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1 Dipartimento di Scienze Fisiologiche-Farmacologiche Cellulari-Molecolari—Sez. di Fisiologia Generale e Biofisica Cellulare, Università di Pavia, 27100 Pavia, Italy; 2 Departments of Otolaryngology, Physiology and Biophysics, and Anatomy and Neurosciences, The University of Texas Medical Branch, Galveston, Texas 77555; 3 Centre for Molecular Biology and Neuroscience and Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
Submitted 23 December 2002; accepted in final form 4 April 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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;
Eatock and Rüsch 1997).
During embryonic development, type I and type II hair cells of the chicken are
contacted by afferent and efferent nervous terminals
(Meza and Hinojosa 1987) and
progressively acquire different types of ionic conductances, each one
specifically shaping the receptor potential
(Masetto et al. 2000). The
different patterns of ionic conductances acquired during maturation account
for the specific sensory transduction process operated in the adult animal by
distinct hair cells.
Here we report an additional voltage-dependent ionic current, identified as
a TTX-sensitive–amiloride-insensitive Na current
(INa). Sodium current expression appears to be a feature
of immature hair cells in acoustic organs of mammals
(Oliver et al. 1997), whereas
it is present in a significant fraction of vestibular hair cells in both
neonatal and adult mammals (Rüsch and
Eatock 1997). In lower vertebrates, INa has
been reported in some mature acoustic and vestibular hair cells
(Evans and Fuchs 1987;
Fuchs and Evans 1988;
Sugihara and Furukawa 1989).
In the chick, some hair cells from in vitro cultured otocysts and from the
semicircular canal cristae of newborn chick express INa
(Sokolowski et al. 1993), but
it is not known when this current appears during in vivo development and if it
is maintained in the adult animal. Moreover, the properties and role of this
current have not been characterized.
To fill these gaps, our studies have investigated INa properties and contribution to the hair cell voltage response from its first appearance in the developing sensory crista up to hatching and in the adult animal. We conclude that in the chicken semicircular canal type I and type II hair cells, INa appears during embryonic development and persists in the adult animal. Its properties in the adult animal appear similar, although possibly not identical, to those found in the embryo. For type II hair cells, moreover, INa expression appears to be related to cell location in the crista both in the embryo and in the adult animal. Possible roles in the sensory transduction process are discussed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Slice preparation
Detailed procedures for semicircular canal dissection and slice preparation
have been reported previously (Masetto et
al. 2000; Weng and Correia
1999). Briefly, for the experiments on chick embryos, fertilized
chicken eggs of the Cobb variety were obtained from a local supplier and
incubated at 38.3°C. At different developmental days, embryos were removed
from the eggs and decapitated and the semicircular canals were excised. For
experiments on adult chickens, domestic and White leghorn hens of variable age
(10 wk to 2 yr) were studied. The semicircular canals with the ampullae were
removed using a lateral approach through the squamosal part of the temporal
bone from animals deeply anesthetized using an intravenous injection of
pentobarbital sodium (10–30 mg) followed by subsequent intramuscular
injections of ketamine hydrochloride (20–60 mg). After removal of the
labyrinths bilaterally, the chicken, while still deeply anesthetized was
decapitated. All procedures used were approved by the Ministero Italiano della
Sanità and by the Animal Care and Use Committee at the University of
Texas Medical Branch-Galveston and are consistent with the American
Physiological Society's Guiding Principles in the Care and Use of Animals.
Immediately after removal, the ampullae were incubated in D-MEM (No. 31600-026, GIBCO BRL-Life Technologies) supplemented with 1.5% newborn calf serum (No. N-4637, Sigma, St. Louis, MO), 24 mM NaHCO3, 15 mM 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 (see Table 1).
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An agar block containing the ampulla and a small portion of the
semicircular duct was immersed in the partially frozen slicing solution and
cut using a vibratome (Campden). Slice thickness varied between 150 and 250
µm. The slices were then transferred to a dish with a glass cover slip
bottom and immobilized using a weighted nylon mesh. The tissue and
microelectrode were viewed using differential interference contrast optics
employing an upright microscope (Zeiss Axioskop, Germany) equipped with
x40 and x63 water-immersion objectives. Additional magnification
(x1.25, x1.6) was obtained by means of a Zeiss Optovar attachment.
For control current recordings, the dish contained a standard extracellular
solution (Extra-std; see Table
1). Slices were not superfused except when testing drugs. In the
latter cases, a gravity fed four-barreled pipette made with a common tip was
used. The tip was immersed in the bath; the slices were perfused with
Extra-std solution through one line, while keeping the other three lines
closed; drugs, dissolved in Extra-std, were administered through the same
pipette by manually switching to a different line. We found that this
procedure minimized mechanical artifacts due to the outflowing solution
reaching the slice, allowing positioning of the perfusion pipette tip very
close to the slice (
200 µm). Tetrodotoxin (Alomone) and amiloride
(Sigma) were tested on chick embryo preparations only.
