This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

1. Cell Culture
Note: All cell culturing protocols must be performed using proper cell culture techniques (for reference see the first 3 Chapters of Cell Biology: A Laboratory Handbook, Volume I 10). HEI-OC1 cells do not require any additional coating or treatment of the cell culture dishes for proper adherence and growth. Very important: do not use glassware dishes for cell culture purposes; the phenotype and biological response of the cells to pharmacological drugs will change (G Kalinec &#38; F Kalinec, unpublished); conventional plastic cell culture dishes are recommended (see Table of Materials/Equipment). Pay special attention to aseptic techniques to avoid contaminations, but never use antibiotics (e.g., ampicillin or streptomycin) with HEI-OC1 cells. If necessary, use amphotericin B. While HeLa and HEK-293 cells have been used as control in previous studies with HEI-OC1 8, any other cell line could be acceptable for this purpose.
<ol>
	<li>Resuscitation of Frozen HEI-OC1 Cells 
	Note: This protocol can also be used with other cell lines from the same transgenic mouse developed in our laboratory, such as OC-k3, from organ of Corti 11,12, and SV-k1, from stria vascularis 13,14.
	<ol>
		<li>Remove a vial of cryopreserved HEI-OC1 cells from liquid N2, and place it in a water bath at 37 &#176;C. Submerge only the lower half of the vial, and allow it to thaw until only a small amount of ice remains in the vial. Wipe the outside of the vial with 70% alcohol.</li>
		<li>Pipette the cells from the vial and deliver the entire volume (~ 1.5 x 105 cells/ml) slowly, drop by drop, into a 15 ml conical tube containing 10 ml of a pre-warmed growth medium, like Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Remove DMSO by centrifuging the tube at 300-500 x g for 5 min, discarding the supernatant and re-suspending the cells in 9 ml of fresh growth medium (DMEM + 10% FBS). Disaggregate clumps or sheets of cells by flux and reflux of the suspended cells, from the pipette to the medium and vice-versa, three to four times with the tip of the pipette touching the bottom of the tube.</li>
		<li>Place the total volume of cells suspended in growth medium in 100 mm-diameter untreated, plastic cell culture dishes.</li>
		<li>Incubate the cells at 33 &#176;C with 10% CO2 (permissive conditions).</li>
	</ol>
	</li>
	<li>Subculture of HEI-OC1 Cells 
	Note: Prior to confluence (~80%) the cells should be brought into suspension and sub-cultured in order to prevent the culture dying. This protocol can also be used with other cell lines developed in our laboratory from the same transgenic mouse, such as OC-k3, from organ of Corti 11,12, and SV-k1, from stria vascularis 13,14.
	<ol>
		<li>Remove old medium and wash the cells with 2 ml of PBS.</li>
		<li>Cover the cell monolayer with a solution of 0.25% trypsin, using 1 ml per 25 cm2 of surface area. To move onto the next step more than 40% of the cells must be detached. Examine the cells using an inverted microscope and, if necessary, &#34;slap or tap&#34; the culture dishes gently to release any remaining attached cells. 
		Note: Take care as trypsinization for long periods can cause cell death; not enough and the transferred cells will insufficient for the planned studies.</li>
		<li>Resuspend the cells, following the procedure described in 1.1.3, and transfer them to a 15 ml conical tube.</li>
		<li>Centrifuge for 5 min at 300-500 x g, discard the medium, collect the cells with fresh growth medium and seed them in, at least, four 100 mm-diameter cell culture dishes. The number of cells per dish will depend on the amount of cells recovered. Incubate the cells at 33 &#176;C with 10% CO2 (P-HEI-OC1) and divide again as many times as necessary for the planned experiments or for generating a new stock.</li>
		<li>For stock preparation, replace 100 mm-diameter dishes by 250-550 ml cell culture flasks; for experiments, smaller dishes or multi-wells plates may be more adequate.</li>
		<li>For differentiation, incubate HEI-OC1 cells at permissive conditions until they reach ~80% confluence; then move the dishes to 39 &#176;C with 5% CO2 (NP-HEI-OC1) for 2 to 3 weeks. 
