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Glutamate induced currents in isolated inner hair cells from guinea-pig cochlea
Brain Research 976 (2003) 135–138 www.elsevier.com / locate / brainres
Short communication
Glutamate induced currents in isolated inner hair cells from guinea-pig cochlea Takashi Kimitsuki*, Mitsuru Ohashi, Yuki Wada, Takumi Okuda, Shizuo Komune Department of Otorhinolaryngology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan Accepted 1 April 2003
Abstract We investigated the direct action of glutamate (Glu) on the membrane current of isolated inner hair cells of guinea-pig cochlea. Glu elicited inward currents at a holding potential of 270 mV. Eight of 13 cells showed a steady inward current, while five of 13 cells showed a fast and rapidly desensitized current. I–V relationships demonstrated that the reversal potential of Glu-induced current was near 0 mV. Glu-induced currents were dose-dependent, where the half maximum concentration (Kd ) was 41 mM and Hill coefficient (n) was 1.75. 2003 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Auditory, vestibular, and lateral line: periphery Keywords: Glutamate; Inner hair cell; Cochlea; Membrane current; Guinea pig
Auditory stimuli are detected by hair cells in the cochlea of inner ear and are transmitted to the brain by way of the auditory nerves. Inner hair cells (IHCs) are primary transducers of sensory information, forming chemical synapses with dendrites of type I spiral ganglion neurons. Immunocytochemistry studies showed that glutamate (Glu) could be found in hair cells of the guinea pig cochlea [1,24]. Whereas functional Glu receptors were also expressed by spiral ganglion neurons with a postsynaptic localization in the hair cell synapse [12,17,21]. These studies indicate that Glu acts as IHC transmitter or precursor of the transmitter. Glu release from hair cells is stimulated by K 1 in a Ca 21 -dependent manner [2,3,5,10]. In the vestibular organ, Glu exerted presynaptic effects on the release of transmitters from the hair cells [19,23,25]. NMDA receptors (NR-1) showed a basolateral location in type I vestibular hair cells [9], AMPA receptors (GluR4) presented on presynaptic membrane of cochlear IHCs [15], and Glu receptor subunit d1 was expressed in hair cells of both the auditory and vestibular systems [22]. Increased intracellular Ca 21 concentration following Glu application *Corresponding author. Tel.: 181-985-85-2966; fax: 181-985-857029. E-mail address: [email protected] (T. Kimitsuki).
was reported in vestibular hair cells [4] and cochlear IHCs [13,14]. These findings indicate that Glu might act on the presynaptic autoreceptor in a positive feedback manner, though there is no study showing the current change by Glu in hair cells. In the present study, we investigated the direct act of Glu on the membrane current of isolated IHCs, using a patch clamp method. Adult albino guinea-pigs (200–350 g) were killed by rapid cervical dislocation and both bullae were removed. The organ of Corti was dissected from all cochlear turns using a fine needle in Ca-free external solution. The organ of Corti was transferred to the recording chamber and treated with trypsin (1 mg / ml, T-4665, Sigma) for 12 min. Gentle mechanical trituration was carried out and the enzyme was rinsed out by perfusing with standard external solution for at least 10 min before starting any experiments. The shape of IHCs is flask-like. The nucleus is centrally located and the mitochondria are scattered. The most important landmarks for identifying IHCs are the tight neck and the angle between the cuticular plate and the axis of the cell [7,11]. The external standard solution contained (mM): 142 NaCl; 4 KCl; 2 MgCl 2 ; 1 CaCl 2 ; 2 NaH 2 PO 4 ; 8 Na 2 HPO 4 ; adjusted to pH 7.4 with NaOH; glutamate ( L-glutamate acid, 12-0880-2, Sigma-Aldrich Japan) was
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02756-2
136
T. Kimitsuki et al. / Brain Research 976 (2003) 135–138
