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TRIGEMINAL EFFERENT PROJECTIONS TO AUTONOMIC NUCLEI

IN RAT SPINAL CORD: A LIGHT AND ELECTRON
MICROSCOPIC STUDY

presented by

LI SA ANN BELT

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TRIGEMINAL EFFERENT PROJECTIONS TO
AUTONOMIC NUCLEI IN RAT
SPINAL CORD: A LIGHT AND

ELECTRON MICROSCOPIC STUDY
By

LISA ANN BELT

A THESIS

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

MASTER OF SCIENCE

Department of Anatomy

1 996

ABSTRACT

TRIGEMINAL EFFERENT PROJECTIONS TO AUTONOMIC NUCLEI IN THE RAT
SPINAL CORD: A LIGHT AND ELECTRON MICROSCOPIC STUDY

By

LISA ANN BELT

Anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) was used to
determine the overall morphology, distribution and synaptic relations of efferent axons
originating from neurons in dorsomedial (DM) and ventrolateral (VL) subdivisions of rat
trigeminal nucleus oralis (V0) and terminating in the sympathetic interomediolateral cell
column (IML) throughout the thoracic spinal cord. Light microscopical ﬁndings reveal
that from cells in each subdivision two morphologically distinct types (Types I and H) of
efferent axons project bilaterally to IML. Type I efferents are a direct continuation of
parent axons in the lateral ﬁrniculus. They enter IML dorsally after sending branches into
lamina V. “Within IML these axons branch into several medially directed thin terminal
strands containing up to six boutons. Parent ﬁbers of Type II efferents enter the thoracic
ventral horn from the anterior funiculus and proceed dorsally through lamina VIII, IML,
and laminae IV and V. In IML each parent axon gives rise to one or two collateral
branches which terminate into thin terminal strands containing one to four boutons.

Electron microscopic analyses reveal that the majority of boutons from Type I eﬂ‘erents

are ﬁlled with closely packed agranular spherical synaptic vesicles among which are
scattered several dense core vesicles. These axonal endings form asymmetrical to
intermediate synaptic junctions on large diameter (2.0 - 5.0 pm) dendritic shaﬁs. Boutons
of Type II eﬁ‘ercnts are ﬁlled with a single population of agranular spherical synaptic
vesicles. These axonal endings form asymmetrical to intermediate junctions on the
neuronal cell bodies as well as on large and small diameter (1.0 -2.0 pm) dendritic shafts.
\Vrthin IML the majority of cell bodies and dendrites receiving VO input are thought to be
preganglionic sympathetic neurons. The results of this study suggest that sensory input
ﬁ'om orofacial regions innervated by the trigeminal nerve may have a signiﬁcant inﬂuence

on sympathetic outﬂow to the entire body.

DEDICATION

To my family, Phil, Sandi and Scott Belt and my ﬁrture
husband, Douglas Lupini

iv

ACKNOWLEDGEMENT

I would like to express my thanks and appreciation to my advisor Dr. William M.
Falls for his patience, support and guidance throughout my graduate studies. I would also
like to thank the members of my graduate committee: Drs. Irena Grofova, Ralph Fax and
Arthur Weber for their support and guidance.

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

Many individuals are remembered for their support and encouragement, however

the love of my family and friends helped me achieve my goals.

TABLE OF CONTENTS

List of Figures ............................................... vii
Abbreviations ................................................ viii
Introduction ................................................. 1
Materials and Methods ......................................... 4
Results ..................................................... 8
Discussion .................................................. 12
Figures .................................................... 19
Bibliography ................................................ 3 1

LIST OF FIGURES

1.

2.

PHA-L Injection Sites ......................................... 19
Type I Eﬁ‘erent Axon Terminal Arbors ............................. 21
Type II Eﬁ‘erent Axon Terminal Arbors ............................ 23

Light Micrographs of Type I and Type II Eﬂ'erent Axon Terminal Arbors . . .25

Electron Micrographs of the Cytology and Synaptic Relations of a Type I

Eﬁ‘erent Axonal Ending ............................. . ......... 27
Electron Micrograph of the Cytology and Synaptic Relations of a Type II

Eﬁ‘erent Axonal Ending ..................................... 27
Electron Micrograph of a Type II Efferent Parent Axon ................. 29

Electron Micrograph of the Cytology and Synaptic Relations of Type H
Eﬁ‘erent Axonal Ending ....................................... 29

Electron Micrograph of the Cytology and Synaptic Relations of Type H
Eﬂ‘erent Axonal Ending ....................................... 29

ABBREVIATIONS

AF

AMB

BZ

DAB 3,3’
dcv
DH

DM

IS

LF
MDH
PF
PHA-L

RT

anterior funiculus

nucleus arnbiguus

astrocytic process

bouton

border zone subdivision
dendrite

diaminobenzidine subdivision
dense core vesicle

dorsal horn

dorsomedial subdivision
horseradish peroxidase
interomediolateral cell column
injection site

lateral ﬁrniculus

medullary dorsal horn
posterior ﬁrniculus

phaseolus vulgaris leucoagglutinin

room temperature

viii

SVT

TBS-TX

TS

§

§

Vo

VO-TS

Vi

soma

spinal trigeminal tract

tris buffered saline with triton X

terminal strand

facial motor nucleus

ventral horn

ventrolateral subdivision

trigeminal nucleus oralis

trigeminal nucleus oralis- trigeminospinal

trigeminal nucleus interpolaris

ix

INTRODUCTION

Clinical outcomes from mechanical manipulation of the head (noxious and
innocuous) using osteopathic craniosacral techniques support the existence of eﬁ‘erent
projections from neurons in sensory trigenrinal nuclei receiving these mechanical inputs
via axons of primary trigeminal neurons to autonomic centers in the brainstem and spinal
cord (26). Autonomic responses, both sympathetic and parasympathetic to stimulation of
trigeminal primary afferents include: sweating on the brow, pupiilary changes (constriction
and/or dilation), nausea, vomiting, increases and decreases in the heart rate, diving reﬂex,
ﬂushing of the skin and feelings of anxiety, to name a few. Knowledge of the circuitries
underlying the myriad of autonomic responses are essential if one is to understand the
ﬁrnctional consequences of osteopathic craniosacral manipulative techniques. The
purpose of the present investigation is to extend previous studies (24,46,53) and to
provide new anatomical evidence for the existence of a trigeminospinal tract linking
neurons in trigeminal nucleus oralis (V o) with the preganglionic sympathetic neurons in
the thoracic interomediolateral cell column (IML).

