Dipartimento di Scienze Fisiologiche-Farmacologiche Cellulari-Molecolari, Sez. di Fisiologia Generale e Biofisica Cellulare, 27100 Pavia, Italy
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ABSTRACT |
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Masetto, S., P. Perin, A. Malusà, G. Zucca, and P. Valli. Membrane Properties of Chick Semicircular Canal Hair Cells In Situ During Embryonic Development. J. Neurophysiol. 83: 2740-2756, 2000. The electrophysiological properties of developing vestibular hair cells have been investigated in a chick crista slice preparation, from embryonic day 10 (E10) to E21 (when hatching would occur). Patch-clamp whole-cell experiments showed that different types of ion channels are sequentially expressed during development. An inward Ca2+ current and a slow outward rectifying K+ current (IK(V)) are acquired first, at or before E10, followed by a rapid transient K+ current (IK(A)) at E12, and by a small Ca-dependent K+ current (IKCa) at E14. Hair cell maturation then proceeds with the expression of hyperpolarization-activated currents: a slow Ih appears first, around E16, followed by the fast inward rectifier IK1 around E19. From the time of its first appearance, IK(A) is preferentially expressed in peripheral (zone 1) hair cells, whereas inward rectifying currents are preferentially expressed in intermediate (zone 2) and central (zone 3) hair cells. Each conductance conferred distinctive properties on hair cell voltage response. Starting from E15, some hair cells, preferentially located at the intermediate region, showed the amphora shape typical of type I hair cells. From E17 (a time when the afferent calyx is completed) these cells expressed IK, L, the signature current of mature type I hair cells. Close to hatching, hair cell complements and regional organization of ion currents appeared similar to those reported for the mature avian crista. By the progressive acquisition of different types of inward and outward rectifying currents, hair cell repolarization after both positive- and negative-current injections is greatly strengthened and speeded up.
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INTRODUCTION |
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Hair cells are the mechanoreceptor cells of the
inner ear. Mechanical stimuli change the amount of current flowing
through mechanotransduction channels located on hair cells'
stereocilia (Hudspeth 1989), thus generating a receptor
potential whose final shape also depends on the interplay of different
types of basolateral ion channels, the latter ones of which vary among
hair cells from different inner ear organs, as well as from different
regions of the same sensory epithelium (Art and Fettiplace
1987
; Fuchs 1992
; Masetto et al.
1994
; Murrow 1994
; Weng and Correia
1999
).
Although the electrophysiological properties of fully developed hair
cells have received considerable attention in the last two decades, far
less is known about their ontogeny. The available data suggest that
different types of ion channels are progressively acquired by
developing hair cells. In the chick cochlea, as early as E10
(embryonic day 10), hair cells located at the basal or at the apical
extremes express a slow or a fast outward K+
current, respectively (Griguer and Fuchs 1996). The
later acquisition (E19) of a Ca-activated
K+ current occurs at a time when auditory
function begins to mature rapidly (Fuchs and Sokolowski
1990
). In the mammalian cochlea, inner hair cells acquire only
postnatally a fast K+ current, which turns them
from spiking pacemakers into high-frequency signal transducers
(Kros et al. 1998
).
As far as vestibular organs are concerned, mouse utricular hair cells
acquire IK, L (the signature current
of type I hair cells) (Correia and Lang 1990;
Eatock et al. 1994
; Rennie and Correia
1994
), and the slow inward rectifying
Ih a few days after birth
(Rüsch et al. 1998
). By contrast, no data are
available about the electrophysiological properties of vestibular hair
cells during in vivo prenatal development, either in avians or in
mammals. We aimed at filling this gap, at least in part, by
investigating the electrophysiological properties of avian vestibular
hair cells as a function of development between E10 and
E21. The chick has been chosen, because a large literature
database is available regarding other aspects of inner ear development
for this animal.
The crista slice preparation used here is a very suitable preparation for the proposed goal because it allows one to correlate electrophysiological data with cell location and morphology.
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METHODS |
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Embryo dissection
Fertilized chicken eggs of the Cobb variety were obtained by a
local supplier and incubated at 38.3°C. Although hatching occurred at
embryonic day 21 (E21), when embryos were staged according to the criteria of Hamburger and Hamilton (1951), we
observed that development consistently lagged behind this standard
atlas up to E19; this is presumably the result of the higher
incubation temperature (39.4°C) used by Hamburger and Hamilton in the
first 9 days of incubation. Table 1 shows
the relationship that we observed between stages of development and
days of incubation.
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Once removed from the eggs, embryos were decapitated and semicircular canals dissected out. The bone surrounding the ampullae was removed in embryos older than E14. The ampullae were incubated in Dulbecco's modified Eagle's medium (DMEM; Catalog number 31600-026, GIBCO BRL, Life Technologies) to which was added 1.5% newborn calf serum (Catalog number N-4637, Sigma, St. Louis, MO), 24 mM NaHCO3, 15 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; Sigma), buffered at pH 7.4 with NaOH, and carboxygenated (95% O2-5% CO2) in a humidity-saturated chamber at 37°C. After an incubation period of 2-6 h, the organ was removed from the culture medium and embedded in 4% agar wt/vol (Sigma) in a slicing solution containing (in mM): 145 NaCl, 3 KCl, 15 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.1 CaCl2, 7.5 MgCl2, and 10 glucose; 50 mg/l ascorbate; pH 7.4 with NaOH.
Slicing procedure
The technique used for obtaining slices of chick embryo
semicircular canal cristae generally followed that reported for slicing the semicircular canal of the pigeon (Masetto and Correia
1997). Briefly, an agar block containing the ampulla and a
small portion of the semicircular duct (surrounded by the bone tissue
until E14), was immersed in the slicing solution (partially
frozen) and cut with a vibratome (Campden, UK). Slice thickness varied between 150 and 250 µm. The slices were then transferred to a dish
with a glass coverslip bottom, and immobilized by the use of a weighted
nylon mesh. The tissue and microelectrode were viewed and photographed
by the use of differential interference contrast optics with the use of
an upright microscope (Zeiss Axioskop, Germany) equipped with ×40 and
×63 water-immersion objectives; additional magnification (×1.25,
×1.6) was obtained by the use of a Zeiss Optovar. For total current
recordings, the dish contained a standard extracellular solution (in
mM: 145 NaCl, 3 KCl, 15 HEPES, 2 CaCl2, 0.6 MgCl2, pH 7.4 with NaOH). For recordings of inward current through Ca2+ channels recordings,
slices were kept in the standard extracellular solution and, once the
whole-cell configuration was achieved, were perfused with the following
solution (in mM): 100 NaCl, 35 tetraethylammonium (TEA)Cl, 2 4-aminopyridine, 5 BaCl2, 3 KCl, 15 HEPES, 5 CsCl, 0.6 MgCl2, pH 7.4 with NaOH. Usually the
slice was changed after each recording because perfusion of the latter extracellular solution accelerated deterioration of unclamped hair
cells, presumably by causing strong and prolonged cell membrane depolarization.
