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The Journal of Neuroscience, September 15, 1998, 18(18):7487-7501
Postnatal Development of Type I and Type II Hair Cells in the
Mouse Utricle: Acquisition of Voltage-Gated Conductances and
Differentiated Morphology
Alfons
Rüsch1,
Anna
Lysakowski2, and
Ruth
Anne
Eatock1
1 The Bobby R. Alford Department of Otorhinolaryngology
and Communicative Sciences, Baylor College of Medicine, Houston, Texas
77030, and 2 Department of Anatomy and Cell Biology,
University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
The type I and type II hair cells of mature amniote vestibular
organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II
cells also have different complements of voltage-gated channels. Type I
cells alone express a delayed rectifier, gK,L, that
is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in
denervated organotypic cultures and in acutely excised epithelia. From
postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells
acquired the type I-specific conductance gK,L and/or a slow
inward rectifier, gh. By P8, the percentages of cells
expressing gK,L and gh were at mature
levels.
To investigate whether the electrophysiological differentiation
correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in
cultures and neonatal organs. The morphological and
electrophysiological differentiation followed different time courses,
converging by P28. At P0, when no hair cells expressed
gK,L, 33% were classified as type I by
ultrastructural criteria. By P28, ~60% of hair cells in acute
preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither
electrophysiological nor morphological differentiation depended on
ongoing innervation.
Key words:
type I hair cell; calyx terminal; voltage-gated
conductance; delayed rectifier; inward rectifier; inner ear
development; vestibular organ; utricle
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INTRODUCTION |
Despite increasing interest in the
development of the inner ear, relatively little is known about the
development of function in the sensory hair cells. Quantitative
ultrastructural studies of inner ear development are also rare. Here,
we combine electrophysiological and ultrastructural methods to
investigate the differentiation of hair cells in the early postnatal
mouse utricle, a vestibular organ that detects linear head movements
and head position.
The vestibular organs of amniotes have two types of hair cells, which
are classified according to the form of synaptic terminal made by
afferent nerve fibers (Wersäll, 1956 ). Type II cells, present in
all vertebrates, receive bouton terminals. Reptiles, birds, and mammals
also have type I hair cells, which in mature organs are enclosed by
cup-like calyx terminals (Wersäll, 1956; Wersäll and
Bagger-Sjöbäck, 1974). Individual afferent fibers frequently innervate both cell types, and thus have both bouton and
calyx endings (Fernández et al., 1988, 1990, 1995).
The unusual morphology of the type I hair cell and calyx and their
relatively late appearance in evolution suggest that they play a
special role in vestibular function. An obstacle to understanding this
role has been the lack of a comprehensive description of what
distinguishes type I from type II hair cells. In addition to the
difference in afferent terminal, they have different cell shapes
(Wersäll, 1956; Lysakowski and Goldberg, 1997), hair bundle geometry (Lapeyre et al., 1992; Peterson et al., 1996), and
K+ conductances. Type I, but not type II, hair cells
have a delayed rectifier conductance that we call gK,L
(Correia and Lang, 1990; Eatock et al., 1994; Rennie and Correia, 1994;
Ricci et al., 1996; Rüsch and Eatock, 1996a). In many type I
cells, gK,L is substantially activated at the resting
potential, greatly reducing the input resistance (Rennie et al., 1996;
Rüsch and Eatock, 1996b). The present study extends the
description of differences between type I and type II hair cells.
The presence of a signature conductance, gK,L, in
type I cells provided a tool with which to follow the
electrophysiological differentiation of the two cell types. We report
here that gK,L was not expressed in any neonatal cells but
was acquired in the latter half of the first postnatal week by a subset
of hair cells. Other changes occurred at the same time. We asked
whether this electrophysiological differentiation was closely linked to
morphological changes in the epithelium. We developed a set of
ultrastructural criteria to allow classification of hair cell type when
calyx endings have not yet formed or have degenerated, as in immature epithelia and denervated cultures. The morphological differentiation that we observed followed a more gradual time course than the electrophysiological changes. Both types of change occurred whether the
hair cells developed in vivo or in cultures that were
denervated before most calyces formed. Clearly, the calyx terminal, the
hallmark of type I hair cells, is not required for their
differentiation.
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MATERIALS AND METHODS |
Electrophysiology
Tissue preparation. Utricles were excised from young
albino mice (ICR outbred strain) (timed pregnant females were
obtained from Harlan Sprague Dawley, Indianapolis, IN). Date of birth
[postnatal day 0 (P0)] in this strain is typically embryonic day 19 (E19), with E0 being the day of vaginal plug formation as determined by
the animal supplier. The animals were killed by cervical dislocation and decapitated. Some utricles (acute preparations) were excised from
1- to 17-d-old mice and studied immediately. Other utricles were
explanted on collagen-coated coverslips on P1 and were grown in
organotypic cultures. Each culture was used for an electrophysiology experiment between 1 and 8 d after explantation, corresponding to
P2 and P9, respectively, and was then discarded. Culturing techniques were as previously described (Rüsch and Eatock,
1996a). Cultures were maintained at 37°C in Minimum Essential Medium
with Earle's salts (Life Technologies, Gaithersburg, MD),
supplemented with heat-inactivated horse serum (1:11; Life
Technologies).
In both types of preparations, the utricular nerve was cut between the
epithelium and Scarpa's ganglion. The nerve fibers in the cultures
presumably degenerated within hours of being cut off from their cell
bodies. Degenerating terminals were seen with the light microscope in
acute preparations. No nerve endings were evident in histological
material of cultures fixed 3 d after they were made, which was the
earliest stage of the cultures that was examined histologically (see
Materials and Methods, Morphology). In several experiments, whole-cell
recordings were obtained from hair cells isolated from adult utricular
epithelia. The utricles were excised from adult mice and mechanically
dissociated after a 30 min treatment with medium containing crude
papain (500 µg/ml; Sigma, St. Louis, MO) and L-cysteine
(300 µg/ml; Sigma).
Recording. The sensory epithelium of the mouse utricle was
mounted in an experimental chamber on the stage of a top-focusing microscope (Axioskop FS; Zeiss, Oberkochen, Germany) and viewed with a
40× water-immersion objective with differential interference contrast
optics. For recordings, a tear was made in the sensory epithelium with
a micropipette (20-30 µm tip diameter), partly exposing the
basolateral surfaces of hair cells. Patch pipettes were sealed onto the
basolateral membranes after cleaning them with a stream of
extracellular solution. All recordings (n = 381 hair
cells) were done in whole-cell voltage-clamp mode. Recordings were
obtained from hair cells from all regions of the epithelium. Recordings
were occasionally obtained from cells with relatively positive resting
potentials ( 32.8 ± 4.4 mV; range, 41 to 26; n = 11) and small voltage-dependent currents: <500 pA
when stepped to 0 mV from the standard holding potential of 64 mV.
