Evaluation of Interstitial Nerve Cells in the Central Nervous System: A Correlative Study Using Acetylcholinesterase and Golgi Techniques

59 Pages·1968·2.98 MB·English

by  Dr. Gopal D. Das

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Prof. Dr. A. BRODAL, Universitetet i Oslo, Anatomisk Institutt, Karl Johans Gate 47
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Prof. W. lIILD, Department of Anatomy, The University of Texas Medical Branch,
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Ergebnisse der Anatomic und Entwicklungsgeschlchte
Advances in Anatomy, Embryology and Cell Biology
Revues d'anatomie et de morphologie experimentale
41·1
Editores
A. Brodal, o.~lo· W. Hild, Galveston· R. Ortmann, Koln
T. H. Schiebler, Wurzburg· G. Tondury, Zurich· E. Wolff, Paris
Gopal D. Das and Georg W. Kreutzberg
Evaluation of Interstitial Nerve Cells
in the Central Nervous System
A Correlative Study Using Acetylcholinesterase
and Golgi Techniques
W itk 38 Figures
Springer-Verlag Berlin Heidelberg New York 1968
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Contents
Introduction. . . . 7
Material and Methods 10
Results . . . . . . 10
I. General Comments 10
2. Internal Capsule 11
a) Interstitial Neurons in Relation to the Globus Pallidus 11
b) Interstitial Neurons of the Entopeduncular Nucleus. 18
3. Anterior Commissure 20
4. Column of Fornix 22
5. Tractus Peduncularis Transversus . 26
6. Restiform Body ........ 28
7. Spinal Tract of the Trigeminus Nerve 33
8. Pyramidal Tract . . . . . . . . . 39
9. Ventral Spinocerebellar Tract 43
10. Some Observations on Other Fiber Bundles. 46
II. Changes in the Interstitial Neurons During Postnatal Development 46
Discussion 48
Summary. 54
References. 56
Index ............................... 59
Introduction
The presence of nerve cells in the white matter of the spinal cord and in the
spinal and cranial nerves has attracted the attention of some researchers in the
past. Because of their location in such unexpected regions, these neurons provided
a rich field of speculation regarding their nature and function. This was partic
ularly true about the nerve cells lying in the spinal white matter.
From phylogenetic considerations, neurons in the spinal white matter are
present more abundantly in amphibians, reptiles and brids than in mammals.
A. brief survey of literature on lower vertebrates indicates that GASKELL (1885,
1889) was the first to describe the displaced neurons in the white matter of the
spinal cord of alligators and various species of birds. In his consideration they were
displaced ganglion cells. In 1902 von KOELLIKER gave an exhaustive account of
such neurons in the white matter of the spinal cord of reptiles and birds. In these
animals he observed clusters of such neurons running in longitudinal columns and
thus was able to group them into nuclei known as "Hofmann's nuclei". Further,
he suggested that these nuclei arise from the mass of the ventral horn and that
they may give rise t.o preganglionic fibers, motor fibers or ventral commissural
fibers. In t.he ensuing years investigation of these nuclei was extended by STREE
TER, KRAUSE, TERNI, HUBER and others (quoted from ARIENS KAPPERS et. aI.,
1960, Vol. I, p. 206-210).
In mammals, according t.o SHERRINGTON (1890), in 1873 BEJSSO first described
the displaced neurons in t.he white matter of t.he spinal cord of the ox. It was
SHERRINGTON (1890) who presented a detailed account of the outlying neurons
in the white matter of spinal cord of various mammals like the mouse, rat, rabbit.,
cat, dog, ox, lion, monkey and man. He distinguished different types of outlying
neurons in the different funiculi on the basis of their cytological characteristics.
In t.he anterior or ventral funiculus he found medium to large size neurons, whose
axons were found to contribute to the ventral whit.e commissure. The small
fusiform neurons in the lat.eral funiculus were considered by him as a part of the
sympathet.ic column, and those in the posterior or dorsal funiculus showed a
close resemblance to the nerve cells of the column of Clark. POLYAK in 1922
described neurons of varying sizes in all the funiculi of t.he spinal cord in man. He
considered most of these nerve cells to be displaced sensory neurons. BERTRAND
and VAN BOGAERT (1923) seemed to agree with SHERRINGTON on cytological
distinctions of the nerve cells lying in different funiculi. Despite t.heir differences
of opinion on the nature of outlying or aberrant neurons, however, they concurred
on one aspect, namely these nerve cells are displaced from the adjacent neural
structures.
