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Scanning electron microscopy of a second type of supraependymal cell in the monkey third ventricle.

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Scanning Electron Microscopy of a Second
Type of Supraependymal Cell in the
Monkey Third Ventricle '
PENELOPE W. COATES
Department of Biological Structure, University of Washington School of
Medicine, Seattle, Washington 981 95
ABSTRACT
The third ventricle of monkeys has been examined with the
scanning electron microscope (SEM). Two populations of supraependymal (SE)
cells were distinguished on the basis of morphology and location. One type has
been previously reported (Coates, '72, '73a,b,c). Another, type 2 SE cell, is now
described.
Type 2 SE cells were found in the third ventricle of both sexes and in all age
groups although the numbers varied highly from animal to animal. The most
common site for type 2 SE cells was the floor and transition zone of the third
ventricle. Visualized with SEM, these cells had variable morphology, but may be
characterized by a small cell body, few non-branching processes some of which
were flared and surface features such as ruffled membranes. Type 2 SE cells
most likely correspond to Kolmer or epiplexus cells originally described in association with the choroid plexus. As such, they are probably phagocytes.
Macrophage-like intraventricular cells
within the cavities of the vertebrate brain
were originally described using light microscopy (Kolmer, '21). These cells were
found on the choroid plexus and named
wandering, Kolmer or epiplexus cells (Kolmer, '21; Kappers, '53). Transmission electron microscope (TEM) studies further
elaborated on the morphology of such
choroid plexus associated cells and their
phagocytic nature (Tennyson and Pappas,
'64; Carpenter et al., '70).
With the advent of the scanning electron
microscope (SEM), wide areas of cerebral
ventricular surfaces have become more
easily accessible for direct observation.
During the course of SEM study of the
third ventricle of monkeys, two different
populations of supraependymal cells (cells
situated above the ependyma) were noted
on ventricular surfaces other than choroidal. This is a report on the second of
two types of supraependymal (SE) cell.
MATERIALS AND METHODS
Third ventricles from the brains of three
species of monkeys (M. nemestrina, M.
m.ulatta and M . fasicularis) were examined in the scanning electron microscope.
ANAT. REC., 182: 275-288.
More than 50 examples from both sexes
of fetal, young, juveniles and adults were
included, although not every species was
represented for all age groups. Brains were
obtained from both normal and experimentally treated animals from a wide variety of other nonrelated studies. Where
relevant this is indicated.
Fixation was achieved by perfusion
through the heart using either 4% paraformaldehyde in 0.1 M phosphate buffer or
a combination of 4% paraformaldehyde
with 0.5% glutaraldehyde and 0.5% dextrose in 0.1 M phosphate buffer. Following
fixation, brains were removed from the
cranial cavity and stored overnight in fresh
fixative in the cold. The hypothalamic region of the third ventricle was dissected
out under a dissecting microscope, sagittally sectioned, and further trimmed into
sizes appropriate for SEM processing.
Tissue destined for SEM was postfixed
in 1.25-2.5% buffered osmium tetroxide
for 2-4 hours after which the blocks were
dehydrated in ascending grades of ethaReceived Dec. 19, '74. Accepted Feb. 13, '75.
1 This work supported by USPHS grants RR-05432
and GM-16598 from the National Institutes of Health
and GSRF 171 funds from the University of Washington Graduate School.
275
276
PENELOPE W. COATES
no1 to 100% ethanol. Occasionally, blocks
were held without ill effect in 70-75%
ethanol in the cold until processing for
SEM could be completed.
Following dehydration, either amyl acetate was substituted for the alcohol followed by critical point drying with liquid
carbon dioxide (Anderson, '51) in an
Aminco apparatus (Aminco, Silver Springs,
Md.) or freon 113 (freon TF) was substituted for the alcohol followed by critical
point drying using freon 13 (Cohen et al.,
'68) in a Bomar instrument (Bomar, Tacoma, Wa.).
The specimens were mounted on aluminum stubs with either silver conducting
paste (Ted Pella Co., Tustin, Calif.) or
silver print conducting paint (GC Electronics, Hydrometals, Inc., Rockford, 111.).
They were coated with carbon followed by
gold-palladium (60-40% ) or by gold alone.
A Cambridge Stereoscan Mark 2A scanning electron microscope and an Etec
Autoscan U-1 scanning electron microscope operating at 20 kv were used for
observation and photography.