These were thought to be supporting cells and were excluded from the
analysis.
Patch pipettes contained 140 KCl mM, 0.1 mM
CaCl2, 5 mM EGTA-KOH, 3.5 mM MgCl2, 2.5 mM
Na2ATP, 5 mM HEPES-KOH, pH 7.4, and 290 mmol/kg. The free Ca2+ concentration is ~2
nM (calculated with EQCAL software; Biosoft, Cambridge,
England). The experimental chamber was perfused at 10 ml/hr 1 with an extracellular solution that
contained 144 mM NaCl, 0.7 mM
NaH2PO4, 5.8 mM KCl, 1.3 mM CaCl2, 0.9 mM
MgCl2, 5.6 mM D-glucose, 10 mM HEPES-NaOH, vitamins and amino acids as in Eagle's
MEM, pH 7.4, and 320 mmol kg1. Channel blockers
were dissolved in this standard extracellular solution and applied
locally through a gravity-fed micropipette. To visualize flow of the
superfusate, we added polystyrene latex beads (Sigma) of ~1 µm in
diameter at a concentration of ~5 × 106
ml-1. Recordings were made at room temperature
(22-25°C).
Analysis. Series resistances (Rs) and
capacitances (C) were calculated from fits of monoexponential functions
to capacitive current transients evoked by 10 mV steps from a holding
potential at which most voltage-gated channels were deactivated (64
mV for type II hair cells; 104 mV for type I hair cells).
Rs and C values were also read from the patch-clamp
amplifier (Axopatch 200A; Axon Instruments, Foster City, CA) after
electronically nulling the current transients. These values were
usually within 10% of the values obtained by fitting the transients.
Rs was between 2.5 and 11 M , 70-90% of which was
electronically compensated by the amplifier. Data were filtered with an
eight-pole low-pass Bessel filter (902; Frequency Devices, Haverhill,
MA), with corner frequencies ranging from 500 Hz for experiments on the
slow conductances gK,L and gh to 10 kHz for
capacitance measurements and experiments on the fast inward rectifier
gK1. The data were digitized at twice the filter
frequency using a 12-bit acquisition board (Digidata 1200; Axon
Instruments) in conjunction with the software package pClamp 6.0 (Axon Instruments) and stored on disk. Voltages have been corrected
off-line for liquid junction potentials (4 mV for the standard
solutions) and, for steady-state activation curves, uncompensated
series resistances. Leak subtraction was not done unless stated. The
linear leak conductance was usually <1 nS. Analyses and fits were done
with the program Origin (Microcal Software, Northampton, MA), which
uses a Levenberg-Marquardt least-squares fitting algorithm.
Steady-state activation (conductance-voltage) curves were generated
from tail currents after steps to various membrane voltages. The tail
currents were fitted, extrapolated to the offset of the iterated
prepulse, and then converted to conductance by dividing by the driving
force. Plots of the conductance values against the prepulse membrane
potential were fitted with Boltzmann functions:
|
(1)
|
where g is conductance, gmax is maximum conductance,
Vm is membrane potential,
V1/2 is the potential at which the conductance is half-maximally activated, and S is the voltage
corresponding to an e-fold change in conductance.
Equation 2 was used to fit the sigmoidal activation kinetics of
gK,L, gDR,I,
gDR,II, gh, and the sigmoidal
deactivation kinetics of gh.
|
(2)
|
where I(t) is the current at time t,
I0 is the current at 0 msec,
I is the current in steady state, and
 1 and 2 are macroscopic time constants
that give the time course of the final approach to peak current and the
sigmoidal onset, respectively.
We measured the reversal potential, Vrev,
of conductances from instantaneous I-V relationships. The
conductance was activated by stepping to a voltage within its
activation range for a fixed period and then stepping through an
iterated series of voltages. The tail currents evoked by the iterated
step were fitted and extrapolated back to the instant of the step. The
extrapolated values were plotted against membrane potential, and
reversal potentials were obtained from linear fits of the data in the
vicinity of the reversal.
Data from hair cells in cultured and acute preparations were similar
and have been pooled, unless stated otherwise. Results are presented as
mean ± SD.
Morphology
To assess the postnatal morphological appearance of the utricle,
we examined its ultrastructure at fixed time points and classified and
counted all the cells in representative samples. Most of the data are
from utricles that were fixed in situ. For comparison, several cultured epithelia were also fixed and studied. In
situ fixations were performed at P0 (n = 4 animals), P3 (n = 1), P4 (n = 3), P7
(n = 3), P10 (n = 3), and P28
(n = 2). Cultures were fixed 3 (n = 1 culture), 6 (n = 1), and 9 (n = 1) d
after they were made on P1. We refer to the cultures by the
corresponding postnatal ages, P4, P7, and P10.
Fixation and post-fixation. For in situ
fixations, ICR mice were first deeply anesthetized with a mixture of
10% 5-5,diallylbarbituric acid, 40% urethane, and 40% monoethyl
urea (0.3 ml/kg1, i.p.) and then perfused
transcardially with 2 ml of a warm heparinized saline, pH 7.4, followed
by 10 ml of a warm trialdehyde fixative (3% glutaraldehyde, 2%
paraformaldehyde, 1% acrolein, and 5% sucrose in 0.08 M
cacodylate buffer) (DeGroot et al., 1987). The same fixative was used
to fix the cultured organs. All utricles were post-fixed for 1 hr in
1% OsO4 in 0.1 M cacodylate buffer,
dehydrated in graded alcohols and propylene oxide, and embedded in
araldite (Durcupan; Fluka BioChemika, Ronkonkoma, NY).
Sectioning. For sectioning, the utricular epithelium was
oriented perpendicular to its long axis so that sections in the middle of each block contained hair cells in each of three regions: the striola, the lateral extrastriola, and the medial extrastriola. The
striola is a narrow strip of hair cells, ~80 µm wide in these organs, which has specialized morphology (Lindeman, 1973;
Fernández et al., 1990) and afferent physiology (Goldberg et al.,
1990). Glass knives were used to cut semithin serial sections, 2 or 5 µm thick, through the first one-third to one-half of a block. The
sections were serially mounted on glass slides. A long series of
ultrathin (75 nm) serial sections was then cut from the middle of the
block with a diamond knife (Delaware Diamond Knives, Wilmington, DE).
