Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

Kitani, Rei; Kalinec, Federico

doi:10.3791/2965

Cited by 3 publications

(8 citation statements)

References 12 publications

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“…Temporal bones were obtained from young albino guinea pigs (Cavia porcellus, 200-300 g) of either sex, euthanized with CO 2 according to procedures approved by House Research Institute's Institutional Animal Care and Use Committee. OHCs, isolated as described in the literature (27,28), were transferred to glass-bottomed experimental dishes containing L-15 medium with 25 mM HEPES (Sigma, St. Louis, MO), observed with Nomarski differential interference contrast (DIC) optics on an Axiovert 135TV inverted microscope with a 63Â/1.2 C-Apochromat objective (Zeiss, Thornwood, NY), and used within 2 h after isolation. Osmolarity was measured with a freezing-point osmometer (mOsmette 5004, Precision Systems, Natick, MA), and adjusted to 305-310 mOsm with D-glucose or distilled water.…”

Section: Isolation Of Ohcsmentioning

confidence: 99%

“…External alternate electrical field A full description of the equipment and procedures is available in the literature (27,28). Briefly, suspended OHCs were placed in the experimental dish between two silver wire electrodes (f ¼ 0.25 mm) with a tip distance of 0.8 mm.…”

Section: Electrical Stimulationmentioning

confidence: 99%

“…Cell longitudinal axes were kept parallel to the applied EAEF by rotating the recording chamber. Illumination provided by a light-emitting-diode light source (High Power LED System-36AD3500, Lightspeed Technologies, Campbell, CA), electrical stimulation, and image capture were digitally synchronized by control circuitry and software developed by House Research Institute's Engineering Core (27,28).…”

Section: Electrical Stimulationmentioning

confidence: 99%

“…Images of isolated OHCs were captured in AVI format at 1000 frames/s (fps) using the ultra-high-speed Photron Fastcam X 1024 PCI camera (Photron USA, San Diego, CA). Cell images were analyzed off-line using ProAnalyst software (Xcitex, Cambridge, MA) and further processed using Excel software (Microsoft, Redmond,WA) (27,28). Statistical analysis, including analysis of variance, contingency, and nonlinear regression techniques, were performed using JMP 9 software (SAS Institute, Cary, NC) and p % 0.05 as the criterion for statistical significance.…”

Section: Data Handlingmentioning

confidence: 99%

“…In previous publications, we provided a general description of OHC motility in response to osmotic challenges (25,26) and electrical stimulation (25)(26)(27)(28). Here, we use polystyrene and carboxylated microspheres (MS) as membrane markers and high-speed video recording to perform a detailed analysis of the local responses of the lateral wall of electrically and osmotically stimulated OHCs.…”

Section: Introductionmentioning

confidence: 99%

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Microdomains Shift and Rotate in the Lateral Wall of Cochlear Outer Hair Cells

Kitani

Park

Kalinec

2013

Biophysical Journal

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Outer hair cell (OHC) electromotility, a response consisting of reversible changes in cell length and diameter induced by electrical stimulation, confers remarkable sensitivity and frequency resolution to the mammalian inner ear. Looking for a better understanding of this mechanism, we labeled isolated guinea pig OHCs with microspheres and, using high-speed video recording, investigated their movements at the apical, mid, and basal regions of osmotically and electrically stimulated cells. After hypoosmotic challenge, OHCs shortened and their diameter increased, with microspheres moving always toward the central plane; iso-osmolarity returned OHCs to their original shape and microspheres to their original positions. Under electrical stimulation, microspheres exhibited robust movements, with their displacement vectors changing in direction from random to parallel to the longitudinal axis of the cells with peak reorientation speeds of up to 6 rad/s and returning to random after 5 min without stimulation. Alterations in plasma-membrane cholesterol levels as well as cytoskeleton integrity affected microsphere responses. We concluded that microspheres attach to different molecular microdomains, and these microdomains are able to shift and rotate in the plane of the OHC lateral wall with a dynamics tightly regulated by membrane lipid composition and the cortical cytoskeleton.

