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Dorsal roots of the rabbit investigated by freeze-substitution.

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Dorsal Roots of the Rabbit Investigated by
Freeze-substitution '
S. K. MALHOTRA AND A. VAN HARREVELD
Kerckhoff Laboratories of the Biological Sciences, California Institute of
Technology, Pasadena, California
ABSTRACT
Dorsal roots from adult rabbits prepared by a freeze-substitution technique and studied by electron microscopy have been compared with those obtained
by standard fixation with 0 ~ 0 4 . The essential features of myelinated and nonmyelinated fibers and their associated Schwann cells are similar in appearance after
both types of fixation. There are, however, slight differences. The extracellular clefts
between the nonmyelinated axons and their associated Schwann cells are wider i n
freeze-substituted roots (200 to 250 A versus 100 to 150 A ) . The major dense lines
in the myelin sheath are wider (45 to 55 A versus about 30 A ) and their radial repeat
period is larger (145 to 155 A versus 100 to 120 A) i n freeze - substituted material
than i n routinely fixed nerves.
Electron microscopy of cerebellar cortex
processed by a freeze-substitution technique revealed considerably more extracellular space i n the molecular layer than
is usually seen in electron micrographs obtained by routine chemical fixation (Van
Harreveld, Crowell and Malhotra, '65). I n
view of these findings it was of interest
to investigate peripheral nervous tissue
by the same technique of freeze-substitution and to compare the results with
those obtained by standard chemical fixation.
MATERIALS AND METHODS
Dorsal roots from adult rabbits have
been used which are similar to peripheral
nerves, but contain less connective tissue
and lack the epineurium found in peripheral nerves (Nathaniel and Pease, '63;
Gamble, '64). The study of frozen material has to be restricted to the superficial
layer of the tissue (about 10 thick) because the fine structure in the deeper parts
is disorganized by ice crystals (Van Harreveld and Crowell, '64). The absence of
a perineurium in the root is therefore a
distinct advantage. Lumbar roots were
taken from rabbits narcotized with pentobarbital (50 mg/kg) and frozen within
1 to 2 minutes after removal from the body.
The procedure for freezing and the subsequent treatment of the tissue have been
described previously (Van Harreveld and
Crowell, '64; Van Harreveld, Crowell and
ANAT. REC., 152: 283-292.
Malhotra, '65). The tissue was frozen by
bringing it in contact with a mirror smooth
silver surface precooled to about -207°C
by liquid nitrogen under reduced pressure.
The root was placed on a small block of
agar kept in a trough of aluminum foil
attached to the apparatus that carried the
tissue to the freezing surface. The frozen
tissue was subjected to substitution fixation for two days at -85°C in acetone containing 2% OsO,. The material was subsequently kept for a few hours a t -25°C
and then warmed to room temperature
while still in the substitution medium. The
excess osmium was removed by bathing
the tissue for 3 to 4 hours in repeatedly
changed acetone before the roots were embedded in Maraglas (Freeman and Spurlock, '62).
For controls, freshly dissected roots were
fixed ex situ in a 1% solution of OsOl in
acetate-Verona1 buffer (pH 7 . 3 to 7.4)
made isotonic by addition of indifferent
salts (Zetterqvist, '56).
The tissue blocks were sectioned on a n
LKB Ultrotome in such a way that the
nerve fibers were cut transversely. Sections
about 1 thick were stained with methylene blue and Azure B (Richardson, Jarett
and Finke, '60) for examination by light
microscopy. Only those blocks of tissue
that met the criteria of satisfactory freezing (absence of ghosts of ice crystals in
1 Supported by a grant from The National Science
Foundation (GB 2055).
283
284
S. K . M A L H O T R A AND A . V A N HARREVELD
the surface layer) were examined by electron microscopy. Ultrathin sections were
stained with lead citrate (Reynolds, '63).
RESULTS
The structure of the dorsal root has been
described previously and is essentially similar to that of peripheral nerves (Nathaniel
and Pease, '63). This study will be restricted to the myelinated and nonmyelinated nerve fibers and the Schwann cells
associated with them. Little attention has
been paid to the connective tissue component, which is not very prominent in the
present electron micrographs. The preservation of cellular structural detail by
freeze-substitution is comparable with that
observed in electron micrographs produced
by standard fixation (Van Harreveld,
Crowell and Malhotra, '65; Malhotra and
Van Harreveld, '65).
Nonmyelinated fibers. The axons of the
nonmyelinated fibers and the Schwann cells
are separated by extracellular spaces which
are of more or less uniform width (figs.