OBSERVATIONS
Two distinct populations of SE cells,
sometimes coexisting in the same animal,
were found in the monkey third ventricle.
They differed with respect to morphological characteristics and localization. One
group, now referred to as type 1, has already been described (Coates, '72, '73a,b,c).
The second group, described below, will
be referred to as type 2. Overall, these cells
were found in both females (fig. 1 ) and
males (fig. 5) of all three monkey species
examined: rhesus (fig. l), pig tail (fig. 7)
and ring tail (fig. 8). Type 2 SE cells were
also found in all age groups: fetal (fig. 7),
young (fig. S), juvenile (fig. 6 ) and adult
(fig. 1 ) .
The most common location of type 2 SE
cells within the third ventricle was on the
nonciliated floor and transition zone between the floor and ciliated wall (figs. 1,
3 ) . Less frequently these cells were found
on the lamina terminalis and within third
ventricular recesses (fig. 4).
There was wide variation in the appearance of type 2 cells as visualized with SEM.
However, the following characteristics generally serve to distinguish type 2 SE cells.
Cell bodies were usually small, averaging
6-12 microns. The shape of the cell body
was often roughly diamond or triangular,
with rounded cell bodies also being observed (figs. 1, 6, 4). When rounded cell
bodies were encountered, invariably there
were numerous ruffled membranes, deep
cytoplasmic folds or pseudopods associated
with them (fig. 8 ) .
Many type 2 SE cells possessed processes in distinction to ruffled membranes.
Usually there were only 3-4 processes per
cell which did not branch extensively, and
did not extend from the cell body much
more than twice its diameter. Some processes flared out broadly in a splayed pattern before terminating (fig. 1). Other
such processes shared additional surface
modification in the form of fringed edges
(fig. 3). Small finger-like projections of cytoplasm were occasionally observed along
the inferior aspect of processes in contact
with the underlying ependymal surface
(fig. 2 ) . Processes and cytoplasmic veils
which were flared out to sufficient thinness
often seemed translucent, while others appeared more dense and even knobby.
Type 2 SE cells most often occurred
singly but occasionally were found in a
collection of scattered similar cells (fig. 5).
If the latter were the case, then the cells,
although grouped together, remained separate from one another, and were only
rarely in contact with each other via their
processes. An impression based on visual
observation was obtained that there was
considerable variation in the total numbers
of type 2 SE cells per third ventricle. While
definitive counts were not done at this
time, the following may provide an example. In a typical region of the floor of the
third ventricle of one monkey ( a n acute
cortically lesioned animal) there were 15
type 2 SE cells (fig. 5). Yet comparable
areas from 3 other monkeys (normal
non-operated) at the same magnification
showed only 5, 3 and 0 type 2 SE cells,
respectively.
DISCUSSION
Two populations of SE cells could be
distinguished in the SEM on the basis of
differences in morphology and location. Although it is possible that type l and 2 SE
cells are merely variations of each other,
TYPE 2 SUPRAEPENDYMAL CELLS
with the former representing a more fixed
element while the latter may be more mobile, nevertheless, the two may be compared in the following way, with morphological and distributional differences
suggesting functional differences as well.
While no one single characteristic alone
may be considered specific, taken as a
whole the evidence suggests differentiating
2 cell types.
Type 2 SE cells were distributed more
extensively throughout the third ventricle
than type 1, with the former characteristically found on the floor and transition zone
of the third ventricle and occasionally elsewhere as well, while the latter were for the
most part limited to the regions of the preoptic and infundibular recesses of the third
ventricle.
Both type 1 and 2 SE cell bodies characteristically were small (< 15 p ) . However,
the shape of type 2 cell body was typically
triangular, diamond or rounded, while the
typical cell body shape for type 1 was
rounded or ovoid only.
Furthermore, most type 1 cells were
relatively smooth surfaced while type 2
cells frequently had extensive surface modifications, consisting of ruffled membranes,
veils of cytoplasm and deep folds. The
knobby areas seen in type 2 probably represent underlying organelles such as mitochondria or membrane bound vesicles such
as lysosomes, while the short cytoplasmic
fingers extending to the underlying ependymal surface probably serve as stabilizing
guy wires.
