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Postnatal development of mitral cell perikaryon in the olfactory bulb of the rat. A light and ultrastructural study

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Postnatal Development of Mitral Cell Perikaryon in the
Olfactory Bulb of the Rat. A Light and
UItrastr uctural Study
DYAL N. P. SINGH AND EDWARD J. H. NATHANIEL
Department ofAnatomy, UniversityofAlbertu, Edmonton,Alberta, Canada l" 2 H 7 d
Department oftlnatomy, Faculty ofMedicine, University ofManitoba,
Winnipeg, Manitoba, C d a R3T2N2
ABSTRACT
The differentiation of the mitral cell perikaryon in postnatal rat
olfactory bulb was studied with the light and electron microscope. At birth the
mitral cell was distinguishable and occupied a definitive position in the mitral
cell layer. The cell contained a large oval nucleus surrounded by a thin rim of cytoplasm. Ribosomes, free and clustered, were scattered in the cell cytoplasm.
Rough endoplasmic reticulum was relatively scarce. The Golgi complexes were
made up of stacks of smooth-surfaced cisternae and associated vesicles. In certain cases the Golgi complexes projected into cellular processes. Mitochondria
were present in all regions of the cytoplasm and contained well developed cristae.
A t the end of the first week, the mitral cell had developed significantly in size,
and the cytoplasm contained well-developed rough endoplasmic reticulum. The
Golgi complexes were made up of several stacks of smooth-surfaced cisternae
with the association of vesicles and electron dense bodies. The apical dendrites of
mitral cells a t this period had increased significantly in length. Subsequently,
during the second and third week, the rough endoplasmic reticulum and Golgi
complexes became well developed. Associated with the Golgi complexes were
electron dense lysosomal bodies. At three weeks and in older cells it was observed
that dense lipofuschin granules increased significantly. It is suggested that the
mitral cell matures and differentiates earlier than cells in the cerebral cortex.
Based on previous studies on the olfactory bulb of prenatal and neonatal mouse by Hinds
bulb in different species of chordates, Andres ('68, '72a,b). These studies described the ori('70) concluded that the cytology remained gin and orientation of mitral cells. Altman
relatively constant from cyclostomes to pri- ('69) showed that there was a migration of
mates. The cat olfactory bulb as described by cells from the ependymal layer of the lateral
Willey ('73) contained six layers, namely: pri- ventricle to the olfactory bulb. However, to
mary olfactory nerve layer, glomerular layer, our knowledge there have been no systematic
external plexiform layer, mitral layer, inter- studies on the development and maturation of
nal plexiform layer and granule cell layer. the mitral cell in postnatal rat olfactory bulb.
Located within these layers are several neuNeuronal differentiation in other regions of
ronal cell types. Of these the mitral cell is the the nervous system has been reported by
largest, and its prominence in a layer of the Bellairs ('59) and Mugnaini and Forstrenen
same name allows for easy identification. In ('67) in the chick embryo; Tennyson ('65) in
this respect mitral cells are suited, among the dorsal root ganglion of the rabbit embryo;
other neurons in the nervous system, for stud- Caley and Maxwell ('68, '71) in the cerebral
ies in which individual results are gathered, cortex of postnatal rats; and Bodian ('66) in
rather than a statistical analysis from a the monkey.
heterogeneous population.
The aim of the present study is to describe
The mitral cell has previously been used to
Received Jan.21, '17. Accepted May 31. ''I?.
study neuronal development in the olfactory
ANAT. REC., 189: 413-432.
413
414
DYAL N. P. SINGH AND EDWARD J. H. NATHANIEL
the development of the mitral cell perikaryon
in postnatal rats, with particular emphasis on
this cell's ultrastructure.
