Brain Research, 607 (1993) 301-306 301 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 18674 Effects of modifications of extracellular and intracellular calcium concentrations on the bioelectrical activity of the isolated frog semicircular canal A. Aubert, C. Bernard and H. Vaudry European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, Unit,ersity of Rouen, Mont-Saint-Aignan (France) (Accepted 27 October 1992) Key words: Vestibular dysfunction; Semicircular canal; Electrophysiological recording; Calcium ionophore A23187; Calcium reduction In the inner ear, calcium appears to play a major role in different processes including mechanoelectrical transduction, adaptation during prolonged stimulation and electrical resonance. The aim of the present study was to investigate the effect of an increase of the cytosolic calcium content and a reduction of the perilymphatic concentration of calcium, on the bioelectrical activity of the isolated frog semicircular canal. Under resting conditions, the spontaneous activity of the afferent fibers and the difference of potential between the endolymphatic and perilymphatic compartments, called endolymphatic potential, were recorded. When the sensory epithelium was mechanically stimulated three additional parameters were investigated: the variations of the endolymphatic potential (ampullar direct current), the variations of the ampullar nerve potential (nerve direct current) and the frequency of the evoked afferent spikes. Increase of the intraceUular calcium concentration by administration of the calcium ionophore A23187 (3× 10 -6 M, 20 min) into the perilymphatic compartment, caused a biphasic effect on the spontaneous activity of the ampullar nerve which increased rapidly, reaching a maximum within 15 min, and then gradually declined to stabilize at 74% of the control 1 h after withdrawal of A23187. A23187 did not induce any modifications of the endolymphatic potential, the ampullar direct current or the frequency of the evoked afferent spikes. In contrast, A23187 induced a significant reduction of the nerve direct current which decreased by 31% of the control 1 h after withdrawal of the ionophore. Gradual reduction of the perilymphatic concentration of calcium (from 2 to 1 mM) induced a dose-dependent increase of the spontaneous activity of the ampullar nerve and the frequency of the evoked afferent spikes. Reduction of the perilymphatic calcium concentration from 1.6 to 1.2 mM caused a transient increase of the endolymphatic potential, while 1 mM Ca 2+ induced a decrease to 88% of the control. The nerve direct current slightly increased for calcium concentrations ranging from 1.8 to 1.4 mM and decreased in the presence of 1.2 mM CaCl 2. These data suggest that an increase of calcium into the cytosol induces an alteration of the mechanisms responsible for the spontaneous release of the afferent neurotransmitter and the electrogenic spreading of the postsynaptic potentials. In contrast, an excess of calcium does not impair the mechanisms involved in the generation of the action potentials. Our results also suggest that reduction of the perilymphatic calcium concentration may lead to modifications of the physical and electrical properties of the cell membranes of the labyrinthine epithelium and/or the ampullar afferent fibers. INTRODUCTION o f t h e hair cell, i n d u c e d by the influx of p o t a s s i u m t h r o u g h t h e t r a n s d u c t i o n channels, leads to an influx o f C a l c i u m is a polyvalent a n d u b i q u i t o u s c e l l u l a r mes- calcium, t h r o u g h v o l t a g e - s e n s i t i v e c a l c i u m c h a n n e l s lo- s e n g e r which is involved in t h e c o n t r o l o f m u l t i p l e c a t e d in t h e b a s o l a t e r a l walls of t h e hair cells. This, in func t i o n s such as h o r m o n a l secretion, r e l e a s e o f n e u r o - turn, triggers t h e r e l e a s e o f t h e a f f e r e n t n e u r o t r a n s m i t - t ra n s m i t t ers , e n z y m e activities, ionic t r a n s p o r t s an d m e m b r a n e stability 9'2°'2t. In t h e v e s t i b u l a r organ, cal- t er f r o m t h e hair cells 26. R e p o l a r i z a t i o n of the hair cell cium a p p