 |
INTRODUCTION |
In chick auditory papilla, there is a correlation
between types of ionic currents in hair cells, hair cell location, and
the frequency of damped membrane oscillations in these cells during extrinsic current injections. In turtle auditory papilla, damped membrane voltage oscillations (resonance) also occur in response to
extrinsic current injections in hair cells (Crawford and
Fettiplace 1981
). The frequency (f) of the
oscillations and the quality factor (Q) of the resonance are
close to the characteristic frequency and Q of the hair cell
linear tuning curve obtained from sound presentations (Crawford
and Fettiplace 1981
). Thus electrical tuning of signals in
turtle auditory hair cells occurs, and apparently it is functionally
important. Electrical tuning also occurs in some hair cells in the
cochleae of chicks (Fuchs 1992
), frogs (Ashmore
1983
; Hudspeth and Lewis 1988
), and alligators
(Fuchs and Evans 1988
). Electrical second filters,
however, may not be necessary for frequency sharpening in mammals
(Narayan et al. 1998
).
Roughly 60% of vestibular hair cells found in the pigeon's
semicircular canal, SCC, demonstrate voltage oscillations
(Correia et al. 1989
) during small extrinsic current
injections (20-100 pA). But when compared with those of frog and
turtle auditory hair cells, the voltage oscillations are of a lower
quality (~1 order of magnitude) and a lower frequency (Correia
et al. 1989
; Ricci and Correia 1999
). With the
exception of one study of toadfish vestibular hair cells
(Steinacker et al. 1997
), low-frequency low-quality
oscillations during small current injections also have been observed in
vestibular hair cells of the frog (Housley et al. 1989
)
and the guinea pig (Rennie and Ashmore 1991
).
Angelaki and Correia (1992)
showed that the equivalent
electrical circuit used to model membrane voltage resonance in cochlea hair cells by Crawford and Fettiplace (1981)
and
modified by Ashmore and Atwell (1985)
was inadequate to
completely describe the membrane voltage oscillation properties of
pigeon vestibular hair cells. Satisfactory fits were obtained only when
a complex admittance (Y0) was
substituted for the resistor and inductor in one of the branches of the
resonant equivalent circuit. To achieve adequate fits, it was necessary
that Y0 be modeled as an underdamped
system in some cases, and in other cases it was necessary that
Y0 be modeled as an overdamped or
critically damped system. But as a rough first approximation, the
parameters f and Q based on the resonant
equivalent circuit (Ashmore and Attwell 1985
;
Crawford and Fettiplace 1981
), describe the voltage
response of vestibular hair cells to small extrinsic currents
(Correia et al. 1989
; Ricci and Correia
1999
). These parameters have been used herein to characterize the membrane oscillatory properties of fast and slow cells in the SCC
and utricle.
With the use of the slice preparation, it has been shown that hair
cells in different regions of the frog (Masetto et al. 1994
) and pigeon (Masetto and Correia 1997a
) SCC
neuroepithelium have different combinations of outward and inwardly
rectifying currents. The present study is an extension of those studies
with a threefold purpose. First, we wished to determine whether
regional differences of ionic currents, noted for the pigeon SCC
(Masetto and Correia 1997a
), were also present in the
utricle. Second, we wished to compare the kinetic properties of the
ensemble of ionic currents in the SCC and the utricle, and finally we
wanted see whether these regional differences were reflected in
differences in the membrane voltage oscillations during extrinsic
current injections. A preliminary report of this work has appeared in abstract form (Weng and Correia 1998
).
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METHODS |
White king pigeons (Columbia livia) of either sex,
weighing from 200 to 350 g and with ages ranging from 3 to 12 mo,
provided the tissue used in the present experiments. All experimental
procedures were conducted after approval by the institutional animal
care and use committee and followed the guidelines set forth by the National Institutes of Health and the American Physiological Society.
Slice preparation
SCCs and utricles were harvested and dissected free of each
other. All tissue was then incubated in Dulbecco's modified Eagle's medium (DMEM) augmented with 24 mM NaHCO3, 15 mM
PIPES, 50 mg/l ascorbate, and 1.5% fetal calf serum. The DMEM and
tissue were maintained at 37°C, pH 7.4, and an osmolarity of 320 mosmol/kg, in a saturated 95% O2-5%
CO2 environment. At varying intervals, tissue was
removed from the incubator, embedded in 4% agar, and quickly covered
by partially frozen vibratome bath solution (Table 1). The utricle, with its roof removed
(Fig. 1A), was sliced, using a
vibratome (Campden, Silby, Loughborough, UK) in planes parallel to its
short axis (Fig. 1, B and C). Slices through the horizontal and vertical semicircular canal cristae were parallel to
long axis of the crista (Fig. 2,
A-C). Individual slices (150-200 µm in thickness) were
then transferred to a dish with a No. 1 glass cover slip bottom, held
in place by a weighted nylon mesh and bathed in an extracellular
solution (Table 1) that had been saturated with oxygen. The dish was
mounted on a microscope stage (Zeiss Axioskop), and the cells were
viewed using differential interference contrast microscopy optics
including an Optovar magnifier and a ×40 water immersion objective.
