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This is to certify that the

dissertation entitled
A Two Part Study: Morphology, Cytology and Synaptic Relations
of Primary Trigeminal Axons Terminating in the Dorsomedial
Subdivision of Rat Trigeminal Nucleus Oralis and Comparison
of the Distribution and Morphology of Neurons in Rat Trigeminal

Nucleus Oralis Projecting to Facial, Hypoglossal and Trigeminal
Motor Nuc 1e 1 . presented by

Deborah J. Sohn

has been accepted towards fulfillment
of the requirements for

 

Ph -D - degree in Ana tomgz

MW

Major professor

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MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

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A TWO PART STUDY:

MORPHOLOGY, CYTOLOGY AND SYNAPTIC RELATIONS OF PRIMARY
TRIGEMINAL AXONS TERMINATING IN THE DORSOMEDIAL
SUBDIVISION OF RAT TRIGEMINAL NUCLEUS ORALIS
AND
COMPARISON OF THE DISTRIBUTION AND MORPHOLOGY OF NEURONS
IN RAT TRIGEMINAL NUCLEUS ORALIS PROJECTING TO FACIAL,
HYPOGLOSSAL AND TRIGEMINAL MOTOR NUCLEI

BY
Deborah J. Sohn

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Anatomy

1994

ABSTRACT
MORPHOLOGY, CYTOLOGY AND SYNAPTIC RELATIONS OF PRIMARY
TRIGEMINAL AXONS TERMINATING IN THE DORSOMEDIAL SUBDIVISION
OF RAT TRIGEMINAL NUCLEUS ORALIS
AND
COMPARISON OF THE DISTRIBUTION AND MORPHOLOGY OF NEURONS IN

RAT TRIGEMINAL NUCLEUS ORALIS PROJECTING TO FACIAL,
HYPOGLOSSAL AND TRIGEMINAL MOTOR NUCLEI

BY
Deborah J. Sohn

Two studies were completed in rat trigeminal nucleus oralis
(Vo) . In the first, anterograde transport of horseradish
peroxidase (HRP) was used to examine the overall morphology,
distribution, cytology and synaptic relations of primary
trigeminal axons terminating in the dorsomedial (DM)
subdivision of V0. Injections into the sensory root of the
trigeminal nerve labeled axons throughout DM. Light
microscopic analyses revealed that DM receives terminal
arborizations of two morphologically distinct types (Type I
and Type II) of primary axons. Type I primary axons were a
direct continuation of parent fibers travelling
dorsomedially in the spinal trigeminal tract (SVT) and
terminating as a sparsely branched, long, medially directed
terminal strand containing up to thirty-three boutons. Type
II primary axons arose as medially directed collaterals from
parent branches ascending dorsomedially in SVT. Within DM
they divided into two to four medially directed terminal
strands containing one to nineteen boutons. Electron

microscopic analyses reveal that primary axonal endings from

Type I afferents are found singly or in a simple glomerulus
while Type II primary endings are found in complex
glomerular arrangements. Type I and Type II primary endings
contain agranular spherical synaptic vesicles and make
axodendritic synapses on dendritic shafts. This suggests
that at least two types of orofacial sensory inputs are
being conveyed to OH neurons. In the second study
retrograde horseradish peroxidase (HRP) transport was used
to examine and compare the overall distribution and
morphology of neurons in V0 projecting to facial (VII),
hypoglossal (XII) and trigeminal (Vmo) motor nuclei.
Injections into VII and Vmo labeled different types of
neurons in ventrolateral (VL) and DM subdivisions throughout
the middle third of V0. Injections in XII labeled nearly
equal numbers of VL and DM neurons in the middle third of
V0. These results suggest that there are morphologically
distinct populations of neurons in the middle third of VL
and DM projecting to VII, Vmo and XII. Vo involvement in

complex orofacial reflexes is suggested by these findings.

DEDICATION

To my sons, Richard and Derek

iv

ACKNOWLEDGEMENT

I would like to express my deepest gratitude and
sincere appreciation to my advisor, Dr. William McKenzie
Falls, for his unwavering patience, support, encouragement
and guidance throughout my graduate studies. I also wish to
thank the members of my graduate committee: Drs. Robert
Bowker, Irena Grofova, John Johnson, Frank Pansino, and
Charles Tweedle for their support and guidance.

I would like to extend my gratitude to the faculty and
staff of the Department of Anatomy well as my fellow
graduate students for their friendship, encouragement and
technical assistance.

Although many individuals are remembered for their
generosity and support, my greatest strength comes from my
loving family and friends whose unconditional love helped me

to achieve my goals.

TABLE OF CONTENTS

List of Figures . . . . . . . . . . . . . . . . . . . .vii
Abbreviations . . . . . . . . . . . . . . . . . . . . . ix
Introduction . . . . . . . . . . . . . . . . . . . . . .1
Chapter I Morphology, Cytology and Synaptic Relations

of Primary Trigeminal Axons Terminating in

the Dorsomedial Subdivision of Rat Trigeminal
NuCIeus oralis. O O O O O O O O O O O I O O O O 3

Introduction. . . . . . . . . . . . . . . . .3
Materials and Methods . . . . . . . . . . . .9
Results . . . . . . . . . . . . . . . . . . . 14
Discussion. . . . . . . . . . . . . . . . . . 21
Figures . . . . . . . . . . . . . . . . . . . 27
Bibliography. . . . . . . . . . . . . . . . . 41

Chapter II Comparison of the Distribution and Morphology
of Neurons in Rat Trigeminal Nucleus Oralis
Projecting to Facial, Hypoglossal and
Trigeminal Motor Nuclei . . . . . . . . . . . 48

Introduction . . . . . . . . . . . . . . . 48
Materials and Methods . . . . . . . . . . . . 53
Results . . . . . . . . . . . . . . . . . . 56
Discussion. . . . . . . . . . . . . . . . . 64
Figures . . . . . . . . . . . . . . . . . . . 73
Bibliography. . . . . . . . . . . . 93

vi

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:
Figure 13:
Chapter II
Figure 1:
Figure 2:

LIST OF FIGURES

Type I Primary Afferent Axon Terminal
“borization O O O O O O O O O O O O O O 0

Type II Primary Afferent Axon Terminal
Arborization . . . . . . . . . . . . . . .

Photomicrograph of Type II Primary Afferent

Photomicrograph of Type I Primary Afferent .

28

30

32

32

Electron Micrograph of Myelinated Parent Axons.32

Diagram of Type I Axonal Endings and Their
Synaptic Connections . . . . . . . . . . . .

Diagram of Type II Axonal Endings and Their
Synaptic Connections . . . . . . . . . . . .

Electron
and Its

Electron
and Its

Electron
and Its

Electron
and Its

Electron
and Its

Electron

Micrograph of Type I Axonal Ending
Synaptic Connections . . . . . . . .

Micrograph of Type I Axonal Ending
Synaptic Connections . . . . . . . .

Micrograph of Type I Axonal Ending
Synaptic Connections . . . . . . . .

Micrograph of Type II Axonal Ending
Synaptic Connections . . . . . . . .

Micrograph of Type II Axonal Ending
Synaptic Connections . . . . . . . .

Micrographs Taken From Serial

Sections Through a Type II Axonal Ending

and Its

Synaptic Connections. . . . . . . .

Horseradish Peroxidase (HRP) Injection Sites.

Schematic Drawings of HRP Injection Sites . .

vii

.34

.34

.36

.36

.36

.38

.38

40

.74

.76

Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure

Figure

Figure

Figure

10:

11:

12:

13:

14:

Distribution of Vo-Vmo Projection Neurons . . .78

Distribution of Vo-VII Projection Neurons . . .80
Distribution of Vo-XII Projection Neurons . . .82
Diagrams of Vo-Vmo Projection Neurons. . . . .84
Diagrams of Vo-VII Projection Neurons . . . . .84
Diagrams of Vo-XII Projection Neurons . . . . .84

Photomicrographs of Vo-Vmo Projection Neurons .86
Photomicrographs of Vo-VII Projection Neurons .88
Photomicrographs of Vo-XII Projection Neurons .88

Somatic Cross-Sectional Area of Vo-Vmo,
Vo-VII, Vo-XII Projection Neurons . . . . . .90

Somatic Cross-Sectional Area of Vo-Vmo,
Vo-VII, Vo-XII Projection Neurons located
in DM 0 O O O O O O O O O O O O O O O O O O O 92

Somatic Cross-Sectional Area of Vo-Vmo,

Vo-VII, Vo-XII Projection Neurons located
in VL O O O O O O O I O O O I O O O O O O O O 92

viii

BZ

CDM

DAB

DM

DMSO

HRP

IS

MDMd

MDMV

PHA-L

P1

P2

SVN

SVT

ABBREVIATIONS

border zone Vo

caudal dorsomedial subdivision
3,3’ diaminobenzidine tetrachloride
dorsomedial Vo

dimethysulfoxide

flattened synaptic vesicles
horseradish peroxidase

injection site

trigeminal nucleus caudalis (medullary dorsal horn)
middle dorsomedial subdivision
middle dorsal zone of DM

middle ventral zone of DM
pleomorphic synaptic vesicles
phaseolus vulgaris leucoagglutinin
primary axonal ending type I
primary axonal ending type II
large round synaptic vesicles
small round synaptic vesicles
rostral dorsomedial subdivision
spinal trigeminal nucleus

spinal trigeminal tract

ix

TSNC
Vi
VI I
VL
Vmo
Vms
Vo

XII

trigeminal sensory nuclear complex
trigeminal nucleus interpolaris
facial motor nucleus

ventrolateral Vo

trigeminal motor nucleus

main sensory trigeminal nucleus
trigeminal nucleus oralis

hypoglossal nucleus

INTRODUCTION

Trigeminal nucleus oralis (Vo), the most rostral
subdivision of the mammalian spinal trigeminal nucleus
(SVN),is thought to play a significant role in the reception,
central processing, modification and the transmission of
nociceptive information to the brainstem and spinal cord from
tooth pulp, oral and, perioral areas as well as
mechanoreceptive information from the face and oral cavity.
Ithas been determined through anatomical and
electrophysiological techniques in a variety of mammals that
axons of primary trigeminal neurons terminate in V0
(1,3,10,19,20,23) . Rat Vo can be divided into three
subdivisions on the basis of several morphological and
functional criteria: ventrolateral (VL), border zone (32), and
dorsomedial (DM) (14,15,16,17,18, 19,23,24,26,34,36).