METHODS AND MATERIALS
Albino rats (Sprague-Dawley) aged 1day, 2,
3 and 5 weeks served as experimental animals. Olfactory bulbs of 40 rats (8for each age
group) were fixed by perfusion through the
left ventricles with a mixture of 3%paraformaldehyde and 0.5% glutaraldehyde in 0.1 M
cacodylate buffer. The right atrium was incised to facilitate drainage. Following perfusion, craniectomy was performed and the olfactory bulb removed and hand sectioned into
pieces approximately 1 mm3 under a stereomicroscope. The tissue specimens were kept a t
4°C for four hours in aldehyde solution for
further fixation. The tissue blocks were then
postfixed in 1%osmium tetroxide in 0.1 M
cacodylate buffer for an hour, dehydrated in
ascending concentrations of ethanol and embedded in Araldite (Ladd).
Sections 0.5 pm thick were stained with toluidine blue and studied with the optical microscope. Ultrathin sections (70 nm) stained
with uranyl acetate and lead citrate were
used for electron microscope studies.
OBSERVATIONS
Light microscopy
Perfused brains and the oval-shaped olfactory bulbs from postnatal rats attain their
adult size a t three weeks postnatally (fig. 1).
In semi-thin sections (0.5 pm) from all age
groups studied, the six layers of the olfactory
bulb were easily identified. However, in neonates and 1-day-old animals the mitral cell
and inner plexiform layers appeared somewhat in continuity. Later, in 1-week-old animals, these layers became more obvious (fig.
3). The increase in size of the olfactory bulb
during the initial three weeks of postnatal development seemed to parallel the increase in
width of the outer plexiform layer, which increased from approximately 45 pm a t birth to
325 pm in 3-week-oldanimals (figs. 2,3). Concomitant with the development of the external plexiform layer is a significant increase in
density of dendrites and blood vessels (Singh
and Nathaniel, '73, '75).
The mitral cell is easily identified by its position and the size of its nucleus and perikaryon. A t birth, the perikaryon is relatively
oval, and contains a large nucleus (fig. 4a).
The cytoplasm forms a narrow margin sur-
rounding the nucleus. A prominent nucleolus
is present within the nucleus. In subsequent
development, the mitral cell increases in size
and shows polarity toward the glomerular
layer; a t the same time the cytoplasmic area
widens (figs. 4b-d). After the third week, a
layer of mature mitral cells is obvious.
Electron microscopy
In neonatal and 1-day-old animals the
mitral cell nucleus is relatively large arid the
chromatin evenly dispersed. The centrally
located nucleolus appears dense. No distinction could be made between pars grartulosa
and pars fibrosa. The cytoplasm appear,s pale
and contains a rich population of single and
clustered ribosomes between which other cytoplasmic organelles seem randomly distributed (fig. 5).
The endoplasmic reticulum consists of a few
strands of cisternae widely scattered in the
cytoplasm. Few ribosomes were observed on
their surfaces. Golgi complexes were often
encountered in short dendritic processes, and
are made up of stacks of smooth-surfaced
cisternae (fig. 5). Associated with these cisternae are smooth-surfaced vesicles. Mitochondria are located in all regions of the cytoplasm. A double membrane surrounds the
matrix and well-developed cristae. The mitochondrial matrix is evenly composed except
for an occasional granule similar to those
found in the Mauthner cell by Billings ('72)
and by Mugnaini and Forstrenen ('67) in undifferentiated neurons of the cerebellum (fig.
5: arrow).
At the end of the first postnatal week,
mitral cells show a remarkable increase in the
rate of development (figs. 6,7). This advanced
state, observed in both light and ultrastructural studies, affects every component of the
perikaryon. A singular feature is the Eignificant increase in size and length of the apical
dendrite, which traverses the external plexiform layer to synapse in the glomerular layer
(fig. 6). Few synapses were observed in the
dendritic surface. Ribosomes occur singly and
in rosette formation.
