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Multiplicity of ionic currents underlying the oscillatory-type activity of isolated goldfish hair cells
Neuroscience Research, Suppl. 12 (1990) $27-$38
$27
Elsevier Scientific Publishers Ireland Ltd.
MULTIPLICITY OF IONIC CURRENTS L%K)~qLYING THE OSCILLATfRY-TYPE ACTIVITY OF ISOLATED ~ S H
HAIR C'RLLq
TARO PURUKAWA AND IZUMI SUGIHARA Department of Physiology, Tokyo Medical and Dental University, School of Medicine, Yushima, Bunkyo-ku, Tokyo, 113 Japan INTRODUCTION Based on their morphology and electrical responses, goldfish hair cells isolated from the sacculus, lagers, and utriculus can be divided into oscillatory- and spike types (19). The oscillatory-type hair cells, mostly short and ovoidal in shape, elicit, in response to an injection of depolarizing current, damped oscillatory membrane potential changes, not dissimilar to those reported in hair cells of turtle (1, 3, 4), alligator (5), and avian (6) cochleas, frog sacculus (8) and amphibian papilla (16). However, the ringing in the membrane potential observed in these goldfish hair cells was of a poor quality as can be seen by the fact that the maximum number of waves elicited was ~nly three. On the other hand, spike-type hair cells, cylindrical or gourd-like in shape, respond to depolarizing stimulus with an all-or-none spike followed by a plateau. The present report aims to clarify further the contribution of different ionic channels to the activity of the oscillatory-type hair cells. Since efforts in this direction have already been made (19), the present analysis is a supplement to these efforts. First, the present article is aimed at investigating the variabilities of electrical responses and ionic channels underlying them, because these variabilities turned out to be unexpectedly large. Another aim is to clarify the contribution of the high threshold subclasses of A-current.
A phenomenon of response augmentation due to
inactivation of the A-current is also briefly describecL METHODS Methods of study have been described elsewhere (19). In brief, saccular, lagenal, utricular and ampullar maculas were taken out from 10 - 15 cm long goldfish, and the hair cells were enzymatically isolated (8 unit/ml papain, Sigma p-3125, in calcium deficient saline). Recordings from these cells were made using the tight-seal whole-cell patch technique with either voltage recording under current clamp or current recording under voltage clamp (14). The recording chamber containing the hair cells was mounted on the stage of an inverted microscope equipped with Nomarski differential interference optics
Presented at the 12th Taniguchi International Symposium on Visual Science, November 27-December 1, 1989 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
$28 (IMT-2, Olympus, Japan). q%le micropipettes for patch clamp had electrical resistance ranging from 6 to 12 megohms when filled with K-citrate solution (see below). As a patch clamp amplifier, List Model EPC-7 (West Germany) was usecL To find the approximate values of cell capacitance and series resistance, dial positions of the slow transient cancellation circuitry (C-slow and G-series) were read, under voltage clamp setting, each time a successful whole-cell recording was made. Cell capacitance values ranging from 20 to 40 Mohm were observeci
In voltage clamp experiments, slowing down of response speed due to
the series resistance was compensated for as far as possible (usually by 40 80 %). A recording was continued typically for 5 - 25 miru The cell was ruptured in voltage-clamp, by the use of the initial holding potential of -70 mV. However, responses were observed first under current-clamp condition, then under voltage-clamp, and again with curent-clamp to observe whether or not the condition of the cell would remain unchangecL The input signal was filtered at 10 kHz within EPC-7 and stored in a PCM data recorder (NF Electric Instruments, model No. PR-880, Japan) with a sampling interval of 17.4 ~s. The playback data were filtered at I0 kHz before being fed into the AD converter of a computer (Nippon Data General, model No. N10). Data were sampled at an interval of 0.1 ms for voltage-clamp records, and at 0.5, I, or 2 ms for current-clamp and were stored on a dis~ Solutions
The normal saline used as the external bath contained (mM): 120
NaCI, 2 KCl, 2 CaCI2, and 5 HEPES. The pH was adjusted to 7.2 with about 1.5 mM NaOH. The recording chamber was continuously superfused at about 2 ml/min with normal saline using a peristaltic pump. This superfusion system was also used to replace the medium with Ca2+-free solutions, to which 2 mM Co 2+ and/or Mg 2+ was addefL To apply drugs, such as tetraethylammonium ions (TEA) or 4aminopyridine (4-AP), local superfusion was made by gently blowing solutions out of thin polyethylene tube (tip diameter: 100 ~un) placed near the cell being examinecL A pipette solution of the following composition (mM) was used in most of the present study: 53 K-citrate, I MgCI2, 10 HEPES, 5 EGTA, pH adjusted to 7.2 with about 20 m M KOH. The citrate solution had a total K + concentration of 179 mM, and its EK was -I 1 3 mY against 2 mM of external K +. RESULTS Oscillatory- and spike-type responses General properties of the oscillatory- and spike-type responses in hair cells from the sacculus, lagena or utriculus have already been described (see Introduction and ref. 19). Fig. 1 compares the typical damped oscillatory
$29 responses of a short, "oscillatory-type" hair cell (A) and the typical all-ornone spike responses of long, "spike-type" hair cells (B & C). These responses were elicited by injecting current in the current-clamp mode. Beside marked differences in voltage responses to depolarizing or hyperpolarizing current pulses, the resting membrane potential also differed (-74 mV in A; -I 02 mV in B; and -93 mV in C). Oscillatory-type hair cells had a resting potential less negative than the value for spike-type hair cells (-76+--8.9mV, n=24, and -101+--7.4 mV, n--42, respectively when measured with K-citrate in the pipette). In the following paragraphs, we describe the ionic currents based on our data obtained from 43 oscillatory-type hair cells, of which 27 were from the sacculus, 9 from the lagena and 7 from the utriculus. Hair cells from these maculas could be classified into oscillatory- and spike-types, based on morphology and functional grounds. (However, the spike type cells are not considered any further.) The ampullar hair cells were different; they
A 200 pA
100 pA
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30 pA - 1 0 pA
- 2 0 pA
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Fig. I. Typical voltage responses in 2 types of hair cells evoked by current injectioru A: oscillatory-type responses obtained in a short hair cell from rostral saccule (averages of 12-20 traces). Resonance frequency about 230 Hz. Traces were shifted vertically to avoid crossing of curves. B and C: spikeplateau-type responses obtained in long slender hair cells from caudal saccule. In C, injection of the same intensity of current (5 pA) either set up a spike response or failecL Broken lines indicate -70 mV in all records. Pipette contained K-citrate solution in A and B, but KCI in C_
$30 responded mostly as oscillatory cells in spite of the long stretched shape of their cell body. Thus, some data from the ampullar hair cells are included for comparison. Three outwardly rectifying K + channels in oscillatory-type hair cells In hair cells in which an electrical tuning is markedly developed, such as those from the frog sacculus (9, 13), and from the turtle (I) and chicken (6, 15) cochlea, a Ca2+-activated K + current (IK(Ca)) constitutes the predominant pathway for the outwardly rectifying current. However, this was not the case in the oscillatory-type hair cells of goldfish ear. Fig. 2 shows a case in which the contribution of Ca2+-activated K + channels was examined by superfusing the cell with a saline which contained 20 mM TEA, a blocker of a variety of potassium channels (17). Before the application of
control
-70mV5
-30mY L
20ram ~" 12o~.