		Note: Cells will progressively stop proliferating and dying. Change the medium every other day to remove dead cells. Depending on the original number of cells, usually after ~ 4 weeks no more cells will be available for experiments. Differentiation starts as soon as the cells are placed at NP conditions, but not every cell differentiate at the same pace. Importantly, prestin expression and membrane localization increases during the differentiation process (see Representative Results, Figure 4), providing a qualitative and quantitative indication of the level of differentiation.</li>
	</ol>
	</li>
</ol>
2. Drug Cytotoxicity Studies
Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies (see Representative Results, Figure 1).
<ol>
	<li>Cell Viability (MTT Assay)
	<ol>
		<li>Collect P-HEI-OC1 cells by following the procedure described in 1.2, and count them with either an automatic cell counter or hemocytometer. Adjust the concentration to 2.0 x 105 cells/ml.</li>
		<li>Seed the cells on 96-well clear flat bottom plates (100 &#181;l per well), incubate overnight for attachment to the substrate, and then treat them with the drugs of interest. Do not forget to include blanks and non-treated cells (control)!</li>
		<li>After drug treatment (usually 24 or 48 hr at 33 &#176;C), perform the MTT assay following the manufacturer&#39;s protocol. In general, the protocols for this assay have the following steps:
		<ol>
			<li>Add 10 &#181;l of MTT reagent to each well.</li>
			<li>Incubate the plate at 37 &#176;C for 2 to 4 hr until purple dye is visible, and then add 100 &#181;l of the MTT Detergent Reagent to each well. Do not shake the plate.</li>
			<li>Cover the plate and leave it in the dark for 2 to 4 hr at 37 &#176;C.</li>
			<li>Use a microplate plate reader to measure absorbance at 570 nm in each well, including the blanks (growth medium alone). Normalize data using the average OD in control cells as 100% of viability.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Caspase 3/7 Activation Assay 
	Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies.
	<ol>
		<li>Collect HEI-OC1 cells by following the procedure described in 1.2, and count them with either an automatic cell counter or a hemocytometer. Adjust the concentration to 2.0 x 105 cells/ml.</li>
		<li>Seed the cells on white-walled 96-well plates (100 &#181;l per well), incubate overnight for attachment to the substrate, and then treat them with the drugs of interest. Do not forget to include blanks and non-treated cells (control)!</li>
		<li>After drug treatment (usually 24 or 48 hr at 33 &#176;C), perform the caspase assay following the respective manufacturer&#39;s protocol. In general, the protocols for this assay have the following steps:
		<ol>
			<li>Prepare the reagents, mix them well, and allow them to equilibrate to room temperature.</li>
			<li>Remove the cells from the incubator and allow the plates to equilibrate to room temperature.</li>
			<li>Add the indicated amount of reagent to each well, being careful to avoid cross-contamination by not touching wells containing different samples with the same pipette tips.</li>
			<li>Cover the plate and mix for 30 sec using a plate shaker at 300-500 rpm.</li>
			<li>Incubate the plate at room temperature for a period of, at least, 30 min. Determine the optimal incubation period empirically. If the room temperature fluctuates use a constant-temperature incubator, because temperature fluctuations will affect luminescence reading.</li>
			<li>Measure luminescence in each well, and normalize values using average luminescence in control cells as 100% of caspase activation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell Division (Proliferation)
	<ol>
		<li>Collect HEI-OC1 cells by following the procedure described in 1.2, and count them with either an automatic cell counter or a hemocytometer. Adjust the concentration to 2.0 x 105 cells/ml.</li>
		<li>Seed the cells on 96-well clear flat bottom plates (100 &#181;l per well), incubate overnight at permissive conditions for attachment to the substrate, and then treat them with the drugs of interest. Don&#39;t forget to include blanks and non-treated cells (control)!</li>
		<li>One hour after treatment, add to each well 1 &#181;l of prepared 100x BrdU (5-Bromo-2&#8242;-deoxyuridine) solution, and return the plates to the incubator at 33 &#176;C.</li>
		<li>After 12, 24 and 48 hr, remove the existing medium from the corresponding plates and wash each well once with PBS.</li>
		<li>Perform the cell proliferation assay following the manufacturer&#39;s protocol. In general, the protocols for this assay have the following steps:
