We thank Vincent Schram from the NICHD microscopy and imaging core for assisting in the confocal image acquisition, and Tsg-Hui Chang for invaluable help with colony management and mice care. This research was supported by the Intramural Research Program of the NINDS, NIH, Bethesda, MD, to K.J.S. A.B. was supported by the Intramural Research Program of the NINDS, NIH, and by a Robert Wenthold Postdoctoral Fellowship from the intramural research program of the NIDCD.

The animal care and experimental procedures were performed following the guidelines for the Care and Use of Laboratory Animals, which were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke (Animal protocol #1336 to KJS).
1. Mice
<ol>
	<li>Set a couple of breeding pairs of C57BL/6J wild-type to breed in the animal facility to control the date of birth of the litters and keep track of the age of the pups.</li>
</ol>
2. Cochleae Dissection
<ol>
	<li>Set a clean space close to a stereomicroscope to perform the dissections (Figure 1A). Use 70% ethanol to clean the space and surroundings and place a clean bench pad. A Medical Pathological Waste (MPW) plastic bag would be required to discard the animal carcasses.</li>
	<li>Prepare several 35 mm dishes with some Leibovitz&#39;s L15 media. 
	NOTE: Leibovitz&#39;s L-15 cell media contains 1&#8211;2 mM Ca2+, which is required to maintain the integrity of the tip-links, and contains essential amino acids, vitamins, and sodium pyruvate to improve cell health and survival. Serum was excluded to avoid experimental variability due to its poorly defined composition and potential interference with the dextran.</li>
	<li>Euthanize postnatal-day-6 (P6) mice by decapitation. 
	NOTE: 6 day old mice are somewhat resistant to inhalant anesthetics. Although isoflurane or prolonged CO2 exposures (up to 50 min) may be used for euthanasia, a secondary physical method is recommended to ensure death.</li>
	<li>Use surgical scissors to remove the skin of the skull by making a superficial cut from the anterior to the posterior end and across the external auditory canals.</li>
	<li>Fold the skin towards the nose to expose the cranium (Figure 2B).</li>
	<li>Make an incision from the back to the front of the skull and across the eye line (Figure 2B-C).</li>
	<li>Separate the skull in two halves and remove the brain with the use of a small spatula to expose the temporal bones (Figure 2C-D).</li>
	<li>With small scissors, cut around the temporal bones, and excise the tissue.</li>
	<li>Place both temporal bones in a 35 mm dish and make sure they are covered with L15 media (Figure 2E). 
	NOTE: The following steps are performed under the stereomicroscope. A black background usually helps to visualize the tissue during the fine dissection steps.</li>
	<li>Under a stereomicroscope equipped with a widefield eyepiece (a 10X magnification power (WF10X) and an external alternating current (AC) halogen light source), remove the surrounding cochleae tissue, semicircular canals, and vestibular organs with surgical forceps (Figure 2F).</li>
	<li>To allow the dextran and L15 media to enter the cochlear duct, perform two puncture bounds on the dissected cochleae, one on the round window and other at the apical cochlear region.</li>
	<li>Add 300 mL of Leibovitz&#39;s L15 media in each well from a 9-well glass depression plate.</li>
	<li>Place at least three dissected cochleae on each well.</li>
</ol>
3. Dextran Labeling
<ol>
	<li>Reconstitute the dextran in Hanks&#39; balanced salt solution without Ca2+ and Mg2+ (HBSS-CFM) at a final concentration of 10 mg/mL. This stock solution must be aliquoted in opaque black tubes (protected from light) and stored at -30 &#176;C until use. 
	NOTE: The use of lysine-fixable dextran is critical for a successful outcome of this protocol.</li>
	<li>Prepare each dextran at a final concentration of 2 mg/mL in 500 mL of Leibovitz&#39;s L15 media.</li>
	<li>Remove the media from the cochlea and add Leibovitz&#39;s L15 media containing the dextran of interest at a final concentration of 2 mg/mL. 
