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Acta Physiologica Sinica, February 25, 2007, 59 (1): 103-110 http://www.actaps.com.cn 103 * 200433 (1) (NS, 10 µl) NS (10 µl) + (kynurenic acid, KYNA, 5 mmol/l, 10 µl) + 15 NS KYNA 110 db SPL 1 h (auditory brainstem response, ABR) (compound action potential, CAP) N1 (2) NS + ABR CAP KYNA + N1 NS + KYNA + / KYNA R764 Role of glutamate receptors in the spiral ganglion neuron damage induced by acoustic noise ZHANG Yan-Min, MA Bei *, GAO Wen-Yuan, WEN Wen, LIU Hai-Ying Department of Physiology, the Second Military Medical University, Shanghai 200433, China Abstract: The aim of the present study was to investigate the role of glutamate receptors in the damage of spiral ganglion neurons (SGNs) induced by acute acoustic noise. This investigation included in vivo and in vitro studies. In vivo, kynurenic acid (KYNA), a broad-spectrum antagonist of glutamate receptors, was applied to the round window of guinea pigs, and its protective effect was observed. The animals were divided into three groups: control (saline, 0.9%, 10 µl), saline (0.9%, 10 µl) + noise and KYNA (5 mmol/l, 10 µl) + noise. Saline and KYNA were applied to the round window membrane with a microsyringe. The animals were exposed to 110 db SPL of white noise for 1 h. Hearing thresholds for auditory brainstem responses (ABRs) and compound action potentials (CAPs) in all animals were measured before and after treatment. The amplitudes of III waveform of ABR and N1 waveform of CAP and the latency of N1 waveform at different stimulation levels (intensity-amplitude and intensity-latency functions) were also measured. The cochleas were then dissected for transmission electron microscopy (TEM) after final electrophysiological measurement. In vitro, the SGNs of the normal guinea pigs were isolated and glutamate (100 µmol/l or 1 000 µmol/l) was added into the medium. The morphology of the SGNs was examined by light microscopy. In vivo results showed that the hearing function and morphology of the inner ear including hair cells and SGNs in the control group were normal. Compared with that in the control group the thresholds for ABR and CAP (click and tone burst) in saline + noise group were elevated significantly. The input-output functions showed that the amplitudes of III waveform of ABR and N1 waveform of CAP decreased and the latency of N1 waveform increased obviously. There was significant difference in the amplitude and latency between saline + noise group and KYNA + noise group (P<0.05). TEM indicated obvious swelling and vacuoles Received 2006-08-03 Accepted 2006-11-30 This work was supported by the National Natural Science Foundation of China (No. 30470419). * Corresponding author. Tel: +86-21-25074321; Fax: +86-21-25074321; E-mail: mabei2004@yahoo.com.cn

104 Acta Physiologica Sinica, February 25, 2007, 59(1): 103-110 at the terminate of dendrites of SGNs in NS + noise group. On the contrary, the afferent dendrites in KYNA + noise group showed normal appearance without swelling and vacuoles. In vitro experiment showed that the isolated SGNs of guinea pigs obviously swelled and even died after application of 100 µmol/l or 1 000 µmol/l glutamate. These results suggest that noise exposure causes hearing impairment, damage of hair cells and hair cell/afferent synapse and death of SGNs. The antagonist of glutamate receptors provides protective effects against hearing loss and SGN damage. It is inferred that excessive release of glutamate from the inner hair cells induced by noise may be responsible for these damages. Glutamate receptors are involved in the degeneration and death of SGNs. Key words: spiral ganglion neuron; hearing loss; glutamate; glutamate receptors; kynurenic acid (noise-induced hearing loss, NIHL) - (spiral ganglion neurons, SGNs) [1,2] SGNs SGNs ( / ) 40~60 nm [3-5] AMPA kainate NMDA [6-9] AMPA Na + Cl AMPA NMDA Ca 2+ Ca 2+ [10-13] 135 db 30 min 1.5 [14] 115 db 60 min 47% SGNs ( ) [15] ; SGNs SGNs SGNs [16,17] ( ) [18] SGNs [19] (115 db, 5 h) NMDA SGNs (auditory brainstem response, ABR) [20] (kynurenic acid, KYNA) NMDA AMPA KYNA [3] KYNA [ABR (compound action potential, CAP) - ] SGNs SGNs 1 1.1 KYNA A Sigma

