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Ultrastructural differentiation of the first hensen cell in the gerbil cochlea as a distinct cell type.

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THE ANATOMICAL RECORD 240:149-156 (1994)
Ultrastructural Differentiation of the First Hensen Cell in the Gerbil
Cochlea as a Distinct Cell Type
SAMUEL S. SPICER AND BRADLEY A. SCHULTE
Department of Pathology and Laboratory Medicine, Medical University of South Carolina,
Charleston, South Carolina
ABSTRACT
Background: The mammalian cochlea contains beneath
and lateral to outer hair cells, several types of supporting cells. The function of these cells has not been explained beyond providing a structural
base.
Methods: The supporting cells of gerbil cochlea were examined by electron microscopy with a view to elucidating their biologic activity on the
basis of cytologic structure.
Results: Ultrastructural examination differentiated the laterally located
Hensen cells from their medial neighbor connected to the third Deiters cell.
The latter cell formed a cover to the outer tunnel between Hensen and
Deiters cells, appeared not to reach the basilar membrane, and exhibited a
denser cytosol and more mitochondria, compared to Hensen cells. In these
respects the cell observed here to cover the outer tunnel, corresponded
with the tectal cell described by Henson et al. (1983) in the mustache bat,
but not heretofore documented in other animals.
Conclusions: This distinctive cell in the gerbil differed in displaying
unique villus-like structures which projected from the basomedial surface
and are referred to as fimbriae. The fimbriae and interspersed filopodia
largely filled outer tunnel space and expanded the cell’s basal surface. The
amplification of basal plasmalemma by fimbriae and their content of mitochondria testify to a role for the tectal cell in ion resorption and an influence on ion content and volume of outer tunnel fluid.
Q 1994 Wiley-Liss,
Inc.
Key words: Cochlea, Supporting cells, Morphology, Ion transport, Ultrastructure, Gerbil, Outer tunnel
The fine structure of the lateral supporting cells in
the mammalian cochlea, including Deiters and Hensen
cells, has been investigated in several studies. Deiters
cells support outer hair cells on a cup-shaped apical
surface and project a phalangeal process apically into
the reticular lamina (Smith and Dempsey, 1957; Engstrom and Wersall, 1958; Iurato, 1961: Kimura, 1975,
1984; Smith, 1978). Dieters cells contain numerous organelles including mitochondria and the rosette complex in their apical compartment (Spicer and Schulte,
1993), and in their basal portion a microtubule stalk
(Angelborg and Engstrom, 1972) which apparently
contributes to the mechanical support of overlying hair
cells.
Hensen cells, located lateral to Deiters cells and the
outer tunnel of Corti, consist of lucent flocculent cytosol enclosing sparse organelles-mainly mitochondria
and osmiophilic bodies (Engstrom and Wersall, 1958;
Kimura, 1975, 1984; Santi, 1988). Hensen cells have
been described as showing club-like or polypoid projections toward the outer tunnel and becoming thin in the
region connecting with the phalangeal process of the
third Deiters cell (Kimura, 1975). Prominent microvilli
(c)
1994 WILEY-LISS, INC
which have been interpreted as indicating a resorptive
function, characterize the apical surface of Hensen
cells (Engstrom and Wersall, 1958; Kimura, 1984).
Consisting of few organelles and mainly a translucent cytosol which can be presumed to be highly hydrated, Hensen cells are difficult to preserve. Presumably, for this reason, there are few published
illustrations of the fine structure of these cells. The
present study aimed a t enquiring further into the
structure of the lateral supporting cells in the gerbil
cochlea. Evidence was obtained that the cell in the area
assigned to the first Hensen cell differs in structure
and presumably in function from the more lateral
Hensen cells and comprises a distinct cell type. This
cell in gerbil cochlea resembles in some but not other
structural features, the cell observed in the mustache
Received February 7, 1994; accepted March 23, 1994.
Address reprint requests to Dr. Samuel S. Spicer, Department of
Pathology and Laboratory Medicine, Medical University of South
Carolina, 171 Ashley Avenue, Charleston, SC 29425.
150
S.S.SPICER AND R.A. SCHULTE
Fig. 1. Toluidine blue stained thick section showing a cell (TC) covering the outer tunnel (OT) and presumed to correspond with the
tectal cell of the bat. Segments and processes of the cell fill much of
the tunnel. Cytosol in the first Hensen cell (HC-1)is lighter than that
in the third Deiters cells (DC-3) it borders closely. Hensen cells lie
below and lateral to the outer tunnel and rest on basilar membrane
(BM). Laterally, the Claudius cells (arrows) overlie Boettcher cells
(BC), basilar membrane, and outer sulcus cells (OSC).4 kHz. x 900.
bat by Henson et al. (1983) and designated the tectal
cell.
window. The cochleas were dissected free and left overnight with fixative, in the refrigerator. The scalae were
subsequently flushed gently with phosphate buffer and
perfused with 1% osmium tetroxide in phosphate
buffer. This postfixation was terminated after 30 min
by flushing the scalae with distilled water.