From each series, 1-2 sets of 32 consecutive sections were chosen.
Each set formed a sample ~2.4 µm thick. Two or three samples (1 or
2 per animal) were obtained at each postnatal stage. Semithin sections
were cut from the remainder of the block. The semithin sections from
either side of the ultrathin section series were examined in the light
microscope and were used to reconstruct the location of the ultrathin
samples.
Ribbons of sections were stained with uranyl acetate and lead citrate
(Reynolds, 1963) and examined in a JEOL 100S electron microscope. Micrographs were taken of every fifth
ultrathin section (375 nm apart), and a photomontage (5,000× final
magnification) was made of the entire epithelium contained in the
section. The hair cells of each sample, covered by eight such
photomontages, were classified as described below, and then cells in
each class were counted by their corresponding nuclei. To avoid double
counting, we used the dissector method (Gundersen, 1986) as described
in detail elsewhere (Fernández et al., 1995; Lysakowski and
Goldberg, 1997).
For epithelia that were fixed in situ, counts were performed
on three samples from two animals each at P0, P4, P7, and P10, and on
two samples from one animal at P28. The same techniques were applied to
one sample each from cultured epithelia at P4, P7, and P10.
Cell classification. We classified each cell in a sample as
a supporting cell or one of several types of hair cell: immature hair
cell, type I hair cell, type II hair cell, and undefined hair cell. The
criteria used to classify cell type are described in Results.
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RESULTS |
Electrophysiology
Delayed rectifiers
Figure 1, A and
B, shows whole-cell current families evoked by our standard
voltage protocol in two hair cells. The data were chosen to illustrate
the two main types of outward currents that we encountered. In
A, there was an outward current at the holding potential
(VH = 64 mV) and instantaneous jumps in
current at the onset of the voltage steps. The onset current reversed
between 74 and 84 mV and showed some decay during steps to 84 and
94 mV. This current was through a large
K+-selective conductance, gK,L,
which was activated at 64 mV and which deactivated at more negative
potentials. In contrast, the cell in Figure 1B had
negligible current at 64 mV and very little onset current at the
start of voltage steps. This cell did not express gK,L. We
used input conductance at 64 mV (gin(64)) as a marker
of gK,L; 162 cells with gin(64) >20
nS and resting potentials negative to 50 mV had
gK,L, and 198 cells with gin(64) <4
nS did not have gK,L. Thirty-six cells with intermediate
input conductances were excluded from analysis.
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Figure 1.
Delayed rectifiers gK,L,
gDR,I, and gDR,II in mouse utricular
hair cells. A, B, Families of current
traces from a cell with gK,L (A, acute
preparation, P9) and a cell without gK,L (B,
culture, P4) in response to voltage steps from
VH = 64 mV. Single presentations.
The voltage steps, corrected for Rs error, are indicated
next to most current traces. Data points between 0 msec and the peak of
each outward current trace were fitted with Equation 2 (solid
lines) and then extrapolated in time, as shown. In
A, the instantaneous currents at the onset of voltage
steps and the decaying inward currents at 94 and 84 mV are through
gK,L. The sigmoidally activating current positive to 64
mV is through gDR,I in A and
gDR,II in B. In B, the small
inward current at 94 mV is through gK1 and
gh. C, Activation curves. Legend in
D applies to C. Data for gK,L
(filled circles) are from the cell in
A, obtained with a different protocol (below). The data
for gDR,I (filled squares) were from
a different cell (culture, P6); gK,L was blocked by
superfusing the cell with 20 mM Ba2+
(Rüsch and Eatock, 1996a). The cell without gK,L
(open triangles) was from a culture at P7. For
gDR,I and gDR,II, the activation curves
were generated with protocols similar to those in A and
B, except that VH = 84 mV.
For the gK,L data, VH = 84 mV,
hyperpolarizing prepulses (250 msec to 124 mV) were used to remove
inactivation of gK,L, the iterated voltage steps
were 800 msec, and tail currents were elicited at 54 mV to avoid
activating gDR,I. Tail currents were fitted with
double-exponential functions. Curves, Normalized fits of
Equation 1. gK,L: gmax = 147 nS;
V1/2 = 73 mV; S = 5.1 mV. gDR,I: gmax = 28 nS;
V1/2 = 29 mV; S = 8.3 mV. gDR,II: gmax = 52.0 nS;
V1/2 = 29.1 mV; S = 6.4 mV. D, Voltage dependence of activation time
constants. Time constants were obtained by fitting activating currents
with Equation 2 and are shown on separate ordinates for clarity:
1, top; 2,
bottom. The time constants for gDR,I are
from the fits to the current traces shown in A. We
assumed that the sigmoidal activating currents positive to 60 mV were
through gDR,I exclusively, because gK,L in this
cell was fully activated at VH = 64 mV
(C). The time constants for
gDR,II are from the fits to the current traces in
B and additional traces. The time constants for
gK,L are from a cell (acutely dissociated, P17) recorded
with an internal solution in which all K+ was
replaced with Cs+. Cs+ permeates
gK,L but not gDR,I. In this cell,
V1/2 for gK,L was 81 mV.
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In mature inner ear organs, including the mouse utricle (Eatock et al.,
1994; Ricci et al., 1996; Rüsch and Eatock, 1996a), most hair
cells with gK,L are type I cells, and most hair cells without gK,L are type II cells. As will be shown, however,
a cell without gK,L in a young mouse utricle may be either
a type II cell or an immature type I cell. For this reason, we often
refer to "cells with gK,L" and "cells without
gK,L" rather than type I and type II cells. Cells with
and without gK,L had similar mean membrane capacitances:
3.5 ± 0.7 pF (n = 82 cells) and 3.6 ± 0.9 pF (n = 126 cells), respectively. gK,L was
associated with significantly more negative resting potentials
(VR): 66 ± 5.3 mV in 158 cells without gK,L and 77 ± 3.7 mV in 86 cells with
gK,L. (VR values positive to 40 mV
were excluded.) The properties of gK,L are summarized in
the next section.
In both cells in Figure 1, A and B, outward
currents activated sigmoidally in response to steps positive to 64
mV. These currents were unaffected by removal of external
Ca2+ (Rüsch and Eatock, 1996a) and had the
kinetics of delayed rectifiers rather than A currents. We call the
conductance gDR,I in type I cells, gDR,II in
type II cells, and gDR,N in neonatal cells (P1-P3).
gK,L. In a previous report on the same
preparation (Rüsch and Eatock, 1996a), we showed that
gK,L is K+ selective and is not
sensitive to removal of external Ca2+. It activates
with slow sigmoidal kinetics [principle time constant (1) at 90 mV was 608 ± 233 msec;
n = 4] (Fig. 1D) and inactivates slowly.