Previous studies (18,19, 21,22,23) have shown that each of the three subdivisions
of rat Vo ; dorsomedial (DM), ventrolateral (VL), and border zone (BZ); receivec
functionally and morphologically distinct groups of primary and non-primary afferent
axons which directly inﬂuence second order VO projection neuron activity. Primary

aﬁ‘erents convey to V0 mechanoreceptive and nociceptive information from oral and

2
perioral areas as well as mechanoreceptive input from the face and oral cavity. Eﬁ‘erent

outﬂow from each Vo sudivision is provided by axons of second order Vo projection
neurons which affect synaptic activity in many target areas along the neuraxis. The
existence of trigeminospinal projections, originating from neurons in all portions Of the
spinal trigeminal nucleus (SVN): Vo, trigeminal nucleus interpolaris (Vi), and medullary
dorsal horn (MDH, trigeminal nucleus caudalis) have been established by previous
investigations in rats (7, 9, 20, 34, 46, 47), cats (7, 8, 14, 29, 32, 37, 38, 39, 56), dogs
(14), hamsters (6), monkey (9) and opossum (10, 16). Retrograde studies of V0
trigeminospinal (VO-TS) projection neurons situated in VL have shown bilateral
projections along the entire length of the spinal cord (34, 46) and their termination in
dorsal and ventral horns as well as intermediate gray (lamina VII) suggest that they
inﬂuence the activity of somatosensory relay neurons, somatic motor neurons, and
preganglionic autonomic motoneurons as well as intemeurons. Based on retrograde
transport studies, rat VL has been shown to contain large multipolar cells that innervate
cervical, thoracic, and lumbar spinal cord levels (53, 56). Anterograde studies using
Phaseolus vulgaris leucoagglutirrin (PHA-L) have conﬁrmed projections of V0 neurons
along the entire length of the spinal cord (53). Terminal arbors of axons of VO.TS
projection neurons were found in dorsal and ventral horn laminae as well as thoracic and
sacral IML. Numerous Vo-TS axons were observed following VL injections. However, a
considerably higher density of ﬁbers was seen following injections into DM. Previous data
(53) show that two morphologically distinct types of efferent ﬁbers emanate from DM

and VL neurons and teminate in IML of the thoracic cord.

3
In the present study PHA-L was injected into V0 with Dm and VL as the principle

targets. Identiﬁcation of V0 injection sites as well as the overall morphology of VO-TS
efferent axons terminating in thoracic IML were analyzed using the light microscope. The
overall axonal morphology was compared to those observed in previous studies (53) in
order to determine ﬁber type. Once the efferent ﬁber types. were identiﬁed at the light
microscopic level they were then examined under the electron microscope in order to

determine their cytology and synaptic relations with neuronal elements in thoracic IML.

MATERIALS AND METHODS

Eighteen male Sprague-Dawley albino rats (200-3 00g) were anesthetized with
intaperitoneal injection of sodium pentobarbitol and placed in a Stereotaxic apparatus. The
cranium was opened unilaterally through the occipital bone and the dura incised.
Ionotophoretic injections were made into DM and VL of V0 unilaterally using glass
micropipettes (inside tip diameter 20-30 um) ﬁlled with 2.5% PHA-L (Vector Labs) in
0.05M sodium phosphate buffered (pH 7.4) saline. Micropipette electrodes were lowered
into DM and VL passing through the cerebellum at an angle of approximately 90° with
respect to the horizontal plane. Stereotaxic coordinates for electrode placement used in
this study were detemrined in previous PHA—L studies of V0 eﬁ‘erents to rat spinal cord
(53, 54). Ionotophoretic parameters were set up to use a current of 5 11A provided by an
alternating current source (Midguard, Transkinetictics Model C83) which was applied at
intervals of 7 seconds on and 7 seconds off for 45-60 minutes.

Aﬁer postinjection periods of 10-14 days the animals were deeply anesthetized and
perfused transcardially with two solutions at 4°C. The ﬁrst solution consisted of 250ml of
lSOmM acatate buffer (pH 6.5) containing 4% parafonnaldehyde. The second solution
consisted of 750ml 65mM borate buffer (pH 9.5) containing 4% parafonnaldehyde and
0.05% gluteraldehyde. Aﬁer perfusion the brain and spinal cord were removed, post-ﬁxed
overnight at 4°C in the last perfusiate buffer and cut serially into 20-30um coronal sections

using and Oxford Vibratome into tris buffered saline (TBS, pH 7.6) at room temperature

5
(RT). After three rinses in TBS with 0.04% Triton X-100 (TBS-TX) at RT sections were

placed in a solution of normal rabbit serum in TBS-TX for 30 minutes at RT and rinsed
once in TBS-TX at RT. Sections were then incubated for 24-48 hours in anti-PHA-L
(122000; Vector Labs) diluted in TBS-TX at 4 C. NO labeling was observed when the
anti-PHA-L was eliminated. To visualize the PHA-L, sections were processed with the
avidin- biotinylated HRP ABC technique using Vecastain ABC Kit (Vector Labs). Final
peroxidase reactions were performed with DAB (100mg in 100ml 0.15M TBS). The
100ml DAB solution also contained 40mg ammonium chloride, 200mg glucose oxidase
and 38mg Imidozole. Sections were incubated in the DAB solution for 45-60 minutes.
After they were irnmunoreacted the sections were cleared through a graded series of
glycerol concentrations (20, 40, 60, 80, and 100% for 5 minutes each), placed on glass
slides in 100% glycerin and coverslipped.

Detailed light microscopic evaluations of each section through V0 and the
rostrocaudal extent of thoracic IML were undertaken in order to determine the injection
site in V0 as well as the overall course and distribution, organization and morphology of
VO-TS eﬂ‘erent axons and their terminal arbors in thoracic cord. Sections not containing
isolated or completely labeled axons suitable for electron microscopic evaluations were
rehydrated through a descending series of glycerin concentrations (80, 60, 40, and 20%),
mounted on glass slides, counterstained with cresyl violet and coverslipped. All light
microscopic evaluations were performed with a Leitz laborlux 12 microscope ﬁtted with a
drawing tube. Detailed drawings of PHA-L labeled VO-TS efferent axons and their

terminal arborizations in IML to be taken for electron microscopic evaluation were made

6
using 100x oil immersion objective lens at a magniﬁcation of 1250X. Photographs to

illustrate speciﬁc morphological features of VO-TS axons, such as their diameters, were
also taken.