The cytoplasm of the perikaryon undergoes
significant changes in that there is now an
abundance of cytoplasmic organelles. 14t this
stage, the rough endoplasmic reticulum is
very extensive. In many regions, continuity
between the cisternae of the rough enldoplasmic reticulum and the perinuclear space could
be seen with greater frequency than in one
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
day old cells (fig. 7: *arrows). Sub-surface cisternae were also observed. In most cases, the
rough endoplasmic reticulum channels maintain uniform width (fig. 61, but in some cells
swellings and constrictions were found along
their length.
The Golgi complexes are composed of many
stacks of smooth membranes distributed in
several cytoplasmic areas. Golgi cisternae are
associated with a larger number of smooth
and coated vesicles. In some cells, Golgi complexes were also found in the proximal part of
the apical dendrites. At this stage dense
lysosomal-like bodies were observed in all regions of the cytoplasm; several were closely
associated with Golgi membranes. Mitochondria also increase in numbers and contain
well-developed cristae and the occasional
granule. Synapses, characterized by thick preand postsynaptic membranes and boutons
containing vesicles, were observed with greater frequency.
Although nuclear chromatin is generally
evenly distributed, some aggregations are
located beneath the nuclear membranes. The
nuclear membranes show gentle irregularity
throughout their contours and only a n occasional deep invagination by cytoplasm. Filamentous tufts project for short distances into
the chromatin and correspond to sites of nuclear pores (fig. 7: arrow). These features did
not change significantly in older cells.
The general morphology of the mitral cell a t
the end of the second postnatal week is one of
near maturity (fig. 8). A t this stage, the nuclear envelope is relatively uniform and infoldings few. The nuclear chromatin remains
evenly distributed, except for the aggregations noted above.
The cytoplasm contains long strands of
granular endoplasmic reticulum. These could
be followed for considerable distances through
the cytoplasm. Frequent interconnections between the granular cisternae were observed.
Even a t this late stage of development, a large
part of the cytoplasm is occupied by clustered
and free ribosomes. This observation was also
made in 3-week- and 5-week-old animals.
The Golgi complexes are now extensive. Associated with the many stacks of smooth
cisternae are large numbers of smooth vesicles and a few lysosomes. Similar larger structures are present apart from the Golgi complexes. Mitochondria intermingle with the
other cytoplasmic organelles.
A t the end of the third postnatal week the
415
mitral cell shows no significant changes from
a cell of a 2-week-old animal. The cell is
slightly larger and lipofuscin granules are
more numerous (fig. 9). These lipofuscin granules are electron-dense bodies and contain eccentric pale areas.
DISCUSSION
The results show that mitral cells develop
a t a rapid during the first postnatal week.
During this period the perikaryon and apical
dendrite significantly increase in size and
come to resemble those of a mature cell. It is
known that vision is not the dominant sense
during the first postnatal week of development; the eyes do not open until the end of the
second postnatal week. Thus the animal relies
largely on the sense of olfaction for its early
nutritional needs.
At birth the olfactory bulb is still an immature structure. The mitral cell, however, is
identifiable, although it shows some degree of
cytological immaturity. This condition is
unlike that in the cerebral cortex, a t this time
made up of undifferentiated cells tightly arranged in vertical columns (Caley and Maxwell, '68). Hinds and Ruffett ('73), using Golgi
impregnation techniques, showed that the
mitral cell layer was present by 15 days of
gestation in mouse fetuses. At this fetal stage,
the mitral cell layer is poorly demarcated, but
it surpasses the development of the cerebral
cortex.
The rate of development of the perikaryon
during the first postnatal week is very rapid,
suggesting that olfaction may encourage the
development of the mitral cell. I t is also
reasonable to suggest that lack of sight may
be a factor on the precocious and rapid maturation of the mitral cell during this period.
Similar investigations on monkey spinal cord
show that reflex activity influences early synaptic development (Bodian, '66). Alternatively, if sensory input to the brain is altered during initial postnatal periods of development,
certain aspects of brain maturation and differentiation are significantly affected (Meisami and Timiras, '71, '74; Riesen, '75).