It therefore falls into the broad class of delayed rectifiers. Compared
with most delayed rectifiers, however, gK,L has more Cs+ permeability and an unusually negative and
variable voltage range of activation. A conductance-voltage
(activation) curve is shown in Figure 1C, fitted with a
Boltzmann function (Eq. 1). V1/2 values ranged
from 88 to 62 mV (Fig. 1C; Table
1). More positive
V1/2 values may occur but are difficult to
measure because of interference by gDR,I.
The mean maximum gK,L was ~75 nS (Table 1), which is very
large for the small size of these cells. The presence of a large conductance that is substantially activated at
VR drastically reduces the input resistance
and therefore the size of voltage responses to injected currents
(Rüsch and Eatock, 1996b).
gDR,I and gDR,II. Activation curves
of gDR,I and gDR,II are shown in Figure
1C. Fits by a Boltzmann function (Eq. 1) to the two
conductances produced similar V1/2 values
(between 25 and 30 mV) and S values (6-8 mV) (Table 1).
Activation curves for gDR,N in neonatal cells had similar
values (Table 1). Variation in V1/2 values was
much lower for these conductances than for gK,L.
The time courses of activation of gDR,I and
gDR,II were well described by Equation 2, as shown by the
fits in Figure 1, A and B. Figure
1D shows the fast and slow time constants from these fits and fits of the same equation to currents through
gK,L. gK,L was slowest, and gDR,I
was slower than gDR,II. The principal (slow) time constant
(1) at 44 mV was 44.2 ± 8.3 msec
(n = 4 cells) for gDR,I and 17.1 ± 6.9 msec for gDR,II (n = 4 cells at P7 or later) (0.01 < p < 0.05, Student's
t test).
gDR,I and gDR,II were K+
selective, as shown by the proximity of their reversal potentials
(approximately 73 mV) (Table 1) to EK. The
value for gDR,I was obtained after blocking
gK,L with 5 mM 4-AP.
We tested the sensitivity of gDR,II to 4-AP and external
Ba2+ for comparison with values reported previously
for gDR,I and gK,L (Rüsch and Eatock,
1996a). Cells were held at 64 mV, and currents were activated by 160 msec voltage steps to 0 mV before and during superfusion with the
blocker. Steady-state current at 0 mV in the presence of a given
concentration of the blocker, [B], was expressed as the
fraction, f, of the current in control conditions. The
dissociation constant was calculated as KD = ([B]) (f/(1 - f)).
Concentrations tested were 5 mM for Ba2+
and between 10 and 100 µM for 4-AP. The estimated
KD values are an order of magnitude lower than
those for gDR,I (Table 1).
The maximum size of gDR,II (see Fig. 4D; Table
1) was obtained from Boltzmann fits of activation curves at 44 mV
(gmax in Eq. 1) for 40 cells between P1 and P17. Estimating
maximum gDR,I was more difficult, because gK,L
was maximally activated at voltages at which gDR,I was
maximally activated (Fig. 1C). We estimated maximum
gDR,I as follows. We measured whole-cell current at 54 mV, at which it is mostly through gK,L, and at 0 mV,
at which it is mostly through gK,L and gDR,I.
To estimate gK,L at 0 mV, we assumed that gK,L
is maximally activated at 54 mV and that its I-V
relationship is linear between 54 and 0 mV. We extrapolated the
current through gK,L at 0 mV from its value at 54 mV and subtracted the result from the total current at 0 mV, giving the current through gDR,I.
The mean maximal gDR,I significantly exceeded the mean
maximal gK,L measured from the same cells and was more than
twice as large as the mean maximal gDR,II (Table 1). The
sum of the delayed rectifier conductances in type I cells often
exceeded 200 nS. In the mouse utricle, type I and type II cells have
similar membrane capacitances and therefore surface areas, so that the
larger conductances of type I cells reflect higher current densities.
Summing the mean maximal gK,L and gDR,I values
and dividing by the mean capacitance gives ~50 nS/pF, approximately
fivefold higher than the specific conductances of mouse cochlear inner
hair cells (Kros and Crawford, 1990), turtle cochlear hair cells (Wu et
al., 1995), and CA1 neurons from the hippocampus (Klee et al.,
1995).
Inward rectifiers
Hyperpolarizing steps from VH = 64 mV
activated fast and slow inward currents that could be isolated
pharmacologically. The fast component was fully blocked by 1 mM external Ba2+, leaving the slow
component primarily unscathed. The slow component was blocked by 1 mM external Cd2+, leaving just the fast
component. Both components were blocked by 1 mM external
Cs+. This pharmacology and the other properties
described below show that the fast and slow components correspond to
the two inward rectifiers gK1 and gh,
described in other hair cells (Fuchs and Evans, 1990; Holt and Eatock,
1995; Goodman and Art, 1996; Sugihara and Furukawa, 1996).
gK1. Figure
2A shows a family of
gK1 currents activated by hyperpolarizing steps from
VH = 64 mV in a cell with neither
gh nor gK,L. The reversal potential (82 mV)
(Table 1) was close to EK, showing that
gK1 was highly K+ selective. The gating
of the gK1 family of inward rectifiers shifts with
EK (Hagiwara et al., 1976). In our solutions
(EK = 84 mV), gK1 activated
negative to 40 mV and was almost completely activated at 120 mV
(Fig. 2B; Table 1).
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Figure 2.
The fast inwardly rectifying conductance
gK1. A, Family of current traces elicited by
steps from VH = 64 mV to potentials shown
to the right of the traces. Traces from 144 to 104
mV are overlaid by single-exponential fits to the data points between 0 msec and the time of the peak current. Cell without gK,L or
gh, culture, P3. B, Activation curve.
Data from a different cell without gK,L or
gh, culture, P5. Solid line, A fit of
Equation 1. gmax = 4.2 nS; V1/2 = 82 mV; S = 11.6 mV. This was not corrected for
a linear leak conductance of 200 pS, measured at
VH = 84 mV after blocking gK1
with 1 mM [Cs+]o.
Inset, Some of the tail current traces used to construct
the activation curve. Tail currents were elicited at 44 mV, near the reversal potential of gh.
The last 300 µsec of the 40 msec activating voltage steps are shown.