	NOTE: Although a proportion of the MET channels are open at rest41,42, the dextran incubation was performed with a gentle shaking of the explants to increase the open probability of the MET channel.</li>
	<li>Incubate at room temperature for 2 h with gentle shaking (25 rpm) by using a 3-dimensional shaker with a tilted angle of 25&#176;. 
	NOTE: Fluorescently labeled dextran must be protected from light when possible. To protect the dextran during the 2 h incubation, place the 9-well glass plate inside a cell culture P150 dish wrapped in aluminum foil.</li>
</ol>
4. Sample Preparation for Imaging
<ol>
	<li>After incubation with the dextran, wash the tissue for 2 min twice with media and once with HBSS.</li>
	<li>Incubate the tissue at room temperature for 30 min with 4% paraformaldehyde in Hanks&#39; balanced salt solution (HBSS). 
	CAUTION: Exposure to formaldehyde can be irritating to the eyes, nose, and upper respiratory tract. In certain individuals, repeated skin exposure to formaldehyde can cause sensitization that may result in allergic dermatitis. Formaldehyde is a known human carcinogen and a suspected reproductive hazard.</li>
	<li>Quickly and gently wash the fixed tissue twice with HBSS to remove the paraformaldehyde. 
	NOTE: Decalcification of the temporal bones is not needed at this developmental stage of the cochlea.</li>
	<li>Remove the spiral ligament and the tectorial membrane with fine tip forceps to dissect the organ of Corti (Figure 2G).</li>
	<li>Remove all the small pieces of tissue and wash the tissue with HBSS.</li>
	<li>Permeabilize the tissue in 0.5% Triton X-100 in PBS containing fluorescently-labeled phalloidin (conjugated to green or red when testing the uptake of TR- or FITC-labeled dextran, respectively) at a 1:200 dilution for 30 min to label F-actin and visualize the actin-based stereocilia.</li>
	<li>Wash the tissue 2&#8211;3 times for 2 min each time with HBSS buffer to remove the excess of triton and phalloidin, and once with PBS to remove the salts.</li>
	<li>Mount the organ of Corti tissues on a microscope slide and cover it with mounting media. 
	NOTE: When mounting the tissue, make sure that the side of the tissue containing the hair cell stereocilia is facing the coverslip.</li>
	<li>Remove any potential bubbles generated during the addition of the mounting media and prevent the generation of new bubbles during the placement of the coverslip. 
	NOTE: Aspirate with a pipette any bubble generated during the addition of the mounting media. To prevent air bubbles from being trapped under the coverslip, place an edge of the coverslip close to the sample and carefully and slowly lower the coverslip over the tissue using forceps or a pipette tip.</li>
	<li>Cover the tissue in mounting media with a glass coverslip (Figure 2H). 
	NOTE: Objectives with a numerical aperture above 0.4 are designed to use #1.5 coverslips (0.17 mm thickness). Using the wrong coverslip may have severe implications for the intensity and quality of the images.</li>
	<li>Incubate the mounted tissue overnight at room temperature to let the mounting media dry and store the slides at 4 &#176;C until imaging.</li>
</ol>
5. Image Acquisition and Image Processing
NOTE: The confocal images were taken with a LSM 880 confocal microscope equipped with a 32 channel Airyscan detector in the super-resolution (SR) mode43 and a 63x objective.
<ol>
	<li>Add a small drop of immersion oil on the objective.</li>
	<li>Place the microscope slide containing the mounted tissue sample in the microscope stage with the glass coverslip facing the immersion oil.</li>
	<li>Focus on the sample and set the imaging parameters using the image acquisition software.</li>
	<li>Use identical image acquisition settings and optimal parameters for x, y, and z resolution for each independent experiment. A piezo-driven focus system is required to quickly and precisely move the objective when acquiring the z-stack of images. 
	NOTE: To image the entire apical region of the hair cells, collect a z-stack of images from the stereocilia to the apical half of the hair cell body using the optimal settings. It is crucial to collect a large z-stack along the hair cell to assure the imaging of the vesicle-like particles.</li>
	<li>Use the image acquisition software to process the raw confocal images using the Airyscan 3D reconstruction algorithm with the automatic default deconvolution filter settings.</li>
	<li>Open the confocal images in an image processing software to adjust the brightness and contrast, add the scale bar, and export the images for the final figures.</li>
</ol>

The authors have nothing to disclose.

