Published August 1, 1965
THE ULTRASTRUCTURE
IN THE BRAINS
ZBIGNIEW
OF G L I O S O M E S
OF AMPHIBIA
SREBRO
From the Institute of Comparative Anatomy "G. B. Grassi," University of Rome, and the Center
of Neuroembryology, Consiglio Nazionale delle Ricerche, Rome, Italy. Dr. Srebro's present
address is Department of Biology and Embryology, Medical Academy, Cracow, Poland
ABSTRACT
INTRODUCTION
Light microscope study of the brain of Xenopus
laevis (16, 17) has revealed that the superficial
white matter adjacent to the meninges contains
numerous granules and droplets which are stainable with acid dyes and for which the term submeningeal acidophilic material (SAM) has been
adopted (17). It was ascertained that the S A M
droplets vary in size from a fraction of a micron to
about 5 microns and that they are most abundant
and largest in the most superficial layers of the
white matter and directly under the pia-arachnoid.
Another characteristic feature of the S A M is its
abundance in the vicinity of meningeal vessels. T h e
acidophilic granules and small droplets can be
found in the submeningeal layers of m a n y different
parts of the brain, but are most a b u n d a n t in certain
areas such as that lying rostral and lateral to the
optic chiasm, the optic tectum, the dorsolateral
diencephalon, and the ventral mesencephalon.
Histochemical data have revealed that the S A M is
a protein and is PAS-negative (17).
In this paper electron microscopic evidence is
presented showing that the S A M is an accumulation of gliosomes. T h e gliosomes, although having
some structural features in common with mitochondria, differ considerably from them and may
be considered a distinct kind of organelle, typical
of the glial cell.
MATERIAL
AND METHODS
Small fragments of the superficial white matter together with the pia-arachnoid or deeper parts of the
brains of adult Xenopus laevis Daud. and Rana esculenta
L. of both sexes were fixed in 2 per cent osmium tetroxide immediately after removal or after prefixation
in 6.5 per cent glutaraldehyde. The fixative was pre-
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Small fragments of superficial neuropil and fragments of dcepcr layers from various regions
of the brains of Xenopus laevis Daud. and Rana esculenta L. were fixed in buffered osmium
tctroxide, cmbcddcd in Vcstopal W or mcthacrylatc, and studied with the elcctron microscope. T h c glial fibcrs and thcir meningeal end-feet contain numerous largc mitochondrionlikc dcnsc bodics for which the term "gliosomc" has been adoptcd. Gliosomcs have a
specific and constant structure charactcrizcd by the presence of a row of pcriphcral and
circular canaliculi and an clectron-opaque fibrous or fincly granular matrix. Also, anothcr
lcss frequently found type of gliosomc is prcsent which contains regular lamcllar structures.
T h e gliosomes vary considerably in sizc and m a y be very large, up to 9/z in lcngth. N u m crous and various intcrmcdiate forms bctwcen mitochondria and gliosomcs can be seen.
Gliosomcs are largest and most numerous in thc distal portions of the glial fibcrs and in
the mcningcal cnd-fcct.
Published August 1, 1965
pared after Millonig (11), formula B, and in addition
2 per cent sucrose was added to it. Fixation was for 1
hour and 30 minutes and was performed in the cold
(0--4°C). The tissues were dehydrated in a graded
series of ethyl alcohols, passed through styrene, and
embedded in Vestopal W. For embedding in methacrylate, the tissues were dehydrated in acetone. The
material was sectioned with a glass knife on LKB
Ultrotome and the sections were stained with lead
after Karnovsky (8). The electron micrographs were
obtained with the Hitachi H U 11 electron microscope
operated at 75 kv and with an objective aperture of
50/z. The mierographs were taken at a direct magnification of 10,000 times.
OBSERVATIONS
FIGUaE 1 Gliosome in optic rectum of Xenopus, showing a ramification (rm) and a
double row of transversely cut eanalieuli (arrows). The limiting membrane is visible at
me; at mi a mitochondrion is shown. Vestopal. X 35,000.
l%~Vl~E ~ Gliosome in mesencephalon of Xenopus. The matrix has a fibrous appearance.
The right arrow indicates a peripheral circular canaliculus cut obliquely; the left arrow,
a longitudinally cut canaliculus. Vestopal. )< 85,000.
FIGURE 8 Another gliosome in optic tectum of Xenopus. The right arrow indicates an
obliquely and the left arrow a transversely sectioned canaliculus; me, limiting membrane.
Vestopal. X 85,000.