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Section: Isolation Of Ohcsmentioning

confidence: 99%

Section: Electrical Stimulationmentioning

confidence: 99%

Section: Electrical Stimulationmentioning

confidence: 99%

Section: Data Handlingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Microdomains Shift and Rotate in the Lateral Wall of Cochlear Outer Hair Cells

Kitani

Park

Kalinec

2013

Biophysical Journal

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PKCα-Mediated Signals Regulate the Motile Responses of Cochlear Outer Hair Cells

Park

Kalinec

2015

Biophysical Journal

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There is strong evidence that changes in the actin/spectrin-based cortical cytoskeleton of outer hair cells (OHCs) regulate their motile responses as well as cochlear amplification, the process that optimizes the sensitivity and frequency selectivity of the mammalian inner ear. Since a RhoA/protein kinase C (PKC)-mediated pathway is known to inhibit the actin-spectrin interaction in other cell models, we decided to investigate whether this signaling cascade could also participate in the regulation of OHC motility. We used high-speed video microscopy and confocal microscopy to explore the effects of pharmacological activation of PKCα, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ with lysophosphatidic acid (LPA) and their inhibition with bisindolylmaleimide I, as well as inhibition of RhoA and Rho-associated protein kinase (ROCK) with C3 and Y-27632, respectively. Motile responses were induced in isolated guinea pig OHCs by stimulation with an 8 V/cm external alternating electrical field as 50 Hz bursts of square wave pulses (100 ms on/off). We found that LPA increased expression of PKCα and PKCζ only, with PKCα, but not PKCζ, phosphorylating the cytoskeletal protein adducin of both Ser-726 and Thr-445. Interestingly, however, inhibition of PKCα reduced adducin phosphorylation only at Ser-726. We also determined that LPA activation of a PKCα-mediated signaling pathway simultaneously enhanced OHC electromotile amplitude and cell shortening, and facilitated RhoA/ROCK/LIMK1-mediated cofilin phosphorylation. Altogether, our results suggest that PKCα-mediated signals, probably via adducin-mediated inhibition of actin-spectrin binding and cofilin-mediated depolymerization of actin filaments, play an essential role in the homeostatic regulation of OHC motility and cochlear amplification.

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Motile responses of cochlear outer hair cells stimulated with an alternating electrical field

Kitani¹,

Kakehata²,

Kalinec³

2011

Hearing Research

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The goal of the present study was to evaluate and characterize the motile responses of guinea pig OHCs, stimulated at frequencies varying from 50 Hz to 4 kHz, using high-definition, high-speed video recording and fully automatic image analysis software. Cells stimulated in continuous, burst and sweeping modes with an external alternating electrical field showed robust fast and slow motility, which were dependent on frequency, mode and intensity of stimulation. In response to continuous stimulation, electromotile amplitude ranged from 0.3 % to 3.2 % of total cell length, whereas cell length usually decreased in amounts varying from 0.1 % to 4.3 %. Electromotile amplitude in OHCs stimulated with square wave's sweeps was near constant up to 200 Hz, progressively decreased between 200 Hz and 2 kHz, and then remained constant up to 4 kHz. In continuous and burst modes electromotility followed cycle-by-cycle the electrical stimulus, but it required 1 to 2 s to fully develop and reach maximal amplitude. Instead, slow cell length changes started about 0.6 s after the beginning and continuously developed up to 3 s after the end of electrical stimulation. Incubation of OHCs with 10 mM salicylate affected electromotility but not slow motility, whereas incubation with 3 mM gadolinium affected both. Thus, combination of external electrical stimulation, high-speed video recording and advanced image analysis software provides information about OHC motile responses at acoustic frequencies with an unprecedented detail, opening new areas of research in the field of OHC mechanics.

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Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

Cited by 3 publications

References 12 publications

Microdomains Shift and Rotate in the Lateral Wall of Cochlear Outer Hair Cells

Microdomains Shift and Rotate in the Lateral Wall of Cochlear Outer Hair Cells

PKCα-Mediated Signals Regulate the Motile Responses of Cochlear Outer Hair Cells

Motile responses of cochlear outer hair cells stimulated with an alternating electrical field

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