, this study also shows the distribution of terminal axonal arborizations from. the 'Vi subdivisional regions to the DCN, including the MED (fastigial), INT (interpositus) and LAT (dentate) nuclei. As was the case for projections from Vi to the GCL of the cerebellar cortex, the most robust projection to the DCN was from dii. Labeled axons of dii neurons gave rise to terminal arborizations .bilaterally in the DCN, with ipsilateral predominance. The terminal arborizations were located bilaterally in widespread portions of MED, INT and LAT. Injections in v1Vipc labeled a relatively small number of terminal arborizations in each of the DCN ipsilaterally, while axons of v1Vimc neurons gave 145 rise to only a few in ipsilateral INT. No labeled terminal axonal arborizations were found in the DCN following brVi injections. It appeared that most of the labeled axons which entered the DCN were collaterals of those destined for the cerebellar cortex. This is in agreement with previous studies (18,62). Although the axons which coursed to the DCN were apparently collaterals of those bound for the cerebellar cortex, not all axons which traveled to the cortex provided collaterals to the DCN; This was demonstrated in the present study where brVi injections labeled MFs in the cortex, but did not label terminal axonal arborizations in the DCN. It is well known.that the DCN receive input from.pontine nuclei (Pn), inferior olive (IO), red nucleus (RN) as well as several other areas (25) . However, information regarding trigeminal projections to the DCN is limited. Stimulation of cutaneous trigeminal branches, including supraorbital, infraorbital and mental nerves, have been shown to activate neurons in the ipsilateral INT (76) . The retrograde HRP technique has revealed input to LAT from the ipsilateral spinal trigeminal nucleus (76) . This technique further demonstrated that Vi is the main contributor of trigeminocerebellar axons to the DCN (86). Previous investigations have provided substantial information regarding cerebellar outflow from the DCN. Efferent projections from MED have been shown to terminate in several target nuclei, including the ventrolateral thalamic 146 nuclei, superior colliculus, Pn, vestibular nuclei, IO and spinal cord (5,12,13,94). The termination of axons from INT have been shown to occur in the ventrolateral and intralaminar thalamic nuclei, RN, central gray (CG), nucleus of Darkschewitsch (DK), IO and spinal cord (28,35,53,89). Efferent connections of LAT also involve several of the same target nuclei including the ‘thalamus, parvocellular' RN, oculomotor nucleus, Edinger-Westphal nucleus, DK, Pn, CG and IO (12,15,19,28,35). It is interesting that the DCN project to the ventrolateral thalamus, Pn, RN, superior colliclus, and spinal cord, because these are areas shown to receive direct Vi input (22,23). Until it can be confirmed that Vi terminal axonal arborizations synapse*with.cells that.project to ‘these same ‘targets, the :meaning' of ‘this alternative pathway cannot be determined. The morphological characteristics of the terminal axonal arborizations in the DCN from extra-cerebellar sources are not well documented. The utilization of the anterograde PHA- L method has allowed the structural features of the terminal axonal arborizations from Vi to be described in the present study. Only one type of terminal arbor was found in DCN following injections in dii, v1Vimc and v1Vipc, which was characterized by thin, branched terminal strands bearing modest numbers of en passant and terminal boutons. The importance of this is two-fold. First, it shows that axons from neurons in Vi give rise to collaterals to the DCN which 147 have terminal arborizations that are morphologically different than the MF endings of their continuations. Secondly, the fact that the terminal arbors found in DCN have a number of swellings, or boutons along their length, is suggestive of synaptic activity in these nuclei. This study provides evidence that Vi provides input to the GCL of the cerebellar cortex and the DCN. In the GCL, MFs activate granule cells, among others, which send processes to the molecular layer to contact Purkinje Cell dendrites (62,63). The Purkinje Cells, in turn, provide the efferents from the cerebellar cortex, with the majority terminating in the central nuclei. This