Numerous anatomical, electrophysiological and electron
microscopic studies have been completed in order to discern
the anatomic and functional organization of V0 (1,6,8,9,15, 16,
17,18,19,20,21,22,23,24,25,29,33,36,53,58,60,64,65,68).
However, this information is still incomplete in regards to
DM. The first part of the present study uses light
microscopy to evaluate the overall morphology of primary

1

trigeminal afferent axons terminating in DM and electron
microscopy to evaluate their cytology and synaptic relations.
Comparisons are made with primary trigeminal afferent axons
terminating in BZ and VL. The second part of this study uses
retrograde transport of horseradish peroxidase at the light
microscopic level to study the morphology and distribution of
V0 neurons projecting to facial (VII), hypoglossal (XII) and
trigeminal (Vmo) motor nuclei. Taken together these studies
add further insight into understanding the anatomical
framework and circuitry through which Vo receives, processes
and transmits orofacial mechanoreceptive and nociceptive

information.

CHAPTER I

MORPHOLOGY, CYTOLOGY AND SYNAPTIC RELATIONS OF PRIMARY
TRIGEMINAL AXONS TERMINATING IN THE DORSOMEDIAL SUBDIVISION

OF RAT TRIGEMINAL NUCLEUS ORALIS

INTRODUCTION

Knowledge of the normal central processing of nociceptive
information from the orofacial region is necessary in order to
help devise methods to decrease and/or eliminate acute and
chronic pain syndromes ‘which accompany craniomandibular/
craniovertebral dysfunction. Neuroanatomical studies and
electrophysiological investigations have shown that the spinal
trigeminal nucleus (SVN) and the main sensory trigeminal
nucleus (Vms) receive, process, modify, and relay primary
afferent somatosensory information from the teeth, facial
skin, oral and nasal cavities, temporomandibular joint.and the
Cornea(2,4,5,6,7,9,16,17,41,43,45,48,49,50,51,54,58,61,62,66).
The mammalian SVN can be divided into three
cytoarchitecturally distinct nuclei: :1 rostrally situated
trigeminal nucleus oralis (Vo), an intermediate trigeminal
nucleus interpolaris (Vi) and a caudally located trigeminal
nucleus caudalis, also referred to as the medullary dorsal

horn (MDH) to reflect its structural and functional

4
similarities to the spinal dorsal horn (32,38,61).

Utilizing anatomical and electrophysiological techniques
in a variety of mammals, central axons of primary trigeminal
neurons have been shown to terminate in V0 (1,3,10,19,20,23,
4, 36,43,44, 54,60,61, 62,68,69) . Rat Vo receives nociceptive as
well as mechanoreceptive primary afferent information and is
involved in the central processing, modification, and
transmission of this information from oral and perioral areas,
face and oral cavity, temporomandibular joint and tooth pulp
(3,4,5,9,16,34,36,50,58,60,61,62,66,69). Nociceptive input
from the tooth pulp, oral and perioral areas is thought to
reach second-order Vo neurons by way of small myelinated Aa
and unmyelinated C primary trigeminal axons while
mechanoreceptive information from the face and peripheral
receptive fields located in the oral cavity are thought to be
conveyed to second-order Vo neurons by way of A0 and larger
myelinated A3 primary axons (14,15,16,17,18,19,20,23,24,25,
26,34,35).

On the basis of several morphological and functional
criteria, rat Vo can be divided into ventrolateral (VL) ,
dorsomedial (DM), and border zone (82) subdivisions, (16,17,
18). By correlating the Golgi method with retrograde
horseradish peroxidase (HRP) labeling, VL has been shown to
contain three morphologically distinct types of neurons which
project to MDH in the rat (18) and cat (37). Retrograde HRP

labeling studies in the rat have identified VL trigeminospinal

5

projection neurons, innervating all spinal cord levels (17,47,
57) and Golgi studies have identified a VL Golgi type II
interneuron (14). Trigeminofacial projection neurons (12,13),
trigeminothalamic neurons, (8,13,27,63) and trigeminotectal
projection neurons (8,39) have also been shown to be present
in VL. Anterograde studies utilizing phaseolus vulgaris
leucoagglutinin (PHA-L) have shown that injections into rat VL
result in labeled terminal axonal arbors in the cervical and
thoracic spinal cord, cerebellum, thalamus, solitary nucleus,
ipsilateral and contralateral SVN, as well as Vmo, VII and XII
motor nuclei (64). In addition, two groups of ascending
intratrigeminal axons originating from MDH neurons (16) and
a population of small myelinated primary trigeminal axons
arborize within VL (20,22) and are in a position to provide
input directly to VL neurons.

Using anterograde transport of HRP, BZ has been found to
receive the terminal arborizations of five distinct
populations of primary trigeminal axons. One population is
derived from small myelinated parent axons and is found
throughout BZ (21,22,23,24) while a second population
originates from unmyelinated parent axons which terminate in
the dorsal one-half of the subdivision (22,23). The three
remaining populations arise from different types of large
myelinated fibers (21). Additional studies have revealed that
after' PHA-L injections into 82, anterograde labeling of

terminal axonal arbors can be found in cervical and thoracic

6

spinal cord, cerebellum, thalamus, VII, Vmo, solitary nucleus
and SVN (64). 82 also contains the terminal axonal arbors of
one group of ascending intratrigeminal axons from MDH neurons
(16). Retrograde HRP studies have shown a variety of
projection neurons to be present in the subdivision” .A single
population of trigeminocerebellar projection neurons is
situated in the dorsal portion of BZ (23) while descending
MDH projection, neurons (16) are located in the ventral
portions of 82. Small populations of morphologically distinct
trigeminotectal (8), trigeminospinal (1J0, and
trigeminothalamic (8) projection neurons are also found
throughout the subdivision.

To date, electron microscopic analyses provide evidence
that at least three:distinct types.of synaptic glomeruli exist
in VL and B2 (22). Each glomerulus contains a primary
trigeminal afferent axonal ending; It is within these
glomeruli that the fundamental principles of structural and
functional organization of VL and B2 are found. Within BZ,
two types of glomeruli have been identified: one containing
the axonal ending of a small myelinated primary trigeminal
axon and the other which includes the terminal of an
unmyelinated primary axon. The primary ending terminating in
32, derived from small myelinated parent fibers, lies
centrally in the glomerulus making at least one axodendritic
synapse with one to two dendritic shafts, while the ending of

the unmyelinated primary axon lies at the periphery of the

7
glomerulus where it forms a single axodendritic synapse on a
central dendrite. Axonal endings of small myelinated primary
trigeminal axons were found to lie centrally in identified
glomeruli in VL and they formed axodendritic synapses on
numerous dendritic shafts and spines. It is believed that the
transfer of information from these three populations of
primary axons directly to the dendritic arbors of second-order
B2 and VL neurons occurs at the asymmetrical axodendritic
synapses. All of the above studies continue to provide
evidence that each Vo subdivision, especially VL and B2, is
morphologically and functionally distinct. However, further
analyses, similar to those presented above, are needed with
regard to DM in order to substantiate the view that all of V0
is an important part.of SVN involved in the reception, central
processing and modification and relay of nociceptive and
mechanoreceptive information and that each subdivision is
morphologically and functionally distincta To date,
retrograde studies have shown that within DM there are five
morphologically distinct types of trigeminocerebellar
projection neurons, all of which innervate one or more of the
orofacial portions of four major tactile areas in the rat
cerebellar cortex (26). Furthermore, injections of PHA-I.into
DM have labeled terminal axonal arbors at all levels of the
rat spinal cord, cerebellum, thalamus, solitary nucleus, SVN

bilaterally as well as Vmo, VII and XII (64).

8
In this anterograde HRP study, light microscopic
techniques were utilized to evaluate the overall morphology of
primary trigeminal afferent axons terminating in DM. In
addition, electron microscopy was used to examine the labeled
axons and synaptic relations of ‘their terminal endings.
Comparisons between. these data and data on the primary
trigeminal axons already identified in VL and 82 is discussed
to help distinguish each subdivision’s relationship to one

another as well as to SVN as a whole.

9

MATERIALS AND METHODS

Five adult male Sprague-Dawley albino rats (250-300
grams) were anesthetized with sodium pentobarbital (35-
40mg/kg) and positioned in a stereotaxic apparatus (Kopf
Instruments). All surgical procedures were performed using
aseptic technique. The cranium was opened unilaterally in the
parietal bone, the dural sheath incised and the cerebral
cortex overlying the sensory root and ganglion of the
trigeminal nerve was removed by suction. Dense connective
tissue and bone covering the sensory root of the trigeminal
nerve was removed to allow full exposure. Approximately 1Cyl
of 30% HRP (Sigma Type VI) in 2% dimethylsulfoxide (DMSO) was
injected manually into the sensory root of each animal.
Under visual guidance, multiple injections (0.05-1.0ul HRP
per injection) were delivered over a 5-minute period using a
31-gauge needle connected to a lOul Hamilton Syringe.
Following termination of the injections, the needle was left
in place in the sensory root for an additional 5 minutes and
then withdrawn. Upon completion of the experiment, gelfoam
(UpJohn Company) was positioned in the opening, the incision
was clamped closed and an application of triple antibiotic
ointment (Rugby Laboratories) was placed over the incision
site. All animals were housed, maintained and cared for
according to federally prescribed guidelines (52) . After

postinjection survival periods of 48-72 hours, the animals

10
were deeply anesthetized and perfused transcardially with a
fixative solution (1000 ml) containing 2% paraformaldehyde and
2% glutaraldehyde in a 0.15M phosphate buffer (pH 7.3). The
brain was removed immediately and postfixed overnight at 4%:.
The following day the brainstem, along with the sensory root
containing the trigeminal sensory nuclear complex (TSNC), was
cut on anLOXford'Vibratome into 100um.thick serial sections in
either the transverse or horizontal planes and collected in
tissue trays containing 0.15M phosphate buffer. All sections
were processed immediately using the cobalt-glucose oxidase
technique (42). Sections were rinsed twice in 0.1M Tris
buffer for 5 minutes each, followed by cobalt chloride
pretreatment (filtered 0.5% CoCl2 solution in 0.1M Tris
buffer) for 10 minutes. After washing the sections (3
minutes) in Tris buffer, they were laced in fresh 0.15M
phosphate buffer. A solution composed of 200 ml of 0.15%
phosphate buffer and 0.1g 3,3' diaminobenzidine tetrachloride
or DAB (Sigma) was mixed and filtered; 0.4g B-D(+) glucose,
0.08g ammonium chloride, and 0.0006g glucose oxidase were
added to the solution. Sections ‘were incubated in this
solution for 30 minutes, then rinsed with 0.15M phosphate
buffer. These steps were repeated for a total of three
incubations of DAB. .After rinsing the sections twice in 0.15M
phosphate buffer, they were cleared through a graded series of
glycerin concentrations (20%, 40%, 60%, 80%, 100%), placed on