		<ol>
			<li>Prepare the reagents, including fixing/denaturing solution, wash buffer, primary antibody detection solution, secondary antibody detection solution, and BrdU solution as indicated in the manufacturer&#39;s protocol.</li>
			<li>Add the fixing/denaturing solution to each well, in the amounts indicated in the manufacturer&#39;s protocol, and keep the plate at room temperature for 30 min.</li>
			<li>Remove the fixing/denaturing solution and add the primary antibody at the concentration indicated in the manufacturer&#39;s protocol. Keep the plate at room temperature for 1 hr.</li>
			<li>Remove the solution with the primary antibody, wash the plate 3 times with wash buffer, and then add the secondary antibody at the concentration indicated in the manufacturer&#39;s protocol. Keep the plate with the secondary antibody at room temperature for 30 min.</li>
			<li>Remove the solution with the secondary antibody, wash the plate 3 times with wash buffer, and add the substrate. After 10 min incubation at room temperature, add the STOP solution. Be careful: Control the change in color; if the solution becomes very dark, stop the reaction prior to the standard development time of 10 min.</li>
			<li>Read absorbance at 450 nm within 30 min of adding the STOP Solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cytotoxicity 
	Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies.
	<ol>
		<li>Incubate at permissive conditions, usually for 24-48 hr, HEI-OC1 cells in cell culture medium alone (control) or medium plus the drugs of interest.</li>
		<li>At the end of the treatment, wash the cells three times with PBS, and detach using 1 ml per 25 cm2 of surface area of a non-enzymatic cell dissociation solution for 3 min.</li>
		<li>After detaching, collect and pellet the cells by centrifugation at 3,000 x g for 5 min, remove the supernatant, and stain the cells for 15 min in the dark with the reagent included in cytotoxicity assay.</li>
		<li>Determine the number of live HEI-OC1 cells by flow cytometry as indicated by the manufacturer of the flow cytometer.</li>
	</ol>
	</li>
	<li>Senescence
	<ol>
		<li>Incubate at permissive conditions, usually for 24-48 hr, HEI-OC1 cells in cell culture medium alone (control) or medium plus the drugs of interest.</li>
		<li>Determine the number of senescence-associated beta-galactosidase positive cells using flow cytometry with the protocol described by Debacq-Chainiaux et al. 15 or other method known to provide reliable results. A brief protocol for flow cytometry is the following:
		<ol>
			<li>At the end of the experimental treatments, wash the cells three times with PBS and then incubate them with 100 nM Bafilomycin A1 in cell culture medium for 1 hr at permissive conditions.</li>
			<li>Add C12FDG at a final concentration of ~33 &#181;M, and continue incubating the cells for 1-2 hr.</li>
			<li>Wash the cells twice with PBS, harvest them using 1 ml per 25 cm2 of surface area of a non-enzymatic cell dissociation solution for 3 min, and pellet them by centrifugation at 3,000 x g for 5 min.</li>
			<li>Resuspend the cells in ice-cold PBS and determine the number of positive HEI-OC1 cells by flow cytometry as indicated by the manufacturer of the flow cytometer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Autophagy
	<ol>
		<li>Collect HEI-OC1 cells control and starved (deprived of serum for 48 hr) by following the procedure described in 1.2, and process them for Western blot following standard protocols. In general, the protocols for Western blot have the following steps:
		<ol>
			<li>Seed HEI-OC1 cells in 6-well plates at a concentration of 1.0 x 105 cells/ml. After 24 and/or 48 hr, wash the cells with ice-cold PBS.</li>
			<li>Lyse with 200 &#181;l of TNESV buffer (1% NP40, 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 2mM EDTA, 1 mM Na3VO4) with protease inhibitor cocktail and 1 mM PMSF for 5 min on ice.</li>
			<li>Collect the cells (by scraping the bottom of each well), transfer to microcentrifuge tubes and centrifuge at 16,800 x g for 5 min at 4 &#176;C.</li>
			<li>Collect the supernatants and quantify the protein concentration using the BCA Assay.</li>
			<li>Add 5x loading buffer and 1 mM DTT to each sample and boil for 5 min to denature the proteins.</li>
			<li>Run the samples using SDS-PAGE on a 4-12% gel, and transfer to PVDF membranes.</li>
