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This is to certify that the

dissertation entitled
MORPHOLOGICAL ANALYSIS OF SUBTHALAMIC NEURONS AND

THE ORGANIZATION OF CORTICOSUBTHALAMIC

PROJECTIONS IN THE RAT BRAIN
presented by

Salman Afsharpour

has been accepted towards fulfillment
of the requirements for

Ph . D. degree in Anatomy

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MORPHOLOGICAL ANALYSIS OF SUBTHALAMIC NEURONS AND
THE ORGANIZATION OF CORTICOSUBTHALAMIC

PROJECTIONS IN THE RAT

/<// - m7

BY

Salman Afsharpour

A DISSERTATION

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

Department of Anatomy

1983

To my dearest mother and mother- and father-in- law.

who made everything possible by their continued support.

To my wife Shukoh, who has devoted her life to

furthering my educational career.

To my daughters Runak and Rujyar, for their patient

support.

ii

ACKNOWLEDGEMENTS

I greatefully thank my adviser, Dr. S.T. Kitai, for
his guidance and tremendous support as well as encouragement
throughout my graduate career. I

I would like to thank the following dear friends, for
their advice and support: Docters W. M. Falls, I. Grofova,
M. R. Park, H. Kita, D. Tanaka, D. A. Steindler, C. J.
Wilson, H. T. Chang, S. Sakai, C. A. Tweedle, H. Imai and P.
Landry.

I am especially in debt to Dr. H. Kita, for his
assistance in writing the dissertation; Dr. W. M. Falls and
Dr. H. T. Chang, for introducing me to the Golgi analysis of
the nervous system.

I wish to thank Dr. M. R. Park for his instruction in
using the computer Which was needed in many aspects of my
dissertation.

I would like to thank Dr. J. P. Donoghue of the
Laboratory of Neurophysiology at NIMH for introducing me to
the science of autoradiographic analysis of the nervous
system.

Finally, I would like to thank the timely
encouragements from my classmates and staff in the
department: Kevin Phelan, Ronald Bradley, Bill Bitzinger,

Carol Meister, Barbara Wheaten.

TABLE OF CONTENTS

List of tables.................................. .........vi
List of figures................................. ........vii
Abstract.............. ........ .................. . ......ix
CHAPTER 1. INTRODUCTION...................................l
A. Gross Anatomy Of Subthalamic Nucleus .......... .1
B. Subthalamic Intrinsic Organization..... .......3
a) Light Microscopic Study Of Nissl
Preparations........................... ...3
b) Light Microscopic Study Of Golgi
Preparations................................5
c) Labeling with Intracellularly Injected
Markers.......................... ..........7
d) Electron Microscopic Studies ........ ... ....8
e) Biochemical, Histochemical and
Immunocytochemical Studies...... . .. ...10
C. Subthalamic Afferent Connections.... .. .....11
a) The Globus Pallidus.............. .... ....12
b) The Cerebral Cortex.............. .........14
c) The Pedunculopontine Nucleus......... ....16
d) The Substantia Nigra.......... ..... ........16
e) The Center Median-Parafascicular Nucleus...17
f) Other Less-studied Subthalamic Afferents...l7
g) Physiology Of Subthalamic Afferents........18
D. Subthalamic Efferent Connections ...... ........19
a) The Pallidum..... .............. ............19
b) The Substantia Nigra. ............. .........?1

'IV

c) The Pedunculopontine Nucleus......
d) Other Less—studied Subthalamic Efferents..
e) Physiology Of Subthalamic Efferents....

E. Research Objectives..

CHAPTER ?.

METHODSOOOOOOI

A. GOlgi StudYOOOOOOOOOOOIO

B. Nissl Study...

C. Autoradiographic Study.

CHAPTER 3.

RESULTS.....

0.0.0.0....

A. Golgi StudYOOOOOOOOOOOOI

B. Nissl Study....

C. Autoradiographic Study...

CHAPTER 4.

A. Golgi Study......

B. Nissl Study....

DISCUSSION........

C. Autoradiographic Study.

CHAPTER 5.

REFERENCES.... ..... ...

CONCLUSION..

O O O O
O O O O
O O I
O O O O O

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.93

..75

..77

..30

.098

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.103

.104

..108

.110

LIST OF TABLES
Table l. The sites, extention and the quantity of
amino acid injections in various cerebral

cortical areaSooooooooooo00000000000000.00000072—73

V‘I

10.

ll.

12.

13.

LIST OF FIGURES

Histogram A, Somata size distribution.................33
Histogram B, number of proximal dendrites.............34
Reconstructions of the representative rat STH neurons
from frontal sections.................................36
Photomicrographs of Golgi impregnated STH neurons

from frontal sections..............................38-4l
Reconstructions of Golgi impregnated rat STH neurons
from sagittal sections................................44
Photomicrograph of Golgi impregnated rat STH neurons
from sagittal section.................................46
Reconstructions of Golgi impregnated rat STH neurons
from frontal sections..............................47-50
Photomicrographs of Golgi impregnated rat STH neurons
from frontal sections..............................Sl-54
Higher magnification photomicrographs of Golgi
impregnated rat STH neurons from frontal sections..55-57
Reconstructions of some Golgi impregnated rat STH
afferents..........................................58—6O
Photomicrographs of STH from Nissl stained

horizontal sections................................62—64
Photomicrographs of STH from Nissl stained frontal
sections...........................................65—67
A low magnification photomicrograph of STH from

Nissl stained sagittal section........................69
Illustration of the site and the extent of the amino

acid injections into the cerebral cortex..............75

vii

14.

15.

16.

17.

18.

19.

Illustration of case #3 (representative of the rostral
part of the medial agranular cortical region)......76—78
Illustration of case #19 (representative of the medial
part of the lateral agranular cortical region).....80-82
Illustration of case #21 (representative of the rostral
part of the lateral agranular cortical region).....R3-85
Illustration of case #24 (representative of the caudal
part of the frontal medial agranular cortical
region)............................................87-89
Autoradiographs illustrating the injection sites

and their extent into the somatosensory, visual

and parietal cortex................................9l-94
Summary diagram of the projections from the agranular

cerebral cortical areas to STH in frontal sections....97

ABSTRACT

MORPHOLOGICAL ANALYSIS OF SUBTHALAMIC NEURONS AND
THE ORGANIZATION OF CORTICOSUBTHALAMIC

PROJECTIONS IN THE RAT

BY

Salman Afsharpour

Neuronal organization of the rat subthalamic nucleus
(STH) was studied using Golgi, Nissl and autoradiographic
techniques. Golgi impregnated STH neurons were analysed in'
sagittal and frontal sections under a light microsc0pe. The
somatic shape was either fusiform, oval, or polygonal. The
cross—sectional areas of the soma (SA) varied between
l40-440umg Some of the cells had a few somatic spines. Two
to six proximal dendrites usually gave rise to tapering
secondary dendrites extending up to 500um in length. These
dendrites were sparsely covered with spines. Statistical
analysis of the somatic size and the number of proximal
dendrites showed unimodal distributions.

Neurons located in the central portion of STH had oval
dendritic fields. Some of these neurons had one or two
dendrites extending beyond the nuclear boundary into the
zona incerta, the lateral hypothalamus or the cerebral
peduncle (CP). Neurons located at the borders of STH had
dendritic fields extending parallel to the borders and were

generally confined within the nucleus except for those

ix

neurons adjacent to CP which had some dendrites extending
into CP.

Two types of afferent fibers were observed entering
STH. One type arose from CP while the other entered STH
from the rostral border after crossing the internal
capsule. .

The cytoarchitectonic characteristics of STH neurons
were studied on 25um thick sections stained with cresyl
violet. The densely packed somata appeared fusiform in
frontal sections (6 to 32um in diameter), and round or
polygonal in horizontal and sagittal sections (10 to 28um in
diameter).

Corticosubthalamic projections were investigated using
autoradiographic tracing techniques. Tritiated amino acids
were injected unilaterally into frontal, parietal and
occipital cerebral cortices. The rostral part of the medial
agranular frontal cortex projected throughout the ventral
two—thirds of the medial half of STH. The lateral agranular
frontal cortex projected to the lateral portion of the
rostral two—thirds of STH. The area between the medial and
the lateral agranular cortex projected mainly to the medial
half of STH. The caudal part of the medial agranular
frontal cortex projected to the dorsolateral part of the
caudal two—thirds of STH. The caudal part of the lateral
agranular cortex projected to the ventral aspect of,the’
middle third of STH. No projection from somatosensory,
visual and association areas to STH were detected.

The results of the present study suggested that 1) The

rat STH is an open nucleus in contrast to other species
(i.e. man, monkey, cat). 2) The rat STH may consist of
only one type of neuron. 3) The somatic size and the shape
of STH neurons are dependent on the plane of section. 4)
The cortico-STH projection is unilateral, arising mainly
from the frontal agranular cortex, and is topographicaly

organized.

xi

CHAPTER 1. INTRODUCTION

Although previous anatomical studies have described
the existence of a number of neuronal types within the
subthalamic nucleus of various species, the morphological
descriptions and classification of these neurons are
inconsistent. Furthermore, although the connections of STH
with other subcortical structures has been intensively
studied, information regarding the topographical
organization of the cerebral cortical projections to this
nucleus is surprisingly lacking. The aims of the present
study were to investigate the morphological characteristics
of subthalamic neurons, and the topographical organization
of the corticosubthalamic projections in the rat at the
light microscopic level. Golgi preparations Were used to
study the neuronal types in the subthalamic nucleus, and
Nissl stained preparations were used to analyze the
cytoarchitectonic features of the nucleus. An
autoradiographic tracer method was used to analyze the
topographical organization of the cortico—subthalamic

projections.

BACKGROUND:

A. Gross Anatomy Of Subthalamic Nucleus

The subthalamic nucleus (STH) was first discerned by
Luys (1865) and was subsequently named "Corpus Luysii" by
Forel (1877) (cited by Yelnik & Percheron, 1979). As

described by Rioch (1930) STH is a lens shaped mass situated

on the dorsomedial aspect of the cerebral peduncule (CP).
Rostrally, it is bounded by the internal capsule (IC);
Caudally, it is separated from the substantia nigra (SN) by
an area consisting mainly of fibers and scattered cells
termed "the accessory nucleus of Luys" (Watanabe & Kawana,
1982). The ventromedial part of the nucleus extends toward
the lateral hypothalamic area (LHA).

Embryologically, STH is considered to arise from a
cell column that extends from the ventral thalamus to the
mammillary nucleus (Shanner, 1930). Kuhlenbeck (1948, 1949)
described the STH as a derivative from the dorsocaudal part
of the lateral hypothalamic cell column. Richer (1965)
described STH as arising from the "subthalamic longitudinal
zone" along with both segments of the pallidum. The anlage
of STH lies caudal to that of both pallidal segments, and is
said to mature before either the globus pallidus or the
substantia nigra (cited by Carpenter et al., 1981).

In humans STH has a volume of approximately 158 mm3
which is about 1500 times the size of rat STH, but its size
relative to the total brain is the same in both species
(Bonin & Shariff, 1951).

Clinico—pathologically, STH is known to play an
important role in motor regulation. Lesions of STH result
in dyskinesia, predominantly a choreoid activity (dancing
movements) or ballism (flinging movements) in the monkey
(Whittier & Mettler, 1949; Carpenter et al., 1950; Carpenter
& Carpenter, 1951; Mettler, 1962; Hammond et al., 1979) and

in man (Martin, 1927; Whittier, 1947: Carpenter, 1955) (see

also literature review by Carpenter & Sutin, 1983). This
dyskinesia is characterized by its violence, persistence and
association with marked hypotonus. The dyskinesia can be
ameliorated and abolished by subsequent stereotaxic lesions
in the medial globus pallidus, the ventral lateral nucleus
of the thalamus, or the motor cortex (Whittier & Mettler,
1949). STH dyskinesia is considered to be a physiological
expression of inhibitory influences Which normally act on
the medial segment of the globus pallidus (Carpenter et al.,
1950). Results from experimental investigations have
suggested that STH is somatotopically organized since
lesions in different parts of the nucleus produced
dyskinesia in different parts of the body (Carpenter &
Whittier, 1950; Carpenter & Carpenter, 1951; Mettler &

Stern, 1962).

B. Subthalamic Intrinsic Organization

a) Light Microscopic Study Of Nissl Preparations

Light microscopic analysis of Nissl stained sections
have revealed that cell bodies of the STH neurons in the
monkey and man are either round, polygonal or fusiform in
shape. The smallest somata were found to be 10 X lOum in
Macaca and 11 X llum in man. The largest were 33 X 6mm in
Macaca and 40 X 9um in man (Yelnik & Percheron, 1979). Both
Luys (1865) and Forel (1877) commented on the masses of
brownish pigment in the cytoplasm of human STH neurons while
such pigment in STH neurons of the rhesus monkey is absent

(cited by Carpenter et al., 1981). Rafols & Fox (1976)

however, have noted the presence of such cytoplasmic
lipofuscin granules in all cell profiles of the principal
STH neurons in the squirrel monkey (Saimiri sciureus) and
pig tail monkey (Macaca menestrina) while in the monkey
(Macaca mulatta) the lipofuscin granules were present in
some nerve cell profiles and absent in others. In Nissl
studies by Whittier & Mettler (1949) in the monkey, STH
neurons appeared to be large with large nuclei that
contained chromatin and a massive nucleolus. Differences in
cell size and concentrations between medial and lateral
parts of the nucleus have also been recognized. Foix &
Nicolesco (1925) were the first to indicate, on Nissl
material in human, that STH neurons look smaller in the
ventral and external parts than dorsal and internal parts of
the nucleus (cited by Yelnik & Percheron, 1979). However,
in subsequent Nissl studies, the opposite observation was
reported by Kodama (1928) in man, and Whittier & Mettler
(1949) in monkey. The latter authors described large cells
within the lateral portions and small cells in medial
portions of STH. Rafols & Fox (1976) localized the smaller
cells of the Macaca in a narrow strip along the ventral
border at the medial end of the nucleus. However, Yelnik &
Percheron (1979) have described STH neurons as having
different sizes and shapes, and being more numerous in the
oral and medial portions than in caudal and lateral portions
of STH. Neurons with different sizes and shapes are
intermingled so that no part of STH contains only one

specific cell size or shape. The latter authors in addition

noted more fusiform appearing somata in sagittal sections
and more round and polygonal appearing ones in frontal

sections.

b) Light Microscopic Studies Of Golgi Preparations

Studies on subthalamic neurons using Golgi techniques
have described a number of neuronal types in several
species. However, the classification and description of
these neurons have remained controversial. In early Golgi
impregnation studies, STH appeared to contain only Golgi
type I neurons in rat and mouse (Kolliker, 1896), in man
(Mirto, l896)and in the kitten (Ramon y Cajal, 1910—1911).
Raman y Cajal was the first to illustrate drawings of four
radiating STH neurons of an 8—day—old kitten. Subsequently,
Golgi impregnated STH neurons of cat and dog similar to
drawings by Cajal were illustrated by Ramon Moliner (1962)
and Leontovich & Zhukova (1963).

