]>HEARES3362S0378-5955(99)00194-X10.1016/S0378-5955(99)00194-XElsevier Science B.V.Fig. 1Example of intracellular calcium concentration changes in a single type I sensory cell induced by iontophoretic applications (arrows) of glutamate (Glu), glycine (Gly), NMDA, aspartate (Asp) and NaCl. Significant, rapid and transient increases in the concentration of intracellular calcium were observed for Glu, Gly, NMDA and Asp. No significant change was induced by NaCl.Fig. 2Effect of 7-chlorokynurenate (7CK) on the calcium response to glycine. The presence of 7CK (10 μM) reduced the calcium response induced by glycine application by about 60% (A and B). APV (10 μM), a competitive antagonist of the NMDA receptor, did not affect the calcium response induced by glycine (B). The addition of 7CK (10 μM) and strychnine (50 μM) together resulted in a larger decrease in the calcium response to glycine stimulation (A).Fig. 3Effect of strychnine, a specific antagonist of the glycine Cl−- channel receptor. The presence of strychnine (50 μM) decreased the calcium response to glycine stimulation by about 60%. Strychnine caused no significant decrease in the calcium response induced by glutamate.Fig. 4Effect of Cl−-free gluconate medium. The presence of gluconate (137 mM) decreased the calcium response induced by glycine. In the same condition, there was no significant decrease in the calcium response induced by glutamate application. The addition of gluconate and 7-chlorokynurenate (7CK, 10 μM) together resulted in a larger decrease in the calcium response induced by glycine stimulation.Fig. 5Effect of chelating Ca2+ in the medium by the addition of EGTA. The calcium response induced by glycine was reduced by adding EGTA (1.5 mM) to the medium. When the cells returned to normal medium, the calcium peak level tended toward the typical calcium levels induced by glycine application.Fig. 6Schematic drawing, representing the putative presence of NMDA and glycine receptors on vestibular type I sensory cells and the hypothesis of a peripheral control of the sensory cell activity by the afferent nerve calyx. An activation of these receptors by glycine could induce changes in [Ca2+]i. The increase in [Ca2+]i could also be due to the activation of ionotropic and metabotropic glutamate receptors after release of glutamate.Table 1Analysis of calcium responses to Glu, Gly, NMDA, Asp and NaCl applications in isolated type I sensory cellsGluGlyNMDAAspNaClHair cells(n)2026557Baselineratio 340/3800.77±0.100.77±0.090.76±0.030.78±0.030.76±0.05Peakratio 340/3801.2±0.231.19±0.221.02±0.131.15±0.070.76±0.06Time to peak(s)7.36±1.219.15±2.347.00±1.418.20±1.64Regulation time(s)19.09±3.5920.58±5.0415.08±3.8719.40±3.36Increase/base(%)565534470[Ca2+]i are means of the fluorescence signal of the whole cell soma. The baseline [Ca2+]i values (mean±S.D.) and the response peak values (mean±S.D.) are expressed as the 340 nm/380 nm fluorescence ratio. The time course of the increase [Ca2+]i was rapid, and then returned to basal levels. The estimated time-to-peak and regulation time are given in seconds (mean±S.D.). The nuber of type I sensory cells (n) tested for each agent is indicated.Table 2Calcium levels following glycine stimulation in the presence of 7-chlorokynurenate (7CK, 10 μM), a selective antagonist of the glycine site on NMDA receptor and of APV (10 μM), a competitive antagonist for the glutamate site on NMDA receptorGluGlu+APVGlyGly+APVGly+7CKNMDANMDA+APVHair cells(n)7171711Baselineratio 340/3800.71±0.100.70.71±0.100.70.71±0.100.70.7Peakratio 340/3801.16±0.110.951.09±0.161.280.87±0.101.030.75Time to peak(s)8±157.14±1.0776.83±1.1755Regulation time(s)17.67±3.061019.71±2.431917.67±3.14165Increase/base(%)6333548022455Decrease/specific(%)4806090The [Ca2+]i baseline values (mean±S.D.) and peak response values (mean±S.D.) are expressed as the 340 nm/380 nm fluorescence ratio. The values are expressed as in Table 1. The percentage decrease was evaluated as the ratio of the amplitude of the increase in [Ca2+]i in the presence of the antagonist to that in the presence of glycine alone.Table 3[Ca2+]i changes following glutamate and glycine stimulations in the presence of strychnine (50 μM)GluGlu+strychn.GlyGly+strychn.Gly+strychn.