The changes observed in the developing
mitral cell perikaryon show similarities to developmental changes previously reported for
other neurons (for example, Bellairs, '59;
Lyser, '64;Tennyson, '65; Meller et al., '66;
Caley and Maxwell, '68; Radouco-Thomas et
al., '71; Billings, '72). In each neuronal type,
certain modifications in structure and physi-
416
DYAL N. P. SINGH AND EDWARD J. H. NATHANIEL
ology lead to individuality. For the mitral cell, well, '71). As development proceeds, an intithis individuality seems to lie, a t least in part, mate association between the smooth-surin a rapid rate of growth and differentiation faced Golgi complexes and the rough-surfaced
endoplasmic cisternae is suggestive of exduring the first postnatal week.
In the young mitral cell, free ribosomes, iso- changes between them. These morphological
lated and clustered, fill the cytoplasm; it is changes, along with the appearance of lysogenerally believed that such a phenomenon somes, may be necessary to balance the acindicates that the cell is producing pro- tions of anabolism and catabolism during the
teinaceous material for its growth (Hay, '63). period of progressive growth.
As development proceeds, a vast elaboration
ACKNOWLEDGMENTS
of rough endoplasmic reticulum occurs. The
reticulum, made up of long cisternae studded
We are indebted to Doctor K. L. Moore for
with few ribosomes, increases sginificantly providing facilities during the course of this
even by the end of the first postnatal week. study and to Doctor K. D. McFadden folr his
Such elaboration suggests that substances critical review of this paper. Secretarial assynthesized on the ribosomal complex are sistance of Miss K. Dawson is gratefully acsoon provided adequate channels for move- knowledged.
This investigation was supported in part by
ment to other areas of the cytoplasm. This interpretation would apply to transport of neu- a M.R.C.Grant to Doctor E. J. H. Nathaniel
rotransmitters, believed synthesized by cyto- and M. R. Grant of the University of Alberta.
plasmic organelles. The reticulum may also be
LITERATURE CITED
involved in segregating elaborated protein
Altman,
J.
1969
Autoradiographic and histological
and directing these products to specific areas
studies of postnatal neurogenesis. IV. Cell proliferation
in the perikaryon and processes.
and migration in the anterior forebrain, with special refOthers have noted subsurface and perinuerence to persisting neurogenesis in the olfactory bulb. J.
clear cisternae in developing neurons (for
a m p . Neur., 137: 433-457.
example, Tennyson, '65; Pannese, '68; Caley Andres, K. H. 1970 Anatomy and ultrastructure of the
olfactory bulb in fish, amphibia, reptiles, birds and mamand Maxwell, '68; Hannah and Nathaniel,
mals. In: Ciba Foundation Symposium on Taste and
'75). It has been suggested that these cisterSmell in Vertebrates. G. E. W. Wolstenholme and J.
nae provide a possible route for the flow of meKnight, eds. J . & A. Churchill, London, pp. 177-196.
tabolites into the cell. In a similar manner, Bellairs, R. 1959 The development of the nervous system in chick embryos, studied by electron microscopy. J.
synthesized or waste products could be transEmbryol. exp. Morph., 7: 94-115.
ported out of the cell. The establishment of
S. M. 1972 Development of the Mauthner Cell
such a system may be necessary for a de- Billings,
in Xenopus laeuis: A light and electron microscopic
veloping cell, in that substances needed to
study of the perikaryon. 2.Anat. Entwickl. Gesch., 136:
168-191.
ensure growth are acquired from adjacent
capillaries, astrocytic end feet and extracellu- Bodian, D. 1966 Development of fine structure of spinal
cord in monkey fetuses. I. The motoneuron neuropil at
lar fluid (Rosenbluth, '62).
the time of onset of reflex activity. Bull. Johns Hopkins
In this study, it was observed that Golgi
Hosp., 119: 129-149.
complexes are well formed in 1-day-old ani- Caley, D. W., and D. S.Maxwell 1968 An electron micro.
scopic study of neurons during postnatal development of
mals, a t a time when the rough endoplasmic
the rat cerebral cortex. J. Comp. Neur., 133: 17-44.
reticulum was poorly developed. The Golgi
1971 Differentiation of the neural elements of
complexes are associated with coated and
the cerebral cortex in the rat. In: Cellular Aspects of
uncoated vesicles but dense lysosomal-like
Neural Growth and Differentiation. D. C. Pease, ed. Unibodies are absent. At the end of the first week,
versity of California Press, Berkeley.