Recently, more Golgi studies have been done on STH and
different cell types have been described in the monkey based
on the somatic shape, size and dendritic features. Rafols
& Fox (1976) described two types of principal neurons and
one type of local circuit neuron. Principal neurons were
referred to as either radiating or elongated fusiform types.
Radiating neurons had a few delicate somatic spines and 5
to 8 dendritic trunks that gave rise to branched, tapering
dendrites extending over 400um. Dendrites of these neurons
were thinner than those seen on pallidal or nigral neurons

and occasionally spines were seen on dendritic trunks.

Elongated fusiform neurons were less numerous than those of
the radiating type and were found mainly near the capsule of
the nucleus. They had polar dendrites. Small local circuit
neurons had relatively few long undulating dendrites, but
axons arising from these neurons have not been identified.
In the kitten, Iwahori (1978) has identified three varieties
of Golgi type I STH neurons based on somatic size. All had
polygonal shaped cell bodies. Axons of these neurons were
found to arise from the soma, but occasionally emerged from
a proximal dendrite. Many axon—collaterals were observed to
arise from the parent axons of STH neurons either
intranuclear or extranuclearly coursing in both rostral and
caudal directions. Iwahori classified afferent fibers
entering STH as 1) ascending afferents, the axon-collaterals
arising from ascending fibers in CP; 2) descending
afferents, the axon—collaterals of descending fibers in CP;
and 3) afferents from Meynert's commissure Which crosses CP.
Yelnik & Percheron (1979) analyzed their Golgi data of STH
neurons by means of both algebraic and geometric statistical
parameters, and suggested that STH is a homogeneous closed
nucleus which consists of only Golgi type I (projection)
neurons that are morphologically identical in the cat,
monkey and man. The cell bodies of these neurons were shown
to be ovoid in shape giving them a fusiform appearance When
observed parallel to their long axis, and round or polygonal
When viewed in other directions. On the average, seven
proximal dendrites were observed originating from various

points of STH perikarya: they branched out in successive

bifurcations giving rise to a mean of 27 dendritic tips.
Axon—collaterals of STH neurons were also observed to course

in medial and lateral directions.

c) Labeling With Intracellularly Injected Markers

The recently developed technique of intracellular
labeling with either the enzyme HRP or fluorescent markers
have enabled direct analysis of the morphology of neurons
Which have been physiologically characterized through
intracellular recording techniques (Kitai et al., 1976).
Intracellular recordings in the rat (Kitai & Deniau, 1981)
has demonstrated that Golgi type I STH neurons were
monosynaptically excited by the cerebral cortex. On the
other hand, STH neurons received inhibitory inputs from the
globus pallidus (Kita et al., 1983).

Light microscopic analysis of HRP labeled neurons has
confirmed and extended previous Golgi studies (Iwahori,
1978, in the kitten; Yelnik & Percheron, 1979, in the
monkey) concerning the extent of the elaborate axonal
collateral arborizations of STH Golgi type I neurons (Kita
et al., 1983). Branches of the main axons were traced into
the entOpeduncular nucleus, the globus pallidus and the
substantia nigra. Kita et a1. (1983) distinguished two
types of STH neurons in the rat based on axonal branching
patterns. 1) STH neurons with intranuclear branching axons.
These neurons were characterized by numerous dendritic
arborization and axons which gave rise to collaterals before

leaving the confines of the nucleus. 2) STH neurons without

 

intranuclear branching axons. This group of STH neurons had

less dendritic branching than the other group.

d) Electron Microscopic Studies

Rafols & Fox (1976) recognized two different STH
neurons in their electron microscopic investigation of
normal material in the monkey. Both of their principal
neurons, radiating and elongated fusiform Golgi type I, were
observed to have the same ultrastructural morphology of
cytoplasmic and nuclear contents. The Golgi type I STH
neurons in the monkey, were frequently characterized by
cytoplasmic lipofuscin granules. The few Golgi type II
(local circuit neurons) neurons were characterized by
funnel-shaped protrusions of the cell bodies at the base of
the primary dendrites, and also a large quantity of free
polyribosomes, profiles of the Golgi apparatus were located
at the base of some dendrites and in the rim of the
cytoplasm. The mitochodria appeared to have a darker
matrix, with slightly thinner cristae than the mitochondria
in the Golgi type I neurons. However, only one type of STH
neuron was observed in the rat (Chang et al., 1983). The
somata were found to be often closely apposed to each other
without any intervening glial processes. Membrane
appositions were also found between some somata and
dendrites as well as dendrites and dendrites. The somata
size were observed to range between 10 and 25um in diameter
and all somata were characterized by an abundance of

organelles. Some of the somata had cilia. The nuclei had

deeply invaginated membranes and pale nucleoplasm with
little heterochromatin. Most of the STH neurons had
filamantous nuclear rods.

Electron microscopic studies have revealed that
several types of terminals synapse on STH neurons.
Following pallidal lesions in the cat (Nakamura & Sutin,
1972; Romansky et al., 1979), in the rat (Kooy et al.,
1981), and in the baboon (Usunoff et al., 1982), degenerated
terminals were found to be associated with symmetrical
synapses formed with large dendrites, dendritic spines,
somata and somatic spines. These terminals were large in
diameter and contained large-sized elliptical vesicles.
Another type of terminal was demonstrated after cerebral
cortical lesions (Romansky et al., 1980). The degenerated
terminals were observed to be associated with asymmetrical
synapses mainly on thin dendrites and their spines. These
terminals were small in diameter and contained medium—sized
round vesicles. In an recent electron microscopic study on
rat STH normal material Chang et al. (1983) described at
least two types of terminals similar to those reported by
Nakamura & Sutin (1972) and Romansky et a1. (1979, 1980)
from lesioned material. However, Hassler et al. (1982) in
an electron microscopic study of the synaptic organization
of the baboon STH, described nine different types of
synapses. Type I (F) was the most frequently encountered
and was involved either in axo-dendritic or axo-somatic
symmetrical synapses. This type was characterized by

scattered flat vesicles and many large mitochondria. The

10

terminals were either elongated, tuble shaped (type IF
enpassent) or spindle shaped (type IFa). The type IF
terminals have been shown to degenerate after pallidal
lesions (Usunoff et al., 1982). Type IIF was a smaller
terminal with flattened vesicles, which showed a tendency of
clustering together. Type III (elongated), IV (star shaped)
and V (oval shaped) all contained small, round vesicles, and
were associated with asymmetrical synapses formed with
dendritic terminals or spines. Type VI (SO) terminals which
contained larger, pleomorphic vesicles made synapses on
dendritic terminals. Type VII (LO) showed a looser
arrangement of vesicles intermingled with more dense core
vesicles. Type VIII was a dendritic terminal with loosely
arranged, flat vesicles and was in contact either with a
type VI or a type VII terminal. Type IX was an axo-spinous
microsynaptic one, which partly degenerated after
contralateral pallidal lesion. The origin of these

terminals except type I (F) have not yet been examined.

e) Biochemical, Histochemical and Immunocytochemical

Studies

Biochemical and histofluorescent studies have revealed
that STH contains dOpamine (DA) receptors, DA—containing
axons as well as catecholaminergic cell bodies. In a study
by Brown & Wolfson (1978) using [14C]deoxyglucose as a
metabolic tracer in rats, it was shown that a DA agonist,
apomorphine, markedly increased glucose utilization in STH.

Based on these findings they suggested a direct action of

11

apomorphine on DA receptors in STH. In a subsequent study
by the same authors (Brown et al., 1979),
catecholamine-containing axons and varicosities were
observed in most parts of STH. However, DA-containing cell
bodies were nOt observed in rat STH, although they have been
described in the caudal part of STH in the cat, using the
Falck—Hillarp histofluorescent technique (Meiback & Katzman,
1979). The latter authors have also found DA-containing
dendrites and terminals in the cat STH.

In an immunofluorescence study, the rat STH was found
among many other nuclei and areas of the central nervous
system to be densely innervated by serotonin nerve terminals
in its caudal portion (Steinbusch, 1981). In the cat, Nauta
& Cuenod (1982) have autoradiographically demonstrated
GABA—containing STH cell bodies following pallidal injection
with tritiated GABA. However, they did not observe any STH
neuron labeled after tritiated D-asparate and tritiated

serotonin injections into the pallidal complex.

C. Subthalamic Afferent Connections

Although it is difficult to determine the connections
of this nucleus because of its location in the brain and
relatively small size, different techniques have been used
to determine the afferent connections of STH in various
species. The main afferents to STH arise from the globus
pallidus and the cerebral cortex. Some afferents also arise
from the pedunculopontine nucleus, the center

median—parafascicular nucleus, and the substantia nigra.

12

(for more detailed review on the subthalamic connections,

see Grybiel & Ragsdale, 1979: Carpenter & Sutin, 1983).

a) The Globus Pallidus

There is general agreement in the literature that the
major afferents to STH arise from the ipsilateral lateral
pallidal segment (LPS), (the globus pallidus in
subprimates). This has been demonstrated in the pig
(Shanner, 1936) using a sensitive silver impregnation
technique; in the monkey (Ranson, 1939; Ranson et al., 1941:
Whittier & Mettler, 1949), in the cat (Fonnum et al., 1978),
and in the rat (Kooy et al., 1981) using degeneration
techniques: in the monkey (Kim et al., 1976; Nauta, 1979)
using autoradiographic tracer techniques; in the monkey and
the cat (Rinvik et al., 1979) using retrograde aXonal
transport of horseradish peroxidase (HRP); and in the rat
Carpenter & Fibiger (1978), and in the monkey (Carpenter et
al., 1981) using combined anterograde and retrograde axonal
transport techniques. Carpenter et a1. (1968) and Rinvik et
al. (1979) using degeneration and HRP techniques in the
monkey and the cat, have shown some fibers arising from the
internal pallidal segment (the equivalent of the
entopeduncular nucleus in subprimates) and terminating on
the most caudal and medial parts of STH. However, Carpenter
et a1. (1981) and Kim et a1. (1976) have noted no projection
from the internal pallidal segment to STH. The largest
number of lateral pallidal neurons projecting to STH lie in

the rostral and central divisions of this segment, however

13

fibers are also derived from the cells in the caudal
division of the LPS. Both the degenerating and the
autoradiographic studies in the monkey indicated that
pallido-STH projections have a rostrocaudal organization
(Carpenter et al.,l968; Carpenter et al., 1981, 1981).
Neurons in the rostral two-thirds of the LPS project to the
rostral two-thirds of STH, and neurons in the caudal
division of the LPS project to the caudal third of STH.

Only the medial and caudal pole of STH appears to be void of
LPS projections (Carpenter et al., 1981). The topographical
arrangement of pallido—STH projections was also shown in the
monkey by Nauta & Mettler (1966) and in the cat by.Grofova
(1969). McBride & Larsen (1980) described a mediolateral
laminar origin of pallido—STH projections. Their
observations suggested that neurons in lateral lamina of the
globus pallidus projected to the lateral portion of STH and
that neurons in medial lamina projected to medial parts of
STH. A study on the pattern of distribution of the
pallido—STH projections in normal and experimental animals
(i.e. lesioning globus pallidus) revealed that the pallido-
STH fibers terminated mainly on dendrites. No particular
part of STH was found to receive a specific type of synapse.
Overall 80.3% of the synapses were axodendritic, 12.3% were
axoaxonic, and 7.4% made axosomatic contacts. No axoaxonic
degenerated terminals were seen after pallidal lesions
(Nakamura & Sutin, 1972). Synaptic vesicles in terminal
boutons varied in shape within the same terminal. Dense

core vesicles were evident in about 20% of the terminals.

14

Investigations by Romansky et a1. (1980) on pallido—STH
synaptic relationships, using the degeneration methods,
revealed that the pallidal inputs terminated on perikarya of
large STH neurons, on proximal dendrites, and rarely on
dendritic spines. On the other hand, they did not observe
any degenerated terminals synapsing on the perikarya of

small STH neurons or the other vesicle—containing profiles.

b) The Cerebral Cortex

The projections from the cerebral cortex to STH were
generally denied in early studies using fiber degeneration
methods in monkey (Levin, 1936, 1949; Verhaart & Kennard,
1940). However, Meyer (1949) in man and Mettler (1945);
Devito & Smith (1964); Petras (1965, 1969) in monkey, dog
and cat and Auer (1956) in cat, using degeneration methods
demonstrated projections from various motor cortical areas
to STH. Siegel et a1. (1971) also found some projections
from the temporal cortex to STH in the cat. Studies by
Kunzle & Akert (1977) and Kunzle (1978) using the
autoradiographic tracing techniques in the monkey, have
found some projections from the area 8 (frontal eye field)
to ipsilateral STH which had been previously denied by
Astruc (1971) using fiber degeneration methods in the same
species. An autoradiographic study by Hartmann Von Monakow
et a1. (1978) demonstrated that the corticosubthalamic
fibers Were somatotOpically organized in the monkey. The
frontal motor cortex, area 4, was shown to project

ipsilaterally to the lateral and dorsal parts of STH, and

15

this projection seemed to display an orderly arrangement
with the face area lying laterally and the leg area
medially. The projection of area 6 of the frontal motor
cortex was relatively circumscribed within the intermediate
portion of STH. Area 8 of the prefrontal cortex projected
to the ventral part of STH, and area 9 ofthe prefrontal
cortex projected to the middle part of the ventral portion
of the nucleus. Confirming previous findings from a
degeneration study by Petras (1965, 1969) and an
autoradiographic study by Kunzle & Akert (1977), Hartman Von
Monakow et al. (1978) also found no projection to STH
following tritiated amino acid injeCtions into various parts
of the post central gyrus. Rinvik et a1. (1979) found some
retrogradely labeled cerebral cortical cell bodies following
HRP injections into the ipsilateral STH in the cat and
monkey. A recent electrophysiological study by Kitai &
Deniau (1981), using a combined technique of intracellular
recording and intracellular labeling of neurons with
horseradish peroxidase, demonstrated that cerebral cortical
neurons exert monosynaptic excitatory actions on STH
projection neurons. Electron microscopic studies, in the
cat, indicate that cortical influences upon STH neurons are
mediated by thin myelinated and unmyelinated axons that
terminate in small round boutons on small dendrites and
spines (Romansky et a1. ,1979).