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b0,,
-50 I:A
~! l ~ l m B
;
IA
[
-70 m V ~ '
,
-30 mV
[ _ _
(low threshold)
,
-110
Fig. 2. Demonstration of 3 components of outwardly rectifying current in an oscillatory-type hair cell from saccule. A and B: current-clamp records obtained in control state and during superfusion with 20 mM T E ~ C and D: voltage-clamp records. In C, records during control and during superfusion with TEA were superimposed (a). Current during superfusion with TEA seems mostly composed of A-current (IA, especially its high threshold subclass). Subtracted record (b) shows the time course of IK[Ca }. In De, a hyperpolarizing pre-pulse gave rise to a fast rising" low-threshold A-current whose time course is shown in subtracted record (b). Obtained during superfusion with T E ~
$31
TEA, this hair cell generated, in response to injection of depolarizing pulses, voltage ringing consisting of ah3ut three waves (A). During the superfusion with 10 m M TEA, the response to depolarizing current increased in amplitude, but became much simpler in shape (B). Tne response then consisted of a peak and a following volley. A similar change was produced by superfusing with a saline in which Ca 2+ was replaced with either Co 2+ or M ~ + (see Fig. 5A of ref. 19). These findings support the idea that the Ca2+-activated K + channels could be important for producing ringing of the membrane potential. Fig. 2 Ca shows the result of a voltage-clamp experiment in which the membrane potential was shifted from the holding potential of -70 mV to -30 mV, then back to -70 inV. The outward current was reduced during the superfusion with TEA to about 3/5 of the original amplitude. ~ne component that was reduced by TEA (Fig. 2 (~) would represent the current carried by the Ca 2+activated K + channel (I, 12, 13). The nature of the remaining current cannot readily be specified. However, as judged from marked effects of 4-AP on outward rectification (see Figs. 3-5), it could represent an A-current, possibly, of the high-threshold subclass (see Discussion). In Fig. 2 De, a hyperpolarizing pre-pulse was applied to remove inactivation of the lowthreshold A-current (7, 9, 13). During the pre-pulse, the membrane potential was kept for I sec at -110 mV. The resultant increase in the outward current could represent the component of low-threshold subclass of A-current (Fig. 2 Db). Although the pre-pulse test was carried out under the action of TEA in Fig. 2D, a similar test carried out in the normal saline gave essentially the same magnitude of the low-threshold A-current (not shown). Superfusion with TEA or Ca2+-free saline was made in a total of 7 cases. Ratios of maximal conductance attributed to the Ca2+-activated K + current to that attributed to the high-threshold A-current ranged from 0.15 to 1.4 (mean: 0.6, n--7). Effects of 10 m M 4-aminopyridiD~ on oscillatory-type hair cells Fig. 3 shows an instance in which the effects of superfusion with 10 mM 4AP, a block~.r of the A-current (13, 20), were studied in the current-clamp mode. As shown in A and Ba, the outward rectification was very markedly observed in this cell. During the superfusion with 10 m M 4-AP, the membrane potential was depolarized by about 20 mV and the outward rectification was greatly reduced (Fig. 3 Bb). Hence, injection of small depolarizing current produced a large potential change. Also, the rate of rise of potential became much slower and ringing of the membrane voltage was no longer observecL The after-hyperpolarization, which was very markedly observed in the control state, was also not obse/ved (Fig. 3 Bb). Effects of 4-AP appeared soon after
$32 the start of superfusion, and was nearly complete within one miru
The washout
also proceeded promptly. Fig. 4 shows the results of voltage clamp experiments on the same hair cell as used in Fig. 3. The current-voltage diagram in Fig. 4A shows the effect of 10 m M 4-AP more clearly than in the current clamp experiment. Outward currents produced in this cell in response to depolarizing voltage steps were roughly recta~
in shape, showing no initial peak or rapid decline. Also, no trace
of the low-threshold subclass of the A-current was detected in a pre-pulse test (leftmost traces of Fig. 4 Ba), thereby indicating that the outwardly rectifiying current in this cell consisted mainly of the high-threshold subclass of the A-current.
A
B
a
150 pA
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. . . . . .