Golgi complexes have become extensive and Hannah, R. S., and E. J. H. Nathaniel 1975 Ultrnstructural studies of postnatal differentiation of neurons in
lysosomes numerous. These features suggest
the substantia gelatinosa of rat cervical spinal cord.
that the cell is no longer a "neuroblast" but
Anat. Rec., 183: 323-338.
has reached a higher state of differentiation. Hay, E. D. 1963 The fine structure of differentiating
In neuroblasts, the Golgi complexes are poorly
muscle in the salamander tail. 2.Zellforsch., 69: 6-34.
developed (Pannese, '68; Tennyson, '65). That Hinds, J. W. 1968 Autoradiographic study of histogenesis in the mouse olfactory bulb. 11. Cell proliferation
the 1-day-old mitral cell possesses relatively
and migration. J. Comp. Neur., 134: 305-322.
well-developed Golgi complexes suggests that - 1972a Early neuron differentiation in the
maturation is advanced beyond that of the
mouse olfactory bulb. I. Light microscopy. J. Comp.
cells of the cerebral cortex (Caley and MaxNeur., 146: 233-262.
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
1972b Earlv neuron differentiation in the
mouse olfactory buli. 11. Electron microscopy. J. Comp.
Neur., 146: 253-276.
Hinds, J. W.,and T. L. Ruffett 1973 Mitral cell development in the mouse olfactory b u l b Reorientation of the
perikaryon and maturation of the axon initial segment.
J. Comp. Neur., 151: 281-306.
Lyser, K. M. 1964 Early differentiation of motor neuroblasts in the chick embryo as studied by electron microscopy. I. General aspects. Develop. Biol., 10: 433-466.
Meller, K., J. Eschner and P. Glees 1966 The differentiation of endoplasmic reticulum in developing neurons of
the chick spinal cord. Z.Zellforsch., 69: 189-197.
Meisami, E., and P. S. Timiras 1971 Influence of early
visual input on the development of brain excitability in
the rat. h e r . J. Physiol., 220: 233-238.
1974 Influence of early visual deprivation on regional activity of brain ATPases in developing rats. J.
Neurochem., 22: 725-729.
Mugnaini, E., and P. F. Forstranen 1967 Ultrastructural
studies on the cerebellar histogenesis. I. Differentiation
of granule cells and development of glomeruli in the
chick embryo. Z.Zellforsch., 77: 115-143.
Pannese, E. 1968 Developmental changes of the endo-
417
plasmic reticulum and ribosomes in nerve cells of the
spinal ganglia of the domestic fowl. J. Comp. Neur., 132:
331-364.
Radouco-Thomas, C., G. Nosal and S. Radouco-Thomas
1971,The nuclear-ribosomal system during neuronal differentiation and development. In: Chemistry and Brain
Development. R. Paoletti and A. N. Davison, eda. Plenum
Press, New York, pp. 291-310.
Riesen, A. H. 1975 The Developmental Neuropsychology
of Sensory Deprivation. Academic h a , New York.
Rosenbluth, J. 1962 Subsurface cisterns and their relationship to the neuronal plasma membrane. J. Cell Biol.,
13: 405-422.
Singh, D. N. P., and E. J. H. Nathaniel 1973 Light microscopic study of postnatal olfactory bulb development of
the rat. Can. Fed. Biol. SOC.,
16: 354.