projection is organized into orderly longitudinal (rostrocaudal) zones, with a medial vermal zone projecting to MED, an intermediate (paravermal) zone projecting to INT and a lateral (hemispheral) zone connecting to LAT (19,32). It has also been shown as a result of several previous studies that the afferent projections of the cerebellar cortex and DCN, as well as cortico-nuclear projections in CB, are also based on a sagittal zonal organization (17,19,34,81,91). In the present study, projections from Vi to CB were examined in sagittal and horizontal sections and there was no obvious zonal distribution of labeled axons and their terminal ramifications in the cortex or DCN. The lack of obvious zonal banding of Vi MFs may have been due to the fact that relatively small PHA-L injections were made in the various Vi 148 subdivisional regions, labeling fewer axons and MFs at one time than would result from large injections in Vi. This is an aspect of the trigeminocerebellar projections that deserves further attention in future studies. Projections to the Spinal Cord In addition to describing trigeminocerebellar projections from ‘Vi, this study also shows that PHA-L injections into four subdivisional regions of Vi resulted in labeled axons and terminal arborizations in the CSC. These findings are in good agreement with previous studies in rat (48,69,78) and cat (54,55). Trigeminospinal axons originating from Vi neurons have been shown to terminate predominantly at upper levels (Cl-2) of the CSC (69), with progressively fewer axons terminating at mid-to-lower cervical and thoracic levels (27). One previous study, which used the retrograde HRP method, described projections from the spinal trigeminal nucleus, including Vi, to all levels of the spinal cord as far as the lumbosacral cord (78). The retrogradely labeled cell bodies in Vi were described as being located ventrally at the transition zone between Vi and MDH. It is possible, due to the description and illustration of the location of the labeled cell bodies, that they were actually located in MDH, since no cytological distinction between Vi and MDH was mentioned. Most of the previous studies which addressed trigeminospinal projections did not 149 provide very much information on the specific Vi subdivisions which projected to the spinal cord. The lack of information regarding the Vi subdivisions which contribute to spinal cord projections was probably due to the fact that the delineation of subdivisional differences in Vi had not been revealed until recently (68). However, one study reported that retrogradely labeled neurons occurred mostly in the rostroventral part of Vi following HRP injections into the spinal cord (48) . A more recent study, which utilized the same method, described a more widespread distribution of these neurons in Vi (69). In this study, injections of HRP into mid-to-lower levels of the CSC labeled neurons in lei and v1Vimc, while RCC injections resulted in a greater density of retrogradely labeled cells in brVi, lei, v1Vipc and v1Vimc. The labeled trigeminospinal neurons in Vi following CSC injections exhibited a wide range in cell sizes, including small, medium and large somata. In the present study, the most substantial input to the CSC was from neurons in v1Vimc and dii (which includes lei). Together with a previous study by Phelan and Falls ('91), it appears that lei and v1Vimc are primary contributors of Vi-CSC projections. Another important aspect of the Vi-CSC projection is the identification of the specific spinal cord laminae which receive Vi input. Previous studies, which utilized retrograde tracing methods, were unable to localize the 150 precise laminae of termination of Vi-CSC projections because of the nature of the injection sites. They were usually too extensive, spreading throughout a large area of spinal gray matter. However, the utilization of PHA-L in this study revealed the specific laminae of termination of Vi axons. Labeled axons from neurons in all four Vi subdivisional regions examined, including v1Vimc, v1Vipc, dii and brVi, coursed caudally through MDH and the spinal V tract to the ipsilateral CSC dorsal horn. There they gave rise to terminal arborizations in laminae III and IV, with sparse labeling in lamina V. Some axons from dii and v1Vimc neurons also decussated in