This protocol describes how to perform uptake experiments in murine organ of Corti explants with 3 kDa dextran Texas Red. The goal of this method is to test whether molecules larger than others previously tested were also able to specifically label auditory hair cells and permeate through the MET channel. Similar experimental protocols have been previously used to evaluate the permeability of hair cells to other fluorescent dyes such as FM1-43 (0.56 kDa)12,19,20 and Texas Red-labeled aminoglycosides (1.29&#8211;1.43 kDa)13,52. The time needed for labeling hair cells using these fluorescent molecules varies with their size. The maximal labeling requires a few minutes for FM1-4313,53, 30-60 min for Texas Red-labeled aminoglycosides13, and 1.5 h for Texas Red-labeled 3 kDa dextran. Thus, long incubation times are crucial for this experiment as we saw only minimal labeling of the hair cell body at short incubation times (Figure 2C). We chose to study P6 mice for these experiments because, at this age, most apical and basal hair cells carry transduction currents and express both TMC1 and TMC2 proteins12,18,54,55. It should be possible to study younger animals as MET currents are present earlier than P6, in particular near the base of the cochlea12. Studying older animals may be more challenging because dissection of the bony cochlea becomes more difficult as this structure continues to ossify56.
There are several critical steps in this protocol. The most crucial one being the successful dissection and preparation of the organ of Corti tissue (Figure 1). The delicate structure of the organ of Corti and encasement in the boney cochlea presents a challenge for histological and biochemical analysis. Several detailed protocols for the successful dissection of this tissue have been previously published in this journal56,57,58 and others59. Disruption of the cell-cell junction and loss of the auditory sensory epithelium integrity may lead to the uptake of dextran of up to 40 kDa and the labeling of the affected cells39,40. In agreement with this, we observed unspecific labeling of hair cells and the surrounding supporting cells when the tissue was unintentionally damaged with the forceps during dissection before dextran incubation (Figure 2B and Figure 3B). There are no tricks or guidelines to reach proficiency at dissecting an intact organ of Corti other than practice and patience. However, the presence of 1&#8211;2 mM Ca2+ during tissue dissection and uptake experiments is crucial to maintain the integrity of the tip-link that transmits the mechanical force to the MET channel and thereby preserve MET channel function. Once the tissue is fixed with 4% PFA, the presence of calcium in the buffer or media becomes trivial.
One of the limitations of this study resides in the heterogeneous nature of the dextran molecules. The dextran elaborated by the Leuconostoc bacteria is a polymer of anhydroglucose composed of approximately 95% alpha-(1&#8211;6) D-linkages (Figure 2A). The remaining linkages account for the branching of dextran, which correlates with its length, so dextran of lower molecular weight are more rod-like and have a narrower size distribution while larger dextrans are polydisperse60. Therefore, the actual molecular weight of the molecules in each preparation of the 3 kDa dextran ranges from 1.5 to 3.0 kDa, including the fluorescent molecule61. Additionally, the fluorescently labeled dextran differ in the number of Texas Red molecules attached to the dextran (degree of labeling). It is advisable to use dextran with a degree of labeling of at least 0.4 since a lower degree of labeling will hinder the ability to detect the fluorescently labeled dextran by microscopy. The fluorescent dye coupled to the dextran and the lysine residues in the fixable versions, will determine the overall charge of the dextran. This is an important consideration since the cationic selectivity of the MET channel would preferably uptake cationic dextran27. Lastly, a lysine-fixable dextran is necessary to reach the resolution displayed in these experiments. A non-fixable dextran will continue to diffuse into the tissue hampering its accurate localization and visualization.