glass slides in 100% glycerin and coverslipped for light

11
microscopic analysis. All sections were analyzed in order to
ascertain the overall morphology of labeled primary trigeminal
axons and where they were terminating in the TSNC as well as
their trajectory through the spinal trigeminal tract (SVT),
the root entry zone and the sensory root proximal to the
injection site. Some sections demonstrating completely
labeled primary trigeminal axons in DM of V0 were processed
for electron microscopic analysis according to the procedure
of Gobel et a1. (32). The remaining sections were rehydrated
through a descending series of glycerin concentrations, (100%.
80%, 60%, 40%, 20%), rinsed in a 0.15M phosphate buffer,
mounted on gelatin-coated glass slides, counterstained with
cresyl violet and coverslipped. These sections were
extensively evaluated to determine the overall course,
distribution, organization and morphology of labeled primary
trigeminal axons at the light microscopic level using a Leitz
Laborlux 12 microscope fitted with a drawing tube. Detailed
drawings of HRP-labeled primary trigeminal axons were made
under a 100X oil immersion objective lens at a magnification
of 1,250X to demonstrate parent branches in SVT, as well as
collaterals and terminal arborizations in DM.
Photomicrographs of corresponding sections were taken to
illustrate morphological details. Those sections containing
completely labeled primary trigeminal axons in DM were trimmed
and processed for electron microscopy. These sections were

transferred back through a descending series of glycerin

12
concentrations, rinsed in 0.15M phosphate buffer, and post-
fixed in 2% osmium tetroxide containing 8.5% dextrose in 0.15M
phosphate buffer for one hour at room temperature. After
rinsing in distilled water, the sections were block stained
with 1% uranyl acetate (in distilled water) for 24 hours at
room temperature. Sections were dehydrated for 60 minutes
through a graded series of ethanol concentrations (50%, 70%,
80%, 90%, 100%) followed by 50 minutes of soaking in propylene
oxide (three changes for 10 minutes, 10 minutes, and 30
minutes). After the sections were soaked in a 1:1 ratio of
propylene oxide and Marglas for 30-45 minutes, they were
infiltrated with 100% Marglas for 24 hours. Shallow molds were
used to flat embed the sections in fresh.Marglas and they were
polymerized at 60°C for 24 hours. The blocks were mounted in
specimen holders of a Sorvall MT-z ultramicrotome and trimmed
to the area containing the labeled primary axons in DM which
were previously drawn. One or two pm thick sections were cut
to further identify and localize the fibers. The blocks were
then serially thin-sectioned with the sections mounted on
Formvar coated slotted grids and viewed with a transmission
electron microscope (CM10). Electron micrographs were made
from serial thin-sections and examined. The cytology and
synaptic relationships of the labeled primary axons were
evaluated. Each axon, its terminal arbors and endings were
analyzed with regard to size, shape and relationship to other

axons, dendrites, and cell bodies as well as synaptic

13
junctional morphology. Cytoplasmic contents were identified
and presynaptic and postsynaptic structures, including site of

termination, were classified.

14

RESULTS

LIGHT MICROSCOPY

Following the injections, many primary trigeminal axons
located in SVT, the root entry zone and the sensory root of
the trigeminal nerve are filled with HRP reaction product.
These regions contralaterally' contain :no labeled fibers.
Throughout Vo primary trigeminal axons form terminal
arborizations in each of the three nuclear subdivisions, VL,
32, and DM. Within DM, primary trigeminal afferent terminal
arborizations are found in each of its four portions: rostral
(RDM), middle ventral zone (MDMv), middle dorsal zone (MDMd),
and caudal (CDM) . 0n the basis of their overall morphology and
manner of termination, primary trigeminal axons terminating in
each portion of DM can be classified into two specific types
(Type I and Type II).

The Type I primary trigeminal afferent axon (Figure 1) is
a direct continuation of its parent fiber traveling caudally
in the dorsomedial portion of SVT. Upon entering Vo, the
axon does not branch as it traverses 82 and terminates in DM
as a long medially directed unbranched terminal strand, 425-
650um in length (Figure 1). Measurements of the diameter of
eighteen parent fibers and terminal strands show that along
their course, these axons vary in thickness from 1-2.5 pm at
their maximum diameter (Figures 1, 4). They could never be

traced back to thicker primary axons in either SVT or the

15

sensory root. Along each terminal strand are fourteen to
thirty-three irregularly spaced boutons with one bouton
always found at the terminal end of each strand (Figures 1,
4). Boutons along any one terminal strand may vary in size
and shape, ranging from 1-4um at their maximum diameter and
displaying spherical to elliptical shapes. Type I primary
trigeminal axons appear to ‘terminate in. quual density
throughout all four portions of DM.

Parent fibers of the Type II primary trigeminal axon are
also confined to and descend in dorsomedial SVT. Unlike Type
I parent fibers,these parent axons travel through the root
entry zone and within SVT give rise to medially directed
collaterals as they descend in SVT. The collaterals traverse
82 and enter DM (Figure 2). Measurements of the parent axons
in the root entry zone and in SVT, as well as the collaterals
in SVT and DM show that they range in thickness from 2-4um and
3-5um, respectively, at their maximum diameter. Upon entering
DM, the collaterals divide into two to four medially directed
terminal strands (Figures 2,3). Each strand extends from 28-
74 pm in length. The resulting cone-shaped dendritic arbors
(with the apex at or near to BZ-DM border) spread out
mediolaterally, rostrocaudally and dorsoventrally for 17-93
pm. Located along each of the collaterals and their terminal
strands are one to nineteen irregularly spaced boutons, with
one always located.at the terminal tip of each strand (Figures

2,3). The boutons vary in shape from spherical to elliptical

16
and measure 1-6um along their maximum diameter. Type II
terminal arborizations are confined to DM and are found in all
four portions of the subdivision, with the majority found in
MDMV.
ELECTRON MICROSCOPY

Electron microscopic analyses of isolated HRP-labeled
parent branches and collaterals of Type I and Type II primary
trigeminal axons, terminating in DM reveal that they are
thinly myelinated (Figure 5). The myelin sheaths of Type I
parent axons measure 0.01 to 0.10 pm in thickness while those
of Type II parent axons and collaterals range from, 0.1 to 1.5
pm in thickness (Figure 5) . The terminal strands of both
populations of primary trigeminal axons are unmyelinated
(Figure 8). These findings substantiate the light
microscopical findings for the diameters of other Type I and
Type II HRP-filled parent fibers and for the Type II
collaterals entering DM as well as for the terminal strands
from both populations of primary trigeminal axons arborizing
in DM.

Throughout the DM neuropil, primary (PR) axonal endings
from Type I afferents are found singly or on occasion in a
simple glomerulus (Figures 6,8,9,10) while all of those from
Type II afferents are found in a complex glomerular
arrangement (Figures 7,11,12, 13). The PR endings of each
afferent type are morphologically different and can be

distinguished from each other on the basis of their cytology

17
and synaptic relations (Figures 6,7).

Type I Primary Endings(P1): The P1 endings present
either an elongated scalloped appearance (Figures 6,8) or a
simple dome shape (Figures 9,10). They are filled with a
single population of agranular spherical (45—60 nm in
diameter) synaptic vesicles which are closely packed and fill
the entire ending (Figures 6,8,9,10). P1 endings form
asymmetrical synaptic junctions with dendritic shafts. These
axodendritic synaptic junctions ‘may' be either single or
multiple on the sameidendritic shaft (Figures 6,8,9,10). A.P1
ending synapses on a single dendritic shaft and does not make
synaptic contact with.multip1e dendrites or dendritic spines.
These dendritic shafts exhibit the cytoplasmic characteristics
of distal dendritic branches, i.e. they are 1.5 pm or less in
diameter and contain neurofilaments, microtubules, agranular
endoplasmic reticulum and mitochondria. Most Type I endings
are completely surrounded by overlapping astrocytic processes
except at the synaptic junction. Unlabeled axonal endings are
also found around the postsynaptic dendritic shaft (Figure
6,9). The most common is a small dome shaped ending
containing small round.synaptiC‘vesicles.(r ending; 35-40nm in
diameter). This ending makes an asymmetrical axodendritic
synapse with the postsynaptic dendritic shaft.

On occasion a Type I ending will be found in a simple
glomerulus (Figures 6,10). Here the Type I ending is not only

presynaptic to a dendritic shaft but is also postsynaptic to

18

a single unlabeled axonal ending (P ending). This P ending
containing scattered pleomorphic synaptic vesicles (40-50nm
in diameter) makes an axoaxonic synaptic contact which is
characterized by a single symmetrical to intermediate type
synaptic junction. The P ending does not synapse on the
dendritic shaft which is postsynaptic to the PR ending. The
PR and P endings are surrounded by astrocytic processes.

Type II PR Endings(PZ): The P2 endings present a
scalloped to dome-shaped outline and each lies at the
periphery of a glomerulus containing a central dendrite,
unmyelinated axons and three different types of unlabeled
axonal endings each with different populations of synaptic
vesicles (Figures 7,11,12,13). .All of these neuronal elements
are surrounded by overlapping thick and sheet-like astrocytic
processes. Type II Primary endings contain agranular
spherical (45 to 60 nm in diameter) synaptic vesicles among
which are scattered several spherical dense core vesicles (85
to 100 nm in diameter; Figures 7,11). The synaptic vesicles
are dispersed. throughout the ‘terminal cytoplasm and are
aggregated at the asymmetrical synaptic junctions associated
with each P2 ending (Figures 12, 13c).In each glomerulus, a
Type II Primary ending makes synaptic contact with the central
dendritic shaft (Figures 7,12,13c). This dendritic shaft
possesses the cytoplasmic characteristics typical of distal
dendritic branches and receives at least one and often

multiple asymmetrical axodendritic synapses from the P2 ending

19

(Figures 7,12,13c). In addition, a single dendritic shaft
receives synapses from multiple P2 endings originating from
the same axon, but each involved in separate glomeruli. In
this study'there‘were no instances where dendritic shafts were
found to contain agranular synaptic vesicles, nor were they
observed to participate in either dendrodendritic or
dendroaxonic synapse. Furthermore, PR endings were never
observed to be in contact with dendritic spines.

Small neuronal processes containing synaptic vesicles are
commonly found in the glomeruli containing P2 endings (Figures
7,12,13). They are considered to be axonal endings because
they arise from many of the unmyelinated axons coursing
through the glomeruli and because they, as well as their
unmyelinated parent branches, have never been observed to be
postsynaptic to the P2 endings. These axonal endings can be
divided into three distinct types on the basis of the size and
shape of their synaptic vesicles and.the morphological type of
synaptic junction they establish with their postsynaptic
neuronal elements.The most prevalent small axonal ending is
called the F ending because it contains predominantly
flattened agranular synaptic vesicles measuring 60x30 run
across their long and short.axes, respectively (Figures 7,13).
0n the average, two I? endings are found in any one
glomerulus. They are dome-shaped, measuring between 1.5 to 2
pm in length, and each participates in the following synaptic

arrangement. When impressed into the surface of the P2

20
ending, F endings usually form two symmetrical to intermediate
synapses: an axoaxonic synapse with the P2 ending and an
axodendritic synapse with the central dendritic shaft that
also receives an axodendritic synapse from the same P2 ending
with which the F ending forms an axoaxonic synapse (Figures
7,13). Two other kinds of small axonal endings commonly enter
the glomeruli. These include .5-1um dome-shaped endings (R
endings) containing small aggregates of large round (50-65 nm
in diameter) synaptic vesicles. These endings are relatively
sparse and form single asymmetrical axodendritic synapses on
the central dendritic shaft that also receives a synapse from
the P2 ending as well as a synapse from an F ending. The
second type of small axonal ending is the r ending. They are
few in number, smaller in size and are impressed into the
surface of the central dendrite (Figures 7,13). The r endings
measure 0.5-0.7 pm in length and contain a cluster of
agranular small round (30 - 45 nm in diameter) synaptic
vesicles at the lone asymmetrical synaptic junction formed by

this terminal with the central dendrite.