Electrical recordings
Borosilicate glass pipettes (Drummond Scientific, Broomall, PA)
were pulled to tip diameters between 0.5 and 1.0 µm, fire-polished, and partially coated with Sylgard (Dow Corning 184, Midland, MI). The
micropipettes were filled with intracellular solutions of different
compositions: for total current (in mM): 134 KCl, 2 MgCl2, 10 HEPES, 1 CaCl2,
and 11 EGTA; pH 7.4 with KOH; for currents through
Ca2+ channels (in mM): 140 N-methyl-D-glucamine (NMDG), 2 MgCl2, 10 HEPES, 1 CaCl2,
and 11 EGTA; pH 7.4 with HCl. Adenosine 5'-triphosphate (ATP; 1 mM) was
freshly added each day of the experiment to both intracellular
solutions. Micropipettes had a resistance in the bath of 2-3 M when
filled with the K-based intracellular solution. The patch-clamp
amplifier was a List L/M-EPC-7 (Germany). Series resistance
(Rs) and cell-membrane capacitance
were read in voltage-clamp mode directly from the amplifier's
compensation dials. After electronic compensation by the amplifier,
residual series resistance was between 1 and 11 M
[mean value = 4.2 ± 2.3 (SE) M
; n = 152]. Nominal
voltages are shown for voltage protocols in figures. All calculated
data in this study and figure diagrams have been corrected for voltage
drop across residual Rs, unless
otherwise stated. The amplifier's filter bandwidth was set at 3 kHz.
Digital sampling frequency of voltage- and current-clamp protocols was
at least 3 times the analogue bandwidth of the signal recorded. Current and voltage were measured and controlled through a DigiData 1200 interface (AD/DA converter; Axon Instruments, Foster City, CA) connected to a personal computer (Pentium PC) running pClamp software (version 6.0.3, Axon Instruments). Resting membrane potential with the
K-based intracellular solution was measured as the zero-current voltage
in current-clamp mode (Vz), by averaging seven
measurements taken at 10-s intervals. Leakage current was measured as
the current in response to 250-ms, 10-mV hyperpolarization from a
holding potential of
60 mV (average of 7 measurements). In hair cells without inward rectifying currents (see RESULTS), or where
they were blocked pharmacologically (Ba2+
extracellular solution), current measurements in the above-mentioned voltage range reflected only passive properties of the hair cell membrane and seal leakage, because other currents activated above
60
mV. This limited the use of leakage corrections to type II hair cells
as noted in figure legends. No leakage correction was performed when
IK, L was present (presumptive type I
hair cells). For minimizing contamination by residual unblocked outward
current and leakage current, Ba2+ currents
through Ca2+ channels were sometimes isolated by
subtracting currents recorded in the presence of 200 µM
CdCl2, which blocked all
Ca2+ channels. Averaging of three to five sweeps
was used to improve the signal-to-noise ratio for currents through
Ca2+ channels.
Current traces were not corrected for residual capacitive artifacts.
Voltages were not corrected for the liquid junction potential with the
K-based intracellular solution (3 mV negative inside the pipette), but
were corrected for the liquid junction potential (5 mV) with the
NMDG-based intracellular solution. For current-clamp traces the true
stimulus pipette current is shown as current protocol, because
patch-clamp amplifiers in current-clamp mode can introduce artifacts
evident as distorted stimulus currents (Magistretti et al.
1996; Masetto et al. 1999
).
Test solutions were applied locally through a gravity-fed multibarreled micropipette. Recordings were made at room temperature (22-24°C).
Morphological criteria
Recordings were made from hair cells in selected regions or
zones of the neuroepithelium of all three (posterior,
horizontal, and anterior) semicircular canals. Figure
1A shows microphotographs of
an E12 vertical and an E20 horizontal canal
crista slice from chick embryos. To maintain consistent nomenclature,
we named the zones in accordance with the pigeon crista (Masetto
and Correia 1997) and bullfrog crista (Myers and Lewis
1990
) regional subdivision on the basis of differences in
components of ionic currents, hair bundle morphology, hair cell
density, and primary afferent arborizations. Given that the crista
increased in size during embryo development, it was not possible to
give a fixed size for each zone. The distinct zones were therefore
defined as follows: in the dumbbell-shaped vertical canal cristae (Fig.
1B, left), zone 1 is the most
peripheral region of the sensory epithelium, contacting the planum
semilunatum (PS), and extending for one third of segment S1;
zone 3 is the most central region, extending for one third
of segment S2 on either slope of the crista apex; zone
2 is an intermediate region, extending for one third of both
segments S1 and S2 (see Landolt et al.
1975
or Weisleder and Rubel 1993
for scanning
electron microscopic pictures of the avian cristae). Because the
horizontal crista (Fig. 1B, right) resembles one
of the symmetrical sides of the vertical cristae, it was similarly
divided into zones 1, 2, and 3 starting from the
PS.
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Analysis
Analysis of traces and results were performed with Clampfit (pClamp version 6.0.3, Axon Instruments), Microcal Origin (version 4.1, Microcal Software Inc., Northampton, MA), SigmaPlot (Jandel Scientific, version 1.02, San Raphael, CA), Microsoft Excel (version 5.0c; Microsoft Corporation, Redmond, WA), and GraphPad Prism (version 2.01). In the text, data are expressed in the following format: mean (± SD; n = number of cases). For statistical analysis, F-test and Student's t-test were used when comparing two groups of hair cells, and one-way ANOVA-Tukey's multiple comparison test to compare pairs of group means when comparing three groups of hair cells. In figures, error bars indicate standard deviation, unless otherwise stated.
