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The tridimensional structure of Nissl bodiesA stereoscopic study in ventral horn cells of rat spinal cord.

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THE ANATOMICAL RECORD 207539-546 (1983)
The Tridimensional Structure of Nissl Bodies: A
Stereoscopic Study in Ventral Horn Cells
of Rat Spinal Cord
ALAIN BEAUDET AND ALAIN RAMBOURG
Montreal Neurological Institute, McGill Uniuersity, Montreal, Quebec,
Canada H3A 2B4 (A.B.) and Dkpartement de Biologie du CEA, C.E.N.,
Saclay, Gifsur-Yvette, 91-191 cedex, France (A. R.)
ABSTRACT
The tridimensional structure of rough endoplasmic reticulum
was examined with both high and low voltage electron microscopes in large
ventral horn cells of rat spinal cord, by combining stereoscopic techniques with
the use of thick sections selectively impregnated with heavy metal salts. In all
neurons examined Nissl bodies appeared as well defined clusters of densely
stained and profusely anastomosed plate-, ribbon-, and thread-like cisternae.
Plate-like cisternae were variable in size, often showed a shallow curvature,
and usually ran in short parallel arrays, separated from one another by fairly
constant intervals. All gave rise at their edges to several ribbon-like extensions
which occasionally decreased in width distally, turning into thin, thread-like
cisternae. Characteristically, these ribbon-like structures would emerge at a n
angle from their plate of origin and smoothly curve away from the plane of the
plate to merge with ribbons or threads arising from adjacent or more distant
plates. Most plate-like cisternae were found a t the periphery of Nissl bodies
and tended to be oriented parallel to their surface. In contrast, the center of
NissI bodies was almost exclusively occupied by a complex network of ribbonand thread-like cisternae. It is suggested that the basic platelribbon association
here described in spinal motoneurons might be a constant feature of Nissl body
architecture in various neuronal types, while the size, orientation, and relative
proportion of plate-like cisternae may vary according to the metabolic state
and/or functional specialization of the cells.
In 1894, Nissl reported the presence of
dense basophilic masses within the perikarya of nerve cells. These Nissl bodies have
long since been shown to correspond in the
electron microscope to compact aggregates of
rough endoplasmic reticulum (RER) cisternae (Palay and Palade, 1955). Information is
still lacking, however, on the precise shape
and spatial arrangement of these RER cisternae, which until recently could only be inferred from the reconstruction of serial thin
sections. Cytochemical techniques have now
been developed (Thiery and Rambourg, 1976;
Tsukita and Ishikawa, 1976; Thiery, 1979)
which provide selective staining of intra-cellular membrane systems, to the exclusion of
other cellular elements such as cytoskeleton
or ribosomes. Such methods make it possible
to examine the endoplasmic reticulum in relatively thick sections and, through the use of
stereoscopic techniques, to directly visualize
its tridimensional architecture.
Cci 1983 ALAN
R. LISS, INC
This approach has already contributed to
our understanding of the topographic organization of smooth endoplasmic reticulum in
axons of rat spinal and chick ciliary ganglia
(Droz et al., 1975; Markov et al., 1976, Rambourg and Droz, 19801, rat neurohypophysis
(Alonso and Assenmacher, 1978, 1979a) and
mice sciatic and phrenic nerves (Tsukita and
Ishikawa, 19761, and of RER in nerve cell
bodies of rat spinal ganglia (Droz et al., 1975;
Thiery and Rambourg, 1976; Rambourg et
al., 19831, supraoptic nucleus (Alonso and Assenmacher, 1979131, nucleus raphe dorsalis
(Gamrani and Galas, 1980; Araneda et al.,
19801, and substantia gelatinosa (Hannah,
1978)., None of these studies, however, has
yielded a comprehensive description of the
tridimensional structure of Nissl bodies. The
present report is a n attempt a t providing
Received February 21, 1983; accepted July 25, 1983
540
A. BEAUDET AND A. RAMBOURG
such a description, using as a model the large
ventral horn cell of rat spinal cord.