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Fig. 3. Effects of superfusion with I 0 mM 4-AP on voltage responses of an oscillatory-type hair cell from the saccule. A: current-voltage relaticn at the end of voltage pulses (based on current-clamp data shown in B). B: current-clamp records during control state (a) and during superfusion with 10 m M 4-AP (b). The trace for 30 pA of hyperpolarizing current in a was distorted because of saturation of amplifier.
$33 Inactivation of the A-current was studied by applying pre-pulses in the depolarizing directioru For example, in the rightmest tracas of Fig. 4 Ba, a test-pulse to -30 mV was applied after the membrane potential had been kept for I sec at -50 mV. Because of inactivation, the amount of current flowing at the end of pre-pulse and during the test-pulse became smaller as compared with those in the control records shown in the inset of Fig. 4~
In Fig. 4 Bb, the
activation observed at the end of pre-pulse (filled circles) and during the test-pulse (open circles) became smaller in about the same amount. Although the inactivation reached about 50 % at the maximum in this case, it may be feasible to obtain higher percentages of inactivation by extending the prepulse duratioru
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Fig. 4. Voltage-clamp experiments on the same hair call as in Fig. 3. A: current-voltage curves ugr~r control state and during superfusion with 10 mM 4-AP. Inset, voltage-clamp records under control stat~ B: results of prepulse tests, a: sample records obtained by applying various hyper- and depolarizing pre-pulses. Pre-pulses had a duration of 1 sec, but only their terminal portions are showru Test-pulses were fixed at -30 mV. In b, membrane currents, at end of pre-pulses (filled circles) and during test-pulses (open circles), were plotted against pre-pulse level on a percentage basis.
$34 Superfusion with 10 m M 4-AP was successfully made in a total of 5 cases of the oscillatory-type hair cells. Although, in three of them, the observation was limited only to the current-clamp experiment, the effect of 4-AP was very marked and
comparable to that shown in Figs. 3-5. These results might cast
doubt on the specificity of action of 4-AP, although 4-AP in many cases does not interfere with the Ca2+-activated K + channel activity (9, 10, 13, 20). This problem needs further elucidation (17). Enhancement of response brought about by inactivation of high-threshold Acurrent Effects produced by inactivation of the A-current were studied under a current-clamp condition in Fig. 5.
Responses of this cell under the control
condition are shown in A and C. In B, a steady current at 10 pA was injected and its effect was tested by applying 80 pA pulses (duration, 60 msec) once every 2 sec. Injection of the weak steady current depolarized the cell by 10-I 5 mV from its resting level. A more marked effect appeared in the potential response to superimposed test pulses.
A
30 sec progressing inactivation
B
control
The response to a test
C
10
control 120
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sec
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Fig. 5. Augmentation in response amplitude produced by inactivation of Acurrent. Current-clamp records from a saccular hair cell. A: control responses. In B, injection of steady depolarizing current at I0 pA brought about successive augmentation in the response amplitude with time following the start of steady current injectioru C: current-voltage relations at the end of test pulses (based on current-clamp data shown in A and B).
$35 pulse increased progressively with time after the start of steady current injecticr~ ~nis result is shown schematically in Fig. 5C. Clearly, augmentation of the response amplitude was produced by a reduction in the outward rectification because of inactivation of the A-current. This enhancement of response was quite markecL It represents an opposite behavior to the adaptaticru A similar enhancement attributable to inactivation of potassium channel has been reported in hippocampal neurones (18). In voltage-clamp measurements on this hair cell, outward current in response to a depolarizing pulse rose as sharply as that in the cell shown in Fig. 4~ A low-threshold A-current was not detected at all in the pre-pulse test. The outward rectification was completely abolished with I0 mM 4-AP in this case too. Variability of the low-threshold A-current The magnitude of the low-threshold subclass of the A-current, such as shown in Fig. 2D, varied from case to case among different oscillatory-type hair cells. In certain hair cells (Figs. 3 - 5), the A-current consisted almost exclusively of its high-threshold subclass. In such instances, a
A 30pA .
.
.
.
.
.
.
.
.
.
.
.
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115 mV ---..~/c13
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-°~ mV
-70 . I V - ~ -lO0 mV
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Fig. 6. Response of an ampullar hair cell. A: current clamp records. B: large low-threshold A-current evidenced by applying hyperpolarizing prepulse. C: current-voltage relation at the end of current pulses (based on current clamp data). Some ampullar hair cells, not all of them though, had a large negative resting membrane potential and responded to injected currents as shown in this figure.
$36 hyperpolarizing pre-pulse contributed nothing to the effect of the test pulse (see Fig. 4 Ba). On the contrary, the contribution of the hyperpolarizing pre-pulse was very marked in certain other hair cells. In such cases, a test pulse (e.g., -30 mV) gave rise to a very large outward current which steeply rose to the peak and decline thereafter with a time constant of 20 msec if it was preceded by a hyperpolarizing pre-pulse. Without such a pre-pulse, the same test pulse produced only a small slowly rising outward current (Fig. 6B). In a pre-pulse test of 18 oscillatory-type hair cells from the sacculus, lagena and utriculus, the low-threshold A-current was predominant in only two cases. On the other ham~, the low-threshold-A type of respcnse seems to be very common among
ampullar hair cells (Fig. 6B), in agreement with a report
on the frog ampullar hair cells (7). The cell shown in Fig. 6 had a large negative resting potential and responded with a slow time course to injected depolarizing current. Note that the outward rectification indicated in the current-voltage curve shown in Fig. 6C seems to arise from the low-threshold subclass of A-current. Certain ampullar hair cells we encountered had a resting potential around -70 mV
aD~ responded to depolarizing current like
the cell shown in Fig. 5 ~ Unlike the cell of Fig. 5, however, these ampullar cells elicited, under voltage-clamp setting, a flow of large low threshold Acurrent in response to a depolarizing pulse when the latter was preceded by hyper-polarization.