FmVRE 4 Gliosome in prosencephalon of Rana. The fibrous character of the matrix is
evident. Arrow indicates one of the peripheral canaliculi transversely sectioned. Methacrylate. X 35,000.
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T h e glial fibers in the brains of b o t h species studied
h a v e a " c l e a r , " e l e c t r o n - t r a n s p a r e n t appearance.
T h e y contain fine filaments a n d electron-opaque
granules of 200 to 400 A. T h e granules sometimes
form small aggregates a n d p r o b a b l y are glycogen.
T h e y are more n u m e r o u s in Rana. I n the meninges
the glial fibers form end-feet which lie on the basem e n t m e m b r a n e of the pia-arachnoid.
T h e m e n i n g e a l end-feet of the glial fibers contain n u m e r o u s m i t o c h o n d r i a v a r y i n g considerably
in size. T h e i r characteristic feature is a h i g h electron opacity of the matrix. I n addition, the glial
fibers a n d their end-feet contain large mitochondrion-like dense bodies of u n i q u e and characteristic
structure which will be referred to as gliosomes.
A typical gliosome is oval or very elongated and
has a n outer limiting m e m b r a n e (Figs. 1 to 7 a n d
11). I n some cases the m e m b r a n e forms conspicuous foldings (Fig. 6). An i n n e r limiting m e m b r a n e
c a n sometimes be seen a n d also a n electron-transp a r e n t space w h i c h corresponds to the electron-
t r a n s p a r e n t i n t e r m e m b r a n o u s layer of the paired
limiting m e m b r a n e s of mitochondria. T h e w i d t h of
this space varies, since the outer limiting m e m b r a n e forms the above m e n t i o n e d foldings. I n
some cases the inner limiting m e m b r a n e is invisible a n d the outer limiting m e m b r a n e adheres
closely to the underlying matrix. A n electrono p a q u e fibrous or finely g r a n u l a r m a t r i x fills
the gliosome (Figs. 1 to 6). Only in the most
superficial p a r t a n d close u n d e r the limiting m e m b r a n e of the gliosome a row of r o u n d or oval,
small e l e c t r o n - t r a n s p a r e n t spaces can be seen (Figs.
2, 5, a n d 11). I n these figures these spaces are
transverse sections of a regularly a r r a n g e d set of
circular a n d concentric canaliculi. I n cases w h e r e
the p l a n e of section passes tangentially across the
gliosome the canaliculi are cut obliquely or longitudinally (Figs. 2 a n d 3). T h e canaliculi are a b o u t
250 A wide, b u t they m a y be larger (Fig. 11), or
m a y be n a r r o w e r in other cases a n d almost invisible. As a rule, the l u m i n a of the canaliculi are
e l e c t r o n - t r a n s p a r e n t or at least of low density so as
to give to the whole the a p p e a r a n c e of a straight
row of small "holes" u n d e r the surface of the
gliosome (Figs. 2, 5, a n d 11). I n some of the smaller
gliosomes a n d in the i n t e r m e d i a t e forms the continuity of the l u m i n a of the canaliculi with the
outer s u b m e m b r a n o u s space is visible (Fig. 6).
T h e first type of gliosome described above is the
one most frequently found. A m o n g gliosomes of
this type the largest show a length of u p to 9/z. A
f r a g m e n t of such a very long a n d n a r r o w gliosome
is presented in Fig. 3. T h e m a x i m u m w i d t h observed was t h a t of the gliosome in Fig. 1, which
was 1.5 # wide. T h e size of the gliosomes varies,
a n d the smallest which still showed the characteris-
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Rana. Gliosomes of the second type are far less
frequent t h a n those of the first type. A gliosome of
the second type contains regularly arranged, parallel, a n d longitudinally oriented lamellae (Figs. 7
a n d 8). I n some cases the continuity of the lamellae
with the internal limiting m e m b r a n e is evident
(Fig. 7). I n Xenopus the interlamellar spaces are
a b o u t 100 A a n d 200 A, respectively, a n d the
narrower space is less electron opaque t h a n
the wider one. Thus, the whole gives the impression of parallel paired m e m b r a n e s regularly stacked. T h e paired lamellae can sometimes
be seen to curve at the point where they arrive at
the limiting m e m b r a n e (Fig. 8). I n Rana the spacings are wider ( a b o u t 150 A a n d 300 A, respectively) a n d the density of the n a r r o w e r layer
is the same as t h a t of the wider one. M i x e d forms
between types I a n d I I of gliosomes are present
(Fig. l 1, gs2 a n d gs3).