the midline at the level of the injection site and.descended in the ventral funiculusu These axons then coursed dorsally through the ventral horn, where they gave rise to terminal arbors in laminae IX, and with some then continuing dorsally to the dorsal horn where they issued terminal arbors to laminae III and IV; Generally, the ipsilateral projections to the dorsal horn were more substantial than. those to the contralateral dorsal and ventral horns. It is well known.that laminae III and IV receive tactile information, by means of a large variety of AB cutaneous mechanoreceptive fibers and mechanosensitive fine myelinated A6 fibers from.the skin and.hair follicles for discriminative touch (14,71). In addition, these laminae also receive afferent fibers from the somatosensory cortex (14,71). 151 Projections from the somatosensory cortex have been shown to be somatotopically organized with those projecting to cervical segments originating mainly from.neurons in neck and posterior head representation areas of the SI cortex (95). It has also been demonstrated that the collaterals of Pacinian corpuscles in glabrous skin have terminal arborizations that occupy the medial one-third of the dorsal horn, similar to where the majority of spinal cord.projecting Vi axons terminate (29). In this region, axon collaterals pass ventrally through laminae I and II, or enter lamina III directly from its medial border and form profuse terminal arbors in laminae III and IV, with smaller secondary terminal arbors in laminae V and VI. In addition to the Pacinian corpuscles, another type of rapidly adapting mechanoreceptor occurs in glabrous skin, the Meissner corpuscle. These are innervated by large diameter myelinated fibers which enter the dorsal or dorsomedial part of the dorsal horn and give rise to terminal arborizations in medial lamina III (29). Sensitive slowly adapting cutaneous mechanoreceptors have also been found to arborize profusely in laminae III, IV and V. Another input to the CSC is from primary trigeminal fibers which innervate orofacial regions. 'Trigeminal primary input from face, intra- and perioral structures have been shown to activate neurons in the dorsal horn (1,2) as well as neck.motor neurons (1,2,50,88). Primary trigeminal input has 152 repeatedly been shown to terminate in laminae III-IV; In one study, large myelinated cutaneous afferent fibers innervating the face were stained intra-axonally with HRP (36) . The terminal arborizations of collaterals given off from.a single parent axon entered not only the spinal trigeminal nuclei, but also laminae III and IV of the cervical dorsal horn. In addition, HRP injections into the trigeminal ganglion labeled primary afferents which terminated most heavily in laminae III-V of the contralateral dorsal horn at C1 and C2 levels (42). It is evident that the vibrissae are, in part, responsible for the activation of dorsal horn cells in the cervical cord. Retrograde and transganglionic transport of HRP revealed that the deep vibrissae nerve, which innervates vibrissae follicles, projects to the C1 dorsal horn and is entirely restricted to laminae III-V (9). Thus, if spinal and trigeminal primary input convey tactile information from cutaneous receptors to laminae III-V, it is suggested that the same kind of information is relayed by Vi to these same laminae, especially III and IV. The purpose of this projection may be involved with the modulation of convergent sensory input bilaterally to the ipsilateral cervical dorsal horn, with greater ipsilateral influence. The functional significance of the projections from v1Vimc and dii to laminae IX of the contralateral ventral horn is probably for the purpose of activating motor neurons which innervate neck musculature during orienting behavior. 