dissolved in external standard solution. To block K current, the patch pipette solution was Cs-based containing (mM): 144 CsCl; 2 MgCl 2 ; 1 NaH 2 PO 4 ; 8 Na 2 HPO 4 ; 0.5 EGTA; adjusted to pH 7.4 with CsOH. Membrane currents were measured by conventional whole-cell voltage-clamp recordings using an EPC-8 (HEKA, Lambrecht, Germany). Data acquisition was controlled by the software PULSE / PULSEFIT (HEKA, Lambrecht, Germany). Patch pipettes were pulled on a two-stage vertical puller (PP-830 Narishige, Tokyo, Japan) using a 1.2-mm borosilicate glass (GC-1.2, Narishige, Tokyo, Japan). Pipettes had a resistance of 5–8 MV in the bath and were coated with ski wax (Tour-DIA, DIAWax, Otaru, Japan) to minimize capacitance. The cell’s capacitance was 7.962.5 pF (n515) and the residual series resistance was 1867.8 MV (n515). Cells were continuously perfused with external saline and all experiments were performed at room temperature (20–25 8C). Under the voltage-clamp condition at 270 mV, 0.1 mM Glu elicited inward currents in the dissociated IHCs (Fig. 1). The external standard solution, applied under the same system, did not generate any currents (data not shown). While eight of 13 cells showed a steady current during Glu application (Fig. 1A-a), five of 13 cells showed a fast onset inward current that was rapidly desensitized (Fig. 1A-b). This response was reversible and showed similar amplitude of inward current by a second administration of Glu after a 5-s interval. The cell that demonstrated a steady current showed a sustained inward current over 15 s during the application of Glu (Fig. 1B). The stable response by Glu permitted us to study the voltage-dependence of Gluevoked currents by applying a voltage ramp from 2120 to 180 mV before, during, and after the application of 0.1 mM Glu. The I–V relationships constructed in this way reversed near 0 mV (Fig. 2). The concentration–response relationship for Glu-induced current is shown in Fig. 3. Initial peak amplitude was measured in both non-desensitizing and desensitizing currents and plotted against the Glu concentrations. The concentration–response relations of Glu-induced response can be represented by the following expression: I 5 Imax / f 1 1sKd f Glu g d n g where I is the observed Glu-induced current, Imax is the maximum value of the current, [Glu] is the Glu concentration, Kd is the dissociation constant and n is the Hill coefficient. The values of Kd and n were determined from the plot of p /(12p), where p is the relative conductance (I /Imax ), against [Glu] on logarithmic coordinate as shown in the inset in Fig. 3. Kd was 41 mM and n was 1.75, indicating that two Glu molecules bind to the Glu receptor site to open the channel. This is the first report showing the Glu-induced current occurred in the isolated IHCs of guinea-pig cochlea (Fig. 1). The reversal potential obtained by the voltage ramp
Fig. 1. Glu-induced current response in IHCs at a holding potential of 270 mV. Horizontal bars indicate the application of 0.1 mM Glu. Dotted lines indicate the zero current level. (A) Glu-induced current showing a steady (a) and a fast onset and rapidly desensitized (b) response. (B) Glu applied for over 15 s.
was near 0 mV (Fig. 2), indicating that this channel permits little selectivity of cationic ion. Mechano-electrical transduction (MET) channels, which open by the deviation of hair bundle following mechanical stimulation, is known to be a non-selective cation channel [18]. The Glu-induced current observed in this study is not likely generated by the MET channel stimulated by jet puff stimuli, since the external standard solution did not generate any currents by applying the same puffing system. The concentration–response relationship demonstrated that the dissociation constant (Kd ) at 270 mV was 4.13 10 25 M (Fig. 3). In spiral ganglion cells, the half maximum value for Glu at 270 mV was 4.0310 24 M [16] and 9.1310 25 M [20], which is larger than that in IHCs. This result indicates that the released Glu from IHCs can act
T. Kimitsuki et al. / Brain Research 976 (2003) 135–138
Fig. 2. I–V relationships derived from data shown in inset by plotting membrane current vs. membrane potential before (a), during (b) and after (c) the application of 0.1 mM Glu. Inset: Glu-induced current showing the timing of ramps from 2120 to 180 mV.