Electrophysiological recordings
In the chick embryo experiments, borosilicate glass pipettes (Drummond
Scientific) were pulled to tip diameters between 0.5 and 1.0 µm,
fire-polished, and partially coated with silicone elastomer (Sylgard; Dow
Corning). In the adult chicken experiments, quartz pipettes (Sutter
Instruments) were used. The pipettes were pulled using a Model 2000 laser
puller (Sutter Instruments) to tip diameters of between 0.5 and 1.0 µm. The
micropipette tips were not fire polished, but the quartz blanks had previously
been acid washed and coated with dimethyldichlorosilane. The micropipettes
were filled with either a standard intracellular solution (Intra-K) or with an
intracellular solution designed to block K channels (Intra-Cs; see
Table 1). Both glass and quartz
micropipettes had a resistance in the bath of 2–3 M
when filled
with Intra-K. The patch-clamp amplifier was a List L/M-EPC-7 for experiments
on chick embryos and an Axopatch 200 (Axon Instruments, Foster City, CA) for
experiments on adult chickens. Series resistance (Rs) and
cell membrane capacitance (Cm) values were read in
voltage-clamp mode directly from the amplifier's compensation dials after
using 5-mV hyperpolarizing steps delivered from –60 mV (type II hair
cells) or –90 mV (type I hair cells). After electronic compensation,
residual series resistance was always measured to be <4 M. Because
peak sodium currents were usually <1 nA, the maximal voltage drop across
Rs due to INa was always <4 mV.
Therefore the nominal voltages in the figures in RESULTS have not
been corrected. The amplifier's filter bandwidth was set at 3 or 5 kHz and the
voltage- and current signals were sampled at least two times the analog
bandwidth of the signals recorded. Current and voltage signals were measured
and controlled through a DigiData 1200 interface (AD/DA converter; Axon
Instruments) connected to a personal computer (Pentium PC) running pClamp
software (Axon Instruments).
Current traces were not corrected for residual capacitive artifacts and for "leakage" current. Recordings were made at room temperature (22–24°C).
Hair cell regional distribution and morphology
Recordings were made from hair cells in selected regions or zones
of the cristae (sensory epithelium) of all three (posterior,
horizontal, and anterior) semicircular canals (see
Masetto et al. 2000). It has
been shown that the electrophysiological properties of hair cells differ
according to their location in the cochlear and vestibular sensory epithelium
of amphibians and avians (Art and
Fettiplace 1987; Fuchs
1992; Masetto and Correia
1997; Masetto et al.
1994; Murrow 1994;
Weng and Correia 1999). In the
mammalian semicircular canal, on the basis of afferent innervation, the
crista appears to be concentrically organized in different regions,
called "peripheral, intermediate, and central"
(Goldberg and Brichta 1998).
By inferring an analogous organization in the chick crista,
Fig. 1A presents a
three-dimensional conceptualization of a vertical semicircular canal
crista of the chick embryo reconstructed from a series of pictures
taken at E20–21. At this developmental stage, a longitudinal slice
through the crista is 1 mm. The drawing shown in
Fig. 1A is for
illustrative purposes only. For comparison,
Fig. 1B shows a
scanning electron microscopic picture of a vertical canal crista from
a pigeon. The typical experimental preparation we used is depicted in
Fig. 1C. The
terms zone 1, zone 2, and zone 3 (see Fig.
1, legend), will be used here instead of peripheral, intermediate,
and central region because we did not investigate serial slices throughout the
transverse axis of the crista: we studied hair cells in randomly cut
longitudinal slices. Hair cells in zone 1–3, respectively, are therefore
located at increasing distance from the planum semilunatum (PS),
which borders zone 1 hair cells. Because the horizontal crista
resembles one of the symmetrical sides of the vertical cristae, it was
similarly divided into zones 1–3 starting from the PS.
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Type I hair cells were identified by their typical amphora shape
(Masetto et al. 2000) and/or
by their characteristic current [IKL,
(Rüsch and Eatock 1996);
IKI (Rennie and
Correia 1994)]. A photomicrograph of a typical type I hair cell as
it appears in the crista slice is shown in
Fig. 1D (the arrow
points at the hair cell neck region).
Analysis
Analyses of traces and results were performed with Clampfit (Axon Instruments), Origin (Microcal Software), and Excel (Microsoft).
Throughout the text, average results are expressed in the following format: means ± SD (n = number of cells). In figures, error bars indicate ±1 SD.
Time-dependent inactivation of INa was best fitted by a
monoexponential function of the form
 | (1) |
is the inactivation time constant and k is a nonzero
delay constant. Time-dependent inactivation fitting was delayed k ms
until the current amplitude had decayed to 90% of the peak value to minimize
contamination from current activation. Calcium current
(ICa) contribution to the peak value was considered
minimal, given the much larger amplitude of INa (see
text).
Steady-state inactivation curves for INa were fitted
with a Boltzmann function
 | (2) |
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Generalities
As reported previously (Masetto et al.
2000), both type I and type II hair cells are present in the chick
embryo sensory crista. Before E15 all hair cells resemble type II
hair cells in shape. From E15 onward, some hair cells with the typical
amphora-shape of type I hair cells start appearing in the crista,
most often at zone 2. But we could record IK,L, the
outward rectifying current specific for type I hair cells
(Correia and Lang 1990;
Eatock and Hutzler 1992;
Rennie and Correia 1994), from
E17 only. It is possible, therefore that in chicken embryo, type I hair cell
morphological differentiation precedes IK,L expression. To
minimize hair cell type misidentification, cells with type I shape were
classified as true type I hair cells only if they expressed
IK,L, i.e., when both morphological and
electrophysiological criteria matched.
