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Journal of Physiology (1990), 430, pp. 585-594 With 5 figure8 Printed in Great Britain

PERILYMPHATIC POTASSIUM CHANGES AND POTASSIUM HOMEOSTASIS IN ISOLATED SEMICIRCULAR CANALS OF THE FROG BY P. VALLI, G. ZUCCA AND L. BOTTA From the Institute of General Physiology, University of Pavia, Via Forlanini 6, I-27100 Pavia, Italy

(Received 13 November 1989) SUMMARY

1. Endolymphatic and perilymphatic potassium concentrations were measured with K+-sensitive microelectrodes in isolated semicircular canals of the frog. K+ levels were evaluated both at rest and during sinusoidal stimulation (0-05 Hz) of the sensory organ. 2. Mechanical stimulation of hair cells was associated with sinusoidal changes (about 0-2 mM) in the perilymphatic K+ concentration. 3. Perilymphatic K+-fluctuations were modified neither by impairment of the synaptic transmission at cyto-neural junctions nor by chronic denervation of the crista ampullaris, thus indicating that K+ ions were actually released by hair cells. 4. Voltage-clamp experiments of the whole sensory organ showed that K+ flows across the crista ampullaris can vary from 3 x 1011 molecules of K+ s-5 at rest up to about 15 x 1011 molecules of K+ s-1 during mechanical stimuli. 5. Measurement of intra-ampullar K+ concentration demonstrated that the amount of K+ transported from the perilymph towards the endolymph can be rapidly altered by modifying its perilymphatic levels. This suggests that vestibular organs are endowed with K+ homeostatic mechanisms able to buffer in a very efficient way the concentration of K+ in both the fluids bathing the crista ampullaris. 6. The possible role of K+ homeostatic mechanisms in hair cell adaptation is discussed. INTRODUCTION

An increasing volume of literature has accumulated during recent years about the central role played by K+ in the functioning of hair cells belonging to the acousticolateralis system. The apical membrane of hair cells faces a K+-rich medium whereas their baso-lateral membranes face an extracellular fluid having a low K+ content (Ilyinsky & Krasnikova, 1971; Russell & Sellick, 1976; Valli & Zucca, 1977; Okitsu, Umekita & Obara, 1978; Bernard, Ferrary & Sterkers, 1986; Zucca & Valli, 1986; Valli, Zucca, Botta & Casella, 1988). It is widely believed that this concentration gradient of K+, often associated with a driving voltage across the sensory epithelium, gives rise to a flow of K+ ions throughout hair cell bodies (the receptor current), which sustains the whole conversion process in inner ear end-organs (Davis, 1965; MS 8070

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Corey & Hudspeth, 1979; Zucca, Valli & Casella, 1982; Russell, 1983; Akoev, Andrianov & Sherman, 1984; Ohmori, 1985). The present study, carried out on isolated semicircular canals of the frog, has two main goals: the first is to substantiate the hypothesis that K+ is the main vestibular current carrier; the second is to investigate more closely the K+ homeostatic mechanisms which guarantee the constancy of the concentration gradient of K+ between the endolymphatic and perilymphatic faces of hair cells (Nakai & Hilding, 1968; Kimura, 1969; Oudar, Ferrary & Feldmann, 1988). METHODS

Experiments were performed on vertical posterior semicircular canals isolated from frogs (Rana e8culenta L.) previously anaesthetized by immersion in 0 1 % MS-222 solution. Isolated preparations were usually placed in a two-compartment chamber (Fig. 1) which made it possible to maintain the endolymphatic and the perilymphatic canal sides in contact with two ionically and electrically separated fluids (Valli & Zucca, 1977). The chambers (capacity 5 ml each) were perfused with, respectively, artificial endolymph (in mM); NaCl, 19-5; KCI, 100; NaHCO3, 1-2; NaH2PO4, 0417; CaCl2, 1P8; glucose, 5-5; pH 7*3 and perilymph (in mM): NaCl, 117; KC1, 2-5; NaHCO3, 1-2; NaH2PO4, 0-17; CaCl2, 1-8; glucose, 5.5; pH 7-3. In one group of experiments (Fig. 5), however, canals were mounted in a single bath (capacity 20 ml) filled with perilymphatic fluid (single-bath experiments).