Coronal sections through thoracic IML containing isolated and completely labeled
Vo-TS eﬁ‘erent axons were processed for electron microscopy. The IML and surrounding
area were dissected out of each section and rehydrated through a descending series of
glycerol concentrations (80, 60, 40, and 20% for 5 minutes each) before being osmicated
in 2% osmium tertoxide, rinsed in distilled water and stained en bloc in 1% aqueous uranyl
acatate and embedded in Marglas. Sections through thoracic IML containing PHA-L
labeled ﬁbers and their terminals were thinly sectioned (many in series). Sections were
oriented so that as much as possible of the axons would be cut transversely. Sections
were mounted on Fromvar-coated grids, stained with lead citrate and examined in an
electron microscope.

Electron micrographs from sections through light microscopically identiﬁed
PHA-L labeled VO-TS efferent axons and their terminal arbors in thoracic IML were
examined to observe their size, shape and relationship to other neuronal structures (soma,
dendrites and axons) as well as their cytoplasmic structure. Labeled Vo-TS eﬁ‘erent
axonal endings were classiﬁed as to their size and shape as well as the size, shape and
compactness of their synaptic vesicles. The type of synaptic junction made with
postsynaptic dendrites and neuronal cell bodies was also classiﬁed. Ninety-six labeled
axonal endings were examined in this study and classiﬁed into two morphologically

distinct types Type I (56 endings) and Type II (40 endings). Diameters of postsynaptic

7
dendrites were also measured. Dendritic diameters were detremined ﬁ'om proﬁles out

either longitudinally or transversely in which neurotubule membranes were clearly visible

and properly oriented.

RESULTS
Injection Sites

PHA-L was injected unilaterally into V0 with the middle of DM and VL as the
principal targets (Figure 1). Areas containing densely ﬁlled neurons within DM and VL
were considered eﬂ‘ective zones which had efferents projections originating from them.
Anterograde axonal labeling was seen following PHA-L deposits into DM and VL with
minimal spillover into adjacent areas. Densely ﬁlled neurons were never observed in
adjacent areas following PHA-L injections into DM and VL.

In this study ten successful experiments resulted in the bulk of the PHA-L being
deposited into DM. Two of the brains are used to illustrate representative injection sites
(Figure 1A, schematically, and Figures 1B, C photographically) from the ten studied. The
DM injection site depicted in Figure 1B was situated in the middle of DM. It was
conﬁned to the subdivision with some spillover into dorsal B2. The injection site, as
measured in transverse serial sections, extended 1180 um rostrocaudally, 910 um
dorsoventrally and 1180 um mediolaterally. The second DM injection site as shown in
Figure 1C also was conﬁned to the subdivision with some spillover into dorsal VL. It
measured 720 um rostrocaudally, 1230 um dorsoventrally and 1090 um mediolaterally.
Eight successful experiments resulted in PHA-L being deposited principally into VL. One
brain is used to illustrate a VL injection site (Figure 1A, schematically and Figure 1D,

photographically). This injection site ﬁlled VL and adjacent BZ and spread into the spinal

9
trigeminal tract (SVT). It did not extend into DM or dorsal BZ. This injection site is

representative of the eight injection sites studied and the results from it are reported in this
study. The injection site, as measured transversely in serial sections extended 1100um
dorsoventrally, 1200 um rostrocaudally, and 1000 um mediolaterally. The combination of
these three injection sites completely ﬁlled DM and VL (Figure 1A) and allowed for

accurate analysis of Vo-TS eﬂ‘erent projections to thoracic IML.

Light Microscopy

Following injections targeted to DM and VL, PHA-L labeled parent Vo-TS axons
and their terminal arbors were visible throughout the rostrocaudal length of the thoracic
spinal cord. Several descending ﬁbers terminated in the IML bilaterally. The overall
morphology and their manner of termination in IML allows for the classiﬁcation of these
Vo-TS eﬂ‘erent axons from both subdivisions into two types; Type I and Type II. These
two types are identical to those found in a previous PHA-L study (53, 54).

Terminal arbors of Type I Vo-TS eﬁ‘erent axons (Figures 2, 4) are a direct
continuation of their parent ﬁbers (1.0 to 2.0 pm in diameter) in the lateral ﬁrniculus. The
parent axons enter IML dorsally after sending branches into lamina V. Upon entering the
IML parent ﬁbers branch into several medially directed thin temrinal strands (0.5 to 1.0
pm in diameter) which are conﬁned to IML (Figure 2B). Along the terminal strands are
one to six irregularly spaced boutons en passant and terminal boutons (Figure 2C, D, 4).

These spherical to elliptical-shaped boutons measure 1.5 to 5.0 pm in diameter.

10
Parent ﬁbers (3.0 to 5.0 pm in diameter) of Type II Vo-TS efferent axons enter the

thoracic ventral horn from the anterior ﬁrniculus (Figure 3B). They proceed dorsally
through the lateral aspect of ventral horn laminae VIII and IV, pass through IML, and
proceed into dorsal horn laminae IV and V. In IML each parent ﬁber gives rise to one or
two collateral branches which undergoes secondary branching (Figures 3B-D). From
these branches emerge thin terminal strands measuring 0.5 to 1.0 pm in diameter (Figures
3D, 5). Some terminal strands emanate directly from the parent ﬁber (Figure 3C). Along
a terminal strand are one to four irregular spaced boutons en passant with a bouton at the
end of each terminal strand. Boutons measure 1.5 to 5.0 pm in diameter and display
spherical to elliptical shapes. The terminal axonal arbor is oval in shape and is conﬁned to

IML (Figure 3B).

Electron Microscopy

Electron microscopic analysis of PHA-L labeled parent ﬁbers of Type I and Type
II Vo-TS efferent axons terminating in thoracic IML show that they are thinly myelinated
(Figure 7). The myelin sheaths of both types of parent axons range from 0.1 to 1.5 pm in
thickness. The terminal strands of both types of Vo-TS efferents are unmyelinated (Figure
6, 9). These ﬁndings substantiate the light microscopical ﬁndings for the diameter of
parent axons and terminal strands of both types of Vo-TS eﬁ‘erents terminating in thoracic
IML.

