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Michigan State
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This is to certify that the

thesis entitled

DIENCEPHALIC PROJECTIONS FROM THE ROJTHAL AND
CAUDAL PARTS OF THE DOHJAL COLJMN NUCLEI
IN THE RAT

presented by
Anthony Constantin Bonduki

has been accepted towards fulfillment
of the requirements for

Ph. D. Psychology and

Neuroscience

/'l .
/
/
/
i
I
1 .
0 ‘

degree in

 

 

       

Major professor
John I. Johnson Jr.

9 August 1977
Date 9

0-7639

 

DIENCEPHALIC PROJECTIONS FROM THE ROSTRAL AND

CAUDAL PARTS OF THE DORSAL COLUMN NUCLEI
IN THE RAT

By

Anthony Constantin Bonduki

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Psychology and Neurosciences Program

1977

ABSTRACT

DIENCEPHALIC PROJECTIONS FROM THE ROSTRAL AND
CAUDAL PARTS OF THE DORSAL COLUMN NUCLEI
IN THE RAT

By
Anthony Constantin Bonduki

Although a number of regions in the thalamus and subthalamus
have been reported to receive projections from the dorsal column
nuclei (DCN), there is no agreement in the literature over the number,
identity, and location of such regions. There are also specific dis-
agreements as to whether the rostral regions of the DCN project to
the same diencephalic targets as do the caudal regions of the DCN
(Hand and Liu: Anat. Rec., '66, l54, 353-354; Lund and Webster:

J. Comp. Neur., '67, 130, 30l-3l2; Boivie: Brain Res., '71, 28, 459-
490; RoBards and Watkins: Neurosci. Abstracts, '75, l, 2l5; Hand and
Van Winkle, '77, l7l, 83-109). The present study was carried out in
order to identify the diencephalic DCN targets in the rat, and to
assess the possible differences in diencephalic projections between
rostral and caudal parts of the DCN.

Lesions were made in the DCN of albino rats, rostral to the
obex, caudal to it, and encroaching on both parts or involving their
entire rostro - caudal extent. Control cases included eye removals,
and sham DCN lesions. Spinal hemisections at (23-64 were also made.

Following survivals of two to nine days, the animals were perfused

Anthony Constantin Bonduki

and the brains were processed according to the Fink-Heimer method
for anterograde degeneration. Terminal degeneration in the thalamus
and subthalamus was charted over projected drawings of the sections,
and counts were made of the silver grains of degenerating terminals
in five observed target regions and in four adjacent non-targets,
both contralateral and ipsilateral to the lesion. Those counts were
cross-checked by independent "judges." The data from the counts was
then subjected to a series of analyses of variance and multiple com-
parisons tests (SNKL).

The quantitative results indicated that: l) the number of
degenerating terminals was l035 percent higher on the side con-
tralateral to the DCN lesion than on the ipsilateral side
( p_< .00l ). 2) The number of degenerating terminals was lOZl per-
cent higher in the DCN lesion groups than in the control group
( p_ < .05 ), and 151 percent higher in the entire DCN lesion group
than in the rostral and the caudal DCN lesion groups ( p_ < .05).

3) Three subsets of nuclei on the contralateral side were different
from each other ( p_.< .05 ): a ventrobasal complex (lateral part:
VBl) subset containing the highest amount of degeneration: 193 per-
cent higher than in the extra-VB target nuclei and 1145 percent
higher than in the non-target nuclei; the second subset was composed
of extra-VB target nuclei: nucleus angularis (M. Rose: Mém. de

l' Acad. Pol. des Sci. et Lett., Cracovie, sér. B, '35, l-108;
Mehler: Ann. N. Y. Acad. Sci., '69, l67, 424-468), the anterior
pretectal nucleus, the magnocellular division of the medial

geniculate body, and the ventral part of the zona incerta;

Anthony Constantin Bonduki

degenerating terminals in the nuclei of this subset were 594 percent
more numerous than in the non-target nuclei; and the third subset con-
sisted of non-targets: the medial geniculate body, the optic/
superficial gray strata of the superior colliculus, the medial part
of the ventrobassal complex, and the dorsal part of the zona incerta.
A DCN projection to the anterior pretectal nucleus in the rat has
been described by Lund and Webster ('67, op. cit.) in its ventral
subdivision only; in the present study, terminals were present
throughout its dorso-ventral extent; however, only the caudal part
of the nucleus as described by Scalia (J. Comp. Neur., '72, 145,
223-258), received DCN projections. Lund and Webster ('67, op. cit.)
also described projections to the lateral part of the zona incerta;
our results, in agreement with Smith (J. Comp. Neur., '73, 148,
423-446), found them in its ventral part. A spinal projection to
VB] was confined to its rostral third, overlapping there with DCN
terminals; this projection was ipsilateral only. Similar results
were obtained by Lund and Webster (J. Comp. Neur., '67, l30, 313-328)
and Mehler ('69, op. cit.).

The target nuclei in the thalamus and subthalamus were the
same for the projections from the rostral and the caudal parts of
the DCN, and no differences were detected in the amounts of degenera-
tion ( p_ > .05 ). Also, the antero-posterior, dorso-ventral, and
medic-lateral extents of terminal degeneration within those nuclei
were almost identical for rostral and caudal lesions. Contrary to a
previous report (Hand and Van Winkle, '77, op. cit.). the amount of

degeneration in VB1 too was similar in rostral and caudal DCN cases.

This dissertation is dedicated to my parents and to my wife

Marilou; their sacrifices for me have been immeasurable.

ii

ACKNOWLEDGMENTS

I would like to express my deep affection and gratitude to my
professor, Dr. John 1. Johnson, Jr. He patiently provided me with
guidance and support, and always come to the rescue when needed. I
also extend my appreciation to the other members of my committee,
Dr. Martin Balaban, Dr. Glenn Hatton, and Dr. Charles Tweedle, for
their guidance and understanding.

I am also ever grateful to my teacher and friend, Professor
Mustapha Ziwar who guided my steps during my undergraduate years,
Dr. Venonika Grimm who introduced me to neuropsychology, Dr. Thomas
Jenkins who introduced me to neuroanatomy, and Dr. Charles R. R.
Watson who helped me master the silver-impregnation method used in
this study.

Neal Brophy and Satchel Garrison assisted with the photog-
raphy, and Michael Peterson with the histological processing of the
tissue blocks containing the lesions. Ray R. Humphrys generously
offered time and assistance with the statistical analysis of the
data. Ms. Adeline Zimmerman plowed through the handwritten manu-
script and typed its original draft. Those contributions are grate-
fully acknowledged.

This research was supported by grants N505982 and RR07049
from NIH, and 6843236 from NSF, and funds from the Department of

Psychology, Michigan State University.

iii

TABLE OF CONTENTS

LIST OF TABLES .

LIST OF FIGURES

LIST OF ABBREVIATIONS
INTRODUCTION
MATERIALS AND METHODS

Subjects .

Surgery

Controls . .
Histological Processing.
Analysis of the Data .

RESULTS

Pattern of Projections
Quantitative Results .