onto IHCs themselves before acting on spiral ganglion cells as a transmitter. Glu antagonist reduced the amount of neurotransmitters released by sound stimulation by acting on the presynaptic site on the saccular hair cells [23]. They concluded that a Glu autoreceptor exists on the saccular hair cells. Our results are consistent with Glu autoreceptor existence in IHCs. Increased intracellular Ca 21 concentration after Glu application was reported in vestibular hair cells [4] and
137
cochlear IHCs [13,14], suggesting that Glu acts on hair cells in a positive feedback manner. Glu receptors are classified into the following categories: ionic NMDA types, ionic non-NMDA types, which includes AMPA and kinate types, and metabotrophic. An AMPA receptor subunit GluR4 presents on the presynaptic membrane of IHCs [15], Glu receptor subunit d1 was expressed in hair cells of both the auditory and vestibular systems [22] and NMDA receptors (NR-1) were located in the basolateral of type I vestibular hair cells [9]. NMDA receptors are highly permeable to calcium ion, and the influx of calcium into the hair cell may play a role in augmenting the release of neurotransmitters from the hair cell (positive feedback). Li [14] reported that D-AP5, a selective NMDA-antagonist, completely suppressed the increase in intracellular Ca 21 concentration by Glu application in IHCs, while CNQX, a selective AMPA-antagonist, partially suppressed the Ca 21 increase. They concluded that the autoreceptors existing on the IHC membrane are mainly of the NMDA type, and there are relatively few AMPA type receptors. Guth et al. [6] reported the putative glutamate autoreceptors that were involved in the evoked neurotransmitter release in the frog vestibular hair cells. Hendricson and Guth [8] described that the possibility of autoreceptor as a metabotropic Glu receptor (mGluR) via IP3 mediated signal transduction and ryanodine / caffeine-sensitive Ca store. The precise subtype of the Glu receptor existing on IHCs remains to be determined.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (13671789) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant from The Sound Technology Promotion Foundation.
References
Fig. 3. Concentration–response relationship for Glu-induced current. Each plot is the average of three to 11 sets of data. Bars indicate the standard deviation. The numbers in parentheses are the number of cells examined. The fitted curve was drawn according to the Hill equation. Kd was 41 mM. Inset: Hill plot of data in Fig. 3; the slope gave a Hill coefficient of 1.75.
[1] R.A. Altschuler, C.E. Sheridan, J.W. Horn, R.J. Wenthold, Immunocytochemical localization of glutamate immunoreactivity in the guinea pig cochlea, Hear. Res. 42 (1989) 167–173. [2] R.P. Bobbin, G. Ceasar, M. Fallon, Potassium induced release of GABA and other substances from guinea pig cochlea, Hear. Res. 46 (1990) 83–94. [3] R.P. Bobbin, G. Ceasar, M. Fallon, Changing cation levels (Mg 21 , Ca 21 , Na 1 ) alters the release of glutamate, GABA and other substances from guinea pig cochlea, Hear. Res. 54 (1991) 135–144. [4] G. Devau, J. Lehouelleur, A. Sans, Glutamate receptors on type I vestibular hair cells of guinea-pig, Eur. J. Neurosci. 5 (1993) 1210–1217. [5] M. Eybalin, Neurotransmitters and neuromodulators of the mammalian cochlea, Physiol. Rev. 73 (1993) 309–373. [6] P.S. Guth, A.A. Aubert, A.J. Ricci, C.H. Norris, Differential
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[7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