			<li>Block the membranes with 5% nonfat dried milk in TBS-T with 0.1% Tween 20 for 60 min at RT.</li>
			<li>Incubate the membranes overnight at 4 &#176;C with primary antibodies.</li>
			<li>The next day, wash the membranes extensively with TBS-T, and incubate with a secondary IgG antibody conjugated to horseradish peroxidase (HRP) (1:1,500) for 1 hr.</li>
			<li>Detect immunoreactivity with an enhanced chemiluminescence (ECL) detection system. Normalize protein expression levels using GAPDH (1:2,000 in TBS-T with in 10% nonfat dried milk) as standard.</li>
		</ol>
		</li>
		<li>In the Western blots investigate the expression of, at least, two different markers of autophagy such as Beclin-1 and LC3B (usually at 1:1,000 dilution in TBS-T with 2.5% BSA). Do not forget to look for a control of protein expression such as GAPDH or actin.</li>
		<li>Evaluate the expression of the protein of interest by densitometry using a digital Blot scanner or computer software such as the public domain NIH Image or ImageJ (http://rsb.info.nih.gov/nih-image/).</li>
	</ol>
	</li>
</ol>
3. Electrophysiology Experiments with HEI-OC1 Cells
<ol>
	<li>Harvesting Cells 
	Note: Use cells growing in 100 mm diameter culture dishes at 50%-80% confluency. Trypsin, Accutase, enzyme-free solution, or phosphate- buffered saline + ethylene diamine tetra acetic acid (PBS-EDTA) may be used for lifting the cells without significant effects on the number and quality of the gigaseals. In some cases, however, trypsin treatment makes the cells more fragile. In these cases, allow the cells to recover for approximately 30 minutes before starting the experiments.
	<ol>
		<li>Wash the cells twice with 10 ml PBS without Ca2+ and Mg2+.</li>
		<li>Add 2 ml of an enzyme-free detacher solution, such as non-enzymatic cell dissociation solution, and incubate the dishes for 3 min at 37 &#176;C with 5% CO2.</li>
		<li>Use an inverted microscope to check the detachment of the cells. If necessary, move the cell culture dish to detach more cells, but do it gently. 
		Note: Do not shake the dish.</li>
		<li>Add 10 ml of DMEM+10% FBS and pipette the cells gently up and down five times with a 10-ml pipette.</li>
		<li>Look at the cells under a microscope. If &#62; 80% of the cells are already separated (single), no further pipetting is needed. If the cells are still in clusters, repeat the gently pipetting until more than 80% cells are single.</li>
		<li>Place the ~10 ml of suspended cells into a 15 ml conical tube, centrifuge for 2 min at 100 x g, and discard the supernatant.</li>
		<li>Use ~200 &#181;l of external recording solution (Leibovitz&#39;s L-15 medium adjusted to 305 - 310 mOsm with distilled water) to resuspend the cells. An optical control of the cells should show many single, round cells with smooth membrane edges.</li>
	</ol>
	</li>
	<li>Patch-clamp and NLC Measurements
	<ol>
		<li>Perform patch-clamp and measurements of voltage-dependent nonlinear capacitance (NLC) using adequate amplifiers and software, as well as standard electrophysiological techniques. As internal (intrapipette) solution use 150 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 2 mM ATP-Mg, 0.1 mM GTP-Na, and 10 mM HEPES; adjust pH to 7.2 with Tris.</li>
		<li>Under the microscope, select a single, healthy cell, with round and smooth membrane edges. Attach the tip of the glass pipette to the cell surface and check membrane resistance. This should be around 3-6 megaOhms, corresponding to an internal diameter (ID) of the tip of the pipette of ~2 &#181;m.</li>
		<li>Gently press the micropipette tip against the cell plasma membrane and then apply suction. A portion of the cell membrane will be suctioned into the pipette, generating an electrical resistance in the order of 10 - 100 gigaOhms (gigaseal).</li>
		<li>Establish the whole-cell condition by disrupting the plasma membrane with suction pulses.</li>
		<li>Use cells that do not express prestin, such as HEK-293, as a control 9. 
		Note: Current responses should be filtered at 5 kHz, and corrections made for the effects of residual series resistance. All data collection and most analyses can be performed using the software usually included with the lock-in amplifiers. Capacitance function can be fitted to the first derivative of a two state Boltzmann function relating nonlinear charge to membrane voltage 16. 
		 