Figure 1D shows a
photomicrograph of a typical type I hair cell as it appears in the slice
preparation. Once recorded from, this cell expressed IK,L
(traces not shown).
As far as type II hair cells are concerned, the pattern of ion channels
appeared mature-like from E19 onward
(Masetto et al. 2000). Those
located in zone 1 always expressed a large A-type potassium current,
IKA, the magnitude of which appears to decrease with
distance from the PS, although increasing again moving from zone 2 to zone 3
(i.e., the contribution of IKA to the macroscopic current
is the least in zone 2 hair cells). Inward rectifying currents conversely
(IK1 and Ih) seemed to follow an
opposite gradient of expression with zone 1 hair cells closest to PS
expressing the smallest (if any) inward rectifying current. Type II hair cells
increased expression of the inward rectifiers in zones 2 and 3 with zone 3
expressing the greatest contribution of IK1 and
Ih. A slow outward rectifying K current
(IKv) was present in all type II hair cells, and its
contribution to the macroscopic current was the least in zone 1 hair
cells.
Voltage clamp
INTRA-K IN THE PIPETTE/EXTRA-STD IN THE BATH. From E14 an
additional depolarization-activated ion current is expressed by most hair
cells. This current went unnoticed until recently because conditioning
voltages up to –80 mV are necessary to unmask it
(Wooltorton et al. 2002). As
shown in Fig. 2,
depolarizations above –60 mV after conditioning pulses below –80
mV produced in many hair cells a fast and transient inward current. The
amplitude of the inward current evoked by the depolarizing voltage steps
increases as levels of prehyperpolarization increase.
Figure 2 illustrates the fast
inward current unmasked by this protocol in a type I hair cell (A)
and in a zone 2 type II hair cell (C).
Figure 2, B and
D, shows lack of expression of a fast inward current in a
zone 2 type I hair cell and in a zone 1 type II hair cell using the same
protocol. Other ionic currents expressed in type I and type II hair cells are
detailed in the figure legends. For most of the data gathered, a test voltage
of –40 mV was chosen because at this potential, outward currents in all
hair cells were small or slow enough to allow recording of the faster inward
current at the very beginning of the test voltage step. At membrane voltages
above –40 mV, outward current amplitude and activation speed increased
rapidly, thus overwhelming the smaller inward current. Moreover,
Ih reversed around –40 mV, where its tail current
was minimal. From E14, the fast inward current evoked by our test protocol was
present in a majority of type II hair cells located at zone 2 and 3 of the
slice (38/51; 74%), but only occasionally in type II hair cells located in
zone 1 (2/29; 7%). The fraction of zone 2 and 3 hair cells expressing the fast
inward current increased with embryonic age: it was in fact 3/12 (25%) from
E14 to E16, but 35/39 (90%) from E17 to E21. Before E14 (between E10 and E13)
it was: 0/10 in zone 1 hair cells and 0/15 in zone 2 and 3 hair cells. From
E17 the fast inward current was also seen in a majority of type I hair cells,
irrespective of their position in the sensory crista (24/33; 73%).
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We tested different concentrations of tetrodotoxin (TTX) on inward currents elicited as above in type I and type II hair cells. An example of the inward current block produced by local perfusion with Extra-std to which 300 nM TTX was added is shown in Fig. 3A, and the dose-response curve for TTX is plotted on a semi-logarithmic scale in Fig. 3B. Fitting the average data points for TTX block at the different concentrations using the Hill binding equation gave an IC50 value of 16.7 nM. The Hill coefficient was very close to 1 (1.16), implying that one toxin molecule is sufficient to block one channel as expected for Na channel block by TTX.
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Given its kinetics and TTX sensitivity, we concluded that the inward current we observed consisted primarily of a voltage-dependent sodium current (INa).
Amiloride 100 µM, an effective blocker of epithelial Na channels
(Alvarez de la Rosa et al.
2000), did not affect INa (n = 3; not
shown).
INTRA-CS IN THE PIPETTE/EXTRA-STD IN THE BATH. As mentioned
before, at voltages above –40 mV, INa in chicken
embryo hair cells is masked by the outward rectifying K currents, which
precluded further characterization. Therefore we replaced K+ in the
patch pipette with Cs+, which is known to block most K channels in
many cell types. This was true for chicken embryo type II hair cells as well.
In two cells tested before E14 (1 from zone 1 and 1 from zone 2),
INa was not found, and a very small sustained inward
current, presumably carried by Ca2+ through
voltage-dependent Ca channels (Masetto et
al. 2000), was the only inward current observed. From E14,
INa was found in 10/12 (83%) zone 2 and 3 hair cells, but
no zone 1 hair cells (n = 6). INa did not appear
to be affected by Cs+ inside the pipette. Ionic currents recorded
with the same conditioning protocol as in the preceding text are shown in
Fig. 4A. In a
sample of cells we measured the time necessary to remove steady-state
inactivation of INa by cyclically increasing the duration
of the conditioning step prior to the test step.