+7CKHair cells(n)62664Baselineratio 340/3800.65±0.060.63±0.030.65±0.060.65±0.060.66±0.06Peakratio 340/3800.98±0.080.96±0.060.94±0.110.76±0.090.72±0.04Time to peak(s)6.33±1.157.5±0.716.83±0.756.83±1.946.25±0.83Regulation time(s)19.33±3.0621±1.4116.17±2.3217±3.5817.25±2.38Increase/base(%)5148451812Decrease/specific(%)66073The values are expressed as in Table 1. The percentage decrease in the calcium response is calculated as the ratio of the peak calcium level in the presence of strychnine to that following glutamate or glycine stimulation alone. The addition of both strychnine (50 μM) and 7CK (10 μM) resulted in a larger decrease, by about 73%, in the calcium response induced by glycine application.Table 4[Ca2+]i changes in Cl−-free gluconate mediumGluGlu+glucoGlyGly+glucoGly+gluco+7CKGly after rinseHair cells(n)336613Baselineratio 340/3800.71±0.060.71±0.060.71±0.060.71±0.060.60.68±0.03Peakratio 340/3801.17±0.100.96±0.251.31±0.160.96±0.140.81.23±0.16Time to peak(s)8±07.33±1.159.5±2.178±1.2677.67±2.08Regulation time(s)21.33±3.2118±4.5823.33±4.520.17±6.052622.33±3.21Increase/base(%)643484351373Decrease/specific(%)19588413Glutamate or glycine were applied in normal or Cl−-free gluconate medium. The values are expressed as in Table 1. After rinsing with normal medium, the peak calcium level tended to be similar to that in control after glycine stimulation. The concomitant absence of Cl− and presence of 7CK resulted in a larger decrease in the calcium response to glycine than the one obtained in the absence of Cl− alone.Table 5[Ca2+]i after glycine application in the presence of EGTA (1.5 mM), a specific calcium chelator, and after rinsing with normal mediumGlyGly+EGTAGly after rinseHair cells(n)444Baselineratio 340/3800.67±0.090.54±0.040.67±0.09Peakratio 340/3800.99±0.280.57±0.180.81±0.12Time to peak(s)7.25±0.965.50±0.716.50±2.12Regulation time(s)17.25±2.0619±1.4117.50±0.71Increase/base(%)47621The values are expressed as in Table 1.Glycine induced calcium concentration changes in vestibular type I sensory cellsGinaDevau*gdevau@univ.montp2.frINSERM U432, Université Montpellier II, place Eugène Bataillon, 34095 Montpellier Cedex 5, France*Tel.: +33 4 67 14 48 30; Fax: +33 4 67 14 36 96AbstractGlutamate is the neurotransmitter of the synapse between vestibular type I hair cells and the afferent nerve calyx. This calyx may also be involved in local feedback, which may modify sensory cell activity via N-methyl-D-aspartate (NMDA) receptors. Glycine is the co-agonist of glutamate in NMDA receptor activation. Both agents have been detected by immunocytochemistry in the nerve calyx. Glutamate and NMDA stimulations cause changes in the intracellular calcium concentration ([Ca2+]i) of isolated type I sensory cells. We investigated the effect of glycine stimulation on [Ca2+]i in guinea pig type I sensory cells by spectrofluorimetry with fura-2. Glycine application to isolated type I sensory cells induced a rapid and transient increase in [Ca2+]i. The fluorescence ratio increased by 55% above the resting level. The peak was reached in 9 s and the return to basal level took about 20 s. A specific antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 μM), decreased the calcium response to glycine by 60%. Glycine may activate NMDA receptors. Glycine may also activate the strychnine-sensitive glycine receptor-gated channel. Strychnine (50 μM) decreased the calcium response to glycine by 60%. Thus, glycine probably induces calcium concentration changes in type I vestibular sensory cells via NMDA receptors and/or glycine receptors.KeywordsN-Methyl-D-aspartateGlycineCalciumVestibular sensory cellGuinea pig1IntroductionIn mammals, vestibular sensory cells are mechano-sensory cells, classified as type I or type II according to morphological criteria (Wersäll, 1956; Wersäll and Bagger-Sjöback, 1974). Type I sensory