Following fixation, the inner ears were trimmed of
excess bone and decalcified by perfusion with and then
immersion in 0.12M EDTA at pH 8.0. EDTA solutions
were changed daily for about three days until decalcification was complete. Specimens were then dehydrated by perfusion and immersion with graded alcohols and propylene oxide, and embedded in Epon
LX-112 resin. Before the resin had completely hardened, the cochleas were bisected lengthwise and cut
into half turns which were re-embedded in Epon. Radial slices were cut with a razor blade from the cochlear
regions encoding 0.5 to 10 kHz (Tarnowski e t al., 1991).
These slices were re-embedded in Epon. Thick sections
and adjacent thin sections were taken a t several levels,
25 pm apart from each epoxy block, and the thick sections were stained with toluidine blue. Ultrathin sections were selected for electron microscopic examination and stained with uranyl acetate and lead citrate
MATERIALS AND METHODS
The six Mongolian gerbils (Meriones unguiculatus)
examined here were housed in a low-noise room from
birth to sacrifice a t 3-6 months of age. Similarly aged
animals from the gerbil colony maintained in this facility have consistently shown normal hearing as
judged by evoked potentials of brain stem and auditory
nerve (Schmiedt, 1989; Mills et al., 1990). Procedures
for the care and use of animals were approved by the
Animal Use Committee of the Medical University of
South Carolina under NIH Grant DC 00422.
The gerbils under urethane anesthesia were perfused with minimal pressure via a cardiac catheter employing 10 ml of room temperature normal saline that
contained 0.1% NaNO,. After exsanguination, they
were perfused with 50 ml of room temperature fixative
fluid containing 4.0% paraformaldehyde and 2.0% glutaraldehyde in 0.1M phosphate buffer, pH 7.4. The bullae were then opened, the stapes was removed, the
round window was perforated, and 1.0 ml of the fixative was injected gently into the scalae via the round
COCHLEAR TUNNEL CELLS
151
Fig. 2. The tectal cell (TC) forms a roof over the outer tunnel and
attenuates toward connection a t a tight junction with the Deiters cell
phalanx (P)a t the lateral limit of the reticular lamina. Filopodia (F)
and fimbriae (arrows) extend from the cell into the tunnel. The fimbriae reach the base of the phalanx and lateral surface of the third
Deiters cell. A rosette complex tRC1 occupies apical cytosol near the
Deiters cell’s contact with fimbriae. 4 kHz. x 5,000.
Flg. 3. In a microscopic field below that of Fig. 2, minute processes
presumably derived from the tectal cell (TC) fill much of the outer
tunnel. These fimbriae reach the lateral surface of the third Deiters
cell IDC) but not the surface of Hensen cells tHC) below. 4 kHz. x
2,500.
when the adjacent thick section showed a longitudinal
view of well-preserved organ of Corti. Sections were
viewed ultrastructurally from six animals a t one or
more mid-frequency regions.
the apicolateral corner of the outer tunnel in the position generally assigned to the first Hensen cell (Figs. 1,
2, and 4).This cell differed, however, from the Hensen
cells lateral to it in both location and cytologic structure, and resembled more closely the tectal cell of the
bat (Henson et al., 1983).It joined a Hensen cell laterally a t an apical tight junction and sent a tapering
process medially to a tight junction with the phalanx of
RESULTS
An irregular cell profile often enclosing a roughly
spherical nucleus with dispersed chromatin, occupied
152
S.S. SPICER AND B.A. SCHULTE
Fig. 4. A segment ( S ) of a cell covering the outer tunnel (OT) contains an apical nucleus and reaches upward toward the reticular lamina out of the field. The tunnel’s cover cells (TC) possess fairly numerous, widely distributed mitochondria and project filopodia, and
numerous fimbriae into the tunnel toward the apicolateral surface of
the lateralmost Deiters cell. Rosette complexes (RC)lie beneath these
surfaces. Fimbriae contain minute, dense mitochondria (arrows). 500
kHz. x 4,250.
COCHLEAR T U N N E L CELLS
153
Fig. 5. Tectal cells (TC) a t the top contain relatively dense cytosol
and extend fimbriae into the outer tunnel (OT) in a tangential section
of the organ of Corti. Deiters cells (DC) under outer hair cells (OHC)
contain the rosette complex (RC) and many mitochondria in their
upper compartment and granular cytosol, and a microtubule stalk (S)
in the bottom part. The first Hensen cell (HCI) and more lateral
Hensen cells display lucent, flocculent cytosol, and infrequent mitochondria and adjoin neighbors closely on a fairly regular boundary.