Subsequent to these publications, after nearly 30 years lapse, DUNCAN (1953)
presented his quant.it.ative findings on the distribution of outlying nerve cells
8 G. D. DAS and G. W. KREUTZBERG:
in the white matter of spinal cord of cynomologous monkey and man. In general,
at cervical, lumbar and upper sacral levels such neurons are found in larger number
than in the thoracic region. Implicitely, he too considered them as displaced
sensory neurons.
It is important to distinguish the nerve cells under question from those found
in the human embryos. YOUNGSTROM (1944) and HUMPHREY (1950) have described
sensory-type neurons in the roof plate of spinal cord of the human embryos and
young fetuses. In the later stages they were found in the dorsal funiculus of
the spinal cord. These nerve cells, according to these authors, disappear as the
fetus grows presumably after having served their function; whereas the outlying
or aberrant neurons, described above, are to be found in adult animals.
Findings on the nerve cells in all regions of the spinal nerves also have a
similar long history. SCHAFFER in 1881 first described them in the ventral roots
of the spinal nerves of the cat. Following him ONODl in 1886 and HOCHE in 1892
reported on their presence in man; TANZI in 1893 described them in cat; and
BECHTEREW in 1900 described them in man (quoted from SOSA and ANDREW,
1947). PILOTI (1930) and WINDLE (1931) found sensory neurons in the ventral
roots of spinal nerves in the human fetus and cat. WINDLE, in the same publication
has brought attention to the finding of such neurons by VON KOELLIKER in 1894,
who thought them to be either sympathetic or sensory-type neurons. In 1944
CONEL saw small-size scattered nerve cells along the ventral margin of the spinal
cord and in the ventral rootlets immediately outside the cord of human infants.
In his consideration they were displaced neurons.
In the dorsal roots of the spinal cord of the cat and dog, DUNCAN and CROCKER
(1939) found sensory neurons. After severing the spinal ganglia from the dorsal
roots, they were able to locate scattered sensory nerve cells in the central ends
of the severed dorsal roots. These observations led them to suggest that these
nerve cells are dispersed from the spinal ganglia. Of significant relevance are the
findings by SOSA and ANDREW (1947) who established the presence of sensory
neurons not only in the central ends of dorsal and ventral roots, but also in the
peripheral portions of the spinal nerves in man. They indicated that these isolated
neurons have their origin in the dorsal horn and migrate to different portions of
the spinal nerves through the dorsal roots. This seems to throw a different light
on the problems of migration and dispersion of nerve cells in the spinal nerves and
also probably in the white matter of spinal cord.
Literature on the occurrence of nerve cells in the cranial nerves indicates
that also in these nerves, neurons are found as frequently as in the spinal nerves.
Almost all the reports show their presence in the central roots of the cranial
nerves. As early as in 1887 THOMSEN found, what he called "Ganglienzellen"
scattered in the central ends of the oculomotor, abducens and facial nerves
which he identified as the sensory neurons. Similar sensory-type nerve cells in
the oculomotor, trochlear and abducens nerves of various mammals were described
by TOZER and SHERRINGTON (1910) and TOZER (1912). NICHOLSON (1924) in his
studies on the cranial nerves of human children found medium to large-size
neurons in the oculomotor and abducens nerves. These authors, without any
exception, identified these nerve cells as the sensory neurons closely resembling
those in the spinal ganglia.
Evaluation of Interstitial Nerve Cells in the Central Kervous System 9
~erve cells in the motor root of trigeminus nerve of the cat and other mammals
were described by TAKEDA (1924, 1925) and ALLE~ (1925). Whereas, PETERS
(1935) in his extensive studies on the trigeminus nerve of the guinea pig, pig,
rabbit, cat, dog, ox and man located medium to large size nerve cells in the motor
as well as sensory components of this nerve. He did not find any order in their
distribution, and commented that they vary in number and may be found
between pons and semilunar ganglion. His observations led him to suggest that
these are the displaced cells from the semilunar ganglion of the trigeminus nerve.