Not all type 2 cells had processes, but if
they did, then type 2 cells generally possessed fewer processes (3-4 per cell) than
type 1 (5-7). Most processes on type 2
cells did not extend great distances from
the cell body, did not branch extensively,
usually did not contact each other nor
show an interwoven network with other
similar processes, which are, however, typical characteristics for type 1. Type 2
cell processes often ended in dramatically
flared out terminal flanges, whereas type 1
cell processes often could not be traced
their entire length, or if traceable, type 1
cell processes often seemed to penetrate
the underlying ependyma.
Type 2 SE cells were most often found
as single units compared to type 1 which
277
usually were grouped together with intertwining processes.
It is interesting that although the numbers of type 2 SE cell were highly variable
by visual observation, the impression was
gained that the type of experimental treatment may have influenced the number of
type 2 cells, as exemplified by the one animal which had received an acute cortical
lesion in which the number of type 2 cells
was much higher in a representative area
of the floor of the third ventricle when compared with similar areas in three other typical non-operated animals. This suggests
that these cells may be associated with response to acute brain injury, making their
way into the cerebral ventricular system.
Attention to cerebral intraventricular
cells in the past has been focused on their
relation to the choroid plexus, i.e., the wandering, Kolmer or epiplexus cells (Kolmer,
'21; Kappers, '53; Carpenter et al., '70).
Their possible existence and function elsewhere in the ventricular system has not
been pursued to any great extent, likely
due to their relatively sparse distribution
in other locations compared to the choroid
plexus, combined with the difficulty and
tediousness required to reconstruct and
analyze large surface areas at the ultrastructural level prior to SEM.
However, it is interesting that Kolmer
('21) briefly noted that wandering cells
occurred in the central canal and on the
surface of the ependyma other than that
associated with the choroid plexus. He
mentioned that such cells were of a greater
variety of types and much less frequently
found than the intraventricular choroid
plexus cells.
Type 2 SE cells as visualized by SEM in
the monkey third ventricle very likely correspond to those alluded to by Kolmer.
Cells similar in appearance and referred to
as Kolmer or epiplexus cells have recently
been reported in SEM studies of the choroid plexus and third ventricle of the rat
brain (Hosoya and Fujita, '73; Hosoya and
Fuse, '73; Chamberlain, '74) and choroid
plexus of the rat lateral ventricle (Peters,
'74).
Kolmer or epiplexus cells associated with
the choroid plexus have been shown to be
phagocytic in experimental light and TEM
studies employing India ink, thorotrast and
278
PENELOPE W. COATES
ferritin (Kappers, ’53; Tennyson and Pappas, ’64; Carpenter et al.,’ 7 0 ) , suggesting
the possibility that type 2 SE cells in monkey third ventricle are similarly phagocytic,
and act as scavengers in the ventricular
system for cell debris and products of various kinds, from both normal turnover and
other processes.
The veils of cytoplasm, deep folds, ruffled membranes and fringes present on
many type 2 SE cells are similar to features revealed with SEM for histiomonocytic cells (Bessis, ’ 7 3 ) , and thought to be
involved in locomotion, pinocytosis and
phagocytosis. Depending upon environment, functional state or material ingested, the appearance of such cells varied. If
type 2 SE cells are also locomotive, pinocytotic and phagocytic, this could explain
the wide diversity of their morphology.
ACKNOWLEDGMENTS
The author wishes to thank Mr. Arnold
Schmidt of the Materials Analysis Laboratory of the Department of Ceramic Engineering at the University of Washington
and Mr. Wyman C. Lane, Director, Applications Laboratory of ETEC Corporation
for their skilled assistance and generous
cooperation during the course of this study.
LITERATURE CITED
Anderson, T. F. 1951 Techniques for the preservation of three-dimensional structures in preparing specimens for the electron microscope.
Trans. N. Y. Acad. Sci., 13: 130-134,
Bessis, M. 1973 Living Blood Cells and Their
Ultrastructure. Translated by Robert I. Weed.
Springer-Verlag, New York, Heidelberg, Berlin.
Carpenter, S. J., L. E. McCarthy and H. L. Borison 1970 Electron microscopic study on the
epiplexus (Kolmer) cells of the cat choroid
plexus. Z. Zellforsch., 110: 4 7 1 4 8 6 .
Chamberlain, J. G . 1974 Scanning electron microscopy of epiplexus cells (macrophages) in
the fetal rat brain. Amer. J. Anat., 139: 443447.