		&#160;</li>
	</ol>
	</li>
</ol>

<ol><li>Jat, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+derivation+of+conditionally+immortall+cell+lines+from+an+H-2Kb-tsA58+transgenic+mouse%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Direct derivation of conditionally immortall cell lines from an H-2Kb-tsA58 transgenic mouse.</a> Proc. Natl. Acad. Sci. 88, 5096-5100 (1991).</li>
<li>Kalinec, G. M., Webster, P., Lim, D. J., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cochlear+cell+line+as+an+in+vitro+system+for+drug+ototoxicity+screening%5BTitle%5D)+AND+%22Audiol.+Neurootol%22%5BJournal%5D)">A cochlear cell line as an in vitro system for drug ototoxicity screening.</a> Audiol. Neurootol. 8, 177-189 (2003).</li>
<li>Devarajan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cisplatin-induced+apoptosis+in+auditory+cells%3A+role+of+death+receptor+and+mitochondrial+pathways%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Cisplatin-induced apoptosis in auditory cells: role of death receptor and mitochondrial pathways.</a> Hear Res. 174, 45-54 (2002).</li>
<li>Chen, F. Q., Hill, K., Guan, Y. J., Schacht, J., Sha, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+apoptotic+pathways+in+the+absence+of+cell+death+in+an+inner-ear+immortomouse+cell+line%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Activation of apoptotic pathways in the absence of cell death in an inner-ear immortomouse cell line.</a> Hear Res. 284, 33-41 (2012).</li>
<li>Hayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+autophagy+pathway+maintained+signaling+crosstalk+with+the+Keap1-Nrf2+system+through+p62+in+auditory+cells+under+oxidative+stress%5BTitle%5D)+AND+%22Cell+Signal%22%5BJournal%5D)">The autophagy pathway maintained signaling crosstalk with the Keap1-Nrf2 system through p62 in auditory cells under oxidative stress.</a> Cell Signal. 27, 382-393 (2015).</li>
<li>Tsuchihashi, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+through+4EBP1+and+AMPK+regulates+oxidative+stress-induced+premature+senescence+in+auditory+cells%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Autophagy through 4EBP1 and AMPK regulates oxidative stress-induced premature senescence in auditory cells.</a> Oncotarget. 6, 3644-3655 (2015).</li>
<li>Youn, C. K., Kim, J., Park, J. H., Do, N. Y., Cho, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+autophagy+in+cisplatin-induced+ototoxicity%5BTitle%5D)+AND+%22Int+J+Pediatr+Otorhinolaryngol%22%5BJournal%5D)">Role of autophagy in cisplatin-induced ototoxicity.</a> Int J Pediatr Otorhinolaryngol. 79, 1814-1819 (2015).</li>
<li>Kalinec, G., Thein, P., Park, C., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HEI-OC1+cells+as+a+model+for+investigating+drug+cytotoxicity%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">HEI-OC1 cells as a model for investigating drug cytotoxicity.</a> Hear Res. 335, 105-117 (2016).</li>
<li>Park, C., Thein, P., Kalinec, G., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HEI-OC1+cells+as+a+model+for+investigating+prestin+function%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">HEI-OC1 cells as a model for investigating prestin function.</a> Hear Res. 335, 9-17 (2016).</li>
<li>Celis, J. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCelis%2C+JE+%22Cell+Biology%3A+A+Laboratory+Handbook%22">Cell Biology: A Laboratory Handbook</a>. 1, 3rd, Elsevier Academic Press. (2006).</li>
<li>Bertolaso, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis+in+the+OC-k3+immortalized+cell+line+treated+with+different+agents%5BTitle%5D)+AND+%22Audiology%22%5BJournal%5D)">Apoptosis in the OC-k3 immortalized cell line treated with different agents.</a> Audiology. 40, 327-335 (2001).</li>
<li>Kalinec, F., Kalinec, G., Boukhvalova, M., Kachar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+characterization+of+conditionally+immortalized+organ+of+corti+cell+lines%5BTitle%5D)+AND+%22Cell+Biol+Int%22%5BJournal%5D)">Establishment and characterization of conditionally immortalized organ of corti cell lines.</a> Cell Biol Int. 23, 175-184 (1999).</li>
<li>Belyantseva, I., Kalinec, G. M., Kalinec, F., Kachar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+differentiation+of+two+immortalized+cell+lines+derived+from+the+stria+vascularis+of+a+transgenic+mouse%5BTitle%5D)+AND+%2221st+Midwinter+Meeting+Association+for+Research+in+Otolaryngology%22%5BJournal%5D)">In vitro differentiation of two immortalized cell lines derived from the stria vascularis of a transgenic mouse.</a> 21st Midwinter Meeting Association for Research in Otolaryngology. 620a, (1998).</li>