Manipulation8 of the perilymphatic fluid In different experiments the perilymphatic fluid was replaced with one of the following media: (1) low-Ca2+-high-Mg2+ solutions (n = 5). Ca2+ concentration was reduced to 0-1 mm and Mg2+ concentration elevated to 15 mM; (2) solutions with TTX 0-1 ,ug ml-' added (n = 5); (3) solutions with ouabain 10-4 M added (n = 7). In single-bath experiments (n = 15) local K+ changes at the ampullar surface were produced by injecting towards the ampulla streams of perilymphatic solution (20 j1 min-' for 2 min) with a modified K+ content (0-5 mM; Valli, Zucca, Prigioni, Botta, Casella & Guth, 1985). Osmotic pressure was kept constant by varying the NaCl content. Control experiments (Fig. 5B) demonstrated that no artifacts were introduced solely by the method of application. Mechanical 8timulation of the sensory organ Hair cell stimulation was performed by producing sinusoidal flows (0 05 Hz) inside the canal by means of a microsyringe whose plunger (diameter 0-5 mm; displacements + 5 ,sm) was operated by a servo-controlled stepper motor (Valli & Zucca, 1976).

Recording of canal activity Compound nerve potentials (slow potentials, due to electronic spreading of EPSPs in afferent fibres (Rossi, Valli & Casella, 1977) and spike discharge) were recorded from the whole nerve by means of a fluid electrode. When necessary, action potential frequency was measured using a window discriminator and a frequency-to-voltage converter. Ampullar potentials, which reflect receptor potential in hair cells (Valli & Zucca, 1976), were recorded by means of electrodes placed in the endolymphatic and perilymphatic compartments. In nature, ampullar potentials become negative during excitatory cupula deflections and positive during inhibitory ones. For ease of comparison between tracings, ampullar potentials are reproduced with inverted polarities, except in Fig. 4.

Voltage-clamp experiments The potential between the inside and the outside of the ampulla was measured by means of electrodes positioned one within the ampulla as close as possible to the crista ampullaris, the other in the perilymphatic fluid at about 100 ,um from its external surface. A second pair of electrodes was used to pass current to clamp the transepithelial potential (Fig. 3). These experiments confirmed previous findings (Schmidt & Fernandez, 1962; Enger, 1964; Corey & Hudspeth, 1983;

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Zucca & Valli, 1986; Riisch & Thurm, 1989) that the potential between the inside and the outside of the crista ampullaris is close to 0 mV (0-4+0-12 mV; n = 10). Therefore in the present study the transepithelial potential was always clamped at 0 mV.

Measurement of K+ concentration in the media K+ concentrations were evaluated by means of K+-sensitive electrodes positioned either close to the external surface of the ampulla (Fig. 1) or just inside the ampulla (Fig. 5). Preparation of K+sensitive electrodes was similar to that described by Neher & Lux (1973). Glass capillaries (Corning 7740) were pulled to form microelectrodes with tip diameters of about 25 ,um. The last 400-500 Wum of the electrode tip was coated with 4% (v/v) dichlorodimethylsilane in carbon tetrachloride. Electrodes were filled with a 0-1 M-KCl solution and their tips immersed in the K+ ion exchange resin (WPI, IE-190) which, by capillarity, entered the salinized tract. Each K+-sensitive electrode was calibrated before and at the end of each experiment by measuring its voltage output in physiological solutions with a known K+ content (0 1, 1, 10, 100 mM). If calibration values differed by more than 10% the experiment was discarded (this occurred three times in sixty-one experiments). The main characteristics of K+-sensitive electrodes may be summarized as follows: tip diameter, 23 ± 1-7 jum; resistance, 167 ± 22 MQ; slope, 55 ± 1-8 mV/decade; selectivity K+/Na+, 92+6-4; rise time,1 1 +0 16 s (n = 25). A conventional microelectrode, filled with the same solution present in the bath and positioned close to the K+-sensitive electrode, was used as reference electrode (Figs 1 and 5). To obtain signals related only to K+ activity, the potentials from the reference electrode and from the K+-sensitive electrode were differentially amplified, to eliminate common-mode field potentials. (K+-sensitive electrodes, owing to their rise time, are ill-suited to revealing rapid stimulus-related K+ changes and therefore a low stimulation frequency (0 05 Hz) of hair cells was adopted.)