e a r s to play a m a j o r role in the p r o cess o f su st ai n ed by t h e activation of calcium-sensitive potassium c h a n n e l s 15-17,19. T h e r e b y , any m o d i f i c a t i o n of cal- m e c h a n o e l e c t r i c a l t r a n s d u c t i o n 7. T h e electrical resonance, wh i ch a c c o u n t s for t h e f r e q u e n c y tunning, is also a c a l c i u m - d e p e n d e n t p r o c e s s 1,15A7. D e p o l a r i z a t i o n during resonance or af t er a positive st i m u la t i on is c i u m h o m e o s t a s i s may cau se i m p o r t a n t c h a n g e s in t he f u n ct i o n s o f the v e s t i b u l a r apparatus. I s c h e m i a strokes Correspondence: H. Vaudry, European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, 76134 Mont-Saint-Aignan, France. Fax: (33) (35) 14 69 46. 302 w h i c h i n d u c e an i n c r e a s e o f t h e c y t o s o l i c c o n c e n t r a t i o n o f c a l c i u m 5"25, c o u l d b e t h e o n s e t o f v e s t i b u l a r d y s f u n c tions. B u r n i e r et al. 5 s u g g e s t e d t h a t this i n c r e a s e m a i n l y results from mobilization of intracellular calcium. Concurrently, during cerebral ischemia, a decrease of the MS +10 1 ('m) 0 -10 I t f e x t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n o c c u r s 3A1'23, w h i c h c a n b e a s c r i b e d e i t h e r to t h e l e a k o f m e t a b o l i t e s c a u s e d by t h e r e d u c t i o n o f t h e e x t r a c e l l u l a r w a t e r c o n t e n t o r to an i n c r e a s e cells 3,18,24. In t h e of present the influx study we of calcium have used into the the -20 +3000 +20 isolated Ipv} s e m i c i r c u l a r c a n a l o f t h e frog to i n v e s t i g a t e t h e e f f e c t s o f m o d i f i c a t i o n s o f i n t r a c e l l u l a r o r e x t r a c e l l u l a r calcium concentrations on the bioelectrical properties of the vestibular neurosensory epithelium. The E~' + 4000 results suggest that administration of the calcium ionophore A23187 or gradual reduction of calcium concentrations in t h e p e r i l y m p h impair the release of the afferent neurotransmitter and modify the physical and electrical p r o p e r t i e s o f t h e cell m e m b r a n e s epithelium and/or of the labyrinthine t h e a f f e r e n t fibers. MATERIALS AND METHODS -400 600 Frequency 400 200 0 The experiments were conducted using the vertical posterior semicircular canal of the frog (Rand ridibunda). The procedures for dissection of the semicircular canal, electrophysiological recordings and data analysis have been previously described 2. Briefly, the canal, isolated with the distal stump of the ampullar nerve, was placed in a bicompartmental chamber which allowed the artificial inner ear fluids to be separately perifused and replaced. The spontaneous discharge of the afferent nerve (spontaneous activity) and the transepithelial potential (endolymphatic potential), were recorded in the absence of mechanical stimulation (Fig. 1). Three additional parameters were investigated during mechanical displacement of the endolymphatic fluid: (1) the ampullar direct current corresponding to the variations of the endolymphatic potential which occur when the hair cell potential is changed by modulation of ionic (mainly potassium) fluxes across the transduction channels, (2) the nerve direct current, corresponding to the variations of the ampullar nerve potential which reflect the summation of the excitatory postsynaptic potentials in response to the evoked release of neurotransmitter from the hair cells, and (3) the frequency of the evoked afferent spikes which corresponds to the amplitude peak-to-peak of the difference in frequency of the afferent firing induced by mechanical stimulation (Fig. 1). The variations of the transepithelial potential and ampullar nerve potential were measured as the total peak-to-peak amplitudes of each signal. Mechanical stimulation of the sensory epithelium was elicited by sinusoidal flow movements of the endolymphatic fluid, generated by the displacement of the plunger of a 5/xl Hamilton syringe (amplitude 12.7/zm peak-to-peak, frequency 0.19 Hz). The syringe, filled with endolymph, was tightly inserted to the cut end of