During the recording session, the slice was usually continuously
superfused with oxygenated extracellular solution at room temperature
(~20°C). The flow rate of the superfusate was 1.2 ml/min.

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Fig. 1.
A: top view of a utricle that has been isolated
and whose roof has been removed. White arrows point at the striolar (S)
Zone. Lateral (L) and medial (M) to the striola are the extrastriolar
regions. The long axis of the utricle is in an anterior (A)-posterior
(P) direction. B: slice (180 µm thick) through the
center of the utricle in a plane perpendicular to the long axis. The
otoconial layer (black covering) is curved and thinner over the bulging
otoconial membrane in the S Zone. Inset: section of the
striolar epithelium in which it is apparent that the tallest
stereocilia of 2 hair cells (pointed to by black arrows) oppose each
other. C: sketch of the utricle indicating the S Zone
and each of the extrastriolar (ES) subzones. In this and Fig.
2C, the number of cells from which recordings were
obtained in each subzone is indicated.
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Fig. 2.
A: slice through the horizontal semicircular canal (SCC)
ampulla in a plane parallel to the long axis of the crista. Zones
I-III are demarcated by the arrowheads in this hemicrista.
B: section through the ampulla of a vertical SCC. The
plane of the section is through the long axis of the crista. The crista
is symmetrical about its apex. The zones of 1/2 of the crista
are indicated by arrowheads. PS, planum semilunatum. C:
sketch of the vertical SCC crista indicating widths of the zones and
the subzones.
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Electrical recordings
Ionic current and membrane voltage recordings were made
using conventional tight-seal whole cell patch-clamp techniques
(Hamill et al. 1981
). Access to the cell interior was
achieved either by rupturing the cell membrane or by the use of a
membrane-perforating agent (Horn and Marty 1988
).
Recordings were obtained using an Axoclamp 2A (Axon Instruments) bridge
amplifier in both the voltage-clamp and the current-clamp
mode. The Axoclamp-2A bridge amplifier was used because it has been
shown (Masetto et al. 1999
) that patch-clamp amplifiers
can introduce significant distortions in the measurement of hair cell
membrane voltage oscillations. In the majority of the experiments,
ruptured patch (RP) recordings were made. Glass capillaries (Garner
Glass No. 7052 or World Precision Instruments No. 1B150F-3) that had
been acid washed (Chromerge, Fischer Scientific) and sterilized were
pulled and fire-polished to a tip diameter of ~1 µm. The tips and
shanks of some of the microelectrodes were covered with silicone
elastomer (Sylgard), whereas others were coated with a 5% silanizing
solution consisting of dimethyldichlorosilane (Sigma, No. D-3879) in
chloroform (EM Scientific No. CX 1055-6). No difference was noted in
the size of the final compensated electrode capacitance artifact using
the two methods. In some experiments, perforated-patch (PP) recordings
were made. Electrodes were pulled and polished to a tip diameter of
~1.5-3.0 µm. The electrodes were back-filled with a solution
containing a perforating agent. The perforating agent (either
Amphotericin B, Sigma No. A-4888 or Nystatin, Sigma No. N3503) was
dissolved completely in dimethyl sulfoxide (DMSO). Five milligrams of
the perforating agent was dissolved in 50 µl of DMSO. This stock
solution was diluted to a final concentration of 250 µg/ml in a back
filling solution [back fill solution (PP), Table 1]. Care was taken
to ensure that the final concentration of DMSO was <0.25%. The tip of
the electrode was filled with a tip filling solution [tip solution (PP), Table 1]. For ruptured-patch recordings the access resistance was between 2 and 6 M
and for perforated-patch recordings the access
resistance varied between 6 and 20 M
. The electrode junction potential and electrode capacitance were compensated using the amplifier's analogue circuitry. No attempt was made to compensate for
series resistance. However, the final series resistance produced a
maximal voltage error of <10 mV, and the clamp speed did not limit the
analysis of the activation kinetics.
Data acquisition
Stimuli were generated and signals were sampled using AD/DA
converters (DigiData 1200, Axon Instruments) that were controlled by a
PC running data acquisition software (Clampex 6.3, Axon Instruments). The bandwidth of the amplifier's filter was set at 3 or 10 kHz depending on the experimental protocol. The digital sampling frequency was two to five times the analogue bandwidth of the recorded signal.