Time-dependent monoexponential inactivation of
IK(V) and
IK(A), and activation of
IK1 were fitted by a monoexponential
function of the form:
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(1) |
Time-dependent sigmoidal activations of
IK(V) and
IK(A) were fitted by a second-order
power equation of the form:
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(2) |
Time-dependent sigmoidal activation of
Ih was fitted by a sum of two
exponential functions of the form:
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(3) |
Steady-state activation and inactivation (current-voltage) curves were
fitted with Boltzmann functions:
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(4) |
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RESULTS |
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General membrane properties of developing crista hair cells
Before E15 all hair cell properties were homogeneous enough to be pooled together; primarily on the basis of morphological criteria, we observed that these immature hair cells resembled type II hair cells. Hair cells with a distinctive morphology, resembling type I hair cells, were recognizable starting from E15, and will be dealt with in a separate section (see below). The membrane properties of immature hair cells changed along with embryonic development from E10 to E21. Figure 2 shows cell membrane input capacitance (Cm), resting potential (Vz), and peak chord conductance for total outward current (Gp), measured in hair cells from all regions of the crista at various embryonic ages. Regression analysis shows that both Cm and Gp increase during development: Cm increased from 3.5 pF (±0.8; mean ± SE; n = 22) at E10-E11 to 5.9 pF (±1.6; n = 33) at E20-E21, whereas Gp for the same cells increased from 6.7 nS (±3.8; n = 22) to 14.5 nS (±5.2; n = 33). Vz varied largely among hair cells at the same embryonic day, but a trend toward more negative values along with development is evident.
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Ionic currents in crista hair cells before E12
The pattern of ionic currents expressed by hair cells changed
during development. At E10 and E11 (Fig.
3), all hair cells, independent of the
crista region, almost exclusively displayed a slow-voltage-dependent
outward current on depolarization (Fig. 3A), and no current
on hyperpolarization (Fig. 3B). The slow outward current
reversed close to the K+ equilibrium potential;
from tail-current measurements in two hair cells at E10 and
E11 the reversal potential resulted in 87 and
85 mV,
respectively (estimated EK =
96 mV).
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Consistent with voltage-clamp results, the cell-voltage response to depolarizing current steps revealed the presence of a slow repolarizing current, whereas it behaved almost passively in response to hyperpolarizing currents (Fig. 3C).
The outward K+ current activated in a sigmoidal
manner, and could be well fitted by a second-order power function
(Eq. 2; Fig. 4A). In a
sample of cells from E10-E12 embryos, the
average activation time constant decreased from 46.8 ms (±14.3;
n = 4) at 40 mV to 10.1 ms (±1.4; n = 4) at
10 mV. The inactivation time course was not voltage
dependent: single-exponential fits from E10-E12 hair cell outward currents (Eq. 1; Fig. 4B) gave
decay time constants of 2.8 ± 0.3 s at
40 mV and 2.7 ± 0.6 s at
10 mV (n = 3). This current
resembles the delayed rectifier K+ current
observed in the pigeon crista type II hair cells, although it activates
and inactivates significantly more slowly (e.g., at
40 mV, the
average activation and inactivation time constants were 12.8 and 191 ms, respectively) (Masetto and Correia 1997
); hence it
will be named IK(V).
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The IK(V) inactivation time course did
not change appreciably during development: in
E19-E21 hair cells, the inactivation time
constant was 2.4 ± 0.1 s at 40 mV and 2.7 ± 1.1 s at
10 mV (n = 5). If the
IK(V) inactivation time course could
be well fitted also in hair cells expressing a small additional fast
and transient current component (see METHODS), the latter
precluded a good fitting of IK(V)
activation time course from its very beginning in some cells. In three
cells among those investigated at E19-E21, in
which IK(V) was apparently the sole
outward current, its activation time constant was 39.7 ± 9 ms at
40 mV and 8.7 ± 2.1 ms at
10 mV, that is, not significantly
different from the values found at E10-E12.
The steady-state activation and inactivation curves for
IK(V) are shown in Fig. 4C.
IK(V) tail currents were measured at
60 mV after 100-ms voltage steps at different voltages (see Fig. 3A legend). IK(V) started
activating less negative than
60 mV, and was fully activated for
voltages more positive than 0 mV. When fitted with a Boltzmann function
(Eq. 4), the IK(V)
activation curve between E10 and E12 gave a
V1/2 value of
24 mV (±0.1;
mean ± SE; n = 6) and a slope factor
(S) of 6.1 mV (±0.1; n = 6). Analogous measurements between E19 and E21 gave
V1/2 =
24 mV (±1.8;
n = 9) and S = 12.3 mV (±5.2;
n = 9). The change in slope factor paralleled a
significant change in the percentage of channels open at most negative
voltages: on average, at E10-E12, only 7% of
KV channels were open at
40 mV. At
E19-E21, 21% were open at the same membrane voltage (P < 0.005; Student's t-test). The
difference observed could reflect a true change of
IK(V) voltage dependence, or a contribution from other K+ currents appearing at
later stages of development (a fast transient K+
current and a Ca-dependent K+ current; see
below). However, a similar shift was observed at E19-E21 in cells without the fast
K+ current (n = 3), and in cells
in which the Ca-dependent K+ current was blocked
by the substitution of Mg2+ for
Ca2+ (n = 3); therefore, the
IK(V) activation curve appears to
change along with development.
Steady-state IK(V) inactivation (Fig.
4C) did not appear complete until positive membrane
voltages, and was fully removed at 110 mV; at
E10-E12, V1/2
was
62 mV (±1.6; n = 4) and S was 14.9 mV. These parameters were not significantly different at later stages
of embryonic development (E19-E21;
n = 3). The area under the crossing curves describing
IK(V) steady-state activation and
inactivation (window current) indicates that a substantial fraction of
KV channels can contribute steadily to the
membrane resting potential, and is permanently available for hair cell repolarization in response to depolarization in the physiological range
(see DISCUSSION).
Acquisition of the transient K+ current
The main change observed after E12 in hair cells' electrophysiological properties was the functional expression of a transient outward current (Fig. 5A).
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The voltage- and time-dependent properties of the fast transient
current differed enough from those of
IK(V) to enable us to isolate it
without pharmacological blockers. From tail-current measurements in two
hair cells, the transient outward current reversed at 83 and
85 mV,
that is, close to the estimated K+ equilibrium
potential (
96 mV). Tail currents (not shown) were measured at
different test potentials after conditioning the cell for 5 ms at
20
mV; in this way, <6% of the slower
IK(V) was activated, whereas the
transient current had reached the peak.
Time-dependent inactivation could be well fitted by a single
exponential (Eq. 1; Fig.
6B); the decay time constant
decreased from 30.4 ± 6.9 ms (mean ± SE) at 40 mV to
7.2 ± 1.8 ms at
10 mV (n = 4). The transient
current therefore inactivated 100 times faster than
IK(V). Time-dependent activation could
be well fitted by a second-order power function similarly to
IK(V) (Eq. 2). Activation time constants decreased from 1.6 ± 0.3 ms at
40 mV
(n = 4) to 0.8 ± 0.2 ms at
10 mV; therefore,
this current activated 10 times faster than
IK(V). Given the above-mentioned
properties and similarities to the transient K+
current described in semicircular canal hair cells of mature pigeons
(Masetto and Correia 1997
), the transient outward
current will be called IK(A).