the canal. The ionic compositions of the artificial endolymphatic and perilymphatic solutions are presented in Table I. When the concentration of calcium in the perilymph solution was reduced, the sodium concentration was augmented proportionally to maintain a constant ionic strength. A23187 (Sigma, USA), initially dissolved in dimethyl sulfoxide (DMSO), was made up in the perilymph artificial solution so that the final concentration of DMSO was 0.2%. Control experiments performed in the presence of DMSO alone indicated that, at the concentration used, DMSO exerted no effect per se on the bioelectrical properties of the canal. All solutions were adjusted to --" 1 second Fig. 1. Time course of the different bioelectrical signals recorded at rest or during one cycle of stimulation. MS, mechanical stimulus; EP, transepithelial potential; Adc, amplitude of the slow (d.c.) variation of the transepithelial potential induced by mechanical stimulation; Ndc, amplitude of the slow (d.c.) variation of the afferent nerve potential recorded during mechanical stimulation; F, amplitude of the variation of the spontaneous activity evoked by mechanical stimulation. pH 7.35 to 7.45 by continuous bubbling with 0 2 / C O 2 mixture (95 : 5). In order to obtain stabilization of the bioelectrical parameters, the preparation was exposed to standard solutions during 90 min before the experimental manipulations started. All bioelectrical signals were recorded every 5 min. In the absence of mechanical stimulation, the spontaneous activity of the ampullar nerve and the endolymphatic potential were sampled at 0.5 ms intervals and the results were expressed as the mean of the values collected during 40 TABLE I Ionic composition of the artificial endolymphatic and perilymphatic fluids NaCI KCI CaCI a MgCI 2 NaHCO 3 NaH2PO 4 KHCO 3 KH2PO 4 Glucose Perilymph Endolymph (raM) (raM) 96.8 1.5 2 0.81 20 0.17 1.7 97 2 0.81 4 20 0.17 4 303 s. The sensory epithelium was mechanically stimulated during 43 s and the ampullar direct current, the nerve direct current and the frequency of the evoked afferent spikes were sampled at 15 ms intervals. Results were calculated as the average of 8 stimulation periods for each signal. The data are expressed as the mean of 6 to 10 independent experiments and the profiles represent percentages of the basal level (mean+S.E.M). Statistical analysis was performed using the nonparametric Mann and Whitney test. ® pZ w ~ ~r uJ o iJu ._1 i- 0 ..j tu '< ¢_ RESULTS 160. A23187 100 =o m uJ Bioelectrical acticities during administration ionophore A23187 > of the E 50 uJ Z A d m i n i s t r a t i o n of A23187 (3 × 10 -6 M, 20 min), into the p e r i l y m p h a t i c c o m p a r t m e n t , caused modifica- ® tions of the s p o n t a n e o u s activity a n d nerve direct current. D u r i n g i n f u s i o n of A23187, the s p o n t a n e o u s activity i n c r e a s e d t r a n s i e n t l y (Fig. 2a) to reach 165 + 26% t~ ® e= IJ. 2oo. < IZ ,,>, 16o. ,~ lOO, ~ 60 ^2s187 ;: A23187 I ~' lOO =~ 60 I ab e'o 9'o T I M E (rain) Fig. 3. Effect of the ionophore A23187 on (a) the nerve direct current and (b) the frequency of the evoked afferent spikes. In this series of experiments, the actual values (corresponding to 100%) were: nerve direct current, 251 +57 p.V (n = 6) and afferent evoked frequency, 815_+102 spikes/s (n = 6). See legend to Fig. 2 for other details. 0 a. ® <_ I-- Z iii I0 I,IJ I D. >, t,U -I I-- •-J 100, ~ 60 ( P < 0.01) of the control level, 15 m i n after the begin- A23187 150- n i n g of A23187 a d m i n i s t r a t i o n a n d then, d e c r e a s e d to 1 74 + 5% ( P < 0.01) of the control, 60 m i n after A23187 withdrawal. T h e e n d o l y m p h a t i c p o t e n t i a l was not significantly modified d u r i n g a d m i n i s t r a t i o n of A23187 (Fig. 2b). T h e nerve direct c u r r e n t decreased by 31% "la. .J 0 Z uJ ( P < 0.05) within 80 m i n after the onset of A23187 a d m i n i s t r a t i o n (Fig. 3a). T h e f r e q u e n c y of the evoked 6 a'o 6'o ~o T I M E (mind Fig. 2. Effect of the ionophore A23187 on (a) the spontaneous activity of the ampullar nerve and (b) the endolymphatic potential. A23187 (3×10 -6 M) was added into the