DISCUSSION

Quantitative Treatment of the Data .
Diencephalic Target Nuclei of DCN Projections
Ventrobasal Complex . . . . .
Zona Incerta . .
Review of the "PO" Region
Nucleus Angularis .
Anterior Pretectal Nucleus .
Magnocellular Division of the Medial Geniculate
Ipsilateral Degeneration . . . . . .
Other Nuclei . .
Rostral and Caudal DCN Comparisons
Cytoarchitecture . . .
Afferents . .
Dorsal Roots Distribution
Peripheral Receptive Fields .
Efferents . .
Overview of Rostral and Caudal DCN Differences

iv

Page
vi
vii

viii

01-9-wa (A)

Page

BIBLIOGRAPHY . . . . . . . . . . . . . . . . 5]
APPENDICES . . . . . . . . . . . . . . . . 59

A. Fink-Heimer Technique . . . . . . . . . . . 60
8. Instructions for Counting Terminal Degeneration . . 63
C. Counts of "Grains" of Terminal Degeneration: Tables. 65

LIST OF TABLES

Table Page

Cl. Counts of silver grains of terminal degeneration in
nine diencephalic nuclei resulting from rostral DCN
lesions, caudal DCN lesions, entire DCN, and mixed
rostral and caudal DCN lesions, and control lesions . 66

vi

LIST OF FIGURES

Figure Page

1. Thionine stained sections, and diagrams, through
different levels of a rostral DCN lesion (RDCN34) . . 6

2. Thionine stained sections, and diagrams, through
different levels of a caudal DCN lesion (RDCN53) . . 8

3. Photomicrographs of sections stained according to the
Fink-Heimer method, showing anterograde degeneration in
diencephalic target nuclei, and control cases . . . 12

4. Camera lucida drawings of sections from the
diencephalon, showing the extent of terminal
degeneration . . . . . . . . . . . . . . l6

5. Schematized representation of the terminal degeneration
in the target nuclei along the antero-posterior, and
the media-lateral axes of the diencephalon . . . . l9

6. Density of terminal degeneration plotted as a total
number of silver grains in a volume of tissue, from
each of nine contralateral nuclei . . . . . . . 25

7. Density of terminal degeneration plotted as a total
number of silver grains in a volume of tissue, from
each of nine ipsilateral nuclei . . . . . . . . 27

8. Coronal section through the diencephalon of the rat, at
a level corresponding to level E (to F) of Figure 4;
thionine stain . . . . . . . . . . . . . . 37

9. Coronal section through the diencephalon of the rat, at

a level corresponding to level K of Figure 4; thionine
stain . . . . . . . . . . . . . . . . . 39

vii

AntiPt,

LIST OF ABBREVIATIONS

nucleus angularis

anterior pretectal nucleus

central intralaminar nucleus (Killackey and Ebner, '72)

central lateral nucleus

cerebral peduncle

dorsal column nuclei (cuneate and gracile nuclei)

fasciculus retroflexus

lateral cervical nucleus

nucleus lateralis posterior

posterolateral nucleus, pars lateralis (Lund and Webster,
'67a and b)

medial geniculate body

medial geniculate body, pars magnocellularis; alSo
MGm: medial division of M68
M61: internal division of M68

medial lemniscus

optic tract

parafascicular nucleus

posterior region, or posterior group:
Pom, PO], Poi: medial, lateral, and intermediate
divisions respectively.
Pop: posterior part of P0 (Berkley, '73)

nucleus reticularis thalami

subthalamic nucleus

superior colliculus

suprageniculate nucleus

substantia nigra, pars compacta

substantia nigra, pars reticulata

ventrobasal complex, pars lateralis; also VPL:
ventral posterolateral nucleus

ventrobasal complex, pars medialis

zona incerta, dorsal part

zona incerta, ventral part

viii

INTRODUCTION

A number of nuclei in the thalamus and subthalamus have been
identified as recipients of projections from the dorsal column nuclei
(DCN: cuneate and gracile nuclei) in a variety of species: opossum
(Hazlett et al., '72; RoBards and Watkins, '75), hedgehog (Jane and
Schroeder, '7l), tree shrew (Schroeder and Jane, '71), rat (Lund and
Webster, '67a), cat (Hand and Liu, '66; Boivie, '7la; Jones and
Powell, '71; Jones and Burton, '74; Hand and Van Winkle, '77), and
macaque monkey (Bowsher, '61). There is a general consensus over the
connection to the ventrobasal complex (VB), but not over the "extra-
VB" nuclei. These have included a central intralaminar nucleus
(CIN), the magnocellular division of the medial geniculate, (Meme),
the parafascicular nucleus (Pf), a posterior thalamic nucleus, a
pretectal nucleus, the suprageniculate nucleus (59), the zona
incerta (21), and a "P0 group" which inconsistently includes some of
these areas. Moreover, Groenewegen et al., ('75) could ascertain a
projectiOn only to V8 in the cat; and Basbaum et al., ('77) did not
find any DCN labeling folloiwng HRP injections in a "posterior group"
in the thalamus of the rat.

Differences in cytoarchitecture have been reported between
various regions of the DCN rostral and caudal to the obex (Kuypers

and Tuerk, '64; Basbaum and Hand, '73). The differences were

reported to be correlated with the sites of termination in the DCN,
of corticofugal afferents (Kuypers and Tuerk, '64), and of primary
afferents from the dorsal funiculus, and of non-primary afferents
ascending in the dorsal and in the dorsolateral funiculi (Tomasulo
and Emmers, '72; Rustioni, '73 and '74; Nijensohn and Kerr, '75).
They have also been reported to correSpond to different patterns of
projections to the thalamus and subthalamus from various portions of
the DCN in the cat: rostral and middle portions (Hand and Liu, '66);
rostral and caudo-ventral, and caudo-dorsal portions (Hand and Van
Winkle, '77): and from rostral and caudal parts in the DCN of the
rat (Lund and Webster, '67a). However, Boivie ('7la) did not find
any difference in projections between rostral and caudal DCN in the
cat, and RoBards and Watkins ('75) reported similar projections from
different regions along the rostro-caudal extent of the DCN in the
opossum.

The purpose of this investigation is l) to identify the
various diencephalic nuclei receiving projections from the dorsal
column nuClei in the rat; 2) their relation to a "P0 region" in the
thalamus; and 3) to investigate the possible differences in
diencephalic projections from the rostral and caudal parts of the

DCN.

MATERIALS AND METHODS

Subjects

Seventy-eight albino rats from the Sprague-Dawley strain were
used in this investigation. They were from both sexes, four to ten

months old, and weighed 250 to 350 gm.

Surgery

Unilateral lesions (sixty seven) were made in different parts
of the dorsal column nuclei: rostral to the obex, caudal to the
obex, encroaching on both rostral and caudal portions (mixed), and
including the entire rostro—caudal extent of the DCN.

Surgery was done under ether anesthesia; a cylindrical cush-
ion was placed under the animal's throat to permit a flexure of the
head in an antero-ventral direction. A longitudinal incision of the
skin was then made along the midline of the back, the muscles were
separated and retracted, and the dura over the foramen magnum was
cut and reflected. A portion of the occipital bone was removed in
rostral DCN, entire DCN, and sham lesions, but not in caudal DCN
lesions. The lesions were done under an Olympus operating micro-
scope; they were made with suction, or with a scalpel, or electroly-
tically with 200-500 uA for lO-l5 secs. through a glass-insulated
tungsten microelectrode as anode which had a 60 um uninsulated tip

length. Following the lesion, absorbable gelatin sponge (Gelfoam)

4

was applied, the wound was closed, the skin sutured, and the animal

was given an IP injection of dextrose (lo-15cc).

Controls

Controls included sham DCN lesions in three animals, unilat-
eral eye removals in three rats, and cervical spinal hemisections in
nine. Eye removals were made by gently pulling the eye out of the
socket, and severing the ocular muscles and the optic nerve. Spinal
hemisections were done at the level of C3-C4; the vertebral column
was exposed, a laminectomy made, the dura reflected, and the cord
was crushed with microforceps for 15 to 20 secs. or was cut with a

scalpel.

Histological Processing

Following a survival period of 2 to 9 days, the animals were
perfused with 0.9% saline followed by l0% formalin, the brains were
extracted and sectioned frozen at 33 um thickness in a coronal plane.
Every tenth section (or fith in a few cases) was stained according
to the Fink-Heimer technique, procedure I (Fink and Heimer, '67;
Heimer, '70) for tracing degeneration (Appendix A). Adjacent sec-
tions were stained with thionine for cytoarchitectonic localization.
A block from the medulla or from the cord containing the lesion was
embedded in celloidin, cut coronally at 25 um and every fifth sec-
tion was stained with thionine to assess the extent of the damage.

Adjacent cord sections were also stained with luxol-blue.

Analysis of the Data

 

Sections from four rostral (Figure l), five caudal (Figure 2),
and ten entire and mixed DCN lesions and from five spinal hemisec-
tions, three eye removals, and two sham DCN lesions were examined
with the light microsc0pe. Terminal degeneration was charted on
projected drawings.

