Membrane traffic in outer hair cells of the adult mammalian cochlea

Kaneko, Toshihiko; Harasztosi, Csaba; Mack, Andreas F.; Gummer, Anthony W.

doi:10.1111/j.1460-9568.2006.04796.x

Cited by 23 publications

(25 citation statements)

References 65 publications

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“…There are several possible sources of the large variations seen in FRAP measurements from the OHC apex. First, the OHC apical pole has been demonstrated to be the site of rapid membrane endocytosis possibly for both the recycling and sorting of membrane constituents (Meyer et al 2001;Griesinger et al 2004;Kaneko et al 2006), and the presence and turnover of vesicles may contribute low levels of fluorescence that affect FRAP measurements. Second, the apical membrane geometry is more complex than either the lateral or basal regions.…”

Section: Discussionmentioning

confidence: 99%

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Organ

Raphael

2009

JARO

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The outer hair cell (OHC) lateral plasma membrane houses the transmembrane protein prestin, a necessary component of the yet unknown molecular mechanism (s) underlying electromotility and the exquisite sensitivity and frequency selectivity of mammalian hearing. The importance of the plasma membrane environment in modulating OHC electromotility has been substantiated by recent studies demonstrating that membrane cholesterol alters prestin activity in a manner consistent with cholesterol-induced changes in auditory function. Cholesterol is known to affect membrane material properties, and measurements of lipid lateral mobility provide a method to asses these changes in living OHCs. Using fluorescence recovery after photobleaching (FRAP), we characterized regional differences in the lateral diffusion of the lipid analog di-8-ANEPPS in OHCs and investigated whether lipid mobility, which reflects membrane fluidity, is sensitive to membrane cholesterol. FRAP experiments revealed quantitative differences in lipid lateral mobility among the apical, lateral, and basal regions of the OHC and demonstrated that diffusion in individual regions is uniquely sensitive to cholesterol manipulations. Interestingly, in the lateral region, both cholesterol depletion and loading significantly reduced the effective diffusion coefficient from control values. Thus, the fluidity of the OHC lateral plasma membrane is regulated by cholesterol levels in a non-monotonic manner, suggesting that the overall material properties of the lateral plasma membrane are optimally tuned for OHC function in the native state. These results support the idea that the cholesteroldependent regulation of prestin function and electromotility correlates with changes in the properties of the lipid environment that surrounds and supports prestin.

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Section: Discussionmentioning

confidence: 99%

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Organ

Raphael

2009

JARO

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“…Indeed, various markers have been used, such as sulforhodamine (Lichtman et al 1985), fluorescently labeled antibodies (Kraszewski et al 1996;Westphal et al 2008), styryl dyes (i.e., FM dyes; Betz and Bewick 1992), and fluorescent dextrans (Holt et al 2003). Among these markers, FM dyes have proved to be valuable in a number of different preparations, including neuromuscular junctions (NMJ) from the frog (Betz and Bewick 1992), snake (Teng et al 1999), lizard (Lindgren et al 1997), and larval Drosophila (Ramaswami et al 1994); hippocampal neurons in culture (Ryan et al 1993) and acute slice (Zakharenko et al 2001); goldfish retinal bipolar cells (Zenisek et al 2000); Caenorhabditis elegans neurons (Kay et al 1999); rat calyx of Held (de Lange et al 2003); inner and outer hair cells (Griesinger et al 2002;Kaneko et al 2006); and a variety of nonneuronal preparations (for a review, see Cochilla et al 1999). …”

Section: Discussionmentioning

confidence: 99%

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

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The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K + ) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. Reagents FM dye(s)Dye concentrations are usually 2-10 µM for FM1-43 or FM4-64, and 25-40 µM for FM2-10. The dyes readily dissolve in water; stocks of 1-5 mM concentration should be prepared and can be stored at 4˚C. Neuronal tissue or cell sample of interest Equipment Imaging setupAn upright epifluorescence microscope equipped with a 40×-60× water-immersion objective is optimal. When working with cell cultures, the advantages of high-resolution oil-immersion objectives (e.g., 100×/1.4 numerical aperture [NA]) can be exploited by using an inverted setup. For FM1-43, maximal fluorescence is at 465-nm excitation/560-nm emission (Henkel et al. 1996); a conventional fluorescein filter set (435-nm excitation) can also be used. Because the major concern in FM imaging is phototoxicity (which appears usually before any clear signs of photobleaching and alters the ability of the preparations to recycle vesicles), keep the illumination to a minimum by including neutral-density filters. For example, when using a 40×/0.75-NA water-immersion objective with a 100-W mercury lamp, 5%-15% transmission is sufficient. On confocal setups, an argon 488-nm laser line is typically used for excitation, but special care has to be taken to avoid phototoxicity. Images are typically acquired by charge-coupled device (CCD) cameras or photomultiplier tubes, which allows for fast acquisition rates and software-based quantitative image analysis.Neuronal stimulator (see Step 3)

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“…The dye was applied to the excised cochlear preparation for 20 s. FM 1-43 dye quickly enters hair cells through non-specific cation channels (Meyers et al 2003). The application time was short to minimize signal contamination by dye entry through apical endocytosis (Gale et al 2001;Griesinger et al 2002;Griesinger et al 2004;Griesinger et al 2005;Kaneko et al 2006;Meyers et al 2003). The dye was then rinsed away under constant perfusion for another 20 s. FM 1-43 fluorescence within the organ of Corti was visualized using the 40Â water immersion objective using a mercury arc lamp and a filter set (XF21, Omega Optical, Brattleboro, VT).…”

Section: Fm 1-43 Dye Applicationmentioning

confidence: 99%

Altered Traveling Wave Propagation and Reduced Endocochlear Potential Associated with Cochlear Dysplasia in the BETA2/NeuroD1 Null Mouse

Xia

Visosky

Cho

et al. 2007

JARO

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The BETA2/NeuroD1 null mouse has cochlear dysplasia. Its cochlear duct is shorter than normal, there is a lack of spiral ganglion neurons, and there is hair cell disorganization. We measured vertical movements of the tectorial membrane at acoustic frequencies in excised cochleae in response to mechanical stimulation of the stapes using laser doppler vibrometry. While tuning curve sharpness was similar between wild-type, heterozygotes, and null mice in the base, null mutants had broader tuning in the apex. At both the base and the apex, null mice had less phase lag accumulation with increasing stimulus frequency than wild-type or heterozygote mice. In vivo studies demonstrated that the null mouse lacked distortion product otoacoustic emissions, and the cochlear microphonic and endocochlear potential were found to be severely reduced. Electrically evoked otoacoustic emissions could be elicited, although the amplitudes were lower than those of wild-type mice. Cochlear cross-sections revealed an incomplete partition malformation, with fenestrations within the modiolus that connected the cochlear turns. Outer hair cells from null mice demonstrated the normal pattern of prestin expression within their lateral walls and normal FM 1-43 dye entry.Overall, these data demonstrate that while tonotopicity can exist with cochlear dysplasia, traveling wave propagation is abnormally fast. Additionally, the presence of electrically evoked otoacoustic emissions suggests that outer hair cell reverse transduction is present, although the acoustic response is shaped by the alterations in cochlear mechanics.

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Membrane traffic in outer hair cells of the adult mammalian cochlea

Cited by 23 publications

References 65 publications

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Altered Traveling Wave Propagation and Reduced Endocochlear Potential Associated with Cochlear Dysplasia in the BETA2/NeuroD1 Null Mouse

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