Instantaneous tail-current measurements (Fig. 6C) show that
IK(A) starts activating at
60 mV and
is completely activated at 0 mV. Boltzmann fit (Fig. 6D)
yielded V1/2 =
36.5 mV (±0.9;
n = 5) and S = 8.2 mV (±0.9;
n = 5). Steady-state inactivation curve (Fig.
6D), obtained by measuring the peak current amplitude at
40 mV after conditioning the cell at different voltages (as in Fig.
6A), shows that IK(A) is
almost fully inactivated (96%) at
30 mV, whereas inactivation is
completely removed at
120 mV (V1/2 =
75 mV; Fig. 6D). There is thus a significant window current contributing to the receptor potential steadily between
60
and
30 mV. IK(A) properties did not
change appreciably throughout embryonic development.
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To illustrate IK(A) contribution to
hair cell currents in the different zones, we plotted the time-to-peak
(tp, Fig. 5C) and steady-to-peak ratio
(Is/Ip,
Fig. 5D) of hair cell total currents versus embryonic day of
development, measured at 20 mV. The parameters considered can reveal
the dominant current component of the total outward current, given the
considerably faster activation and inactivation kinetics of
IK(A) versus
IK(V). From these considerations, tp would be smaller in hair cells
with a significant IK(A), and larger
in hair cells dominated by IK(V). On
average, from E12, when
IK(A) first appears, to
E21, tp was 14.1 ms (±26;
n = 54) in zone 1 hair cells, 60.2 ms
(±36.8; n = 42) in zone 2 hair cells, and
32.6 ms (±40.1; n = 24) in zone 3 hair
cells. These values were statistically significantly different among
zones (P < 0.05 between all pairs of zones; one-way
ANOVA-Tukey). The time-to-peak was <6.6 ms (the median for all hair
cells from E12 up to E21) in 44/54 (81.5%)
zone 1 hair cells, in 6/42 (14.2%) zone 2 hair cells, and in 10/24 (41.6%) zone 3 hair cells, investigated
from E12 to E21.
As far as
Is/Ip
is concerned, both Is and
Ip were measured at 20 mV after
having conditioned the cell at
120 mV for 100 ms to remove most
IK(A) inactivation;
Is was measured at the end of the test
potential, lasting 100 ms; that is, when
IK(A) had almost completely
inactivated, whereas Ip was measured
in the first 6 ms of the test potential, when
IK(A) had already reached the peak.
The ratio
Is/Ip,
therefore, will be 1 if the maximal current amplitude attained in the
first 6 ms of the test-voltage step equals the current amplitude
attained at the end of the test-voltage step. Because
IK(A) reaches the peak in the first 6 ms at
20 mV, whereas IK(V) is much
slower (we estimated that at
20 mV on average <20% of
KV are open in the first 6 ms of the voltage step), an
Is/Ip
value close to 1 would indicate a similar contribution to the total
outward current from IK(A) and
IK(V); this ratio will be <1 if the
total outward current declines from the peak attained in the first 6 ms
to a lower steady level (total outward current dominated by
IK(A)), and otherwise >1 if the
steady outward current is greater than the current amplitude attained
in the first 6 ms (total current dominated by the slow
IK(V)). From E12 to
E21,
Is/Ip
was on average 0.6 (±0.3; n = 48) in zone 1 hair cells, 2.4 (±1.4; n = 35) in zone 2 hair cells, and 1.4 (±1.1; n = 22) in zone
3 hair cells. These values were statistically significantly
different among zones (P < 0.05 between all pairs of
zones; one-way ANOVA-Tukey). The preceding results indicate that
IK(A) is not evenly expressed across
the crista: indeed it was by far the predominant current in zone
1 hair cells from E12 on, but less important in
zone 3 and very small (or even absent) in zone 2 hair cells.
Another aspect worth considering is the relative importance of IK(A) along with development. From Fig. 5D it can be seen that for IK(A) hair cells the ratio Is/Ip does not change significantly once IK(A) is expressed. In fact, it was 0.7 ± 0.5 (mean ± SE) at E13-E14 (n = 11) and 0.6 ± 0.3 (n = 17) at E20-E21 (the difference is not statistically significant). Because Is density (Is/Cm) in the same cells did not change significantly during embryonic development (data not shown), both IK(V) and IK(A) appear to increase in amplitude roughly proportionally to the increase in hair cell membrane surface area. The same is not as easy to evaluate for the other crista zones, in which IK(A) is more variable (even absent).
The voltage response of hair cells to depolarizing currents was
profoundly modified by the presence of
IK(A). This current rapidly
counteracted the depolarization produced by positive-current steps,
producing a very fast depolarizing peak followed by a few clear
oscillations (Fig. 5D). In some cells the damped voltage oscillations could be slightly distorted by the patch-clamp amplifier (Masetto et al. 1999) (see pipette current stimulus in
Fig. 5D). However, in other cells the oscillation observed
was the true voltage response of the hair cell, because no pipette
current artifacts were detectable (see, for example, Fig.
7). Short-lived membrane-voltage
oscillations disappeared when the resting potential was depolarized by
a positive-holding current (Fig. 7), which induced inactivation of
KA channels. Moreover, membrane-voltage oscillations were much less pronounced in hair cells without
IK(A) (see, for example, Fig.
9C). Similarly, in mature pigeons the quality of membrane
oscillations was greater in zone 1 hair cells, which
expressed the largest IK(A), compared
with zone 3 hair cells (Weng and Correia
1999
).
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Ca-activated K+ current
The presence of a Ca-activated K+ current
(IKCa) was tested by perfusing the
cells with a low-Ca/high-Mg extracellular solution (see slicing
solution in METHODS), where Ca2+ was
replaced by Mg2+ in an approximately 1:3 ratio to
minimize the otherwise expected change in surface membrane potential
(Blaustein and Goldman 1968).
This substitution had a complex effect: before E14 it
produced a small outward current increase. This increase was most
likely because of the blockade of an inward Ca current
(ICa) produced by
Mg2+ ions blocking Ca2+
channels (Carbone et al. 1997). Starting from
E14, Ca2+/Mg2+
substitution could produce variable effects, including either a small
increase or a small decrease of the total current, or both (with
current increasing for more negative potentials, and then decreasing;
Fig. 8A). When both increase
and decrease were observed, the currents obtained by subtracting
current in the presence of low-Ca/high-Mg solution from the control
current (Fig. 8, B and C) revealed the block of
an inward current (likely ICa) at
negative potentials, and of an outward current (likely
IKCa) at more positive potentials.