Recordings Signals were sampled digitally using an analog-to-digital converter coupled to a personal computer, analysed and plotted on paper. In some experiments, owing to the length of observation periods, responses were recorded on a chart recorder (Figs 4 and 5). Denervated canal preparations Denervated canals (n = 5) were obtained by cutting the vertical posterior nerve close to the ampulla, in MS-222 anaesthetized frogs. Two weeks afterwards, the animals were killed and the canals mounted in the usual two-compartment chamber. RESULTS

Preliminary remarks Perilymphatic K+ changes were evident only if the K+-sensitive electrode was positioned close (about 50 ,um) to the region where the ampullar nerve enters the ampulla and decreased rapidly when the electrode was pulled back (at about 150-200 /sm from the ampulla no changes were detected), or when it was moved laterally along the ampullar surface. It was also observed that the K+ content in the fluid bathing this area was slightly higher (0-02-0-03 mM) than in that bathing the remaining parts of the canal. This slight increase in K+ concentration was in fact normally utilized as an index of good positioning of the K+-sensitive electrode in the zone where the highest perilymphatic K+ changes take place during stimulation. Control experiments (n = 5) demonstrated that K+ changes were related to the sensory organ's activity. In fact K+ fluctuations disappeared in about 15 min after poisoning of the preparation with 2,4-dinitrophenol (5 mM). The fact that the fluid in contact with the outer side of the crista ampullaris is slightly enriched in K+ indicates that there is an outflow of K+ from the sensory organ. This K+ outflow can be modulated by sinusoidal cupula deflections (Fig. 1).

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During excitatory half-periods, K+-sensitive electrodes revealed positive shifts indicating an increase in K+ content (0415-0-20 mM; 0 16 ± 002 mM; n = 35) whereas changes of opposite polarities were observed during inhibitory half-periods. The decrease in K+ content below its resting level may be better observed when, instead

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of the usual excitatory half-period (Fig. IB), the sinusoidal stimulation starts with an inhibitory half-period (Fig. 1 C). From Fig. 1 it may also be noted that K+ tracings are clearly 'phase-delayed' (5-6 s) in comparison with both canal responses and stimulation, thus indicating that a diffusion process is involved. To verify whether K+ changes at the ampullar surface were almost exclusively related to hair cell activity, the activation of postsynaptic nerve elements was progressively impaired (Fig. 2). It may be noted that neither TTX-induced spike suppression (Fig. 2 A2), nor synaptic blockade, obtained by replacing the perilymphatic fluid with a low-Ca2+ - high-Mg2+ solution (Fig. 2 B2), nor chronic denervation of the sensory organ (Fig. 2 C) prevent nearly normal perilymphatic K+ changes from taking place during stimulation. The close correlation between ampullar potentials and perilymphatic K+ changes indicates that K+ changes are chiefly due to modulation of the receptor current. To obtain a direct measure of this current, voltage-clamp experiments were performed (Fig. 3). These experiments showed that the peak-to-peak intensity of the receptor current was of about 300 nA (271 +19 nA; n = 10). Assuming that the receptor current goes close to zero during inhibitory cupula deflections, it can be inferred from current-tracings that the intensity of the resting receptor current was of about 50 nA (52 + 3.4 nA; n = 10). Voltage-clamp experiments also confirmed that in the absence of K+ from the endolymphatic fluid (replaced either by Na+ or by different mixtures of Ca2+ and Na+), a condition which leads to the suppression of any canal activity