Various i n t e r m e d i a t e forms between type I gliosomes a n d m i t o c h o n d r i a can frequently be found.
T h e m i t o c h o n d r i a in glial fibers have a dense m a trix a n d relatively few cristae w h i c h in some cases
m a y be very short or dilated, as shown in Figs. 6
a n d 12. T h e gliosome in Fig. 9 m a y also be classified as a n intermediate form.
FIGURE 5 Gliosome in preoptic area of Xenopus. Note the finely granular matrix and
the peristaltic wavelike coutour of the gliosome. Arrows indicate transverse sections of
peripheral canaliculi. Vestopal. )< g5,000.
I~GURE 6 Fragment of a glial fiber in optic tectum of Xenopus. A gliosome (gs) with a
double row of mostly transversely sectioned canaliculi (arrows at right center) and two
mitochondria (mi, rail) are visible. Mitochondrion mi has long dilated cristae, the walls
of which are continuous with the internal limiting membrane (arrows at top right). Mitochondrion rail is an intermediate form with indistinct cristae and peripheral canaliculi.
The lumen of the peripheral canalieulus appears continuous with the submenlbranous
space (left arrow). At mfa folding of the limiting membrane of the gliosonae, Vestopal.
X 35,000.
FIGUr~E 7 Gliosome of type II in prosencephalon of Rana. Note the regular packing of
parallel membranes which appear continuous with the internal limiting membrane (arrow). Methacrylate. X 35,000.
FIGUllE 8 Xenopus, optic rectum. In the right part of the figure is a gliosome of type
II; arrows indicate the points of flexion of the paired lamellae. To the left is a fragment of a gliosome of type I. Vestopal. X 85,000.
I;kGURE 9 A branching intermediate form of gliosome. Xenopus, dorsolatcral diencephalon. Vestopal. X 35,000.
FIGURE 10 Another branching gliosome. At the point of ramification the limiting membranes are visible (arrow). Xenopus, optic rectum. Vestopal. X 35,000.
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tic structure were less t h a n I # long. Also, the shape
of the gliosomes is v a r i a b l e ; they m a y be round,
oval, very long a n d narrow, or r a m i f o r m a n d irregular in shape. T h e shape of the gliosome shown
in Fig. 5 is interesting in t h a t its outline resembles a
series of peristaltic waves.
I n spite of the great variability in size a n d shape
of the gliosomes, their characteristic structural elements, i.e. the external limiting m e m b r a n e , the
fibrous or g r a n u l a r dense matrix, a n d the peripheral canaliculi, can always be found. T h e fibers of
the m a t r i x are a r r a n g e d longitudinally in the direction of the long axis of the gliosome. I n some of
the gliosomes the m a t r i x is fibrous b u t a n external
layer of g r a n u l a r a p p e a r a n c e is present.
T h e large gliosomes sometimes show a n internal
row or two of canaliculi (Figs. 1 a n d 6). T h e gliosome in Fig. 1 has two such internal rows a n d also
shows a ramification, the point of the ramification
being at the level of the more peripheral row of
canaliculi. I n Figs. 9 a n d 10 the gliosomes also
present ramifications. T h e gliosome in Fig. 10
shows, in addition, the presence of limiting m e m branes at the point of the ramification.
Besides the form of gliosomes described above,
a n o t h e r type is also present in b o t h Xenopus a n d
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DISCUSSION
The present findings show that also in amphibia
the glial fibers contain the large " a b n o r m a l mitochondria" which have been described in mammals
(3, 5, 7, 13) and have been found in reptiles (4, 6,
12). In amphibia, however, they are especially
large and numerous and accumulate in the meningeal end-feet of the glial fibers. The " a b n o r m a l
mitochondria" present in glial prolongations certainly are what the light microscopists described
as gliosomes. Considering the peculiar and constant structure of the " a b n o r m a l mitochondria,"
it seems justifiable to restrict the term gliosomes to
these structures. Although gliosomes have some
structural features in common with mitochondria,
such as their form, the paired limiting membranes,
and, in some cases, the presence of crista-like projections, they appear to be a distinct kind of glial
cell organelle. It would be of great interest to ascertain whether the "mitochondrial" enzymes such
as succinic dehydrogenase or cytochrome oxidase
are also present in gliosomes.
Although the mitochondrial origin of gliosomes
cannot be proved solely on the basis of the present
findings, the presence of numerous and various intermediate forms seems to corroborate such an
assumption. Assuming that gliosomes derive from
mitochondria, it would be possible to understand
the mechanism of formation of the peculiar peripheral canaliculi present in these organelles. The
canaliculi could easily form from the cristae mitochondriales as these are pushed to the periphery of
the organelle by the accumulating matrix and
eventually lose their continuity with the limiting
membrane.