T. Kimitsuki et al. / Brain Research 976 (2003) 135–138 modulation of spontaneous and evoked neurotransmitter release from hair cells: some novel hypothesis, Hear. Res. 56 (1991) 69–78. D.Z.Z. He, J. Zheng, R. Edge, P. Dallos, Isolation of cochlear inner hair cells, Hear. Res. 145 (2000) 156–160. A.W. Hendricson, P.S. Guth, Transmitter release from Rana pipiens vestibular hair cells via mGluRs: a role for intracellular Ca 11 release, Hear. Res. 172 (2002) 99–109. G. Ishiyama, I. Lopez, A. Ishiyama, Subcellular immunolocalization of NMDA receptor subunit NR-1 in the chinchilla vestibular periphery, Brain Res. 851 (1999) 270–276. Y. Kataoka, H. Ohmori, Activation of glutamate receptors in response to membrane depolarization of hair cells isolated from chick cochlea, J. Physiol. 477 (1994) 403–414. T. Kimitsuki, M. Nishida, H. Kawano, A. Haruta, K. Matsuda, S. Komune, Membrane current possessing the properties of a mechanoelectric transducer current in inner hair cells of guinea-pig cochlea, Brain Res. 915 (2001) 101–103. H. Kuriyama, O. Jenkins, R.A. Altschuler, Immunocytochemical localization of AMPA selective glutamate receptor subunits in rat cochlea, Hear. Res. 80 (1994) 233–240. X. Li, J. Sun, N. Yu, Y. Sun, Z. Tan, S. Jiang et al., Glutamate induced modulation of free Ca 21 in isolated inner hair cells of the guinea pig cochlea, Hear. Res. 161 (2001) 29–34. X. Li, J. Sun, N. Yu, Y. Sun, Z. Tan, S. Jiang et al., D-AP5 blocks the increase of intracellular free Ca 21 induced by glutamate in isolated cochlear IHCs, Chin. Med. J. 115 (2002) 89–93. A. Matsubara, J.H. Laake, S. Davanger, S. Usami, O.P. Ottesen, Organization of AMPA receptor subunits at a glutamate synapses: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti, J. Neurosci. 16 (1996) 4457–4467. T. Nakagawa, S. Komune, T. Uemura, N. Akaike, Excitatory amino
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
acid response in isolated spiral ganglion cells of guinea pig cochlea, J. Neurophysiol. 65 (1991) 715–723. A.S. Nieldzielski, R.J. Wenthold, Expression of AMPA, kainite and NMDA receptors subunits in cochlear and vestibular ganglia, J. Neurosci. 15 (1995) 2338–2353. H. Ohmori, Mechano-electrical transduction currents in isolated vestibular hair cells of the chick, J. Physiol. 359 (1985) 189–217. I. Prigioni, G. Russo, P. Valli, S. Masetto, Pre-and postsynaptic excitatory action of glutamate agonists on frog vestibular receptors, Hear. Res. 46 (1990) 253–259. J. Ruel, C. Chen, R. Pujol, R.P. Bobbin, J.L. Puel, AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig, J. Physiol. 518.3 (1999) 667–680. S. Safieddine, M. Eybalin, Coexpression of NMDA and AMPA / kainite receptor mRNA in cochlear neurons, NeuroReport 3 (1992) 1145–1148. S. Safieddine, R.J. Wenthold, The glutamate receptor subunit d1 is highly expressed in hair cells of the auditory and vestibular systems, J. Neurosci. 17 (1997) 7523–7531. P.A. Starr, W.F. Sewell, Neurotransmitter release from hair cells and its blockade by glutamate-receptor antagonists, Hear. Res. 52 (1991) 23–41. S. Usami, K.K. Osen, N. Zhang, O.P. Ottersen, Distribution of glutamate-like and glutamine-like immunoreactivities in the rat organ of Corti: a light microscopic and semiquantitative electron microscopic analysis with a note on the localization of aspartate, Exp. Brain Res. 91 (1992) 1–11. P. Valli, G. Zucca, I. Prigioni, L. Botta, C. Casella, P.S. Guth, The effect of glutamate on the frog semicircular canal, Brain Res. 330 (1985) 1–9.