Uncompensated capacitive currents during the first 150 µsec after the
step to 44 mV are not shown. Tail currents were fitted by
single-exponential functions and extrapolated back to the instant of
the step to 44 mV; the fit is shown for the 134 mV trace
(dashed line). C, Voltage dependence of
the activation time constants from the fits in A.
Solid line, A fit to the data of the following equation:
= 0.1 + (4.1 × exp(Vm/15.5)). is in msec and
Vm is in mV.
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gK1 activated with single-exponential kinetics (Fig.
2A). At 100 mV, the time constant of activation was
~4 msec and decreased e-fold with 15.5 mV of
hyperpolarization (Fig. 2C). Positive to 110 mV,
gK1 did not inactivate. Inactivation at more negative potentials may reflect block by Na+ (Ohmori,
1984).
gmax (~4 nS) (Table 1) was estimated from the current
3-5 msec after the onset of a step to 124 mV. This method did not eliminate leak conductances, which were between 0.1 and 1.5 nS.
gh. In frog saccular hair cells (Holt and
Eatock, 1995), gh contributes to the input conductance and
resting potential and causes a transient voltage response to
hyperpolarizing current steps. The latter effect is also seen in mouse
utricular hair cells (Rüsch and Eatock, 1996b). The activation
and kinetic properties of gh in the mouse utricle also
resemble those in the frog saccule. Figure
3A shows current through
gh activated by hyperpolarizing steps from 64 mV.
gh started to activate near the mean resting potential of
cells without gK,L (67 mV) and was fully activated by
140 mV (Fig. 3B).
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Figure 3.
The slow inwardly rectifying conductance
gh. A, Family of current traces elicited by
the voltage protocol shown. VH = 64 mV.
Cell without gK,L and with gK1, culture,
P5. Current through gK1 was completely blocked by 1 mM [Ba2+]o, except
for small transients at the onset of the voltage steps. Average of
three presentations is shown. Tail currents were elicited at 84 mV,
close to the reversal potential of gK1. Currents during the
steps to 84 mV are not shown. The currents in response to the voltage
steps activated and deactivated with sigmoidal time courses that were
fitted with Equation 2. Examples of the fits for both activation and
deactivation are shown for the current trace at 148 mV as
smooth lines superimposed on the data. Tail currents
after steps to 124 and 136 mV were omitted for clarity. Parameters
of the fit to current activation: I0 = 17
pA; I = 125 pA; 1 = 32 msec; 2 = 0.19 msec. A small linear component
(24.8 fA/msec) was added to improve the fit. Parameters of the fit to
the tail current: I0 = 60 pA;
I = 27 pA; 1 = 237 msec; 2 = 50 msec. Corrected for 160 pS linear leak
conductance, measured in 1 mM
[Ba2+]o. B, Activation
curve. Data were measured from the tail currents at 84 mV shown in
A and additional traces. Tail currents were fitted with
Equation 2, as shown for one trace in A, extrapolated to
the instant of the step to 84 mV, converted to conductance (assuming
Vrev = 43.5 mV), and plotted against the
prepulse potential. Solid line, A fit of Equation 1.
gmax = 1.6 nS; V1/2 = 98 mV;
S = 9.9 mV. C, Time constants of
fits to the activation of currents through gh.
1, filled circles;
2, open circles. Recordings in
standard (Ba2+-free) extracellular solution from a
different cell with gK1 but no gK,L (culture,
P8). No additional linear component was used for the fits. Because
gK1 activation (Fig.
2A,C) was 10-fold faster at
all potentials than the fast component of gh,
gK1 was accommodated in these fits by
I0 of Equation 2.
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gh activated and deactivated with sigmoidal kinetics, which
were fit by Equation 2 (Fig. 3A). The two activation time
constants reached maximal values of ~220 and 30 msec close to 98
mV, at which gh was half-activated (Fig. 3B).
gh did not inactivate within 500 msec (data not shown) at
any membrane potential. gh also had sigmoidal deactivation
kinetics in frog saccular hair cells.
To measure maximal gh in a large number of cells, we took
the current evoked by a step from 64 mV to 124 mV and subtracted the current at 10 msec from the current at 400 msec. At 10 msec, the
current through gh is not substantially activated
(principal of ~60 msec at 124 mV) (Fig. 3C), but
currents flowing through the linear leak conductance and
gK1 have reached steady state. Therefore, the difference
current is exclusively through gh. The mean conductance
(~1 nS) (Table 1) was similar for cells with and without
gK,L.
The reversal potential of gh (approximately 44 mV) (Table
1) was measured in five cells that lacked gK,L and in which
gK1 was blocked with 1 mM external
Ba2+. If we assume that the current is carried just
by K+ and Na+, we calculate from
the reversal potential a permeability coefficient (PNa/PK) of
0.14, similar to the value for gh in other hair cells (Holt
and Eatock, 1995; Sugihara and Furukawa, 1996).
Changes with postnatal age
On P1-P2, hair cells in the mouse utricle expressed
gDR,N and gK1 but neither gK,L nor
gh. The first cell with gK,L was encountered in
a cultured epithelium on P3 (Fig.
4A), after which the
percentage of cells with gK,L increased rapidly to a
plateau of 50-75% by P6. The progression in acute preparations (Fig.
4B) was comparable but noisier, presumably reflecting
the smaller numbers of measurements per day. When data obtained after
P7 were pooled, similar percentages of cells expressed gK,L
in the cultures (70%) and in the acute preparations (63%). The
postnatal mouse utricle is primarily postmitotic (Ruben, 1967), so the
change represents differentiation of cells present at birth rather than
birth of a new cell type.
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Figure 4.
The delayed rectifiers gK,L and
gDR as functions of age. A,
B, Changes in incidence with age in cultures
(A) and acute preparations
(B). In this and subsequent figures, the
number of cells from which recordings were obtained at
each stage is given at the top of the columns.
C, D, Maximal sizes of the conductances as
functions of age. Zero values are not included. Values calculated as
described in Materials and Methods and Results.
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In Figure 4, C and D, the fully activated
conductances (gmax) for gK,L (Fig.
4C), gDR,I, and gDR,II (Fig.
4D) are plotted against postnatal day.
gmax values did not vary systematically with age. At all
ages, gDR,I exceeded gDR,II. The relationships between gDR,I, gDR,II, and the
delayed rectifiers in the neonatal cells are not known. However, cells
that acquired gK,L simultaneously experienced a large
increase in the non-gK,L delayed rectifier (gDR,I). Recall that cells with gK,L
have similar membrane capacitances (surface areas) to those without
gK,L. Moreover, capacitance did not change significantly in
comparisons of data from P0, P4, P7, P10, and P17. Thus, current
density increased dramatically in type I cells relative to neonatal and
type II cells.