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IKCa was found from E14
onward in 29% (2/7) of zone 1 hair cells, and in 50% (3/6)
of zone 2 and zone 3 hair cells tested, and its
amplitude was always very small (<10% of the total outward current at
20 mV). The small amplitude of IKCa
appears to be a common feature of semicircular canal hair cells
(Lang and Correia 1989
; Masetto et al.
1994
; Sokolowski et al. 1993
), although
IKCa might be more pronounced in vivo
than observed with the ruptured whole-cell recording mode. When
high-Mg/low-Ca solution was tested in current-clamp mode on two cells
expressing IKCa it produced a 3- and a
7-mV reversible depolarization of the cell resting membrane potential, respectively.
Inward rectifying currents
At later stages of development, hyperpolarizing voltage steps from
a holding potential of 60 mV activated a slow inward rectifying current in many hair cells (Fig.
9A). This current was often
very small: at
120 mV it was, on average, 90.7 pA (±49.7; mean ± SE; n = 9, zones 2 and 3 hair
cells at E16-E18). By comparison, in a sample of
hair cells in which a time dependence of the inward current was not
evident, the presumed leakage current at
120 mV was 49.5 pA (±13;
n = 6, zone 1 hair cells at
E16-E18), that is, not very different in
amplitude. Moreover, the presence of a slight inward rectification of
the inward current at more negative voltages often appeared as a
consequence of hair cell deterioration at these low voltages (i.e.,
leakage increase); therefore, we used a combination of current-clamp
(see below) and voltage-clamp experiments to increase our confidence
that an inward rectifying current was actually present. In this way, we
could find this slow inward rectifying current already at
E14-E15, although only in 2/11 hair cells.
Incidentally, of these 11 cells, 5 were from zone 1, and
none of them expressed it. However, it is only from E16 that
the majority of hair cells from zones 2 and 3 clearly expressed this slow inward rectifying current: from
E16 up to E18, in fact, it was found in 9 of the
10 zone 2/zone 3 hair cells investigated. Conversely, it was
absent in all zone 1 hair cells investigated at the same
developmental stages (n = 6). This current reversed at
voltages more positive than
60 mV (as indicated by the presence of
inward tail currents at
60 mV at the end of hyperpolarizing steps),
suggesting that it is not strongly selective for
K+ ions.
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These features resemble those of the
Ih described in adult pigeon
semicircular canal hair cells (Masetto and Correia
1997). According to Hestrin (1987)
,
Ih could be well fitted by a sum of
two exponential functions (Eq. 3). In a sample of cells
s (the slow-time constant) was 81.9 ms (±18;
mean ± SE; n = 5) at
120 mV, a value very close
to that measured in mature canal hair cells of the pigeon (92.4 ms at
120 mV) (Masetto and Correia 1997
), and decreased with
increasing hyperpolarization. As anticipated, current-clamp experiments
helped to reveal the presence of Ih. This current in fact produced a clear sag in the voltage response to
hyperpolarizing currents steps (Fig. 9B). Moreover, because its reversal potential is more positive than average
Vz of hair cells (likely close to
40
mV) (Holt and Eatock 1995
),
Ih also produced a transient
afterdepolarization at the end of hyperpolarizing current steps. The
afterdepolarization produced by deactivating Ih could be emphasized by
hyperpolarizing the cell resting membrane potential. In the example of
Fig. 9C, the cell was hyperpolarized from
69 to
90 mV by
a holding current of
60 pA. At the end of the negative-current step,
a large afterdepolarization developed at voltages close to
80 mV, at
which a greater driving force for Ih
should exist. Furthermore, because K+ and
Ca2+ currents are not active at this potential,
they do not affect the voltage response produced by
Ih. Figure 9C also shows
that the time course of the afterdepolarization (lasting about 200 ms
at
90 mV) is very similar to that of the afterhyperpolarization observed after a positive-current step of the same amplitude. In the
cell shown, the afterhyperpolarization was most likely the result of
the slow deactivation of IK(V) and/or
IKCa, because IK(A) was not evident in this cell.
From E19 Ih was often obscured by a larger and faster inward rectifying current, IK1 (Fig. 10A). As for Ih, this current did not appear abruptly from E19: in fact, it was found in 3/16 hair cells from E16 to E18 (of these 16 cells, 5 were from zone 1, and none of them clearly expressed IK1). However, from E19 this fast inward rectifying current was found in most zone 2 and zone 3 hair cells (13/20 cells expressed IK1, plus possibly a small Ih, whereas the remaining 7 cells displayed Ih only), although in very few zone 1 hair cells (2/15 expressed a small IK1, plus possibly a small Ih, and 2/15 expressed only a very small Ih).
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The time-dependent activation of this current could be well fitted by a
single-exponential function (Eq. 1); the activation time
constant was 3.1 ms (±0.8; mean ± SE; n = 3) at
120 mV (i.e., about 20 times smaller than that of
Ih) and decreased by increasing hyperpolarization. The preceding value is very close to that of IK1 described in mature pigeon
vestibular hair cells (2.9 ms at
120 mV) (Masetto and Correia
1997
). IK1 accelerated and
compressed the voltage response to hyperpolarizing-current steps (Fig.
10B). For example, from E10 to E15
negative-current steps of 40 pA amplitude drove the hair cell membrane
potential from its resting value up to an average value of
149.9 mV
(±37.6; n = 22, all zones pooled); from E19
to E21 the same current step injection applied to zone
2/zone 3 hair cells produced a significantly smaller
hyperpolarization:
85.5 mV (±10.7; n = 16). Probably
because of the low expression of IK1
in zone 1 hair cells even at E19-E21,
these cells generated relatively large voltage responses to
negative-current steps: for example, 40 pA negative-current steps
hyperpolarized zone 1 hair cells from their resting value up
to an average value of
118.1 mV (±34.8; n = 8).
IK1 was accompanied by a more negative
Vz; from E19 to
E21, Vz was on average
63.1 mV (±13.5; mean ± SE; n = 17) in hair cells lacking IK1, and
73.7 mV
(±11.1; n = 15) in hair cells displaying
IK1 (P < 0.05;
Student's t-test).
Inward currents through voltage-dependent Ca2+ channels
In the presence of blockers of K+ currents
and Ih (see METHODS),
depolarization evoked voltage-dependent inward currents in the majority
of hair cells tested. The inward current was better recorded with
Ba2+ instead of Ca2+ in the
extracellular medium, because with Ca2+ it was
smaller and largely contaminated by unblocked residual outward currents
(not shown). Ba2+ currents
(IBa) were present from the earliest
developmental stage investigated (E10), and increased
considerably with development (Fig. 11,
A and B). For example,
IBa peak amplitude measured at 25 mV
was 12.3 pA (±12.2; mean ± SE; n = 7) at
E10-E12, and 44.4 pA (±24.9; n = 7) at E19-E21. The difference in
IBa amplitude between earlier and
later developmental stages was statistically significant
(P < 0.01; Student's t-test); however, if
the Ba2+ current density is computed
(IBa/Cm),
no significant differences are found.