2 and 3). Measurements at places where
the membranes are sharp and parallel to
each other give values of 200 to 250 A for
these extracellular clefts. An extracellular
space of similar width is found between
the membranes forming the mesaxons in
Schwann cells enclosing nonmyelinated
fibers. Occasionally, however, the two apposing membranes come closer together
and even touch each other over a small
distance (fig. 3, arrows), where they may
form five layered tight junctions (Farquhar
and Palade, '63; Karlsson and Schultz, '64).
A few nonmyelinated fibers can be seen in
figures 1 and 2 which are not associated
with Schwann cells.
In the deeper regions of the root, ice
crystals are formed which disorganize the
fine structural details (fig. 8). Ghosts of
ice crystals can be recognized as light
(unstained) areas bounded by sharp dark
outlines which represent concentrated protoplasm. The plasma membrane of the
nonmyelinated fiber and the Schwann cell
membrane are at places separated by a n
intervening space which may be as wide as
300 A or more. At other locations the intervening space is completely obliterated and
the two apposing membranes form a five
layered tight junction over large distances.
The prevalence of tight junctions in the
deeper regions in contrast to their rare occurrence in the superficial layers differs
from the findings of Elfvin ('63) who observed many tight junctions in the surface
layers of frozen-dried preparations of
splenic nerve. The tight junctions observed
in the present material are most likely artifacts without functional significance as
contrasted to e.g., electrical synapses (Bennett et al., '63; Dewey and Barr, '64).
Myelinated fibers. The myelin sheath
after freeze-substitution (figs. 5 and 6 )
shows essentially the same pattern as described for instance by Robertson ('58)
and Sjostrand ('60) in material prepared
by routine fixation in OsO, (fig. 7) or by
freeze-drying (Elfvin, '63). The well defined major dense lines, which are formed
by the intimate apposition of the dense
layer of the Schwann cell membrane that
faces the cytoplasm, measure between
45 A and 55 A. Each major dense line is
separated from the next by a distance of
90 A to 100 A; and the distance from the
middle of one major dense line to the middle of the next is about 150 A, which is the
repeating period of the major dense lines
in freeze-substituted material (figs. 5 and
6 ) . The about 100 A wide pale area between two major dense lines is bisected by
a n inconspicuous intraperiod line, which
is not very well preserved in tissues fixed by
OsO+ In a few electron micrographs, some
of the major dense lines are locally resolved into two thin dense lines separated
by a lighter line (fig. 6 ) . Such a n appearance in the myelin sheath which has also
been found by Robertson ('57) and Sandborn et al. ('64) could be due to incomplete
fusion of the apposed cytoplasmic surfaces
of the Schwann cell membranes.
Differences between roots fixed by freezesubstitution and by standard methods.
Although the preservation of myelinated
and nonmyelinated fibers by freeze-substitution and direct Os04 fixation is essentially alike, there are a few differences.
The clefts between nonmyelinated axons
and Schwann cells in roots fixed e x situ by
OsOl are in general 100 to 150 A wide
(fig. 4 ) . Similar values have been reported
in the literature for peripheral nerves (see
e.g., Sjostrand, '60; Elfvin, '61, '63; Finean,
'61; Gray, '64; Peters, '64; Robertson, '64).
FREEZE-SUBSTITUTED DORSAL ROOTS
The extracellular cleft between the nonmyelinated axon and the Schwann cell and
the space in the mesaxons is generally 50
to 100 A wider in freeze-substituted material than in routinely fixed tissue. A direct
comparison of this difference in the width
of the clefts around the nonmyelinated
fibers can be made from figures 3 and 4.
The major dense lines in the myelin
sheath are slightly wider in freeze-substituted nerve fibers (45 to 55 A versus 30 A ) .
The repeat period of the major dense lines
is 145 to 155 A after freeze-substitution
which is a little larger than the 100 to
120 A period determined in material fixed
with OsO,. Figures 6 and 7 which are at
the same magnification demonstrate these
differences in the myelin sheaths. This discrepancy can be explained by different degrees of shrinkage of the myelin sheath
during freeze-substitution and routine fixation. The repeat period for the major dense
lines obtained by freeze-substitution and by
freeze-drying (Elfvin, '63) is closer to the
radial repeat period of 180 A to 185 A
determined by x-ray diffraction in fresh
mammalian nerve (Schmitt, Bear and
Palmer, '41 ; Fernandez-Moran and Finean,
'57) than the 100 A to 120 A repeat period
observed in standard electron micrographs
(Finean, '61; Finean and Robertson, '58).