<li>Gratton, M. A., Meehan, D. T., Smyth, B. J., Cosgrove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strial+marginal+cells+play+a+role+in+basement+membrane+homeostasis%3A+in+vitro+and+in+vivo+evidence%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Strial marginal cells play a role in basement membrane homeostasis: in vitro and in vivo evidence.</a> Hear Res. 163, 27-36 (2002).</li>
<li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocols+to+detect+senescence-associated+beta-galactosidase+%28SA-betagal%29+activity%2C+a+biomarker+of+senescent+cells+in+culture+and+in+vivo%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nat Protoc. 4, 1798-1806 (2009).</li>
<li>Santos-Sacchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+inhibition+of+voltage-dependent+outer+hair+cell+motility+and+capacitance%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Reversible inhibition of voltage-dependent outer hair cell motility and capacitance.</a> J. Neurosci. 11, 3096-3110 (1991).</li>
<li>Fink, S. L., Cookson, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis%2C+pyroptosis%2C+and+necrosis%3A+mechanistic+description+of+dead+and+dying+eukaryotic+cells%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells.</a> Infect Immun. 73, 1907-1916 (2005).</li>
<li>Majno, G., Joris, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis%2C+oncosis%2C+and+necrosis.+An+overview+of+cell+death%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Apoptosis, oncosis, and necrosis. An overview of cell death.</a> Am J Pathol. 146, 3-15 (1995).</li>
<li>Vanden Berghe, T., Linkermann, A., Jouan-Lanhouet, S., Walczak, H., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulated+necrosis%3A+the+expanding+network+of+non-apoptotic+cell+death+pathways%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Regulated necrosis: the expanding network of non-apoptotic cell death pathways.</a> Nat Rev Mol Cell Biol. 15, 135-147 (2014).</li>
<li>Sun, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+kind+of+cell+suicide%3A+mechanisms+and+functions+of+programmed+necrosis%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">A new kind of cell suicide: mechanisms and functions of programmed necrosis.</a> Trends Biochem Sci. 39, 587-593 (2014).</li>
<li>Chan, F. K., Luz, N. F., Moriwaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmed+necrosis+in+the+cross+talk+of+cell+death+and+inflammation%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">Programmed necrosis in the cross talk of cell death and inflammation.</a> Annu Rev Immunol. 33, 79-106 (2015).</li>
<li>Vercammen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+signaling+of+the+Fas+receptor%3A+initiation+of+both+apoptotic+and+necrotic+cell+death+pathways%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.</a> J Exp Med. 188, 919-930 (1998).</li>
<li>Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging%2C+cellular+senescence%2C+and+cancer%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Aging, cellular senescence, and cancer.</a> Annu Rev Physiol. 75, 685-705 (2013).</li>
<li>Bian, S., Koo, B. W., Kelleher, S., Santos-Sacchi, J., Navaratnam, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+highly+expressing+Tet-inducible+cell+line+recapitulates+in+situ+developmental+changes+in+prestin%27s+Boltzmann+characteristics+and+reveals+early+maturational+events%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">A highly expressing Tet-inducible cell line recapitulates in situ developmental changes in prestin's Boltzmann characteristics and reveals early maturational events.</a> Am J Physiol Cell Physiol. 299, C828-C835 (2010).</li>
<li>Abe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+expression+of+the+outer+hair+cell+motor+prestin+in+the+mouse%5BTitle%5D)+AND+%22J+Membr+Biol%22%5BJournal%5D)">Developmental expression of the outer hair cell motor prestin in the mouse.</a> J Membr Biol. 215, 49-56 (2007).</li>
<li>Oliver, D., Fakler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+density+and+functional+characteristics+of+the+outer+hair+cell+motor+protein+are+regulated+during+postnatal+development+in+rat%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Expression density and functional characteristics of the outer hair cell motor protein are regulated during postnatal development in rat.</a> J Physiol. 519 Pt 3, 791-800 (1999).</li>
<li>Tsunoo, M., Perlman, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cochlear+Oxygen+Tension%3A+Relation+to+Blood+Flow+and+Function%5BTitle%5D)+AND+%22Acta+Otolaryngol%22%5BJournal%5D)">Cochlear Oxygen Tension: Relation to Blood Flow and Function.</a> Acta Otolaryngol. 59, 437-450 (1965).</li>
</ol>