At all stages examined, most hair cells that did not express
gK,L did express gK1 (Fig.
5). Neither the incidence nor size of
gK1 varied systematically with postnatal age. The fate of
gK1 in type I cells is less clear. Most cells that acquired
gK,L must have expressed gK1 between P1 and P3
(Fig. 5A), because the epithelium is primarily postmitotic
by P3. Whether gK1 is retained as these cells mature cannot
be determined by inspection of the currents evoked by hyperpolarizing
steps in control solutions (Fig. 2A). Activation of
gK1 would be obscured by deactivation of the much larger
gK,L, and steady-state gK1 is difficult
to distinguish from a leak conductance. One test for inward rectifiers
is whether extracellular Cs+ blocks the steady-state
inward current at deeply hyperpolarized potentials at which
gK,L is not activated. In four cells with gK,L
and no detectable slow inward rectifier (gh),
extracellular Cs+ (1 or 5 mM) reduced
the steady-state current at 124 mV to 28 ± 16% of its control
value (162 ± 84 pA). Thus, a substantial fraction of the small
steady-state current is through a Cs+-sensitive
conductance rather than a leak conductance. If we assume that the
Cs+-sensitive conductance is entirely
gK1, then we calculate a mean conductance of 2.7 nS,
which is well within the normal range for cells without
gK,L (Fig. 5B).
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Figure 5.
gK1 did not vary with postnatal age.
Data were pooled from cultures and acute preparations.
A, Percentage of the cells without gK,L that
expressed gK1 (filled bars) as a
function of age. B, Chord conductances measured as
described in Materials and Methods and Results. Zero values are
not included.
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gh was not present in neonatal cells but was acquired in
the same period as gK,L. Figure
6 shows the incidence
(A,B) and size (C,D) of gh as functions of
postnatal day for cells with gK,L (A,C) and cells without
gK,L (B,D). Data from
cultures and from acute preparations are pooled. gh was
first detected on P3, and by P4 its incidence had increased
dramatically. In cells without gK,L, the incidence
continued to increase until ~P8. gh was present in 88%
of cells without gK,L sampled after P7. In cells with
gK,L, there was no clear trend with age.
gh was present in 34% of all cells with gK,L
in the sample.
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Figure 6.
gh as a function of postnatal age.
Data from cultures and acute preparations were pooled.
A, B, Percentages of cells that expressed
gh and gK,L (A) or
gh and no gK,L
(B). C, D, Chord
conductances in cells with gK,L (C)
and without gK,L (D). See Materials
and Methods and Results for details. Zero values are not
included.
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The size of gh varied from 0.11 to 5.7 nS (Fig.
6C,D) but showed no systematic
dependence on postnatal day.
Figure 7 summarizes our observations from
this and other studies on conductances in neonatal and older hair cells
in the mouse utricle. Neonatal cells have a mechanosensitive
conductance (gmet) (Rüsch and Eatock, 1996b;
Holt et al., 1997), a voltage-gated Ca2+ conductance
(gCa) (A. Rüsch and R. A. Eatock, unpublished
observations), a voltage-gated Na+
conductance (gNa) (Rüsch and Eatock, 1997), a
delayed rectifier K+ conductance that activates
positive to 55 mV (gDR,N), and a fast inward
rectifier (gK1). Mature type I cells differ most
obviously from neonatal cells by the expression of a large negatively
activating delayed rectifier (gK,L) and the very
large total delayed rectifier conductance (gK,L plus
gDR,I). Mature type II cells differ from neonatal
cells in a more subtle way, by the expression of the slow inward
rectifier gh. Approximately one-third of type I cells also
acquire gh. It is not clear whether they continue to
express gK1. gNa was seen in just 1 of 84 type
I cells (Rüsch and Eatock, 1997), and we are not sure whether it
is retained in adult type II cells.
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Figure 7.
Schematic overview of the conductances in neonatal
and differentiated type I and type II hair cells of the mouse
utricle.
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Morphology
Does the electrophysiological differentiation of type I and II
hair cells coincide with their morphological differentiation? Previous
morphological studies of rodent vestibular organs suggested that some
maturational changes, e.g., calyx formation and increases in hair
bundle height, occur after birth (Dechesne et al., 1986; Anniko, 1990),
but these studies have not provided a systematic ultrastructural
description of changes during the period of acquisition of
voltage-gated conductances just described. We have examined the
ultrastructure of utricular epithelia fixed in situ at five time points between P0 and P28 and of cultured utricles on P4, P7, and
P10. We classified cells into different types, as described below, and
then counted each type at each stage, as described in Materials and
Methods.
The sensory epithelia of otolith organs have a distinctive band called
the striola. In material fixed in situ, the striola can be
distinguished from the extrastriola because the otoconia are relatively
small, the hair bundles reverse orientation, and in epithelia from
animals older than P4, complex calyces (enveloping >1 hair cell) are
common (Lindeman, 1973). Recognition of these regions in the cultured
organs was more difficult, because the otoconia were removed at the
time of culturing, no calyces were present, and hair bundles were often
disturbed by mechanical removal of the otolithic membrane so that it
was difficult to ascertain their orientation. Therefore, we did not
attempt to subdivide the cultures into striolar and extrastriolar
zones.
Cell type classification
The electron micrographs in Figures
8 and 9
illustrate the features that we used to classify cells.
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Figure 8.
Ultrastructure of the neonatal and early postnatal
mouse utricle. A, P0; B, P3;
C, P4. The epithelia shown in A and
B were fixed in situ. The epithelium
shown in C was excised and cultured on P1 and fixed
3 d later. A, Adjacent to the striolar region (in
the juxtastriolar region) (Fernández et al., 1990).
B, C, Striolar region. A,
Immature (Imm) hair cells had some features of
supporting cells (SC, see C), including
microvilli (MV) instead of stereocilia, and
secretory granules (SG). They were distinguished from
supporting cells by their longer microvilli and more apical nuclei, and
because they had a few large secretory granules rather than many small
ones. Unlike more mature hair cells, they lacked cuticular plates and
had fine elongate microvilli instead of stereocilia. Even at P0, it is
possible to classify some hair cells as type I or type II. The cell
labeled I in its nucleus (left) has an
immature partial calyx (PC). The left
cell (II) has finer stereocilia
(S) and smaller mitochondria
(M), both traits of mature type II hair
cells. The hair cell in the middle has relatively thick
stereocilia and large mitochondria, consistent with it being type I. Large afferent endings on it may be an early stage of calyx formation.