From previous anatomical studies of the cortico—STH
projection, it is clear that there has been no report

concerning the organization of this projection in the rat,

16

while the majority of physiological and anatomical data on
the function of the basal ganglia and its related nuclei
(i.e STH) have been obtained in the rat. Thus, the data
from the present investigation could be correlated with
physiological data obtained in previous studies and should
contribute to a better understanding of the role of STH in

basal ganglia function.

c) The Pedunculopontine Nucleus

Studies utilizing' both retrograde HRP trasport and
autoradiographic tracing techniques suggested that only a
small number of scattered peduncuIOpontinal afferents end in
STH. This has been demonstrated in cat (Nomura et al.,
1980; Rinvik et al., 1979) and in monkey (Carpenter et al.,
1981; Rinvik et al., 1979). Tritiated amino acid
injections in pedunculopontine nucleus produced labeling in
only the ventromedial part of rat STH (Veazey et al., 1980).
However, the synaptic relationships and the physiology of

this input to STH has not been investigated.

d) The Substantia Nigra

Although SN has been demonstrated to receive
projections from STH in various species (see Subthalamic
efferent to SN in following pages), there is little evidence
that this connection is reciprocal. No STH afferents were
observed to arise from either part of SN in degeneration
studies in monkey, cat and rat (Nauta & Mettler, 1964;

Carpenter et al., 1964; Carpenter & Peter, 1972; Faull &

17

Carman, 1968) as well as autoradiographic studies in monkey
(Carpenter et al., 1975; Carpenter et al., 1976).
Physiological data in the rat also did not support the
existence of a nigro-STH projection (Hammond et al., 1978).
Afifi & Kaelber (1965) in a degeneration study in the cat,
and Rinvik et a1. (1979) in a retrograde HRP axonal
transport study in the cat and the monkey, are the only
authors to report of nigral projections to STH, although
nigral efferents may traverse or bypass STH (Deniau et al.,
1980: Carpenter et al., 1976; Carpenter et al., 1981). The
projections they found were not limited to any specific part

of STH (Afifi & Kaelber, 1965).

e) The Centre Median—parafascicular Nucleus

The projection from the centre median—parafascicular
nucleus was first described by Sugimoto et a1. (1982) using
both anterograde and retrograde axoplasmic transport
techniques in the cat and the rat. Tritiated amino acid and
HRP injections in the centre median-parafascicular complex
revealed dense labeling in the ipsilateral STH, over its
rostral one-third portion. A few scattered centre
median-parafascicular neurons were retrogradely labeled

following HRP injection into the cat STH.

f) Other Less-studied Subthalamic Afferents
Studies based on retrograde HRP axonal transport
techniques have described various other STH afferent areas,

including the locus coeruleus (Rinvik et al., 1979;

18

Carpenter et al., 1981), dorsal raphe, the hypothalamus and
the central nucleus of amygdala (Rinvik et al., 1979).
However, since these pathways have not been confirmed by
further studies using other anatomical techniques (i.e

autoradiography), their true nature remains unclear.

g) Physiology Of Subthalamic Afferents

Previous electrophysiological studies (Frigyesi &
Rabin, 1971, in the cat; Ohye & Feger, 1976, in the monkey),
have indicated that striatal stimulation induces excitatory
responses in STH neurons. However, in a recent
intracellular recording study by Kita et a1. (1983) of rat
STH neurons after decortication globus pallidus stimulations
induced only short latency monosynaptic IPSPs. This result
suggested that the excitations reported in earlier studies
were most likely due to the activation of cortico-STH fibers
coursing through the caudate-putamen and the pallidum. In
an electrophysiological study in the cat by Tsubokawa &
Sutin (1972), it was found that most STH neurons which
showed spontaneous unit activities were inhibited by the
stimulation of the globus pallidus or the ventral tegmental
area of Tsai.

The neurotransmitter of the pallidosubthalamic pathway
was biochemically demonstrated to be gamma-amino butyric acid
(GABA) (Fonnum et al., 1978). Evidence from physiological
data have supported the above finding that GABA might be the
neurotransmitter of the pallido—STH pathway (Rouzaire—Dubois

et al., 1980). However, Kooy et a1. (1981) have denied the

19

possibility of either GABA or acetylcholine as
neurotransmitters in pallido-STH fibers using histochemical
techniques. Thus, the nature of the neurotransmitter of
pallido-STH fibers is still controversial.

I'The cerebral cortical activity on STH neurons was
studied by Kitai & Deniau (1981), using a combined technique
of intracellular recording and labeling with HRP in the rat.
They demonstrated that cerebral cortical neurons exert
monosynaptic excitatory actions on STH projection
neurons.

No physiological studies concerning the effects of the
substantia nigra, the pedunculopontine, the centre
median-parafascicular nucleus and other sparse STH afferents

were carried out.

D. Subthalamic Efferent Connections

The major STH efferent targets are the pallidum, the
substantia nigra, and to a lesser extent the
pedunculopontine nucleus, the cerebral cortex and other

nuclei.

a) The Pallidum

Subthalamic projections to the medial pallidal segment
(MPS) were first described by Kolliker (1896). A subsequent
study by Mirto (1896) confirmed this projection and also
postulated that STH projects to both pallidal segments using

a Golgi method in human material. Other studies also

20

indicated that STH projects to both segments of the
pallidum. This has been demonstrated in the monkey (Glees &
Wall, 1946} Whittier & Mettler, 1949; Carpenter et al.,
1967) using degeneration techniques, in the monkey (Devito
et al., 1980) using retrograde HRP tracing teChnique, in the
monkey (Nauta & Cole, 1974, 1978); in the rat (Ricardo,
1980); and in the monkey and the rat (Carpenter et al.,
1981) using autoradiographic tracing techniques, and in the
rat (Deniau et al., 1978; Kita et al., 1983) using
intracellular recording and labeling with HRP. Axons of STH
neurons were found to send collaterals to the entopeduncular
'nucleus, the globus pallidus and the substantia nigra (Kita
et al., 1982). Autoradiographic studies in the monkey
demonstrated the massive nature of the STH-pallidal
projection, and indicated that terminals end in arrays
parallel to the medullary lamina suggesting a topographical
organization (Nauta & Cole, 1978; Carpenter et al., 1981).
Although profuse terminations were found in both pallidal
segments, the number of arrays and the silver grain density
were much greater in the medial pallidal segment (Carpenter
et al., 1981). In the monkey, rostromedial parts of STH
were found to project to the rostral and the medial portions
of LPS, while lateral parts of STH projected to the central
division of LPS. In the cat, STH-pallidal projections cover
both the entopeduncular nucleus and the globus pallidus, but
did not exhibit a laminar pattern (Nauta & Cole, 1978). The
STH projection to the cat entopeduncular nucleus was also

greater than that to the globus pallidus. Although the MPS

21

receives a profuse projection from STH, the connections
between STH and pallidum in the primate are not totally
reciprocal (Carpenter et al., 1981). On the other hand, the
interconnections between STH and LPS appear to be reciprocal
and organized topographically in a rostrocaudal sequence

(see also Perkins & Stone, 1980).

b) The Substantia Nigra

Subthalamonigral projections have long been suggested,
(Kolliker, 1896). In subsequent experimental studies using
degeneration techniques several authors have described
STH-SN projections in various species (Morgan, 1928; Glees &
Wall,l946; Whittier & Mettler, 1949). Injection of HRP in
various parts of SN revealed labeling of cells in STH
(Kanazawa et al., 1976). The latter authors found that the
largest number of HRP positive cells in STH were associated
with HRP injected predominantly into the pars compacta.
However, most of the evidence suggests that STH-SN
projections are confined mainly to the pars reticulata
(Nauta & Cole, 1978; Deniau et al., 1978; Carpenter et al.,
~1981). Antidromic and orthodromic stimulations of STH-SN
fibers support the view that these projections end mainly on
cells of the pars reticulata (Hammond et al., 1978). Nauta
& Cole (1978), in their autoradiographic study of STH I
efferent fibers, noted that the labeled fibers converged on
the medial part of STH and entered SN directly from the
dorsal side, or via fibers interposed between CP and SN.

Anatomical and physiological evidence in the rat indicated

22

that a single STH neuron supplied axonal collaterals to both
the pallidum and the SN (Deniau et al., 1978; Hammond et

al., 1978; Kooy et al., 1980; Kita et al., 1983). Recent
data from the anterograde and retrograde axoplasmic transport
studies (Ricardo, 1980; McBride & Larsen, 1980; Carpenter et
al., 1981; Carpenter et al., 1981) have supported the

existence of STH-SN projections.

c) The Pedunculopontine Nucleus

A subthalamo—pedunculopontine fiber pathway was
suggested by Woodburn (1946). Both retrograde and
.anterograde axoplasmic transport studies in the monkey and
the cat suggested that PPN may receive relatively small
projections from STH (Nauta & Cole, 1974, 1978; McBride &
Larsen, 1980; Numora et al., 1980; Jackson & Crossman,
1980). Based on their autoradiographical data in the cat
and the monkey, Nauta & Cole (1978) concluded that the
lateral part of STH projects to the pars compacta of PPN.
This projection was confined to both dorsal and ventral
aspects of the brachium conjuctivum at the rostrocaudal
level of the trochlear nucleus. Using a combined retrograde
HRP transport and autoradiograpgic tracing techniques, in
the rat, Jackson & Crossman (1980) noted an appreciable
projection from STH to PPN. The number of cells labeled in
STH by retrograde HRP transport was heavier than the number

of labeled cells in entopeduncular nucleus.

d) Other Less-studied Subthalamic Efferents

23

Studies based on retrograde HRP axonal transport
techniques have described a direct projection from STH to
the cerebral cortex in the rat (Jackson & Crossman, 1981).
The retrogradely labeled STH neurons were located only on
the ipsilateral side and were restricted to the lateral half
of the nucleus. A few STH neurons were labeled from HRP
injection in the rat insular cortex (Afsharpour et al.,
unpublished). These STH neurons were confined to the
lateral, but more dorsal aspect of STH. Nauta & Cole
(1978), in an autoradiographic study of STH efferents, noted
sparse projections to the ipsilateral putamen, the ventralis
lateralis and the ventralis anterior complex of the
thalamus, and the midbrain. Although certain studies
suggested that STH influences the excitability of the
midbrain reticular formation (Adey et al., 1959; Shibazaki
et al., 1980), there is little information regarding the
pathways from STH to this region.

From previous studies on the connections of the STH
with the motor cortex, basal ganglia related nuclei (e.g.
pallidal complex, substantia nigra), the peduncu10pontine
nucleus, the locus coeruleus, and the centre median
parafascicular nucleus, one can speculate‘that the STH most
likely integrates these inputs and subsequently conveys this
information to the pallidum and the substantia nigra. Since
the output of the basal ganglia appears to be through either
the pallidal complex or the substantia nigra, the control
exerted by STH over basal ganglia function may be of a

powerful nature. In view of this, it is not surprising that

24

lesions of STH results in the most extreme of movement
disorders (hemiballism)—a consequence of basal ganglia

dysfunction.

e) Physiology Of Subthalamic Efferents

Data from the physiological study of Yoshida (1971),
suggests that STH may be the origin of the entopeduncular
inhibition in the cat. Following STH stimulation,
suppression of activity was detected in mOSt entopeduncular
neurons projecting to both the lateral habenula (73%) and
the ventralis anterior of the thalamus (15%) (Larsen &
Sutin, 1978) (see also Perkins & Stone, 1980). In the rat,
STH stimulation produced three types of responses in the
entopeduncular nucleus: 1) a short latency inhibitory
response, 2) a short latency activation and 3) long latency
inhibitory responses (Shibazaki et al., 1980). These
authors suggested that STH neurons inhibit entopeduncular
neurons projecting to both the lateral habenula and the
pedunculopontine nucleus. In an attempt to determine the
transmitter or transmitters involved in the
subthalamopallidal pathway, Nauta & Cuenod (1982), using an
autoradiographic technique, have suggested that the
neurotransmitter of this pathway is mainly GABA. The
possibility that the neurotransmitter in the
subthalamopallidal pathway of glycine origin has been
suggested by Yoshida (1974).

The response of SN neurons to STH projecting fibers was

studied by Hammond et a1. (1978). They demonstrated

25

excitatory responses of SN neurons in both pars compacta and

pars reticulata following STH stimulation in the rat.

E. Research Objectives

Although the rat has been a widely used animal in
recent experimental studies of the basal ganglia and its
related nuclei, there have been very few studies concerned
with the intrinsic anatomical organization of the rat STH.
For example, there have been no studies concerned with the
classification of cell types or the organization of afferent
inputs such as the cortico-STH fibers. Thus, the
elucidation of the morphology of rat STH neurons, the
cytoarchitectural features of the nucleus and the
relationShip of STH with various cerebral cortical areas at
the light microscopic level should serve to clarify some of
the controversies with respect to the anatomical

organization of STH.