In the hypoglossal nerve of human fetuses and adults, PEARSON (1943, 1945)
detected sensory neurons. He found that in the early stages these neurons are
seen in the intramedullary course of the nerve near the hilus of inferior olive,
and they appear bipolar. During development these nerve cells are scattered and
take a unipolar shape.
In comparison to the wealth of findings and speculations on the nerve cells in
the spinal white matter and in the spinal and cranial nerves, there is extremely
meager information available on such neurons in the white matter of other regions
of the central nervous system. It may possibly be due to the fact that in the central
nervous system it is difficult to draw a sharp line between white matter and the
adjacent nuclei. Even when occasional neurons in the fiber tracts are seen, they
are regarded as displaced cells. WI~KLER and POTTER (1914) in the cat and FOIx
and XICOLESCO (1925) in man have described nerve cells in the internal capsule
and considered them to be the aberrant or displaced neurons from the globus
pallidus. But R.UI6x Y CAUL (1911), who saw scattered as well as grouped nerve
cells in various fiber bundles of the central nervous system, apparently did not
subscribe to this viewpoint. Instead he called them interstitial nerve cells. 'Yith
Golgi technique, he was able to distinguish the morphological peculiarities of these
neurons more adequately than others who used routine basophillic stains. It
seems proper to defer his observations which will be treated in detail in other
sections of this presentation.
Kumerous designations have been given to these nerve cells situated in the
fiber bundles, such as: displaced, aberrant, heterotopic, paragriseal, intra·
medullary, intercalated and interstitial. Irrespective of the name given to them,
however, they are always regarded as part of the adjacent or neighboring neural
structures. They are assumed to have been displaced into the white matter by
mechanical forces operating during the growth and development of the nervous
system. These assumptions have neither been proved nor disproved. The studies
cited above are based on the material stained with routine basophillic stains
which can at best provide limited data. In addition to this, lack of information
on the morphological changes that these neurons undergo during development
restricts their evaluation.
Although the literature reviewed above shows that almost all the studies are
confined to the spinal cord and the spinal and cranial nerves, they have established
the fact that presence of neurons in these regions is not an exceptional phenom.
enon. Furthermore, they point to the possibility of the existence of such nerve
cells in the fiber bundles in higher regions of the central nervous system. How
can such neurons be identified and distinguished from those of the adjacent
nuclei? To what extent would neurons situated in the higher regions of the nery·
10 G. D. DAS and G. W. KREUTZBERG:
ous system bear a similarity or dissimilarity to those in the spinal white matter
and the spinal and cranial nerves 1 These issues can be more adequately resolved
by histochemical and Golgi techniques than by standard histological staining
methods. Therefore, the focus of the present investigation was centered upon
those regions of the central nervous system that are rostral to the upper-most level
of the cervical spinal cord.
In our histochemical studies on the postnatal development of the central
nervous system, neurons in various fiber bundles showed a characteristic acetyl
cholinesterase activity and a systematic pattern of distribution. The postnatal
developmental changes in the fiber bundles, where they are situated, seemed to
have a marked influence on the morphology of these neurons. These observations
prompted replication of the study with a large number of animals of various
ages in order to gain an insight into the postnatal influences on these neurons.
It also appeared imperative to look into their morphological peculiarities and
similarities in relation to the neighboring neural structures, and for this purpose
a parallel study with Golgi method of impregnation was conducted. The following
presentation is the result of an attempt to correlate the findings on the nerve
cells of white matter of the central nervous system by these two methods.
Material and Methods
Rabbits and guinea pigs of the following ages were used for acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE) reactions: newborn, 3, 6, 9, 12, 16,20,25,30 and 40 days,
3 and 6 months. The animals were anesthetized by ether prior to sacrificing them. The unfixed
brains were removed very quickly, cut into 3 or 4 blocks depending upon the size of the brain,
and these blocks were frozen with carbon dioxide on freezing stages. On a Dittes-Duspiva
cryostat 16 I.L thick sections were cut serially. Every fifth section was saved at those levels,
where on the basis of previous observations the interstitial neurons in compact fiber bundles
were expected to be present. At other levels every 20th section was saved. Most of the sections
were used for AChE reaction and some representative sections from each level were used for
BChE reaction.