DISCI/SSION Damped membrane potential oscillations in the goldfish oscillatory-type hair cells, elicited by injection of depolarizing current, are of a much poorer quality than those reported in frog or turtle hair cells (19). This difference may be due to the different ionic channels used by these hair cells for outward rectificatioru Whereas the outward rectification arise almost exclusively from activation of IK(Ca ) in frog or turtle hair cells, it was served in goldfish oscillatory-type hair cells by mixed activation of IK(Ca ) and IA, especially of its high-threshold sublass. M o r ~ ,
contribution of
IK(Ca ) and high-threshold IA differed greatly among different hair cells. Perhaps, outward rectification in goldfish oscillatory-type hair cells functions mostly to make the cell's time constant short and to enable the cell to follow each sound wave up to high frequencies such as 500 - 1,000 Hz (see Fig. 12 of ref. 1 9 for the mode of operation of goldfish hair cells). A similar interpretation has been offered regarding the outward rectification in the inner hair cells of mammalian cochlea (12). Furthermore, the large variablity of ionic channels in the oscillatory-type hair cells contributing
S37 to the outwardly rectifying action might be responsible for different response speed observed among different hair cells. About the low- and hiqh-threshold subclasses of ~-current Originally, the A-current is a fast, transient K current which is activated by depolarizing steps from holding potentials negative to the resting potential. The low-threshold subclass of A-current roughly meets the above definition (see Figs. 2D, 4 Ba and 6B), whereas the high-threshold subclass considerably departs from it. We included the latter in the A-current as a special subclass because
(I) it was susceptible to blockade by 4-AP, (2) it
showed a voltage-dependent inactivation, though of a slower time constant, and (3) a similar non-typical A-current has been reported in which voltagedependent activation and inactivation takes place at more depolarized levels (17). The functional role played by the low-threshold subclass of A-current in hair cells remains unclear. %'nis current is almost completely inactivated at membrane potentials close to -70 mV. It remains not inactivated in cells whose resting potential was much more negative (see Fig. 6). Furthermore, the lowthreshold A-current is much better developed in the ampullar hair cells than in hair cells from the sacculus, lagena and utriculus. On the other hand, as mentioned above, the high-threshold subclass of Acurrent, together with IK(Ca), plays an important role in characterizing the response of oscillatory-type hair cells. The high-threshold A-current, in certain cases of cell, produced a powerful outwardly rectifying action. It operated at a very high speed, and its inactivation brought about enhancement of response (see Fig. 4). About the resting membrane potential Besides differences in the ionic channels, the resting membrane potential is an important o~ntributing factor in bringing about differences in the response between the oscillatory- and spike-type hair cells (Fig. I ). In the oscillatory-type hair cells, the resting potential was mostly set around -70 mV, while it was generally more negative in the spike-type hair cells. However, some oscillatory-type hair cells had more negative resting potentials, i.e., near -100 mV. In such cases, injection of small depolarizing current produced a much larger depolarization than was usually the case with oscillatory-type hair cells with resting potential near -70 mV (Fig. 6). Without doubt, such an observation is related to the presence of a low slope oonductance region in the I-V curve (see control trace of Fig. 5C) inserted between the two inflection points attributable to the outward and inward rectification channels (2).
$38 The mechanism of how the resting potential of each hair cell is determined is unknown (11). However, the presence of two rectification channels should influence the level of resting potential. When a tendency for a depolarization is dominant in a given hair cell, the resting potential of that cell should settle around the inflection point produced by the outward rectification, as any further depolarization is preventecL On the other hand, the resting membrane potential should settle at a more negative level if the tendency of the cell is for a hyperpolarizatioru A~q~3wI~X~ITS This work was supported by grants from The Ministry of Education, Science, and Culture, Japan.
R ~ ~
J. Physiol. 400:237-274
10. Hudspeth AJ, Lewis RS (1988)
J. Physiol. 400:275-297
11. Jones SW (1989) Neuron 3:153-161 12. Kros CJ, Crawford AC (I989) In: Wilson JP, Kemp DT (eds.) Cochlear Mechanisms, Plenum, New York, pp 189-I 95 13. Lewis RS, Hudspeth AJ (1983)
Nature 304:538-541
14. Marty A, Neher E (1983) In: Sakmann B, Neher E (eds) Single-Channel Recording, Plen~m, New York, pp 107-122. 15. Ohmori H (1984) J. Physiol. 350:561-581 16. Pichford S, Ashmore JF (1987) Hearing Res. 27:75-83 17. Rudy B (1988) Neuroscience 25:729-749 18. Storm JF (1988) Nature 336:379-381 19. Sugihara I, Furukawa T (1989) J. Neurophysiol. 62:1330-1343 20. Thompson SH (1977) J. Physiol. 265:465-488
$27
Elsevier Scientific Publishers Ireland Ltd.