The LSM 880 confocal microscope equipped with an Airyscan detector used to image the organ of Corti samples provided us with twice the resolution, and better signal-to-noise ratio (SNR) compared to a conventional confocal microscope62. Therefore, this microscope allowed us not only to observe the robust dextran accumulation at the cell body but also to pinpoint the accumulation of the dextran at the stereocilia tips and the vesicle-like pattern at the cell body. A conventional confocal microscope would allow for the visualization and quantification of the accumulation of fluorescent dextran at the cell body, but it would difficult to distinguish the three rows of stereocilia and the precise localization of the dextran at the tips of the shorter stereocilia rows. A similar resolution to the one achieved with the LSM 880 Airyscan confocal could be reached using a conventional confocal microscope with oversampling and deconvolution, although the SNR would be lower than the one reached with the Airyscan detector.
In addition to the use of 3 kDa dextran-TR to assay for functional MET channels, this protocol could be further used to study endocytic processes in hair cells since all the dextrans tested reveal an intracellular vesicle-like pattern resembling endocytosis. This process was not the focus of our work, and additional studies would be required to fully characterize this phenomenon.

The hair cells of the inner ear are the sensory cells that detect sound and covert the mechanically stimuli in electrical signals, which are ultimately interpreted by our brain. These cells have a staircase-shaped bundle of three rows of actin-based filaments, known as stereocilia, which protrude from their apical region1,2. The mechanical stimuli deflect the stereocilia filaments toward the longest row and trigger the opening of the mechanotransduction (MET) channels3. The opening of the MET channels leads to an influx of cations that depolarizes the cell and consequently signals the release of synapse vesicles at the basal region of the hair cell.
The biophysical properties of the MET channel essential for hearing have been extensively characterized. Among other properties, these channels are cationic selective and have a relatively large conductance (150&#8211;300 pS in low Ca2+)4,5,6,7,8,9,10. Remarkably, large fluorescent molecules such as FM1-43 and Texas Red-labeled aminoglycosides are permeant blockers of the MET channel, resulting in their accumulation in the hair cell body that can be visualized using fluorescence microscopy11,12,13,14. Conversely, the molecular identity and the structure of the MET channel and its permeation pathway have remained elusive. Increasing experimental evidence indicates that the transmembrane-like channel protein 1 (TMC1) is a component of the MET channel in mature hair cells15,16,17,18,19. Mutations in the transmembrane-like channel 1 (TMC1) alter the MET channel properties19,20,21,22 and cause deafness. In addition, TMC1 localizes to the site of the MET channel18,23 and interacts with the tip-link responsible for transmitting the mechanical force to the MET channel24,25. Furthermore, recent bioinformatics analysis has identified the TMC proteins as evolutionary related to the mechanosensitive channels TMEM63/OSCA proteins and the TMEM16 proteins, a family of calcium-activated chloride channels and lipid scramblases26,27,28. A structural model of TMC1 based on the relationship between these proteins revealed the presence of a large cavity at the protein-lipid interface27. This cavity harbors the two TMC1 mutations that cause autosomal dominant hearing loss (DFNA36)27,29,30,31,32, and selective modification of cysteine mutants for residues in the cavity alter MET channel properties28, indicating that it could function as the permeation pathway of the MET channel. The large size of this predicted cavity in TMC proteins could explain the ability of large molecules to permeate the MET channel. To test the prediction that the MET channel contains an unusually large permeation pathway and to push the limits of the size of the cavity observed in TMC1, we developed a protocol to perform uptake experiments in the organ of Corti explants with a larger molecule, 3 kDa dextran fluorescently labeled with Texas Red.
Dextran is a complex branched polysaccharide composed of many D-glucose molecules bound by alpha-1,6 glycosidic linkages. Its high solubility in water, low cell toxicity, and bioinertity make it a versatile tool to study several cellular processes. In addition, dextran is available in a wide range of sizes and fluorescently labeled with fluorophores of several colors. Fluorescently labeled dextrans are commonly used in cell and tissue permeability research33,34, to study endocytosis in multiple cellular systems35,36, and for neural tracing37,38. In the auditory field, dextran molecules have also been used to assess the disruption of the cell-cell junction and loss of the auditory sensory epithelium integrity after exposure to intense noise in the chinchilla organ of Corti39,40.