Figure 4B shows two
examples for the conditioning voltages of –80 and –120 mV. The
average removal time constant for the inward current was 6.8 ± 1.6 ms
(n = 10) at –120 mV, 22.1 ± 11.2 ms (n = 3) at
–100 mV, and 41.6 ± 10.1 ms (n = 3) at –80 mV. In
2/5 hair cells with INa tested with the preceding voltage
protocols, even prolonged conditioning at –80 mV did not recruit
INa at –40 or –20 mV. In some cells therefore, all
INa is completely inactivated at –80 mV.
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At –40 mV, INa was usually the only ionic current
present in zone 2/3 type II hair cells, although a very small inward sustained
current was observed sometimes (see also
Fig. 4C, red
trace), presumably a Ca2+ current flowing through
voltage-dependent Ca channels (Masetto et
al. 2000). Intracellular Cs+ blocked outward K currents
0 mV at which point a slow outward (Cs+) current appeared and
increased rapidly with further depolarization. Therefore
INa kinetics and the current-voltage relationship
(I-V) were investigated 0 mV. Because INa was
much larger than ICa, INa properties
were investigated without blocking ICa.
Figure 4C shows
INa recorded from a type II hair cell at the different
membrane voltages shown. From the magnified inset, it is obvious that
INa increases up to –20 mV and both activation and
inactivation speed increase with depolarization.
In type I hair cells, intracellular Cs+ was not as an effective blocker of all potassium currents as in type II hair cells. A large inward current component of IK,L carried by K+ was still present up to –50 mV (Fig. 5A). Because IK,L is unique to type I hair cells, the presence of this Cs+-insensitive component of IK,L allowed us to identify electrophysiologically type I hair cells even when morphology did not. INa was found in 7/10 type I hair cells.
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With Cs+ in the pipette, IK,L became outward
at membrane voltages more than –50 mV due to a more positive shift of
the reversal potential of the mixed K+/Cs+ current
relative to that with standard internal solution (Intra-K). The shift in
reversal potential, from about –90 to about –50 mV, is likely the
consequence of the lower permeability to Cs+ compared with
K+ in the channels carrying IK,L
(Griguer et al. 1993; Rennie
and Correia 1994,
2000;
Rüsch and Eatock 1996).
Moreover, the contaminating outward component of IK,L was
significantly smaller with Cs+ in the pipette, and
INa could thus be studied relatively well 0 mV in type
I hair cells (Fig.
5B).
The steady-state inactivation curve, based on an average of currents for four type II and one type I hair cells, and the current-voltage relationship, based on an average of currents for six type II hair cells and three type I hair cells, obtained with intracellular Cs+, are shown in Fig. 6, A and B, respectively. INa was completely inactivated at –60 mV, and its inactivation was largely removed at –120 mV. Fitting with Eq. 2 (see METHODS) gave a half-inactivation value of –96 mV and 9% INa available at –80 mV. Therefore a very low fraction of Na channels is available at membrane voltages of –80 mV and above. Similar results were obtained with Intra-K inside the pipette.
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INa activated between –70 and –60 mV, reached a peak at –20 mV (Fig. 6B), and reversed at positive membrane voltages (not shown).
Time-to-peak (tp) and decay time constant
(d) for INa (average for 6 type II and 4
type I hair cells) are shown in Fig.
6C. They were both voltage dependent, decreasing
with depolarization.
TTX (300 nM) completely blocked INa (data not shown), whereas it did not affect the small sustained inward current (presumably ICa, ref. Fig. 4C). Perfusion with Extra-std solution, to which 100 µM Cd2+ was added, completely blocked the presumed ICa and also reversibly blocked INa almost completely (n = 3; data not shown).
Current clamp
INTRA-K IN THE PIPETTE/EXTRA-STD IN THE BATH. During steady-state conditions, at membrane voltages above –70 mV, INa is almost completely inactivated. Because INa starts activating above –70 mV, the steady-state inactivation curve and the activation curve do not intersect; in other words, no sodium window currents are expected in either type I or type II hair cells. Therefore it is probable that INa can contribute only transiently to the receptor potential, provided that hair cells are depolarized from voltages more negative than –70 mV. To determine the quality and quantity of INa contribution to the type I and type II hair cell voltage-response, we undertook current-clamp (CC) experiments.
In hair cells investigated from E17, on average Vz was –63 ± 11 mV (n = 12) for zone 1 type II hair cells, –71 ± 8 mV (n = 30) for zone 2/3 type II hair cells, and –73 ± 8 mV (n = 33) for type I hair cells.
In a first set of experiments, we confirmed that INa does not contribute to the hair cells resting membrane potential (Vz) since TTX 300 nM had no effect on Vz (data not shown).
As already shown in a previous paper on chicken embryo
(Masetto et al. 2000),
depolarizing current steps delivered to type II hair cells from their
Vz produced a small peak depolarization, which increased
monotonically and then decreased to finally reach a steady level. Different
types of outward rectifier K channels are responsible for membrane
repolarization (Masetto et al.
2000). Peak amplitude increased gradually by increasing the
depolarizing current step amplitude. This general behavior, depicted in
Fig. 7A, was
observed in all (n = 42) type II hair cells investigated in CC
(E17–E21). The same was true for most (31/33), although not all, type I
hair cells (Fig. 7B).