cells are surrounded by an afferent nerve calyx. Type II sensory cells are contacted only by button-shaped afferent endings. Physiological and pharmacological studies have shown that glutamate is the principal excitatory neurotransmitter at the cytoneuronal synapse between sensory cells and primary afferent nerve fibers, in vestibular hair cells (Annoni et al., 1984; Drescher et al., 1987; Drescher and Drescher, 1992; Guth et al., 1988; Soto and Vega, 1988; Zucca et al., 1992) and in cochlear hair cells (Anson and Ashmore, 1994; Kataoka and Ohmori, 1996). Different glutamate receptor types are involved in vestibular neurotransmission between the sensory cells and the primary afferent neurons. Non-N-methyl-D-aspartate (NMDA) receptors have been detected mostly by electrophysiological and pharmacological studies in the vestibular system of batracians (Soto and Vega, 1988; Prigioni et al., 1990, 1994), although NMDA receptors have also been shown to be present (Zucca et al., 1993; Soto et al., 1994). In mammals, immunocytochemical and in situ hybridization studies have implicated the GluR2/R3 and GluR4 subunits of AMPA receptors (Demêmes et al., 1995; Niedzielski and Wenthold, 1995; Rabejac et al., 1997), and the NR1 and NR2A–D subunits of NMDA receptors in vestibular neurotransmission (Fujita et al., 1994; Niedzielski and Wenthold, 1995). Moreover, presynaptic glutamate receptors may also interact as autoreceptors (Valli et al., 1985; Prigioni et al., 1990; Guth et al., 1991, 1998b). The presence of NMDA receptors has been detected on isolated type I sensory cells from guinea pig by calcium spectrofluorimetry with fura-2 (Devau et al., 1993). Vestibular hair cell neurotransmission has been reviewed by Guth et al. (1998a).Type I sensory cells have the particularity that they are surrounded by an afferent nerve calyx. Various studies support the hypothesis of a local control of type I sensory cell activity by this afferent nerve calyx (Scarfone et al., 1988, 1991). In particular, the calyx contains synaptic-like microvesicles and proteins involved in the synaptic vesicle cycle (synaptophysin, synapsin I and rab3a, Dechesne et al., 1997) suggesting that neuroactive substances may be released. This local control could involve glutamate (Demêmes et al., 1990) and/or a neuropeptide, substance P (Scarfone et al., 1996).The NMDA receptors on isolated type I sensory cells may act as presynaptic receptors in the active zone of neurotransmitter release or may be involved in the local control by the afferent nerve calyx. Glutamate and glycine are the co-agonists that activate NMDA receptors (Johnson and Ascher, 1987; Langosch et al., 1988; Corsi et al., 1996). Glycine and glutamate have both been detected by immunochemistry, co-localized in the thick calyceal afferent fibers of the vestibular epithelium (Reichenberger and Dieringer, 1994; Straka et al., 1996a,b; Bäurle et al., 1997). Glycine may have a modulatory effect: possibly co-released with glutamate from the nerve calyx, glycine may activate NMDA receptors or act independently on putative glycine receptors.To test whether there was local control of sensory cells involving the afferent nerve calyx, we investigated the action of glycine on isolated type I sensory cells of guinea pig by measuring variations in intracellular calcium concentration ([Ca2+]i) by spectrofluorimetry with the sensitive dye fura-2. We show that glycine can act on at least two different sites: the glycine site of the NMDA receptor selectively antagonized by 7-chlorokynurenic acid (Kemp et al., 1988; Kemp and Leeson, 1993), and the glycine receptor, which is strychnine-sensitive and highly permeable to chloride (Van den Pol and Gorcs, 1988; Vandenberg et al., 1992; Zafra et al., 1997; Breitinger and Becker, 1998).2Materials and methods2.1Vestibular type I sensory cell preparationIsolated vestibular sensory cells were prepared as previously described (Devau et al., 1993). Briefly, young pigmented guinea pigs (200–250 g) were anesthetized and then decapitated. The labyrinths