153 Although this hypothesis cannot be confirmed without further electrophysiological and electron microscopic studies, previous studies support a trigeminospinal projection which effects the activity of neck. motorneuronal output (1,2,50,88,92). Several studies have indicated a close functional relationship between the trigeminal system and upper cervical cord motor neurons. Stimulation of several trigeminal primary afferents have been shown to excite cells in the ventral horn of the upper cervical cord. Abrahams et. al. ('79) reported that neck motor neurons are most powerfully excited by stimulation of the infraorbital (IO) nerves. Sumino and Nozaki ('77) have demonstrated that the neck motor neurons also receive input from the inferior alveolar, lingual and masseter nerves, suggesting that the trigeminospinal reflex is induced not only by cutaneous input from the face region, but also by input from intraoral structures. It has also been observed that the IO and supraorbital (SO) nerves, which innervate the nose and forehead regions, respectively, elicit different response patterns from the cervical motor neurons and muscles (77). The response of neck muscle motorneurons to IO nerve stimulation is consistent with a movement which involves raising the animal's head in an avoidance posture to a stimulus applied to the nose. On the other hand, 80 nerve stimulation results in a complex movement involving twisting of the head away from the stimulus and raising of the head 154 towards the stimulus. Such a movement is similar to, and thought to be directly involved with, suckling and orientation reflexes. Although the projections from.Vi have been shown to primarily terminate in the rostral cervical cord, with fewer projections to the cervical enlargement, it has also been previously suggested that the presence of secondary trigeminal fibers at caudal cervical and thoracic levels might also be related to reflex swallowing mechanisms, i.e. , that sensory stimulation of the oral cavity might reflexly inhibit phrenic and intercostal activity (16). This study described the morphological characteristics of terminal arborizations of axons from Vi neurons in the CSC. These terminal arbors were derived from.two major groups of axons. One group, which were located in the ipsilateral dorsal horn, originated from.thin.parent fibers which coursed from Vi caudally through MDH mainly to laminae III and IV. The second group of terminal arbors were derived from thicker parent axons which crossed the midline from the injection site, and descended in the ventral funiculus. From the ventral funiculus, this second group of axons coursed dorsally through the ventral horn to the dorsal horn. These two paths to the CSC are also utilized by axons of V0 trigeminospinal projection neurons (85), and the morphological characteristics of their terminal arborizations are similar to those from Vi axons. The terminal axonal arborizations that were labeled in the cervical cord 155 following Vi injections were rostrocaudally oriented in the spinal gray matter. The predominantly rostrocaudal orientation of Vi terminal arborizations matches those of primary spinal afferent terminal arborizations in laminae III and IV (71,73), and parallels the longitudinally oriented dendrites of cells in these laminae (72,73). Finally, it appears that SC may play a role in contributing to an alternative, indirect pathway for information from Vi to the spinal cord. It has been shown in the cat and rat (60), as well as in the hamster (75), that neurons mainly located in the intermediate laminae of the lateral SC project primarily to the cervical cord. Tectospinal neurons projecting to the rostral segments of the cervical spinal cord are found over a larger extent of the contralateral SC than those terminating in the cervical enlargement. The tectospinal projection to the cervical enlargement in both rats and cats arise almost exclusively from the caudolateral quadrant of the contralateral SC, whereas the tectal projection.to the rostral (upper) cervical spinal cord originates, in cats, from almost the entire extent of the colliculus and, in rats, from its greater part. Most of the tectospinal neurons were located in the intermediate laminae. No evidence for tectospinal projections to the thoracic or lumbar levels of the spinal cord was found in the cat or rat. Because the retrograde HRP technique was used to determine the location of tectospinal 156 neurons in these experiments, the specific cervical cord laminae in which the tectospinal axons terminated was not determined. Intracellular recording and horseradish peroxidase injection techniques were used to delineate the structural and functional characteristics of the SC cells in the hamster, which could.be antidromically activated from the first cervical segment of the spinal cord (75). Most of the tectospinal cells