In two type I hair cells showing INa in VC, a large and
fast depolarizing peak was observed, distinctively characterized by an
increase in slope of the depolarizing phase of the membrane voltage at around
–50 mV (Fig.
7C). The voltage response resembled an action potential
with a suddenly recruited increase in amplitude but without the overshoot. In
these two cells, Vz was –83 and –84 mV.
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VC experiments showed that negative potential prepulse conditioning rapidly and substantially removed INa inactivation. The same strategy was employed in CC experiments. Hair cells were prehyperpolarized to different levels by mean of negative current steps prior to a depolarizing current test pulse. Voltage responses to brief (100 ms) and long (5 s) conditioning current steps were recorded.
Black traces in Fig. 8 show the typical voltage responses of a type I(top) and of a type II (bottom) hair cell, both expressing a large INa during VC recordings. When the same hair cells were prehyperpolarized conveniently, the depolarization produced by the test current step showed an inflection and accelerated slope as the potential approached –55 mV (red traces). This was observed in all type II hair cells (n = 17) and most (17/22) type I hair cells expressing INa in VC and tested with the conditioning CC protocol. On the contrary, no acceleration was seen in hair cells without INa in VC regardless of the CC protocol. Consistently, the change in slope of the depolarizing phase was greatly reduced by TTX at the concentration of 300 nM (Fig. 9). Besides significantly reducing the slope increase of the depolarizing voltage, TTX also produced a slight decrease in the amplitude of the peak. TTX did not modify the voltage response of hair cells that did not express INa.
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Thus Na channels can be recruited in type I and type II hair cells of the chicken embryo. Using appropriate experimental protocols, INa, when recruited, causes an acceleration in the rising slope and an increase in the peak depolarization elicited by a positive current step. However, most hair cells had to be prehyperpolarized below –90 mV, and sometimes below –100 mV, to show an INa contribution to the voltage response. Currently, it is not clear how such negative membrane voltages can occur physiologically (see DISCUSSION). To test whether conditioning current steps of longer duration could recruit INa at more positive voltages, we applied 5-s duration preconditioning pulses. Many hair cells did not tolerate such long hyperpolarizations and demonstrated sudden changes of the membrane voltage randomly distributed during the negative steps. Moreover the slow inward rectifying current, Ih, strongly repolarized the cell membrane during the 5-s conditioning steps. Consequently, larger negative current steps were needed to hyperpolarize the cell membrane potential to levels achieved with the short pulses. From these experiments, it appears that holding voltages more negative than –80 mV are necessary to recruit INa in the voltage response of chicken embryo hair cells (n = 7, 4 type II and 3 type I hair cells). One of these cells however (zone 2, type I, E21), did show an action-potential like response when depolarized from membrane voltages up to –82 mV.
Adult chicken
The adult chicken semicircular canal type I and type II hair cells showed similar ionic currents and regional distribution of their components as in the embryo at late developmental stages (E19–21). At the same time, some differences necessitate further investigation. For example Ih seemed faster and larger in the adult compared with the embryo. Present experiments were undertaken to verify if in the adult animal the Na current was still expressed, if so, was its regional distribution the same, and, finally, were its properties and contribution to the voltage response analogous to those described in the embryo.
INa was found in all (18/18) zone 2/3 type II hair cells (see for example Fig. 10A), and no (0/7) zone 1 type II hair cells (see for example Fig. 10B). INa was found in 5/7 type I hair cells (see for example Fig. 10C), all located primarily in zone 2/3. As in the embryo, hyperpolarizations below –80 mV were necessary to substantially remove INa inactivation, and cells had to be depolarized above –60 mV to activate it. An estimate of INa steady-state half-inactivation in type II hair cells gave an average value of –89 mV (data not shown); on average, 21 ± 19% Na channels (n = 10) were available from –80 mV (vs. 9% in the chick embryo).
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The time course of activation and inactivation was found to be very similar to that observed in the embryo (compare Fig. 10A with Fig. 2C). In adult type II hair cells, time to peak at –40 mV, after conditioning for 100 ms at –100 mV, was 0.9 ± 0.2 ms (n = 10) versus 1.2 in the chick embryo (see Fig. 6C).
INa peak amplitude at –40 mV, after conditioning at –120 mV, was on average 0.44 ± 0.47 nA (n = 19) in type II hair cells, and 0.99 ± 0.61 nA (n = 5) in type I hair cells, versus 0.22 ± 0.15 nA (n = 43) and 0.36 ± 0.32 nA (n = 16) for zone 2/3 type II hair cells and type I hair cells, respectively (at E17–21). Statistical analysis indicated that variances (F test) and means (Welch-corrected t-test) were significantly different (P < 0.01). Average membrane capacitance (Cm) for the same groups of cells did not differ significantly in the chick embryo versus the adult chicken. It was in fact: 4.7 ± 3.8 pF (n = 19) in adult type II hair cells versus 5.5 ± 1.3 pF (n = 43) in E17–21 zone 2/3 type II hair cells, and 6.3 ± 1 pF (n = 5) in adult type I hair cells versus 5.2 ± 1.7 pF (n = 16) in E17–21 zone 2/3 type I hair cells. Because INa amplitude increases significantly in the adult, whereas the hair cell surface area (as estimated from Cm) does not, the density of INa results to be significantly greater in the adult compared with the embryo semicircular canal hair cells. Single-channel analysis is necessary to resolve whether an increase in Na channels density or Na channel elementary conductance account for INa density increase.