MULTIPLICITY OF IONIC CURRENTS L%K)~qLYING THE OSCILLATfRY-TYPE ACTIVITY OF ISOLATED ~ S H
HAIR C'RLLq
TARO PURUKAWA AND IZUMI SUGIHARA Department of Physiology, Tokyo Medical and Dental University, School of Medicine, Yushima, Bunkyo-ku, Tokyo, 113 Japan INTRODUCTION Based on their morphology and electrical responses, goldfish hair cells isolated from the sacculus, lagers, and utriculus can be divided into oscillatory- and spike types (19). The oscillatory-type hair cells, mostly short and ovoidal in shape, elicit, in response to an injection of depolarizing current, damped oscillatory membrane potential changes, not dissimilar to those reported in hair cells of turtle (1, 3, 4), alligator (5), and avian (6) cochleas, frog sacculus (8) and amphibian papilla (16). However, the ringing in the membrane potential observed in these goldfish hair cells was of a poor quality as can be seen by the fact that the maximum number of waves elicited was ~nly three. On the other hand, spike-type hair cells, cylindrical or gourd-like in shape, respond to depolarizing stimulus with an all-or-none spike followed by a plateau. The present report aims to clarify further the contribution of different ionic channels to the activity of the oscillatory-type hair cells. Since efforts in this direction have already been made (19), the present analysis is a supplement to these efforts. First, the present article is aimed at investigating the variabilities of electrical responses and ionic channels underlying them, because these variabilities turned out to be unexpectedly large. Another aim is to clarify the contribution of the high threshold subclasses of A-current.
A phenomenon of response augmentation due to
inactivation of the A-current is also briefly describecL METHODS Methods of study have been described elsewhere (19). In brief, saccular, lagenal, utricular and ampullar maculas were taken out from 10 - 15 cm long goldfish, and the hair cells were enzymatically isolated (8 unit/ml papain, Sigma p-3125, in calcium deficient saline). Recordings from these cells were made using the tight-seal whole-cell patch technique with either voltage recording under current clamp or current recording under voltage clamp (14). The recording chamber containing the hair cells was mounted on the stage of an inverted microscope equipped with Nomarski differential interference optics
Presented at the 12th Taniguchi International Symposium on Visual Science, November 27-December 1, 1989 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
$28 (IMT-2, Olympus, Japan). q%le micropipettes for patch clamp had electrical resistance ranging from 6 to 12 megohms when filled with K-citrate solution (see below). As a patch clamp amplifier, List Model EPC-7 (West Germany) was usecL To find the approximate values of cell capacitance and series resistance, dial positions of the slow transient cancellation circuitry (C-slow and G-series) were read, under voltage clamp setting, each time a successful whole-cell recording was made. Cell capacitance values ranging from 20 to 40 Mohm were observeci
In voltage clamp experiments, slowing down of response speed due to
the series resistance was compensated for as far as possible (usually by 40 80 %). A recording was continued typically for 5 - 25 miru The cell was ruptured in voltage-clamp, by the use of the initial holding potential of -70 mV. However, responses were observed first under current-clamp condition, then under voltage-clamp, and again with curent-clamp to observe whether or not the condition of the cell would remain unchangecL The input signal was filtered at 10 kHz within EPC-7 and stored in a PCM data recorder (NF Electric Instruments, model No. PR-880, Japan) with a sampling interval of 17.4 ~s. The playback data were filtered at I0 kHz before being fed into the AD converter of a computer (Nippon Data General, model No. N10). Data were sampled at an interval of 0.1 ms for voltage-clamp records, and at 0.5, I, or 2 ms for current-clamp and were stored on a dis~ Solutions
The normal saline used as the external bath contained (mM): 120
NaCI, 2 KCl, 2 CaCI2, and 5 HEPES. The pH was adjusted to 7.2 with about 1.5 mM NaOH. The recording chamber was continuously superfused at about 2 ml/min with normal saline using a peristaltic pump. This superfusion system was also used to replace the medium with Ca2+-free solutions, to which 2 mM Co 2+ and/or Mg 2+ was addefL To apply drugs, such as tetraethylammonium ions (TEA) or 4aminopyridine (4-AP), local superfusion was made by gently blowing solutions out of thin polyethylene tube (tip diameter: 100 ~un) placed near the cell being examinecL A pipette solution of the following composition (mM) was used in most of the present study: 53 K-citrate, I MgCI2, 10 HEPES, 5 EGTA, pH adjusted to 7.2 with about 20 m M KOH. The citrate solution had a total K + concentration of 179 mM, and its EK was -I 1 3 mY against 2 mM of external K +. RESULTS Oscillatory- and spike-type responses General properties of the oscillatory- and spike-type responses in hair cells from the sacculus, lagena or utriculus have already been described (see Introduction and ref. 19). Fig. 1 compares the typical damped oscillatory
$29 responses of a short, "oscillatory-type" hair cell (A) and the typical all-ornone spike responses of long, "spike-type" hair cells (B & C). These responses were elicited by injecting current in the current-clamp mode. Beside marked differences in voltage responses to depolarizing or hyperpolarizing current pulses, the resting membrane potential also differed (-74 mV in A; -I 02 mV in B; and -93 mV in C). Oscillatory-type hair cells had a resting potential less negative than the value for spike-type hair cells (-76+--8.9mV, n=24, and -101+--7.4 mV, n--42, respectively when measured with K-citrate in the pipette). In the following paragraphs, we describe the ionic currents based on our data obtained from 43 oscillatory-type hair cells, of which 27 were from the sacculus, 9 from the lagena and 7 from the utriculus. Hair cells from these maculas could be classified into oscillatory- and spike-types, based on morphology and functional grounds. (However, the spike type cells are not considered any further.) The ampullar hair cells were different; they
A 200 pA
100 pA
4o~
30 pA - 1 0 pA
- 2 0 pA
-
-
-
~
~ ~
C
20 mV I 1 0 50pAl
L20 ms
ms
lOpA,
Fig. I. Typical voltage responses in 2 types of hair cells evoked by current injectioru A: oscillatory-type responses obtained in a short hair cell from rostral saccule (averages of 12-20 traces). Resonance frequency about 230 Hz. Traces were shifted vertically to avoid crossing of curves. B and C: spikeplateau-type responses obtained in long slender hair cells from caudal saccule. In C, injection of the same intensity of current (5 pA) either set up a spike response or failecL Broken lines indicate -70 mV in all records. Pipette contained K-citrate solution in A and B, but KCI in C_
$30 responded mostly as oscillatory cells in spite of the long stretched shape of their cell body. Thus, some data from the ampullar hair cells are included for comparison. Three outwardly rectifying K + channels in oscillatory-type hair cells In hair cells in which an electrical tuning is markedly developed, such as those from the frog sacculus (9, 13), and from the turtle (I) and chicken (6, 15) cochlea, a Ca2+-activated K + current (IK(Ca)) constitutes the predominant pathway for the outwardly rectifying current. However, this was not the case in the oscillatory-type hair cells of goldfish ear. Fig. 2 shows a case in which the contribution of Ca2+-activated K + channels was examined by superfusing the cell with a saline which contained 20 mM TEA, a blocker of a variety of potassium channels (17). Before the application of
control
-70mV5
-30mY L
20ram ~" 12o~.