Data analysis
REGIONS STUDIED.
The long axes of the vertical SCC cristae were partitioned into three
zones, named as before (Masetto and Correia 1997a
) and illustrated in Fig. 2, B and C. Zone I, the
peripheral region, extends ~60 µm from the planum semilunatum (PS).
Zone III, the central region, extends ~160 µm on either side of the
apex. Zone II, the intermediate region is ~135 µm and is between
Zones I and III (Fig. 2C). The horizontal SCC crista is a
hemicrista, and there is only one of each of the three zones (Fig.
2A). Zone III in the vertical SCC cristae was further
partitioned into two subzones (IIIa and IIIb), of roughly equal size,
bisected by a ridge on the surface of the epithelium. Hair cells
residing in the different subzones were studied. A thickening in the
otolithic membrane defined the striolar (S) Zone. A high-power
magnification of the hair cells in this zone revealed ciliary bundles
tapered in opposite directions (Fig. 1B, inset,
black arrows). Type I hair cells were not found in any other
region of the utricle (Jorgensen and Anderson 1973
). The
lateral extrastriolar (ES) region was demarked by the edge of the S
region and the end of the neuroepithelium (ESa, Fig. 1C).
The medial ES region was roughly subdivided into three equally sized
subregions (ESb-d, Fig. 1C). Cells were studied in each of
these subregions. Only type II hair cells were studied, and they were
identified on the basis of their morphology (Correia et al.
1989
; Ricci et al. 1997a
,b
).
HAIR CELLS STUDIED.
Four hundred seven hair cells produced recordings. Of these, 272 hair cells were selected as the total sample because these cells met
three criteria: 1) their zero current potential
(Vz) was more negative than
40 mV,
2) they produced recordings that lasted at least 10 min, and
3) they produced both voltage-clamp and current-clamp
recordings. The distribution of the number of cells by zone was: 39 hair cells were from Zone I; 71 hair cells from Zone III; 116 hair
cells from the ES Zone and 46 from the S Zone. The number of cells
studied in each subzone is indicated in Figs. 1C and
2C.
The total sample of cells was further sorted into fast and slow cells
by comparing the time-to-peak (Tp) of
the net current (Lang and Correia 1989
). To study the
properties of the ionic currents in hair cells residing in different
zones of the SCCs (Zone I and Zone III) and the utricle (S Zone and ES
Zone), 10 cells from each zone were randomly selected from the total
sample as representative and analyzed as follows.
PARAMETERS CHARACTERIZING MEMBRANE VOLTAGE AND CURRENT
RESPONSE.
Input impedance (Rin), input
capacitance (Cin), and membrane time
constant (
in) were calculated from the
measurement of the voltage (Vss)
response to a
20-pA current pulse (250 ms long) from
Vz (see Table
2). Rin
was determined from the solution of the equation
Rin = Vss (mV)/20 (pA) where
Vss = steady-state voltage measured
just before the off time of the pulse. A single exponential function
[Vss + Vexp
(t
]
was fitted to the charging portion of the voltage response. The input
capacitance, Cin, was calculated from
the equation Cin =
in/Rin. A
model cell with a 33-pF capacitor (Cin) in parallel with a 0.5-G
resistor (Rin) and in series with a 10 M
resistor (to simulate the resistance of the patch electrode) was
tested using the above method. The calculated value of
Cin was in error by <6%.
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Table 2.
Summary of mean parameters from detailed analysis of 10 hair cells from
each zone of the SCCs and utricle
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The activation time constant (
a) of the
outward net current was estimated by fitting Eq. 1 to the
rising phase of the current trace in response to a voltage pulse. The
voltage pulse was 200 ms wide and varied from
60 to 0 mV.
|
(1)
|
where I0 is the initial
current, I is the steady-state current, and
a is the activation time constant. The
parameter n was varied from 1 to 4, but n = 3 gave the best fits and therefore was used in the final analysis.
Other parameters were calculated to quantify the outward current.
These included peak current (Ip), peak
conductance (gp), steady-state
conductance (gss), the ratio of
gp to
gss, and the time-to-peak current
(Tp).
The membrane voltage response to a current pulse (250 ms wide) was
fitted with a sinusoidal function that decays to a plateau (Ricci and Correia 1999
). The magnitude of the pulse
varied from 20 to 120 pA.
|
(2)
|
where Vss is the steady-state
plateau voltage, Vp is the peak
voltage above the plateau,
Q is the time
constant of the envelope decay, f is the frequency of the
oscillation,
=3.14, and
is the sinusoidal phase angle.
