Summary

Исследование космического подвижности ее клеток волос с Сочетание внешнего переменного электрического поля и стимулирование Высокоскоростной анализ изображений

Published: July 18, 2011
doi:

Summary

Надежный метод для исследования внешней волосковых клеток (OHC) подвижных ответов, в том числе electromotility, медленный моторики и изгиб, описывается. OHC подвижность, вызванные стимуляцией с внешнего переменного электрического поля, и метод использует преимущества высокоскоростной записи изображения, светодиодной подсветки, а в прошлом поколении анализа изображений программное обеспечение.

Abstract

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name “hair cells” derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape —elongate, shorten and bend— in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.

OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.

The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies.

We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

Protocol

1. Выделение OHCs Начните эту процедуру уборки височных костей из морских свинок, мышей или вашей модели млекопитающих животных. Затем откройте височных костей использованием молоточек щипцы, чтобы разоблачить улитки, и погрузить их в Лейбовиц L-15. Удалить кости избыток аккура…

Discussion

Экспериментальный метод, представленные здесь дает оценки OHC подвижных ответы в кГц без каких-либо ограничений на движение клетки. Различные протоколы стимуляции, дополнительных маркеров (микросферы), а также изменения в ориентации ячейки по отношению к электрическому полю, позволяют…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

Работа выполнена при поддержке Национального института здоровья Гранты R01DC10146/R01DC010397, NIDCD P30 DC006276 исследований Core, и вузов. Ее содержание исключительно ответственности авторов и не обязательно отражают официальную точку зрения NIH или вуза. Авторы заявляют ни одна из существующих или потенциальных конфликтов интересов.

Materials

Name of the reagent Company Catalogue number Comments (optional)
Leibovitz’s L-15 Gibco 21083  
Collagenase (Type 4) Sigma-Aldrich C5138 1mg/mL in L-15

Referencias

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  8. Kitani, R., Kakehata, S., Kalinec, F. Motile responses of cochlear outer hair cells stimulated with an alternating electrical field. Hearing Research. , (2011).
  9. Dallos, P., Evans, B. N. High-frequency outer hair cell motility: corrections and addendum. Science. 268, 1420-1421 (1995).
  10. Frank, G., Hemmert, W., Gummer, A. W. Limiting dynamics of high-frequency electromechanical transduction in outer hair cells. Proc. Natl. Acad. Sci. 96, 4420-4425 (1999).
  11. Santos-Sacchi, J. On the frequency limit and phase of outer hair cell motility: effects of the membrane filter. J. Neurosci. 12, 1906-1916 (1992).
  12. Inoué, S. . Video Microscopy. , (1986).

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Citar este artículo
Kitani, R., Kalinec, F. Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis. J. Vis. Exp. (53), e2965, doi:10.3791/2965 (2011).

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