Within the thoracic IML neuropil axonal endings of both Types I and Type II

VO-TS are found singly (Figures 5,6,8,9). However, the endings of each type are

ll
structurally different and can be distinguished form each other on the basis of their

cytology and synaptic relations (Figure 5,6,8,9).

The majority of the endings of Type I VO-TS efferent axons have an elongated
appearance and display either a scalloped (Figures 5A, B) or smooth contour (Figures 6A,
B). They are ﬁlled with closely packed agranular spherical synaptic vesicles (40-60 nm in
diameter) among which are scattered several spherical dense core vesicles (85-100 nm in
diameter; Figure 6B). These axonal endings form single or multiple asymmetrical to
intermediate synaptic junctions on a single dendritic shaft. These dendritic shafts measure
2.0 to 5.0 um in diameter and contain some granular endoplasmic reticulum as well as
neuroﬁlarnents, neurotubules,agranular endoplasmic reticulum and mitochondria (Figures
5, 6). Most endings of Type I Vo-Ts efferent axons are completely ensheathed by
astrocytic processes except at their presynaptic membrane ( Figures 5,6 ).

Endings of Type II Vo-TS eﬂ‘erents axons present a dome-shaped appearance and
are ﬁlled with a single population of agranular spherical (40-50 nm in diameter) synaptic
vesicles (Figures 8,9). The synaptic vesicles aggregate at the one or two asymmetrical to
intermediate synaptic junctions associated with each tenninal. Endings of Type II Vo-TS
efferents make synaptic contact with the cell bodies (Figure 8), large diameter dendrites
(2.0 to 5.0 pm ) as well as smaller dendritic shafts measuring 1.0 to 2.0 pm in diameter .
A Type II ending synapses on a single dendritic shaft. Type II axonal endings along the
same terminal strand have been observed to synapse on large as well as smaller diameter
dendritic shafts. Type II terminals are ensheathed by overlapping astrocytic processes

except at their presynaptic membrane (Figures 8,9).

DISCUSSION

Light Microscopy

The present anatomical study demonstrates that IML of rat thoracic spinal cord
receives bilaterally terminal arborizations of two morphologically distinct types of Vo-TS
eﬂ‘erent axons; Type I and Type 11. These ﬁndings support and extend previous light
microcopical analyses (55) by showing that both types of axons arise from neurons in DM
as well as VL. The exact morphology ofDM and VL neurons giving rise to Type I and
Type II Vo-TS efferent axons innervating IML have yet to be determined but they are part
of the structurally and ﬁmctionally diverse neuronal populations making up DM and VL.
Type I Vo-TS efferents enter IML ﬁ'om the lateral funiculus while Type II Vo-TS
eﬂ‘erents traverse the anterior ﬁrniculus and enter IML after giving off collaterals in the
ventral horn. Axons exiting the anterior and lateral funiculi and terminating in IML have
been identiﬁed by Ramon Y Cajal (44) in Golgi preparations in the newborn mouse. The
axons appear to display morphologies similar to Types I and II Vo-TS efferents described
in this study as well as in previous PHA-L investigations (54). Taken together these data
suggest that in the rat there are two separate efferent tracts, both originating from two
different Vo subdivisions, that are involved in conveying orofacial sensory input to IML.
By virtue of these connections with IML, Vo must play an important role in the expression

of reﬂexes involved in the intergration of trigeminal and sympathetic systems.

l 3
Methodological Considerations

The anterograde PHA-L method, which labels the axons and their terminal
arborizations by injecting PHA-L at sites which contain the parent cell bodies and
dendrites, was used to identify the overall morphology, cytology and synaptic relations of
VO-TS efferent axons projecting ﬁ'om DM and VL to IML of rat thoracic cord. The
uptake and transport of PHA-L appears only in the parent cell bodies and dendrites and
not in ﬁbers of passage, although in other neuronal systems retrograde transport of
PHA-L as well as anterograde transport of lectin through ﬁbers of passage have been
demonstrated (12, 52). The PHA-L injection sites in this study were relatively small
which allows for complete ﬁlling followed by detailed characterization of VO-TS efferent
axons and their terminal arbors at the light microscopic level as well as their cytology and
synaptic relations at the electron microscopic level. Injection sites were only considered
eﬁ‘ective if they ﬁlled a signiﬁcant portion of the nucleus with little spillover into
surrounding areas and contained densely ﬁlled neurons. Extensive labeling has been
thought to be the result of increased uptake secondary to axonal damage produced by the
administration of PHA-L by a continuous current (not a pulse every 7 seconds). When
using a small micropipette and pulsed current as was done in this study, the number of
labeled VO-TS efferent axons was low (49). The differences observed in the morphologies
of VO-TS efferent axons and their terminal arborizations in IML arose from cells located
within DM and VL PHA-L injection sites and not ﬁ'om ﬁbers of passage. In this study
PHA-L labeled neurons were not observed in areas outside the boundaries of DM and VL

which contained either no PHA-L reaction product or was only lightly stained.

l4
Recognition of morphological features of the terminal axonal arbors of Vo-TS efferent

axons at the light microscopic level shows the great advantage that the PHA-L method has
over other anterograde techniques such as radioactive amino acids, horseradish peroxidase
(HRP) or wheat germ agglutinin conjugated to HRP (W GA-HRP) and Golgi (25, 61). In
this study the labeling presented by PHA-L allows for recognition of two morphologically

distinct types of Vo-TS efferents innervating thoracic IML.