The best fitted values of
Q and f
in Eq. 2 were subsequently used to calculate an electrical
resonance quality factor (Q) using Eq. 3
(Crawford and Fettiplace 1981
).
|
(3)
|
Rectified sinusoidal currents, 50 pA in magnitude and at
discrete frequencies (0.05, 0.5, 5.0, 50, and 217 Hz), were injected into 10 hair cells from the S Zone. Gain (impedance magnitude) and
phase were calculated between current injection and voltage membrane
potential. Rectified sine wave current injection has been used in the
past to simulate the waveform of the mechanoelectric transduction
current and study the membrane potential changes of type II
(Sugihara and Furukawa 1989
) and type I hair cells (Correia et al. 1996
).
Instead of modeling the membrane voltage response to the injected
rectified sinusoidal currents in terms of resistive, capacitive, and
inductive components of a resonant electrical circuit (Ashmore and Attwell 1985
; Crawford and Fettiplace 1981
),
the admittance, Y(s), or impedance,
Z = 1/Y(s), of the membrane was
described by parameters that more directly relate to the properties of
the membrane (Angelaki and Correia 1991
; Correia
et al. 1989
; Mauro et al. 1970
). Thus the
impedance magnitude and phase values resulting from rectified sine wave
current injections were fitted by an equation that describes a resonant
circuit in terms of membrane properties
|
(4)
|
where s =
+ j
,
j = (
1)1/2 and
= 2
f. The input conductance
Gin = (Rin)
1. The
magnitude of the active conductances are
Gi and their relaxation time
constants,
i. In the final analysis, the
impedance magnitude and phase were fitted by a two conductance model. A
one-conductance model did not provide an adequate fit, and a
three-conductance model did not greatly improve the fit.
Curve fitting of Eqs. 1 and 2 was done using
nonlinear regression algorithms in Origin 5.0 (Microcal Software). Gain
and phase values of the impedance function were calculated using the
program Sinefit (University of Texas Medical Branch). Curve fitting of Eq. 4 was done using the program Scientist 2.01 (Micromath).
Values are generally presented as means ± SD unless otherwise
noted in which case the standard error of the mean (SE) is used. Statistical significance and lack thereof were accessed when
P < 0.05 and P > 0.05, respectively. Exceptions to these probability values are explicitly
stated in the text.
 |
RESULTS |
Figure 3 presents histograms of log
Tp measured for 272 cells residing in
either Zone I or Zone III of the SCC or the ES Zone or the S Zone of
the utricle. Average values for Tp are
also presented. A statistically significant difference
(P < 0.001, repeated measures ANOVA) existed between
the values of Tp for cells in the
different zones. The values of Tp were
statistically significantly smaller for cells from the ES Zone
(t-test, P < 0.001) and Zone I
(t-test, P < 0.001) when compared with
cells from Zone III and the S Zone. The median value of
Tp for all cells was 3.83 ms. This
Tp value separated cells into two
groups, which we have designated as "fast" and "slow" cells.
Thus the ES Zone of the utricle and Zone I of the SCC contained
predominately fast cells, whereas the S Zone of the utricle and Zone
III of the SCC contained mostly slow cells. There was no statistically
significant difference between values of
Tp for the subzones of Zone III of the
SCCs and the subzones of the ES Zone so the data were pooled.

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Fig. 3.
Three-dimensional histogram comparing log time-to-peak for cells from
Zones I and III of the SCC and the S Zone and ES Zone of the utricle.
Mean ± SD values for Tp is presented
on the back wall of the histogram.
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A random sample of 10 cells from each zone was chosen for further
analysis. Normalized outward current traces from these cells are
presented in Fig. 4A (cells
from the SCC) and in Fig. 4B (cells from the utricle). The
current traces from cells of Zone III and the S Zone show slower
activation, slower inactivation and delayed peaks. These
characteristics of the current traces did not change with repeated
testing. Ninety percent of the cells from Zone III and 100% of the
cells from the S Zone were classified as slow (dotted lines) using the
Tp criterion while 100% of the cells from Zone I
and 90% of the cells from the ES Zone were classified as fast (solid
lines).

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Fig. 4.
A: normalized current traces obtained from 20 SCC hair
cells (10 from Zone I and 10 from Zone III). B:
normalized current traces obtained from 20 utricular hair cells (10 from the S Zone and 10 from the ES Zone). Dotted curves identify traces
with Tps >3.83 ms and represent responses
from Zone III and S Zone hair cells. Solid curves identify traces with
Tps <3.83 ms and represent responses from
Zone I and ES Zone hair cells. Inset: voltage
protocol. Black arrow indicates a
Tp of 3.83. Percentages of cells that were
classified as fast and slow cells for each zone are presented in the
legends.