21

DISCUSSION

The present anatomical study demonstrates that all four
subdivisions of DM of rat Vo receive the terminal
arborizations of two morphologically distinct types of primary
trigeminal axons. Based on light and electron microscopic
data, both types of axons are of the small myelinated.variety.
Previous light and electron microscopical analyses have shown
that rat Vo receives two additional morphologically distinct
types of small-diameter myelinated primary trigeminal axons
(26). One type arborizes in VL and the other type terminates
throughout 82. The parent branches of the four groups of small
myelinated axons terminating in V0 display the morphology and
are in the size range generally accepted for A0 primary axons
(26). Based on previous electrophysiological data, these Aa
fibers most likely respond to light tactile and/or noxious
stimuli from the orofacial region, (3,11,33,34,36,46,53,56,
59,60). Taken together these data suggest that within rat Vo
therelare at least four separate synaptic circuitries involved
in the processing of orofacial pain and/or tactile input from
small myelinated primary trigeminal axons. Two are located in
DM with one each in B2 and VL. Autoradiographic,
degeneration, and anterograde transganglionic HRP transport
studies in the cat and rat agree that primary trigeminal axons
terminate in V0 and are somatotopically organized

(1,2,10,43,44,49,50,54,68,69) . By comparing the locations of

22

the suggested subdivisions in rat V0 to the regions of
termination of primary afferent axons belonging to branches
associated with each of the three divisions of the rat
trigeminal nerve (2,10,43,50), it can be concluded that DM
would be receiving the terminal arborizations of a great many
of the jprimary ‘trigeminal. neurons innervating :mandibular
orofacial receptive fields. Furthermore, DM also extends a
short distance into the dorsal portion of the intermediate
one-third of V0, especially laterally. In this location, DM
is in a position to receive terminal arbors of several of the
primary trigeminal neurons whose peripheral processes course
in branches of the maxillary division of the trigeminal nerve
(43). The two HRP-labeled populations of small myelinated
primary trigeminal axons analyzed in this study form their
terminal arborizations throughout DM. Taken together, these
data strongly suggest that the two populations of primary
axons in this study are derived from primary trigeminal
neurons whose peripheral receptive fields are located in
orofacial regions (e.g. oral cavity) innervated by mandibular
and maxillary divisions of the trigeminal nerve.

The findings of the present study suggest that at least
two types of orofacial primary afferent input are being
conveyed to neurons in all portions of DM. The primary
endings from each of the two types of small-diameter primary
trigeminal axons display differences in their cytology and

synaptic relations. The single axodendritic synapse typical

23
of most of the P1 endings is in marked contrast to more
complex glomerular synaptic arrangements involving the P2
endings. The synaptic arrangements of the primary endings in
DM from both Type I and Type II primary afferents, are
different from those of primary endings belonging to primary
afferent axons terminating in glomeruli in other trigeminal
sensory nuclei (28,29,30,31,40). However, the P2 ending
does display a glomerular arrangement similar to that found
for HRP-filled Primary endings in B2 belonging to
unmyelinated primary trigeminal afferent axons (22). Primary
endings of both Type I and Type II afferents make asymmetrical
axodendritic synapses on a single dendritic shaft. According
to previous ultrastructural studies (28,29,30,40) on synaptic
circuitry in primary trigeminal afferent glomeruli,
axodendritic synapses are the site where small-diameter
primary axons deliver their inputs directly to the dendrites
of second—order Vo neurons resulting in the activation of
these cells. The neuronal origin of the dendritic elements
cannot be determined absolutely, but the most likely sources
are neurons whose dendrites are either confined to or enter DM
where the.two types.of small-diameter primary axons form.their
terminal arborizations. The most probable neuronal sources or
the dendrites in DM receiving Type I and Type II afferent
input include trigeminocerebellar projection neurons located
in the three portions of DM, (CDM, MDM, RDM; 26), as well as

descending MDH projection neurons (18), trigeminospinal, SVN

24

and solitary projection neurons (64) and neurons projecting
to Vmo, VII, and XII (65). In a previous study (22), distal
dendritic shafts were occasionally found in VL glomeruli
involving primary endings from small myelinated primary
trigeminal axons, however, proximal dendritic shafts appeared
to receive primary endings from Type I and Type II afferents
in these studies. In addition, Type I and Type II primary
endings did not synapse on dendritic spines. This is
substantiated by information from Golgi and retrograde HRP
studies (26) that show that the dendritic shafts of many DM
neurons emit relatively few, widely scattered dendritic
spines.

Non-primary axonal endings are components of small
glomeruli in DM involving P2 endings and some P1 endings.
However, synaptic vesicle-containing dendritic shafts and
spines that have been observed in many primary afferent
glomeruli in MDH (28,29) to be presynaptic to nonsynaptic
vesicle-containing dendritic shafts and spines as well as to
primary endings have not been observed in the glomeruli in
these studies. Previous electron microscopic studies
analyzing glomeruli involving PR endings from small myelinated
axons in VL and 32 (22) have suggested that the transfer of
input from the primary endings and the subsequent activation
of second-order ‘Vo neurons can be affected within each
glomerulus by populations of axons from nonprimary neuronal

sources. In this study the axons would be giving rise to the

25

P endings in some Type I glomeruli and the F endings in all
Type II glomeruli. Their position and synaptic arrangement
strongly suggests that both P and F endings play a major role
in influencing small-diameter primary afferent transmission in
DM. Both F and P endings exhibit symmetrical to intermediate
synaptic contacts, thought to belong to axons derived from at
least two sources that could inhibit or diminish the firing
rate of second order Vo neurons in response to small-diameter
primary input. In the case of the F endings, this would be
accomplished either presynaptically through their axoaxonic
synapses directly on the P2 endings and/or postsynaptically
through their axodendritic synapses on the central dendritic
shaft. In the case of the P ending this would be accomplished
only presynaptically through their axoaxonic synapses directly
in the P1 ending. Numerous studies have shown that terminals
containing flattened synaptic vesicles like those observed in
this study, which form similar synaptic connections around
primary endings, are a common component of primary afferent
glomeruli found in other trigeminal sensory nuclei and are
probably'derived from.interneurons, (22,28, 29,40). Whether or
not interneurons are present in DM has yet to be described.

Based on the above findings and the results of the
present study most P1 endings probably transfer their small
myelinated.primary afferent input directly to second-order DM
neurons without having it be affected by axons from nonprimary

neuronal sources. In contrast, the transfer of small

26
myelinated primary afferent input from P2 endings to second
order DM neurons most likely is affected substantially by at
least one non-primary neuronal source. This Type I input
reaches second order DM neurons unchanged from the periphery
while Type II input to second order DM neurons undergoes
significant modification before being relayed to other nuclei

along the neuraxis.

27

Figure 1: Type I Primary Afferent Axon Terminal Arborizations

Composite drawings from horizontal sections showing the
terminal arborizations in the dorsomedial (DM) subdivision
ofrat trigeminal nucleus oralis (Vo) of one morphologically
distinct type of HRP-filled primary trigeminal afferent axon
(Type I). The parent fiber (P) travels caudally in the
dorsomedial portion of the spinal trigeminal tract (SVT),
entering Vo by traversing through the border zone subdivision
(B2) and terminating in DM as a long medially directed
unbranched terminal strand, 425-650um in length. Along the
terminal strand (ts) are 14-33 irregularly spaced boutons (b)
with one bouton always found at the terminal end of each
strand. Bar = 10pm. Vmo, trigeminal motor nucleus; MDMv,
middle ventral zone.

28

TYPE I PRIMARY AFFERENT

v

'U'U

m
N
o --
.3
"CT
‘77)"

  

 

 

Figure 1

29

Figure 2: Type II Primary Afferent Axon Terminal
Arborizations

Composite drawings from transverse sections showing the
terminal arborizations in DM of rat Vo of the second
morphologically distinct type of HRP-filled primary trigeminal

afferent axon (Type II). Parent fibers are confined to and
descend in dorsomedial SVT where they give rise to medially
directed collaterals (C). These collaterals traverse BZ

unbranched and enter DM dividing into two to four medially
directed terminal strands (ts). Each strand extends from 28-
74 pm in length, Along the terminal strand are 1-19
irregularly space boutons, with one always located at the
terminal tip of each strand. Bar=10um. VII, Facial motor
nucleus; VL, ventrolateral subdivision; MDMd, middle dorsal
zone.

C)

m
N
O
,3
p...

IT

30

TYPE n PRIMARY AFFERENT

 

Figure 2

 

31

Figure 3: Photomicrograph of Type II Primary Afferent

Photomicrograph that documents the morphological
characteristics of the Type II primary afferent highlighting
one of the terminal strands (ts) and one of the irregularly
spaced boutons (b). x 1,824

Figure 4: Photomicrograph of Type I Primary Afferent

Photomicrograph that documents the morphological
characteristics of the Type I primary afferent highlighting
one of the terminal strands (ts) and one of the irregularly
spaced boutons (b). x 1,686

Figure 5: Electron. Micrograph of Type I and Type II
Myelinated Parent Axons

Photomicrograph that documents the HRP labeled
morphological characteristics of Type I and Type II parent
fibers with their surrounding myelin sheaths in SVT. x 22,080

32

 

Figures 3, 4, 5

33

Figure 6: Diagram of'Type Ileonal Endings and their Synaptic
Connections

A schematic drawing taken from sets of serial thin
sections (Figures 8,9,10) of the neuronal processes and
synaptic connections in DM of Type I axonal endings. Primary
axonal endings (P1) containing agranular synaptic vesicles
form asymmetrical synaptic junctions with dendritic shafts
(D). On occasion a P1 ending will be found in a simple
glomerulus where it is postsynaptic to a single unlabeled
axonal ending(P) making a symmetrical to intermediate synaptic
junction and containing pleomorphic synaptic vesicles.
Unlabeled axonal endings containing round (r) synaptic
vesicles make asymmetrical axodendritic synapses with
dendritic shafts. Appreciable portions of the Type I endings
and their synaptic connections are surrounded by astrocytic
processes (thick black lines). d, small dendritic shaft.