were exclusively somatosensory and gave rapidly adapting responses to deflection of vibrissae and/or guard hairs, and greater than 50% of the cells were located in the lateral portion of the intermediate laminae. These cells were located in.the same general region that receive Vi input (22) . These findings indicate that in addition to direct input to the CSC, Vi may utilize an alternative pathway through the neurons in SC to convey somatosensory information from orofacial regions to the cervical cord to contribute to the regulation of head and neck.movements which orient the animal toward stimuli in its environment. Collateral Projections Previous studies have indicated that axons of some Vi projection neurons send collaterals to more than one target nucleus along the neuraxis. Double retrograde fluorescent labeling techniques in the rat have shown a significant number of neurons in Vi which project to both the thalamic ventrobasal complex and CB (65,84). This has been disputed 1F ~¢ he.“ .. 157 by antidromic collision 'techniques, combined electrOphysiological recording and antidromic stimulation techniques, and double retrograde transport of Fast Blue and HRP, which have revealed that very few, if any, Vi neurons project to both the ventrobasal thalamus and the CB (43,51,97). Thus, collateralization of trigeminothalamic axons to the CB is debatable. A more recent study revealed that at least some of the axons of Vi cells are collateralized (69). When HRP was injected into the thalamus, 80% of the large multipolar neurons were labeled, 50% of these neurons were labeled after cerebellar injections, and 60% were labeled following spinal cord injections. These data indicate that there must be collateralization of at least 30% of the large multipolar neurons in Vi. It has been suggested by Phelan and Falls ('90) that since the collateralization of trigeminothalamic and trigeminospinal cells has not previously been reported, the collateralization must exist between trigeminothalamic and trigeminocerebellar neurons. Similarities and Differences in Vi Subdivisional Projections to the Cerebellum and Spinal Cord The present study demonstrates that there are similarities and differences between the projections from four Vi subdivisional regions to the cerebellum and spinal cord. Regarding projections to CB, the four Vi subdivisional 7...; ‘.. I file. ‘d‘ ‘- E 158 regions were similar in that they all projected to ipsilateral lobule 9a of the vermis and Crus I, and all but brVi projected to ipsilateral SIM, Crus II and PML. Thus, the major CB cortical projections were similar for all regions. In addition, axons from.neurons in all regions gave rise to complex MFs in the GCL, however, most simple MFs resulted from v1Vimc injections. Neurons in dii differed from neurons in all other Vi subdivisional regions in that they also projected to contralateral lobule 9a, Crura I and II, PML and SIM. The projections from the four Vi subdivisional regions to the DCN were all different. Injections of PHA-L into dii produced labeled terminal axonal arborizations in all nuclei bilaterally, including MED, INT and LAT. Injections in v1Vipc produced labeled terminal arbors in all of the nuclei ipsilaterally, and only a few labeled terminal arbors were found in ipsilateral INT following injections into v1Vimc. No labeled terminal arbors were found in the DCN following brVi injections. The four Vi subdivisional regions were also similar in that they all projected to the CSC, with decreasing density from RCC to CE. In all cases, the major projection was ipsilateral to laminae III and IV with similar types of rostrocaudally oriented terminal arborizations. However, v1Vimc and dii differed from v1Vipc and brVi in that they also projected to contralateral CSC, specifically to laminae 159 III, IV and IX. Furthermore, the axons that coursed to contralateral CSC were thicker that those that projected ipsilaterally. Although some significant differences were found in the CB and spinal cord projections from v1Vimc, v1Vipc dii and brVi, it is possible that injections into dii may have masked more subtle.differences between.the three subdivisions which comprise it, including lei, chi and irVi. These three dorsal subdivisions have been shown to be morphologically' distinct based. on. cyto- and myeloarchitectural studies (68), as well as being different based on afferent input from primary trigeminal neurons innervating orofacial regions (44,90) ., Attempts to make precise, well-contained injections into these dorsal subdivisions were unsuccessful due to their relatively small size. Thus, differences in the projections from the six Vi subdivisions are possibly greater than those revealed by the present study; Further efforts to make micro-injections into these regions and define their specific projections will be reserved for future studies. Ill. __tto'-.v‘_.u ..