Thus INa is present in the adult chicken type I and type II hair cells where it shows similar properties and topographical distribution. It should be mentioned, however, that no experiments with Intra-Cs have been made on the adult animals. It is possible therefore that in zone 1 hair cells the fast IKA masked INa. At –40 mV, after conditioning at –120 mV for 100 ms, IKA peak amplitude was 0.38 ± 0.2 nA (n = 7), while IKA time to peak in the same cells was 3.94 ± 0.85 ms (n = 7). A sodium current with similar features as that described above for zone 2/3 type II hair cells and type I hair cells should have been noted. Nonetheless, the possibility that a smaller, and perhaps slower, INa was obscured by outward currents in the adult animal exists.
Finally, CC experiments on zone 1 type II hair cells did not reveal any clue of INa contribution to the voltage response (e.g., change in slope of the depolarizing phase or action potential like responses).
One of 11 (9%) of type II hair cells expressing INa in VC showed an action-potential-like response after depolarization from Vz in CC (Fig. 10D). Note in Fig. 10D that the resting membrane potential in this cell was around –72 mV and that a damped oscillation is present after the action-potential-like response. From the average steady-state INa inactivation curve very few Na channels (7%) should be open at –72 mV. This apparently paradoxical result is discussed. An acceleration of the depolarizing phase of the membrane voltage was observed in three of the other type II hair cells expressing INa in VC when the cell membrane was preconditioned below –80 mV. Average Vz was –71 ± 12 mV (n = 15) in zone 2/3 type II hair cells, and –60 ± 11 (n = 4) in zone 1 type II hair cells.
Average Vz in adult chicken type I hair cells was
–72 ± 6 mV (n = 6). In adult chicken type I hair cells,
current steps of the same amplitude as those used in the embryos produced only
very small voltage changes as found in most adult species. Consistently, the
average quasi-instantaneous input slope conductance for type I hair cells,
calculated 1 ms from the beginning of the voltage step at –80 mV
delivered from –60 mV, was 50 ± 47 M (n = 7) in
the adult versus 106 ± 113 M in the embryo (n = 33).
Statistical analysis indicated that variances (F test) and means
(Welch-corrected t-test) were significantly different (P
< 0.05). As to what could be responsible for the differences in the
preceding text, Ih and IK,L appeared
larger in the adult.
With the exception of the expression of INa, the
activation and deactivation kinetics of the macroscopic outward current
(Fig. 10C) is typical
of mature type I hair cells in other species
(Rennie and Correia 1994;
Rennie et al. 1996;
Rüsch and Eatock
1996).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Comparison of hair cells general properties between the embryo and adult chicken
During the course of inner ear development, hair cells gradually acquire
different types of voltage-dependent ionic currents
(Eatock and Rüsch 1997;
Fuchs and Sokolowski 1990;
Kros et al. 1998;
Masetto et al. 2000;
Sokolowski et al. 1993).
However, the progression of ionic currents expression from immature to mature
hair cells may not be linear: in hair cells from the mammalian cochlea for
example, certain K+ currents are expressed transiently during
development (Marcotti and Kros
1999; Marcotti et al.
1999), suggesting a role for them in hair cell maturation. Sodium
current expression as well has been related to immature stages of the
mammalian cochlea (Oliver et al.
1997) and utricle (Lennan et
al. 1999). Here we show that in chicken semicircular canal hair
cells INa is first expressed in immature epithelia and
then maintained in the adult animal. Similar results have been reported for
the mouse utricle hair cells (at least for type II hair cells)
(Rüsch and Eatock 1997).
Thus although it is possible that INa is involved in the
maturation process of hair cells/synapse, this current should also play a role
in the fully differentiated hair cells. The latter appears to be true in (at
least some) lower vertebrates as well given that in reptiles
(Evans and Fuchs 1987), fishes
(Sugihara and Furukawa 1989),
and amphibians (Perin et al.
2001), INa is also expressed by a
subpopulation of hair cells in the adult animal. It is intriguing that in all
reports so far INa appears to be expressed by a fraction
only of the hair cells from a given sensory epithelium.
Interestingly, INa has not been reported in another
avian, the pigeon—in either adults
(Lang and Correia 1989;
Masetto and Correia 1997;
Ricci et al. 1996) or embryos.
It should be noted, however, that no appropriate experiments (intracellular
solution designed to block K channels in combination with holding or
conditioning voltages more negative that –60 mV) are available in the
literature concerning pigeon semicircular canal hair cells, and thus
INa could have been missed.
Finally, it should be pointed out that present results show that INa gross properties and contribution to the CC response appear similar in the chick embryo and adult, but the less negative value found in the adult animal for INa V1/2 inactivation (–89 vs. –96 mV in the embryo) suggests that INa in the adult might not be identical to INa in the embryo.