For acetylcholinesterase reaction GOMORI'S modification of KOELLE and FRIEDENWALD'S
technique was employed (GOMORI, 1952). It was found that 3 hours incubation gave satis·
factory results. Using butyrylthiocholine iodide as substrate, some sections were treated for
BChE reaction. Incubation period in this case was 24 to 30 hours.
Guinea pigs of 1, 3, 6, 9, 12, 15, 20, 30 and 90 days age, and rabbits of 1 and 4 months age
were used for Golgi method. Modification of Cox method according to RAM6N-MoLINER (1958)
was employed. Transverse sections of 80--100 I.L thickness were cut serially and every section
was saved. After preliminary evaluation of the interstitial neurons, drawings of the most
representative of these nerve cells were made with the aid of a Wild microscope and its
drawing-tube arrangement at a magnification of 400 to 500 x. This permitted an accurate
recording of details of the interstitial neurons and their dendrites. At such a high magnification
it was not possible to follow an axon in its entirety without overlooking the numerous breaks
in it. Therefore only the initial segments ofaxons, where clearly identifiable, were sketched
upto the first break.
Results
1. General Comments
In the present study interstitial neurons only in those fiber bundles of the
central nervous system are described, which in transverse sections appear well
delineated from the neighboring neural structures and compact in their organi
zation. These restrictions permitted investigation of the fiber bundles only at
Evaluation of IllterRtitial Nerve Cells ill the Central Nervous Hystem 11
certain levels. Thc findings are presented under the headings of the fiber bundles
where the interstitial neurons werc studied.
The tcrmintcrstitial nerve cells is chosen in preference to others, for it seems
to make no assumptions regarding thc origin, affiliations and the nature of
displaccment of these nerve cells.
The interstitial nerve cells, to be described, in various fiber bundles are
characterized by moderate to intcnse AChE activity. With this enzyme reaction
thcir somas and processes are often clcarly identifiable. These nerve cells do not
show any non-specific cholincsterase activity hydrolysing the substrate butyryl
thiocholinc. Whercas, the fiber bundles, within which these neurons are located,
show an intcnse Beh E activity. With thcsc considerations only the findings with
AChE technique arc described.
Morphological interprctation of the interstitial neurons, with the Golgi method,
is essentially along the lines of classification proposed by RAMON-MoLlNER (1962)
and RAMON-MoLIN1<lR and NAUTA (1966). On the basis of dendroarchitectonics
they have attcmptcd to classify the neurons in three large categories, namely
isodendritic, allodendritic and idiodendritic neurons.
These neurons by virtue of the fact that they are located in the compact fibcr
bundles, seem to undergo great changes during the postnatal development.
Essential details on this aspect are given separately. However, the main descrip
tion of the interstitial neurons is confined to the early developmental stages of the
animals, when these nerve cells are amenable to a clear-cut evaluation.
2. Internal Capsule
The interstitial nervc cells in the internal capsule are found spread over wide
arcas. For the eonvenience of description they arc treated separately in two
categories, those in relation to the globus pallid us and those which form the
entopeduncular nucleus.
a) I nterstitial Neurons in Relation to the Globus Pallidus
AChliJ Characteri8tic8. At the region of genu the interstitial neurons surrounding
the globus pallidus are detectable with very intense AChE activity, which
distinguishes them from the neighboring neural structures. At the levels, where
the globus pallidus is largest in its size (WINKLER and POTTER, 1911, Plates VII
to X), these neurons are found in the internal capsule arching over the globus
pallidus.
Medially they appear to extend into the substantia innominata of Reichert
and laterally into the external medullary lamina of striatum (Fig. 1 c). In the
latter region thesc neurons, with an intense AChE activity, are found juxtaposed
to the lateral boundary of globus pallidus. Closely spaced serial sections reveal that
this arch of the interstitial neurons is present at all levels of the globus pallidus.
At the rostral most level of globus pallidus (WINKLER and POTTER, 1911, Plate IV)
these neurons abound with characteristic intense AChr~ activity in the region of
globus pallidus as well as in the internal capsule (Fig. 1 a). Similarly at its caudal
most level (\VINKLER and POTTER, 1911, Plate XII) these neurons are seen where
the caudal end of globus pallidus would be identified and also in the ansa lenti
cularis (Fig. Ld ).