The results were also quantified in two rostral lesions, two
caudal lesions, two both rostral and caudal (one entire DCN, and one
mixed), and two control (one eye removal, and one sham DCN) lesions.
Animals with 3 to 5 days survivals were selected where terminal
degeneration was at an optimum level in all the nuclei. Counts of
"grains" of terminal degeneration were made under oil immersion
(objective x 40) in 5 identified target nuclei and in 4 adjacent non-
target nuclei, both contra- and ipsilateral to the lesion, and in
the contralateral target nuclei the counts were done on two consecu-
tive sections (300-330 um apart). In each nucleus of each section
counts of terminal degeneration "grains” were done in six squares
chosen randomly from a total of sixteen squares in one quadrant of a
Whipple-Hausser ocular micrometer. The random selection was
obtained by taking the last two digits from six consecutive entries
in a table of random numbers; the digit before last was considered
0 (zero) when even, and l (one) when odd. Duplicate numbers and
numbers higher than 16 were discarded. Counts made on the contra-
lateral side of the two rostral cases and the two caudal cases, and
in some of the ipsilateral nuclei, were cross-checked by ten inde-

pendent judges unfamiliar with the Fink-Heimer procedure and unaware

Figure l.

Left column: thionine stained sections through
different levels of a rostral DCN lesion (RDCN34).
The top photograph represents the most anterior
section showing tissue damage, the bottom photograph
represents the most posterior level showing tissue
damage, and the center photograph is of a section in
the middle of the antero-posterior extent of the lesion.
Right column: diagrams of the corresponding photo-
graphs on the left; damaged region is shaded in black.
Scale for all the sections is the same as in the top
photograph.

 

FIGURE l

Figure 2.

Left column: thionine stained sections through differ-
ent levels of a caudal DCN lesion (RDCN53). The
sequence of photographs is similar to that of Figure l.
The diagrams in the right column correspond to the
adjacent photographs; damaged region is shaded in
black. Scale for all the sections is the same as in
the top photograph.

 

 

 

 

FIGURE 2

10

of the location where they were counting; they were all given a simi-
lar set of instructions (Appendix B). The data from the various
counts was subjected to statistical analysis (see Results and Dis-

cussion sections).

RESULTS

Pattern of Projections

In all the cases of DCN lesions, terminal degeneration was
present on the side contralateral to the lesion (Figure 3), in
l) the lateral part of the ventrobasal complex (V81), 2) nucleus
angularis (Ang), 3) throughout the dorso-ventral extent of the
anterior pretectal nucleus (Ant.Pt), 4) the magnocellular division
of the medial geniculate (MGmc)’ and 5) the ventral portion of the
zona incerta (ZIV).

Apart from slight background staining, no degeneration was
observed on the side ipsilateral to the lesion. Ipsilateral termin-
als were present in all the target nuclei however, when some of the
caudal lesions encroached on the other side (Figure 2); and in one
animal with a rostral lesion (RDCN34), ipsilateral degeneration was
observed in Ant.Pt and MGmC only (Figure 7).

The pattern of projections to the diencephalon was similar
in all DCN lesions (Figure 3); rostral, caudal, mixed and entire.
The same target nuclei were involved, and the rostro-caudal, medio-
lateral, and dorso-ventral extents of terminal degeneration in each
nucleus were almost identical in all the DCN lesion groups (Figures
4 and 5). Figure 4 shows on projected drawings of the sections the
various levels of the diencephalon at which terminal degeneration

was found. Figure 5 shows a schematized representation of the

ll

Figure 3a.

12

Photomicrographs of sections stained according to
the Fink-Heimer method, showing anterograde
degeneration in diencephalic target nuclei, and
control cases.

A.
B.
C.

D.

Photomicrograph from Ang; rostral DCN lesion.
Photomicrograph from Ang; caudal DCN lesion.
Photomicrograph from Ang; eye removal
(control lesion).

Photomicrograph from Ant.Pt; rostral DCN
lesion

The scale below 8 is for all the sections.

 

 

FIGURE 30

 

 

 

 

 

 

 

 

 

Figure 3b.

14

Photomicrographs of sections stained according to

the Fink-Heimer method, showing anterograde degenera-
tion in diencephalic target nuclei, and control cases.
Degeneration seen in VB] (E, F) was denser than that
in other nuclear regions.

Photomicrograph from VB]; rostral DCN lesion.
Photomicrograph from VB]; caudal DCN lesion.
Photomicrograph from VB]; sham DCN lesion
(control case).

Photomicrograph from MGmc; rostral DCN lesion.
Photomicrograph from ZIV; caudal DCN lesion.

cale is the same as in Figure 3a.

Viz-c: m‘nrr'l

 

 

 

FIGURE 3!)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4.

16

Camera lucida drawings of sections from the
diencephalon (A: anterior, M: posterior), showing
the extent of terminal degeneration (stippling) from
1) a rostral DCN lesion (R), 2) a caudal DCN lesion
(C), and 3) a whole DCN lesion (W). The levels A - M
are from sections 330 um apart.

l7

 

FIGURE 40

18

 

MGmc

FIGURE 4b

Figure 5.

19

Schematized representation of the terminal degenera-
tion in the target nuclei. Degeneration in each
nucleus was plotted at its widest extent along the
media-lateral axis of the diencephalon, at successive
antero-posterior levels (A-M). The result is a dia-
grammatic projection on a horizontal section. R, C,
W: same as Figure 4. l mm = 50 um.

21

degeneration in the antero-posterior and medic-lateral axes of the
diencephalon. The degeneration extended from level A to level G in

VB In nucleus angularis and the zona incerta, it extended from C

l'
to H. The terminals in nucleus angularis were slightly more ventral
at their caudalmost level (H). In the zona incerta only the ventral
portion was involved, and the field of degeneration grew medio-
laterally at caudal levels along with the enlargement of the nuclear
area of 21. Degeneration in the anterior pretectal nucleus and in
the magnocellular medial geniculate extended from level J to M. A
small field of terminals is present at level I in Ant.Pt of the
entire DCN case illustrated because it represents a slightly more
posterior plane than in the illustrated rostral DCN and caudal DCN
cases. Similarly, degeneration is less extensive at level J of
Ant.Pt and MGmc in the rostral case illustrated because it is
slightly more anterior than its counterparts in the other two cases
represented. Both large and smaller cells in MGmc were involved in
the field of terminals. The medialmost boundary of MGmc however, is
somewhat arbitrary, since its cells large and small are scattered

medially among fibers from the medial lemniscus, and terminals get

mixed with degenerating fibers.

Quantitative Results

 

Counts of “grains" of terminal degeneration were made in the

five target nuclei (Ang, Ant.Pt, MG VB], and 21v), and in four

mc’
adjacent non-target muclei: the ventral division of the medial

geniculate body (MGB), the medial part of VB(VBm), the

22

optic/superficial gray strata of the superior colliculus (SC), and
the dorsal part of 21 (ZId). The density of DCN terminals in each
of those nine nuclei on the contralateral side is plotted in Figure 6.
Counts of "grains" in the ipsilateral nine nuclei are plotted in Fig-
ure 7.
Discrepancies between these counts and the counts made by
the independent judges did not exceed 9%, with a mode of 2 to 3% in
the contralateral target nuclei, and did not exceed one "grain" in
the contralateral non-targets (Appendix C).
Fmax tests for homogeneity of variances (Winer, '62) were
computed separately for the group of targets, and the group of non-
targets, contralaterally, then ipsilaterally, for each of the lesion
cases. The results indicated homogeneity.
In order to determine if any statistical differences were
present in the data, a three-factors analysis of variance (lesions
x sides x nuclei) with repeated measures on two factors (sides x
nuclei), was computed (Winer, '62); all the main effects were signifi-
cant ( p_' s < .00l ) : sides, f.( 1,72 ) = 276.53; lesions, 5
( 3,72 ) = 67.89; nuclei, f_( 8.72 ) = 61.08. Two-way interactions
( p_< .00l ) and a 3-way interaction ( p_< .00l ) were present.
Differences between the lesion groups were then tested with
a two-factors (lesions x nuclei) with repeated measures on one factor
(nuclei) analysis of variance. The analysis was made on the con-

tralateral target nuclei in two rostral, two caudal, one entire DCN,

23

and two control cases.* The result was E_( 3,34 ) = 24.68, p_< .001
This was followed by multiple comparisons using the Student-Newman-
Keuls procedure (SNKL) (Winer, '62), it indicated that:
l. The control group differed from the other three groups
(2<.05)
2. the entire DCN case differed from the other three
groups ( p_< .05 )
3. the rostral and the caudal groups did not differ
from each other ( p_> .05 )
Differences between the nuclei were tested in the same way:
a two-factors (lesions x nuclei) with repeated measures on one factor
(lesions) analysis of variance was computed for all nine (targets
and non-targets) contralateral nuclei on two rostral, two caudal, and
two general (one mixed and one entire DCN) cases. The result was
f_( 8,53 ) = 53.82, p_< .001. Three subsets significantly different
from each other ( p < .05) emerged from the SNKL test: 1) VB];