The Specific Aims Of This Study Are:

1) To perform a detailed light microscopic analysis
of morphological and cytoarchitectural features of rat STH
neurons using classical Golgi impregnation techniques and
Nissl staining procedures.

2) To determine the tOpographical organization of
the cerebral cortical projections to the subthalamiC‘nucleus
using the tritiated amino acid autoradiographic tracing

technique.

26

CHAPTER 2. METHODS

A. Golgi Study

Aproximately 200 Sprague-Dawley rats (5-75 days old)
weighing 5-400 grams were used in this study. The rats were
anesthetized with intraperitoneal administrations of
Avertine (0.015ml/gm body weight) for neWborn and Nembutal
(50mg/kgm body weight) for adults. The animals were
perfused intraventricularly with saline (0.9% Nacl) followed
by a fixative (e.g. 3% glutaraldehyde in 0.15M phosphate
buffer; 2% glutaraldehyde with 2% paraformaldehyde in 0.15M
phosphate buffer(pH 7.3); 10% formaldehyde in saline; or
chloral hydrate). Dissected brains were post-fixed for
12-72 hours. Blocks 3—5mm thick which contained STH were
processed for the Golgi impregnating methods(e.g. rapid
Golgi "Valverde, 1970"; Golgi Kopsch "Gobel, 1978a"; Chloral
hydrate "Moliner's modification, 1957" and mixing rapid
Golgi with Golgi KOpsch method). Frontal or sagittal
80—120um thick sections were cut using an Oxford Vibratome.
The serial sections were dehydrated in two changes of 100%
ethanol for 10 minutes, cleared in two changes of fresh
Xylene for 5 minutes each, mounted on glass slides with a
synthetic neutral medium (Protex or Histoclad), and then
analysed by light microscopy.

Well impregnated STH neurons were selected and
detailed tracings of these elements were made using an
Olympus microscope and a drawing tube. They were also

photomicrographed using a Leitz Orthoplan microscope.

27

The somatic cross—sectional areas were measured from
previously drawn neurons with the aid of a graphic analyzer
attached to a PDP—ll computer. The existence of somatic
spines and the density of dendritic spines and dendritic
appendages were noted. The number of primary dendrites
and the branching pattern forming the dendritic field were
analysed. Any correlation between the location of the

neurons in STH and their dendritic shape was also noted.

B. Nissl Study

The cytoarchitectural characteristics of rat STH were
investigated on 25 to 30nm thick sections cut in frontal,
sagittal and horizontal plans. Sections were first
dehydrated in graded alcohols (70%, 95%, 100%), defatted in
fresh Xylene, rehydrated in graded alcohols (100%, 95%, 70%).
rinsed in distilled water and then stained with cresyl
violet. The sections were dehydrated, cleared in fresh
Xylene and cover slipped. Selected samples from frontal,
sagittal and horizontal planes were photographed using a
Leitz Orthoplan microscope. The cytoarchitectonic features

of STH in different planes of sections were then analyzed.

C. Autoradiographic Study
Twenty male Sprague-Dawley rats weighing 230-4889 were
anesthetized with sodium pentobarbital (Nembutal, SOmg/kg

i.p.). A mixture (1:1) of two amino acids of the following

28

were stereotaxically injected into the various agranular and
granular regions of the frontal, parietal and occipital
cerebral cortex: 3H-Proline(L-[5—3HJ-Proline, specific
activity 54 ci/mmole), 3H-Leucine (L-[3,4-3H(n)]—Leucine,
specific activity 58 ci/mmole and 3H—Lysine
(L-[4,5-3H(n)-Lysine, specific activity 10 to 51 ci/mmole)
(from New England Nuclear). The original solutions of
tritiated amino acids were first evaporated then diluted to
concentrations of 15 to 20uci/ul with 0.9% saline.
Injections of 0.25ul to 0.4ul of this concentration were
carried out by a Hamilton 1 microliter syringe driven by a
micromanipulator over a period of 15 to 30 minutes. The
needle was left for 10 to 15 minutes at the injection sites
after the end of the injections. Table 1 lists the
injection areas, injected radioactive amino acid quantities,
concentrations and survival times following the injections.
After a survival times of either 4 or 7 days, the animals
were deeply anesthetized and perfused with 0.9% saline,
followed by either 10% formaldehyde in 0.9% saline or 4%
formaldehyde in phosphate-buffered saline (pH 7.2). Brains
were dissected and post fixed in 10% formaldehyde with 30%
sucrose for 12 to 24 hours. Frozen sections 25 to 30um in
thickness were cut in the frontal (15 cases), sagittal (2
cases) or horizontal planes (3 cases). Two sets of
alternating serial sections were mounted on subbed glass
slides, air dried, dehydrated through graded alcohol,
defatted by fresh xylene, rehydrated, and then air dried.

The slides were dipped in Kodak NTB2 nuclear emulsion

'29
diluted 1:1 with distilled water. The individual slides
were allowed to dry 3 hours or overnight in a humid
atmosphere to prevent emulsion cracking. They were stored
in light-tight plastic slide boxes containing a small amount
of Drierite, and exposed for 6 to 15 weeks at 4 degrees
centigrade . The properlyexposed slides were developed with
full strength D-l9 (Kodak) at 18 to 20 degrees centigrade for
2 to 5 minutes, washed in tap water for 30 seconds, fixed in
rapid fix for 5 minutes and washed in tap water for l to 3
hours. The sections were subsequently counterstained with
cresyl violet and examined with both light and dark—field
microscopy. Micrographs were taken by a light and
dark—field microscope, Wild—M400. Camera lucida drawings
from serial sections were also made using an Olympus
microscope. Cerebral cortical areas were identified
according to the cytoarchitectonic observations made from
Nissl stained sections in frontal and sagittal planes
(Donoghue & Wise, 1982; Hall & Lindholm, 1974). Subcortical
nuclear regions were identified according to the atlas of

Pellegrino et a1. (1979).

30

CHAPTER 3. RESULTS

A. Golgi Study

In the present Golgi study the quantity and quality of
impregnation of neurons in the rat STH varied considerably
from block to block of brain tissue as well as from method
to method. The Goli Kopsch method gave the majority of the
present results with the rest from a mixed Golgi
Kopsch-rapid Golgi technique. The fixative also influenced
the amount of impregnation. Three percent glutaraldehyde
appeared to be the best for light microsc0pic Golgi
impregnation studies. Although the Golgi methods are
undoubtedly capricious, and the small size of the rat STH
which is enwrapped by bundles of passing fibers make it
difficult for the chemicals to penetrate into the nucleus,
we have succeeded by staining enough neurons which stand out
sharply on a clean background. ‘Some double impregnated
sections that were stained with the mixed Golgi method gave
a better STH outline, thus sharply delineating it from the
structures bordering it.

The rat STH is a disk shaped nucleus that lays
obliquely on the dorsomedial surface of CP. Two roughly
equidistance axes (l—l.5mm long) of STH are directed
rostrocaudally and dorsolateral-ventromedially. The
intermediate portion is the thickest part (250-270um width)
of the nucleus (Fig.5 and 7). The STH is separated
rostrally from the entopeduncular nucleus by the internal

capsule; caudally, it is separated from the substantia nigra

31

by an area consisting mainly of fibers with scattered cells.
The lateral part of the posterior hypothalamus borders STH
ventromedially (Fig.5 and 7).

In the present study the distributions of the somatic
cross-sectional areas (SA) and the number of proximal
dendrites were statistically analysed. The results showed a
unimodal distribution of each parameter (Fig.1) and
indicated that rat STH consists of only one type of neuron.
These neurons are distributed rather randomly within the
nucleus. Some dendrites of almost all neurons located at
the lateral border of STH cross over this border into CP (H
in Fig.4 and B, J, K, M in Fig.6).. Some dendrites of
centrally located STH neurons spread out beyond the nucleus
either dorsomedially into 21 and/or ventrolaterally into CP
(C, G in Fig.4 and E'in Fig.6) or some of the dendrites
cross over the dorsal or the dorsomedial boundaries of STH
to extend into ZI only (B, E in Fig 4). Other dendrites of
these neurons spread out beyond the ventromedial border of
STH into the lateral hypothalamic area (C in Fig.6).
Neurons similar to STH neurons are also observed embedded
within the fibers of CP (D in Fig.4). None of the neurons
which are located at the dorsomedial, medial or the
ventromedial borders of STH had dendrites Which cross these
borders (F in Fig.6).

In the frontal or sagittal planes, three different
varieties of neurons were distinguishable on the basis of
the origin and the number of proximal dendrites. One

variety possessed three to five proximal dendrites and were

32

Figure l.
Histograms: A, Somata size distribution of 45
Golgi impregnated rat STH neurons. B, number of proximal

dendrites per neuron.

No.of neurons

33

 

 

Somata cross-sectional areas

. 2
In um

No. ofcells

20
15
10

l

l

 

34

 

No.of prox.dendri tes

 

 

35

the most frequently impregnated neurons (see histogram B in
Fig.1). They were medium in size (average: 225nm). The
main characteristic feature of this variety was that one of
the dendrites originated from the mid-section of the cell
body (A in Fig.2 and 3; E, F in Fig.4; C, E, F, J, K, N in
Fig.6) while the remaining dendrites arose mainly from
opposite poles of the cell body. The other two varieties
were less frequently impregnated and were characterized by
having either two proximal dendrites originating from
opposite poles of a fusiform shaped cell body (C in Fig.2, 3
and 4; M in Fig.6), or four to six proximal dendrites
originating from the cell body without any specific
orientation (B in Fig.2 and 3; A, G, H in Fig.4; A, G, L in
Fig.6). All of these neurons were randomly distributed
within the nucleus without showing any specific pattern of
arrangement. Somatic cross-sectional areas (SA) ranged
between 1401111? and 440nm? (see histogram A in Fig.1). The
somata were fusiform, oval, polygonal round or multipolar
shaped. Some of the neurons had delicate somatic spines (C
in Fig.2 and 3; A, B, D in Fig.4; I, K in Fig.6). The
diameters of proximal dendrites were between 1.4um and
3.6um. Bifurcation of the proximal dendrites occurred at
variable distances (3 to 90um) from the cell body (see
neuron C in Fig.6). Frequently, the secondary dendrites
further bifurcated at distances between 18 to lOOum from the
cell body, and extended to well over 500nm from the point of
branching (C in Fig.6). Somtimes there were long tapering

secondary dendrites that extended approximately 400nm from

 

 

 

 

36

Figure 2.

Reconstruction of the representative rat STH neurons:
A. The most frequently impregnated neuron with oval shaped
cell body and variable dendritic spines. Note the single
dendrite originates from the mid-region of the cell body.
Arrowhead points to a filiform process. B. A radiating
neuron with an ovoid shaped cell body and dendrites studded
with variable amounts of spines. a, axon; arrowheads point
to filiform processes; 3, points to a dendritic spine with
two heads. C. A bipolar neuron with fusiform—shaped cell
body and a few dendritic spines. ArroWheads point to

filiform processes; 5, somatic spines.

37

 

 

38

Figure 3.
Photomicrographs of Golgi impregnated STH neurons

illustrated in Fig. 2. Scale bar = SOum.

39

m .
\

 

40

 

 

 

41

 

 

42

the branching point without futher bifurcation (F in Fig.6).
Occasionally, a single unbranched dendrite, 1.3 to Bum in
diameter, originated from the cell body and extended over
410nm (B in Fig.4). Beadings in some of the distal
dendrites were observed in all Golgi impregnated rat STH
neurons. Dendrites which crossed beyond STH borders were
usually beaded (see Fig.4 and 6). The dendrites of all STH
neurons were covered by spines. The quantity of the spines
varied from neuron to neuron. Usually, proximal dendrites
had few spines, while secondary and tertiary dendrites had
sparse to moderate amounts of spines (Fig.2, 4, 6). Even
within the same neuron, some dendrites could have more
spines than others (Fig.2). Dendritic spines were
pedunculated. Spine shafts were 1 to 3um long, ending with
a small bulbous head. Dendritic appendages were also
frequently seen in rat STH neurons. These filiform—shaped
processes measuring 3 to 60um long and less than lum in
diameter, frequently arose from the distal dendrites, and
occasionally from proximal dendrites (see arrow heads in
Fig.2, 4 and 6).

In the present study, the axons of rat STH neurons
were not impregnated beyond their initial segments. They
arose from either the cell body (A, E, G in Fig.4; B, N in
Fig.6) or from a proximal dendrite (B in Fig.2 and 3; D in

Fig.4; K in Fig.6).

Afferent fibers:

Quite a few sagittal sections that were prepared by

43

Figure 4.

Reconstructions of Golgi impregnated rat STH neurons
from sagittal sections. Rostral is to the left, dorsal is
at the tOp of the figures. Note the position of different
varieties of STH neurons with respect to the shape of the
nucleus (A-H). Also note that centrally located neurons A, F
and H extend some of their dendrites into CP; neurons B and
E extend some dendrites into ZI; C and G, extend dendrites
into both ZI and CP. Neuron D has its cell body and about
half of its dendrites completely embedded within CP. a,
points to axons; arrow heads point to filiform processes; 3,
somatic spines. CP, cerebral peduncle; EP, entopeduncular
nucleus; OT, optic tract; SN, substantia nigra; STH,

subthalamic nucleus; TH, thalamus; ZI, zona incerta.

44

 

 

 

 

 

 

IL

45

Figure 5.

Photomicrograph of Golgi impregnated STH neurons some
of which are illustrated in Fig. 4 (i.e. neuron B corresponds
to neuron B, and neuron H to neuron H in Fig. 4). In this
figure dendrites which extended beyond the confine of STH to
ZI, IC and CP and also some of ZI neurons' dendrites
extending into STH (arrow heads) are visible. CP, cerebral
peduncle; EP, entopeduncular nucleus; IC, internal capsule;
SN, substantia nigra; STH, subthalamic nucleus; TH,

thalamus; ZI, zona incerta. Scale bar = 200um.

46

 

47

Figure 6.