Short communication
Glutamate induced currents in isolated inner hair cells from guinea-pig cochlea Takashi Kimitsuki*, Mitsuru Ohashi, Yuki Wada, Takumi Okuda, Shizuo Komune Department of Otorhinolaryngology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan Accepted 1 April 2003
Abstract We investigated the direct action of glutamate (Glu) on the membrane current of isolated inner hair cells of guinea-pig cochlea. Glu elicited inward currents at a holding potential of 270 mV. Eight of 13 cells showed a steady inward current, while five of 13 cells showed a fast and rapidly desensitized current. I–V relationships demonstrated that the reversal potential of Glu-induced current was near 0 mV. Glu-induced currents were dose-dependent, where the half maximum concentration (Kd ) was 41 mM and Hill coefficient (n) was 1.75. 2003 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Auditory, vestibular, and lateral line: periphery Keywords: Glutamate; Inner hair cell; Cochlea; Membrane current; Guinea pig
Auditory stimuli are detected by hair cells in the cochlea of inner ear and are transmitted to the brain by way of the auditory nerves. Inner hair cells (IHCs) are primary transducers of sensory information, forming chemical synapses with dendrites of type I spiral ganglion neurons. Immunocytochemistry studies showed that glutamate (Glu) could be found in hair cells of the guinea pig cochlea [1,24]. Whereas functional Glu receptors were also expressed by spiral ganglion neurons with a postsynaptic localization in the hair cell synapse [12,17,21]. These studies indicate that Glu acts as IHC transmitter or precursor of the transmitter. Glu release from hair cells is stimulated by K 1 in a Ca 21 -dependent manner [2,3,5,10]. In the vestibular organ, Glu exerted presynaptic effects on the release of transmitters from the hair cells [19,23,25]. NMDA receptors (NR-1) showed a basolateral location in type I vestibular hair cells [9], AMPA receptors (GluR4) presented on presynaptic membrane of cochlear IHCs [15], and Glu receptor subunit d1 was expressed in hair cells of both the auditory and vestibular systems [22]. Increased intracellular Ca 21 concentration following Glu application *Corresponding author. Tel.: 181-985-85-2966; fax: 181-985-857029. E-mail address: [email protected] (T. Kimitsuki).
was reported in vestibular hair cells [4] and cochlear IHCs [13,14]. These findings indicate that Glu might act on the presynaptic autoreceptor in a positive feedback manner, though there is no study showing the current change by Glu in hair cells. In the present study, we investigated the direct act of Glu on the membrane current of isolated IHCs, using a patch clamp method. Adult albino guinea-pigs (200–350 g) were killed by rapid cervical dislocation and both bullae were removed. The organ of Corti was dissected from all cochlear turns using a fine needle in Ca-free external solution. The organ of Corti was transferred to the recording chamber and treated with trypsin (1 mg / ml, T-4665, Sigma) for 12 min. Gentle mechanical trituration was carried out and the enzyme was rinsed out by perfusing with standard external solution for at least 10 min before starting any experiments. The shape of IHCs is flask-like. The nucleus is centrally located and the mitochondria are scattered. The most important landmarks for identifying IHCs are the tight neck and the angle between the cuticular plate and the axis of the cell [7,11]. The external standard solution contained (mM): 142 NaCl; 4 KCl; 2 MgCl 2 ; 1 CaCl 2 ; 2 NaH 2 PO 4 ; 8 Na 2 HPO 4 ; adjusted to pH 7.4 with NaOH; glutamate ( L-glutamate acid, 12-0880-2, Sigma-Aldrich Japan) was
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02756-2
136
T. Kimitsuki et al. / Brain Research 976 (2003) 135–138