Electron Microscopy

The ultrastructure portion of this study indicates that the axonal endings of Types I
and II Vo-TS efferents in thoracic IML display differences in their synaptic relations and
cytology. Axonal endings of both types of Vo-TS eﬂ‘erents synapse on dendrites while
axonal endings of Type II Vo-TS efferents also synapse on neuronal cell bodies. The
majority of neurons in IML belong to sympathetic preganglionic neurons as determined in
retrograde HRP studies in the cat (11), dog (43), and rat (48). In addition, sympathetic
preganglionic cells have also been observed in the lateral ﬁmiculus and the middle region
of the intermediate gray adjacent to IML in a variety of mammals (42). The Golgi studies
of Ramon Y Cajal (44) in the newborn mouse provide insight into the overall morphology
of these sympathetic preganglionic neurons. Those neurons whose cell bodies are situated
within IML have dendritic ﬁelds conﬁned primarily to the region while neurons whose cell
bodies are located in either the lateral funiculus or the intermediate gray send dendritic
branches into IML. When taken together the results of these studies, along with the

ﬁndings in the present investigation, strongly suggest that the axonal endings of Types I

15
and II Vo-TS eﬂ‘erents are most likely synapsing on the dendrites and cell bodies of

sympathetic preganglionic neurons. Within IML, axonal endings of Type I Vo-TS efferent
axons make synaptic contact with large diameter dendrites while terminals of Type II
Vo-TS eﬁ‘erents synapse on the same size dendrites as well as cell bodies and smaller
diameter dendritic branches. Based on these ﬁndings axonal endings of Type I efferents
are most likely synapsing on sympathetic preganglionic neurons conﬁned to IML while
terminals of Type II eﬁ‘erents are probably synapsing on these same neurons as well as
sympathetic preganglionic whose cell bodies are in the lateral ﬁrniculus and the middle
portion of the intermediate gray by way of connections with their smaller diameter ,
dendritic branches projecting into IML. Without taking into account the density of each
type of axonal ending synapsing on IML neurons, it would appear that the axonal endings
of Type II Vo-TS efferents, with their placement along the entire length of dendritic
shafts Of IML neurons as well as the cell bodies, would have a more important role in
controlling the activity of sympathetic preganglionic neurons in IML than would the
terminals of Type I VO-TS efferents which synapses only on large diameter dendrites of
the same neurons. It has long been suggested that axonal endings that contain spherical-
shaped agranular synaptic vesicles and establish asymmetrical to intermediate synaptic
contacts (axonal endings of both Type I and II Vo-TS efferents) may be associated with
an excitatory ﬁrnction (57,58). The fact that all of the synaptic contacts observed
throughout this study are of this type may therfore indicate a preponderance of excitatory
synaptic inputs impinging on sympathetic preganglionic neurons in IML ﬁ'om second—

order DM and VL neurons. In addition to spherical agranular synaptic vesicles, axonal

l6
endings of Type I Vo-TS efferents also contain large dense core vesicles (> 80 nm in

diameter). Although it is not possible to identify the neurotransmitter from the
ultrastructural appearance of synaptic vesicles in the presynaptic terminal, large dense core
vesicles have long been associated with neuroactive peptides within the central nervous
system. The fact that they are found in the terminals of Type I Vo-TS efferents suggests
that peptides may play an important role in the activation of IML preganglionic neurons by
orofacial stimulation. Immunocytochernical studies (1) support this assumption by
indicating that IML receives aﬁ‘erent input from axons stained for somatostatin and
oxytocin. Neuropeptide somastatin-like activity has been illustrated (5) while substance P
and Met-enkephalin reactivity have been observed ( personal communication with R
Bowker) for neurons in DM and VL, the same subdivisions whose neurons give rise to

Types I and II VO-TS efferents terminating in the IML of this study.

Functional Considerations

Vo appears to have the strongest projection to the spinal cord of the three major
subdivisions of the rat spinal trigeminal nucleus (24) and these projections arise from
neurons in DM and VL subdivisions (54). Previous light and electron microscopical data
have shown that DM and VL receive the temrinal arbors of small myelinated (A ) primary
trigeminal axons ( 24, 55). Based on previous electrophysiological data, these A ﬁbers
most likely respond to light tactile and/or noxious stimuli from the orofacial region
(4,17,27,28,29,33,40,45,50,51). Taken together these data suggest that DM and VL

neurons are involved in the processing of orofacial pain and/or tactile input from small

17
myelinated primary trigeminal axons. Autoradiographic, degeneration, and anterograde

transganglionic HRP transport studies in cat and rat agree that primary trigeminal axons
terminating in V0 are somatopically organized (2,3,13, 30,31,35,36,4l,59,60). By
comparing locations of the rat DM and VL subdivisions to the regions of termination of
primary afferent axons belonging to branches associated with each of the divisions of rat
trigeminal nerve (2,13,31,35), it can be concluded that DM and VL would be receiving the
terminal arborizations of primary trigeminal neurons innervating mandibular, maxillary,
and ophthalmic orofacial receptive ﬁelds. From these data it appears that Types I and II
Vo-TS efferents originating from second-order neurons in DM and VL are probably
conveying to thoracic IML orofacial pain and/or tactile information from the entire
peripheral receptive ﬁeld of the trigeminal nerve. The importance of this assumption is
that mechanical and/or noxious stimulation of the orofacial regions innervated by the
trigeminal nerve probably have a marked inﬂuence on sympathetic outﬂow from IML of
the thoracic cord.

The sympathetic portion of the autonomic nervous system, for the most part, is
activated by exterorecptive inputs arriving by way of somatic primary afferent ﬁbers (e.g.
trigenrinal primary afferent input). Theseinputs are initiated by favorable and unfavorable
changes in the environment. Clinical outcomes from mechanical manipulation of the head
(noxious and innocuous) using both intra- and extraoral techniques support existence of
efferent projections fom Vo neurons, which receive these primary trigeminal mechanical
inputs, to IML neurons in the thoracic spinal cord (26). Manual manipulations of the

cranium can either alleviate observed symptoms or produce the onset of new symptoms.

l 8
Sympathetic reﬂex responses throughout the body to mechanical stimulation of trigeminal

primary aﬂ‘erents include: sweating, pupillary dialtion, decreased salivation (dry mouth),
vasoconstriction in the skin and viscera, increased blood pressure, feelings of anxiety,
increased heart rate, decreased intestinal movements, contraction of vesical and rectal
sphincters, deepened respiration, and increased blood ﬂow to skeletal muscles, heart,
lungs, and brain. The V0 projections to IML provide an important link in a pathway by
which exteroreceptive primary trigeminal inputs from the orofacial region can contribute
to the expressions of reﬂexes involved in the integration of the trigeminal and sympathetic
systems throughout the body. Though the trigemino-sympathetic pathway, Vo may be the
portion of the spinal trigeminal nucleus which participates in the manifestation of
sympathetic responses which have been observed clinically following manual

manipulations of the head.