2) extra-VB targets (Ang, Ant.Pt, MG ZIV); and, 3) non—targets

mc’
(MGB, Sc, VBm, ZId).

 

*In all analyses of variance, the count of contralateral
SC in the eye removal control case (Figure 6) was substituted by
zero.

 

DISCUSSION

Quantitative Treatment of the Data

An initial apprehension at quantifying Fink-Heimer results was
not borne out. The procedure proved to be simple and reliable; it
does, however, require "clean" sections, relatively free from arti-
facts such as "dust," and with not too much staining of normal fibers.
Also, it is necessary to account for the nuclei being considered when
terminal degeneration in each of them is at its optimum.

Three necessary assumptions for computing analyses of vari-
ance were met: 1) samples (sampled squares) were drawn at random;
2) variances of the several subgroups (targets, non-targets, on dif-
ferent sides, and in different lesion cases) were homogeneous, as
determined by the Fmax test; and 3) the amount of degeneration in
each nucleus was assumed independent from that in the other nuclei.

The interactions obtained in the three-factors analysis of
variance may be accounted for by the inclusion of data from nuclei
free of terminal degeneration: l) ipsilateral target and non-target
. nuclei in all cases; 2) contralateral non-target nuclei in the DCN
lesion cases; and 3) contralateral target and non-target nuclei in
the control cases.

Degeneration resulting from either rostral or caudal DCN
lesions may be considered "moderate" in the extra-VB target nuclei

and "heavy" in VB; that resulting from entire DCN lesions may be

24

Figure 6.

25

Density of terminal degeneration plotted as total
number of silver grains in a volume of tissue

(103 x 5.5 um3), from each of nine contralateral
nuclei. R and r: rostral DCN lesions; C and c:
caudal DCN lesions; M: mixed lesion (partial
rostral and caudal); W: entire DCN lesion: E: eye
removal (control); S: sham DCN lesion (control).

The different groups identified by the analyses
of variance and the multiple comparisons tests (SNKL)
may be visualized in Figures 6 and 7: l) at the bottom
of the graphs rest the contralateral non-target nuclei,
the nine ipsilateral nuclei, and all the nuclei (both
sides) in the control cases (except for contralateral
SC in the eye removal case). Then 2) at a high level
come the contralateral extra-VB target nuclei (Ang,
Ant.Pt, MGmc, ZIV) in the lesion cases, they overlap
with each other in density of terminals so that they
all fall in one category of density. No difference
appears between caudal and rostral cases either since
they are very close to each other and they overlap in
all the nuclei. Next, 3) at a higher level, degen-
eration in VB] from rostral and caudal lesions forms a
special sub roup, with caudal and rostral again over-
lapping. 4? Projections from the entire DCN are
consistently more dense than from rostral or caudal
DCN in all the contralateral target nuclei. Finally,
5) it appears that the projection from the entire
DCN to V8] and the retinal projection (eye removal) to
SC are in a further different category of density.

26

 

 

" u _
E _
m m m
u n u
u m m (C Rc ME
" — . ws
_ _ _
. . _
_ . _
. u c . E
u u R M .l C u S
u _ _
m m M
n W n M C " ES
m h u
u n n
n w n C FC " SE
n . .
u u n
. _ u
u u u M rCR E
n u u s w
. _ _
n _ _
_ u n
u . M c C rR _ S E
_ n u
m u u
u u " SE
_ C TC
m m m
m u W
W m M C u u S E
u u u
_ . _
_ _ .
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m m. w w m m w m w m m
”.22. on x n m._<z_2mme no 69232 w
>><m1 >mm> >><MI mhdmmoO—z Oznomoxodm

SC

ANG ANTPT MGmc MG

Zlv Zld
FIGURE 6

vem

v13|

Figure 7.

27

Density of terminal degeneration plotted as total
number of silver grains in a volume of tissue
(103 x 5.5 um3), from each of nine ipsilateral
nuclei. Symbols same as in Figure 6.

 

28

 

110-1
1004
90‘
n .
280
3,
[0
L6
x70:
"’9
Z
meo-
.1
4
g
2'
3550. c
l.—
u.
C
{5404
m
2
:3
Z c
IN301 c
C
R
c R
20., c c c
c
C
O F
I 1 w
3 WS w C 3 WI 1' CC GRrE
5i: ch 1515 $ch W's SE («~55 R cs
M M r Mr M'E MR SME M
7 f 1 1 1 M # «rw 1
VB. v13m ZIV 21d ANG ANTPT MGmc MGB 50

FIGURE 7

29

considered "heavy" in the extra-VB nuclei and "very heavy" in VB. To
the latter category would also belong the degeneration in SC after

eye removal.

Diencephalic Target Nuclei of DCN Projections

 

Ventrobasal Complex

Projections from the DCN to V8] have been demonstrated in
the opossum (Hazlett et al., '72; RoBards and Watkins, '75), hedgehog
(Jane and Schroeder, '7l), tree shrew (Schroeder and Jane, '71), rat
(Lund and Webster, '67a; Basbaum et al., '77), cat (Matzke, '51;
Hand and Liu, '66; Boivie, '7la; Jones and Burton, '74; Groenewegen
et al., '75; Hand and Van Winkle, '77), raccoon (Welker and Johnson,
'65), and macaque monkey (Walker, '38; Bowsher, '61). The pattern
of termination is an "onion skin" type of somatotopic concentric
laminae: gracile fibers terminating laterally, and cuneate fibers
medially in the lateral division of V8 (Welker and Johnson, '65;
Lund and Webster, '67a; Boivie, '7la); and the medial division of V8
receiving projections from the main sensory trigeminal nucleus (Lund
and Webster, '67b; Smith '73). In this study, one caudal lesion
(RDCN53) encroached slightly on the other side damaging the nucleus
of Bischoff (Figure 2) mostly where the tail is represented (Johnson
et al., '68); ipsilateral degeneration resulted in VB], which was
confined to a small area lateralmost in VB].

Hand and Van Winkle ('77) reported that lesions rostral
to the obex produced less degeneration in VB1 in the cat than caudal

lesions, even when the former were larger. Such a difference was not

30

found in this study: density of degeneration in VB1 was similar in
rostral and caudal cases, and so were the spatial dimensions of the
area covered by the terminals. Degeneration was denser, however, when
lesions involved the entire rostro-caudal extent of the DCN (Fig- .
ure 6), and in all DCN lesions it was significantly ( p_< .05 )

denser in VB1 than in other target nuclei.