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(Valli, Zucca & Casella, 1979; Zucca et al. 1982) both the receptor current and the perilymphatic K+ fluctuations were completely suppressed. The effects of the abovementioned solutions were almost completely irreversible even after protracted washing of the preparation with normal solutions. This might indicate that high concentrations of Na+ and Ca2+ in the endolymph cause permanent damage of the hair cells, thereby blocking transduction. These experiments, which only confirm results already reported, are not presented in detail here, for the sake of brevity. The results presented above demonstrate that sensory activity in vestibular organs is associated with intense outflows of K+ from the basal pole of hair cells. This observation supports the concept that a very efficient K+ homeostatic mechanism,

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mainly based on the Na+-K+-ATPase (see Sterkers, Ferrary & Amiel, (1988) for a review), operates in semicircular canals. To demonstrate the importance of this process, the Na+-K+-ATPase was blocked by means of ouabain (Fig. 4). It may be noted that the drug produced a rather complex effect on both canal and K+ tracings: (i) the potential across the crista ampullaris rapidly decreased, settling, in about 5 min, at a new level lower than resting level by about 2 mV (1 7+0-37 mV; n = 7); (ii) a marked spike discharge, from 142 + 36 to 537 + 44 impulses s-' (n = 7), associated with a pronounced nerve depolarization (0-58 + 0-08 mV; n = 7) ensued in afferent fibres; (iii) a considerable increase in K+ concentration (0-5-48 mM; 0-68 + 0-16 mM; n = 7) took place in the perilymphatic medium. Ouabain-induced K+ changes remained constant for 10-15 min and then slowly decreased. These experiments confirmed that K+ is released almost exclusively from the baso-lateral membranes of the hair cells. In fact, only in the fluid bathing the crista ampullaris was it possible to observe a medium enriched in K+ after ouabain treatment. The marked rise in perilymphatic K+ concentration observed after administration of ouabain suggests that, even at rest, the amount of K+ crossing the crista ampullaris is very high and that, in normal conditions, nearly all K+ ions are caught by the Na+-K+-ATPase and pumped back into the endolymphatic space. This K+ transport has been evidenced in single-bath experiments (Fig. 5). It can be noted that, at the beginning of the experiment, the endolymphatic K+ content was close to its physiological concentration (about 100 mM) and then exponentially decreased until, in about 15-20 min, it reached steady-state conditions (12-15 mM; 12-9 + 0-8 mM; n = 15). Graphs in Fig. 5 show that, after the equilibration period, injection of normal perilymphatic solutions (K+, 2-5 mM; Fig. 5B) produced almost no change in intra-ampullar K+ level whereas injection of K+-enriched solutions (K+, 5 mM; Fig. 5C) produced an increase in K+ concentration (from 13-4+ 1-1 mm to 19-4 + 1-2 mM; n = 5); K+-free solutions (Fig. 5D) had an opposite effect (from 12-7 + 0-4 mm to 8-6 + 0-5 mm; n = 5). K+-induced endolymphatic K+ changes disappeared in about 5 min. Graphs (C) and (D) also show that, after ouabain administration, the concentration gradient of K+ vanished within 10-15 min. Thereafter the injection of K+-modified solutions had almost no effect. K+ concentration values depended on the position of the electrodes. In fact when the electrodes were advanced towards the crista ampullaris, higher K+ levels, but dispersed throughout the range 28-64 mm, were observed. The increase in K+ concentration was constantly associated with a parallel increase (0-2-0-5 mV) in the intra-ampullar potential. DISCUSSION

The present experiments clearly demonstrate that mechanical stimulation of canal organs is associated with fluctuations of K+ in the perilymphatic fluid which roughly parallel the time course of the receptor current flowing across the crista ampullaris. Perilymphatic K+ fluctuations were not appreciably modified either by impairment of postsynaptic nerve elements or after degeneration of nerve fibres innervating the sensory organ, but they disappeared following ion manipulations that led to the suppression of the receptor current. It is therefore reasonable to conclude that