The authors declare no existing or potential conflict of interest.

In this report we describe how to culture HEI-OC1 cells and use them to evaluate mechanisms of drug-induced cytotoxicity and to investigate functional properties of prestin, the molecular motor of cochlear OHCs. The technical procedures, however, are general enough to be easily adapted to different studies.
All the protocols described here require the correct use of well-established cell culture techniques 10. Just like with any other cell line, working with HEI-OC1 cells requires a...

House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are derived from the auditory organ of a transgenic mouse 1,2. Incubation of any cell from this transgenic mouse at 33 &#176;C/10% CO2 (permissive conditions) induces expression of an immortalizing gene that triggers de-differentiation and accelerated proliferation; moving the cells to 39 &#176;C/5% CO2 (non-permissive conditions) lead to decreased proliferation, differentiation and, at least in the case of HEI-OC1, cell death 2,3.
HEI-OC1 cells were cloned and characterized in our laboratory over a decade ago, and initial studies indicated that they express specific markers of cochlear hair cells, such as prestin, myosin 7a, Atoh1, BDNF, calbindin and calmodulin, but also markers of supporting cells like connexin 26 and fibroblast growth factor receptor (FGF-R) 2. Therefore, it was suggested that HEI-OC1 could represent a common progenitor for sensory and supporting cells of the organ of Corti 2. Parallel studies provided strong evidence that archetypal ototoxic drugs like cisplatin, gentamicin and streptomycin induced caspase-3 activation in these cells, while drugs considered non-ototoxic, like penicillin, did not 2,3. Therefore, this cell line was proposed as an in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs. It is estimated that HEI-OC1 cells have been used in more than one hundred and fifty studies published in the last ten years.
Whereas looking at the potential pro-apoptotic effect of different drugs was the major goal of most of the studies involving this cell line, other important cell processes like autophagy and senescence have just started to be investigated in HEI-OC1 cells4-7. In a recent study from our laboratory 8, we used HEI-OC1 cells to collect a comprehensive set of data about cell death, survival, proliferation, senescence and autophagy induced by different pharmacological drugs frequently used in the clinic. We also compared some of the responses of HEI-OC1 cells with those from HEK-293 (human embryonic kidney cells) and HeLa (human epithelial cells) receiving identical treatment. Our results indicated that HEI-OC1 cells respond to the each drug in a characteristic way, with a distinctive dose- and time-dependent sensitivity to at least one of the mechanisms under study. We also emphasized in that study that a correct interpretation of the experimental results will require performing parallel studies with more than one technique 8.
In a different study we investigated the use of HEI-OC1 cells to evaluate the functional response of prestin, the motor protein of cochlear outer hair cells (OHCs) 9. We reported flow cytometry and confocal laser scanning microscopy studies on the pattern of prestin expression, as well as nonlinear capacitance (NLC) and whole cell-patch clamping studies in HEI-OC1 cells cultured at permissive (P-HEI-OC1) and non-permissive (NP-HEI-OC1) conditions. Our results indicated that both total prestin expression and plasma membrane localization increase in a time-dependent manner in NP-HEI-OC1 cells. Interestingly, we also found that the increase in prestin localization at the plasma membrane of NP-HEI-OC1 cells correlated with a decrease in Na+K+ATPase, which translocated from the plasma membrane to the cytoplasm without significant changes in total cell expression. In addition, we demonstrated that P-HEI-OC1 cells have a robust NLC associated to prestin motor function, which decreased when the density of prestin molecules present at the plasma membrane increased. Altogether, these results strongly support the usefulness of HEI-OC1 cells to investigate auditory proteins.
In this video article we describe how to culture HEI-OC1 cells, why it is convenient to use cells growing at permissive conditions (P-HEI-OC1) for cytotoxicity studies, how to evaluate the mechanism/s of drug-induced cytotoxicity and how to perform electrophysiological studies (e.g., patch-clamp, non-linear capacitance (NLC)) to investigate functional properties of prestin, the molecular motor of cochlear OHCs.