DISCUSSION
The clefts between the Schwann cell
membranes and nonmyelinated fibers
are somewhat wider in freeze-substituted
than in routine OsO, fixed tissue. It h a s
been suggested that the water distribution
i n the living tissue may be better preserved
by freeze-substitution than by routine
chemical fixation (Van Harreveld, Crowell
and Malhotra, '65). The larger radial repeat period in the myelin sheath in freezesubstituted material, which approaches the
repeat period observed with x-ray diffraction in fresh nerve, seems to support this.
I n the cerbellum the large extracellular
space observed in freeze-substituted material was not uniformly distributed i n the
tissue but was found especially between
the axons of granular layer cells which are
present in groups (Van Harreveld, Crowell
and Malhotra, '65). These axons are not
closely associated with glia cells. Dendritic
elements and presynaptic endings showed
285
in general a close relationship with glia
from which these elements were in most instances separated by the usual narrow (100
A ) clefts. In the dorsal root the nonmyelinated fibers are associated with the
Schwann cells which can be compared
with the glia of the central nervous system
and from which the nerve fibers are separated by 200-250 A wide clefts. The relationship of associated cells and nervous
elements is in the dorsal roots as well as
in the cerebellar cortex characterized by a
separation of the elements by relatively
narrow clefts.
ACKNOWLEDGMENTS
We greatly appreciate the valuable technical help of Mrs. Josephine Pagano and
Miss Judy Schwaffel.
LITERATURE CITED
Bennett, M. V. L., E. Aljure, Y . Nakajima and
G. D. Pappas 1963 Electronic junctions between teleost spinal neurons: Electrophysiology
and ultrastructure. Science, 141: 262-264.
Dewey, M. M., and L. Barr 1964 A study of the
structure and distribution of the nexus. J. Cell
Biol., 23: 553-585.
Elfvin, L. G . 1961 Electron microscopic investigation of the plasma membrane and myelin
sheath of the autonomic nerve fibres in the cat.
J. Ultrastr. Res., 5: 388-407.
1963 The ultrastructure of the plasma
membrane and myelin sheath of peripheral
nerve fibres after fixation by freeze-drying. J.
Ultrastr. Res., 8: 283-304.
Farquhar, M. G., and G . E. Palade 1963 Functional complexes i n various epithelia. J. Cell
Biol., 18: 375-412.
Fernandez-Moran, H., and J. B. Finean 1957
Electron microscopic and low angle x-ray diffraction studies of the nerve myelin sheath.
J. Biophys. Biochem. Cytol., 3 : 725-748.
Finean, J. B. 1961 Chemical ultrastructure in
living tissues. Charles C Thomas, Springfield,
Illinois.
Finean, J. B., and J. D. Robertson 1958 Lipids
and the structure of myelin. Brit. Med. Bull.,
14: 267-273.
Freeman, J. S . , and B. 0. Spurlock 1962 A new
epoxy embedment for electron microscopy. J.
Cell Biol., 13: 437-443.
Gamble, H. J. 1964 Comparative electron microscopic observations on the connective tissues
of a peripheral nerve and a spinal nerve root
i n the rat. J. A.nat. London, 98: 17-25.
Gray, E. G. 1964 Electron microscopy of cell
surface. Endeavour, 23: 61-65.
Karlsson, U., and R. Schultz 1964 Plasma membrane apposition in the central nervous system
after aldehyde perfusion. Nature, 201 : 12301231.
286
S. K. MALHOTRA AND A. VAN HARREVELD
Malhotra, S. K., and A. Van Harreveld 1965
Some structural features of mitochondria in
tissues prepared by freeze-substitution. J.
Ultrastr. Res., 12: 4 7 3 4 8 7 .
Nathaniel, E. J. H., and D. C. Pease 1963 Degenerative changes i n rat dorsal roots during
Wallerian degeneration. J. Ultrastr. Res., 9:
511-532.
Peters, A. 1964 An electron microscope study
of the peripheral nerves of the hag fish (Myxine
glutinosa, L.). Quart. J. Exp. Physiol., 49:
35-42.
Reynolds, E. S. 1963 The use of lead citrate at
high pH as an electron opaque stain in electron
microscopy, J. Cell Biol., 17: 208-213.
Richardson, K. C., L. Jarett and E. H. Finke
1960 Embedding in epoxy resins for ultrathin
sectioning in electron microscopy. Stain Technol., 35: 313-323.
Robertson, J. D. 1957 The ultrastructure of
frog muscle spindles, motor endings and nerve
fibres. J. Physiol., 137: 6P-8P.
- 1958 Structural alterations i n nerve
fibres produced by hypotonic and hypertonic
solutions. J. Biophys. Biochem. Cytol., 4: 349364.