Figure 7: Diagram of Type II Axonal Ending and Their Synaptic
Connections

A schematic drawing taken from sets of serial thin
sections (Figures 11, 12, 13) of the neuronal processes and
synaptic connections in DM Type II primary trigeminal afferent
glomeruli. A primary axonal ending (P2) containing agranular
synaptic vesicles and dense core vesicles forms an
asymmetrical synaptic junction with a central dendritic shaft
(D). Unlabeled axonal endings containing large (R) and small
(r) round synaptic vesicles make asymmetrical axodendritic
synapses with the central dendritic shaft. Unlabeled axonal
endings containing flattened (F) synaptic vesicles form two
symmetrical to intermediate synapses, an axoaxonic synapse
with the P2 ending and an axodendritic synapse with the
central dendritic shaft that also receives an axodendritic
synapse from the same P2 ending with which the F ending forms
an axoaxonic synapse. Appreciable portions of the glomerulus
are surrounded by astrocytic processes (thick black lines).

34

TYPEI PRIMARY AFFERENT

SYNAPTIC CONNECTION

 

 

TYPE II PRIMARY AFFERENT

SYNAPTIC CONNECTIONS

 

Figures 6,7

35

Figure 8: Electron Micrograph of Type I Axonal Ending and Its
Synaptic Connections

Two P1 endings surrounded by glial processes, suggest
formation of asymmetrical synapses (arrows) on the adjacent
dendritic shaft (D). ts, terminal strand; x 52,250

Figure 9: Electron Micrograph of Type I Axonal Ending and Its
Synaptic Connections

A dome-shaped P1 ending wrapping around a dendritic shaft
(D) and forming an asymmetrical axodendritic synapse (arrow).
An unlabeled axonal ending with small round (r) synaptic
vesicles also synapses on the dendrite, D, by forming an
asymmetrical synapse (arrow). x 52,250

Figure 10: Electron Micrograph of Type I Axonal Ending and Its
Synaptic Connections

On occasion a P1 ending will be found in a simple
glomerulus where it is postsynaptic to a single unlabeled
axonal ending (p). The p endings form symmetrical to
intermediate synaptic junctions (arrow) and contain
pleomorphic synaptic vesicles. x 52,250

36

 

Figures 8, 9, 10

37

Figure 11: Electron Micrograph of Type II Axonal Ending and
Its Synaptic Connections

Representative thin section through a P2 axonal ending
in the DM neuropil containing agranular spherical synaptic
vesicles among which are scattered several dense:core vesicles
(DCV). The P2 ending probably synapses (arrow) on the adjacent
dendritic shaft (D). x 48,460

Figure 12: Electron Micrograph of Type II Axonal Ending and
Its Synaptic Connections

A glomerulus is formed by a central dendritic shaft (D)
and multiple surrounding neuronal processes. The P2 axonal
ending forms an asymmetrical synapse (arrow) with the central
dendrite. An unlabeled axonal ending with large round (R)
synaptic vesicles also synapses on the central dendrite by
forming an asymmetrical synapse (arrow). x 34,920

38

 

1
J.

Figures 11,

39

Figure 13e,b,c,d: Electron Micrographs Taken From Serial
Sections Through a Type II Axonal Ending
and Its Synaptic Connections

Serial sections through a glomerulus showing the P2
axonal ending and its asymmetrical synapse (arrow) on a
central dendrite (D). Multiple surrounding neuronal processes
are clearly demonstrated. An unlabeled axonal ending with
small round (r) synaptic vesicles forms an asymmetrical
synapse (arrow) with the central dendrite. Two unlabeled
axonal endings, each containing flattened (F) synaptic
vesicles, form two symmetrical to intermediate synaptic
junctions, one on the central dendrite and the other on the P2
ending. x 34,920

40

 

Figure 13

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peroxidase. Brain Res. 204:415-420.

Hoffman, D. S., R. Dubner, R. L. Hayes, and T. P. Medlin
(1981) Neuronal activity in medullary dorsal horn of
awake monkeys trained in a thermal discrimination task.
I. Responses to innocuous and noxious thermal stimuli.
J. Neurophysiol. 46:409-427.

Huerta, M. F. A. Frankfurter, and J. K. Harding (1983)
Studies of the principal sensory and spinal trigeminal
nuclei of the rat: projections to the superior
colliculus, inferior olive and cerebellum. J. Comp.
Neurol. 220:147-167.

Ide, L.S. and Killackey, H.P. (1985) Fine structural
survey of the rat's brainstem sensory trigeminal complex.
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Ishidori, H., T. Nishimori, Y. Shigenaga, S. Suemune, Y.
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of upper and lower primary teeth in the trigeminal
sensory nuClear complex in the young dog. Brain Res.
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Itoh, K., A. Konishi, S. Nomua, N. Mizuno, Y. Nakamura,

and T. Sugimoto. (1979) Application of coupled oxidation
reaction to electron microscopic demonstration of
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Jacquin, M. F., K. Semba, M. D. Egger, and R, W. Rhoades.
(1983) Organization of HRP-labeled trigeminal mandibular
primary afferent neurons in the rat. J. of Comp.
Neurol. 215:397-420.

Jacquin, Mark F. K. Semba, R. W. Rhoades, and M. David
Egger. (1982). Trigeminal primary afferents project
bilaterally to dorsal horn and ipsilaterally to
cerebellum, reticular formation, and cuneate, solitary,
supratrigeminal and vagal nuclei. Brain Res. 246:285-
291.

Keller, 0., M. Kalina, E. Ujec, A. Zivnand L. Vyklicky
(1981) Projection of tooth pulp afferents to the
brainstem and the cortex in the cat. Neuro. Letts.
25:233-237.

Khayyat, G.F., Yu. Y.J. and King, R. B. (1975) Response
patterns to noxious and non-noxious stimuli in rostral
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Leong, S. K., J. Y. Shieh and W. C. Wong (1984)
Localizing spinal-cord-projecting neurons in adult albino
rats. J. Comp. Neurol. 228:1-17.

Liu, D. and Y. Hu (1988) The central projection of the
great auricular nerve primary afferent fibers -- an HRP
transganglionic tracing method. Brain Res. 445: 205-210.

Marfurt, C. F. (1981) The central projections of
trigeminal primary afferent neurons in the cat as
determined by transganglionic transport of horseradish
peroxidase. J. of Comp. Neurol. 203:785-798.

Marfurt, C. F. and D. F. Turner (1984) The central
projections of tooth pulp afferent neurons in the rat as
determined by the transganglionic transport of
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Marfurt, C. F. and S. F. Echtenkamp (1988) Central
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Nord, S. G. (1976) Response of neurons in rostral and
caudal trigeminal nuclei to tooth pulp stimulation.
Brain Res. Bull., 1:489-492.

Panneton, W. M. and H. Burton. (1981) Corneal a n d
periocular representation within the trigeminal sensory
complex in the cat studied with transganglionic transport
of horseradish peroxidase. J. of Comp. Neurol. 199:327-
344.

Paxinos, G. and C. Watson (1986) The Rat Brain
Stergtaxic Coordinates 2d Edition. New York.

Rowe, M. J. and Sessle, B.J. (1972) Responses of
trigeminal ganglion and brain stem neurons in the cat to
mechanical and thermal stimulation of the face. Brain
Res., 42:367-384.

Ruggiero, D. A., C. A. Ross and D. J. Reis (1981)
Projections from the spinal trigeminal nucleus to the
entire length of the spinal cord in the rat. Brain Res.
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Sessle, B. J. (1987) The neurobiology of facial and
dental pain: present knowledge, future directions. J.
Dent. Res. 66:962-981.

Sessle, B. J. and Greenwood, L. F. (1976) Inputs to
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innocuous and noxious stimuli. Brain Res., 117:211-226.

Sessle, B.J. and Hu, J.W. (1981) Raphe-induced
suppression of the jaw-opening reflex and single neurons
in trigeminal subnucleus oralis, and influence of
naloxone and subnucleus caudalis, Pain, 10:19-36.

Shigenaga, Y., T. Okamoto, T. Nishimori, S. Suemune, I.D.
Nasution, I.C. Chen, K. Tsuru, A. Yoshida, K. Tabuchi, M.
Hosoi, and H. Tsuru. (1986a). Oral and facial
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Shigenaga, Y., S. Suemune, M. Nishimura, T. Nishimori, H.
Sato, H. Ishidori, A. Yoshida, K. Tsuru, Y. Tsuiki, Y.
Dateoka, I.D. Nasution, and. M. Hosoi. (1986b)
Topographic representation of lower and upper teeth
within the trigeminal sensory nuclei of adult cat as
demonstrated by the transganglionic transport of
horseradish peroxidase. J. Comp. Neurol. 251:299-316.

Silverman, J. D., and L. Kruger (1985) Projections of the
rat trigeminal sensory nuclear complex demonstrated by
multiple fluorescent dye retrograde transport“ iBrain
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Smith, L.A. and Falls, W.M. (1994) Topographical
Distribution and Morphological Features of Second Order
Projections from identified subdivisional Areas of Rat
Trigeminal Nucleus Oralis. Ph.D. Thesis. Dept. of
Anatomy, Michigan State University, East Lansing, MI.

Sohn, D.J. and Falls, W.M. (1994) Comparison of the
Distribution and Morphology of Neurons in Rat Trigeminal
Nucleus Oralis Projecting to Facial, Hypoglossal and
Trigeminal Motor Nuclei. Ph.D. Thesis. Dept. of Anatomy.
Michigan State University, East Lansing, MI.

Sugimoto, T. and M. Takemura. (1993) Tooth Pulp Primary
Neurons: Cell Size Analysis, Central Connnection, and
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Projections from dental structures to the brain stem
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trigeminal nuclei in cat. Exper. Neurol. 74:787-799.

CHAPTER II

COMPARISON OF THE DISTRIBUTION AND MORPHOLOGY OF NEURONS IN
RAT TRIGEMINAL NUCLEUS ORALIS PROJECTING TO FACIAL,

HYPOGLOSSAL AND TRIGEMINAL MOTOR NUCLEI

INTRODUCTION

Trigeminal nucleus oralis (Vo), the most rostral
subdivision of the mammalian spinal trigeminal nucleus (SVN),
is thought to play a major role in the overall circuitry
underlying orofacial function, dysfunction and reflexes (e.g.
corneal reflex, orienting reflex, pinnae orienting reflex,
jaw-opening reflex, etc.). V0 is considered a significant
contributor to the reception, central processing, modification
and relay of nociceptive and mechanoreceptive information from
the facial skin, oral and nasal cavities, temporomandibular
joint, cornea and tooth pulp. These activities suggest that
Vo may play an important role in numerous acute and chronic
pain states (e.g. trigeminal neuralgia). On the basis of
several morphological and functional criteria, rat Vo can be
divided into three subdivisions: ventrolateral (VL),
dorsomedial (DM), and border zone (82), (6,8,9,10,14,15,26).