105 Hanks Gibco 1.2 45 250~300 g (NS, 0.9%, 10 µl) NS (0.9%, 10 µl) + KYNA (5 mmol/l, 10 µl) + 15 1.3 Khan [21] 2% (40 mg/kg, i.p.) (38±0.5) 10 min (120 cm 60 cm 60 cm) 40 db SPL (B&K 1405) (FJG600-1) 4145 B&K 2610 124 db ±1 db 4 khz 110 db SPL 1 h 1.4 1.4.1 ABR Spirit 2000 (Nicolet ) ABR 2% (40 mg/ kg ) (click) (tone burst)(2 4 6 8 khz) THD-39P Click 0.1 ms 11.1 /s 0.1 ms 10 ms 100~3 000 Hz 128 ABR ABR 1.4.2 CAP click (2 4 6 8 khz) CAP N1 CAP N1 CAP CAP (N1 latency) 1.5 1% 2% 1:1 4 Epon812 700 Å 1.6 SGNs 20% Hanks 0.1 mg/ml 10 min Hanks A 1.7 means±sem EXCEL t P<0.05 2 2.1 KYNA ABR CAP NS NS + click (2 4 6 8 khz) ABR 30~39.5 db CAP 29~36.5 db KYNA + ABR 7.5~11 db CAP 6.5~13.5 db NS + ABR CAP NS KYNA + (P<0.05)( 1) NS NS ABR CAP / SGNs 2.2 KYNA ABR CAP NS click ABR CAP N1 NS + 90 80 70 60 50 40 30 db click ABR CAP N1 - KYNA + click ABR CAP N1

106 Acta Physiologica Sinica, February 25, 2007, 59(1): 103-110 1. KYNA Fig. 1. Effect of KYNA on noise-induced threshold shifts of the auditory brainstem response (A) and compound action potential (B). * P<0.05 vs NS group and KYNA + noise group. 2. KYNA Fig. 2. Effect of KYNA on noise-induced amplitudes of the auditory brainstem response (A) and compound action potential (B). * P<0.05 vs KYNA + noise group. NS + (P< 0.05)( 2) 2.3 KYNA CAP N1 NS CAP N1 0.8~1.6 ms NS + 90 80 70 60 50 40 30 db click N1 - KYNA + click CAP N1 NS + (P<0.05) (P>0.05)( 3) 2.4 KYNA / / NS (outer hair cells, OHCs) / 3. KYNA N1 Fig. 3. Effect of KYNA on noise-induced N1 latency of compound action potential. * P<0.05 vs KYNA + noise group. ( 4) NS + OHCs /

107 4. / Fig. 4. Transmission electron microscopy pictures of one outer hair cell and the hair cell/afferent synapse in the saline group. A: The outer hair cell displayed normal appearance and structure. Scale bar, 5 µm. B: The hair cell/afferent synapse showed normal structure without swelling and intracellular vacuoles. Scale bar, 2.5 µm. 5. NS + / Fig. 5. Transmission electron microscopy pictures of the outer hair cell and the hair cell/afferent synapse in NS + noise group. A: The outer hair cell showed swelling with small vacuoles beneath the membrane (arrows). Scale bar, 2.5 µm. B: A large vacuole appeared in the cytoplasm of one outer hair cell. Scale bar, 2 µm. C: Some relative large vacuoles in the afferent dendrites. Scale bar, 2.5 µm. D: The afferent dendrites had a severe swelling appearance. Scale bar, 2.5 µm. E: The mitochondria in the afferent dendrites showed edema with vacuoles inside. Scale bar, 0.5 µm. ( 5) KYNA + OHCs / NS ( 6) 2.5 SGNs SGNs 5~6 h 100 µmol/l 1 000 µmol/l 1 SGNs SGNs ( 7)

108 Acta Physiologica Sinica, February 25, 2007, 59(1): 103-110 6. KYNA + / Fig. 6. Transmission electron microscopy pictures of the outer hair cells and the hair cell/afferent synapse in KYNA + noise group. A, B: The outer hair cells displayed normal appearance without swelling and vacuoles inside. The hair cell/afferent synapse showed normal structure. Scale bar, 5 µm and 2.5 µm, respectively. C: There was no swelling and vacuoles in the dendrites and the mitochondria had normal structure. Scale bar, 0.4 µm. 7. Fig.7. The toxic effect of glutamate on the isolated spiral ganglion neurons of guinea pigs. A: The isolated spiral ganglion neurons in the normal medium showed normal appearance. Scale bar, 25 µm. B: Spiral ganglion neurons showed swelling and vacuoles 1 h after application of glutamate in the medium. Scale bar, 25 µm. C: Degeneration and death of spiral ganglion neurons after application of glutamate. Scale bar, 25 µm. 3 SGNs [22] SGNs (1) SGNs - SGNs [2] (2) SGNs [23]

109 [24,25] SGNs NS + click (2 4 6 8 khz) ABR 30~39.5 db CAP 29~36.5 db ABR CAP KYNA ABR 7.5~ 11 db CAP 6.5~13.5 db NS + (P<0.05) KYNA NIHL NIHL CAP CAP N1 / N1 (0.5 ms) N1 0.8~1.6 ms NS + click N1 / AMPA AMPA Na + Cl KYNA + click N1 / KYNA SGNs CAP - SGNs [26] NS + 90 80 70 60 50 40 30 db click CAP N1 ABR SGNs SGNs KYNA + click CAP N1 NS + KYNA SGNs SGNs NS + OHCs KYNA KYNA + OHCs OHCs OHCs OHCs KYNA SGNs SGNs 20 h 100 µmol/l 1 000 µmol/l SGNs SGNs SGNs (1 µmol/l, 10 µmol/l) ( )

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