Spinothalamic fibers terminate throughout the rostro-caudal
extent of VB1 in tree shrew (Schroeder and Jane, '71) and in primates
(Mehler et al., '60; Mehler, '62 and '69). In the chimpanzee, macaque
and man-but not in subprimates-Mehler described the terminals of
fibers from the spinal anterolateral funiculus to V8] as occurring
only in some cell clusters in what he called “bursts," overlapping
with the DCN terminals in an "archipelago" fashion. A similar pattern
of terminals in the macaque's VB1 has also been reported for the
ventral Spinothalamic tract (Kerr '75). In the Opossum, ascending
afferents from all segments of the cord terminate in the most ventral
part of V8 (Hazlett et al., '72): and, in the hedgehog, no spinal
fibers were found terminating in V8 (Jane and Schroeder, '71). In
the cat, Spinothalamic fibers (except from the lateral cervical
nucleus: LCN) do not terminate in VB1 but in the ventral lateral
nucleus and do not overlap with the DCN projection to V8 (Boivie,
'7lb; Jones and Burton, '74); projections from the LCN, however,
terminate throughout VB1 but are concentrated dorsolaterally
(Boivie, '70). In the rat, a Spinothalamic projection has been
described as terminating in the rostral third of V8, overlapping

there with DCN terminals (Lund and Webster, '67b; Mehler, '69). Our

31

results are in agreement with such a description; degeneration from
spinal hemisections at C3-C4 was present in VB1 in the most rostral
two (levels A and B in Figure 4) of the levels where DCN terminals

were found in V8 (levels A to G). Lund and Webster ('67b) observed
an additional field of degeneration in the caudolateral portion of

V8], which occurred after high cervical cord hemisections (C1-C2)

but not at C or below; they attributed it to the LCN. The rat's

3
homologue of an LCN, however, appears to extend throughout the seg-

ments of the cord (Gwyn and Waldron, '68).

Zona Incerta

A projection from DCN to zona incerta has been reported in
the opossum (Hazlett et al., '72; RoBards and Watkins, '75), the
hedgehog (Jane and Schroeder, '71), and the cat (Hand and Liu, '66;
Hand and Van Winkle, '77). In the latter species, Boivie ('7la)
described it as occupying the medial portion of 21, while Spinothala-
mic terminals (other than LCN's) occupied the lateral portion. In
the rat, however, Lund and Webster ('67a) traced DCN fibers to the
lateral part of 21. A ventral division of 21 (ZIV) has also been
reported as the recipient of projections from the DCN in the hedgehog
(in: Schroeder and Jane, '71), and from the principal sensory
trigeminal nucleus in the rat (Smith, '73).

In the present study, terminal degeneration extended through-
out the medio-lateral extent of the ventral region of 21. More dor-
sally, there seemed to be a slight amount of degeneration, but when

counted it did not rise significantly above the background (Figure 6),

32

and statistical analysis placed the dorsal region (ZId) in the non-
target nuclei subset. At the most then, there may be a few scattered
'ML fibers terminating in ZId. The dorsal boundary of the degenera-
tion in ZIv however, varied slightly from section to section (Fig-

ure 4).

Review of the "P0" Region

0f the four diencephalic nuclei (Ang, Ant.Pt, MGmc, ZIV)
which were identified in this study as a subset of extra-VB targets,
three (Ang, Ant.Pt, and MCmC) have often been included in a "P0"
region. A "posterior nuclear group" was described in the cat by Rose
and Woolsey ('58) as composed of the suprageniculate nucleus ($9),
the area extending anteriorly from the medial geniculate to V8, and
MGmc which they considered a differentiated portion of P0. Poggio
and Mountcastle ('60) extended it to include the posterior nucleus
of Rioch ('29) and the ventral portion of his lateral posterior
nucleus (LP). They reported a heterogeneity of cellular character-
istics in this zone (P0): some cells were found to be modality spe-
cific, responding only to auditory, vestibular, light mechanical, or
noxious stimuli: other cells were found to be multimodal, responding
to more than one stimulus modality. All somatic sensory responses
were to bilateral stimulation, a fact they attributed to bilateral
termination of afferents from the anterolateral column of the spinal
cord. The bilaterality was also reported by Whitlock and Perl in the
cat ('59) and monkey ('61); they, however, restricted their P0 region

to MGmc and 59. Furthermore, Poggio and Mountcastle ('60) believed

33

that DCN projections do not terminate in P0, but that their fibers
"terminate wholly within VB."

Moore and Goldberg ('63) subdivided the P0 region in the cat
into: 1) a medial portion (Pom) receiving afferents from the ascend-
ing somatic sensory pathways; and 2) a lateral portion (P01) which
they described as an anterior extension of MGmc’ reaching
dorsolaterally over the caudal end of V8 and separating it from the
external medullary lamina, and which received projections from the
inferior colliculus. MGmc was dealt with separately, apparently
excluded from the P0 group. In the rabbit, however, Tarlov and
Moore ('66) could not identify a similar P01; instead, they referred
to a P0 homologous to the cat's Pom, and equated the internal divi-
sion of the medial geniculate (MGi = MGmc) with the cat's P0]. And
in the kangaroo rat, Carey and Webster ('71) did not identify a P01
but instead, they described inferior colliculus projections to
"medial posterior thalamic nuclei" and to "lateral posterior thalamic
nuclei."

Jones and Powell ('68) forther defined a ''P0 group" in the
cat similar to that of Goldberg and Moore ('63), but added to it
"adjacent portions of the pretectum" and the dorsal part of M68.

The dorsal lobe of M68 had previously been included in P0, but had
been specifically mentioned only by Tarlov and Moore ('66) in the
rabbit. Diamond et al., ('69) then subdivided the cat's P0m into an
intermediate P0 (Poizbetween P0m and P01) and a somewhat shrunken
Pom. .While PO1 received afferents from the interior colliculus and

from auditory neocortical areas AI and A11; Poi received projections

34

from the lateral tegmental system and from the posterior ectosylvian,
insulo-temporal, and suprasylvian fringe auditory areas of the neo-
cortex. P0m in the cat has also been subdivided by Berkley ('73)
into: 1) an anterior portion (Pom) between VB and the intralaminar
nuclei, where few cells possessed wide peripheral receptive fields
and displayed multimodal responses; and 2) a posterior portion (Pop)
composed of MGmc and 39, where many cells displayed such characteris-
tics.

Some authors, however, have expressed discontent with the P0
concept and its inconsistencies. Graybiel ('72) favored replacing
the P0 term and its subdivisions with Rose and Woolsey's "posterior
nuclear group," and addressing nuclear entities such as dorsal M68
and 59; she also relegated the rostral portion of P0m back to the LP
nucleus. And Jones and Burton ('74) dropped 89 and (most of) MGmc
from the PD group, but they retained the term and its pom, P0], and.
P0, subdivisions. In the present study, discrete loci of degenera-
tion were observed in the posterior region of the thalamus, they are
best described in specific nuclei: nucleus angularis, the anterior
pretectal nucleus, and the magnocellular division of the medial

geniculate body.

Nucleus Angularis

A region composed Of large and small cells, capping the
dorsomedial aspect of V8 and intercalated between VBm and the central
lateral nucleus (CL) was included by Poggio and Mountcastle ('60) in

the cat's posterior nuclear group. This region which corresponds to

35

the ventral portion of Rioch's ('29) LP nucleus, has emerged as the
major component of P0m (Moore and Goldberg, '63; Diamond et al., '69;
Boivie, '71a and b: Jones and Powell, '71; Berkley, '73); it appears
to correspond to nucleus angularis of the present study.

Projections to this area (Ang or Pom) in the cat have been
reported from the DCN (Boivie, '7la; Jones and Powell, '71; Jones
‘ and Burton, '74; Hand and Van Winkle, '77), the Spinothalamic pathway
(Boivie, '7lb; Jones and Powell, '7l; Jones and Burton, '74), the
lateral cervical nucleus (Boivie, '70), and neocortical areas SI and
511 (Jones and Powell, '68 and '71).-

In the opossum, Hazlett et al., ('72) described DCN, spinal
and cerebellar (dentate and interposed nuclei) projections to a
thalamic region they identified as the caudo-lateral portion of the
ventral lateral nucleus of Oswaldo-Cruz and Rocha-Miranda ('67).
’Similar results were found by Walsh and Ebner ('73) who reported also
"additional afferents from the ascending reticular formation."
Killackey and Ebner ('72) expanded the area to include "nucleus C" of
Oswaldo-Cruz and Rocha-Miranda ('67) and the central lateral nucleus.
They named this expanded area "central intralaminar nucleus" (CIN).
RoBards and Watkins ('75) then reported DCN projections in the
opossum to CIN.