Each Vo subdivision has been shown to receive
morphologically and functionally distinct groups of primary

48

49

and non-primary afferent axons which are in a position to
directly influence the activity of second order Vo projection
neurons (8,11,12,14,15,26,45). These neurons have most of
their dendritic arbors confined to the subdivision and thus
provide the efferent outflow from each Vo subdivision
affecting the synaptic activity within numerous locations
along the neuraxis.

Retrograde transport studies in the rat (10) and cat
(31)have shown that VL contains three morphologically distinct
types of small neurons which project to medullary dorsal horn
(MDH). In addition, VL trigeminospinal projection neurons
innervate all levels of the rat spinal cord (9,34,40) .
Trigeminofacial projection neurons (4,5); trigeminothalamic
projection neurons, and(3, 5, 27,43) ; trigeminotectal projection
neurons (3,32) , have also been shown to be present in VL.
Anterograde studies utilizing phaseolus vulgaris
leucoagglutinin (PHA-L) have shown that injections into rat VL
result in labeled terminal axonal arbors in the cervical and
thoracic spinal cord, cerebellum, thalamus, solitary nucleus,
and SVN bilaterally (44). Anterograde studies have
revealed that after PHA-L injections into 82, labeling of
terminal axonal arbors was found in cervical and thoracic
spinal cord, cerebellum, and thalamus, as well as, solitary
nucleus and SVN bilaterally (44) . Retrograde studies have
shown a variety of projection neurons to be present in B2. A

single population of trigeminocerebellar projection neurons is

50
situated in the dorsal portion of 82, (14) as well as
descending MDH projection neurons (8) located in the ventral
portion. Small populations of morphologically distinct
trigeminotectal. (3), trigeminospinal, (9), and
trigeminothalamic (3)projection neurons are also found
throughout 82.

Within DM, utilizing the retrograde horseradish
peroxidase (HRP) method, five morphologically distinct types
of trigeminocerebellar projection neurons have been
identified, all of which innervate one or more of the
orofacial portions of four major tactile areas in the rat
cerebellar cortex (26). Injections of PHA-L into DM
anterogradely labeled terminal axonal arbors at all levels of
the rat spinal cord, in the thalamus, solitary nucleus,
bilateral SVN, as well as oculomotor (III), trochlear (IV) and
abducens (VI) cranial nerve nuclei and the Edinger-Westphal
nucleus (44).

Of particular interest are the efferent projections from
Vo neurons to the cranial nerve motor nuclei, as these neurons
are believed to provide a route for V0 involvement in complex
reflexes (e.g. corneal reflex) elicited by stimulation of the
skin of the face, oral and nasal mucous membranes and.muscles,
tendons and bones of the jaw and facet IPresently, information
is lacking on the precise distribution, density and morphology
of these projection neurons in relation to V0 subdivisions.

In a recent anterograde PHA-L study in the rat, the overall

51

pattern, organization and morphology of efferent axons arising
from projection neurons in each Vo subdivision and forming
terminal axonal arborizations in trigeminal (Vmo), facial
(VII), and hypoglossal (XII) motor nuclei were examined (44).
Following injections into DMZ and. VL, labeled axons and
terminal arbors were observed bilaterally in VII, XII and Vmo.
Injections confined to Bz produced labeling of axons and
terminal arbors ipsilaterally in ‘Vmo and bilaterally in VII.

The results of this study (44) suggest that each
subdivision of rat Vo may be capable of influencing motor
output from these cranial nerve nuclei. The terminal arbors of
V0 efferents in Vmo, VII and XII were morphologically
different suggesting that different populations of V0 neurons
project to each nucleus. In Vmo, a single population of
efferent axons was found” In ‘VII, two :morphologically
distinct types of V0 efferent terminal axonal arborizations
were noted. Two morphologically distinct populations of
efferent axons were also seen to terminate in XII.
Furthermore, at least four of these five types of terminal
axonal arbors were morphologically distinct from each other.
The present light microscopic study continues to examine the
efferent connections of projection neurons in V0 subdivisions
to Vmo, VII, and XII. Utilizing the retrograde HRP
technique, regional distribution and morphology of labeled
trigeminofacial, trigeminohypoglossal and trigeminomotor

neurons in rat V0 is examined and compared following

52
injections of HRP into Vmo, VII and XII. The density,
distribution and somatodendritic morphology, when evident of
these neurons is evaluated and compared throughout the

rostrocaudal extent of V0.

53

MATERIALS AND METHODS

Six adult male Sprague Dawley albino rats (250-300 g)
were anesthetized with sodium pentobarbital (35-40 mg/kg)and
positioned in a stereotaxic apparatus (Kopf). Aseptic
technique was employed for all surgeries. The scalp was
incised and a small opening was made in the cranium overlying
the cerebellar cortex (cases #1-4) and in the left parietal
bone (cases #5-6). The dural sheath was incised. A 0.05%
solution of HRP (0.0005 g HRP in 10 pl 0.9% saline) was placed
in a glass micropipette (inside tip diameter 20-30 pm) and
iontophoretically delivered by a pulsed (7 seconds on, 7
seconds off) positive current of 5 mA (Midgard, Transkinetics
model CS3) into either VII, 1XII, or‘Vmo utilizing coordinates
obtained from the stereotaxic atlas of Paxinos and
Watson( ’86) . Injections were made at an angle of approximately
90°, with respect to the horizontal plane, for VII and XII and
an angle of approximately 78“ with respect to the horizontal
plane, for Vmo. Following termination of the current, the
micropipette was left.in.place for aniadditional.5 minutes and
then withdrawn. Upon completion of the experiment, gelfoam
(UpJohn Company) was positioned in the opening, the incision
was clamped closed, and an application of triple antibiotic
ointment (Rugby Laboratories) was placed over the incision
site. All animals were housed, maintained and cared for

according to federally prescribed guidelines (38).

54
Forty-eight hours later, the animals were perfused according
to the procedure described previously (Chapter 1). Sections
of the brainstem containing the trigeminal sensory nuclear
complex (TSNC) were blocked and sectioned on an Oxford
Vibratome into 50pm thick sections in the transverse plane.
Procedures for processing the tissue for HRP activity
utilizing the cobalt-glucose oxidase method as well as section
preparation for initial light microscopic examination has been
described previously in Chapter 1. The full extent of the
injection site was determined under brightfield illumination.
Representative camera lucida drawings and photomicrographs
(25X objective) of HRP-filled target sites were made(Figures
1a,b,c; 2a,b,c). In order to preserve relative cell
morphology, the positions of retrogradely labeled neurons
within Vo were plotted from serial sections throughout the
rostrocaudal extent of the nucleus using a drawing tube and a
40X objective attached to a Leitz Laborlux 12 microscope
(Figures 3,4,5) . Retrogradely labeled neurons were identified
by their content of HRP granules distributed throughout the
cytoplasm. Only those profiles exhibiting a distinct nucleus
were plotted. Camera lucida drawings of individual
retrogradely labeled neurons throughout the rostrocaudal
extent of the nucleus were made using a 40X objective
(Figures 9, 10, 11). Photomicrographs of representative
retrogradely labeled neurons from each subdivision were taken

at 40X (Figures 9, 10, 11) . The somatic cross-sectional areas of

55
retrogradely labeled neurons were measured using a Bioquant
Image Analysis System which computed these areas from tracings
of the perimeters of labeled neurons drawn with the aid of a
camera lucida using a 40X objective. Retrogradely labeled
neurons were grouped according to their position within the
various cytoarchitecturally distinct Vo subdivisions defined
in previous studies (7,8,11,12,13,14,15,26). These
measurements were grouped into block intervals of 25 um’ and
plotted as frequency histograms for each of the proposed
subdivisions (Figures 13, 14) and used for the compilation of
the total somatic‘cross-sectional.area.histograms (Figure 12).
The mean and standard deviations were calculated for all sets
of data. Relative frequencies of labeling of specific
projection neurons were generated from the number of cells
measured in each region. Histograms were drawn using

Freelance Graphics on IBM-PC.

56

RESULTS

Following unilateral HRP injections into Vmo, VII, and
XII motor nuclei, the morphology and distribution of
retrogradely labeled trigeminomotor, trigeminofacial, and
trigeminohypoglossal projection neurons were examined
throughout the rostrocaudal extent of V0, bilaterally. In
each case, the intranuclear regional distribution of labeled
neurons was plotted and illustrated (Figures 3,4,5) by a
series of selected line drawings taken from four
representativelcoronal sections through.ratUVo relative to the
regions of DM, one section each through rostral (RDM) and
caudal (CDM) DM and two sections through middle (MDM) DM. Vo
extends from the caudal pole of VII where it exhibits an
overlap with trigeminal nucleus interpolaris (Vi), to the
rostral pole of VII, where itigradually overlaps with the main
sensory trigeminal nucleus (Vms). All three Vo subdivisions
extend the entire rostrocaudal length of the nucleus. BZ is
a long, thin subdivision located along the lateral border of
the nucleus, immediately adjacent to SVT. VL is located
medial to the ventral two-thirds of 82, and occupies the
largest area of the nucleus. DM is located dorsal to VL and
medial to the dorsal one-third of 82.

NEURONS PROJECTING TO THE TRIGEMINAL MOTOR NUCLEUS
Unilateral HRP deposits were localized within Vmo (Figure

1a). The schematic diagram in Figure 2a shows the locations of

57

two injection sites in two different animals in Vmo and the
extent of overlap. The photomicrograph in Figure 1a
illustrates the spread of HRP in one of the animals following
a single injection centered in Vmo. This injection filled the
majority of the rostro-caudal extent of the nucleus while
filling' three-fourths, of the :dorsal-ventral extent. It
occupies an area measuring 1,335um rostrocaudally, 1,120um
dorsoventrally and 1,160um mediolaterally. The injection in
the second animal is somewhat larger than in first animal. The
nucleus is almost filled completely with reaction product
with some spread into the neighboring peritrigeminal and
intertrigeminal nuclei. The injection site occupies an area
measuring 1,500um rostrocaudally, 1,360um dorsoventrally and
1,240um mediolaterally.

Figure 3 is series of line drawings from coronal sections
through the brainstem containing Vo showing the location of
retrogradely labeled neurons as a result of HRP injection into
Vmo in the second animal. One hundred twenty-two neurons are
filled with HRP reaction product. The majority of labeled Vmo
neurons are located in ipsilateral VL and DM regions with a
few labeled cells in B2. Contralaterally, DM contains two
labeled neurons. Although labeled neurons were found
throughout the rostrocaudal extent of V0, approximately two-
thirds were located in the middle one-third of the nucleus
with the other one-third of labeled cells found near the

rostral and caudal poles of V0. The rostral pole of V0

58
contained the least number of labeled neurons. Labeled VL
neurons outnumbered those in DM by a 2:1 ratio.

Two major types of retrogradely labeled trigeminomotor
projection neurons were found.in‘VLland DM (Figures 6,9). The
cell bodies of these neurons were basically spherical in shape
with a few pyramidal-shaped neurons scattered throughout VL.
The labeled cells could be grouped into small (< 25pm in
diameter) and large (>25 pm) varieties with somatic cross-
sectional areas of <300um’ and >300um’, respectively. Their
average cross-sectional areas were 175nm2 for the small cells
and 407nm2 for the large neurons. (Figures 13,14) .

