1996 Fiscal Year Final Research Report Summary
Optical Recording of Membrane Potential on Inner Ear Cells Using a Voltage-Sensitive Dye.
| Project/Area Number |
07457407
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Otorhinolaryngology
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| Research Institution | Kansai Medical University |
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Principal Investigator |
YAMASHITA Toshio Kansai Medical University, Otolaryngology, Professor and Chairman, 医学部, 教授 (10077654)
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| Co-Investigator(Kenkyū-buntansha) |
OHNISHI Sumio Kansai Medical University, assistant, 医学部, 助手 (80257914)
HARADA Narinobu Kansai Medical University, assistant, 医学部, 助手 (00198920)
TOMODA Kohichi Kansai Medical University, associate professor, 医学部, 助教授 (50164041)
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| Project Period (FY) |
1995 – 1996
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| Keywords | optical recording / voltage-sensitive dye / inner ear cells / membrane potential |
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| Research Abstract |
1. Optical measurement of membrane potential is a method to quantify changes in the absorption of light by voltage-sensitive dyes or immunofluorescence corresponding to membrane potential changes evoked in cells. An optical measurement system (ARGUS-50/PDA ; Hamamatsu Photonics K.K., Hamamatsu, Shizuoka) was set up and used for recording a membrane potential.
2. An absorptional dye for membrane potential measurement (NK3041 ; Nippon Kankoh Shikiso Kenkyusyo, Okayama, Japan) was used as the voltage-sensitive dye. To stimulate cells into depolarization, a high K^+ solution was used as the perfusion.
3. We used acutely dissociated outer hair cells from guinea pigs initially, but responses to the stimuli could not detected because of its motility while depolarizing. When cultured spiral ganglion cells (SGCs) and vestibular ganglion cells (VGCs) from newborn mice were tested, responses were detectable. Cultured cells were considered to be stable in the perfusion system, allowing measurement.
4. Ultimately, it was in only 22.2% of the cells tested that we were successful in detecting optical changes. The occurrence of substantial noises in the optical measurement with stimulation by replacement of the physiological standard solution and that the change in absorbance of NK3041 as the signal is as little as 0.3%, leading to an extremely small S/N ratio, are possible factors that would seem to account for this low success rate. This results indicated that it would be difficult to detect minute membrane potential changes under the conditions of measurement in isolated SGCs and VGCs with the optical measurement system using NK3041.
5. Further study is necessary in order to establish sensitive dyes that show greater optical reactions to membrane potential changes. We had also fried to investigate to detect the spacial patterning of the voltage change producted by electrical stimulation of the auditory nerve in brain stem slices using optical measurement.
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