Figure 1: PHA-L Injection Sites

Representative transverse sections (A-D) showing the location of Phaseolus vulgaris
leucoagglutinin (FHA-L) injection sites in rat trigeminal nucleus oralis (V o). A.
Composite schematic drawing of the three injection sites shown photographically in B-D.
B. A PHA-L injection site located in the dorsomedial subdivision of V0 (DM) and
extending dorsally into the dorsal portion of the border zone subdivision of V0 (BZ) as
well as a short distance ventrally into the dorsal portion of the ventrolateral subdivision of
V0 (VL). C. A PHA-L injection site located in DM which extends into dorsal BZ as well
as a short distance ventrally into dorsal VL. D. A PHA-L injection site located in VL
completely ﬁlls the subdivision as well as adjacent BZ and the spinal trigeminal tract
(SVT) but does not extend into DM or dorsal BZ. VH, facial motor nucleus: AMB,
nucleus ambiguus.

l9

 

Figure 2: Type I Efferent Axon Taminal Arbors

Composite schematic drawings (A-D) showing the location and morphology of
terminal arborizations of PHA-L labeled Type I Vo-trigeminospinal (TS) axons in the
interomediolateral cell column (IML) of the thoracic spinal cord. A. Thoracic cord
laminae. B. Terminal arborizations in IML of a Type I Vo-TS axon. The parent ﬁber
enters the IML dorsally from the lateral funiculus (LF) after sending a branch into Lamina
V and gives rise to medially directed terminal strands (TS). C and D. Schematic drawings
of the terminal strands containing one to six irregularly spaced boutons en passant and
terminal boutons (B). DH, dorsal horn.

21

LF

 

DH

9:20
IML/ /
VH
100Em

 

A

TYPE I EFFERENT

22

«a?

Figure 3: Type II Eﬁ‘erent Axon Tenninal Arbors

Composite schematic drawings (A-D) showing the terminal arborization of PHA-L
labeled Type II VO-TS eﬁ‘erent axons in IML of the thoracic spinal cord. A Thoracic
spinal cord laminae. B. A Type II parent ﬁber traverses the lateral portion of the ventral
horn (VI-I) from the anterior funiculus (AF), enters IML ventrally, and proceeds into the
base of the dorsal horn. \VIthin IML the parent ﬁber gives rise to a collateral which forms
a terminal arborization. C. Terminal arborization in IML. Some terminal strands (TS)
emanate ﬁ'om the parent ﬁber (PF) directly. All terminal strands have irregular spaced
boutons (B) with a terminal bouton at the end of the strand. D. Tenninal arborization in
IML. Most terminal strands arise ﬁom collateral banches with boutons en passant along
their length and terminal boutons at the end of each strand.

23

 

A
TYPE I: EFFERENT

(-PF

 

 

24

Figure 4: Light Micrographs Of Type I and Type II Efferent Axon Terminal Arbors
Photomicrographs (A, B) which document morphological characteristics of Type I

(A) and Type II (B) Vo-TS efferent axons terminating in thoracic IML. These
photomicrographs highlight the terminal strands (TS) and one bouton (B) each.

25

szmmuu—m = mn>._.

 

hzmmmuu—m _ um>h

L»:

26

Figure 5: Electron Micrographs of the Cytology and Synaptic Relations of a Type I
Efferent Axonal Ending

Adjacent sections (A, B) through an ending (E1) of a Type I Vo-TS efferent axon.
This elongated ending has a scalloped contour and forms an asymmetrical to intermediate
synaptic junction (arrows) with a dendritic shaft (D). The ending is completely ensheathed
by astrocytic processes (AP) except at the presynaptic membrane. X 35,308.

Figure 6: Electron Micrographs of the Cytology and Synaptic Relations of a Type I
Eﬁ‘erent Axonal Ending

Adjacent sections (A, B) through an ending (E1) of a Type I Vo-Ts efferent axon.
This ending is elongated with a smooth contour and is ﬁlled with closely packed agranular
spherical synaptic vesicles ( 40—60 nm in diameter) and a few dense core vesicles (dcv; 85-
100 nm in diameter). The ending forms an asymmetrical to intermediate synaptic junction
(arrows) with a dendritic shaft (D). The ending is completely ensheathed by astrocytic
processes except at the presynaptic membrane. X 35,308.

27

 

28

Figure 7 Electron Micrograph of a Type 11 Parent Axon

PHA-L labeled parent ﬁber of a type II Vo-TS efferent axon terminating in
thoracic IML. Parent axons are thinly myelinated with a myelin sheath measuring 0.1 to
1.5um in thickness. X 34,920.

Figure 8: Electron Micrograph of the Cytology and Synaptic Relations of a Type II
Eﬁ‘erent Axonal Ending

In this electron micrograph a dome-shaped ending (E2) of a Type H Vo-Ts
efferent axon forms an intermediate axosomatic synaptic junction (arrow) with a soma (S).
The ending contains agranular spherical ( 40-50 nm in diameter) synaptic vesicles. The
ending is ensheathed by an astrocytic process (AP) except at it’s presynaptic membrane.

X 35,307

Figure 9: Electron Micrographs of the Cytology and Synaptic Relations of Type II
Eﬁ‘erent Axonal Endings

Two adjacent sections through two axonal endings (E2) connected by a terminal
strand (TS) of a Type II Vo-TS eﬁ‘erent axon. The endings, ﬁlled with agranular spherical
synaptic vesicles each form asymmetrical to intermediate synaptic junctions (arrows) with
a large (D1), and a smaller dendritic (D2) shaft. The endings are ensheathed by astrocytic
processes (AP) exept at the presynaptic membrane. X 34,920.

29

 

30

BIBLIOGRAPHY

1. Appel, N. M., and R P. Elde (1988) The interomediolateral cell column of the
thoracic spinal cord is comprised of target-speciﬁc subnuclei: Evidence from
retrograde transport studies and immunohisochemistry. J. Neurosci. 8(5):

1767-1775.
2. Arvidsson, J. and Gobel, S. 1978) Further observations Oftransganglionic
degeneration trig ' primary sensory neurons. Brain Res. 4: 1-12.

3. Arvidsson, I. and Gobel, S. (1981) An HRP study of the central projections of
primary trigeminal neurons which Innervate tooth pulps In the cat. Brain Res.
210: 1- 16.