MATERIALS AND METHODS
The central nervous system of adult male
Sprague-Dawley rats was fixed by intra-aortic arch perfusion of a 3.5% glutaraldehyde
solution in 0.1 M sodium cacodylate buffer.
The spinal cord was dissected out of the vertebral canal and sliced a t the level of the
cervical enlargement into 1-mm-thick slabs
which were post-fixed for 1 hr in the same
fixative. Small tissue blocks (1mm3) including the ventro-lateral aspect of the anterior
horn were cut out under a dissecting microscope, rinsed in cacodylate buffer, and stained
for 1 hr in a 5% aqueous uranyl acetate solution, at 42°C. Following several rinses in
distilled HzO, the specimens were poststained in a lead citrate, copper sulfate, and
trisodium citrate solution for 1 h r a t 42°C
(Ur-Pb-Cu stain, Thiery and Rambourg,
1976).The blocks were then washed again in
distilled H20, post-fixed overnight in a 1%
aqueous solution of osmium tetroxide a t 4"C,
dehydrated in graded ethanols, and embedded in Epon. Thick (2-5 pm) and semi-thin
(0.5-1 pm) sections were cut from each block
on a Porter Blum ultramicrotome, deposited
on Formvar-coated copper grids, and respectively examined a t high (1000 kV) and low
(100 kV) voltage in the electron microscope.
Large multipolar neurons were systematically photographed a t magnifications ranging from 2800 to 22,000. Most of these cells
undoubtedly corresponded to motoneurons,
but the possibility that our sampling also
comprised a number of large propriospinal
neurons (Cajal, 1909-1911; Aitken and
Bridger, 1961)cannot be formally excluded.
For stereoscopy, two photographs of the
same field were taken a t plus and minus 7"
angles from the original 0" position, by tilting the goniometric stage of the electron microscope. Tridimensional visualization was
obtained by looking at properly adjusted
pairs of such pictures with a stereoscopic binocular lens.
RESULTS
At low magnification using either conventional or high voltage electron microscopes,
the perikaryon shows well individualized aggregates of heavily stained and profusely anastomosed lamellar elements (Fig. 1).These
aggregates, which correspond to the blocklike basophilic structures identified as Nissl
bodies in the light microscope, present
roughly circular or oval outlines in 0.5 pmthick sections but are found to be ovoid in
shape when stereoscopically examined with
the high voltage electron microscope in 3-5
pm-thick sections. Their internal lamellar
constituents were shown in a previous publication (Rambourg et al., 1983) to correspond
to rough ER cisternae filled up with heavy
metal precipitate.
The Nissl bodies are separated from one
another by strands of cytoplasm containing
densely stained mitochondria, stacks of Golgi
saccules and scattered lysosomal granules.
In between these organelles, smooth ER cisternae form a pervasive network of delicate,
filamentous elements which are continuous
with the rough ER cisternae of the Nissl
bodies (Figs. 1-3). Although generally well
demarcated by these strands of cytoplasm,
Nissl bodies may occasionally be confluent
and occupy large, irregular regions of the
perikaryon (Figs. 1-21.
Within each Nissl body, the Ur-Pb-Cu impregnated RER cisternae appear as either
plate-, ribbon-, or thread-like structures (Figs.
3-61, The plate-like cisternae are variable in
size and shape; they usually show a shallow
curvature and may be partially twisted (Fig.
5). These plates are not fenestrated, but give
rise a t their edges to several ribbon-like extensions which usually decrease in width
distally, becoming narrow, thread-like connecting cisternae. Such ribbon-like structures emerge a t a n angle from their plate of
origin, curving smoothly above or below the
plane of the plate to merge with adjacent
plate-like or ribbon-like cisternae (Figs. 3-5).
In each Nissl body, plate-, ribbon-, or
thread-like cisternae assume a characteristic
tridimensional arrangement, illustrated by
the drawing in Figure 4. The plate-like cisternae usually run in short parallel arrays of
two or three elements, separated from one
another by fairly constant intervals of approximately 200 nm (Figs. 3-5). These stacks
of cisternae are frequently, but not exclusively, oriented parallel to the surface of the
Nissl body, with the concavity of the plates
facing inward. Such a concentric organization can be best appreciated in sections passing through the center of the Nissl body (Figs.