The hair cell to its right is undefined
(U), because it has relatively thick stereocilia
(a type I trait) but small mitochondria (a type II trait).
CP, Cuticular plate. B, C,
Hair cells grown in culture (C) acquired
differentiated traits similar to those seen in situ
(B). Note the thicker stereocilia
(S) and larger mitochondria
(M) in the type I hair cells relative to
the adjacent type II cells. The type I nuclei had more clumps of dark
heterochromatin than did the type II nuclei. Type I nuclei are often
more apical than type II nuclei at this stage but not later, after the
neck forms (Fig. 9B,D).
SC, Supporting cell labeled in its nucleus. Scale bar, 5 µm.
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Figure 9.
Type I morphologies in the early postnatal mouse
utricle. A, B, P4, extrastriolar region
fixed in situ. C, P7, striolar region
fixed in situ. D, Juxtastriolar region
cultured on P1 and fixed 6 d later (P7). On P3-P4, some type I
hair cells lacked calyces (Fig. 8C), and others had
partial calyces (A, PC) or full calyces
(B, I). Some type I cells
(B) had the necks that are characteristic of
mature type I cells, and others did not (A). The
neck is not a response to the calyx, because it was sometimes seen in
cultures (D). As the type I cells developed
necks, their nuclei tended to move toward the base of the epithelium;
compare the adjacent type I cells in D. Complex calyces,
which surround more than one type I cell (C) and
are common in the mature striola, were first seen on P7. The bulging
apical surfaces of the type I cells in D are a secondary
trait. In B, the apical reticular meshwork
(RM) of a supporting cell is labeled. Other
labels are as in Figure 8. Scale bar, 5 µm.
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Supporting cells were relatively narrow cells with dark
(electron-dense) cytoplasm and located between the lighter hair cells. Supporting cells had many small- to medium-sized secretory granules (~15-20 in a single section) (Figs.
8A,B, 9C), short apical
microvilli, and an apical reticular meshwork (Fig.
9B, RM) (Wersäll,
1956) instead of a cuticular plate. Their nuclei were located in a
layer at the base of the epithelium (Fig. 8C,
SC).
Hair cells were distinguished from supporting cells by the more apical
location of the nucleus and by lighter cytoplasm and nucleoplasm. Hair
cells were further classified as immature, type I, type II, or
undefined.
Immature hair cells appeared to be intermediate between supporting
cells and mature hair cells. Li and Forge (1997) observed comparable
cells in guinea pig utricular epithelium after aminoglycoside-induced damage and interpreted them as showing transdifferentiation of supporting cells to hair cells. The immature cells in our specimens typically had one to five large secretory granules per section (Figs.
8A, 9A,C,
SG), in contrast to none in mature hair cells and more
numerous smaller granules in supporting cells. The apical microvilli of
immature hair cells had lengths and diameters that were intermediate
between those of supporting cell microvilli and those of true
stereocilia (Fig. 8A,C,
MV). There was no apical reticular meshwork and
frequently either no cuticular plate or one that was poorly defined
(Fig. 8A). The electron density of the cytoplasm
ranged from dark, as in supporting cells (Fig. 8A, left), through intermediate (Fig. 8A,
right) to light, as in more mature hair cells (Fig.
8C, right). The immature hair cells with the
darkest cytoplasm had more basal nuclei and often retained their
attachments to the basement membrane, as observed in regenerating epithelia by Li and Forge (1997).
The primary attribute used to classify hair cells as type I or type II
has been the form of the afferent synaptic contact. Because
synaptogenesis is incomplete in neonatal utricles and the cultures were
denervated, we also used a set of secondary attributes to distinguish
type I from type II hair cells. These attributes have been noted in the
course of ultrastructural examination of mature vestibular organs from
chinchillas (Lysakowski, 1996; Lysakowski and Goldberg, 1997).
Hair cells were classified as type I if they received a full or partial
calyx ending. A full calyx is an afferent nerve terminal that surrounds
the basolateral membrane of the hair cell nearly up to the tight
junctions that segregate apical and basolateral cell surfaces (Fig.
9B,C). We defined an afferent
terminal to be a partial calyx rather than a large bouton terminal if
the appositional length of its contact with the hair cell exceeded 5 µm and/or if the parent branch extended in both directions around the
base of the hair cell (Figs. 8A, 9A,
PC). Hair cells were also considered to be type I if they
lacked calyces but fulfilled two or more of the following secondary
criteria: a constricted region (neck) below the cuticular plate (Fig.
9B-D) (Wersäll, 1956; Correia et al., 1989);
electron-dense patches of clumped heterochromatin in the nucleus (Figs.
8B,C, 9B) (Lysakowski,
1996); an apical surface that bulges into the endolymphatic lumen (Fig. 9D) (Favre, 1986; Lysakowski, 1996); and, relative to
neighboring type II cells, thicker stereocilia (Fig.
8A-C, S) (Lysakowski, 1996) and
larger-diameter mitochondria (Figs. 8A-C,
9B, M) (Lysakowski, 1992).
Type II hair cells included hair cells that were not immature, that had
no calyx, and that fulfilled two or more of the following secondary
criteria: they did not have necks and, relative to neighboring type I
cells, their stereocilia were thinner (Fig. 8A-C,
S); mitochondria had smaller diameters (Figs.
8A-C, 9B, M); and
nuclear heterochromatin was more uniform (Figs.
8B,C, 9B).
Test of the secondary attributes in mature tissue. In many
cells, not all of the secondary attributes followed the patterns described. When secondary attributes were ambiguous or in conflict, we
called the cells undefined hair cells. This category also included hair
cells for which only part of the cell was in the sample (Fig. 8A, far right U). Despite these
exceptions and the qualitative nature of the secondary attributes, a
test of the method in mature tissue suggests that they are good
predictors of cell type. We classified the hair cells of one P28 sample
twice, once according to the presence or absence of calyces and a
second time using the secondary attributes but ignoring the presence of
any calyx ending on the hair cells. Using the primary attribute alone
(calyx or no calyx), 60 of 94 hair cells were classified as type I, 27 as type II, and 7 were undefined, i.e., we could not determine whether
a calyx was present because only a small part of the cell was present.
Using secondary attributes alone, we classified 42 as type I, 16 as
type II, and 36 as undefined. Thus, secondary attributes are not always
able to identify cell type as defined by the afferent ending.