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Traces of outward currents, inward currents and membrane voltages from
a fast cell from Zone I of the SCC and a slow cell from Zone III are
shown in Figs. 5, A-F.
Stimulus protocols used throughout the study for all cells are
presented in the top panel of each column. The activation
and inactivation of the net outward current is faster for the fast cell
(Fig. 5A) as compared with the slow cell (Fig.
5B). Mean parameters quantifying these kinetic differences
for the random sample of 10 cells, whose outward currents are presented
in Fig. 4, are summarized in Table 2. The mean peak current
(Ip) and mean peak chord conductance
(gp), are statistically significantly
larger, whereas the mean steady-state conductance (gss),
Tp, and the mean activation time
constant (
a) are statistically significantly
smaller for fast cells from Zone I and the ES Zone when compared with
equivalent parameters for cells from Zone III and the S Zone.

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Fig. 5.
Illustration of the stimulus protocols (top panel) used
to obtain the voltage-clamp and current-clamp responses in this study
(unless otherwise specified). Middle panel: outward
(A) and inward (C) currents and a
membrane voltage response (E) to these protocols from a
fast cell located in Zone I of the SCC. Calibration bars above this
panel apply to the traces in both the middle and
bottom panels. Bottom panel: traces
(B, D, and F) that are comparable to
those found in the middle panel but for a slow cell from
Zone III of the SCC. Although traces are not shown for the utricle, the
qualitative responses from the ES Zone and S Zone were not different
from those of Zone I and Zone III, respectively.
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Inwardly rectifying currents found in both fast and slow cells were of
two types (Masetto and Correia 1997a
): a fast
inwardly rectifying current, IRIK1, (Fig. 5C) that
relaxed at very hyperpolarized potentials (>120 mV) and a slowly
activating inwardly rectifying current, Ih, (Fig. 5D).
In a given hair cell, IRIK1 or Ih could be
present either singly or together. In some hair cells, no inwardly rectifying currents could be detected. Table
3 summarizes the percentages and
distributions of cells with fast and slow outward currents, IRIK1 and
Ih inwardly rectifying currents in Zones I and III of the SCCs and the
S and ES Zones of the utricle. IRIK1 occurs singly 40% of the time in
zones (Zone I and ES Zone) containing fast cells but only 21% of the
time in zones (Zone III and the S Zone) containing primarily slow
cells. The current Ih occurs singly in 22% of the cells in Zone I and
the ES Zone and singly in 39% of the cells in the S Zone or Zone III.
Thus IRIK1 occurs almost twice as often in fast cells and
Ih occurs almost twice as often in slow
cells (see Fig. 5, C and D, for
examples).
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Table 3.
Ratio (percentage) of the distribution of types of outward and inwardly
rectifying currents in different regions of the SCCs and utricle
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Figure 5, E and F, illustrates membrane
voltage responses for fast and slow cells, respectively. The voltage
responses for fast and slow cells depended on the composition of the
ensemble of outward and inward conductances. In Fig. 5E,
it can be seen that the inactivation of the fast outward conductance
produced a continuous voltage depolarization during the step at higher levels of current injection. The slow conductance produced a plateau of
voltages during the duration of the pulse (Fig. 5F). The
hyperpolarized response for the fast cell, which contained IRIK1, had a
more rapid activation, and the voltage plateaued during the pulse
duration. The hyperpolarized response of the slow cell containing
Ih (Fig. 5F) activated more
slowly and repolarized during the duration of the pulse. The onset of
repolarization was keyed to the activation of
Ih. Membrane depolarization and
repolarization following pulse onset occur faster in the fast cell
(Fig. 5E) than in the slow cell (Fig.
5F). This difference in membrane oscillation frequency was quantified by curve fitting Eq. 2 to membrane
voltage responses to extrinsic current injections (Figs.
6, B and D)
and then by comparing the mean best fitted parameters (presented in
Table 2). For current injections ranging from 20 to 120 pA, mean
frequency of oscillation, f, and the quality of
resonance, Q were statistically significantly greater
for fast cells from Zone I and the ES Zone when compared with slow
cells from Zone III and the S Zone.

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Fig. 6.
Curve fits of current traces and oscillatory voltage responses for a
fast cell from Zone I (A and B) and for a
slow cell from Zone III (C and D). The
equations fitted and best fit lines (dark solid lines) are shown in
each graph. A 2 estimate of goodness-of-fit is also
shown. Outward currents (A and C) were
produced by a 60 mV ( 60 to 0 mV to 60 mV) voltage pulse. The
starting points of the curve fits were t = 20 ms,
the start of the voltage pulses. The voltage responses
(B and D) were produced by an 80-pA
current pulse injection. The starting points of the curve fits were
t = 25 ms, the start of the current pulses.