2-4).
Each of these plate-like cisternae is connected to several neighbouring plates from
either the same or adjacent stacks by means
of their lateral ribbon- or thread-like exten-
3-D STRUCTURE OF NISSL BODIES
Fig. 1. The perikaryon of a large motoneuron from
the anterior horn of rat spinal cord. 0.5 pm-thick, UrPbi
Cu-stained section-100 kv, x 10,000. The Nissl bodies
(Nb)appear as well delineated clusters of densely stained
and profusely anastomosed elements. Between the Nissl
541
bodies, pale strands of cytoplasm exhibit mitochondria
(MI, Golgi stacks (G),lysosomes (L), and a loose network
of tenuous, thread-like structures which corresponds to
the smooth endoplasmic reticulum (arrows).
542
A. BEAUDET AND A. RAMBOURG
543
3-D STRUCTURE OF NISSL BODIES
sions (Figs. 3-5). As described above, these
ribbon- or thread-shaped cisternae characteristically leave the plane of the plate from
which they arise to either directly reach the
plate located above or below, or to merge
with ribbons or threads originating from
more distant plates of the same or adjacent
stacks (Figs. 3-6). The consequence of this
arrangement is that in the space between
two adjacent stacks, the ribbon- or threadshaped anastomotic cisternae show a zig-zag
or criss-cross pattern (Figs. 3-51.
In some instances, and particularly towards the center of the Nissl body, narrow
ER cisternae may accumulate, branching and
interconnecting in all directions to form a n
elaborate tridimensional network (Fig. 6).
The cisternae never seem to interlace or interlock like the links of a chain, but instead
appear to be arranged in such a way as to be
separated from one another by a fairly constant distance equivalent to that separating
the plate-like cisternae within the stacks ke.,
approximately 200 nm).
Figs. 2-6. These figures are stereopairs. Each pair is
composed of photographs of the same field taken at two
different angles (+7” from the original 0” position of the
goniometric stage). A single stereoscopic image may be
obtained by using a stereoscopic binocular lens, the distance between the central axes of the two lenses being
adjusted to 65-70 mm. All figures are taken from 0.5
jim-thick sections of the ventral horn of rat spinal cord
impregnated with heavy metals (Ur-Pb-Cu).
Fig. 2. Section from the perikaryon of a large motoneuron showing a collection of interconnected Nissl bodies. Some are directly confluent (e.g., Nb 1and 2);others
are separated by a strand of cytoplasm (e.g., Nb 1and 31,
yet connected by means of thin, thread-like cisternae of
smooth endoplasmic reticulum (arrows). All Nissl bodies
are made up of short plate-, ribbon-, and thread-like
cisternae anastomosed in all directions. Note that most
plate-like cisternae tend to be perpendicular to the plane
of section in Nissl bodies cross sectioned in their center
(Nb 1,3), but parallel to that plane in Nissl bodies intersected at their periphery (Nb 2). x 6,500.
Fig. 3. The overall spatial arrangement of RER cisternae may be appreciated in this tridimensional overview of a single, ovoid Nissl body. Plate-like cisternae
are organized into a number of short parallel arrays
(large arrows). Characteristically, plates of the same or
adjacent stacks are interconnected through flat and narrow, ribbon-like cisternae (small arrows). These ribbonlike extensions occasionally decrease in width, giving
rise to thin, thread-like cisternae (arrowheads). Most
plate-like cisternae are found at the periphery of the
Nissl body, the center being almost exclusively occupied
by a complex network of anastomosed ribbon- and threadlike cisternae. x 12,500.
DISCUSSION
The present study provides a new outlook
on the shape and tridimensional organization of RER cisternae within Nissl bodies. In
contrast to previous descriptions, which were
based on the analysis of serial thin sections
(Palay and Palade, 1955; for review, see Peters et al., 1976), the model proposed here
relies on direct stereoscopic examination of
RER in thick sections, using either high or
low voltage electron microscopes.