Reconstructions of Golgi impregnated rat STH neurons
from frontal sections: A, Rostral. B, Intermediate. C,
Caudal. Lateral is to the right, dorsal is at the top of
the figures. Note the positions of different varieties of
rat STH neurons with respect to the shape of the nucleus
(A—N). Some dendrites of neurons A,B,D,E,J,K,M,N, extended
into CP. A dendrite of neuron C extended into lateral part
of the posterior hypothalamus, and dendrites of a centrally
located neuron E, extended into both ZI and CP. a, point to
axons; arrow heads, point to filiform processes. CP,
cerebral peduncle; HTH, hypothalamus; OT, optic tract; STH,

subthalamic nucleus; TH, thalamus; ZI, zona incerta.

48

 

 

 

 

 

 

 

 

 

 

51

Figure 7.

Photomicrographs of Golgi impregnated STH neurons some
of which are illustrated in Fig. 6. A, Rostral. B,
Intermediate. C, Caudal. Neurons C in A, G & I in B, and L
in C of these figures are illustrated as C in A, G & I in B,
and L in C of Fig.6 respectively. Note the intermingling
pattern between the dendrites of both STH and ZI neurons
(arrow head in C). CP, cerebral peduncle; LHA, lateral
hypothalamic area; OT, optic tract; STH, subthalamic

nucleus; ZI, zona incerta. Scale bar = 200um.

<
I
._l

 

 

 

53

 

fr

 

 

 

54

 

 

 

 

55

Figure 8.

Higher magnification photomicrographs of Golgi
impregnated rat STH neurons from frontal sections. Lateral
is to the right, dorsal is at the top of the figures. A,
Illustrates the frontal part of STH. Note Golgi impregnated
STH neurons extending some of their dendrites beyond the
cytoarchitectural border of the nucleus and entering ZI
dorsomedially (upper neuron), and ventromedially (lower
neuron). B, Illustrates the midportion of STH. Note the
dendrite of the upper STH neuron extends beyond the
dorsomedial border of the nucleus and enters 21. CP,
cerebral peduncle; STH, subthalamic nucleus; ZI, zona

incerta.

 

 

56

 

 

57

 

230m

 

 

 

 

4::T_________________________________________________________________________T:%‘1

58

‘Figure 9.

Afferents to rat STH as seen from sagittal sections.
Rostral is to the right, dorsal is at the top of the
figures. A, Reconstructions of some Golgi impregnated rat
STH afferents. The majority of the afferents are
axon—collaterals of CP fibers. Some of them are derivatives
of CP fibers which are directed caudorostrally (a, b, c),
and others of rostrocaudally directed fibers (d, e, f).

Some descending fibers running through the internal capsule
also emit axon—collaterals toward STH (g). Another type of
STH afferents are fibers Which enter the nucleus from
rostral aspect after crossing IC fibers (h, i, j, k). B,
Photomicrograph of some Golgi impregnated STH afferents that
are illustrated in A. CP, cerebral peduncle; IC, internal
capsule; STH, subthalamic nucleus; ZI, zona incerta. Scale

bar = 200um.

59

 

 

 

 

6O

 

61

the mixed Golgi Kopsch—rapid Golgi procedure, had well
impregnated afferent fibers. A large number of axon
collaterals were derived from fibers within CP. They were
usually directed dorsocaudally to enter STH (d, e, f in
Fig.9A; c, d in 98). Some of the axon collaterals which
originated from fibers within CP at caudal levels of STH
were directed dorsorostrally to enter STH (a, b, c in
Fig.9A; a, b in 93). Rostrally, there were two kinds of)
fibers that entered STH: l) Axon collaterals and fibers
which were relatively smaller in diameter from others,
originated from IC fibers and directed toward STH (g in
Fig.9A; e in 98), and 2) fibers that were relatively thicker
in diameter crossed IC toward STH (h, i, j, k‘in Fig.9A; g,
h, i, j in 98). In addition, there were fibers passing

through STH without giving rise to any axon collaterals.

B. Nissl Study

The appearance and distribution of the somata of rat
STH neurons in various parts of the nucleus are shown in
Fig.10, ll, 12. Rat STH cell bodies were densely packed
within the confines of the nucleus except at the
dorsolateral and more rostral parts of STH. Some scattered
cells in CP which could belong to STH were also visible (see
arrow in Fig.10A). In frontal sections, STH somata appear
to be more elongated at the dorsolateral and the dorsomedial
parts of the nucleus. The longest, thinnest and the widest

STH cell bodies observed from the frontal sections were

 

 

 

 

 

 

T_T_—_________77T_____________________________________::a

62

Figure 10.

Photomicrographs of STH from Nissl stained 25um thick
horizontal sections. Lateral is to the right, dorsal is at
the top of the figures. A, Low magnification
photomicrograph showing the STH and the surrounding
stractures. Note that STH is packed with somata compared to
the other adjacent areas. CP, cerebral peduncle; EP,
entopeduncular nucleus; IC, internal capsule; SN, substantia
nigra; STH, subthalamic nucleus; ZI, zona incerta. Scale
bar = 0.5mm. B, Higher magnification photomicrographs of A.
The top photomicrograph is rostral part, lower left is
middle part, and the lower right is the caudal part of STH.
Note the polygonal or round shaped somata of STH neurons in
this plane of section. Many adjacent STH neurons which have
somatic membrane relationships are also visible . Scale bar

= SOum.

63

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65

Figure 11.

Photomicrographs of STH from Nissl stained 25um thick
frontal sections. Lateral is to the right, dorsal is at the
top of the figures. A, Low magnification photomicrograph
showing the STH and the zona incerta. Note that STH is.
packed with somata. CP, cerebral peduncle; STH, subthalamic
nucleus; ZI, zona incerta. Scale bar = 320nm. B, Higher
magnification photomicrographs of A. top photomicrograph is
the lateral part, lower left is the middle part, and the
lower right is the medial part of STH. Note the fusiform
shaped somata of STH neurons in this plane of section,

especially at the lateral part of STH. Scale bar = SOum.

66

 

67

 

68

Figure 12.

A low magnification photomicrograph of STH from Nissl
stained 25um thick sagittal section. Rostral is to the
left, dorsal is at the top of the figure. Note the round or
polygonal shaped somata of STH neurons in this plane of
section. Some fiber bundle sections are also visible. CP,
cerebral peduncle; STH, subthalamic nucleus; ZI, zona

incerta. Scale bar = l/3mm.

69

 

70

approximately 31pm, 6um and 16.6um, respectively. However,
in the horizontal or sagittal sections, STH somata were more
polygonal or round and measured between 10 to 28nm in
diameters. All STH somata within the confines of the
nucleus were intermingled in a manner such that no specific
part of STH consisted of neurons with a particular size or

shape.

C. Autoradiographic Study

Injection sites

The injection sites produced by representative amino
acids injections into the various regions of the cerebral
cortex are shown in Fig.13. Around the injection sites were
uniformly distributed fields of label in the cortical
neuropil that resulted in an apparent enlargement of the
injection sites when observed under dark field illumination
(Fig.13, 14A, 15A, 16A, and 17A). These fields of labels
are probably due in large part to the local collateral
arborizations of cortical cells (Cajal, 1911). From the
cytoarchitectural point of View, the injection sites of all
cases appeared to be restricted to the frontal agranular
cortex except for CXSTH 23, 25 and 26. In the latter cases
the injection sites appear to be limited to the caudal part
of the frontal granular, occipital and parietal cerebral
cortices respectively (Fig.13, 14A, 15A, 16A, 17A and cases
23, 25 and 26). Labeled projection axons arising from the

injection sites were seen coursing caudally within fascicles

71

through the striatum, the internal capsule and the cerebral
peduncle (e.g. Fig.13, CXSTH 12). Labeled fibers arising
from IC and CP bundles were observed to reach the
ipsilateral STH and resulted in either diffusely distributed
silver grains or dense accumulations of labeling in a
relatively restricted part of STH. Contralateral labeling
was not observed.

Based on the cortical projection pattern into STH, the
injection sites of the frontal agranular cortex (Fig.13)

haVe been divided into four regions:

1. Rostral part of the medial agranular cortex

Figure 14C shows the light and diffuse labeling
pattern of CXSTH 3, a representative case in which the
”radioactive amino acids were injected into the rostral part
of the medial agranular cortex (Donophue & Wise, 1982), the
eye & eyelid representations areas of Hall and Lindholm
(1974). As seen from Fig.14C, E and 19A labeling was
confined to the ventral two-thirds of the medial half of STH
and extended rostrocaudally. The projection displayed a
mediolateral ordered arrangement with some overlap. The
rostral part of the eye & eyelid field projected
ventromedially (cases CXSTH 1 and 2) and the caudal part of
the field projected to the ventrolateral aspect of the

medial half of STH( case CXSTH 5).

2a. Rostral part of the lateral agranular cortex

The projections from the lateral agranular cerebral

Table 1.

Injection sites and parameters of the

radioactive labeling used for tracing corticosubthalamic

projections (see also fig.13).

Case

numbers

CXSTH,1
CXSTH,2
CXSTH,3
CXSTH,4
CXSTH,5
CXSTH,6
CXSTH,7
CXSTH,8
CXSTH,9
CXSTH,10
CXSTH,11
CXSTH,12
CXSTH,19
CXSTH,20
CXSTH,21
CXSTH,22
CXSTH,23

CXSTH,24

Injection

site

Bragma point

(APHML)
+6 +1
+5 +1
+3.5 +1.5
+0.5 +1
+2 +1
-O.5 +1.5
—1 +2.5
-O.5 +3
+0.5 +3.2
+5.8 +1.9
+5.5 +2.8
+5 +3.5
+4.2 +2.8
+2 +2.6
+4.3 +4
+2.3 +4.5
-2.5 +6
-3 +1.5

Radioactive
lable
Leucine
Proline
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Lys.
Leu. Lys.
Leu. Lys.
Leu. Pro.
Leu. Pro.
Leu. Pro.
Leu. Pro
Leu. Pro.
Leu. Pro

Total

Ouant.

ul

0.25
0.3

0.3

0.3

Concent.

uCi/ul

20
20
20
2O
20
20

20

20
20
20
20
15
15
15
15
15

15

Survival

time/day

 

 

 

 

 

CXSTH,75

CXSTH,26

-5 & +5

Leu.

Leu.

73

& Pro.

& Pro.

0.3

0.3

15

15

rt '
I
I
I
r

 

74

Figure 13.

Illustration of the site and the extent of the amino
acid injection into the various regions of the rat cerebral
cortex. Black areas represent the injection site. The
stippled areas represent the corona surrounding the center
of the injection site which probably corresPonds to
diffusely spread amino acids. Case 4 and 12 were cut into
sagittal sections; 6,7 and 11 horizontal sections; and the
rest frontal sections. The schematic drawing of rat
cerebral hemisphere at the center of the figure shows the
topographical placement of the injection sites. B
corresponds to the bregma. One mm spaced lines are drawn in
order to show the relative coordinate of various injection

sites.

75

 

 

   

 

 

 

 

 

 

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//F/l7’7///
///////
7/////

 

 

 

 

76

Figure 14.

Illustration of case #3 (representative of the rostral
part of the medial agranular cortical region injections). A
& B, Dark and light—field photomicrographs of the injection
site. The center of the injection site and its extent is
shown in B. Note the diffuse pattern of silver grains
surrounding the center of the injection site shown in A.
Scale bar = 2mm. C, Dark—field photomicrograph showing the
labeling in STH. Note the light and diffuse labeling within
the medial half of STH. D, light-field photomicrograph
shows the cytoarchitectural borders of STH. Scale bar =
0.5mm. E, Drawing of the projection pattern in frontal
sections. Labelings are observed in the entire rostral
caudal extent of the nucleus. Fiber-like silver grain
accumulations in CF are indicated by heavy stipples. Fibers
which were cut in cross section in GP or STH are indicated
by stippled circles indicated by arrow heads. The uneven
but diffuselly distributed silver grains (stipples) within
STH probably represent terminal fields. E1, rostral and E4,

caudal. Scale bar = 1mm.

77

a“!
, {4.31.

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_' 1 ~‘!.:
5‘9“ NB

 

78

 

 

 

 

79

cortex, (Ml) (Donophue, 1982), corresponding to the mouth,
jaw and the tongue representations area (Hall and Lindholm,
1974) were considerably more dense than that of the medial
agranular area (Fig.16C and E). A comma shaped accumulation
of silver grains was found in the lateral two-thirds of the
rostral two-thirds of STH (Fig.19C). The rostrocaudal parts
of this cortical region displayed some superimposed labeling
in STH such that the rostral parts of the cortex projections
were confined to the lateral two—thirds of the rostral
two-thirds with more concentration at the dorsolateral tip
of STH, and the caudal parts of the cortex projections were
mostly restricted to the dorsolateral tip and the dorsal
aspect of the lateral half of STH (Fig.19C CXSTH 11 & 12 and

21 & 22 respectively).

2b. Medial part of the lateral agranular cortex
Generally, the projections from this region occupied
almost the same part of STH as that of the medial granular
cortex. Although overlapping between adjacent projections
are seen, the pattern of labeling appears different than
those of the other cortical regions (Fig. 13, 15 and 198).
As seen from these figures, the amino acid injections in
this cortical area resulted in a band-shaped labeling of
STH. The band—shaped labeling was confined to the ventral
aspect of rostral STH, and tended to shift dorsally at the
caudal part of STH. The projections from the rostrocaudal
extent of the cortex Which correspond to the rhinarium,

vibrissa and forelimb representations areas respectively

80

Figure 15.

Illustration of case #19 (representative of the medial
part of the lateral agranular cortical region injections).
A & B, Dark and light field photomicrographs of the
injection site Scale bar = 2mm. C, Dark—field
photomicrograph showing the labeling within STH. Note the
dense and relatively wide band of labeling extends from the
dorsolateral to the ventromedial aspect of STH. D,
light-field photomicrograph showing the cytoarchitectural
borders of STH. Scale bar = 0.5mm. E, Drawing of the
projection in frontal sections. Labelings are seen in
rostral through caudal sections. Note the shifting of the
terminals from the ventral STH in the rostral sections to
the dorsal aspect in the caudal sections. E1, rostral and

E4, caudal. Scale bar = 1mm.