INa expression in relation to animal age and crista zones
In the present work, sodium currents have been found in the majority of type I and type II hair cells.
As far as type I hair cells are concerned, no correlation was found between
cell location or age and INa expression. For example, not
all (22/32; 69%— data pooled from Intra-K and -Cs experiments) type
I hair cells in zone 2 of the chicken embryo crista displayed
INa and not all (3/5; 60%) of the type I hair cells
in the adult chicken crista zone 2 expressed INa. This
argues that a proportion of type I hair cells simply do not express
INa either during embryological development or as an
adult. It would be interesting to determine if the expression of
INa is transcriptionally downregulated in 30–40% of
type I hair cells or not found in a subclass of type I hair cells, for
example, those found as multiple hair cells within a single calyx.
Regarding the correlation between INa expression, age,
and location of type II hair cells on the crista, the results are clear. The
overwhelming majority of type II hair cells from zone 2/3 demonstrated
INa at late developmental stages (E17–21, 35/39 in
Intra-K + 8/9 in Intra-Cs = 43/48, 90%), whereas very few from zone 1
demonstrated INa in the same developmental period (2/16 in
Intra-K + 0/3 in Intra-Cs = 2/19, 10%). However, a minority of type II
hair cells from zone 2/3 demonstrated INa at earlier
developmental stages (E14–16, 3/12 in Intra-K + 2/3 in Intra-Cs = 5/15,
30%), versus 0/13 in Intra-K + 0/3 in Intra-Cs zone 1 hair cells in the
same period. Before E14 0/10 in Intra-K + 0/2 in Intra-Cs zone 2/3 type II
hair cells and 0/15 in Intra-K + 0/2 in Intra-Cs zone 1 hair cells
demonstrated INa. Thus INa appears in
the sensory crista at E14 in a minority of type II hair cells in zone 2/3,
where its incidence increases over the time span from E14 to E21. Of course,
hair cells from zone 1 could start expressing INa after
birth, but this hypothesis seems unlikely because in the adult chicken 18/18
(100%) of the type II hair cells from zones 2/3 expressed
INa, whereas none out of 7 from zone 1 demonstrated
INa.
In the mammalian cochlea, sodium current expression peaks in outer hair
cells progressively from the base to the apex of the cochlea
(Oliver et al. 1997). The
authors pointed out a correlation between efferent innervation of outer hair
cells in the apical and basal regions and INa maximum
expression. In the chick embryo semicircular canal, efferent terminals appear
in the crista around E14, and the efferent synapse is presumably functional
(judging from choline-acetyltransferase activity) from E18
(Meza and Hinojosa 1987). It
is not possible to definitively correlate efferent synapse formation and
INa expression in the chicken embryo crista
because so many neural processes are simultaneously developing between E10 and
E21. Interestingly, the afferent calyx terminal surrounds type I hair cells
from E17. At that time, efferent terminals are precluded from directly
contacting type I hair cells. Because we found that most type I hair cells
from E17 to E21 express INa, it is presumable that
INa expression is not related to direct efferent
innervation of premature type I hair cells.
As in mammalian vestibular hair cells
(Eatock and Rüsch 1997),
INa expression anticipates that of Ih
and IK,L (appearing at E16 and E17, respectively)
(Masetto et al. 2000). It is
not known if INa is expressed in mammalian hair cells
before birth. It should be noted, however, that in mammals inner ear
development lags behind that of birds (IK,L for example
appears before birth in the chick but only after birth in the rat and mouse
utricle) (Rüsch et al.
1998).
INa pharmacology
INa in chick embryo hair cells was blocked by
submicromolar concentrations of TTX. Similarly, TTX (100 nM) completely
blocked INa in rat utricule hair cells
(Lennan et al. 1999), rat
outer hair cells (Witt et al.
1994), and alligator tall cochlear hair cells
(Evans and Fuchs 1987). Higher
TTX concentrations were required to completely block INa
in another study of rat outer hair cells (Kd = 474 nM)
(Oliver et al. 1997), and in
mouse utricle hair cells (Kd = 348 nM)
(Rüsch and Eatock 1997).
Thus sensitivity to TTX by hair cell Na channels is in between the two
extremes of the classic axonal Na channels (Kd 1 nM)
and the cardiac "resistant" Na channels (Kd
> 1 µM). In addition, we found that INa is
reversibly blocked by Cd2+ (100 µM), at a
concentration that blocked TTX-resistant cardiac Na channels but not
TTX-sensitive skeletal muscle Na channels
(Backx et al. 1992). The
inverse relation between block by Cd2+ and TTX suggested
that toxins and Cd2+ may compete for the same binding
site. Amiloride (100 µM), known to block epithelial sodium channels
(Alvarez de la Rosa et al.
2000), had no effect on Na channels in chicken hair cells.
Nine alpha-subunit sodium channel isoforms, named
NaV1.1–NaV1.9, have been identified up to now
which are functionally expressed (Goldin
et al. 2000). NaV1.1, NaV1.2,
NaVv1.3, and NaV1.7 are highly TTX-sensitive, broadly
expressed in neurons, NaV1.4 is abundant in skeletal muscle and is
sensitive to nanomolar concentration of TTX. NaV1.6 is expressed
primarily in the CNS, NaV1.5, NaV1.8, and
NaV1.9 are TTX-resistant to varying degrees and expressed in heart
and dorsal root ganglion (DRG) neurons. All the isoforms demonstrate fast
inactivation, are blocked by TTX and splice variants exist.