IBa activated rapidly, and did not
show evident time-dependent inactivation, at least for the 160-ms
depolarizing-step duration (Fig. 11A). No voltage- or
Ba-dependent inactivation of IBa was observed conditioning the cell membrane voltage between
30 and
110
mV for 250 ms before step to
10 mV (n = 2; not
shown). No significant differences were observed in
IBa voltage-dependent parameters (Fig.
11B) between early and late developmental stages: on
average, IBa activated at
50 mV
(±0.9; n = 7) and peaked at
23.4 mV (±1.9;
n = 7) in E10-E12 embryos,
versus
53 mV (±1.8; n = 7) and
26.4 mV (±4;
n = 7), respectively, in E19-E21
embryos. IBa was completely blocked
(in a partially reversible fashion) by adding 200 µM
CdCl2 to the external medium (Fig.
11B). This feature was used to isolate in some cells the net
Ba2+ current from the contaminating leakage and
outward current, by subtracting from the control current the current
remaining after Cd2+ block. In seven cells in
which Cd2+ subtraction was compared with the
usual leakage subtraction (see METHODS),
IBa obtained after
Cd2+ subtraction was found to peak 3 mV less
negative, and to reverse more positive [more positive than 40 mV, vs.
an average value of 11.9 mV (±10.3; n = 7)]. No
differences were observed in voltage threshold of
IBa activation, consistently with the
observation that the Cd-insensitive outward current just started
activating around
50 mV, thereafter increasing monotonically with
depolarization (Fig. 11B). The preceding results are
consistent with previous reports of Ba2+ current
flowing through voltage-dependent Ca2+ channels
in mature (Lang and Correia 1989
) and developing
(Sokolowski et al. 1993
) avian hair cells.
|
Ionic currents in type I hair cells
Starting from E15, some hair cells in the slices
displayed morphological features typical of mature type I hair cells:
they had a large apical region bearing a short bundle of stereocilia, and a very constricted neck (Fig.
12D, left panel), compared
with a typical type II hair cell (Fig. 12D, right panel). We
were able to record from cells showing the aforementioned morphology
from E17; all these cells (n = 11) displayed
a large outward current considerably active at 60 mV, as shown by the
presence of inward tail currents in response to hyperpolarizing voltage
steps and of substantial instantaneous currents in response to
depolarization (Fig. 12A). This outward current was almost
completely deactivated at the holding potential of
80 mV (Fig.
12B). This low-voltage-activated outward current resembles
IK, L described in avian and mammalian mature vestibular type I hair cells (Rennie and Correia
1994
; Rüsch and Eatock 1996
). In most
cells we found it difficult to fit with a single Boltzmann function the
activation curve of the total outward current, because it appeared to
reach a first plateau around
50 mV, and then to increase again to
reach a second plateau around
10 mV (Fig. 12C, diamonds).
A second outward current component, activating at less negative
voltages than IK, L, but partially overlapping to it, can explain the observed inflection in the current-voltage relationship. However, in one cell
IK, L activated more negative, and its
activation curve could be better resolved (variability of
IK, L activation curves among type I
hair cells has already been reported) (Rüsch and Eatock
1996
). The activation curve of IK,
L for this single cell is shown in Fig. 12C
(triangles): Boltzmann fit (Eq. 4) gave a
V1/2 value of
82 mV and a slope factor (S) of 8.4 mV. IK, L
started activating around
110 mV, and was almost completely activated
at
60 mV, where outward current began to increase again, presumably
because of the activation of a different channel population. For this
reason, saturation for IK,L activation curve is
not shown. We did not investigate the nature of the second outward
current component, although the absence of a fast inactivation allowed
us to rule out that it was IK(A).
Additional outward rectifying currents besides IK,
L and activating less negative than
55 mV have been
reported in type I hair cells (Rennie and Correia 1994
;
Rüsch and Eatock 1996
).
|
As far as type I hair cell outward current kinetics is concerned, a
comparison with type II hair cells is possible only at potentials less
negative than 60 mV, because in type II hair cells outward currents
just start activating at
60 mV. From this comparison, type I hair
cell outward currents appear to activate significantly more slowly than
type II hair cell outward currents. The average time-to-peak for total
outward currents elicited at
20 mV was, in fact, 164.8 ms (±33;
n = 9) in zone 2 E19-E21 type I
hair cells, and 55.5 ms (±40; n = 13) in zone 2 E19-E21 type II hair cells (P < 0.0001).
The average time-to-peak for total outward currents in the same type I
hair cells measured at
60 mV was 161.6 ms (±38.2; n = 9).
Among type I-like hair cells we recorded from, 10/11 were located at
zone 2, and 1/11 at zone 1. In zone 2,
therefore, there appears to be a maximal density of type I-like hair
cells, intermingled with type II hair cells. Because of
IK, L, zone 2 type I-like hair cells display many differences once compared with zone
2 type II hair cells of the same embryonic age
(E17-E21). For example, the average peak chord
conductance, measured at 60 mV (stepping from a holding potential of
80 mV) was 18.7 nS (±13.5; n = 10) in type I-like
hair cells, versus 2.2 nS (±1; n = 9) in type II hair
cells (P < 0.01; Student's t-test). The
difference in the average peak chord conductance between the two cell
samples was significant also when measured at
20 mV (55.8 nS ± 1.9 in type I-like hair cells vs. 9.9 nS ± 1.7 in type II hair
cells; P < 0.001; Student's t-test).
Because the average membrane capacitance (see Fig. 2A) of
the two previous samples was not significantly different (5.8 pF ± 1.4; n = 10 for type I-like hair cells vs. 5.2 pF ± 1.8, n = 9 for type II hair cells), the
difference observed in chord conductance is not the result of
differences in type I hair cell size versus type II hair cell size.
Given its low-voltage-activation range, IK,
L is likely to contribute to the negative
Vz of type I-like hair cells (73.6 mV ± 5.1; n = 11).
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DISCUSSION |
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In the present work we describe for the first time the electrophysiological properties of semicircular canal hair cells during in vivo embryonic development. Our results show that developing hair cells acquire different types of ion channels, following a precise temporal sequence. Each appearing ion current shapes hair cell voltage response in a specific manner. The main findings are discussed in this section, also in relation to other developmental parameters.