Mehler ('66) considered the caudal subdivision of Olszewski's
('52) central posterolateral nucleus (VPLC) to be the primate homo-
logue of P0. DeVito ('71) equated the suprageniculate nucleus (59)
with P0 in the macaque and squirrel monkey, and she reported that the

descending somatic projections were to a region similar to the cat's

36

P0, which she then identified as oral pulvinar and the rostral part of
medial pulvinar.

In the rat, the region was included in nucleus "lateralis
thalami" by Gurdjian ('27). Lund and Webster ('67a and '67b) referred
to it as "posterolateral complex, pars lateralis" (LPL), and reported
that DCN and Spinothalamic fibers terminate in a large-celled dorsal
portion. Cowan et al., ('72) described a similar descending connec-
tion from the somatic sensory neocortex in the mouse, but they
labelled the region "ventral lateral" (VL) nucleus. Mehler ('69)
also found spinal and cerebellar projections to this area in the rat:
he suggested that it corresponds to the lateral subdivision of
nucleus angularis described by M. Rose ('35) in the rabbit, while its
medial subdivision corresponds to the central lateral nucleus.

In the present study, the region in question has been iden-
tified as nucleus angularis (Figure 8), and terminal degeneration has
been observed in it in all DCN lesions. The locus of degeneration
moves to a slightly more ventral position at its caudalmost level
(level H). Jones and Burton ('74) reported that in the cat, P0m
extends caudally to include a group of cells usually considered part
of MGmc' In the present study, loci of degeneration in the anterior
pretectal nucleus and in MGmc were separated from that in nucleus
angularis by 450 to 600 um in the anterio-posterior axis (Figures 4

and 5).

37

 

Figure 8. Coronal section through the dience halon of
the rat, at a level corresponding to level E to F) of
Figure 4; thionine stain.

38

Anterior Pretectal Nucleus

 

This is the largest of the pretectal nuclei in a variety of
mammals including the rat (Figure 9) (Rose, '42; Scalia, '72); in
the cat, however, it is much smaller and less clearly demarcated
(Rose, '42), and the most prominent of the pretectal nuclei is the
posterior (Kanaseki and Sprague, '74). The anterior pretectal nucleus
(Ant.Pt) corresponds to the anterior portion of the pretectal nucleus
and to the deep pretectal nucleus of Bucher and Nauta ('54). Unlike
the other pretectal nuclei, Ant.Pt was found to be free of terminal
degeneration following eye removal in a number of investigations in
the rat and other species (Hayhow et al., '62; Scalia, '72; Kanaseki
and Sprague, '74) and in control cases in the present study.

Ant.Pt also corresponds to the nucleus identified by Ramon
y Cajal ('09-'11) as "posterior thalamic nucleus;" this label was.
also used by Gurdjian ('27) and has since been retained for rodents
and "lower mammals." It is different, however, from the posterior
nucleus described by Rioch ('29) which has been identified in the cat
as inferior pulvinar, and shown to receive heavy projections from
neocortical areas 17, 18, and 19, and the area of Clare-Bishop
(Kanaseki and Sprague, '74; Kawamura el al., '74). Jones and Powell
('71) and Robertson and Rinvik ('73) considered the anterior pretectal
nucleus identified by Berman ('68) in the cat to be nucleus limitans,
and it was then included in the suprageniculate nucleus (59) by
Diamond et al., ('69), and by Jones and Powell ('71).

Ramon y Cajal ('09-'11) first described collaterals from the

"medial ribbon of Reil" (medial lemniscus) to the "posterior thalamic

39

 

Figure 9. Coronal section through the diencephalon
of the rat, at a level corresponding to level K of
Figure 4; thionine stain.

4o

nucleus" in the mouse. Hazlett et al., ('72) found a DCN projection
to the oppossum's "pretectal nucleus," which is considered a homologue
of the nucleus posterior thalami of Cajal (Oswaldo-Cruz and Rocha-
Miranda, '67). Hand and Van Winkle ('77) reported cuneate projections
to a suprageniculate nucleus in the cat which probably included Ant.
Pt. Jane and Schroeder ('71) illustrated terminal degeneration in the
"pretectal nucleus" of the hedgehog following DCN lesions, but they
did not discuss it. In the rat, Lund and Webster ('67a) described

DCN projections to the ventral portion (pars reticularis: Scalia, '72)
of the anterior pretectal nucleus; and Smith ('73) reported projec-
tions from the principal sensory trigeminal nucleus in the rat to a
"posterior thalamic region" which he also referred to as "posterior
thalamic nucleus," it appeared to involve a part of Ant.Pt. Libouban
('64) recorded from the anterior pretectal nucleus in the rat which
she identified as "pretectal nucleus" or "posterior thalamic nucleus
of Cajalfi'she found that medium-sized elements (approximately 40% of
the total population) were responsive to stimulation of the body
surface and face and to displacement of the vibrissae. Davidson

('65) recorded from a "posterior nuclear complex" or "P0" in the rat;
he reported a bilateral somatic representation with a rather poor
topographical organization: a tendency of the hindleg and tail
representation to be confined to a smaller lateral area. From his
diagrams it would appear that this "posterior nuclear complex" mainly
comprised the anterior pretectal nucleus, with some involvement of

MGmc and of Gurdjian's ('27) nucleus lateralis thalami.

41

In the present study, terminal degeneration was observed in
the anterior pretectal nucleus. The degeneration was not confined to
the ventral subdivision of the nucleus as previously reported (Lund
and Webster, '67a), but it was uniformly present throughout its dorso-
ventral extent. (It seemed more dense laterally.) It did not, how-
ever, extend to the rostral levels of the nucleus as deliminted by
Scalia ('72), i.e., the anterior portion of the pretectal nucleus of
Bucher and Nauta ('54) which Scalia included in Ant. Pt, did not
have any degeneration in it.

Spinothalamic afferents to the "pretectal region" have been
described in the macaque by Walker ('38). Also, in the macaque,
squirrel monkey and chimpanzee, Mehler ('69) traced projections from
the anterolateral funiculus of the spinal cord to what he suggested
may be the "posterior pretectal nucleus." He had previously found a
similar connection in man (Mehler, '62) and had identified the area
as "nucleus posterior of Spalteholz." In the hedgehog, Jane and
Schroeder ('71) illustrated spinal terminals in a "pretectal nucleus,"
and Lund and Webster ('67b) reported a spinal projection to (the
ventral portion of) the anterior pretectal nucleus.

Corticofugal projections were reported in the cat, from the
area of Clare—Bishop (in the lateral suprasylvian gyrus) and from
areas 7 and 21 (of the middle suprasylvian gyrus) to the anterior
pretectal nucleus (Kawamura et al., '74); and from all parietal areas
to nucleus limitans (=Berman's Ant.Pt) (Robertson and Rinvik, '73).
Projections from the somatic sensory neocortex have also been

reported to the “pretectal region" in the macaque and squirrel monkey

42

(DeVito, '71); and to the "ventral pretectum" (Price and Webster,
'72) and the posterior thalamic nucleus (Valverde, '61) in the rat.
Magnocellular Division of the
Medial Geniculate

Afferents from the anterolateral funiculus of the spinal cord
have been described to terminate in MGmc in the monkey (Mehler et al.,
'60), and in man (Mehler, '62). In the latter, Mehler observed that
only large cells in the dorsomedial region of MGmc were involved.
In the cat, Boivie ('7lb) reported a dense projection from the
anterolateral funiculus, relating to large cells in the medial part
of MGmC; Jones and Burton ('74), however, reported that although some
degeneration was associated with the large cells located medially,
most of the degeneration related to smaller more caudal cells belong-
ing to Pom. They suggested that "reports of spinal terminals in
MGmc proper result from failure to recognize the full extent of Pom."
Those caudally located P0m cells occupy an area in the cat's thalamus
(Figure 2 in: Jones and Powell, '71) similar to the ventral region
of Ant.Pt in the rat. Projections to MGmc were also reported from
the spinal cord in the rat (MGm: Lund and Webster, '67b), from the
lateral cervical nucleus in the cat (Boivie, '70), and from the ven-
tral Spinothalamic tract in the macaque (Kerr, '75).