dissolved in external standard solution. To block K current, the patch pipette solution was Cs-based containing (mM): 144 CsCl; 2 MgCl 2 ; 1 NaH 2 PO 4 ; 8 Na 2 HPO 4 ; 0.5 EGTA; adjusted to pH 7.4 with CsOH. Membrane currents were measured by conventional whole-cell voltage-clamp recordings using an EPC-8 (HEKA, Lambrecht, Germany). Data acquisition was controlled by the software PULSE / PULSEFIT (HEKA, Lambrecht, Germany). Patch pipettes were pulled on a two-stage vertical puller (PP-830 Narishige, Tokyo, Japan) using a 1.2-mm borosilicate glass (GC-1.2, Narishige, Tokyo, Japan). Pipettes had a resistance of 5–8 MV in the bath and were coated with ski wax (Tour-DIA, DIAWax, Otaru, Japan) to minimize capacitance. The cell’s capacitance was 7.962.5 pF (n515) and the residual series resistance was 1867.8 MV (n515). Cells were continuously perfused with external saline and all experiments were performed at room temperature (20–25 8C). Under the voltage-clamp condition at 270 mV, 0.1 mM Glu elicited inward currents in the dissociated IHCs (Fig. 1). The external standard solution, applied under the same system, did not generate any currents (data not shown). While eight of 13 cells showed a steady current during Glu application (Fig. 1A-a), five of 13 cells showed a fast onset inward current that was rapidly desensitized (Fig. 1A-b). This response was reversible and showed similar amplitude of inward current by a second administration of Glu after a 5-s interval. The cell that demonstrated a steady current showed a sustained inward current over 15 s during the application of Glu (Fig. 1B). The stable response by Glu permitted us to study the voltage-dependence of Gluevoked currents by applying a voltage ramp from 2120 to 180 mV before, during, and after the application of 0.1 mM Glu. The I–V relationships constructed in this way reversed near 0 mV (Fig. 2). The concentration–response relationship for Glu-induced current is shown in Fig. 3. Initial peak amplitude was measured in both non-desensitizing and desensitizing currents and plotted against the Glu concentrations. The concentration–response relations of Glu-induced response can be represented by the following expression: I 5 Imax / f 1 1sKd f Glu g d n g where I is the observed Glu-induced current, Imax is the maximum value of the current, [Glu] is the Glu concentration, Kd is the dissociation constant and n is the Hill coefficient. The values of Kd and n were determined from the plot of p /(12p), where p is the relative conductance (I /Imax ), against [Glu] on logarithmic coordinate as shown in the inset in Fig. 3. Kd was 41 mM and n was 1.75, indicating that two Glu molecules bind to the Glu receptor site to open the channel. This is the first report showing the Glu-induced current occurred in the isolated IHCs of guinea-pig cochlea (Fig. 1). The reversal potential obtained by the voltage ramp
Fig. 1. Glu-induced current response in IHCs at a holding potential of 270 mV. Horizontal bars indicate the application of 0.1 mM Glu. Dotted lines indicate the zero current level. (A) Glu-induced current showing a steady (a) and a fast onset and rapidly desensitized (b) response. (B) Glu applied for over 15 s.
was near 0 mV (Fig. 2), indicating that this channel permits little selectivity of cationic ion. Mechano-electrical transduction (MET) channels, which open by the deviation of hair bundle following mechanical stimulation, is known to be a non-selective cation channel [18]. The Glu-induced current observed in this study is not likely generated by the MET channel stimulated by jet puff stimuli, since the external standard solution did not generate any currents by applying the same puffing system. The concentration–response relationship demonstrated that the dissociation constant (Kd ) at 270 mV was 4.13 10 25 M (Fig. 3). In spiral ganglion cells, the half maximum value for Glu at 270 mV was 4.0310 24 M [16] and 9.1310 25 M [20], which is larger than that in IHCs. This result indicates that the released Glu from IHCs can act
T. Kimitsuki et al. / Brain Research 976 (2003) 135–138
Fig. 2. I–V relationships derived from data shown in inset by plotting membrane current vs. membrane potential before (a), during (b) and after (c) the application of 0.1 mM Glu. Inset: Glu-induced current showing the timing of ramps from 2120 to 180 mV.