Amorphous material separates upper and lower fibrous bands in the
basilar membrane (BM). 4 kHz. x 2,250.
Fig. 6. Fimbriae of a tunnel cover cell enclose lucent cytosol and are
bordered closely by round, dense profiles of presumed microvillar projections from the fimbrial surface (arrows).10 kHz. x 12,500.
the third Deiters cell, thus delimiting the outer tunnel
space apically (Figs. 1 and 2). The lateral surface of
segments of the cell covering the outer tunnel in the
gerbil fitted against Hensen cells (Figs. 1 and 5). Profiles of these cells were not observed in continuity from
a n apical front on endolymph, to a contact with the
basolateral surface of the third Deiters cell or with
basilar membrane. Instead, sections showing the presumed tectal cell at the roof of the tunnel also disclosed
a segment of a cell with structural features of a Hensen
cell adjoining the third Deiters cell along its basolat-
era1 plasmalemma and resting on the basilar membrane (Fig. 5 ) . The cell covering the outer tunnel in
the gerbil possessed denser cytosol than the more laterally situated Hensen cells, and disclosed, moreover,
more numerous mitochondria which were distributed
throughout the cytoplasm rather than a t the margins
(Figs. 4 and 5).
The most distinctive feature of the gerbil’s tunnel
cover cell, however, concerned its highly expanded
basal and medial surface (Figs. 1-91. The cell extended
numerous villus-like projections, referred to as fim-
Flg. 7. Elongated profiles of longitudinally sectioned fimbriae protrude into the outer tunnel (OT) toward the third Deiters cell (DC).
TC, tectal cells. x 2,250.
154
S.S.SPICER AND R.A. SCHULTE
Fig. 8. Fimbriae infiltrate tunnel space between a tectal cell and
Deiters cell. The filamentous meshwork (arrow) of a rosette complex
together with marginated mitochondria, lie beneath the Deiters cell
plasmalemma contacted by fimbriae. 4 kHz. x 7,500.
Fig. 9. Profiles of fimbriae enclose membrane limited bodies, possibly lysosomes (arrowheads),and minute mitochondria. The mitochondria reveal cristae separated by sparse matrix. Microvillar extensions
protrude from some fimbriae (arrows). 1 kHz. x 33,750.
briae, along with irregular filopodia, into the outer
tunnel. The fimbriae and filopodia filled much of the
tunnel space reaching the apicolateral surface of the
third Deiters cell, often over a n underlying rosette
complex (Figs. 2, 4,and 8). Minute cell processes presumed to be microvillar extensions from the surface,
bordered some fimbriae (Fig. 6). The diameter of the
fimbriae ranged mostly between 0.2 and 0.5 pm but
occasionally reached up to 1.0 pm. The fimbriae enclosed lucent cytosol, sparse membrane limited bodies,
and dense, exceptionally small mitochondria (Figs. 6
and 9) which measured about 0.2 pm in diameter.
A full profile of the most medial (first) Hensen cell
was rarely observed, presumably because of a n arched
contour and orientation a t a n angle to that of Deiters
cells. The few full images obtained showed that the
presumed first Hensen cell maintained extensive basomedial contact with the basolateral plasmalemma of
the third Deiters cell, basal contact through basement
membrane with basilar membrane, and apicomedial
contact with segments of the tectal cell or outer tunnel
space, while also fronting apically on the scale media.
More often, apical segments of Hensen cells were observed bordering tectal cells, and separate basal seg-
ments (Fig. 5) were encountered in approximation to
Deiters cells and basilar membrane. Although not in
continuity, these profiles appeared similar cytologically, and were assumed to represent upper and lower
regions of the first Hensen cell. The first Hensen cell
thus formed the basal limit and part of the lateral
boundary of the outer tunnel. The somewhat irregular
lateral plasmalemma of the second Hensen cell approximated its neighbors’ surface closely except a t occasional focal separations.
DISCUSSION
Several structural features afford evidence for differentiating the cell in the first Hensen cell area of gerbil
cochlea a s a separate cell type. The body of this cell
joined laterally to a Hensen cell and its thin medial
process reached the reticular lamina forming a cover to
the outer tunnel a s shown schematically in Figure 10.
The cell also displayed denser cytosol and more numerous and widely distributed mitochondria compared
with Hensen cells. In these respects, the cell covering
the outer tunnel in gerbil cochlea resembled that observed by scanning and transmission electron micros-
155
Cells
Deiters Cells
Fig. 10. Diagramatic representation of cells delimiting lateral tunnel spaces in the organ of Corti.
copy by Henson et al. (1983) in the mustache bat, Pteronotus p . parnellii, and referred to as the tectal cell.