81

 

 

 

82

 

83

Figure 16.

Illustration of case #21 (representative of the rostral
part of the lateral agranular cortical region injections).
A & B, Dark and light-field photomicrographs of the
injection site Scale bar = 2mm. C, Dark-field
photomicrograph showing the labeling within STH. Note the
heavy projection in the dorsal aspect of the nucleus. D,
light field photomicrograph shows the cytoarchitectural
borders of STH. Scale bar = 0.5mm. E, Drawing of
projection in frontal sections. Labelings are seen in the
rostral two—thirds of STH. Note the widespread labeling in
the rostral part while the labeling becomes restricted to
the more dorsal aspect of STH toward the middle third of the
nucleus. No labeling is seen in the caudal third of the

nucleus. E1, rostral and E4, caudal. Scale bar = 1mm.

84

 

85

 

 

 

 

 

 

86

(Hall and Lindholm, 1974) were confined to the medial half

of rostral STH, rostrally, and extended to the medial part

of the lateral half of STH caudally.

3. Caudal part of frontal medial agranular cortex

Figure 17C and E show the faint projections of this
region, corresponding to the caudal portion of eye & eyelid
and the trunk representations areas (Hall and Lindholm,
1974). Silver grains were confined to the dorsolateral
portion of the caudal two-thirds of STH. The overlapping
pattern in these projections can be contrasted with the
projections from the lateral agranular region, in Which the
superimposed labeling occurs rostrally and the partial
overlap occurs caudally. Whereas the superimposed labeling
in this case was seen caudally and the partial overlap

occurred rostrally.

4. Caudal part of the lateral agranular cortex cortex
Figure 19E shows the labeling from the caudal part of
the lateral agranular cortex, which corresponds to the
hindlimb representations area (Hall and Lindholm, 1974).
Labeling was observed in the ventral aspect of the middle
third of STH (see also table 1 and Fig.13 for injection»

site).

5. Other cerebral cortical areas
When the injections were confined to the frontal

granular (CXSTH 23), occipital (CXSTH 25) and parietal

 

87

Figure 17.

Illustration of case #24 (representative of caudal
part of the frontal medial agranular cortical region
injections). A & B, Dark and light-field photomicrographs
of the injection site Scale bar = 2mm. C, Dark-feld
photomicrograph showing the labeling within STH. Note the
diffused labelings in the lateral two-thirds of STH. D,
light field photomicrograph shows the cytoarchitectural
borders of STH. Scale bar = 0.5mm. E, Drawing of
projection pattern in frontal sections. Labeling covers
almost the entire nucleus in the caudal sections and becomes
restricted to the ventral portion in the middle sections.
Labeling is not seen in the rostral third of STH. E1,

rostral and E4, caudal. Scale bar =lmm.

88

 

89

 

 

9O

(CXSTH 26) areas (see Fig.18 and table 1), no labeling was

found within STH.

In summary, the medial frontal agranular cortex
projections to STH were faint and less in density than the
more pronounced lateral frontal agranular projections.
Caudally spaced injections of each region produced orderly
overlapped or superimposed projections to STH. Each
cortical region showed some topographically arranged
projections in STH. The projection from the whole frontal
agranular cortex fills all parts of STH. Injections in the
frontal granular, parietal and occipital cortex did not

result in labeling within STH.

 

91

Figure 18.

Dark—field (D) and light-field (L) photomicrographs
illustrating the injection sites and the extent of the amino
acid injections into the somatosensory, visual and parietal
cortex (cases #23, #25 and #26). No labelings were seen in

STH in these cases. Scale bar = 2mm.

92

 

 

93

 

 

94

 

 

F .—

96

(case #7 in Fig.13). Labeling is in the ventral aspect of
the middle third of STH. No labeling is seen in the rostral.

or the caudal sections. Scale bar for all groups = 1mm.

 

 

95

Figure 19.

Summary diagram of the projections from the agranular
cerebral cortical areas to STH shown in frontal sections.
Rostrocaudal extent of the nucleus is indicated from right
to left in each row. A, Projections from the rostral part
of the medial agranular cortical areas (cases #1, 2, 3 and 5
in Fig.13). Labeling is in the medial half of the nucleus
except at the rostral portion of STH. The dorsal portion of
the medial half of STH is void of labeling. B, Projections
from the medial part of the lateral agranular cortical areas
(cases #10, 19, 20, 9 and 8 in Fig.13). Labeling is in the
medial half of STH, and extends to the ventral portion of
the lateral half of the nucleus. C, Projections from the
rostral part of the lateral agranular cortical areas (cases
#11, 12, 21 and 22 in Fig.13). Labeling occupies the entire
nucleus at the rostral portion of STH. In the middle
sections, the labeling becomes sparse in the medial half of
the nucleus. In the caudal sections, the labeling becomes
restricted to the lateral half of STH and eventually at the
most caudal sections there is no labeling. D, Projections
from the caudal part of the frontal medial agranular
cortical areas (cases #4, 6 and 24 in Fig.13). The pattern
of labeling is almost mirror image of that in C. Almost
entire nucleus is labeled in the caudal sections and no
labeling in the rostral sections. In the middle sections,
labeling is predominantly in the lateral half and the
ventral portion of the medial half of STH. E, Projections

from the caudal part of the lateral agranular cortical areas

97

 

9&8 #5 22 :4
V M

 

T’I

 

 

 

98

CHAPTER 4. DISCUSSION

A. Golgi Study

General Comments On Neuronal Types:

Many of the previous Golgi studies on STH neurons have
either not shown illustrated figures (Kolliker, 1896; Mirto,
1896), or have presented only drawings (Ramon y Cajal,
1910—1911; Ramon Moliner, 1962; Leontovich & Zhukova, 1963;
Iwahori, 1978; Yelnik & Percheron, 1979). Rafols & Fox
(1976) were the only ones to illustrate their data with both
drawings and photomicrographs. In the present study we
attempted to illustrate cell morphology with both drawings
and photomicrographs of STH neurons, so that more adequate
comparisons could be made concerning the varieties of cell
morphology described in this study.

In the few recent Golgi studies a number of STH
neuronal types have been described. Rafols & FOX (1976)
demonstrated two varieties of principal neurons, radiating
and elongated fusiform, in addition to a local Golgi type II
interneuron in the monkey. Iwahori (1978) distinguished
three neuronal types in the kitten: Type I are the main
constituent of the nucleus, having medium sized oval or
polygonal cell bodies. Type II are large with multipolar
or polygonal cell bodies. And type III are small with
polygonal cell bodies. However, Yelnik & Percheron (1979)
emphasized that STH consists of only one type of neurons
(Golgi type I) in cat, monkey and man. And in a most recent

intracellular labeling study by Kita et al., (1983), two

 

 

99

types of STH meurons were distinguished, STH neurons with
and without intranuclear collaterals.

In our Golgi material axons were rarely and only
partially impregnated. So we have classified rat STH
neurons on the basis of their somatic and dendritic
morphological characteristics. The results of the
statistical analysis of somatic size and the number of
proximal dendrites suggested that rat STH consisted of only
one type of neuron. This type can be further subdivided

into three morphologically different varieties.

l. Subthalamic Neurons With 3 to 5 Proximal Dendrites:
Neurons which are morphologically similar to this
variety have been illustrated by Kita et al. (1983) in the
rat STH. Moreover, they have provided more axonal
information on the distribution and morphology of axons in
their intracellular HRP labeling of STH neurons. These
cells are also similar to those illustrated by Leontovich &
Zhukova (1963) in their figure 12, in the dog; Rafols & Fox
(1976) in their figures 2 and 9 as one of the principal
neurons of the adult monkey STH; Iwahori (1978) in his
figures 2C, 4A,I in the cat, and Yelnik & Percheron (1979)
in their figures 6E, 7D and 8B,C in the cat, monkey and man.
In the present study this variety of rat STH neuron was the
most frequently impregnated and has been found to be usually
medium in somatic size, with an elongated ovoid shaped soma.
The quantity of this type of neuron however, was less than

the other types demonstrated by Rafols & Fox (1976) and

 

100

Iwahori (1978). The latter authors have observed more
radiating STH Golgi impregnated neurons than the elongated
ovoid ones. This type of STH neuron was not even mentioned
in earlier studies (Kolliker, 1896; Mirto, 1896; Ramon y

Cajal,.l910-l9ll).

2. Subthalamic Neurons With 4 to 6 Proximal Dendrites:

This variety of rat STH neuron appears to correspond
to the radiating principal neurons of Rafols & Fox (1976) in
their figures IC, D; 10; 13 in the monkey; type I neurons of
Iwahori (1978) in his figures 2A; 4B, C, D, E, F, G, H, J,
K, L in the kitten; some of the Golgi type I neurons of
Yelnik & Percheron (1979) in their figures 6A, B, C, D in
the cat; 6F, G, H, I in the monkey; 6K, L, M in the baboon
and 7C, 8D in the human, and also to some neurons
illustrated by Kita et a1. (1983) in the rat. In the
present study the feature which characterized the morphology
of this variety of neurons is the origin and number of
proximal dendrites that reflects its radiating dendritic
field. This variety of rat STH neuron was less frequently
impregnated than the elongated ovoid ones mentioned above.
On the other hand, in previous Golgi studies, radiating
neurons were either the only ones illustrated (Ramon y
Cajal, 1910-1911; Ramon Moliner, 1962; Leontovich & Zhukova,
1963) or considered to be the predominant neuron in STH
(Rafols & Fox, 1976; Iwahori, 1978; Yelnik & Percheron,

1979).

 

.. .
I, I“

.r‘ . . ..I

. .a
.- .

101

3. Subthalamic Neurons With Only Two Proximal Dendrites:

This variety of rat STH neurons were characterized by
a fusiform shaped soma and only two proximal dendrites.
Like the radiating variety, these neurons were infrequently
impregnated. This variety has not been previously obServed
in other species or in the rat using the intracellular HRP
labeling technique (Kita et al., 1983; Hammond et al.,
1981), with the exception of one cell illustrated in figure
7 of Yelnik & Percheron (1979).

Golgi type II local interneurons described in detail
by Rafols & Fox (1976) have not been observed in any other
Golgi or intracellular labeling study of STH. In the
present study, despite the use of several Golgi impregnating
techniques and a large number of rats, none of the observed
neurons were seen to have the morphological characteristic

features of Golgi type II neurons.

Dendritic Appendages Of Rat Subthalamic Neurons:

In the present study, in addition to dendritic spines,
we have observed on a majority of rat STH neurons thin
dendritic appendages or filiform processes which are
variable in length (3 to 60um long) and end with a round
head. These processes have not been described on principal
neurons in previous Golgi studies in other species. Yelnik
& Percheron (1979) in their figure 5H, illustrated one small
human STH neuron in which the dendrites were characterized
by thin processes with intermittent swellings. Rafols & Fox

(1976) in their figure 3, have also shown a Golgi type II

 

 

L

102

neuron characterized by bulbous dendritic appendages and
beaded axon-like processes. The presence of this type of
dendritic appendages was observed in intracellularly HRP
labeled rat STH neurons (Kita et al., 1983).

The question of Whether STH is an open or closed
nucleus has been discussed by Yelnik & Percheron (1979). An
open nucleus is is defined as one where dendrites of neurons
within the nucleus intermingle with dendrites of neurons in
adjacent structures after they have crossed beyond the
cytoarchitectonic limits of the nucleus (Mannen, 1960). In
our observations we have found dendrites of some centrally
located STH neurons to extend beyond the confines of the
nucleus and terminate in Z1 and LHA. None of the previous
Golgi studies in different species (Kolliker, 1896 in rat
and mouse; Mirto, 1896 in man; Ramon y Cajal, 1910-1911 in
mice and cat; Ramon Moliner, 1962 in cat; Leontovich &
Zhukova, 1963 in dog; Rafols & Fox, 1976 in monkey; Iwahori,
1978 in kitten; Yelnik & Percheron, 1979 in cat, monkey and
man) have indicated any such STH neurons' dendrites
spreading out of the dorsal, dosomedial or the ventromedial
borders of the nucleus. However, in recent studies by
Hammond et a1. (1981) and Kita et a1. (1983) using the
intracellular HRP labeling technique, some rat STH neurons'
dendrites were shown to extend beyond the borders of the
nucleus and terminate in ZI, IC and CP. Since our
Golgi-impregnation confirm the findings of Hammond et al.
(1981) and Kitai et a1. (1983) concerning dendritic

extension beyond the confines of the rat STH, we suggest

 

Inn-115311

153“:

in:

 

103

that the rat STH be considered as an open nucleus.

Afferent Fibers 0f Subthalamic Nucleus:

One of the main afferent fibers to STH arises from the
ipsilateral globus pallidus (the homolog of the lateral I
pallidal segment in primate). This has been demonstrated in
a variety of species using different techniques. Cerebral
cortical afferent to STH have also been demonstrated. A few
afferents from the pedunculopontine nucleus have been
reported as well as a nigrosubthalamic projection.

Kolliker (1896), Mirto (1896), Ramon y Cajal
(1910—1911), and Iwahori (1978) have mentioned that STH
afferents were mainly axon-collaterals of fibers that
descended within CP. In the present study axon-collaterals
were also obServed to arise mainly within CP coursing toward
STH. Thy were of two directions: 1) axon—collaterals from
fibers directed rostrocaudally in CP and 2) axon-collaterals
of fibers directed caudorostrally in CP. In addition, a
number of axon—collaterals from IC fibers were seen to enter
the nucleus after crossing IC rostrally. The latter fibers
were found to arise from the large cells of the globus
pallidus (Shanner, 1936). Some fiber bundles passing STH

without emiting any axon—collateral were also seen.

B. Nissl Study
The cytoarchitecture of STH has been described in

man (Foix & Nicolesco, 1925) to have STH neurons that

 

104

appeared larger in the dorsal and internal parts of the
nucleus than in ventral and external parts. However
opposite observations were reported by Kodama (1928) in
‘man, and by Whittier & Mettler (1949) in the monkey. The
latter authors observed large STH somata in the lateral
parts and the smallar ones in the medial parts of the
nucleus. Rafols & Fox (1976) have also localized the
smaller somata of the monkey to a narrow strip along the
ventral border at the medial end of the nucleus. Yelnik &
Percheron (1979) have revealed that different shapes of
somata were intermingled so that no part of the nucleus
'contained any particular shape of neuron. In spite of the
unimodal distribution of STH neurons, the latter authors
found more fusiform somata in sagittal sections and more
round or polygonal somata in frontal sections of samples
taken from monkey and man.