perilymph solution and perfused during 20 rain. Results are expressed as percentages of the basal level. In this series of experiments, the actual values (corresponding to 100%) were: spontaneous activity, 396+98 spikes/s (n = 7) and endolymphatic potential, 2.9+0.9 mV (n = 5). The dotted lines represent control profiles recorded in the absence of A23187. afferent spikes (Fig. 3b) a n d the a m p u l l a r direct curr e n t (results n o t shown) were n o t i m p a i r e d by administ r a t i o n of A23187. Effect of gradual reduction of calcium concentration in the perilymphatic compartment W h e n the isolated semicircular canal was perifused with d e p l e t e d calcium solutions (from 2 to 1 mM), a d o s e - d e p e n d e n t increase of the s p o n t a n e o u s activity of the a m p u l l a r nerve was observed (Fig. 4a). W i t h i n 25 304 min after the onset of calcium reduction to 1 mM, the signal reached 233 + 29% of the control ( P < 0.01). The spontaneous activity returned to nearly normal values within 30 min after re-establishement of a physiological calcium concentration. Reduction of the perilymphatic calcium concentration from 1.6 to 1.2 m M caused a transient increase of the endolymphatic potential (Fig. 4b). In contrast, when Ca 2÷ concentration was reduced to 1 mM, the endolymphatic potential decreased to 88 + 2% of the control ( P < 0.01). Calcium reduction caused a biphasic effect on the nerve direct current (Fig. 5a). For concentrations ranging from 1.8 to 1.4 mM, a slight and gradual increase of the nerve direct current was observed, while perifusion of m e d i u m containing 1.2 m M Ca 2+ caused a significant inhibition of the signal. During the 30 rain following infusion of the lowest Ca 2+ concentration tested (1 ® 1.8 mMCICI2 160] 1,8 n~ GaOl2 1'0 t [ - - I 100 : .o1 60 160 • 1.emildCIGI2 .J UJ > UJ .J ,.J < a0 ,< m 60 ~ 4( tO ~ll 1.4 mM O I O I 2 E O Bi 6 o ~ "°tr w n,, 100 160 1 6O E W u ' "°1;"°'°i 80 100 6O eo -J ~ ~ I1~ ~' 150 ~ 1.4 ~ ClICI2 - 0 < 1.2 mM OIIICl~ :~ - ! ~. 100 ~.~ eo • 160 z 0 ~I 100 0 , Z 6O U,I eo TIME(1111111 1,4 rnMCICI2 0 O 80 60 160 z ~ 0 Fig. 5. Effect of gradual reduction of the calcium concentration in the perilymph solution on (a) the nerve direct current and (b) the frequency of the evoked afferent spikes. In this series of experiments, the actual values (corresponding to 100%) were: nerve direct current, 174_+45 pN (n = 10) and afferent evoked frequency, 712_+ 119 spikes/s (n = 10). See legend to Fig. 4 for other details. > W ,.I . 260 1.0mMCIGI2 100 -1 < im 50 U. 4C 1601 [ - - I ~ 100 .~ WI 1.0 mid GICI2 180~ , ~ 1.11mMGIGI2 " : ; .... Z TIME(rain) .J . 1.ZmidGIGI2 "> . io •.J W ~ L 1,8 mM C I C I 2 :::1' 100 ~ 1.2~ CaCl2 | a 1.4 n~l GaGI2 ,.o] [ - - I Z Ut 13 . : 1.8~ C|CI2 ~- I-Z w z ® ® ® 1.2mMGaOl2 100 ; ~ ; : 60 1.0mid OlOl2 1.0 ~ C|CI2 mM), the nerve direct current increased to 124%. T h e frequency of the evoked afferent spikes increased when calcium concentrations were below 1.4 m M (Fig. 5b). T h e signal increased to 114 + 4% of the control ( P < 0.01), within 25 min after the onset of infusion of a 1 m M calcium solution. T h e recovery was totally achieved after the calcium concentration was restored to 2 mM. Reduction of calcium concentrations into the perilymphatic c o m p a r t m e n t had no effect on the ampullar direct current (results not shown). DISCUSSION 60 60 ; io e'o T~ (rain) 6 io eb TIME (e~e) Fig. 4. Effect of gradual reduction of the calcium concentrations in the perilymph on (a) the spontaneous activity of the ampullar nerve and (b) the endolymphatic potential. Perilymph solutions containing low calcium concentrations were perfused for 25 min; then the concentration of calcium was restored to 2 raM. Results are expressed as percentages of the basal level. In this series of experiments, the actual values (corresponding to 100%) were: spontaneous activity, 235 + 67 spikes/s (n = 8) and endolymphatic potential, 3.1 -+ 0.4 mV (n = 10). The dotted lines represent the control profiles recorded during perifusion with a perilymph solution containing 2 mM of calcium. In the present study, we have used the frog semicircular canal as an in vitro preparation to investigate