DCN and spinal fibers have been reported to terminate in the
caudal portion of a posterior thalamic nucleus in the opossum
(Hazlett et al., '72), which may be the small medial division of MGB

identified by Morest ('65) in Golgi preparations in this animal.

43

DCN and spinal projections have also been described to a "P0 complex"
in the hedgehog (Jane and Schroeder, '71) and tree shrew (Schroeder
and Jane, '71); this area appears closely ralated to MGB in the
figures.

DCN fibers were reported terminating in MGmc in the cat (Hand
andLiu, '66; Hand and Van Winkle, '77). Boivie ('7la) however, did
not observe them in MGmc’ and Jones and Burton ('74) were uncertain
as to their termination there. In the rat, Lund and Webster ('67a)
reported DCN projections to MGmc ("MGm“). In the present study DCN
terminals were observed in MGmc; they appeared related to both the
large and small cells.

Smith ('73) reported a projection from the principal sensory
trigeminal nucleus in the rat to a posterior thalamic region which
included MGmc' Fibers from nucleus caudalis of the trigeminal com-
plex have also been reported to terminate in MGmc in the rat (Lund
and Webster, '67b), the cat (Stewart and King, '63), and marmoset
(Dunn and Matzke, '68). However, Tiwari and King ('74) attributed
this thalamic connection (in baboon and squirrel monkey) to a projec-

tion from the reticular nuclei adjacent to nucleus caudalis.

Ipsilateral Degeneration

RoBards and Watkins ('75) reported some degeneration in
extra-VB target nuclei ipsilateral to the DCN lesion in the opossum.
In the present study the degeneration in all the target nuclei was
strictly contralateral to the DCN lesion; ipsilateral degeneration

was only encountered when the lesion encroached on the other side,

44

as in some of the caudal lesions (Figure 2). However, in one case
with a rostral DCN lesion confined to one side (RDCN36), a light
amount of degeneration was present in the ipsilateral Ant.Pt. and
MGmc (Figure 4). It may be due to a slight encroachment of the
lesion on the inferior vestibular nucleus. Vestibular responses
have been recorded from this thalamic region (Mickle and Ades, '54;

Poggio and Mountcastle, '60: Wepsic, '66).

Other Nuclei

A DCN projection has been reported to the parafascicular
nucleus (Pf) in the opossum (Hazlett et al., '72; RoBards and Watkins,
'75). In the present study, degeneration was observed in Pf only
occasionally, and a DCN projection to it cannot be ascertained.

A projection from the DCN has been reported to the deep
layers of the superior colliculus in the opossum (Hazlett et al.,
'72: RoBards and Watkins, '75), rat (Lund and Webster, '67a), and
cat (Hand and Liu, '66: Hand and Van Winkle, '77). Some degeneration
was also observed in the present investigation, in the deep layers
of the caudal levels of the superior colliculus, following DCN

lesions.

Rostral and Caudal DCN Comparisons

Cytoarchitecture

 

Two different patterns of cytoarchitecture have been described
by Kuypers and Tuerk ('64) in various regions of the dorsal column
nuclei of the cat: 1) clusters composed of round cells with bushy

and intertwined dendrites and whose perikarya are organized in

45

circles with cell-free cores. These clusters occupy the dorsal
region of the DCN caudal to the obex. 2) Rostral to the obex, and in
the ventral region caudal to it, the cells are triangular and multi-
polar and they are diffusely organized; they are of the radiating
type, sparsely ramifying and starting to ramify closer to the soma
then the clusters cells. Basbaum and Hand ('73) also described two
cytoarchitectonic subdivisions in the rat's cuneate nucleus: 1) a
region caudal to the obex made up of round cells arranged in "bricks,"
extending throughout the medic-lateral and dorso-ventral extents of
the nucleus, except in its ventralmost aspect, and isolated from each
other by fiber bundles; and 2) a region rostral to the obex, composed
of round, spindle shaped and multipolar cells, and which does not
display the "slab“ architecture of the caudal region. This "slab"
architecture, however, simply represents the "resorting zones" where
the scrambled dorsal root fibers from various peripheral fields
“become reshuffled" into fiber laminae to produce a somatotopic
organization: in raccoons, and probably in other species as well,
those "bricks" represent the digits of the forepaw (Johnson et al.,

'68).

Afferents

Neocortical descending projections to the DCN of the cat were
reported to terminate mostly in the regions of diffuse cellular
organization, while the cell clusters region received only few fibers
from the cerebral neocortex (Kuypers and Tuerk, '64).

In addition to their main input from the primary fibers in the

dorsal funiculus (from the dorsal roots), and to their corticofugal

46

afferents, the cuneate and gracile nuclei have been reported to
receive fibers ascending in the lateral funiculus in the opossum
(Hazlett et al., '72), and in the dorsolateral funiculus (DLF) in the
cat (Gordon and Grant, '72). In the latter species, DLF fibers and
non-primary fibers ascending in the dorsal funiculus have been found
to terminate mainly in the regions of diffuse cytoarchitecture in the
DCN (Rustioni, '73 and '74). The DLF projection to the gracile
nucleus of the rat was also reported to terminate principally in the
rostral portion (Tomasulo and Emmers, '72), and so was that to the

cuneate and gracile nuclei in the macaque (Nijensohn and Kerr, '75).

Dorsal Roots Distribution

Dorsal root fibers in the cat were found to form dense terminal
plexuses in relation to specific cell clusters, while in the region
of diffuse cytoarchitecture, dorsal roots terminals were diffuse and
displayed a greater intersegmental overlap than in the clusters
regions (Kuypers and Tuerk, '64). This overlap was also greater in
the rostral than in the caudal portions of the rat's DCN (Basbaum

and Hand, '73).

Peripheral Receptive Fields

 

Gordon and Seed ('61) have reported that most of the cells in
the rostral portion of the cat's gracile nucleus possess large periph-
eral fields (51.1cm2), while those of the middle region displayed
samll fields (4.5cm2) and mutual inhibition. However, these charac-
teristics are more understandable in terms of somatotopic organiza-

tion: the large peripheral fields are from proximal parts of the

47

body, while the distal parts possess small peripheral fields and
their DCN neurons display mutual inhibition. In addition, each
particular receptive field is represented unchanged throughout its
rostro-caudal representation in the DCN (Winter, '65; Johnson et al.,

'68).

Efferents

Some authors have identified more numerous diencephalic targets
from the rostral DCN than from their other portions: Hand and Liu
('66) reported projections from the rostral gracile nucleus in the
cat to MGmc’ the "posterior thalamic complex," the ventral
posterolateral complex (VPL=VBI), and 21; while the middle portion of
the gracile projected only to MGmC and VPL. Hand and Van Winkle ('77)
further observed in the cat that the rostral portion of the cuneate
nucleus as well as its caudal portion and the ventral zone of its

middle portion projected to the fields of Forel, MG P0], Pom, Sg,

mc’
VPL, and 21: while the dorsal zone of its middle portion projected
only to MGmc’ $9, and VPL. They suggested that the former DCN regions
represented a paleolemniscal system and the latter a neolemniscal
system. In the rat, Lund and Webster ('67a) reported projections from
the portion of the DCN rostral to the obex to the anterior pretectal
nucleus, "lateral posterolateral complex" (LPL), MGm (equivalent to
MGmc)’ VPL, and 21; but only to MGm and VPL from the DCN region caudal
to the obex. However, similar diencephalic projections were reported

from various rostro-caudal levels of the DCN in the opossum (Hazlett

et al., '72: RoBards and Watkins, '75), and the hedgehog (Jane and

48

Schroeder, '71) and from the caudal and rostral portions of the DCN
in the cat (Boivie, '7la).