‘ .-S 1 160 Figure 1. PHA-L Injection Sites. Schematic drawings (E-H) of representative transverse sections (A-D) through PHA-L injection sites (IS) in four subdivisional regions of rat trigeminal nucleus interpolaris (Vi). These regions are: ventrolateral magnocellular (leimc;A,E), ventrolateral parvocellular (v1Vipc;B,F), dorsomedial (dii;C,G) and border region (brVi;D,H). The injection sites were, for the most part, confined to the respective subdivisional area of rat Vi. Amb, ambiguus nucleus; IO, inferior olive; svt, spinal trigeminal tract. 161 em 1.... whoa. .... (ehhw ..mv a. brh .w. . V ...»... ....) a . Figure l 162 Figure 2: Cerebellar Projections from dii. A-F. Schematic drawings of representative sagittal sections through the mediolateral extent of the ipsilateral cerebellum (CB), showing the distribution of labeled axons and terminal arbors, in the granule cell layer (GCL) and deep cerebellar nuclei (DCN), following the injection of PHA-L into dii. In the GCL, labeled mossy fibers (MFs) were found predominantly in lobule 9a, Crura I and II, paramedian lobule (PML) and simple lobule (Sim). In the DCN, labeled terminal arborizations were located in the medial (Med), intermediate (Int) and lateral nuclei (Lat). Numerals 1-10 indicate lobules. Cop, copula pyramis; ICF, intercrural fissure; PCF, preculminate fissure; PrF, primary fissure; PSF, posterior superior fissure; SF, secondary fissure. 163 Figure 2 hand my ceusu I onus! Pill. 164 Figure 3: Cerebellar Projections from dii. A-F. Schematic drawings of representative sagittal sections through the mediolateral extent of the contralateral CB, showing the distribution of labeled axons and terminal arbors, in the GCL and DCN, following the injection of PHA-L into dii. In the GCL, labeled MFs were found predominantly in lobules 6a, 9a, Crura I and II, and PML. In the DCN, labeled terminal arborizations were located in Med, Int and Lat. There were fewer labeled. MFs and terminal axonal arborizations in contralateral CB than there were in ispilateral CB following dii injections. 165 PML Cop ive s A D contra. 6808 I I ORUS ll :tion 7. predflfi m In :3 5 PML 4 1 ”ed: 3 Cap mini: 5 E wfl ‘ hand [cp . causu Figure 3 166 Figure 4: Cerebellar Projections from v1Vimc. A-F. Schematic drawings of representative sagittal sections through the mediolateral extent of the ipsilateral CB, showing the distribution of labeled axons and terminal arbors, in the GCL and DCN, following the injection of PHA-L into v1Vimc. In the GCL, labeled.MFs were found predominantly in lobule 9a, Crus II and Sim. In the DCN, labeled terminal arborizations were located in Int. 167 CRUS ll PML Cop PML Cap PML Figure 4 168 Figure 5: Cerebellar Projections from v1Vipc. A-F. Schematic drawings of representative sagittal sections through the mediolateral extent of the ipsilateral CB, showing the distribution of labeled axons and terminal arbors, in the GCL and DCN, following the injection of PHA-L into v1Vipc. In the GCL, labeled MFs were found predominantly in lobule 9a and Crura I and II. In the DCN, a few labeled terminal arborizations were located in Med, Int, and Lat. 169 CRUS ll PML Cop CRUS ll Cap hand ICF 6808 l PML Figure 5 170 Figure 6: Cerebellar Projections from brVi. A-F. Schematic drawings of representative sagittal sections through the mediolateral extent of the ipsilateral CB, showing the distribution of labeled MFs in the GCL following the injection of PHA-L into brVI. Labeled MFs were found predominantly in lobule 9a and Crus I. No labeled terminal axonal arborizations were found in the DCN. 171 CRUSH PML CRUSII PML Cap PML Figure 6 172 Figure 7: Drawings of Mossy Fibers in CB Granule Cell Layer. Representative drawings of the two types of MFs located in the GCL. Some were simple MFs (asterisks), characterized by an axon that gave rise to irregular enlargements that were simple and fusiform, with smooth globular contours and a few short, finger-like projections. The majority were complex MFs, characterized by an axon with irregular swellings that were more complicated in shape than the simple MFs, displaying large convoluted expansions out of which.projected several tapering or filiform appendages (arrows). chars: rents 1'1 :ouIS 1“ I were 3 . . swell,"3 .;&y‘f in.