Consistent with this, the fraction of undefined hair cells was higher
in the denervated cultures than in material fixed in situ
(Fig. 10B-D). On the
other hand, just 3 of 94 cells were classified as type II by one method
and type I by the other method.
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Figure 10.
Percentages of cell types as functions of
postnatal age. The numbers of cells for all samples at
each stage are shown at the top of the columns.
A, Percentages of hair cells and supporting cells. All
data are from epithelia fixed in situ: two samples at
P28 and three samples at each of the other stages. Percentages were
separately calculated for each sample at each age and then averaged
together. B-D, Percentages of hair cell types as
functions of postnatal age. The key in B applies to all
three histograms. Type I cells were identified by the presence of a
full calyx or partial calyx or, in the absence of any calyceal contact,
by the secondary attributes described in Results. The lines
joining adjacent columns separate the type I, type II, and other
(immature plus undefined) categories. The percentage of cells that
could be classified as type I or type II increased with age.
B, Utricles that were cultured on P1 and fixed 3, 6, or
9 d later. Counts were made from one sample at each stage.
C, D, Counts in the striolar
(C) and extrastriolar (D)
regions of utricles that were fixed in situ. Counts were
made from three samples each at P0, P4, P7, and P10 and two samples at
P28. Pooled in situ data from the striolar and
extrastriolar regions (data not shown) resemble the data from the
extrastriolar region, which is ~90% of the total area.
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In summary, the secondary attributes provide a way to classify cell
type when afferents are missing. Use of the secondary attributes alone
results in more undefined cells but is reasonably accurate with respect
to the cells that are classified as type I or type II.
Changes with postnatal age
In Figure 10A, cells from epithelia fixed
in situ are divided into supporting cells and hair cells and
are shown as percentages of the total number of cells at the five
sampled postnatal days. The percentage of hair cells increased from
~40% on P0 to ~60% by P28. This is not likely to reflect birth of
new cells, because Ruben (1967) showed that terminal mitosis of all but
a small percentage (<3%) of mouse utricular hair cells precedes
birth. Consistent with this, we observed a few mitotic figures at P0
and none later. It appears that between P0 and P28, some supporting
cells transformed into hair cells. This is supported by the
transitional appearance of the immature hair cells.
Figure 10, B-D, shows changes with postnatal day in
the percentages of each hair cell type in the cultures
(B) and in the striolar (C) and
extrastriolar (D) regions of utricles fixed in
situ (supporting cells are not included). The following trends can
be discerned.
Cell type. In cultures and in vivo, the
percentages of type I and type II cells increased with postnatal age at
the expense of the immature and undefined categories. In
situ, 50% of cells were classified as type I or type II on P0
(Fig. 10C,D, data from both regions pooled). This
increased by P10 to ~90% in situ and ~70% in culture
(Fig. 10B). The cultures had more undefined hair cells, presumably because only secondary attributes are available. In
the epithelia fixed in situ, the percentage of undefined
cells was similar at P10 and P28 (9% pooled across both regions),
suggesting that this represents the small parts of cells at the edges
of the samples rather than immaturity. At P28, the percentage of type I
hair cells was 65% in the extrastriolar region, 54% in the striolar
region, and 63% when data from both regions were pooled. These
percentages are in close agreement with the percentages of cells with
gK,L at P6 and later and not very different from the
percentage of type I hair cells in the mature guinea pig utricle and
chinchilla crista (51% in both) (Lindeman, 1969; Fernández et
al., 1995).
Although similar final percentages of type I cells were obtained by
morphological and electrophysiological criteria, the morphological changes were more gradual than the electrophysiological changes. At P0,
there were cells that were morphologically type I that did not express
gK,L, and after P6 some cells that were not fully differentiated in appearance expressed the mature complement of voltage-gated channels.
Striola versus extrastriola. Differentiation in the
striola appeared to lead differentiation in the extrastriola; in the
striola the proportions of type I cells changed very little
postnatally. Other studies have shown that the frequency of terminal
mitoses peaks earlier in the striola (E14) than in the extrastriola
(E15) (Sans and Chat, 1982) and that the central zone of the utricular epithelium, which includes the striola, leads peripheral zones in
both the development of hair bundles (Mbiene et al., 1984) and
synaptogenesis (Sans and Dechesne, 1987).
Calyx formation. On P0, only one full calyx was seen, but
29% of striolar hair cells and 21% of extrastriolar hair cells were contacted by partial calyces. In a sample not included in the counts in
Figure 10, partial calyces contacted two immature hair cells,
consistent with cells committing to a type I identity at an early
stage. Full calyces were first found in appreciable numbers at P4, when
they were exclusively simple. They were present on a higher percentage
of hair cells in the striola than in the extrastriola.
Cell shape. Cell shape was not a reliable marker at early
stages. Partial calyces contact cells with no necks in Figures
8A (P0) and 9A (P4). The distinctive flask
shape (Fig. 9B-D) of mature type I cells was more
frequently seen beginning at P7. The flask shape did not depend on the
calyx (Fig. 9D).
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DISCUSSION |
Between P4 and P7, type I cells differentiated from neonatal cells
by greatly increasing the density of delayed rectifier K+ channels of at least two types and in some cases
acquiring gh. Type II cells differentiated by acquiring
gh. No specific morphological events correlated in time
with the electrophysiological changes. Morphological differentiation
was more gradual, with some cells recognizable as type I or II by P0,
and some continuing differentiation past P7 (Fig. 10). Results with
denervated cultures showed that neither calyx formation nor sustained
postnatal innervation of any kind was required for electrophysiological
and morphological differentiation of type I and type II hair cells.
Thus, despite the concurrence of calyx formation and gK,L
acquisition in vivo, these distinguishing marks of the
mature type I cell are independently acquired. The existence of cells
with ultrastructural traits that are intermediate between supporting
cells and hair cells suggests that during normal development cells
postmitotically transform from supporting cells to hair cells.
Postnatal changes in the proportions of the two cell types support this
interpretation.
Electrophysiological differentiation of hair cells
Conductances similar to those found in neonatal mouse
utricular hair cells occur early in development in hair cells from
other organs. In vestibular hair cells in cultured chick otocysts
(Sokolowski et al., 1993), delayed rectifier, fast inward rectifier,
and voltage-gated Na+ and Ca2+
conductances have all been identified at relatively early stages. Neonatal mouse cochlear hair cells transduce (Kros et al., 1992) and
express a slow delayed rectifier and gNa and
gCa (Kros et al., 1993 |
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Abstract
Introduction