Tectal cells have apparently not been demonstrated or
referred to in a n animal other than the bat.
No single profile of this distinctive cell yet observed
has revealed a surface exposed to the scala media, contact with the basolateral plasmalemma of the third Deiters cell, and apposition to the basilar membrane. The
cell in the gerbil like that in the bat can therefore be
presumed not to reach basilar membrane a s do Hensen
cells. However, Hensen cells were only rarely observed
in full length profile from scala media to basilar membrane because of curvature and orientation. Nevertheless, it appears doubtful that a cell profile facing scala
media and interpreted as a tectal cell actually possessed continuity out of the plane of section with a
basal cell profile bordering a Deiters cell and the basilar membrane and interpreted as a Hensen cell. The
reason for this view lies in the invariably greater cytosolic density and abundance of organelles in profiles
of the tunnel covering cell, compared with the presumed Hensen cell segment at the base.
Conversely, a question arose whether a partial cell
profile bordering the base of the third Deiters cell and
resting on basilar membrane, and a separate profile
bordering the tectal cell and fronting on scala media,
represented basal and apical segments of Hensen cells
connected a t a level outside that of the plane of section.
Ultrastructural findings of Henson et al. (1982) suggested that the basally located cell profiles in the bat
constitute a separate cell-type termed the tunnel floor
cell. Present observations in the gerbil favor interpreting the cell contacting the third Deiters cell and basilar
membrane a s part of a Hensen cell that reaches the
scala media a t another level. This interpretation rests
on the content of similar lucent, flocculent cytosol and
sparsity of organelles other than a few mitochondria in
both the lower and the upper segments, and the infrequent observation of a first Hensen cell extending from
basilar membrane to scala media.
The fimbriae decorating the basomedial surface were
the most definitive structures characterizing the cells
covering the outer tunnel. In this respect, the cover cell
a t the roof of the tunnel in the gerbil cochlea differed
from the bat’s tectal cell (Henson e t al., 1983) which
lacked such plasmalemmal amplification. To our
knowledge, this specialized surface adaptation has not
been encountered on any other cell. Such a fimbriated
cell appears not to have been described heretofore, in
cochlea, possibly because of differences between gerbils
and the other genera examined.
Expansion of the plasma membrane inward or outward from the surface supplies increased area for flux
of fluid and solute to or from the cell. Surface expanded
by infoldings of apical or basal plasmalemma, a s for
example in gastric parietal cells or renal and salivary
gland ductal epithelium, commonly utilizes a n ATPase
pump to increase efflux of a specific ion against a gradient. However, the cells a t the roof of the outer tunnel
lacked a cytochemically detectable level of Na,KATPase (Schulte and Adams, 1989) or H,K-ATPase
(S.S. Spicer, A.J. Smolka, and B.A. Schulte, unpublished data) and thus failed to yield evidence of such
pump activity.
On the other hand, amplified plasmalemma in the
form of microvilli protruding from the cell can facilitate a less energy dependent resorption down a gradient. The extensive evaginations of the fimbriated cell’s
plasmalemma into the lumen of the outer tunnel points
to a probable similar function a t this site. The fimbriae
differ from microvilli of brush border cells, however, in
their larger dimensions and content of abundant cytosol and exceptionally minute mitochondria. The mitochondria in the fimbriae testify to a transport process
with an ATP requirement intermediate between that
in striations of renal and glandular duct cells and that
in microvilli of intestinal or renal brush borders.
The fimbriated surface of cover cells provides evidence of resorption by these cells, implying they affect
ion content and volume of fluid in the outer tunnel.
156
S.S. SPICER AND B.A. SCHULTE
The fimbriae can be viewed in this light as resorbing
an ion, possibly K + from outer tunnel fluid for transport to Hensen cells and ultimately Claudius cells
through the gap junctions that have been demonstrated between inner ear supporting cells (Iurato e t
al., 1976; Nadol, 1978; Santos-Sacchi, 1987). This K'
uptake could counter balance the sound induced efflux
of K' from outer hair cells and nerves into the outer
tunnel (Johnstone et al., 1989) and impede K' leakage
from endolymph into the tectal cells and Hensen cells.
Largely lacking apical microvilli, the fimbriated cells
are presumably not concerned, as are neighboring
Hensen cells, with resorption from endolymph, and for
this reason and their apparent failure to reach basilar
membrane appear not to contribute directly to fluid or
ion translocation between endolymph and perilymph.
ACKNOWLEDGMENTS
The authors appreciate the valuable technical and
secretarial assistance of Mrs. Nancy Smythe and Mrs.
Leslie Harrelson. This work was supported by NIH
Grants DC 00422 and DC 00713.
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