The vast majority of labeled neurons belonged to the
small cell population. Although each Vo subdivision contained
both populations of neurons, large size cells were found more
frequently in‘VL, while small size neurons predominated.in DM.
The average cross-sectional area of large and small labeled
cells in VL were 145nm2 and 425um’, respectively, while the
average cross-sectional area of large and small labeled cells
in DM were 225nm2 and 442nm“, respectively. At the caudal pole
of V0, labeled neurons were located almost exclusively in the
medial one-half of both DM and VL. Only one neuron was found
in dorsal BZ. There were no labeled neurons found in the
caudal pole of contralateral Vo. All of the labeled neurons
belonged to the small cell population and had spherical
somata. A few small, pyramidal shaped neurons were found in

VL. Portions of the dendritic fields were visible on the most

59

densely filled neurons. DM neurons presented a bipolar
appearance while those cells in VL had thin radiating
dendrites or a bipolar appearance (Figures 6,9). The number
of labeled neurons increased in the middle one-third of V0,
both in DM and VL (Figure 3). 82 continued to show very few
labeled neurons and.they were:generally located in its ventral
half. Contralateral DM also contained a few labeled small
cells. Moving from caudal to rostral, labeled neurons were
most prominent in the medial one-half of V0 caudally, and the
lateral one-half rostrally. Labeled neurons were most dense
dorsally in DM and ventrally in VL leaving a conspicuous
absence of neurons ventrally in DM and dorsally in VL. Some
labeled DM and VL neurons were small bipolar-shaped cells
with spherical shaped cell bodies, while, a number of large
size neurons with spherical shaped somata were present in DM
and VL, as well as a few pyramidal-shaped cell bodies in VL.
Neurons with bipolar-shaped dendritic fields were observed in
DM while neurons with multipolar-shaped dendritic fields were
present in both VL and DM (Figures 6,9).

At the rostral pole of‘Vo there was a striking paucity of
labeled neurons (Figure 3). Of the few cells present in VL,
most had large spherical-shaped somata while those scattered
cells in DM possessed small spherical-shaped cell bodies.
Neurons in both subdivisions appeared to have more bipolar-
shaped dendritic fields (Figures 6,9). There were no labeled

neurons found in BZ and the rostral pole of V0

60

contralaterally contained no labeled cells.

NEURONS PROJECTING TO FACIAL MOTOR NUCLEUS

Unilateral HRP injections were ‘made in 'VII in ‘two
animals. The schematic diagram in Figure 2b illustrates the
two injection sites and the spread of HRP in each while the
photomicrograph in Figure 1b illustrates the spread of HRP in
the second animal. This injection site measures 1,300um
rostrocaudally, 1,080um dorsoventrally and 660nm
mediolaterally and fills intermediate and lateral portions of
VII. The injection site from the first animal illustrated in
Figure 2b fills the lateral and intermediate portions of the
nucleus and measures 1,455um rostrocaudally, 1,210um
dorsoventrally and 690um mediolaterally. There is some spread
into the perifacial zone and intermediate reticular region
dorsally.

Figure 4 show a series of selected line drawings taken
from coronal sections through Vo (injection in first animal)
showing the location of ninety-one labeled Vo-VII projection
neurons. The majority of labeled cells were in VL and DM and
occupied the middle one-third of ipsilateral Vo. Labeled
cells in VL outnumbered those in DM by a 2:1 ratio. A few
labeled neurons were observed in 82. Contralateral VL and DM
also contained.a few labeled cells. Injections in VII labeled
VL and DM neurons characterized by spherical-shaped somata.

These cell bodies could be characterized as medium-

61

sized (between 20am and 30pm in diameter) with somatic cross-
sectional areas of 100-550um’, and average cross-sectional
areas of 289nm“ (Figure 7,10) . Although there were fewer
neurons labeled in the caudal pole as compared to the middle
of V0, VL neurons continued to outnumber those in DM 2:1. BZ
contained Only one labeled cell which occupied its ventral
half. Contralaterally, a few labeled neurons were located in
VL. The cell bodies gave rise to bipolar dendritic fields
(Figure 7,10). In the middle one-third of V0, DM neurons were
located ventromedially in the caudal most sections, whereas
they became situated dorsolaterally as one moves rostrally
(Figure 4). Labeled VL neurons were located more ventrally in
caudal sections but filled the middle of the subdivison more
rostrally. A few labeled neurons were found in the ventral
and dorsal most regions of 82. Contralaterally, there were a
few labeled neurons in DM and‘VL. All of the Vo-VII projection
neurons displayed basically bipolar-shaped dendritic fields
(Figure 7,10).

At the rostral pole of‘Vo, DM and‘VL were sparcely filled
with labeled neurons (Figure 4). No labeled neurons were
found in B2. Only one labeled cell was found contralaterally

in VL.

NEURONS PROJECTING TO THE HYPOGLOSSAL NUCLEUS
Unilateral HRP deposits were localized within XII in two

animals. The schematic drawing in Figure 2c illustrates two

62

injection sites while the photomicrograph in Figure 1c
illustrates the injection site from the first animal. It
measures 1,250um rostrocaudally, 1,400um dorsoventrally and
1,210um mediolaterally as it fills the dorsoventral and
rostrocaudal extent while filling approximately the medial
three-fourths of the nucleus. The injection site in the
second animal illustrated in Figure 2c fills the lateral half
of the nucleus throughout its dorsoventral and rostrocaudal
extent. The spread of HRP in this injection site measures
1,190um rostrocaudally, 1,575um dorsoventrally and 1,140
mediolaterally. There is some spread into the gigantocellular
reticular region.

Figure 5 show a series of line drawings from coronal
sections through Vo showing the location of labeled neurons as
a result of the HRP injection described above. Fifty-nine
neurons were labeled and distributed in nearly equal numbers
in ipsilateral VL and DM in the middle one-third of V0. In
addition, a few labeled neurons were observed in ipsilateral
Bz as well as contralateral DM. These labeled neurons in each
of the subdivisions exhibited spherical-shape somata. (Figure
8,11) They could be identified as being small (< 25 pm in
diameter) neurons with somatic cross-sectional areas of 100 to
450 [.11112 and average cross-sectional areas of 185nm2 (Figure
8,11).

At the caudal pole of V0, labeled neurons were sparse and

widely scattered but appeared to be evenly distributed between

63

VL and DM (Figure 5). One labeled neuron was found in the
ventral one-half of BZ. No neurons were located
contralaterally. Small Vo-XII projection neurons, distributed
equally throughout the nucleus, displayed both bipolar and
multipolar-shaped dendritic fields (Figure 5).

The middle one-third of V0, contained the majority of Vo-
XII projection neurons. Distributed evenly between VL and DM,
these labeled neurons were located in the medial portion of DM
and throughout VL. A few labeled neurons were found
contralaterally in DM with one labeled cell located in the
ventral one-half of BZ.

The rostral pole of V0 was basically devoid of labeled
neurons (Figure 5). Two labeled cells were found along the

medial border of VL.

64

DISCUSSION

These anatomical studies show that Vo has predominantly
ipsilateral efferent projections to Vmo, VII, and XII. Vo
projection neurons are situated primarily in the middle third
of the nucleus with the majority of them located in VL and DM
subdivisions with only a few cells in BZ. Vo neurons
innervating Vmo, VII and XII fall into three size ranges with
small neurons projecting to Vmo and XII, medium size cells
innervating VII and large-sized neurons forming terminal
arbors in Vmo. These findings coupled with previous anatomical
studies that show that DM, VL and B2 neurons receive
morphologically and functionally distinct types of primary
trigeminal afferents (6,7,8,9,10,11,12,45), suggest that Vo
efferents to vmo, VII, and XII provide multiple projections
carrying different types of primary trigeminal afferent input
to these cranial nerve motor nuclei. Projections to Vmo, VII,
and XII from DM, VL and B2 most likely provide a route for
Vonvolvement in complex reflexes (e.g., corneal reflex,
orienting reflexes, jaw opening reflex) elicited by
stimulation of different types of primary trigeminal afferents
innervating the skin of the face, oral and nasal mucous

membranes and muscles, tendons and bones of the jaw and face.

INJECTION SITES

Unilateral HRP injection sites in Vmo, VII and XII were

65

only considered effective if they filled a significant portion
of the nucleus, with little spread into surrounding areas and
if Vo contained densely filled neurons. The Vmo injection
site in the first animal filled a significant amount of the
nucleus without spread into surrounding areas. However, the
number of labeled neurons in V0 was less than in the second
animal which not only filled the nucleus but had some spread
into adjacent intertrigeminal and peritrigeminal areas.
Having fewer labeled cells in V0 after the injection in the
first animal was probably due to the lack of HRP reaction
product in the most dorsal and ventral extremes of the
nucleus. It is important to note that the distribution of the
labeled Vo cells was generally the same in both cases.

Injection sites in VII, in both animals, filled the
dorsoventral and rostrocaudal extents of the nucleus, however,
neither injections totally filled the medial aspect of the
nucleus. These two cases showed similar distributions of
labeled Vo neurons with fewer labeled cells present in the
second animal. Injection sites which would include the most
medial aspect of VII most likely would result in an increase
in the number of labeled Vo cells but the distribution of
labeled neurons would probably be similar.

For XII, the dorsoventral and rostrocaudal extent of the
nucleus was filled by combining the data from the injection
site in the second animal,which filled only the lateral one-

half of the nucleus and the injection site in the first animal

66

which filled the medial two-thirds of the nucleus.

VO PROJECTIONS TO Vmo

The organization of efferent projections from V0 to Vmo
has been well established in previous anatomical studies
(37,41,42,46) However, the specific morphological features of
o-Vmo projection neurons, their locations in V0 subdivisions,
as well as their distributions, have not been elucidated. A
previous anatomical and edectrophysiological study (41,42)
demonstrated the existence of two distinct types f Vo
projection neurons to cat Vmo. The first cell type displayed
a cell body similar in size and shape to the large size Vo-
Vmo projection neuron found in the present studyu The second
neuronal type was smaller than the first, and appeared to be
morphologically similar to the small size Vo-Vmo projection
neuron in this study. These findings substantiate those of
the present study that two distinct types of Vo-Vmo projection
neurons are present in V0. The present study extends these
findings by showing the two types of cells are found in DM and
VL of rat V0. The anatomical and electrophysiological study
of Shigeniga, et al,(41,42) correlated size of Vo-Vmo
projection neurons with low and high threshold
mechanoreception and inhibitory and excitatory postsynaptic
potentials suggesting that Vo-Vmo neurons play an important
role as excitatory or inhibitory interneurons on masticatory

‘motoneurons and.may be involved in jaw reflexes related to jaw

67

movement or avoidance reactions. The large Vo-Vmo projection
neurons received input from low-threshold mechanoreceptor
afferents while the small Vo-Vmo projection neuron received
nociceptive input from afferents innervating tooth pulp. The
study of Shigeniga, et al (41,42) indicated that Vo-Vmo
projection neurons form terminal arbors in the dorsolateral
subdivision of Vmo and contain inhibitory or excitatory
interneurons for jaw-closing motoneurons. The same study
further suggested that due to differences in latencies,
activation of large and small Vo-Vmo projection neurons may
elicit fast and slow inhibitory postsynaptic potentials in
masseter motoneurons which may cause inhibition of jaw
reflexes in relation to the jaw movements or avoidance
reactions.