Coates, P. W. 1972 Scanning electron rnicroscopic studies of third ventricle from infant
monkey brains disclose supraependymal cells.
J. Cell Biol., 55: 47a.
1973a Supraependymal cells in recesses of the monkey third ventricle. Amer. J.
Anat., 136: 533-539.
19731, Supraependymal cells: light and
transmission electron microscopy extends scanning electron microscopic demonstration. Brain
Res., 57: 502-507.
1973c Supraependymal cells and surface specializations on the floor of monkey
third ventricle: scanning electron microscope
studies. Anat. Rec., 175: 294 (abstract).
Cohen, A. C . , D. P. Marlow and G . E. Garner
1968 A rapid critical point method using fluorocarbons (“freons” ) as intermediate and transitional fluids. J. Microscopie, 7: 331-342.
Hosoya, Y., and T. Fujita 1973 Scanning electron microscope observations of intraventricular macrophages (Kolmer cells) in the rat brain.
Arch. Histol. Jap., 35: 133-140.
Hosoya, Y., and S. Fuse 1973 Scanning electron microscopic observations on the third ventricular wall of the rat. Acta Anat. Nippon, 48:
276-289.
Kappers, J. Ariens 1953 Beitrag zur experinientellen Untersuchung von Funktion und Herkunft der Kolmerschen Zellen des Plexus chorioideus beim Axolotl und Meerschweinchen. Z.
Anat. Entwick., 117: 1-19.
Kolmer, W. 1921 uber eine eigenartige Beziehung von Wanderzellen zu den Chorioidealplexus des Gehirns der Wirbeltiere. Anat. Anz.,
54: 15-19.
Peters, A.
1974 The surface fine structure of
the choroid plexus and ependymal lining of the
rat lateral ventricle. J. Neurocytol., 3: 99-108.
Tennyson, V. M., and G. D. Pappas 1964 Fine
structure of the developing telencephalic and
myelencephalic choroid plexus in the rabbit.
J. Comp. Neur., 123: 379-412.
PLATES
PLATE 1
EXPLANATION O F FIGURES
Normal adult female M. mulatta.
280
1
Type 2 supraependymal (SE) cell on the nonciliated floor of the third
ventricle. Extending from the small diamond shaped cell body are
four processes, three of which flare out broadly before terminating in
ruffled edges. The hexagonal pattern formed by leaflets and microvilli
on the underlying ependymal cells is characteristic for monkey third
ventricular floor. x 2,280.
2
A variation of type 2 SE cell, this time located near the infundibular
recess of the third ventricle. Its processes are knobby. A few slender cytoplasmic fingers extend to the underlying ependymal surface.
x 4,560.
TYPE 2 SUPRAEPENDYMAL CELLS
Penelope W. Coates
PLATE 1
281
PLATE 2
EXPLANATION OF FIGURES
Normal adult female M. mulatta.
282
3
Type 2 SE cell i n the transition zone of the third ventricle seems suspended above the surface. The edges of the processes are fringed.
x 2,280.
4
A rounded up type 2 SE cell in the infundibular recess. The cell body
is covered with ruffled membranes and short cytoplasmic membranous
extensions. X 4,560.
TYPE 2 SUPRAEPENDYMAL CELLS
Penelope W. Coates
PLATE 2
283
PLATE 3
EXPLANATION O F FIGURES
Juvenile male M. mulatta. Left cortical lesion.
284
5
Low power SEM shows many type 2 SE cells on the floor of the third
ventricle. The cells are separated and not clustered closely together.
X 712.
6
Higher magnification of one SE cell from the above figure. The small
cell body is triangular from which three processes emerge. There is a
red blood cell nearby. x 2,850.
'TYPE 2 SUPRAEPENDYMAL CELLS
Penelope W. Coates
PLATE 3
285
PLATE 4
EXPLANATION OF FIGURES
7 There is considerable membrane ruffling on this type 2 SE cell. Normal fetal female M. nemestrina. x 2,375.
8
286
Cytoplasmic veils, ruffled membranes and pseudopod-like extensions
occur on this type 2 SE cell located on the floor of the third ventricle.
Nine-month male M. fasicularis. Normal except left eyelid suture two
weeks after birth. x 4,275.
TYPE 2 SUPRAEPENDYMAL CELLS
Penelope W. Coates
PLATE 4
287
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