the effects of modifications of calcium concentrations on the electrophysiological properties of the labyrinthine epithelium. T h e increase of cytosolic calcium levels, generally observed during ischemia, appears to be the cause of various metabolic disturbances such as inhibition of mitochondrial respiration, degradation of plasma m e m b r a n e s and cytoskeleton, modifications of 305 enzymatic metabolism and generation of oxygen free radicals9'2°'21. In our model, administration of the calciun ionophore A23187, a drug known to induce an increase of the cytosolic concentration of calcium22, into the perilymphatic compartment caused modifications of the spontaneous activity and the nerve direct current. In contrast, the endolymphatic potential, the ampullar direct current and the frequency of the evoked afferent spikes were not significantly modified during infusion of A23187. The relative stability of the endolymphatic potential and the ampullar direct current indicates that the mechanisms responsible for the generation of the endolymphatic potential and for mechanoelectrical transduction are independent from the intracellular concentration of calcium. Within the first 15 minutes of infusion of A23187, the spontaneous activity was markedly increased. During this period, the nerve direct current and the frequency of the evoked afferent spikes, recorded during the mechanical stimulation of the sensory epithelium, remained unchanged. These observations suggest that the evoked release of the afferent neurotransmitter from the hair cells and the physical and electrical properties of the membranes of the afferent fibers are not altered by the rise of intracellular calcium. Thus, the increase of the spontaneous activity can be attributed to an increase of the amount of neurotransmitter normally released at rest. Fifteen minutes after the onset of A23187 infusion, the spontaneous activity and the nerve direct current decreased, while the frequency of the evoked afferent spikes remained stable. According to Valli and Zucca 26, the nerve direct current is caused by electrotonic spreading, along the afferent fibers, of excitatory postsynaptic potentials generated at the cytoneural junctions after the release of neurotransmitter from the hair cells 26. The stability of the amplitude of the frequency of the afferent spikes suggest that the amount of neurotransmitter released during stimulation of the sensory epithelium and the mechanisms responsible for the generation of the action potentials were not impaired. Thus, the decrease of the nerve direct current may result from an alteration of the mechanisms involved in the electrotonic spreading of the signal. The decrease of the spontaneous activity of the ampullar nerve, which is likely due to an inhibition of the tonic release of the afferent neurotransmitter, may result from various phenomena including hyperpolarization of the cells induced by activation of calcium-sensitive potassium channels 14A6'17, regulation of the intracellular calcium homeostasis, depletion of the pool of neurotransmitter a n d / o r alteration of the mechanisms involved in the process of neurotransmitter release. Owing to the sus- tained influx of calcium induced by the ionophore, the amplitude and duration of the effect of A23187 and the relative stability of the frequency of the evoked afferent spikes, only an alteration of the mechanisms responsible for the transport and release of the neurotransmitter may account for the decrease of the spontaneous activity of the ampullar nerve. Recent studies have shown that, during ischemia of the central nervous system, the extracellular concentration of calcium decreases 11'1824. We have thus investigated the electrophysiological impairments induced by reduction of the perilymphatic calcium concentration. All the bioelectrical activities, except the ampullar direct current, were altered by reduction of calcium in the perilymph. The modifications of the endolymphatic potential induced by reduction of the extracellular calcium concentration can be ascribed either to an alteration of the mechanims of secretion of the endolymph or to a modification of the transepithelial resistance 