were removed, opened and the cristae ampullaris quickly dissected in Hanks’ balanced salt solution (HBSS, Sigma, France). This medium contained 137 mM NaCl, 5.4 mM KCl, 1.7 mM Ca2+, 0.4 mM MgSO4, 0.31 mM Na2HPO4, 0.46 mM KH2PO4 and 5.5 mM D-glucose (Sigma, France). HBSS solution was buffered to pH 7.4 with 5 mM HEPES and adjusted to 290 mOsm. Hair cells were dissociated enzymatically by incubating the sensory epithelium in HBSS containing collagenase (1 mg/ml, Sigma, France) for 10 min at room temperature (20°C). After three rinses in HBSS, hair cells were mechanically dissociated by gentle trituration with fine iron microelectrodes. The isolated hair cells were then transferred onto slides coated with poly-L-lysine (0.5 mg/ml, Sigma, France).Type I hair cells were identified by their characteristic amphora shape, which was preserved after isolation. An elongated neck region separated the spherical basal region (containing the nucleus) from the cuticular plate, into which the intact hair bundle was inserted. The type I sensory cells were selected on the following criteria: smooth plasmalemma and no granules within the cytoplasm. This morphological examination also verified that there was no shape abnormality, for example turgescent swelling or plasmolytic shrinking due to osmotic pressure imbalance. This preparation was used for a patch clamp study (Griguer et al., 1993).The care and the use of animals in this study were approved by the French Ministère de l’Agriculture et de la Forêt (authorization number 04889).2.2Calcium measurements[Ca2+]i was measured using the fluorescent dye fura-2. The cells were loaded with 2.5 μM fura-2/AM (Molecular Probes, Eugene, OR, USA) in HBSS containing 0.02% pluronic-DMSO for 30 min at room temperature. Experiments were performed with an inverted microscope (Axiovert 10, Zeiss, Le Pecq, France) equipped with a fast fluorescence photometer controlled by MSP 69/AIS. Excitation light was provided by a xenon lamp (XBO 75 W) and was passed through filters to select the two excitation wavelengths, 340 and 380 nm. Light at both excitation wavelengths was dimmed by about 90% by a neutral density filter (green UG11) to protect the cells from phototoxicity and bleaching. The emission signals were passed through a 510 nm narrow bandpass filter. A circular 10 μm diaphragm was used to limit fluorescence measurement to a field containing a single sensory cell and centered on the soma. Each fluorescence ratio value was determined by an 80 ms time resolution of the measurement. Changes in fluorescence ratio (free dye 340 nm/bound dye 380 nm) were used to estimate changes in free cytosolic calcium concentration as previously described (Grynkiewicz et al., 1985).2.3Pharmacological applications2.3.1Iontophoretic application of glutamate, NMDA, aspartate and glycineIsolated sensory cells were stimulated by focal iontophoretic application of various agents. Glutamate, NMDA, aspartate and glycine were applied via a micropipette with the tip situated about 2 μm from the basolateral hair cell membrane. Microelectrodes were filled with one of the following solutions: glutamate (1 M, pH 8), NMDA (0.4 M, pH 8), aspartate (1 M, pH 8) and glycine (1 M, pH 8) (Sigma, France). The tip diameter of the micropipettes was less than 1 μm and their resistance when they were filled with any of the various solutions was about 25 MΩ. Electrolytes were ejected using a negative pulse (−3 μA) lasting 5 s. Breaking currents of opposite polarity were used to prevent cation leakage from iontophoretic electrodes. Control tests of the iontophoretic procedure were performed by ejection of Cl− using a negative current or of Na+ using a positive current from microelectrodes filled with sodium chloride (NaCl, 1 M).The electric charge ejected through the pipette was estimated from the Faraday equation Q=It/F where I is the amplitude of the current applied, t the duration of the application and F the Faraday constant (96 500 C). After ejection, the diffusion of the agent depends on several factors