In all neurons examined, Nissl bodies were
found to be made up of lamellar or “platelike” cisternae and narrower “ribbon-like”
and “thread-like” cisternae. These observations broadly conform with those of Palay
and Palade (1955) who originally described
“tubules” and “large flattened vesicles” designated as cisternae, as basic components of
the Nissl bodies membrane system. As correctly forseen by these and other investigators (for review, see chapter V in Peters et
al., 19761, all of these elements are interconnected in a continous ti*idimensional network. Our findings, however, reveal a spatial
organization which differs in several respects
from that proposed by Palay and Palade
(1955). This can be best appreciated by comparing their model of the tridimensional arrangement of RER in Nissl bodies of
motoneurons (text Fig. 1 in Palay and Palade, 1955) with our own drawing in Figure
4. In both diagrams, flattened or plate-like
cisternae are of different sizes and shapes
and form regularly spaced parallel arrays.
However, in our diagram, plate-like cisternae are neither fenestrated nor involved in
the formation of “reticular sheets,’’ i.e., interconnected with other plates running in
the same plane. In fact, ribbon- and threadlike connecting cisternae are never found to
remain in or leave at a perpendicular angle,
the plane of their plate of origin.
A recent analysis of the fine structure of
six types of neurons in rat dorsal root ganglia
(Rambourg et al., 1983) suggests that the
basic platelribbon association here described
in spinal motoneurons may be a constant
feature of Nissl body architecture. The combination of “tubular” and “lamellar” elements visualized with the same technique in
neurons of rat supraoptic nucleus (Alonso and
Assenmacher, 1979b) is also in keeping with
this interpretation. In contrast, the overall
spatial arrangement of RER cisternae appears to vary, not only from one cell type to
544
A. BEAUDET AND A. RAMBOURG
Fig. 4. Ink drawing of a Nissl body as seen with a
stereoscope in the underlying stereo pair. Note the
roughly concentric orientation of plate-like cisternae at
the periphery of the Nissl body and, within its core, the
complex tridimensional organization of ribbon- and
thread-like elements. x 17,000.
3-D STRUCTURE OF NISSL BODIES
Fig. 5. The structural relationships between plateand ribbon-like cisternae seen at high magnification.
Three parallel plate-like cisternae, roughly perpendicular to the plane of section, occupy the center of the field.
Each gives rise to a number of ribbon-like extensions,
which gently curve away from the plane of the plate and
establish connections with neighbouring plate- (arrows)
or ribbon-like (arrowheads)cisternae. ~ 5 0 , 0 0 0 .
545
Fig. 6. The center of a Nissl body seen at high magnification. It is almost exclusively composed of ribbon(arrows) and thead-like (arrowheads) cisternae, anastomosed in all directions. Note that all cisternae remain
separated by a fairly constant interval (approx. 200 nm),
similar to that separating plate-like cisternae within a
stack. X22,OOO.
546
A. BEAUDET AND A. RAMBOURG
another, but also within a given category of
neurons. Thus the differences in the size, orientation, and relative proportion of plate-like
cisternae observed betwen lower motor and
various types of primary sensory neurons
(Rambourg et al., 1983) or the shift from “lamellar” to “tubular” configurations reported
to occur upon dehydration in cells of rat supraoptic nucleus (Alonso and Assenmacher,
1979b). Such variations presumably reflect
differences in the functional specialization
and/or metabolic activity of the cells.
ACKNOWLEDGMENTS
This investigation was supported by the
Commissariat a l’energie atomique (France),
the Medical Research Council (Canada), and
a France-Quebec exchange program. The authors are indebted to Dr. Yves Clermont for
his inspiring review of the manuscript. They
also thank Dr. Alain Marraud and his colleagues for their help with the work a t the
high voltage electron microscope (CNRSONERA, Ch5tillon-sous-Bagneux, France),
Miss Eva Schneider for preparing the drawing in Figure 4, and Miss Beverley Lindsay
for typing the manuscript.
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