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Current-voltage (I-V) plots for mean
Ip and mean Iss
from 10 hair cells in each of the two zones of the SCCs and each of the two zones of the utricle are shown in Fig.
7, A and C,
respectively. Comparable voltage-current (V-I) plots are
shown in Fig. 7, B and D. The data points
in the I-V plots were not corrected for cell size
because it can be seen from Table 2 that there was no statistically
significant difference between values of Cin for cells in different zones.

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Fig. 7.
Current-voltage (I-V) plots for mean peak and mean
steady-state values of the outward currents of 10 hair cells from each
of the 2 zones of the SCC (A) and each of the 2 zones of the
utricle (C). Voltage-current (V-I) plots
of mean peak and mean steady-state membrane potentials for the same 10 cells from the SCC (B) and the utricle
(D). Slopes for the best-fitted straight lines through
the peak (steady state) membrane voltages over the range from 40 to
+20 pA in Fig. 6, B and D are 1.00 (0.92)
mV/pA for the cells in Zone I, 0.77 (0.61) mV/pA for the cells in Zone
III, 1.16 (1.06) mV/pA for the cells in the ES Zone, and 1.02 (0.89)
mV/pA for the cells in the S Zone. The correlation coefficients
(Rs) for the fits ranged from 0.96 to 0.99. In this and
subsequent figures the error bars represent ±1 SD unless otherwise
specified.
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The V-I plots in Fig. 7, B and
D, indicate that the steepest change in membrane
potential occurs around rest over the range of current injection from
40 to +20 pA. Rectification begins at +20 pA for cells from all zones
but less so for the peak voltage from cells of the S Zone of the
utricle. The slopes of linear curve fits over the range
40 to +20 pA
were about the same order of magnitude (~1 mV/pA or 1 G
). However,
the slow cells from Zone III and the S Zone were the least sensitive,
i.e., had the smallest slopes (see values in the legend of Fig. 7).
Although the peak membrane voltage response was not statistically
significantly different for cells from Zone I and Zone III, it was
statistically significantly different for cells from the S Zone and the
ES Zone (P < 0.01, repeated measures ANOVA). The
opposite was true for the steady-state response. Lack of rectification
of the peak voltage response contributed to the difference between
cells from the S Zone and the ES Zone. Rectification of the
steady-state response for the cells from Zone III contributed to the
difference between cells from Zone I and Zone III.
Figure 8 is a plot of the activation time
constants,
as, for cells in each zone over the membrane
potential range from
30 to 40 mV. Generally, the values of
a declined to an asymptote as the membrane potential
increased. The time constant of the decay,
'a,
was determined by fitting the equation for a single exponential shown
in the figure. The best-fitted parameters of the equation for each zone
are presented in the figure legend. The activation time constants for
cells in the S Zone (100% slow) are statistically significantly slower
than those for other zones and are more than 3 times those of the other
zones at
30 mV and more than 1.75 times at 40 mV. Moreover, the time
constant of decay value,
'a, for cells in the S
Zone is nearly 1.5 times that of the cells in the ES Zone and 2-3
times that of cells in the 2 zones of the SCCs. Thus the hair cells in
the S Zone have long activation time constants that remain long over a
large voltage range.

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Fig. 8.
Plot of mean activation time constant, a, as a function
of membrane potential. Means are based on 10 observations except where
as could not be reliably measured in all cells. Smaller
numbers of observations are presented in parentheses. Evaluation of the
equation shown in the figure provided good fits to the data (for all
zones, R2 0.98, P < 0.001). The best-fitted parameters for the
different zones were as follows: Zone I, a0 = 0.30, a1 = 0.59, V0 = 30, 'a = 20.00; Zone III,
a0 = 0.65, a1 = 0.41, V0 = 30,
'a = 28.00; ES Zone,
a0 = 0.19, a1 = 0.55, V0 = 30,
'a = 42.62; and S Zone,
a0 = 0.00, a1 = 3.67, V0 = 30,
'a = 66.20.