including the viscosity of the medium and the size of the molecule.2.3.2Microperfusion of glutamate and glycine antagonistsAntagonist microperfusion was started 30 s before stimulation with glutamate or glycine. Antagonists were dissolved in HBSS at a concentration of 50 μM for strychnine (Sigma, France) and 10 μM for 7-chlorokynurenate (Sigma, France) and DL-2-amino-5-phosphonovaleric acid (APV, Sigma, France). Microperfusion of antagonist alone did not affect the resting calcium concentration.2.4Data analysisCells loaded with fura-2 responded to the application of glutamate agonists or glycine by an increase in fluorescence intensity ratio corresponding to an increase in [Ca2+]i. The fluorescence ratio is expressed in terms of molarity using the equation of Grynkiewicz et al. (1985): [Ca2+]i=(R−Rmin/Rmax−R)αKD, where Rmin=0.4; Rmax=11.3; α=11.9; KD=224 nM.Fluorescence ratio changes are presented rather than changes in absolute [Ca2+]i because many factors (e.g. viscosity of the medium, diffusion, etc.) can affect the absolute [Ca2+]i. The mean basal [Ca2+]i was calculated for all the cells tested in the different experiments. Data are expressed as the peak increase in [Ca2+]i from initial basal levels. The calculated mean amplitude and duration of the calcium responses of each hair cell were averaged for each agonist and antagonist tested. Values are expressed as means±S.D.3Results3.1Calcium concentration changes induced by glutamate, glutamate agonists and glycine in type I vestibular sensory cellsThe calcium fluorescence ratio for type I sensory cells was 0.75±0.09 (n=40) at rest, corresponding to a calcium concentration of 88.4 nM. This value is consistent with the satisfactory maintenance of viable cells in vitro. Applications of glutamate, NMDA and aspartate induced rapid and transient increases in fluorescence ratio of 56, 34 and 47% respectively (Fig. 1). Glycine application induced a similar increase of 55% (Fig. 1). The delay between application and response was about 1 s. The peak value was reached in about 9 s and the return to resting level took about 20 s, indicating that regulatory processes were occurring (Table 1). The shapes of response curves were similar for all cells tested.The specificity of the calcium response induced by iontophoretic applications was checked by ejection of a different anion (Cl−) or cation (Na+). Neither caused any change in fluorescence ratio (Fig. 1).3.2Effect of 7-chlorokynurenic acid on the calcium response to glycine in sensory cellsTwo consecutive applications of glycine, separated by a rinse of 5 min, induced similar calcium responses (Fig. 2A,B), with similar profiles and peak amplitudes.A selective antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 μM), reduced the calcium response evoked by glycine stimulation by about 60% (Fig. 2A,B). The presence of APV (10 μM), a competitive antagonist of the glutamate site on the NMDA receptor, had no significant effect on the calcium response evoked by glycine application (Fig. 2B), whereas the presence of APV decreased the calcium response induced by glutamate by 48% and that induced by NMDA by 90% (Table 2).3.3Effect of strychnine on the calcium response induced by glycine in type I sensory cellGlycine may also activate the ionotropic glycine receptor, which is selectively antagonized by strychnine. The calcium response evoked by glycine was therefore evaluated in the presence of strychnine (50 μM): it was inhibited by 60% (Fig. 3, Table 3). The calcium response induced by glutamate application was not affected by the presence of strychnine (Table 3).Strychnine and 7-chlorokynurenate, applied together, reduced the calcium response induced by glycine by 73% (Fig. 2, Table 3).3.4Effect of extracellular chloride ions on the calcium response induced by glycineThe ionotropic glycine receptor is selectively permeable to chloride ions. We tested whether there was a relationship between chloride influx and changes in [Ca2+]i. The chloride ions of the medium were replaced with gluconate which