P. VALLI, C. ZUCCA AND L. BOTTA perilymphatic K+ changes are almost exclusively related to hair cell activity. This group of experiments therefore provides direct evidence that K+ is the main carrier of the receptor current in vestibular hair cells. Voltage-clamp experiments allowed a rough estimate of the amount of K+ crossing the crista ampullaris. In fact, if the receptor current is carried almost exclusively by K+, it can be calculated that about 3 x 1011 molecules of K+ s-1 are needed to carry the resting receptor current (intensity about 5 x 10-8 coulomb s-1), an amount that may vary from zero up to 12 x 101l-15 x 1011 molecules of K+ s-1 during mechanical stimulation of the sensory organ. Because of such an intense K+ outflow at least two problems must be solved concomitantly by vestibular end-organs. The first is the necessity to prevent perilymphatic K+ accumulation. The second is that, to maintain the correct K+ gradient at both ends of hair cells the endolymphatic fluid must rapidly regain an amount of K+ equivalent to that lost during receptor current flow. Dark cells, responsible for the transport of K+ from the perilymph towards the endolymph (Sterkers et al. 1988), are likely to be those most directly concerned in these K+ homeostatic processes. Hair cells should not be involved.-In fact, if K+ were carried back through hair cell bodies, a K+ current would be produced which, by flowing in the opposite direction, would tend to cancel the receptor current. Moreover a build up of K+ on the baso-lateral surfaces of hair cells might result in a reduction in the driving voltage for the receptor current and in a depolarization of the afferent nerve terminals. According to Glynn & Karlish (1975) active transport of K+ across plasmatic membranes can be stimulated by a rise, or depressed by a reduction, of the K+ content in the extracellular fluid. Consequently the amount of K+ which is transported by dark cells can vary according to its perilymphatic level. Single-bath experiments have provided convincing evidence that this process occurs in vestibular organs. In fact any variation in perilymphatic K+ concentration produces a prompt consensual change in the transport of K+ towards the endolymph. This process, therefore, may actually buffer the concentration of K+ in both the fluids bathing hair cells. In addition to dark cells, also glial, but mainly supporting cells, might be of importance in preventing perilymphatic accumulation of K+, especially in the narrow clefts surrounding hair cells. Studies on the cochlea (Johnstone, Patuzzi, Syka & Sykova, 1989), on the central nervous system (Sykova & Orkand, 1980) and on the retina (Coles & Orkand, 1983) support this idea. Glial, and possibly also supporting cells, in fact can operate as K+ stabilizers. Glial cell membranes are almost exclusively permeable to K+ and highly sensitive to extracellular K+ changes (Kettenmann, Sonnhof & Schachner, 1983). Glial and supporting cells therefore might constitute a system able to sequester K+ when its concentration in the extracellular medium increases or to release this ion in the opposite case. The vestibular K+-buffer system might also have, together with other possible mechanisms (Hudspeth, 1989), a role in hair cell adaptation to long-lasting stimuli (Lowenstein, 1955; Kalmijn, 1974; Precht, 1974; Precht, 1976; Akoev, Ilyinsky & Zadan, 1976; Valli, Caston & Zucca, 1984; Valli et al. 1988). In fact any event which produces changes in receptor current intensity will activate the K+ buffer system which, in turn, tends to restore basal K+ levels, i.e. the concentration gradient of K+ that normally sustains resting activity in hair cells. 592

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We thank Professor Vanni Taglietti and Professor Ivo Prigioni for their helpful comments on this manuscript. The work was supported by grants from the Consiglio Nazionale delle Ricerche and from the Ministero della Pubblica Istruzione. REFERENCES

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Perilymphatic potassium changes and potassium homeostasis in isolated semicircular canals of the frog.

1. Endolymphatic and perilymphatic potassium concentrations were measured with K(+)-sensitive microelectrodes in isolated semicircular canals of the f...
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