Hair cells' electrophysiological properties change along with development
During embryonic development, from E10 up to
E21, crista hair cells increased in size, ion current
amplitude, and ion channel variety. Because at E10 most
chick cochlear hair cells are postmitotic (Katayama and Corwin
1989), and provided that the same is true for crista hair
cells, then ionic currents expressed after E10 mostly should
reflect their acquisition by preexisting hair cells, and not the
appearance of new hair cells with different electrophysiological profiles. For the same reason it should be assumed that, although at
early stages all hair cells morphologically resemble mature type II
hair cells, some of these cells will differentiate as type I.
At the earlier stages here examined (E10-E12),
all hair cells expressed only ICa and
IK(V). As far as
ICa is concerned, in mature hair cells
this current is mainly involved in afferent transmitter release, most
likely of glutamatergic nature (Ottersen et al. 1998).
However, it is not known when inner ear afferent synapses become
functional during development. Interestingly, in the chick cochlea
developing cochlear ganglion neurons express functional glutamate
receptors even before connections between cochlear hair cells and
cochlear neurons exist (Jiménez and Núñez 1996
). In the chick semicircular canal, the earliest afferent synapselike contacts are observed around E7 (stages
28 and 29), although these early contacts could
represent either synaptic precursors or nonsynaptic adherent junctions
(Ginzberg and Gilula 1980
). On the basis of
morphological criteria, afferent innervation can be considered
maturelike from E14-E15 (stage 39)
(Meza and Hinojosa 1987
). Therefore
Ca2+ channels, here reported to be functionally
expressed from (at least) E10, could also play additional
roles during the development and maturation of the crista afferent
innervation; this is interesting in light of the recognition that
transmitters play a role in shaping the developing pattern of neuronal
connectivity (Spitzer 1991
).
IK(V) is likely to be important for
counteracting the regenerative depolarizing action of
ICa; in fact, both currents activate between 60 and
50 mV, and IK(V)
inactivates very slowly and incompletely up to 0 mV.
In principle immature hair cells could be depolarized by the transducer
current. At E10 hair cells already display short hair bundles, on which tip links have been observed (Tilney et al. 1986), but it is not known whether the mechanotransduction
apparatus is actually working. If this is the case, mechanotransduction currents would be able to strongly affect hair cell membrane potential: in fact, present current-clamp experiments show that positive-current steps of 100 pA (in the range of the receptor current in newborn chicks) (Ohmori 1985
) can steadily depolarize the hair
cell membrane voltage to
20 mV. Depolarization by transducer current
and/or Ca2+ influx through voltage-dependent
Ca2+ channels may be important during
development: for example, sustained membrane depolarization has been
suggested to facilitate insertion of membrane proteins, such as
receptors for neurotransmitters (Ohmori and Sasaki
1977
). At later stages of development, with the acquisition of
IK(A) (at E12) and
IKCa (at E14), hair cells' voltage response to depolarizing current becomes more rapid and compressed: for example, 100-pA positive-current steps depolarize hair
cells between
50 and
40 mV.
Voltage responses to negative current steps were purely passive until
the appearance of Ih, followed by
IK1. Both inward rectifying currents
were also likely to contribute to the membrane resting potential,
although differently, given their different reversal potential. It is
well known that mature hair cells can be hyperpolarized by negative
deflection of the hair bundles and by the activation of the inhibitory
efferent system (Guth et al. 1998). In the chick semicircular canal, the latter is presumably functional from
stage 43 [judging from choline acetyltransferase (ChAT)
activity, corresponding here to E18] (Meza and
Hinojosa 1987
), that is, at a time when hair cells have already
started expressing Ih and
IK1, which therefore could play a role
in the efferent action.
From E17 an additional ion current, termed
IK, L, can be found in some hair cell,
which is particularly important because it is considered peculiar for
type I hair cells (Correia and Lang 1990; Eatock
and Hutzler 1992
; Rennie and Correia 1994
). From a morphological point of view, chick crista type I hair cells have been
previously reported to differentiate at stage 39 (our E14-E15), when nerve afferent terminals shaped
as half-chalices also appear (Meza and Hinojosa 1987
).
In close agreement, we could distinguish type I-like hair cells and
the associated calyx synapse from E15. We also found that
these cells typically expressed (at least from E17) a
low-voltage-activated delayed rectifier current resembling
IK, L, thus further supporting their
type I nature (Correia and Lang 1990
; Eatock and
Hutzler 1992
; Rennie and Correia 1994
). The
precocious differentiation of type I hair cells in chick vestibular
hair cells compared with mammals (where it occurs around the 4th
postnatal day with the acquisition of IK,
L) (Eatock and Rüsch 1997
; Rüsch et al.
1998
) is not surprising because, as already recalled, inner ear
development in mammals lags behind that of avians.
The chicken sensory epithelia are essentially mature at hatching
(Forge et al. 1997), even regarding the nervous afferent activity (Jones and Jones 1995
, 1996
;
Manley et al. 1991
). In our experiments, we found that,
close to hatching, the assortment of ion channels is very similar to
that of mature avian vestibular hair cells (at least for type II)
(Lang and Correia 1989
; Masetto and Correia 1997
).
Nonetheless, ion current kinetics, voltage-dependence, and pharmacology
(Griguer and Fuchs 1996
), might still be different from
those of adult animals. Unfortunately, comparable data on adult chicken
are not available, so the closest counterpart would be the pigeon. When
compared with mature pigeon crista hair cells (Masetto and
Correia 1997
; Weng and Correia 1999
), chick
embryo crista ion currents do show some differences (e.g., total ionic currents for the chick embryo are slower for all crista zones); however, this could be the result of either developmental or
interspecies differences.
A schematic drawing summarizing our findings about type I and type II hair cells differentiation during chick crista embryonic development is shown in Fig. 13. At E10 all hair cells are quite small, display a short and immature hair bundle, and have already been reached by immature afferent nerve fibers; when recorded from, these cells express a voltage-dependent Ca2+ current (ICa) and a voltage-dependent slow outward K+ current (IK(V)). From E12, most zone 1 and zone 3 hair cells additionally express a fast and transient outward rectifying K+ current (IKA). By this time, morphological features typical of true synaptic contacts (pre- and postsynaptic membrane thickenings, synaptic bars surrounded by vesicles and facing the afferent nerve terminals) are commonly observed throughout the sensory crista. By E14, when afferent synaptic contacts appear to be morphologically mature and efferent contacts start to be observed in type II hair cells, some hair cells acquire a Ca-dependent K+ current (IKCa).