4. Azerad, 1., Woda, A, and Albe-chsard, D. (1982) Physiological roperties of
neurons in diﬂ‘erent parts of the cat trigeminal sensory comp ex. Brain Res.
246: 7-21.

5. Bowker, R M., L. C. Abbott, and R P. Dilts (1988) Peptidergic neurons in the
nucleus raphe magnus and the nucleus gigantocellularis: Their distributions,
interrelationships, and projections to the spinal cord. Prog. in Brain Res. 77:
95-127.

6. Bruce, L. L., J. G. McHafer and B. E. Stein (1984) Organization of the
. hamster trigeminal complex: Projections to superior colliculus, thalamus,
and spinal cord. Soc. Neurosci. Abstr. 10 :483.

7. Burton, H. and A. D. Loewy (1976) Descending projections ﬁ'om the marginal cell
layer and other regions of the monkey spinal cord. Brain Res. 116 :485491.

8. Burton. H. and A D. Jr. Craig, D. A Poulos, and Molt (1979) Efferent projections
ﬁom temperature-sensitive recordin loci within the marginal zone of the
nucleus caudalis of the spinal trigeminal complex of the cat. J. Comp. Neurol.
183: 753-778.

9. Burton, H., and A. D. Loewy (1977) Projections to the spinal cord ﬁ'om the
medullary somatosensory relay nuclei. J. Comp. Neurol. 173: 773-792.

10. Cabana, T. and G. F. Martin (1984) Developmental sequence in the origin of
descending spinal pathways. Studies using retrograde transport techniques in
the north arnerican opossum (Didelphis virginiana). Dev. Brain Res. 15:247-
263.

ll. Chung, J. M., K. Chung, and R. D. Wurster (1975) Sympathetic preganglionic
neurons of the cat spinal cord: Horseradish peroxidase study. Brain Res. 91:
126-13 1.

12. Clilfer, K. D. and G. J. Geisler, Jr. (1988) PHA-L can be transported anterogradely
through ﬁbers of passage. Brain Res. 485: 185-191.

31

32

13. Contreras, R J., R. M. Beckstead and R Norgren (1982) The central projections of
the trigeminal, facial, glosso pharyngeal and vagus nerves: An autoradiographic
study In the rat. J. Autononuc Ner. Sys. 6: 303-322.

14. Craig, A D., Jr. (1978) Spinal and medullary input to the lateral cervical nucleus. J.
Comp. Neurol. 1812729-744.

15. Crutcher, K. A, A O. Humbertson and G. F. Martin (1978) The origin Ofthe
brainstem-spinal pathways In the north american opossum (Didelphis
vir gInIarIa.) Studies using horseradish peroxidase method. J. Comp. Neurol.
179: 169-194

16. Darian-Smith, I. (1973) The trigeminal system. In: Handbook of Sensory
Physiology. Vol II. A. Iggo, ed. Spnnger-Verlag, Berlin, pp.27l-3 14.

17. Falls, W. M. (1983) Light and EM analysis of identiﬁed trigerninospirrl
projection neurons in rat trigeminal nucleus oralis. Anat. Rec. 205 :55A.

18. Falls, W. M. (1984a) Termination In trigeminal nucleus oralis of ascending
intratrigenrinal axons originating ﬁ'om neurons on the medullary dorsal horn:
An HRP study in the rat employing light and electron microscopy. Brain Res.
290: 136-140.y

19. Falls, W. M. (1983b) A Golgi type II neuron in trigeminal nucleus oralis: A
Golgi study in the rat. Neurosci. Letts. 41:11-17.

20. Falls, W. M. (1984b) Axonal endings terminating on dendrites of identiﬁed large
trigenrinospinal projection neurons in rat trigeminal nucleus oralis. Brain
Res. 324: 1279-1298.

21. Falls, W. M. (1984c) The morphology of neurons in trigeminal nucleus oralis
projecting to the medullary dorsal horn (trigeminal nucleus caudalis): A
retrograde horseradish peroxidase and Golgi study. Neuroscience 13: 1279-
1298.

22. Falls, W. M, R. E. Rice, and J. P. Van Wagner (1985) The dorsomedial portion
of trigeminal nucleus oralis (Vo) In the rat: Cytology and projections to the
cerebellum. Somatosens. Res. 3: 89-1 18.

23. Falls, W. M. .(1986) Morphology and synaptic connections of myelinated

primary axons in the ventrolateral region of the rat trigeminal nucleus
oralis. J. of Comp. Neurol. 244: 96-1 10.

24. Gerfen, CR, and PE. Sawchenko (1984) An anterograde neuroanatomical
tracing method that shows the detailed morphology of neurons, their axons and
terminals: Immunohistological localization of the axonally transported plant
lectin, phaseoulus vulgaris leucoagglutinin (PHA-L). Brain Res. 2902219-238.

25. Greenmarr, P. E. (1996) Principles of manual medicine. 2nd ed. Williams and
Wilkins Baltimore, MD pp. 159-172

26. Greenwood, F. (1973) An electrophysiological study of the central connections for
primary afferent ﬁbers from the dental pulp in the cat. Arch. Oral Biol.
17:701-709.

33

27. Hayashi, H. (1982) Differential terminal distribution of single large cutaneous
afferent ﬁbers in the spinal trigeminal nucleus and in the cervical spinal dorsal
horn. Brain Res. 244:173-177.

28. Hayashi, H., R. Sumino and B. J. Sessle (1984) Functional organization of the
trigeminal subnuclcus interpolaris: Noeiceptive and innocuous afferent inputs,
projections to thalamus cerebellum, and spinal cord, and descending
modulation ﬁ'om periaqueductal gray. J. Neurophysiol. 51:890-905.

29. Jacquin, Mark F. K. Samba, R W. Rhoades, and M. David Egger (1982)
Trigeminal pri afferents project bilaterally to dorsal horn and ipsilaterally to
cerebellum, reticu ar formation, and cuneate, solitary, supratrigeminal and vagus
nuclei. Brain Res. 246:285-291.

30. Jacquirr, M. F., K. Semba, M. D. E er, and R W. Rhoades (1983) Organization
Of HRP-labeled trigeminal man ' ular primary aﬂ’erent neurons in the rat. J.
Comp. Neurol. 215: 397-420.

31. Kuypers, H. G. J. M., and V. A Maisky (1975) Retrograde axonal transport of
horseradish peoxidase from spinal cord to brain stem cell groups in the cat.
Neurosci. Letts. 1: 9-14.