It cannot be decided, at the moment, whether
the accumulations of tubules and vesicles, observed
in the proximal portions of glial fibers and close to
the apparently forming gliosomes, have any significance in the formation of these organelles.
T h e often observed branching of gliosomes is a
finding which needs attention, since it m a y mean
their fusion or, alternatively, their division.
In accord with the hypothesis that gliosomes are
organelles different from mitochondria are the
light microscope observations of Srebro and
Borz~dowska (17). The authors showed that the
SAM, which the present findings have demonstrated to be identical with gliosomes since the
size and the localization of the S A M droplets and
of the gliosomes are the same, can be demonstrated
after fixation in Bouin's fluid. This fixative con-
l~otmE 11 Terminal (submeningeal) part of glial fiber in preoptie area of Xenopus.
Single granules of glycogen, gliofilaments (gf), and gliosomes (gsl, gs:, gs~) are present.
Part of a marginal cell (rag) is sho~m. Vestopah X 35,000.
I~GVRE 1~ Another glial terminal in preoptic area of Xenopus. A dense material (dg;
outline shown by dotted line), probably a disintegrating gliosome, lies close to the marginal cell (rag). The canalieuli of the gliosome are still visible (arrows). Also, mitochondria (mi) with a dense matrix and gliofilaments (gf) are present. Vestopal. X 85,000.
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The meningeal end-feet contain gliofilaments,
mitochondria, intermediate forms between mitochondria and gliosomes, and gliosomes (Figs. 11
and 12). In the submeningeal region, numerous
marginal glial cells are present. They lie directly on
the basement membrane of the pia-arachnoid or
deeper in the submeningeal neuropil. The mar' ginal cells have a " d a r k " cytoplasm with numerous
small electron-opaque granules. Their short prolongations are present between the glial endfeet, some of the latter being partially or completely surrounded by the cytoplasm of the marginal cell (Fig. 11). In some of the end-feet a dense
malerial that seems to be the remnants of disintegrating gliosomes can be observed close to the
marginal cell cytoplasm (Fig. 12, dg).
Glial fibers in the deeper parts of the brain contain tubules 300 to 400 A wide which sometimes
are intricately convoluted (Fig. 13). Some of the
tubules contain a moderately electron-opaque material while others form wide cisternae (Fig. 14).
Besides the accumulations of tubules, agglomerates
of vesicles can be seen which aggregate into electron-opaque masses (Fig. 14, va). Gliosomes in
• various stages of formation are present in this region (Figs. 13 and 14, gsO. In Fig. 13 a gliosome is
shown which forms a tubule-like protrusion on the
side of the above described accumulation of
tubules.
Gliosomes are larger and more numerous in
Xenopus than in Rana.
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Using the bromphenol blue staining method and
the Sakaguchi reaction for arginine, Srebro and
Borz~dowska (l 7) showed that the SAM is a protein. It is also PAS-negative. Since the present results have demonstrated that the S A M is an accumulation of gliosomes, it seems justifiable to
assume that the matrix forming their bulk is proteinaceous. The site of the elaboration of this material and the significance of its deposition and
accumulation are yet to be determined. Equally
unknown is the significance of the submeningeal
and perivascular localization of gliosomes. W h a t ever the physiological role of these peculiar structures ultimately turns out to be, they certainly appear to be a distinct kind of cell organelle, limited
to the glial cell, but widely distributed among
different vertebrate species.
The author wishes to thank Professor Alberto
Stefanelli for advice received and all facilities made
available, and Dr. Bruno Bertolini for review of the
manuscript and helpful criticism.
This study was supported by a grant from the National Research Council, Rome.
Received for publication, August 5, 1964.
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FIGURE 13 Fragment of proximal part of glial fiber in preoptic area of Xenopus. Numerous gliosomes (gs) in various stages of formation are present, some of which are very
small (gsl). At tu are agglomerates of tubules. Numerous gliofilaments can be seen in the
lower part of the figure. Vestopal. X 35,000.
FIGURE 14 Another fragment of proximal part of glial fiber of Xenopus. A larger gliosome (gs) and several small, probably forming, gliosomes (gsl) are present. Numerous
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tains acetic acid, which is known to destroy mitochondria.
Against the assumption that gliosomes are only
a kind of mitochondrion is the fact that the cristae
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