In the present study, lesions rostral or caudal to the obex
produced similar patterns of degeneration in the diencephalon: the
same target nuclei were involved, the amount of degeneration was
similar in the various nuclei in rostral and caudal cases and the
terminals extended over virtually identical antero-posterior, dorso~
ventral, and media-lateral dimensions in the particular nuclei in
rostral and in caudal lesions. Lesions including the entire DCN
produced denser degeneration in all the target nuclei but did not
affect its spatial extents within the nuclei. It is possible that a
lesion restricted to the dorsal portion of the DCN caudal to the
obex could have produced a different pattern. However, the quanti-
tative results obtained argue against such a possiblity: if the
dorsal portion of the caudal DCN had restricted diencephalic projec-
tions, the total amount of degenerating terminals in the various
target nuclei resulting from caudal DCN lesions would have been
smaller than that resulting from rostral DCN lesions.

Overview of Rostral and Caudal
DCN Differences

Cells throughout the gracile nucleus of the cat were fired
by antidromic stimulation of the medial lemniscus (Gordon and Seed,
'61). Injections of HRP in the cat's thalamus extending over VB
and portions of most of the extra-VB targets produced labelling in
the clusters cells but also in the multipolar and triangular cells of

the other DCN regions (Berkley, '75; Cheek et al., '75). However,

49

40% of the cells in the rostral gracile region did not respond to
antidromic stimulation in the medial lemniscus (Gordon and Seed, '61),
and some DCN cells remained unlabelled after the thalamic HRP injec-
tions, particularly small cells in the rostral region (Berkley, '75;
Cheek et al., '75). These cells may have other projections, for
example, to the inferior olive as suggested by Berkley ('75) or to
the cerebellum where HRP injections in the anterior lobe labelled
cells in the rostral DCN (Cheek et al., '75). However, at least some
of the cerebellar projections are collaterals of fibers in the medial
lemniscus (ML): Gordon and Seed ('61) reported that some gracile
units in the cat responded to antidromic stimulation in both the
cerebellum and the medial lemniscus; and Johnson et al., ('68)
described large cells in the M-T area of the cuneate, which responded
to deep stimulation only, and atrophied only after lesions of both
the cerebellum and the medial lemniscus. It is also probable that
the small cells in the DCN which remain unlabelled after thalamic
HRP injections and which do not respond to ML antidromic stimulation
are interneurons: they may be the units responding trans-synaptically
to ML antidromic stimulation (Gordon and Seed, '61), and they may be
the recipients of the corticofugal projections to the DCN, of fibers
from the dorsolateral funiculus and of non-primary afferents from the
dorsal funiculus. These interneurons may be scattered throughout the
DCN but concentrated in the rostral region.

In summary, the DCN in the rat projects to a number of dis-

crete nuclear regions in the thalamus and subthalamus; they include:

50

the ventrobasal complex (pars lateralis), nucleus angularis, the
anterior pretectal nucleus, the magnocellular division of the medial
geniculate, and the ventral part of the zona incerta. No differences
were observed in the present study in diencephalic projections from
rostral and caudal DCN. The difference between DCN regions rostral

to the obex and DCN regions caudal to it may only be in a preferential
concentration of interneurons rostrally, and/or in additional brain-

stem projections from the rostral portion.

BIBLIOGRAPHY

51

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APPENDICES

59

APPENDIX A

FINK-HEIMER TECHNIQUE

60

APPENDIX A

FINK-HEIMER TECHNIQUE

Cut frozen sections at 30 um and place them in 3% formalin.
Preliminary:
1. Maximum 4 sections (rat brain size) in each crucible
2. Wash in DW (dist. water) #1 3-5 mn
Wash in DW #2 3-5 mn
(Wash in DW #3)

3. Potassium permanganate (0.05%) 5-20 mn
agitate frequently

4. Wash in DW

5. Oxalic acid (1%) 1 mm or less

Hydroquinone (1%) 1:] ratio until decolorized
6. Wash in DW #1 3 mn
Wash in DW #2 3 mn
Wash in DW #3 3 mn
Impregnation:
1. 0.5% Uranyl nitrate 88 m1 1 hr. or more
2.5% Silver nitrate 99 m1
2. 0.5% Uranyl nitrate 80 m1 30-60 mn
2.5% Silver nitrate 120 ml
3. Wash in DW #1 3 mn
Wash in DW #2 3 mn
Wash in DW #3 3 mn

Exposure: Ammoniacal silver
agitate constantly 2-4 mn

lst Ammonium hydroxide (conc.) 4.2-4.4 ml
2nd Sodium hydroxide 5.4 ml

mix well
3rd Silver nitrate (2.5%) 90 m1

pour slowly

mix well 61

Developing:

Reducer 54 ml
54 m1

180 m1

1600 ml

1. 3 reducer changes #1
#2
#3.
2. Wash in DW
Fixing:
1. Sodium thiosulfate (1%)
2. Wash in DW #1

Wash in DW #2
Wash in DW #3

Mount in Albrecht's alcohol gelatin

1.5% aqueous gelatin
80% alcohol

1:1 ratio

1% citric acid
10% formalin

100% alcohol

DW
2-5 sec
15 sec
45 sec-2 mn

1 mn

30 sec

3-5 mn
3-5 mn
3-5 mn

Dry at room temperature, or for 10-30 mn at 58°C

Dehydration: 5-10 mn in each

70% alcohol
95% alcohol
100% alcohol
100% alcohol
Xylene #l
Xylene #2

mm‘wa-d
o o o o o o

Coverslip

APPENDIX B

INSTRUCTIONS FOR COUNTING TERMINAL DEGENERATION

63

APPENDIX 8

INSTRUCTIONS FOR COUNTING TERMINAL DEGENERATION

l. The counting I need you to do is to cross-check the one I did.

2. First, be careful not to move the table: and do not, at any time,
move the eyepiece: it would move the ocular micrometer, and
would change the area we are counting from.

3. There is a grid (an ocular micrometer) in the eyepiece; its upper
left hand-side quadrant has 16 squares in it; we are going to
count grains in 6 (random) of them; we are numbering the squares
from left to right, top row first.

4. Move the fine focus knob so that the section gets slightly out of
focus one way, then in focus, then slightly out Of focus the
other way; and count all the round black grains you will see.
Look throughout the squares and check their corners too. If a
grain is on the boundary line of the square, count it if it pro-
trudes at all inside, even if most of it is outside. 00 not
count line-like things even if they are tiny; and make sure the
grains you count are black, not dark gold or brown.

5. Count as "coarse," the grains that are half the size or larger Of
a (or: of this) nucleolus; when less than half its size, count
them as "small."

* Counting the grains as "coarse" and "small" helped prevent
overlooking the smaller fibers especially when there were many
large ones.

6. Do not count from strings of black grains or from series of
stubby lines.

If "dust" was Observed (occasionally). additional instructions were
given:
You may see a "carpet" Of fine grains uniformly distributed
throughout the section (or most of it); you can still see
them when the section is totally out of focus and blurred.
Those are not terminals, do not count them.

64

APPENDIX C

COUNTS OF "GRAINS" OF TERMINAL DEGENERATION

65

66

Appendix Table Cl.

Counts of silver grains of terminal degeneration in nine
diencephalic nuclei resulting from rostral DCN lesions, caudal
DCN lesions, entire DCN, and mixed rostral and caudal DCN lesions,
and control lesions (eye removal, and sham DCN lesion).

Letters: R, r, C, c, W, M. E, and S are the abbreviations
used in Figures 6 and 7.

In each lesion case, the contralateral counts (contra.),
ipsilateral counts (ipsi.), and contralateral counts made by the
judges, were all on the same section (section 1) for the particu-
lar nucleus.

Counts were also made on a second section in the five
contralateral target nuclei (contra. in section:2). Those
contralateral counts from two sections were added and averaged
(M). The average was used in Figure 6, but all the counts used
in the analyses of variance were from section 1 only.

67
APPENDIX C

COUNTS OF "GRAINS" OF TERMINAL DEGENERATION

 

 

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