'
b0,,
-50 I:A
~! l ~ l m B
;
IA
[
-70 m V ~ '
,
-30 mV
[ _ _
(low threshold)
,
-110
Fig. 2. Demonstration of 3 components of outwardly rectifying current in an oscillatory-type hair cell from saccule. A and B: current-clamp records obtained in control state and during superfusion with 20 mM T E ~ C and D: voltage-clamp records. In C, records during control and during superfusion with TEA were superimposed (a). Current during superfusion with TEA seems mostly composed of A-current (IA, especially its high threshold subclass). Subtracted record (b) shows the time course of IK[Ca }. In De, a hyperpolarizing pre-pulse gave rise to a fast rising" low-threshold A-current whose time course is shown in subtracted record (b). Obtained during superfusion with T E ~
$31
TEA, this hair cell generated, in response to injection of depolarizing pulses, voltage ringing consisting of ah3ut three waves (A). During the superfusion with 10 m M TEA, the response to depolarizing current increased in amplitude, but became much simpler in shape (B). Tne response then consisted of a peak and a following volley. A similar change was produced by superfusing with a saline in which Ca 2+ was replaced with either Co 2+ or M ~ + (see Fig. 5A of ref. 19). These findings support the idea that the Ca2+-activated K + channels could be important for producing ringing of the membrane potential. Fig. 2 Ca shows the result of a voltage-clamp experiment in which the membrane potential was shifted from the holding potential of -70 mV to -30 mV, then back to -70 inV. The outward current was reduced during the superfusion with TEA to about 3/5 of the original amplitude. ~ne component that was reduced by TEA (Fig. 2 (~) would represent the current carried by the Ca 2+activated K + channel (I, 12, 13). The nature of the remaining current cannot readily be specified. However, as judged from marked effects of 4-AP on outward rectification (see Figs. 3-5), it could represent an A-current, possibly, of the high-threshold subclass (see Discussion). In Fig. 2 De, a hyperpolarizing pre-pulse was applied to remove inactivation of the lowthreshold A-current (7, 9, 13). During the pre-pulse, the membrane potential was kept for I sec at -110 mV. The resultant increase in the outward current could represent the component of low-threshold subclass of A-current (Fig. 2 Db). Although the pre-pulse test was carried out under the action of TEA in Fig. 2D, a similar test carried out in the normal saline gave essentially the same magnitude of the low-threshold A-current (not shown). Superfusion with TEA or Ca2+-free saline was made in a total of 7 cases. Ratios of maximal conductance attributed to the Ca2+-activated K + current to that attributed to the high-threshold A-current ranged from 0.15 to 1.4 (mean: 0.6, n--7). Effects of 10 m M 4-aminopyridiD~ on oscillatory-type hair cells Fig. 3 shows an instance in which the effects of superfusion with 10 mM 4AP, a block~.r of the A-current (13, 20), were studied in the current-clamp mode. As shown in A and Ba, the outward rectification was very markedly observed in this cell. During the superfusion with 10 m M 4-AP, the membrane potential was depolarized by about 20 mV and the outward rectification was greatly reduced (Fig. 3 Bb). Hence, injection of small depolarizing current produced a large potential change. Also, the rate of rise of potential became much slower and ringing of the membrane voltage was no longer observecL The after-hyperpolarization, which was very markedly observed in the control state, was also not obse/ved (Fig. 3 Bb). Effects of 4-AP appeared soon after
$32 the start of superfusion, and was nearly complete within one miru
The washout
also proceeded promptly. Fig. 4 shows the results of voltage clamp experiments on the same hair cell as used in Fig. 3. The current-voltage diagram in Fig. 4A shows the effect of 10 m M 4-AP more clearly than in the current clamp experiment. Outward currents produced in this cell in response to depolarizing voltage steps were roughly recta~
in shape, showing no initial peak or rapid decline. Also, no trace
of the low-threshold subclass of the A-current was detected in a pre-pulse test (leftmost traces of Fig. 4 Ba), thereby indicating that the outwardly rectifiying current in this cell consisted mainly of the high-threshold subclass of the A-current.
A
B
a
150 pA
___
o control
150
. . . . . .
-,
. . . .
-10
I
o
I
mm
o
100
-30 pA
I
o
I I
o
.50
o
4-AP
I
-180 -140 -i00 o
J ~-Y=,
i
"b"'°• 1oi-80 -40
0
I ,
,,,
I
o
-50
-30 ~z~
Fig. 3. Effects of superfusion with I 0 mM 4-AP on voltage responses of an oscillatory-type hair cell from the saccule. A: current-voltage relaticn at the end of voltage pulses (based on current-clamp data shown in B). B: current-clamp records during control state (a) and during superfusion with 10 m M 4-AP (b). The trace for 30 pA of hyperpolarizing current in a was distorted because of saturation of amplifier.