onto IHCs themselves before acting on spiral ganglion cells as a transmitter. Glu antagonist reduced the amount of neurotransmitters released by sound stimulation by acting on the presynaptic site on the saccular hair cells [23]. They concluded that a Glu autoreceptor exists on the saccular hair cells. Our results are consistent with Glu autoreceptor existence in IHCs. Increased intracellular Ca 21 concentration after Glu application was reported in vestibular hair cells [4] and
137
cochlear IHCs [13,14], suggesting that Glu acts on hair cells in a positive feedback manner. Glu receptors are classified into the following categories: ionic NMDA types, ionic non-NMDA types, which includes AMPA and kinate types, and metabotrophic. An AMPA receptor subunit GluR4 presents on the presynaptic membrane of IHCs [15], Glu receptor subunit d1 was expressed in hair cells of both the auditory and vestibular systems [22] and NMDA receptors (NR-1) were located in the basolateral of type I vestibular hair cells [9]. NMDA receptors are highly permeable to calcium ion, and the influx of calcium into the hair cell may play a role in augmenting the release of neurotransmitters from the hair cell (positive feedback). Li [14] reported that D-AP5, a selective NMDA-antagonist, completely suppressed the increase in intracellular Ca 21 concentration by Glu application in IHCs, while CNQX, a selective AMPA-antagonist, partially suppressed the Ca 21 increase. They concluded that the autoreceptors existing on the IHC membrane are mainly of the NMDA type, and there are relatively few AMPA type receptors. Guth et al. [6] reported the putative glutamate autoreceptors that were involved in the evoked neurotransmitter release in the frog vestibular hair cells. Hendricson and Guth [8] described that the possibility of autoreceptor as a metabotropic Glu receptor (mGluR) via IP3 mediated signal transduction and ryanodine / caffeine-sensitive Ca store. The precise subtype of the Glu receptor existing on IHCs remains to be determined.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (13671789) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant from The Sound Technology Promotion Foundation.
References
Fig. 3. Concentration–response relationship for Glu-induced current. Each plot is the average of three to 11 sets of data. Bars indicate the standard deviation. The numbers in parentheses are the number of cells examined. The fitted curve was drawn according to the Hill equation. Kd was 41 mM. Inset: Hill plot of data in Fig. 3; the slope gave a Hill coefficient of 1.75.
[1] R.A. Altschuler, C.E. Sheridan, J.W. Horn, R.J. Wenthold, Immunocytochemical localization of glutamate immunoreactivity in the guinea pig cochlea, Hear. Res. 42 (1989) 167–173. [2] R.P. Bobbin, G. Ceasar, M. Fallon, Potassium induced release of GABA and other substances from guinea pig cochlea, Hear. Res. 46 (1990) 83–94. [3] R.P. Bobbin, G. Ceasar, M. Fallon, Changing cation levels (Mg 21 , Ca 21 , Na 1 ) alters the release of glutamate, GABA and other substances from guinea pig cochlea, Hear. Res. 54 (1991) 135–144. [4] G. Devau, J. Lehouelleur, A. Sans, Glutamate receptors on type I vestibular hair cells of guinea-pig, Eur. J. Neurosci. 5 (1993) 1210–1217. [5] M. Eybalin, Neurotransmitters and neuromodulators of the mammalian cochlea, Physiol. Rev. 73 (1993) 309–373. [6] P.S. Guth, A.A. Aubert, A.J. Ricci, C.H. Norris, Differential
138
[7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