cannot pass through the glycine receptor channel or the plasmalemma. In the presence of sodium gluconate (137 mM), the calcium resting level was slightly lower (Fig. 4) and the calcium response evoked by glycine was 58% smaller (Fig. 4, Table 4). The addition of both gluconate and 7-chlorokynurenate decreased the calcium response to glycine by 84% (Fig. 4, Table 4).3.5Effect of extracellular calcium ions on the calcium response induced by glycineWe tested whether external calcium affects the changes in calcium concentration induced by glycine by adding a specific calcium chelator EGTA (1.5 mM) to the medium (HBSS). In the presence of EGTA, calcium resting levels in the cell decreased by about 20% and the increase in calcium variation by glycine was only 6% of the basal calcium level measured in presence of EGTA (Fig. 5, Table 5). Therefore, extracellular calcium seemed to be the major source of the calcium concentration changes.4Discussion4.1Calcium concentration changes induced by glycine in type I vestibular sensory cellVestibular sensory cells are mechano-transducers, encoding and sending sensory messages by the afferent nerve to the vestibular nuclei. In isolated type I vestibular sensory cells, the low and stable [Ca2+]i before each stimulation indicated that the cells in our preparations were not altered or excited. The [Ca2+]i in type I sensory cells was similar to that observed in other cells in vitro such as cochlear ganglion neurons (Harada et al., 1994), vestibular ganglion neurons (Rabejac et al., 1997), hippocampal neurons (Murphy and Miller, 1988), and Purkinje neurons (Hockberger et al., 1989).Glycine application induced a rapid and transient increase in [Ca2+]i. Increases in [Ca2+]i were also evoked by glutamate and by two NMDA receptor agonists, aspartate and NMDA. The response curve following glycine stimulation was similar to those obtained after glutamate and aspartate stimulation. After stimulation, [Ca2+]i returned to basal levels. The [Ca2+]i is regulated by processes involving calcium binding proteins, the Ca-ATPase pump in the plasmalemma and intracellular membrane of the endoplasmic reticulum, and the Na/Ca exchanger (Tucker and Fettiplace, 1995; Tucker et al., 1996). Intracellular calcium is a messenger in fast event cascades in various cellular processes. Electrophysiological and confocal microscopy studies have shown that [Ca2+]i increases particularly in microdomains in which the calcium concentration reaches about 100 μM. These microdomains are located near the basolateral membrane in frog vestibular sensory cells (Lenzi and Roberts, 1994; Issa and Hudspeth, 1994; Tucker and Fettiplace, 1995; Tucker et al., 1996). This site corresponds to active zones of the sensory cells characterized by the presence of voltage-activated Ca2+ channels and Ca2+-activated K+ channels (Hudspeth and Lewis, 1988) and synaptic bodies involved in neurotransmitter release, which requires the rapid regulation of Ca2+ levels (Parsons et al., 1994; Fuchs, 1996). It has also been suggested that changes in [Ca2+]i at the apex of sensory cells may affect the adaptation of the mechano-transduction channels in the hair bundle, adjusting the operating range of the transducer (Denk et al., 1995).4.2Effect of glycine on NMDA/glycine receptorsGlycine may activate a glycine site on the NMDA receptor in type I vestibular sensory cells. We found that a selective antagonist of this site, 7-chlorokynurenate, reduced the increase in [Ca2+]i induced by glycine. Glycine is a co-agonist with glutamate to activate the NMDA receptors (Moriyoshi et al., 1991; Hollmann and Heinemann, 1994; Mori and Mishina, 1995; Corsi et al., 1996; Sucher et al., 1996). However, in some NMDA receptor subunit configurations, heteromeric NMDA receptors may be activated by glycine alone. The presence of the NR1 subunit associated with NR2A or NR2B or NR2C subunits in Xenopus oocytes results in the induction of a small but clear inward current induced by 10 μM glycine alone (Meguro et al., 1992; Kutsuwada et al., 1992). In