1975 Postnatal development of blood vessels
(capillaries) in the rat olfactory bulb. A light and ultrastructural study. Neurosci. Letters, 1: 203-208.
Tennyson, V. M. 1965 Electron microscopic study of the
developing neuroblasts of the dorsal root ganglion of the
rabbit embryo. J. Comp. Neur., 124: 267-318.
Willey, T. J. 1973 The ultrastructure of the olfactory
bulb. J. Comp. Neur., 152: 211-232.
PLATE
1
EXPLANATION OF FIGURES
1 Demonstrationof perfused rat brains to show the increase in size of the olfactory bulb
with age (actual size).
2 Histogram of the width of the external plexiform layer at different ages. Adult size
was attained at three weeks postnatal.
418
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 1
EXTERNAL P L E X I F O R M LAYER GROWTH
1614-
-
in
12A
?
’”
v
-
10-
2
1
O
day
wk
wk
AGE
i
3
5
wk
wk
AGE
WIDTH OF EXTERNAL
PLEXIFORM LAYER ( X 1 5 p )
1 day
2 weeks
3055f0154
6 7 0 0 k 0 420
1 1 50 k 0 273
3 weeks
15 305 0 366
5 weeks
16 331. 0 527
1 week
419
PLATE 2
EXPLANATION OF FIGURES
3 Photomicrographs of the olfactory bulb of rats aged 1 day, 1, 2 and 3 weeks. Each micrograph extends from the fiber to the granular layer and indicates the increase in
size of the olfactory bulb with age. X 36.
4 Photomicrographs of mitral cells in rats aged: 1 day, 4a; 1 week, 4b;2 weeks, 4c; 3
weeks, 4d. The mitral cell (M)shows a large nucleus and scanty cytoplasm a t birth
(4a). Subsequent development shows an increase in the cytoplasmic area (4b-d).
X 360.
420
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 2
42 1
PLATE 3
EXPLANATION OF FIGURE
5 Part of a mitral cell at birth showing a dendritic process. The nucleus (N) is located in
the lower part of the micrograph. Note the developed Golgi complex (GI and the predominance of r i b m e s in the cytoplasm. A mitochondrion is shown to contain a
granule (arrow).x 19,710.
422
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 3
PLATE 4
EXPLANATION OF FIGURE
6 Electron micrograph of a mitral cell (M)and its apical dendrite (D)from a rat one
week of age. The cytoplasm contains well established rough endoplasmic reticulum
(er) and Golgi complexes (G). A spinous process (DS)is observed projecting from the
dendrite. x 12,330.
424
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 4
PLATE 5
EXPLANATION OF FIGURE
7 Part of a mitral cell (MI from 1-week-oldrat. Continuity between the perinuclear
space and rough endoplasmic cisternae is shown (*arrows).Site of a nuclear pore is
shown (arrow). The nuclear membrane is relatively uniform and the chromatin is
evenly dispersed. A granule cell (Gr) is located adjacent to the mitral cell. X 12,600.
426
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 5
PLATE 6
EXPLANATION OF FIGURE
8 Mitral cell from a 2-week-oldrat. An area of part of the mitral cell perikaryon shows
the distribution of the rough endoplasmic reticulum (er) and Golgi complexes (GI.
Note the presence of small dense lysosomal bodies (arrow) in proximity to the Golgi
complexes. Large numbers of ribosomes are arranged in polysomal clusters (r).
X 27,720.
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 6
PLATE 7
EXPLANATION OF FIGURE
9 Mitral cell from a 3-week-old rat. Electron micrograph shows the distribution of
rough endoplasmic reticulum (er) and several Golgi complexes (G).
The presence of
lipfuscin granules (L)is observed. Note the presence of nuclear pores (arrow) on the
nuclear membrane. X 15,570.
430
MITRAL CELL DIFFERENTIATION IN RAT OLFACTORY BULB
Dyal N. P. Singh and Edward J. H. Nathaniel
PLATE 7
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