Our results generally confirmed the findings of Yelnik
& Percheron (1979). However, the rat STH somata were
observed to be more fusiform specifically in dorsolateral
parts of the nucleus When observed in frontal sections, and
more polygonal or round When observed in sagittal or
horizontal sections. Thus the results of the present
studies (Golgi and Nissl) suggest that the size or shape of
STH neurons seem to be linked most likely to the plane of

section.

C. Autoradiographic Study

 

r-I

 

 

105

Two amino-acid injections were confined to the rostral
part of the medial frontal agranular cortex, and the other
caudally placed two injections extended into the lateral
agranular (Agl) regions. These injections resulted in only
faint labeling in STH. This may indicate a minor projection
from this particular part of the frontal cortex to STH.
However, the projections from the lateral and intermediate
agranular regions to STH were more dense than that of the
medial agranular part. This indicated that a large number
of projection neurons to STH are located in these regions of‘
the agranular cortex. Interestingly, Donoghue & Wise (1982)
have shown that the Agl of the frontal cortex contains more
cortico-spinal projection neurons than that of the medial
agranular (Agm) of the frontal cortex. The similarity of the
results in these two studies and previous STH Golgi studies
(Cajal, 1910—1911; Iwahori, 1978) as well as the present Golgi
study of rat STH demonstrating some axon collaterals entering
STH from IC and CP fibers, suggests that the cortico— STH
fibers are axon collaterals of cortico-spinal fibers.

Hartmann—Von Monakow et a1. (1978) have shown that
the monkey precentral and frontal motor cortex (area 4)
contain more projection neurons to STH compared to areas 6, 8
and 9 of the prefrontal cortex. These projections were
topographically organized within the lateral half of monkey
STH. In the present study we have also demonstrated that
the rat cortico—STH projections are topographically
organized. However, in the rat the arrangement of the face,

forelimb and hindlimb representation area projections were

 

 

106

different from that of the monkey. In the rat, the jaw, lip
and the tongue representation areas project to the dorsal
aspect of STH, the forelimb area projects to the ventral
aspect of the lateral half of STH, and the hindlimb area
projects to the ventral aspect of the middle third of STH.
0n the other hand, in the monkey, the face area projects to
the lateral, the leg area to the medial aspect of the
lateral part of STH, and the arm area projection was found
in between the face and the leg areas (Hartmann-Von Monakow
et al., 1978). Another interesting point which was revealed
from this study was that the projection from the eye and
eyelid representation area to STH in the rat was found to
have similar characterestics to the monkey area 8
(prefrontal eye field )projection to STH. The similarities
included 1) Light and diffuse labeling; 2) The labeling is
confined to the ventral part of the medial half of STH
(Kunzle & Akert, 1977 and Hartmann —Von Monakow et al.,
1978). Our observations have also shown that the
projections from rat frontal agranular cortex filled the
entire STH. However, in a study in the monkey, the
cumulative plot of labelings of all injection cases in
frontal and prefrontal cortex failed to cover STH
completely(Hartmann-Von Monakow et al., 1978). The
difference in results may be due to either a species
difference or that the amino-acids injections in the various
frontal and prefrontal cortices in the monkey did not cover
the entire projection areas. The present study confirmed a

point suggested by all of the previous studies that the

 

P,

 

107

somatosensory or the postcentral cortex has no projections
to STH (Petras, 1965, 1969; Kunzle, 1977 and Hartmann-Von
Monakow et al., 1978). In addition to the somatosensory
area, we have shown that-the rat parietal and occipital
cortices also have no projections to STH.

In conclusion, the cortico—STH projections in the rat
are somatotopically organized with some overlapping. These
projections, (with moderate labeling from the lateral
agranular cortex and light labeling from the medial

agranular cortex),fills the entire ipsilateral STH.

 

I:—

108

CHAPTER 5. CONCLUSION

Nissl and Golgi studies on rat STH revealed the
following: Rat STH is densely packed with neurons of
variably shaped somata. Statistical analysis of somatic
size and the number of proximal dendrites, suggested that
STH consists of a single type of neuron with some varieties.
Generally two to six proximal dendrites originated from the
soma and gave rise to sparsely-spined tapering secondary and
tertiary dendritic branches extending up to 500um in length.
The dendritic fields of individual STH neurons were usually
large. Some neurons located in the central part of STH had
some of their dendrites crossing the borders of the nucleus
into the zona incerta, the lateral hypothalamic area or the
cerebral peduncle. This suggested that STH was an open
nucleus, and that some STH neurons may recieve inputs not
only from afferents which terminate within the nucleus, but
also from those terminating in the surrounding areas. The
cortico—STH projections were studied by an autoradiographic
technique in the rat. The cortico—STH projections were
found to be ipsilateral and arose from the frontal agranular
cerebral cortex in which heavier projections were from
lateral and relatively lighter from medial frontal areas.
The overall cortical projections filled the entire STH,
however these projections exhibit a fine topographical
organization. The above findings suggested that a single

STH neuron with a large dendritic field may receive

 

1.;

109

overlapped cortical afferents. For example, a centrally
located STH neuron which had dendrites directed
dorsomedially and ventrolaterally may receive cortical
terminals originating from both lateral and medial agranular
regions on respective dendrites, or a neuron located at the
cerebral peduncle may receive cortical terminals originating
from both rostral and caudal parts of the medial agranular

region.

 

110

REFERENCES

Adey, W. R. and D. F. Lindsly (1959) On the role of
subthalamic areas in the maintenance of brain—stem
reticular excitability. Exp. Neurol. 1, 407-426.

Afifi, A. and W. W. Kaelber (1965) Efferent connections
of the substantia nigra in the cat. Exp. Neurol. 11,
474-482.

Albe-Fessard, D., Stutinsky, F. and S. Libouban (1966)
Atlas sterotaxique du Diencephale du rat. Blanc,
Edition du CNRS, Paris.

Astruc, J. (1971) Corticofugal connections of area 8
frontal eye field in macaca mulata. Brain Res.

Auer, J. (1956) Terminal degeneration in the Diencephalon
after ablation of frontal cortex in the cat. J.
Anatomy, 90, 30-41.

Bonin, G. V. and G. A. Shariff (1951) Extrapyramidal
nuclei among mammals. A quantitative study. J. Comp.
Neurol. 94, 427-438.

Brown, L. L. and L. I. WOlfson (1978) Apomorphine icrease
glucose utilization in the usbatantia nigra,
subthalamic nucleus and corpus striatum of rat.
Brain Res. 140, 188—193.

Brown, L. L., Makman, M. H., Wolfson, L. I., Dvorkin, B.,
Warner, C. and R. Katzman (1979) A direct role of
dopamine in therat subthalamic nucleus and an adjacent
interpedunculararea. Science; 206, 1416-1418.

Carpenter, M. B., Whittier, J. R. and F. A. Mettler
(1950) Analysis of choreoid hyperkinesia in the rhesus
monkey. J. Comp. Neurol. 92, 293-332.

Carpenter, M. B. and C. S. Carpenter (1951) Analysis of
somatotopic relations of the corpus Luysi in man and
monkey. J. Comp. Neurol. 95, 349-370.

Carpenter, M. B.(1955) Ballism associated with partial
destruction of the subthalamic nucleus of Luys.
Neurology; 5, 479-489.

Carpenter, M. B. and R. E. McMasters (1964) Lesions of
the substantia nigra in the rhesus monkey efferent fiber
degeneration and behevioral observations. American
Journal of Anatomy; 114, 293-319.

Carpenter, M. B. and N. L. Strominger (1967) Efferent fibers
of the subthalamic nucleus in the monkey. A comparison of

 

111

the efferent projections of the subthalamic nucleus,
substantia nigra and globus pallidus. American Journal
of Anatomy; 121, 41-71.

Carpenter, M. B., Fraser, R. A. R. and J. E. Shriver (1968)
The organization of pallidosubthalamic fibers in the
monkey. Brain Res. 11, 522-559.

Carpenter, M. B. and P. Peter (1972) Nigrostriatal and
nigrothalamic fibers in the rhsus monkey. J. Comp.
Neurol. 144, 93-116.

Carpenter, M. B, Nakano, K. and R. Kim (1975)
Nigrothalamic projections in the monkey demonstrated by
Autoradiographic techniques. J. Comp. Neurol. 165,
401-416.

Carpenter, M. B. (1976) Anatomical organization of the
corpus striatum an d related nuclei. Assoc. Res. Nerv.
Ment. Des. Prec. ss, 1-36.

Carpenter, M. B., Batton, R. R. III, Carleton, S. C. and J.
T. Keller (1981) Interconnections and organization of
pallidal and subthalamic nucleus neurons in the monkey.
J. Comp. Neurol. 197, 579—603.

Carpenter, M. B., Carletton, S. C., Keller, J. T. and P.
Conte (1981) Connections of the subthalamic nucleus in
the monkey. Brain Res. 224, 1—29.

Carpenter, M. B. and J. Sutin (1983) The corpus striatum.
Williams Wilkins, Baltimore-London, Page 599—603.

Carter, D. A. and H. C. Fibiger (1978) The projections of
the entopeduncular nucleus and globus pallidus in rat as
demonstrated by Autoradiography and Horseradish
peroxidase Histochemistry. J. Comp. Neurol. 177,
113—124.

Chang, H. T., Kita, H. and S. T. Kitai (1983) The fine structure
of the rat subthalamic nucleus: An electron microscopic
study. J. Comp. Neurolo. in press.

Chan-Paley, V. (1977) Cerebellar dendate nucleus
organization, cytology and transmitters. Berlin
Heidelberg, New York pg. 501-507.

Cowan, W. M., Gotlieb, Henddrickson, A. B., Paice, J. L. and
T. A. Woolsey (1972) The autoradiographic demonstration
of axonal connection in the central nervous system.
Brain Res. 37, 21-51.

Deniau, J. M., Hammond, C., Chevalier, C. and J. Feger
(1978) Evidence for branched subthalamic nucleus
projections to substantia nigra, entopeduncular nucleus

 

 

112

and globus pallidus. Neuroscience letter; 9, 117-121.

Deniau, J. M., Grofova, I., Steindler, D. and S. T. Kitai
(1980) Axonal arborizations of nigrothalamic and
nigrotectal cells: Anatomical and electrophysiological
studies. Soc. Neurosci. Abst. 5, 807.

Devito, J. L. and O. A. Smith, JR. (1964) Subcortical
projections of the prefrontal lobe of the monkey.
J. Comp. Neurol. 123, 413-423.

Devito, J. L., Anderson, M. E. and K. E. Walsh (1980) A
horseradish peroxidase study of afferent connections of

the globus pallidus in Macaca Mulata. Exp. Brain Res.
38, 65-73.

Donophue, J. P. and S. T. Wise (1982) The motor cortex of the
rat: Cytoarchitecture and microstimulation mapping.
J. Comp. Neurol.

Fairen, A., Peters, A. and J. Saidanha (1977) A new procedure
for examining Golgi impregnated neurons by light and
electron microsc0py. J. Neurocytology; 6, 311-337.

Faull, R. L. M. and J. B. Carman (1968) Ascending projections
of the substantia nigra in the rat. J. Comp. Neurol.
132, 73-92.

Fonnum, F., Grofova, I. and E. Rinvik (1978) Origin and
distribution of glutamate decarboxylase in the nucleus
subthalamicus of the cat. Brain Res. 153, 370-374.

Fox, C. A., Anderson, A. N., Qui, I. J. and J. A. Rafols (1974)
Primate globus pallidus. A Golgi and electron
microsc0pic study. J. Furhirn Forschung; 15, 75-93.

Frigyesi, T. L. and A. Raisin (1971) Basal Ganglia-Diencephalon
synaptic relations in the cat. III. An intracellular
study of ansa lenticularis, lenticular fasciculus and
pallido-subthalamic projection activities. Brain Res.

35, 67-87.

Glees, P. and P. D. Wall (1946) Fiber connections of the
subthalamic region and the centro-median nucleus of
the thalamus. Brain; 69, 195-209.

Gobel, S. (1978) Golgi studies of the neurons in layer I of
the dorsal horn of the medulla (Trigeminal Nucleus
Caudalis). J. Comp. Neurol. 180, 375-394.

Graybiel, A. M. and C. W. Ragsdale, jr (1979) Fiber connections
of the Basal Ganglia. Progress In Brain Res. 51,
239-283.

Grofova, I. (1969) Experimental demonstration of a topical

 

 

 

113

arrangement of the pallidosubthalamic fibers in the
cat. Psychiat. Neurol. Neurochir. 72, 53-59.

Hall, R. D. and E. P. Lindholm (1974) Organization of motor
and somatosensory neocortex in the albino rat.
Brain Res. 66, 23-38.

Hammond, C., Deniau, J. M., Rizk, A. and J. Feger (1978)
Electrophysiological demonstration of an excitatory

subthalamo-nigral pathway in the rat. Brain Res.
151, 235-244.

Hammond, C., Feger, J., Bioulac, B. and J. P. Souteyrands
(1979) Experimental hemiballism in the monkey produced
by unilateral kainic acid lesion in corpus Luysii.
Brain Res. 171, 577-580.

Hammond, C., Yelnik, J., Deniau, J. M., Kalia, M. and S. T.
Kitai (1981) Three dimentional quantitative morphology
of subthalamic neurons intracellularly labeled with HRP.
64-14 Abst. Society forNeuroscience.

Hartmann-Von Monakow, Akert, K. and H. Kunzle (1978)
Projections of the precentral motor cortex and other
cortical areas of the frontal lobe to the subthalamic
nucleus in the monkey. Exp. Brain Res. 33, 395-403.