Wooltorton and Eatock
(2002) have performed RT-PCR
on the vestibular epithelia and ganglia using Na channel 
subunit
primers. TTX-sensitive NaV1.1 and NaV1.2 subunit
gene product and TTX-insensitive NaV1.5 subunit gene
product was detected in both epithelia and ganglia. More experiments on
chicken hair cells will be required, although the relatively low TTX
sensitivity and block by submillimolar Cd2+ make cardiac
Na isoforms interesting candidates. Nav1.5, in particular, shows a
more negative voltage dependence of steady-state inactivation (see
Goldin and Allan 1999).
INa properties and operational range
INa activated close to –60 mV and showed voltage-
and time-dependent activation and inactivation. A peculiar property of the
INa we investigated, compared with INa
of excitable cells, is its steady-state inactivation, which is complete around
–60 mV. We found that membrane voltages more negative than –80 mV
were necessary to significantly remove inactivation. An
INa with similar features has been reported in rat outer
hair cells (Oliver at al.
1997; Witt et al.
1994), in rat type I crista hair cells
(Bao et al. 1999), and in mouse
utricle type II hair cells (Rüsch and
Eatock 1997). Because of the negatively shifted inactivation
curve, the contribution of INa to hair cell receptor
potential has been questioned. The same conclusion appears quasi-true
for the hair cells we investigated. If in fact we expect that inhibitory
efferent system (presumably acting through IKCa
activation) (Fuchs 2002) or
negative hair bundle deflection can hyperpolarize the hair cell at most up to
the value for the K+ reversal potential, then in vivo hair cells
would not experience membrane voltages significantly more negative than
–85 mV. This value assumes an estimated intracellular K+
concentration of 140 mM and a perilymphatic K+ concentration of 5.8
mM and at 37°C (Soto et al.
2002). A contribution of INa to the voltage
response was seen in most hair cells only if they were conditioned
below – 80 mV, which should be a border-line condition in
vivo.
Nonetheless, in some hair cells from the chick embryo and the adult chicken slice of the epithelium, INa did contribute to the membrane depolarization from their resting membrane potential (which in these cells was around –80 mV). Therefore the possibility must be entertained that under certain conditions INa is not "physiologically silent" in the operating range of the hair cell. This possibility is strengthened because INa is not downregulated but expressed by a large fraction of type I and type II hair cells both during development and in the adult animal.
In 4/65 (6%) hair cells investigated in the embryo and adult chicken
demonstrating INa in VC, an action-potential-like response
to a current step from Vz was observed in CC, and this low
incidence precluded a detailed investigation of the phenomenon. A
posteriori, no macroscopic features differentiated these cells from the
others. It is possible that the ability to recruit Na channels from
Vz depended on some particular combination of factors like
the precise value of the resting membrane potential plus the relative
contribution of different types of ion channels. Ca channels could play an
important role because it is known that they activate around –60 mV and
have very rapid activation kinetics. They would boost cell membrane
depolarization and thus reinforce regenerative activation of Na channels.
ICa amplitude can vary significantly among hair cells and
even in a same cell during the recording due to its fast run-down.
Unfortunately, it is difficult to compare ICa and the CC
response in the same hair cell. Experiments using the perforated patch variant
of the patch-clamp technique could shed light on this topic.
Because of the negatively shifted steady-state inactivation curve for
INa, relative to Vz, a large number of
Na channels are unavailable around Vz. However, it is
possible that the inactivation curve could be right-shifted by some type of
modulation. For example, it has been reported in cardiac cells that protein
kinase C shifted the steady-state half-inactivation voltage for
INa 9 mV in the depolarizing direction
(Watson and Gold 1997). Thus a
study of the possible modulation of Na channels by protein kinases A or C, or
G protein, as reported for other cellular systems
(Bevan and Storey 2002;
Sen et al. 2002) would be an
interesting extension of our studies, particularly because in the chicken the
Na+ current is expressed in a large fraction of hair cells.
Recently it has been reported that in neonatal rat
(Lennan et al. 1999) and
neonatal mouse (Wooltorton et al.
2002) utricular hair cells, a significant INa
can be elicited on depolarizing the cell from –60 mV. At the same time,
it had been previously reported that in neonatal mouse utricule hair cells,
INa was fully inactivated at –60 mV
(Rüsch and Eatock 1997).
These conflicting results have recently been reconciled by suggesting that
multiple subunit isoforms may be present in different hair cells in
the epithelium (Wooltorton and Eatock
2002). It is also possible that there may be time-dependent level
of Na channel modulation in different hair cells. This could explain why the
same current step delivered to the same cell but at different times during the
recording sometimes evokes an all-or-none peak depolarization as shown here
(Fig. 7C).
INa role
In excitable cells, INa is responsible for action
potential activity. Hair cells of adult mammals do not appear to generate
action potentials, although spiking activity does characterize a distinct
developmental stage of mammalian cochlear hair cells (Kros et al.
1993,
1998) and is present in adu