rat neocortical neurons, nerve terminals, which release cholecystokinin and somatostatin, possess NMDA receptors, the channels of which can be operated by glycine or D-serine alone with no apparent activation of the glutamatergic co-agonist site (Paudice et al., 1998). The functional properties of the NMDA receptor channel are determined by the NR2 subunit (Kutsuwada et al., 1992; Molinoff et al., 1994; Paudice et al., 1998). The composition of NMDA subunits, in the vestibular epithelium, is unknown but the NR1 subunit has been detected in vestibular sensory cells (Devau et al., 1997). Glycine may act as a co-agonist with residual glutamate released by the sensory cell near the soma before rapidly spreading (schematic drawing Fig. 6). This contamination by glutamate may account for the NMDA receptor activation. The presence of APV (Watkins and Olverman, 1987), a competitive antagonist of the glutamate site on NMDA receptors, decreased the calcium response to NMDA by 90% and that to glutamate by 48% but did not alter the calcium response to glycine. This suggests that NMDA receptors sensitive to glycine are present on vestibular sensory cell. The almost complete inhibition of NMDA by APV, and only about 50% inhibition by glutamate alone suggest that glutamate acts on both ionotropic and metabotropic receptors (Devau et al., 1993; Guth et al., 1998a,b)However, 7-chlorokynurenate, a selective antagonist of the glycine site of NMDA receptors, contributes to the partial inhibition of the calcium response induced by glycine.4.3Effect of glycine on strychnine-sensitive glycine receptorsGlycine also activates ionotropic glycine receptors, which are specifically permeable to chloride ions and sensitive to strychnine. Strychnine reduced the increase in [Ca2+]i after glycine application by 60%, but decreased by only 6% the calcium response induced by glutamate. Strychnine interacts with the nicotinic receptors of acetylcholine as a potent antagonist. In frog vestibular hair cells, strychnine interacts with nicotinic-like receptors (Guth et al., 1994; Guth and Norris, 1996). The α9 subunit of the nicotinic receptor is very sensitive to strychnine and mRNA encoding the α9 subunit has been detected in the adult rat peripheral vestibular system (Hiel et al., 1996). Acetylcholine is the principal neurotransmitter of the efferent system and cholinergic receptors are present in type II sensory cells. However, the type I sensory cells of mammals are surrounded by afferent nerve calyces and are not in contact with any efferent nerve endings. Thus, the cholinergic receptors are not responsible for the type I sensory cell response to strychnine.The stimulation of glycine receptors resulted in chloride permeability. In the hair cell, chloride ions contribute greatly to the maintenance of cell volume (Rennie et al., 1997). In low chloride medium, the outer hair cells of the cochlear epithelium first shorten, then rapidly increase in length. The cells then collapse due to an efflux of Cl− followed by K+ and water (Cecola and Bobbin, 1992). In Cl−-free gluconate-HBSS, resting calcium levels were low in isolated type I sensory cells. Transferring the cells to normal HBSS resulted in a return to normal calcium resting levels. In the medium, gluconate may be associated with sodium or calcium. In many preparations, gluconate has been used to substitute for chloride ions in experiments with isolated vestibular type I hair cells (Rennie et al., 1997). However, gluconate may chelate external calcium and partially reduce the glycine effect.In other systems, for example, at some stage of development in chick embryo ciliary ganglion cells, activation of the ionotropic glycine receptor triggers an increase in [Ca2+]i, which is inhibited by strychnine (Sorimachi et al., 1997). Activation of the embryonic ionotropic glycine receptor causes depolarization and calcium transients in other neurons such as rat dorsal horn neurons (Wang et al., 1994). In adult neurons and smooth muscle cells, calcium activates chloride channels (Marsh et al., 1997; Wang and Kotlikoff, 1997). We found that in adult vestibular type I sensory cells, activation of the strychnine-sensitive glycine receptor induced an increase in [Ca2+]i, but the biochemical steps linking these events are unknown.The addition of strychnine and 7-chlorokynurenate together strongly decreased the calcium response suggesting that there are two components involved in the calcium transient. In the presence of an agonist, the activity of a ligand-gated channel such as an NMDA or glycine receptors may be modified by desensitization, which may in turn affect cell excitability (Jones and Westbrook, 1996). After washing for 5 min, a second glycine stimulation induced a calcium response with an amplitude similar to that of the first. Thus, we found no evidence of desensitization in type I vestibular sensory cells.4.4Glycine and amino acid transportersGlycine may also directly activate glycine transporters. These transmembrane proteins have 12 transmembrane segments like other members of the transporter family, and their function depends on the cotransport of electrogenic sodium and chloride (Attwell and Mobbs, 1994; Olivares et al., 1997; Zafra et al., 1997). The detection and the cellular localization of glycine transporters in the vestibular sensory epithelium could be determined by an immunocytochemical study, with specific antibodies directed against the glial glycine transporter (GLYT1) and the neuronal glycine transporter (GLYT2). The distribution of GLYT2 is correlated with that of strychnine-sensitive glycine receptors in most of the central nervous system except cerebellum (Nelson, 1998). They may be responsible for the residual response in the presence of 7-chlorokynurenate and strychnine. The sodium and chloride flux induced by glycine uptake in vestibular sensory cells may cause a change in [Ca2+]i contributing to the overall calcium response to glycine stimulation. To measure the sodium concentration changes induced by activation of glycine transporters a specific sodium-sensitive dye, SBFI, could be used.4.5Calcium source: influx of extracellular calcium and mobilization of intracellular calcium storesThe increase in [Ca2+]i induced by glycine may result from the entry of external calcium or from release from internal stores or both. Calcium may enter through the highly sensitive calcium channel, of the NMDA receptor, or may be released from the internal calcium store following signal transduction. Activation of the NMDA receptor may also depolarize the membrane and calcium influx may be increased by opening of the L-type calcium channels present in both types of vestibular sensory cells (Boyer et al., 1998). The presence of nickel and cadmium ions (Ni2+/Cd2+), which block voltage-dependent calcium channels, decreased by 30% the calcium response induced by glycine (personal observation). Calcium channel specific blockers (such as dihydropyridines for L-type or ω-conotoxin-GVIA for N-type) could be used to determine the type of the voltage-dependent calcium channels involved in the calcium concentration changes in vestibular sensory cell after glycine application. However, only L-type calcium channels have been shown in type I vestibular sensory cell (Boyer et al., 1998). In the presence of EGTA, a specific calcium chelator, the calcium response to glycine application was very small indicating that an influx of external calcium was the major component of the response. However, a residual response persisted demonstrating that internal calcium stores were not totally empty.In conclusion, the application of glycine induced an increase in [Ca2+]i in isolated type I vestibular sensory cells from guinea pig. Changes in calcium concentration involved at least two receptors: (i) heteromeric NMDA receptors, which are highly sensitive to glycine, and (ii) the strychnine-sensitive glycine receptor. Changes in calcium concentration may affect neurotransmitter release in the basolateral region of the cell. 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