Hassler, R., Usunoff, K. G., Romansky, K. V. and J. F. Christ
(1982) Electron microsc0piy of the subthalamic
nucleus in the baboon. I. Synaptic organization of
the subthalamic nucleus in the baboon. J. Hirnforsch.
23, 597-611.

Iwahori, N. (1978) A Golgi study on the subthalamic nucleus
of the cat. J. Comp. Neurol. 182, 383-398.

Jackson, A. and A. R. Crossman (1981) Subthalamic nucleus
efferent projection to the cerebral cortex. Neurosc.
6, 2367-2377.

Jackson,A . and A. R. Crossman (1981) Subthalamic projection
to nucleus Tegmenti pedunculopontins in the rat.
Neuroscience Letter; 22, 17-22.

Kanazawa, 1., Marshall, G. R. and J. 8. Kelly (1976) Afferents
to substantia nigra studied with Horseradish peroxidase
with special reference to fibers from the subthalamic
nucleus. Brain Res. 115, 485-491.

Kim, R., Nakano, K., Jayarama, A. and B. Carpenter (1976)
Projections of the globus pallidus and adjacent
structures: An autoradiographic study in the monkey.
J. Comp. Neurol. 169, 263-290.

Kita, H., Chang, H. T. and S. T. Kitai (1983) The morphology

 

 

of intracellularly labeled rat subthalamic neurons:
A light microscopic analysis. J. Comp. Neurol. in
press.

Kita, H., Chang, H.T. and S.T. Kitai (1983) Pallidal inputs
to subthalamus: Intracellular analysis. Brain Res.
264, 255-265.

Kitai, S. T., Kocsis, J. D., Preston, R. J. and M. Suginori
(1976) Monosynaptic input to caudate neurons
identified by intracellular injection of horseradish
peroxidase. Brain Res. 109, 601-606.

Kitai, S. T. and J. M. Denau (1981) Cortical inputs to the
subthalamus: Intracellular analysis. Brain Res.
214, 411-415.

Kodama, S. (1928) Beitrage zur normalen Anatomie des corpus
Luysi beim Menschen. Arb.anat.sendai 13, 221-254.

Koeing, J. F. R. and R. A. Klipple (1963) The Rat Brain:
A stereotaxic atlas of the forebrain and lower partes
of the brain stem. Baltimore: Williams and Wilkins.

Kolliker, V. (1896) Handbuch der Gewebelehre des menschen.
Bd. 2, Aufl. 6, Engelmann, Leipzig.

Kooy Van Der, D. and T. Hattori (1980) Single subthalamic
nucleus neuronsproject to both globus pallidus and
substantia nigra in rat. J. Comp. Neurol. 192, 751-768.

Kooy Van Der, D., Hattori, T., Shannak, K. and O. Horykiewicz
(1980) The pallidosubthalamic projection in the rat.
Brain Rse. 204, 253-268.

Kuhlenbeck, H. (1948) The Derivatives of the thalamus
ventralis in the human brain and their relation to
the so-called subthalamus. Military Surgeon;

102, 433-447.

Kuhlenbeck, H. and W. Haymaker (1949) The derivatives of the
hypothalamus inthe human brain, their relation to
the extrapyramidal and anatomicsystems. Military
Surgeon; 105, 26—52.

Kunzle, H. (1975) Notes on the application of radioactive
amino acids for the tracing of neuronal connections.
Brain Res. 85, 267-271.

Kunzle, H. and K.A kert (1977) Efferent connectiions of
cortical, area 8 (frontal Eye field) in Macaca
fasciculata. A reinvestigationusing the
autoradiographic technique. J. Comp. Neurol.

173, 147-164.

 

 

 

115

Kunzle, H. (1978) An autoradiographic analysis of the
efferent connections from premotor and adjacent
prefrontal regions (area 6 and 9) in Macaca
fasciculata. Brain Behav.Evol. 15, 185-234.

Larsen, K. D. and J. Sutin (1978) Output organization of the
Feline entopeduncular and subthalamic nuclei.
Brain Res. 157, 21-31.

Leontovich, T. A. and G. P. Zhukova (1963) The specifity of the
neuronal structure and tOpography of the reticular

formation in the brain and spinal cord of carnivora.
J. Comp. Neurol. 121, 347-379.

Levin, P. M. (1936) The efferent fibers of the frontal lobe
of the monkey, macaca mulata. J. Comp. Neurol. 63,
369-419.

Levin, P. M. (1949) Efferent fibers in: The precentral motor
cortex. (ed. p. c. BUCY), pg. 133-148. Urbana:
University of Illinois Press.

Martin, J. P. (1927) Hemichorea resulting from a local lesion

of the brain (the syndrome of the body of Luys).
Brain; 50, 637-651.

McBride, R. L. and K. D. Larsen (1980) Projections of the
feline globus pallidus. Brain Res. 189, 3-14.

Meiback, R. C. and R. Katzman (1979) Catecholaminergic
innervation of the subthalamic nucleus: Evidence for
a rostral connection of the A9 (substantia nigra)
dopaminergic cell group. Brain Res. 173, 364-368.

Mettler, F. A. (1945) Fiber connections of the corpus striatum
of the monkey and baboon. J. Comp. Neurol. 82, 169-204.

Mettler, F. A. (1947) Extracortical connections of the primate
frontal cerebral cortex. J. Comp. Neurol. 86, 119-165.

Mettler, F. A. and G. M. Stern (1962) Somatotopic localization
in rhesus subthalamic nucleus. Arch. Neurol. 7,

Meyer, M. (1949) A study of efferent connections of the
frontal lobe inthe human brain after leucotomy.
Brain; 72, 265-299.

Mirto, D. (1896) Sulla fina anatomia delle regioni
peduncolare e subthalamica vell, vomo.

Morgan, L. O. (1928) Symtoms and fiber degeneration following
experimental lesions in the subthalamic nucleus of
Luys in the dog. J. Comp. Neurol. 44, 379-403.

 

 

116

Nakamura, S. and Sutin (1972) The pattern of termination of
pallidal axons upon celle of the subthalamic nucleus.
Exp. Neurol. 35, 254-264.

Nauta, H. J. W. and W. R. Mehler (1964) The ascending effernt
projections of the substantia nigra. Trans. Amer.
Neuro. Ass. 89, 74-78.

Nauta, H. J. W. and W. R. Mehler (1966) Projections of the
lentiform nucleus in the monkey. Brain Res. 1, 3-42.

Nauta, H. J. W. and M. Cole (1974) Efferent projections of
the subthalamic nucleus. Trans. Amer. Neuro. Ass. 99,

Nauta, H. J. W. and M. Cole (1978) Efferent projections of the
subthalamic nucleus: An autoradiographic study in the
monkey and cat. J. Comp. Neurol. 180, 1-16.

Nauta, H. J. W. (1979) Projections of the pallidal complex.

An autoradiographic study in the cat. Neuroscience;
4, 1853-1873.

Nomura, S., Mizuno, N. and T. Gimoto (1980) Direct projections
from the pedunculopontine tegmental nucleus to the
subthalamic nucleus in the cat. Brain Res. 196,

223-227.

Nauta, H. J. W. and M. Cuenod (1982) Perikaryal cell labeling
in the subthalamic nucleus following the injection of
3H-Gama-Aminobutyric Acid into the pallidal comlex:

An autoradiographic study in cat. Neuroscience;
7 No: 11, 2725-2734.

Ohye, C., Guyader, C. L. E. and J. Feger (1976) Responses of
subthalamic andpallidal neurons to striatal
stimulation: An extracellular study on awake monkey.
Brain Res. 111, 241-252.

Pellegrino, L. J., Pellegrino, A. S. and A. J. Cushman (1979)
A stereotaxic atlas of the rat brain. Plenum Press.
New York and London.

Perkins, M. N. and T. W. Stone (1980) Subthlamic projections to
the globus pallidus: An electrophysiological study in
the rat. Exp. Neurolo. 68, 500-511.

Petras, J. M. (1965) Some fiber connections of the precentral
and post central cortex with the basal ganglia,
thalamus and subthalamic nucls. Trans. America. Neurol.
Ass. 90, 274-279.

Petras, J. M. (1969) Some efferent connections of the motor
and somatosensory cortex of simian primates and felid,

 

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117

canid and procyonid carnivores. Annals N. Y. Academy of
science; 167, 469-505.

Petras, J. M. (1969) Some fiber connections of the precentral
cortex (area 4 and 6) with diencephalon in the monkey
(macaca mulata). Anat. Res. 163, 322.

Rafols, J. A. and C. A. Fox (1976) The neurons in the primate
subthalamic nucleus: A Golgi and electron microsc0pic
study. J. Comp. Neurol. 168, 75-112.

Ramon-Moliner, E. (1962) An attempt at classifying nerve cells
on the basis of their dendritic patterns. J. Comp.

Ramon-Moliner, E. (1970) The Golgi-Cox technique.
Cont. Res. Meth. Neuroanatomy; page, 12-31.

Ramon y Cajal (1910-1911) Histologie du systeme nerveux de I
homme et des vertebres. Tome Z. Instituto Ramony Cajal,
Madrid (1952 reprint),pg. 438-444.

Ranson, S. W. (1939) Pallidofugal fibers in the monkey.
Arch. Neurol. and Psych. 42, 1059-1067.

Ranson, S. W., Ranson, S. W. jr. and M. Ranson (1941)
Fiber connections of corpus striatum as seen in marchi
preparations. Arch. Neurol. and Psych. 46, 230-249.

Ricardo, J. A. (1980) Efferent connections of the subthalamic

region in the rat. I. The subthalamic nucleus of Luys.
Brain Res. 202, 257-271.

Rinvic, E., Grofova, 1., Hammond, C., Feger, J. and J. M. Deniau
(1979) A study of the afferent connections to the
subthalamic nucleus in the monkey and the cat using the
Horseradish peroxidase technique. Advan. Neurol. 24,
53-69.

Rioch, D. M. K. (1930) Studies on the diencephalon of
carnivora. J. Comp. Neurol. 49, 121-153.

Rogers, A. W. (1973) Techniques of autoradiography.
Elsevier Scient. Co. N. Y.

Romansky, K. V., Usunoff, K. G., Ivanov, P. 0. and G. P. Galabor
(1979) Cortico-subthalamic projection in the cat. An
electronmicroscopic study. Brain Res. 163, 319-322.

Romansky, K. V., Usunoff, K. G., Ivanov, P. O. and R.
Hassler (1980) Pallido-subthalamic projection in the
cat. Anat. Embryol. 159, 163-180.

Rouzaire-Dubois, B., Hammond, C. Hamon, B. and J. Feger (1980)
Pharmacologicalblockade of the globus pallidus-induced

 

118

inhibitory response of subthalamic cells in the rat.
Brain Res. 200, 321-329.

Shaner, R. F. (1936) Development of the finer structure and
fiber connections of the globus pallidus, corpus of
Luys and substantia nigra, in the pig. J. Comp. Neurol.
64, 213-232.

Shibazaki, T., Hammond, C. Rouzaire, D. B. and J. Feger (1980)
Subthalamic inhibition of identified entopedunculal
output neurons in intact and lesioned rats. Neurosc.
Lett.Suppl. 5, S38.

Siegel, A., Lovis, A. and J. P. Assoni (1971) Fiber connections
of the temporal lobe with the corpus striatum and
related structures in the cat.Exp. Neurol. 33, 130-146.

Steinbusch, H. W. M. (1981) Distribution of
Serotonin-Immunoreactivity in the central nervous system

of the rat cell bodies and terminals. Neuroscience;
6’ NO. 4’ 557-6180

Sugimoto, T., Hattori, T., Mizuno, N., Itoh, K. and M. Sato (1982)
Direct projections from the centre median—parafascicular

complex to the subthalamic nucleus in the cat and rat.
J. Comp. Neurolo. 214, No. 2, 209-216.

Tsubokawa, T. (1968) Subthalamic neuron: Response to joint
movement. Brain Res. 10, 463-466.

Tsubokawa, T. and J. Sutin (1972) Pallidal and tegmental
inhibition of oscillatory slow waves and unit activity
in the subthalamic nucleus. Brain Res. 41, 101-118.

Usunoff, K. G., Hassler, R., Romansky, K. V., Wagner, A. and
J. F. Christ (1982) Electron microscopy of the
subthalamic nucleus in the baboon. II. Experimental
demonstration of pallido-subthalamic synapses.
J.Hirnforsch. 23, 615-625.

Valverde, F. (1970) The Golgi method. A tool for comparative
structural analysis. Conte. Res. Meth. in Neuroanatomy;
Pg. 12-31.

Veazey, R. B. and C. M. Severin (1980) Efferent projections of
the deep mesencephalic nucleus (pars lateralis) in the

Verhaart, W. J. C. and M. A. Kennard (1940) Corticofugal
degeneration following themocoagulation of areas 4,6
and 45 in macaca mulata. J. Anatomy; 74, 239-254.

Watanabe, K. and E. Kawana (1982) The accessory nucleus of
Luys in the rat. Okajimas Folia Anat. Jpn., 58 (4-6):
859-874.

 

119

Whittier, J. R. (1947) Ballism and the subthalamic nucleus
(nucleus hypothlamicus; corpus Luysi). Arch. Neurol.
Psych. 58, 672-692.

Whittier, J. R. and F. A. Mettler (1949) Studies on the
subthalamus of the rhesus monkey. J. Comp. Neurol.
90, 280-317

Whittier, J. R. and F. A. Mettler (1949) Studies on the
subthalamus of the rhesus monkey. J. Comp. Neurol.

Woodburn, R. T. (1946) The major efferent connections of
the Basal Ganglia. Anat. Res. 94, 507.

Yelnik, J. and G. Percheron (1979) Subthalamic neurons in
primates: A quantitative and comparative analysis.
Neurosci. 4, 1717-1743.

Yoshida, M., Rabin, A. and M. Anderson (1971) Two types of
monosynaptic inhibition of pallidal neurons produced
by stimulation of the diencephalon and substantia nigra.
Brain Res. 30, 235-239.

Yoshida, M. (1974) Functional aspects of, and role of
transemitter in, the Basal Ganglia. Confin. neurol.

 

 

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