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|
Figure 9, A-D,
graphically summarizes, for cells in each zone, the membrane
oscillation responses to extrinsic current injections. Figure
9A shows that the oscillation frequency,
f, increases toward an asymptote as the magnitude of
current injection increases. The time constants are faster for fast
cells in Zone I and in the ES Zone. Furthermore, the ranges of
oscillation frequencies are higher for the cells of the ES Zone and
Zone I. The mean frequencies at each current level are statistically
significantly different for cells from Zone I compared with Zone III
(P < 0.01, repeated measures ANOVA) and for cells
from the S Zone compared with cells from the ES Zone
(P < 0.001, repeated measures ANOVA). Except for
the lowest current injection, there is an ordering of the values of
oscillation frequencies for a given level of current injection. The S
Zone shows the lowest f, Zone III the next lowest, Zone
I the next lowest, and the ES Zone has the highest f
value. For the lowest current injection (20 pA), the oscillation
frequencies range from ~30 for the cells from the S Zone to ~65 for
cells from Zone I and the ES Zone. As pointed out previously
(Correia et al. 1989
), however, these values could be
underestimates by as much as 2 octaves because the recordings were made
at 20° below the pigeon's usual body temperature (40°C). Figure
9B is a plot of the time constant of damping,
Q, of the membrane potential oscillations as a function
of the current injection magnitude. The damping time constant,
Q, declines to an asymptote as the magnitude of current
injection increases. At each level of current injection the
Qs are not clearly separated. The parameters of the
best-fitted exponential functions are given in the figure legend.

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Fig. 9.
A: mean frequency of oscillation (f) as a
function of injected current magnitude. The test protocol is shown in
Fig. 5, top panel, column 3. The family
of curves represents data from hair cells in different zones. Curves
rise to an asymptote. Data points were fitted with an equation:
f = f0 + f1e (I I0/ ).
The smallest value of R2 = 0.97. The best-fitted values were as follows: Zone I,
f0 = 70, f1 = 122, = 0.12; Zone III,
f0 = 38, f1 = 57, = 0.10; ES Zone,
f0 = 68, f1 = 119, = 0.14; S Zone,
f0 = 47, f1 = 119, = 0.17. B: plot of the mean time constant of the damped
oscillation ( Q) of the membrane potential as a function
of injected current. Solid lines represent evaluation of a single
exponential function. The family of curves overlap illustrating small
variation in Q (6.3 < Q < 9.7) for hair cells in different zones. C and
D: plots of quality factor, Q, derived
from Eq. 3, vs. frequency for cells from the 2 zones of
the SCCs (C) and the 2 zones of the utricle
(D). While the data from the cells (mostly slow) in Zone
III and S Zone show statistically significant negative correlations
relating Q and f, the cells
(predominately fast) in Zone I and the ES Zone vary about a constant,
Q ~ 2.2.
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|
Equation 3 implies that the quality of resonance,
Q, is a function of the frequency of oscillation,
f and the oscillation damping time constant,
Q. As in auditory hair cells, the slow cells found in
Zone III of the SCCs and the S Zone of the utricle demonstrated a
statistically significant negative correlation between Q
and f (Fig. 9, C and D).
But, like fast cells in the pigeon lagena (Ricci and Correia
1999
), fast cells in Zone I of the SCC and the ES Zone of the
utricle express currents with Q values that are
constant about a level of Q ~ 2.2, particularly when f >100 Hz. A statistically
significant difference exists between the mean values of
Q for cells from Zone I compared with cells from Zone
III and between the mean values of Q for cells from the
ES Zone compared with cells from the S Zone except for the two highest
frequencies. These results suggest that the quality of resonance is
"clamped" to a constant value at higher frequencies for fast
vestibular hair cells in the SCC and utricle as in the lagena
(Ricci and Correia 1999
).
Figure 10 presents means ± SE
(n = 10) impedance magnitude and phase values for
membrane voltage responses to 50-pA extrinsic rectified sine wave
current injections of various frequencies. The solid lines represent
evaluation of Eq. 4 (with 2 outward conductances) using
best-fitted parameters. The best-fitted parameters are given in the
figure caption. This plot shows little evidence of resonance in the
response. That is, there is no peak in the impedance magnitude with a
corresponding 90° phase shift. The frequency response does resemble
that of a resonant circuit with a complex admittance having overdamped
characteristics. Such a model has been used to describe inactivation of
the Na+ current in the squid giant axon (Mauro et
al. 1970
) and the admittance properties of the membranes of SCC
hair cells (Angelaki and Correia 1991
).

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Fig. 10.
Plot of mean ± SE impedance (Z) magnitude and
phase based on rectified sine wave injections into 10 cells from the S
Zone. Solid line represents the best-fitted parameters evaluating
Eq. 4. They are Cin = 16 pA, Rin = 250 M ,
G1 = 0.89 nS, 1 = 0.1 ms, G2 = 0.99 nS, and
2 = 2.5 ms. SE values for mean Z
magnitude are obscured by the symbols.