$33 Inactivation of the A-current was studied by applying pre-pulses in the depolarizing directioru For example, in the rightmest tracas of Fig. 4 Ba, a test-pulse to -30 mV was applied after the membrane potential had been kept for I sec at -50 mV. Because of inactivation, the amount of current flowing at the end of pre-pulse and during the test-pulse became smaller as compared with those in the control records shown in the inset of Fig. 4~
In Fig. 4 Bb, the
activation observed at the end of pre-pulse (filled circles) and during the test-pulse (open circles) became smaller in about the same amount. Although the inactivation reached about 50 % at the maximum in this case, it may be feasible to obtain higher percentages of inactivation by extending the prepulse duratioru
A
B
I
+1o 0
" " - - ' " ~ ~-
ISO0 pA
2000
L
1800
•
5 m@
-i0
°i
_~o
/
-30
/0
-EO mY
4-
/v / trol
7
.~
10 Nvl 4...~
-150 -130-110-90 -70 -50 -30 -10 +10
mV
a
1600 1400
_ . C - ~ s o J - - - L _ 7 0 my
-9O __
-110 - 1200 %
1000
100
8OO
8O
6oo
b 6o
4OO
4O
2OO
20
0
0
O--O~O~
\ i
|
|
|
•
-II0 -90 -70 -50 -30 mV
(1 see)
Fig. 4. Voltage-clamp experiments on the same hair call as in Fig. 3. A: current-voltage curves ugr~r control state and during superfusion with 10 mM 4-AP. Inset, voltage-clamp records under control stat~ B: results of prepulse tests, a: sample records obtained by applying various hyper- and depolarizing pre-pulses. Pre-pulses had a duration of 1 sec, but only their terminal portions are showru Test-pulses were fixed at -30 mV. In b, membrane currents, at end of pre-pulses (filled circles) and during test-pulses (open circles), were plotted against pre-pulse level on a percentage basis.
$34 Superfusion with 10 m M 4-AP was successfully made in a total of 5 cases of the oscillatory-type hair cells. Although, in three of them, the observation was limited only to the current-clamp experiment, the effect of 4-AP was very marked and
comparable to that shown in Figs. 3-5. These results might cast
doubt on the specificity of action of 4-AP, although 4-AP in many cases does not interfere with the Ca2+-activated K + channel activity (9, 10, 13, 20). This problem needs further elucidation (17). Enhancement of response brought about by inactivation of high-threshold Acurrent Effects produced by inactivation of the A-current were studied under a current-clamp condition in Fig. 5.
Responses of this cell under the control
condition are shown in A and C. In B, a steady current at 10 pA was injected and its effect was tested by applying 80 pA pulses (duration, 60 msec) once every 2 sec. Injection of the weak steady current depolarized the cell by 10-I 5 mV from its resting level. A more marked effect appeared in the potential response to superimposed test pulses.
A
30 sec progressing inactivation
B
control
The response to a test
C
10
control 120
~ , . . . ~ 0.I
pA
sec
] ~
_.;
[
I
_
I - 7 0 mV
......
-40 pA
~f . inactivation
~u m s
.................. ~\_~
~20 mV
l~OmV
~ms 1o~-J
.o.
C'I
' !
,~" 4-AP
8O
:::1"::;/"
,li,,,s, aJt iiJ,,, t , s
,:,,,,.:..~.
60 4O
a L~'~,;. ,,[{'~',~/
_12o-
I00
•
20
oo
'
"~/d
'.f----~'"~' °"
'
4o 20 o 2o 4o
mV
0 -20 -4O
control
Fig. 5. Augmentation in response amplitude produced by inactivation of Acurrent. Current-clamp records from a saccular hair cell. A: control responses. In B, injection of steady depolarizing current at I0 pA brought about successive augmentation in the response amplitude with time following the start of steady current injectioru C: current-voltage relations at the end of test pulses (based on current-clamp data shown in A and B).
$35 pulse increased progressively with time after the start of steady current injecticr~ ~nis result is shown schematically in Fig. 5C. Clearly, augmentation of the response amplitude was produced by a reduction in the outward rectification because of inactivation of the A-current. This enhancement of response was quite markecL It represents an opposite behavior to the adaptaticru A similar enhancement attributable to inactivation of potassium channel has been reported in hippocampal neurones (18). In voltage-clamp measurements on this hair cell, outward current in response to a depolarizing pulse rose as sharply as that in the cell shown in Fig. 4~ A low-threshold A-current was not detected at all in the pre-pulse test. The outward rectification was completely abolished with I0 mM 4-AP in this case too. Variability of the low-threshold A-current The magnitude of the low-threshold subclass of the A-current, such as shown in Fig. 2D, varied from case to case among different oscillatory-type hair cells. In certain hair cells (Figs. 3 - 5), the A-current consisted almost exclusively of its high-threshold subclass. In such instances, a
A 30pA .
.
.
.
.
.
.
.
.
.
.
.
j o mv
115 mV ---..~/c13
C / o 2O o/° _T~ O ~ i , -I00 -80 -60 -40 -20 o-
S L
~[__~ ~
-°~ mV
-70 . I V - ~ -lO0 mV
-30 mV
0 -20
[__
Fig. 6. Response of an ampullar hair cell. A: current clamp records. B: large low-threshold A-current evidenced by applying hyperpolarizing prepulse. C: current-voltage relation at the end of current pulses (based on current clamp data). Some ampullar hair cells, not all of them though, had a large negative resting membrane potential and responded to injected currents as shown in this figure.