T. Kimitsuki et al. / Brain Research 976 (2003) 135–138 modulation of spontaneous and evoked neurotransmitter release from hair cells: some novel hypothesis, Hear. Res. 56 (1991) 69–78. D.Z.Z. He, J. Zheng, R. Edge, P. Dallos, Isolation of cochlear inner hair cells, Hear. Res. 145 (2000) 156–160. A.W. Hendricson, P.S. Guth, Transmitter release from Rana pipiens vestibular hair cells via mGluRs: a role for intracellular Ca 11 release, Hear. Res. 172 (2002) 99–109. G. Ishiyama, I. Lopez, A. Ishiyama, Subcellular immunolocalization of NMDA receptor subunit NR-1 in the chinchilla vestibular periphery, Brain Res. 851 (1999) 270–276. Y. Kataoka, H. Ohmori, Activation of glutamate receptors in response to membrane depolarization of hair cells isolated from chick cochlea, J. Physiol. 477 (1994) 403–414. T. Kimitsuki, M. Nishida, H. Kawano, A. Haruta, K. Matsuda, S. Komune, Membrane current possessing the properties of a mechanoelectric transducer current in inner hair cells of guinea-pig cochlea, Brain Res. 915 (2001) 101–103. H. Kuriyama, O. Jenkins, R.A. Altschuler, Immunocytochemical localization of AMPA selective glutamate receptor subunits in rat cochlea, Hear. Res. 80 (1994) 233–240. X. Li, J. Sun, N. Yu, Y. Sun, Z. Tan, S. Jiang et al., Glutamate induced modulation of free Ca 21 in isolated inner hair cells of the guinea pig cochlea, Hear. Res. 161 (2001) 29–34. X. Li, J. Sun, N. Yu, Y. Sun, Z. Tan, S. Jiang et al., D-AP5 blocks the increase of intracellular free Ca 21 induced by glutamate in isolated cochlear IHCs, Chin. Med. J. 115 (2002) 89–93. A. Matsubara, J.H. Laake, S. Davanger, S. Usami, O.P. Ottesen, Organization of AMPA receptor subunits at a glutamate synapses: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti, J. Neurosci. 16 (1996) 4457–4467. T. Nakagawa, S. Komune, T. Uemura, N. Akaike, Excitatory amino
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
acid response in isolated spiral ganglion cells of guinea pig cochlea, J. Neurophysiol. 65 (1991) 715–723. A.S. Nieldzielski, R.J. Wenthold, Expression of AMPA, kainite and NMDA receptors subunits in cochlear and vestibular ganglia, J. Neurosci. 15 (1995) 2338–2353. H. Ohmori, Mechano-electrical transduction currents in isolated vestibular hair cells of the chick, J. Physiol. 359 (1985) 189–217. I. Prigioni, G. Russo, P. Valli, S. Masetto, Pre-and postsynaptic excitatory action of glutamate agonists on frog vestibular receptors, Hear. Res. 46 (1990) 253–259. J. Ruel, C. Chen, R. Pujol, R.P. Bobbin, J.L. Puel, AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig, J. Physiol. 518.3 (1999) 667–680. S. Safieddine, M. Eybalin, Coexpression of NMDA and AMPA / kainite receptor mRNA in cochlear neurons, NeuroReport 3 (1992) 1145–1148. S. Safieddine, R.J. Wenthold, The glutamate receptor subunit d1 is highly expressed in hair cells of the auditory and vestibular systems, J. Neurosci. 17 (1997) 7523–7531. P.A. Starr, W.F. Sewell, Neurotransmitter release from hair cells and its blockade by glutamate-receptor antagonists, Hear. Res. 52 (1991) 23–41. S. Usami, K.K. Osen, N. Zhang, O.P. Ottersen, Distribution of glutamate-like and glutamine-like immunoreactivities in the rat organ of Corti: a light microscopic and semiquantitative electron microscopic analysis with a note on the localization of aspartate, Exp. Brain Res. 91 (1992) 1–11. P. Valli, G. Zucca, I. Prigioni, L. Botta, C. Casella, P.S. Guth, The effect of glutamate on the frog semicircular canal, Brain Res. 330 (1985) 1–9.