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DISCUSSION |
One of the major findings of this paper is that hair cells with
outward currents that have fast activation kinetics
(Tp,
a, Table
2) are found primarily in one region of the SCCs
and the utricle, whereas hair cells with slow activation
kinetics are found in a different region. Cells with fast
activation kinetics (fast cells) are found in the peripheral zone (Zone
I) of the SCC and the extra-striolar zones of the utricle. Cells with
slow activation kinetics (slow cells) are found in the central zone (Zone III) of the SCC and the striolar zone (S Zone) of the utricle. This result extends previous observations (Ricci and Correia
1999
) that fast and slow cells are present in the pigeon's
vestibular neuroepithelium and that they are regionally distributed in
the pigeon SCC cristae (Masetto and Correia 1997a
). A
regional distribution of cells, with different mixtures of ionic
currents, has also been noted previously in frog crista (Masetto
et al. 1994
). Also, inwardly rectifying currents in vestibular
type II hair cells are regionally distributed and paired
with fast and slow cells (Table 3). IRIK1 occurs ~2 times more
frequently in Zone I (predominantly populated by fast hair cells) than
in the hair cells in Zone III (predominantly populated by slow hair
cells). Ih was found in the cells of
Zone III ~2 times more frequently in the slow cells of Zone III than
in the fast cells of Zone I. Similar, but less pronounced, differential
distributions were found in the utricle. IRIK1 was found 1.6 times more
often in the hair cells of the ES Zone than in the hair cells of the S
Zone. Ih was found 1.3 times more
often in cells in the S Zone than the ES Zone (Table 3). Thus fast
cells are more likely to have the inward rectifier current IRIK1, and
the slow cells are more likely to have the inward rectifier current
Ih.
Striolar cells were strikingly different from cells in the other
regions, particularly those from the ES Zone. First, the activation
time constant,
a, for striolar cells was
statistically significantly longer and decayed slower as a function of
membrane potential than for cells in the other regions (Fig. 8). For
example, the value of
a for striolar cells in
response to a step from
60 to 0 mV was on average ~5 times that of
cells from the ES region (Table 2). Second, the time constants of
inactivation were longer for cells from the striolar region. This fact
is reflected by the statistic
gp/gss,
which for striolar cells was approximately one-third that of cells from
the ES Zone (Table 2). These comparisons suggest that the fast
activating-fast inactivating current found in pigeon vestibular hair
cells [presumably an A-type K+ current
(Lang and Correia 1989
)] is less prominent in striolar cells. Finally, striolar cells and cells from Zone III of the SCC (slow
cells) showed oscillations to small current injections that were lower
in frequency than those from fast cells from Zone I and the ES Zone.
Ricci and Correia (1999)
have recently suggested that
the low-quality oscillations noted in some dissociated lagenar hair
cells during extrinsic sinusoidal and pulse current injections are not
resonant frequencies but are the cutoff frequencies of the low-pass
membrane filter. When the sinusoidal frequency response cutoff
frequency was regressed against the oscillation frequency resulting
from pulse stimulation, a straight line with a slope of one was
obtained. The data in Figs. 9 and 10 of the present study lend further
support to this notion. In Fig. 10, the mean admittance magnitude for
10 striolar cells begins to decrease near 50 Hz. This value corresponds
to an interpolated value of 50 Hz resulting from analysis of membrane
voltage oscillations in response to pulse current injections into 10 striolar cells (Fig. 9A). It is possible that hair cells in
different regions of the SCC and utricle neuroepithelia may act as
low-pass filters with different corner frequencies, which may be tuned
at a given membrane potential by the mixture of activated ionic
currents. These different filtering properties may result from
differential topographical gene expression, because following complete
loss of hair cells due to streptomycin ototoxicity, new hair cells with
the same mixtures of ionic currents repopulate the same regions of the
neuroepithelium as their predecessors (Masetto and Correia 1997a
,b
).
There is evidence for systematic regional variation in the response
properties of both SCC and otolith organ afferents (Boyle et al.
1991
; Goldberg 1991
; O'Leary and Dunn
1976
). Also, discrete groups of vestibular efferents project to
different regions of the epithelia of the otolith organs and SCCs
(Purcell and Perachio 1997
). The results of the present
study suggest that the different filtering properties of hair cells in
different regions of the utricle and SCC must also be considered also
in the interpretation of responses from the vestibular periphery.
We gratefully acknowledge the splendid technical support of M. E. Pacheco and W. E. Little. We thank Dr. K. J. Rennie for comments on the manuscript.
This work was supported by National Institute on Deafness and Other
Communication Disorders Grant DC-01273 (Claude Pepper investigator
award to M. J. Correia).
Address for reprint requests: M. J. Correia, Rm. 7.102, Medical
Research Building, University of Texas Medical Branch, Galveston, Texas
77555-1063.
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.