$36 hyperpolarizing pre-pulse contributed nothing to the effect of the test pulse (see Fig. 4 Ba). On the contrary, the contribution of the hyperpolarizing pre-pulse was very marked in certain other hair cells. In such cases, a test pulse (e.g., -30 mV) gave rise to a very large outward current which steeply rose to the peak and decline thereafter with a time constant of 20 msec if it was preceded by a hyperpolarizing pre-pulse. Without such a pre-pulse, the same test pulse produced only a small slowly rising outward current (Fig. 6B). In a pre-pulse test of 18 oscillatory-type hair cells from the sacculus, lagena and utriculus, the low-threshold A-current was predominant in only two cases. On the other ham~, the low-threshold-A type of respcnse seems to be very common among
ampullar hair cells (Fig. 6B), in agreement with a report
on the frog ampullar hair cells (7). The cell shown in Fig. 6 had a large negative resting potential and responded with a slow time course to injected depolarizing current. Note that the outward rectification indicated in the current-voltage curve shown in Fig. 6C seems to arise from the low-threshold subclass of A-current. Certain ampullar hair cells we encountered had a resting potential around -70 mV
aD~ responded to depolarizing current like
the cell shown in Fig. 5 ~ Unlike the cell of Fig. 5, however, these ampullar cells elicited, under voltage-clamp setting, a flow of large low threshold Acurrent in response to a depolarizing pulse when the latter was preceded by hyper-polarization.
DISCI/SSION Damped membrane potential oscillations in the goldfish oscillatory-type hair cells, elicited by injection of depolarizing current, are of a much poorer quality than those reported in frog or turtle hair cells (19). This difference may be due to the different ionic channels used by these hair cells for outward rectificatioru Whereas the outward rectification arise almost exclusively from activation of IK(Ca ) in frog or turtle hair cells, it was served in goldfish oscillatory-type hair cells by mixed activation of IK(Ca ) and IA, especially of its high-threshold sublass. M o r ~ ,
contribution of
IK(Ca ) and high-threshold IA differed greatly among different hair cells. Perhaps, outward rectification in goldfish oscillatory-type hair cells functions mostly to make the cell's time constant short and to enable the cell to follow each sound wave up to high frequencies such as 500 - 1,000 Hz (see Fig. 12 of ref. 1 9 for the mode of operation of goldfish hair cells). A similar interpretation has been offered regarding the outward rectification in the inner hair cells of mammalian cochlea (12). Furthermore, the large variablity of ionic channels in the oscillatory-type hair cells contributing
S37 to the outwardly rectifying action might be responsible for different response speed observed among different hair cells. About the low- and hiqh-threshold subclasses of ~-current Originally, the A-current is a fast, transient K current which is activated by depolarizing steps from holding potentials negative to the resting potential. The low-threshold subclass of A-current roughly meets the above definition (see Figs. 2D, 4 Ba and 6B), whereas the high-threshold subclass considerably departs from it. We included the latter in the A-current as a special subclass because
(I) it was susceptible to blockade by 4-AP, (2) it
showed a voltage-dependent inactivation, though of a slower time constant, and (3) a similar non-typical A-current has been reported in which voltagedependent activation and inactivation takes place at more depolarized levels (17). The functional role played by the low-threshold subclass of A-current in hair cells remains unclear. %'nis current is almost completely inactivated at membrane potentials close to -70 mV. It remains not inactivated in cells whose resting potential was much more negative (see Fig. 6). Furthermore, the lowthreshold A-current is much better developed in the ampullar hair cells than in hair cells from the sacculus, lagena and utriculus. On the other hand, as mentioned above, the high-threshold subclass of Acurrent, together with IK(Ca), plays an important role in characterizing the response of oscillatory-type hair cells. The high-threshold A-current, in certain cases of cell, produced a powerful outwardly rectifying action. It operated at a very high speed, and its inactivation brought about enhancement of response (see Fig. 4). About the resting membrane potential Besides differences in the ionic channels, the resting membrane potential is an important o~ntributing factor in bringing about differences in the response between the oscillatory- and spike-type hair cells (Fig. I ). In the oscillatory-type hair cells, the resting potential was mostly set around -70 mV, while it was generally more negative in the spike-type hair cells. However, some oscillatory-type hair cells had more negative resting potentials, i.e., near -100 mV. In such cases, injection of small depolarizing current produced a much larger depolarization than was usually the case with oscillatory-type hair cells with resting potential near -70 mV (Fig. 6). Without doubt, such an observation is related to the presence of a low slope oonductance region in the I-V curve (see control trace of Fig. 5C) inserted between the two inflection points attributable to the outward and inward rectification channels (2).
$38 The mechanism of how the resting potential of each hair cell is determined is unknown (11). However, the presence of two rectification channels should influence the level of resting potential. When a tendency for a depolarization is dominant in a given hair cell, the resting potential of that cell should settle around the inflection point produced by the outward rectification, as any further depolarization is preventecL On the other hand, the resting membrane potential should settle at a more negative level if the tendency of the cell is for a hyperpolarizatioru A~q~3wI~X~ITS This work was supported by grants from The Ministry of Education, Science, and Culture, Japan.
R ~ ~
J. Physiol. 400:237-274
10. Hudspeth AJ, Lewis RS (1988)
J. Physiol. 400:275-297
11. Jones SW (1989) Neuron 3:153-161 12. Kros CJ, Crawford AC (I989) In: Wilson JP, Kemp DT (eds.) Cochlear Mechanisms, Plenum, New York, pp 189-I 95 13. Lewis RS, Hudspeth AJ (1983)
Nature 304:538-541
14. Marty A, Neher E (1983) In: Sakmann B, Neher E (eds) Single-Channel Recording, Plen~m, New York, pp 107-122. 15. Ohmori H (1984) J. Physiol. 350:561-581 16. Pichford S, Ashmore JF (1987) Hearing Res. 27:75-83 17. Rudy B (1988) Neuroscience 25:729-749 18. Storm JF (1988) Nature 336:379-381 19. Sugihara I, Furukawa T (1989) J. Neurophysiol. 62:1330-1343 20. Thompson SH (1977) J. Physiol. 265:465-488