4. Recently, Cereijido et al. 6, using a model of nephron epithelium, showed that a decrease of the extracellular concentration of calcium induces an important fall in the resistance of the tight junctions. According to these observations, the decrease of the endolymphatic potential, observed in the presence of 1 mM CaCI2, may be due to an increase of the ionic conductance of the para-cellular junctions. Modulation of the amount of calcium into the perilymph did not induce any effect on the mechanisms involved in the mechanoelectrical tranduction. In contrast, the synaptic transmission appears to be highly dependent on the perilymphatic concentration of calcium. Reduction of Ca 2÷ concentration caused a dose-related increase of the spontaneous activity of the ampullar nerve and frequency of the evoked afferent spikes. Similar results have been observed by Drescher et al. 8 and Guth et al. 10, using the model of the lateral line organ of the Xenopus laevis. Using the patch-clamp technique, Hudspeth et al. 17 have recorded a depolarization of the bullfrog saccular hair cell when the perilymphatic calcium concentration is reduced from 4 to 0.5 mM. This depolarization may induce activation of voltage-dependent calcium channels, which in turn would cause an influx of calcium leading to the release of the afferent neurotransmitter. Thus, the increase of the spontaneous activity and the frequency of the evoked afferent spikes may be due to a stimulation of neurotransmitter release in response to an increase of the intracellular concentration of calcium. Reduction of the external concentration of calcium may also be effective on the afferent fibers where it can exert a charge screening effect on voltage-sensitive sodium channels ~3. This hypothesis, previously proposed for 306 the the model of the lateral line 8'1°, is supported by the high sensitivity of the afferent firing to small modifications of the perilymphatic concentration of calcium. Indeed, Hansen et al. 12 have observed that an increase of extracellular calcium induces an elevation of the electrical excitability threshold of myelinized nervous fibers resulting from membrane hyperpolarization. Depolarization of the postsynaptic membranes may induce a decrease of the excitability threshold. These data indicate that an excess of calcium in the cytoplasm alters the spontaneous release of the afferent neurotransmitter. Moreover, our data suggest that a modification of the physical properties of the afferent fibers may account for the decrease of the electrogenic spreading of the postsynaptic potentials. In contrast, an excess of calcium in the cells of the afferent fibers does not affect the mechanisms involved in the generation of the action potentials. Our results also suggest that reduction of the perilymphatic concentration may lead to modifications of the physical and electrical properties of the cell membranes of the labyrinthine epithelium a n d / o r the ampullar afferent fibers. Acknowledgements. Supported by grants from the Minist~re de la Recherche et de l'Enseignement Sup6rieur (86C 0305) and CNRS (URA 650). A. Aubert was a recipient of a doctoral fellowship from the Conseil R6gional de Haute-Normandie and ORIL Laboratories. REFERENCES 1 Art, J.J. and Fettiplace, R., Variation of membrane properties in hair cells isolated from the turtle cochlea, J. Physiol., 385 (1987) 207-242. 2 Aubert, A., Bernard, C., Clauser, P., Harpey, C. and Vaudry, H., Effect of phenazine methosulfate on electrophysiological activity of the semicircular canal: antioxidant properties of trimetazidine, Eur. Z Pharmacol., 174 (1989) 215-225. 3 Bondareff, W., Extracellular space in the aging cerebrum. In R.D. Terry and S.G. 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Somjen (Ed.), Mechanisms of Cerebral [schemia and Stroke, Plenum Press, New York, 1988, pp. 91-107. 25 Szekeres, L., Balint, Z. and Tosaki, A., Ion shifts in myocardial cells during ischemia and reperfusion and the effect of membrane stabilizing agents on ischemic ion transport, Biomed. Biochim. Acta, 46 (1987) $527-$533. 26 Valli, P. and Zucca, G., The origin of slow potentials in semicircular canals of the frog, Acta Oto-Laryngol., 81 (1976) 395-405.