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It is difficult to precisely set the timing of appearance of inward
rectifying currents: in fact, a small minority of hair cells started
expressing Ih at E14, and
IK1 at E16 (see
RESULTS). However, only by E16 and
E19 functional expression of
Ih and
IK1, respectively, was observed in
most if not all zones 2/3 hair cells. By E19 a
rise in ChAT activity indicates the ability of efferent fibers to
produce acetylcholine (Ach), which appears to play a major role in the
efferent transmission of the inner ear end organs (Guth et al.
1998).
Starting from E15, hair cells morphologically resembling
type I (large cell body and apical surface, with a very constricted neck) and contacted by large afferent terminals shaped as half chalices
were observed, preferentially in zone 2. We currently do not
know which ionic currents are expressed by these immature type I-like
cells. By E17 clear synaptic contacts between efferent endings and the afferent calyx, which now completely surrounds the
basolateral region of type I hair cells, are found. By this time type
I-like hair cell express IK, L, the
signature current of type I hair cells, in addition to
Ih (at least some cells) and to a
second unidentified outward rectifying current
(IK(X)), activating less negative than
60 mV, and possibly analogous to IKCa described in avian and mammalian
crista type I hair cells (Rennie and Correia 1994
) or to
IDR, I described in mouse utricle type
I hair cells (Rüsch and Eatock 1996
;
Rüsch et al. 1998
).
So far it is not known when type I hair cells commit to their fate, and
little is known also on the possible factors regulating the expression
of diverse ion currents by hair cells. Neurotrophic factors recently
have been described to modulate the expression of hair cell potassium
currents in vitro (Sokolowski et al. 1999). Because
multiple signaling molecules affect different stages of inner ear
development (Don et al. 1997
; Montcouquiol et al.
1998
; Pirvola et al. 1997
; Ylikoski et
al. 1993
), it is conceivable that regionally and sequentially
expressed factors contribute to both regional and developmental
expression of hair cell ionic currents. The present results provide a
baseline for future studies intended to investigate the role of altered
environmental conditions on hair cell electrophysiological profile, for
example, by blocking the expression/function of specific factors at
given developmental stages.
Topographical distribution of ionic currents
From the time of their first expression, some ionic currents were
preferentially, although not exclusively, expressed in different positions in the crista: for example, zone 1 hair cells
always expressed a large IK(A), a
small IK(V), and small or no inward rectifying currents. Hair cells from zone 2 and zone
3 always expressed a large IK(V),
and at least one inward rectifying current (Ih and/or
IK1).
IK(A) was also expressed by a
significant fraction of zone 3 hair cells, but very few
zone 2 hair cells. No comparable data are available for
mature chickens; previous data from another avian show strong
similarities (Masetto and Correia 1997; Masetto et al. 1994
; Weng and Correia 1999
), but in
which the main difference is the lower incidence of
IK1 we observed in zone 1 hair cells (Weng and Correia 1999
). However, because
zone 1 appears not to be homogeneous with respect to inward
rectifier expression (Marcotti et al. 1999
),
cell-sampling biases may be responsible for this difference.
From the preceding discussion it comes out that, similarly to what was
observed in the chick developing cochlea for
IK(A) and
IK(V) (Fuchs and Sokolowski
1990; Griguer and Fuchs 1996
), chick crista hair
cell ionic currents during development are expressed at similar
positions as in the mature animal; therefore, those factors determining
the mature topographical distribution of ion channels in the inner ear
operate early during hair cell differentiation.
In vivo versus in vitro development
In a previous in vitro study of the whole chick otocyst
(Sokolowski et al. 1993), the electrophysiological
properties of hair cells after 3 wk in culture resembled those of
crista type II hair cells isolated from 2- to 3-wk-old chicks. When
compared with the present results, in both studies
ICa and
IK(V) were already present in the
earliest recordings, and were followed by
IK(A) and
IKCa (which appeared with a few days
lag in vitro). Conversely, IK1
appeared in vitro significantly earlier (at E12, vs.
E19 here); this might suggest that some hair cells from the
cultured otocyst develop with an utricular-like rather than with a
canal-like pattern (in the mouse utricular hair cells
IK1 appears earlier than
Ih) (Eatock and Rüsch
1997
).
Ih was not reported in vitro (possibly
because of the too-short voltage protocol used) as
IK, L, although hair cells
morphologically resembling type I hair cells were occasionally noted.
On the other side, some hair cells from the chick cultured otocyst and
2- to 3-wk-old chick cristae (Sokolowski et al. 1993)
expressed a Na+ current, not reported here. If
hyperpolarizing prepulses were necessary to remove
INa inactivation, as reported for
developing mammalian outer hair cells (Oliver et al.
1997), then we could have missed this current because in our
experiments on Ba2+ current (i.e., in the
presence of K+ channel blockers)
depolarizing-voltage steps were usually delivered from a holding
potential of
60 mV. At the same time, no Na+
currents active in the range of the cell resting membrane potential, like those recently described in the rat developing utricle hair cells
(INa half inactivation =
23 mV)
(Lennan et al. 1999) appear to be present in chick in
vivo developing crista hair cells.
Development versus regeneration
After the discovery that hair cells in higher vertebrates can
regenerate after noise or ototoxic trauma (Corwin and Cotanche 1988; Forge et al. 1993
; Ryals and Rubel
1988
; Warchol et al. 1993
; Weisleider and
Rubel 1993
), considerable interest has focused on the
modalities of hair cell regeneration. Shortly after injury, new hair
cells appear in the neuroepithelium, bearing a striking resemblance to
immature hair cells, and their sequence of hair bundle differentiation
parallels the embryonic one (Cotanche 1987
). Type I hair
cells reappear only at a later stage. As far as type II hair cell
properties are concerned, in the pigeon crista early-regenerating hair
cells display lower membrane capacitance and smaller and slower total
outward currents compared with control and fully regenerated hair cells
(Masetto and Correia 1997
). These findings could be
explained, assuming that cell and ion current sizes increased during
regeneration, and also that the expression of the fast IK(A) was delayed in respect to that
of the slower IK(V). The present
results show that the preceding sequence of events is also observed
during chick crista embryonic development; it is therefore possible
that hair cell damage could trigger this same differentiation program
in the adult animal.
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ACKNOWLEDGMENTS |
---|
We thank M. J. Correia, PhD, for valuable comments on the manuscript.
This work was supported by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST), Rome, Italy.
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FOOTNOTES |
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Address for reprint requests: S. Masetto, Dipartimento di Scienze Fisiologiche-Farmacologiche Cellulari-Molecolari, Sez. di Fisiologia Generale e Biofisica Cellulare, Via Forlanini 6, 27100 Pavia, Italy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 September 1999; accepted in final form 4 January 2000.
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REFERENCES |
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