<table><tbody><tr><td>HEI-OC1 cells</td><td></td><td></td><td>ALL THE ASSAY KITS, EQUIPMENTS&amp;nbsp;</td></tr><tr><td>Class II Biological Safety cabinet</td><td>The Baker Company</td><td>Sterilgard III</td><td>AND COMPANIES INDICATED IN THE</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td>PREVIOUS 2 COLUMNS ARE ONLY</td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>Axiovert 25</td><td>EXAMPLES, AND ANY OTHER SIMILAR</td></tr><tr><td>Waterbath</td><td>Stovall</td><td>HWB115</td><td>PRODUCT COULD BE USED.</td></tr><tr><td>Cell counter</td><td>Nexcelom</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Two (2) Cell incubators, one at 33 &amp;deg;C/10% CO&lt;sub&gt;2&lt;/sub&gt; and other at 39 &amp;deg;C/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Forma Scientific</td><td>3110</td><td></td></tr><tr><td>Cell culture dishes, PS, 100 mm x 20 mm with vents</td><td>Greinier Bio-One</td><td>664-160</td><td></td></tr><tr><td>Cell culture dishes , PS,&amp;nbsp; 60 mm x 15 mm with vents&amp;nbsp;</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>Cellstar tissue culture flasks&amp;nbsp; 250 ml</td><td>Greiner Bio-One</td><td>658-175</td><td></td></tr><tr><td>Cellstar tissue cultur&amp;nbsp; flasks 550 ml</td><td>Greiner Bio-One</td><td>660-175</td><td></td></tr><tr><td>&amp;nbsp;6 well cell culture plate, with lid-Cellstar</td><td>Greiner Bio-One</td><td>657-160</td><td></td></tr><tr><td>Microtest Tissue culture plate, 96 well, flat bottom with lid</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Micro-Assay-Plate, Chimmey, 96-well white, clear bottom</td><td>Greiner Bio-One</td><td>655098</td><td></td></tr><tr><td>50 ml Polypropylene conical tube with cap Cellstars</td><td>Becton Dickinson&amp;nbsp;</td><td>352070</td><td></td></tr><tr><td>15 ml Polypropylene conical tubes with cap-Cellstars</td><td>Greiner Bio-One</td><td>188-271</td><td></td></tr><tr><td>PBS pH 7.4 (1x)&amp;nbsp;</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Life Technologies</td><td>11965-084</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Hyclone</td><td>SH10073.1</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>Gibco/Invitrogen</td><td>21083-027</td><td></td></tr><tr><td>Trypsin, 0.25%&amp;nbsp;</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>TACS MTT Cell Proliferation Assay Kit</td><td>Trevigen</td><td>4890-25-K</td><td></td></tr><tr><td>Caspase-Glo 3/7 Assay&amp;nbsp; kit</td><td>Promega</td><td>&amp;nbsp; G8091&amp;nbsp;</td><td></td></tr><tr><td>BrdU Cell Proliferation Assay Kit&amp;nbsp;</td><td>Cell Signaling</td><td>6813</td><td></td></tr><tr><td>Non-enzymatic cell dissociation solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C5789</td><td></td></tr><tr><td>Cell-Tox Green Cytotoxicity Assay Kit</td><td>Promega</td><td>&amp;nbsp; G8741&amp;nbsp;</td><td></td></tr><tr><td>FACSAriaIII instrument&amp;nbsp;</td><td>BD Biosciences</td><td>&amp;nbsp;FACSAriaIII</td><td>With 488 nm excitation (blue laser)</td></tr><tr><td>Digital Blot Scanner</td><td>LI-COR</td><td>C-DiGit</td><td></td></tr><tr><td>Electrophoresis and Blotting Unit</td><td>Hoefer</td><td>SE300 miniVE</td><td></td></tr><tr><td>Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software</td><td>Molecular Devices</td><td>SpectraMax 5</td><td></td></tr><tr><td>Patch-clamp amplifier</td><td>HEKA</td><td>EPC-10</td><td></td></tr><tr><td>Puller for preparing patch electrodes</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr></tbody></table>

working with auditory hei-oc1 cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 &#176;C to stimulate cell proliferation and incubation at 39 &#176;C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

In a couple of recent publications we reported a comprehensive set of studies aimed at evaluating the response of HEI-OC1 cells to several commonly used pharmacological drugs as well as investigating prestin function 8,9. In these studies we made use of all the protocols described in the previous sections.
One of the results of these previous studies was that HEI-OC1 cells cultured at non-permissive conditions (39 &#1...

Watch this Scientific Journal Video about Working with Auditory HEI-OC1 Cells at JoVE.com

Working with Auditory HEI-OC1 Cells

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic ...

Research

JoVE Journal

Biology

15.0K Views.  University of California, Los Angeles. House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

Video: Working with Auditory HEI-OC1 Cells

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Developmental Biology

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Neuroscience

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

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

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 &#x2014;elongate, shorten and bend&#x2014; 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.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Medicine

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

Working with Auditory HEI-OC1 Cells

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