&‘r afl- Purkinjoccll body \ 4 173 Figure 7 arm: _.. av {Tax- at-.. 174 Figure 8: Drawings of Terminal Axonal Arborizations in DCN. Representative drawings of the type of terminal axonal arborizations found in the DCN. They consisted of thin, branched terminal strands with numerous widely spaced boutons. b, bouton; pf, parent fiber; ts, terminal strand. 175 ratios: tenirzli . j .3 y ' SiSeEd 9‘ widely? .rminal =7 20 um Figure 8 “Ti 176 Figure 9: Spinal Cord Projections from v1Vipc and v1Vimc. A,B. Schematic drawings of transverse sections through C2 and C5 of the cervical spinal cord (CSC) showing the distribution of labeled axons and terminal arborizations following the injection of PHA-L into v1Vipc. The projection from v1Vipc to the CSC was ipsilateral and predominantly terminated in laminae III and IV of the ipsilateral dorsal horn. Some terminal branches extended ventrally to lamina V. No labeled axons gave rise to terminal arborizations in the ventral horns or contralateral dorsal horn. C,D. Shows the distribution of labeled axons and terminal arborizations in C2 and C5 following the injection of PHA-L into v1Vimc. The projection to the CSC was bilateral. Ipsilaterally, terminal arborizations were dense in laminae III and IV, with a few in lamina V. Contralaterally, labeled terminal arbors branched in lamina IX of the ventral horn, and laminae III and IV of the dorsal horn. i, ipsilateral. 178 Figure 10: Spinal Cord Projections from brVi and dii. A,B. Schematic drawings of representative transverse sections through C2 and C5 of the cervical spinal cord (CSC) showing the distribution of labeled axons and terminal arborizations following the injection of PHA-L into brVi. The projection from brVi to the CSC was weak, and confined to laminae III and IV of the ipsilateral dorsal horn. C,D. Shows the distribution of labeled axons and terminal arborizations in C2 and C5 following the injection of PHA-L into dii. The projection to the CSC from.dm i was bilateral. Ipsilaterally, terminal arborizations were relatively dense in laminae III and IV, with a few in lamina V. Contralaterally, labeled terminal arbors branched in lamina IX of the ventral horn, of the rostral cervical cord, and laminae III and IV of the dorsal horn. i, ipsilateral. 180 Figure 11: Drawings of Terminal Axonal Arborizations in CSC. Representative drawings of the type of terminal axonal arborizations found in the CSC. They consisted of thin, branched terminal strands with multiple en passant and end boutons. The terminal arbors spanned considerable distances (up to 580 um) rostrocaudally. 181 izaticrs; torrid. sisted if passant u .erable J Figure 11 182 Figure 12: Photomicrographs of Mossy Fibers in CB. Photomicrographs that show'portions of the PHA-L labeled MFs located in the GCL of the CB. A,B. Photomicrographs of complex types of MFs showing the massive convoluted central expansions (large arrows) out of which several tapering or filiform appendages projected (small arrows). C. End of a. simple type of HF with an axon bearing an irregular, fusiform enlargement, with smooth globular contours. D. A MF with a central expansion from ‘which a thin filamentous strand coursed to the Purkinje Cell layer and terminated in a bouton in close approximation to a Purkinje Cell body (PCb). Figure 12 183 184 Figure 13: Photomicrographs of Terminal Axonal Arborizations in DCN and CSC. A,B. Photomicrographs that show portions of Pfikd; labeled terminal axonal arborizations located in the DCN. C,D. Photomicrographs that show portions of the type of puma; labeled terminal axonal arborizations located in the CSC, specifically from.ipsi1ateral lamina IV (c) and contralateral laminae IX (d). . Arbarll ions! xii” em“ M" 160W 185 Figure 13 10. BIBLIOGRAPHY Abrahams, V.C., G. Anstee, F.J.R. 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