An anterograde PHA-L study in the rat at the light
microscopic level (44) showed that Vo-Vmo efferent axons
displayed terminal axonal arbors of a single morphological
type suggesting that small and large DM and VL neurons exhibit
terminal arbors of similar morphology in Vmo. Based on these
findings, further analyses including electron
microscopy,evaluating the cytology and synaptic connections
of Vo-Vmo efferents, are needed to ascertain whether or not
Vo-Vmo projection neurons in different subdivisions of V0 have
morphologically similar terminations in Vmo. It may be that
these efferents differ in their synaptic vesicle size and

shape; and synaptic junction morphology as well as in their

68

postsynaptic targets in Vmo.

VO PROJECTIONS TO XII

A Vo-XII projection in the rat, shown in the present
study, confirms those of the retrograde transport studies of
Borke, et a1, (2). In that study Borke, et al (2) described
the Vo-XII projection as arising from neurons situated only in
the dorsal portion of V0 where primary afferents from the
mandibular division of the trigeminal nerve, innervating
perioral and intraoral structures, terminate.
Electrophysiological studies substantiate this view by showing
a disynaptic pathway to XII from various branches of the
mandibular nerve (33). The present study extends the findings
of Borke, et a1 (2) by showing that approximately equal
numbers of Vo-XII projection neurons are found dorsally in V0
(DM) as well as ventrolaterally (VL) in regions of V0 known to
receive primary afferent input from maxillary and opthalmic
divisions of the trigeminal nerve. Thus, it appears that, in
the rat, Vo-XII projection neurons receive primary afferent
input from the entire orofacial region. Both the present
study and those of Borke, et al (2) agree that only one size
of V0 neuron projects to XII.

Anterograde PHA-L studies (44) have reported two distinct
types of V0 efferents.larborizing in XII, while in the present
study only one size of Vo-XII projection neuron was

identified. The retrograde transport technique used in the

69

present study does not allow one to see the entire dendritic
fields of labeled neurons. Therefore, it is possible that
Vo-XII projection neurons with similar cell body size have
quite different dendritic fields morphologically as well as
morphologically different terminal axonal arborizations in
XII. The fact that XII receives two types of V0 efferents
suggests that at least two different types of orofacial
sensory information are being transmitted to XII.

Mammalian XII receives afferent information from the
nucleus of the tractus solitarius and the sensory trigeminal
nuclei for reflex movements of the tongue in swallowing,
chewing and sucking in response to gustatory and other stimuli
from the oral and pharyngeal mucosae ( 1) . Some of this
trigeminal input undoubtedly is from Vo. Corticobulbar
afferents send information directly to XII as well as
indirectly through interneurons in the neighboring reticular
formation. This input is processed to modulate motor output to

the intrinisic and extrinsic muscles of the tongue (1).

VO PROJECTIONS to VII

Mammalian VII has been described as having perioral and
periorbital motoneurons in its intermediate subdivisions,
innae and neck motoneurons in its medial subdivisions and
proboscis motoneurons situated in its lateral subdivisions
(64) . The present study demonstrates the results of injections

into lateral and intermediate VII subdivisions with slight

70
spread into medial subdivisions. The more lateral injection
revealed the same distribution as the more intermediate-medial
injection except that fewer total numbers of neurons were
labeled. This finding suggests that the Vo-VII projection has
a greater influence on pinnae and neck motoneurons in the rat
than on perioral and periorbital motoneurons, although both
inputs appear to be substantial.

Previous anterograde studies (44) have revealed that Vo
projects to VII. Two morphologically distinct types of
terminal axonal arborizations were found to be present in VII
after a V0 injection. However, the present retrograde
anatomical study suggests that there is one population of Vo-
VII projection neurons based on cell body size. This situation
could be the same as seen for'Vo-XII projection neurons“ That
is, although there is one soma size, the cells may have
structurally different dendritic fields and terminal axonal
arborizations making the cells morphologically different from

each other.

DUALITY OF VO PROJECTIONS TO Vmo, VII AND XII.

The results of the present study suggest that some‘VL and
DM neurons may project to more than one cranial nerve motor
nucleus. Vo-XII projection neurons and several Vo-Vmo
projection neurons had similar sized cell bodies as well as
number of cells projecting to these nuclei. Based on these

findings its possible that one small DM or VL neuron projects

71
to both Vmo and XII. The anterograde studies of Smith and
Falls (64) also contribute to this theory as one type
morphological type of terminal axonal arbor was found in XII
and VII after a V0 injection, which suggests that one Vo

neuron could also be projecting to both VII and XII.

FUNCTIONAL CONSIDERATIONS

The circuitry described above suggests that Vo plays an
important role in not only reflexes dealing with Vmo, VII and
XII but also in fine tuning oral motor function, i.e.
mastication, deglutition, vocalization, and facial expression.
As mechanoreceptive and nociceptive information from the
facial skin, oral and nasal cavities, and teeth are received
in V0, it is important for this information to be sent to
VII. For example, a Vo-VII route projection for the corneal
reflex is important to protect the eye, as well as causing
changes in facial expression secondary to pain or'touch, 'This
latter function is evident in humans with oral or facial pain
where facial expression is affected significantly. A Vo-VII
projection also must underlie chewing or sucking responses to
placing food in the mouth. A Vo-XII projection must underlie
the masticator reflex as well as mastication,deglutition and
vocalization. A Vo-Vmo projection may be essential for reflex
modulation such as in the jaw-opening reflex in which the
contractions of the masseter, temporalis, and medial pterygoid

muscles are inhibited as a result of painful pressure applied

72
to the teeth. As information is collected and processed in V0,
it can have a significant influence on the gross and fine

motor control of the oral facial region.

73

Figure 1a,b,c: Horseradish peroxidase (HRP) Injection Sites

Representative transverse sections (a-c) of HRP injection
sites (IS) in trigeminal motor nucleus (VMO), facial
nucleus(VII), and hypoglossal nucleus (XII). The injection
sites are, for the most part, confined to their respective
nuclear areas.

 

75

Figure 2a,b,c: Schematic Drawings of HRP Injection Sites

Schematic drawings of representative transverse sections
(a-c) showing two HRP injection sites (IS) each in Vmo, VII,
and XII. The injection sites were, for the most part,
confined to their respective nuclear areas. Vp, principal
sensory nucleus; sol, solitary nucleus; svt, sensory
trigeminal tract; Vi, nucleus interpolaris; Vo,nucleus oralis.

76

 

77

Figure 3: Distribution of Vo-Vmo Projection Neurons

Schematic drawings of representative transverse sections
through the entire rostrocaudal length of V0 demonstrating the
distribution of Vo-Vmo Projection Neurons. Drawings are from
caudal to rostral including all the DM subdivisions: caudal
dorsomedial subdivision (CDM); Middle dorsomedial subdivision
dorsal zone (MDMd) and ventral zone (MDMv); and rostral
dorsomedial subdivision (RDM). Vo-Vmo Projection neurons are
distributed primarily throughout ipsilateral V0, with the
majority located in the MDMv and MDMd as well as VL. BZ,
border zone subdivision; svt, spinal trigeminal nucleus.

79

Figure 4: Distribution of Vo-VII Projection Neurons

Schematic drawings of representative transverse sections
through the entire rostrocaudal length of V0 demonstrating the
distribution of Vo-VII Projection Neurons. Vo-VII projection
neurons are distributed primarily throughout ipsilateral V0
with the majority located in the MDMv and MDMd as well as VL.

 

81

Figure 5: Distribution of Vo-XII Projection Neurons

Schematic drawings of representative transverse sections
through the entire rostrocaudal length of V0 demonstrating the
distribution of Vo-XII Projection Neurons. Vo-XII projection
neurons are distributed primarily throughout ipsilateral V0
with the majority of located in MDMv and MDMd as well as VL.

 

83

Figure 6: Diagrams of Vo-Vmo Projection Neurons

Schematic drawings of representative neurons in VL and DM
projecting to Vmo. Two morphologically distinct types of large
and small neurons were identified projecting to Vmo.

Figure 7: Diagrams of Vo-VII Projection Neurons

Schematic drawings of representative neurons in VL and DM
projecting to VII. One distinct type of medium sized
projection neuron was identified projecting to VII.
Figure 8: Diagrams of Vo-XII Projection Neurons

Schematic drawings of representative neurons in DM and VL

projecting to XII. One distinct type of small projection
neuron was identified projecting to XII.

84

Vo-Vmo Projection Neurons

   

 

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Figures 6,7,8

85

Figure 9a,b,c,d,e,f: Photomicrographs of Vo-Vmo Projection
Neurons

Photomicrographs were taken.of representative HRP-filled
Vo-Vmo projection neurons. Two morphologically distinct Vo-Vmo
projection neurons (Fig 7) were identified. In 9a and b are
small neurons while in 9 c-f large cells size are shown.
Cells of both sizes are found in DM (a,c,d) and VL (b,e,f).

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87

Figure 10 e,b: Photomicrographs of Vo-VII Projection Neurons

Photomicrographs of medium-sized HRP-filled Vo-VII
projection neurons in DM (a) and VL (b) of V0.

Figure 11 a,b: Photomicrographs of Vo-XII Projection Neurons

Photomicrographs of the one distinct small sized Vo-XII
projection neuron in DM (a) and VL (b).

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89

Figure 12: Somatic Cross-Sectional Areas of Vo-Vmo, Vo-VII
and Vo-XII Projection Neurons

The percentage of cells identified in all of the cases
involved.in this study'are:graphically displayed to show their
somatic cross-sectional area. The graph displays all three
motor nuclei where neurons from Vo are projecting with the
number of neurons per each nucleus shown in the box.

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91

Figure 13: Somatic Cross-Sectional Area of Vo-Vmo, Vo-VII,
and Vo-XII Projection Neurons in DM

Graph showing the percentage of DM cells with their
cross-sectional areas at 25pm intervals projecting to each
motor nucleus.

Figure 14: Somatic Cross-Sectional Area of Vo-Vmo, Vo-VII,
Vo-XII Projection Neurons in VL

Graph showing the percentage of VL cells with their
cross-sectional areas at 25pm intervals projecting to each
motor nucleus.

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Figures 13,14

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10.

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GQN STATE UNI V.

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