- 1964 Unit membranes: A review with
recent new studies of experimental alterations
and a new sub-unit structure in synaptic membranes. In: Cellular Membranes in Development. Edited by M. Locke. Academic Press,
New York, pp. 1-81.
Sandborn, E., P. F. Koen, J. D. McNabb and
G. Moore 1964 Cytoplasmic microtubules i n
mammalian cells. J. Ultrastr. Res., 11: 123-138.
Schmitt, F. O . , R. S. Bear and K. J. Palmer 1941
X-ray diffraction studies o n the structure of the
nerve myelin sheath. J. Cell. and Comp.
Physiol., 18: 31-42.
Sjostrand, F. S. 1960 Electron microscopy of
myelin and nerve cells and tissues. In: Modern
Scientific Aspects of Neurology. Edited by
J. N. Cummings, Edward Arnolds, London, pp.
188-231.
Van Harreveld, A., and J. Crowell 1964 Electron microscopy after rapid freezing on a metal
surface and substitution fixation. Anat. Rec.,
149: 381-386.
Van Harreveld, A., J. Crowell and S. K. Malhotra
1965 A study of extracellular space in central nervous tissue by freeze-substitution. J.
Cell Biol., 25: 117-137.
Zetterqvist, H. 1956 The ultrastructural organization of the columnar absorbing cells of the
mouse jejunum. Karolinska Institute, Stockholm.
PLATE I
EXPLANATION O F FIGURES
1
Dorsal root prepared by freeze-substitution. The surface of the root
shows on the bottom of the figure. Nonmyelinated and most of the
myelinated fibers are well preserved. A thin layer of Schwann cell
cytoplasm surrounds the myelinated axons. The nonmyelinated axons
are present i n bundles generally invested by Schwann cell cytoplasm.
Calibration line 14.
2
Shows a group of nonmyelinated fibers prepared by freeze-substitution.
Most of the fibers are associated with Schwann cell cytoplasm but a
few in the upper half of the figure do not contact Schwann cells. But
for a few small regions the spaces between axons and Schwann cells
or between apposed Schwann cell membranes are of uniform width.
In the upper corner of the micrograph part of a myelinated fiber is
present. Calibration line 0.5 p .
FREEZE-SUBSTITUTED DORSAL ROOTS
S. K. Malhotra and A. Van Harreveld
PLATE 1
PLATE 2
EXPLANATION O F FIGURES
288
3
A magnified view of a small group of nonmyelinated fibers enclosed
by Schwann cell cytoplasm ( freeze-substitution). The cleft between
nerve fiber and Schwann cell is of uniform width except for one small
area where the two apposing membranes are separated by a very narrow space (arrow 1). Arrow 2 shows apposing Schwann cell membranes in close contact. The figure also shows a small part of a well
preserved myelin sheath with a thin layer of Schwann cell cytoplasm.
Calibration line 0.5 p.
4
Shows dorsal root prepared by ex situ fixation in isotonic osmium
tetroxide. The results are essentially similar to those observed in
well frozen tissue. The spaces between nonmyelinated axons and
Schwann cell membranes and in the mesaxon are more or less
uniformly wide. Part of a myelinated fiber is present on the right.
Calibration 0.5 p.
FREEZE-SUBSTITUTED DORSAL ROOTS
S. K. Malhotra and A. Van Harreveld
PLATE 2
289
PLATE 3
EXPLANATION O F FIGURES
290
5-6
Show typical appearance of the myelin sheath in freeze-substituted preparations. Intraperiod lines are faintly visible. The
axonal membrane (arrow 1) appears slightly thicker than the
Schwann cell membrane (arrow 2 i n fig. 5). The basement
membrane is exceptionally well preserved i n figure 5. Arrow in
figure 6 indicates split i n a major dense line. Calibration line
indicates 0.1 ,u.
7
Typical appearance of the myelin sheath fixed ex situ in isotonic
osmium tetroxide. Calibration line is 0.1 p .
FREEZE-SUBSTITUTED DORSAL ROOTS
S . K. Malhotra and A. Van Harreveld
PLATE 3
291
FREEZE-SUBSTITUTED DORSAL ROOTS
S. K. Malhotra and A. Van Harreveld
8
292
PLATE 4
Is taken at some distance from the surface of a dorsal root subjected to freeze-substitution. The ghosts of ice crystals are obvious. A comparison with figures 2 and 3 shows
that the spaces between axons and Schwann cells have become irregular, wider at
some places and showing no space over large areas at others (tight junctions). Calibration line is 0 . 2 ~ .
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