32. Khayyat, G. F. , Yu, Y. J. and King, R. B. (1975) Response patterns to noxious and
non-noxious stimuli in rostral trigeminal nuclei. Brain Res. 97: 47-60.

33. Leong, S. K, J. Y. Sheih and W. C. Wong( 1984)Loca1izing spinal-cord-
projection neurons in adult albino rats. J .Comp. Neurol. 228: 1-17.

34. 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
horseradish peroxidase. J Comp. Neurol. 223: 525-547.

35. Marfurt, C. F. (1981) The central projections of trigeminal primary afferent
neurons in the cat as determined by transganglionic transport of horseradish
peroxidase. J. Comp. Neur01223 : 535-547.

36. Matsushita, M., M. Ikeda, and N. Okado ( 1982) The cells of origin of the
trigeminothalanric, trigeminospinal and trigeminocerebellar projections in the
cat. Neuroscience 7: 1439-1454.

37. Matsushita, M., N. Okado, M. Ikeda and Y. Hosoya (1980) Identiﬁcation of spinal
projecting neurons in the cat trigeminal spinal and mesencephalic nuclei using
the retrograde horseradish peroxidase technique. In : Integrative Control
Functions of the Brain , Vol. III. Ito, M., N. Tsukahara, K. Kubota, and K.
Yagi (eds). Amsterdam: Elsevier North Holland Biomedical Press. pp. 161-163.

38. Matsushita, M., N. Okado, M. Ikeda and Y. Hosoya (1981) Descending projections
form the spinal and mesencephalic nuclei of the trigeminal nerve to the spinal

cord in the cat. A study with the horseradish peroxidase technique. J. Comp.
Neurol. 196: 173-187.

39. Nord, S. G. (1976) Response of neurons in rostral and caudal trigeminal nuclei to
tooth pulp stimulation. Brain Res. Bull. 1: 489-492.

4o

41

42

4

N

44

45

46

47

34

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

. Petras, J. M., and J. F. Cummings (1972) Autonomic neurons in the spinal cord
Of the rhesus monkey: A correlation of the ﬁndings of cytoarchitectonics and
sympathectomy with ﬁber degeneration following dorsal rhizotomy. J.
Comp. Neurol. 146:189-218.

. Petras, J. M., and A I. Faden (1978) The origin of sympathetic preganglionic
neurons in the dog. Brain Res. 144:353-357.

. Ramon y Cajal, S. (1972 reprint) Histologie du Systeme Nerveux dc l'Homme et
des Vertebres. Vol. 1, Institute Ramon y Cajal, Madrid.

. Rowe, M. J. and Sessle, B. J. (1972) Responses of the trigeminal ganglion and
brain stem neurons in the cat to mechanical and thermal stimulation of the
face. Brain Res. 42:367-3 84.

. Ruggiero, D. A,C. A. Ross, and D. J. Reis 1981) medians ﬁ'om the spinal
trigeminal nucleus to the entire length 0 the sp' cord in the rat. Brain Res.
225:225-233.

. Satoh, K. (1979) The origin of reticulospinal ﬁbers in the rat: A HRP study. J.
Hirnforsch. 20: 13 1-332

. Schrarnm, L. J ., J. R. Adair, J. Stribling, and L. Gray (1975) Preganglionic
innervation of the adrenal gland of the rat: A study using horseradish
peroxidase. Exp. Neurol. 49:540-553.

48. Scoﬁeld, B. R. (1990) Uptake of phaseoulus vulgaris leucoagglutinin (PHA-L)

49

50

51

52

53

54

by axons of passage. J.Neurosci. Methods. 35:47-56.

. Sessle, B. J. and Greenwood, L. F. (1976) Inputs to trigeminal brain stem neurons
ﬁ'om facial, oral, tooth pulp and pharyngolaryneal tissue. I. Respnses to
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 subnuclcus oralis, and inﬂuence of
naloxone and subnuclcus caudalis. Pain 10:19-36.

. Shu, S. Y. and G. M. Peterson (1988) Antero ade and retrograde axonal
tranrgport of phaseoulus vulgaris leucoa utinin (PHA-L) from the globus
palli .us to the striatum of the rat. J. Neurosci. Meths. 25: 175-180.

. Smith, L. A, and W. M. Falls (1988) Efferent projections to rat spinal cord from
trigeminal nucleus oralis utilizing the method of PHA-L. Soc. Neurosci. Abstr.
14:697.

 

. Smith, L. A and Falls, W. M. (1993) Descending trigeminospinal
projections underlying manual manipulations of the head. J. Amer.
Osteo. Assoc.94:949.

. Sohn, D. J. and Falls, W. M. (1990) Primary trigeminal axons terminating in the
dorsomedial subdivision of rat nucleus oralis. In Michigan Chapter: Society for
Neuroscience Abstracts; Vol. XXI.

35

55. Tohyarna, M., K. Sakai, M. Touret, D. Salvert, and M. Jouvet (1979) Spinal
mojections from the lower brain stem in the cat as demonstrated by the
rseradish peroxidase technique. 11. Projections from the dorsolateral pontine
tegrnentum and raphe nuclei. Brain Res. 176:215-223.

56. Uchizono, K. (1965) Characteristics of excitatory and inhibitory synapses in the
central nervous system of the cat. Nature (London) 207:642-643.

57. Uchizono, K. (1967) Synaptic organization of the Purkinje cell in the cerebellum of
the cat. Exp. Brain Res. 4:97-113.

58. Westrum, L. E., Candﬁeld, R C., and O'Connor, T. A. (1980) Projections from
dental structures to the brain stem trigeminal complex as shown by
transganglionic transport of horseradish peroxidase. Neurosci. Lett., 20:31-36.

59. Westrum, L. E., R. C. Canﬁeld and T. A O'Connor (1981) Each canine tooth
projects to all brain stem trigeminal nuclei in the cat. Exper. Neurol. 74:
787-799.

60. Wouterlood, F. G., and H. J. Groenewegen (1985) Neuroanatomical tracing by
use of Phaseoulus vrtgfaris leucoagglutinin (PHA-L): Electron Microscopy of
PHA-L ﬁlled neuro somata, dendrites, axons and axon terminals. BraIn
Res. 326:188-191.

     

    

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253