In this work, we exploited the properties of some of the smallest (3 and 10 kDa) fluorescent dextrans to perform uptake experiments in murine inner ear hair cells and explore the size of the permeation pathway of the inner ear hair cell MET channel. In addition, we used a laser-scanning confocal microscope (LSM) 880 equipped with an Airyscan detector to visualize and localize fluorescent dextran at the stereocilia and the cell body of auditory hair cells.

<table><tbody><tr><td>#1.5 glass coverslips 18mm</td><td>Warner Instruments</td><td>64-0714</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>ThermoFisher</td><td>A12379</td><td></td></tr><tr><td>Alexa Fluor 594 Phalloidin</td><td>ThermoFisher</td><td>A12381</td><td></td></tr><tr><td>alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective</td><td>Carl Zeiss</td><td>420780-9970-000</td><td></td></tr><tr><td>Amiloride hydrochloride</td><td>EMD MILLIPORE</td><td>129876</td><td></td></tr><tr><td>Benchwaver 3-dimensional Rocker</td><td>Benchmarks scientific</td><td>B3D5000</td><td></td></tr><tr><td>C57BL/6J wild-type mice</td><td>strain 000664</td><td>The Jackson Laboratory</td><td></td></tr><tr><td>Cell impermeant BAPTA tetrapotassium salt</td><td>ThermoFisher</td><td>B1204</td><td></td></tr><tr><td>Dextran, Fluorescein, 10,000 MW, Anionic, Lysine Fixable</td><td>ThermoFisher</td><td>D1820</td><td></td></tr><tr><td>Dextran, Texas Red, 10,000 MW, Lysine Fixable</td><td>ThermoFisher</td><td>D1863</td><td></td></tr><tr><td>Dextran, Texas Red, 3000 MW, Lysine Fixable</td><td>ThermoFisher</td><td>D3328</td><td></td></tr><tr><td>Formaldehyde Aqueous Solution EM Grade</td><td>Electron microscopy science</td><td>15710</td><td></td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>ThermoFisher</td><td>14025</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>ThermoFisher</td><td>14170</td><td></td></tr><tr><td>Image J or FIJI</td><td>NIH</td><td>&lt;a href=&quot;http://fiji.sc/&quot; target=&quot;_blank&quot;&gt;http://fiji.sc/&lt;/a&gt;</td><td></td></tr><tr><td>Immersol 518F oil immersion media</td><td>Carl Zeiss</td><td>444970-9000-000</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, GlutaMAX Supplement</td><td>ThermoFisher</td><td>31415029</td><td></td></tr><tr><td>neomycin trisulfate salt hydrate</td><td>Sigma</td><td>N6386</td><td></td></tr><tr><td>PBS (10X), pH 7.4</td><td>ThermoFisher</td><td>70011069</td><td></td></tr><tr><td>Phalloidin-CF405M</td><td>Biotium</td><td>00034</td><td></td></tr><tr><td>ProLong Diamond antifade mounting media</td><td>ThermoFisher</td><td>P36970</td><td></td></tr><tr><td>superfrost plus microscope slide</td><td>Fisherbrand</td><td>22-037-246</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Zen Black 2.3 SP1 software</td><td>Carl Zeiss</td><td>&lt;a href=&quot;https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html&quot; target=&quot;_blank&quot;&gt;https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html&lt;/a&gt;</td><td></td></tr></tbody></table>

dextran labeling and uptake in live and functional murine cochlear hair cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

We observed robust and specific labeling of hair cells after 2h incubation of organ of Corti explants from wild-type postnatal-day-6 (P6) mice with 3 kDa dextran fluorescently labeled with Texas Red (dextran-TR) (Figure 2A-B). Dextran labeling was observed in both inner and outer hair cells (IHC and OHC) at the basal, middle, and apical regions of the organ of Corti (Figure 2B).
Fluorescently labeled phalloidin was used to counterstain filamentous actin (F-actin) and visualize the actin-based hair cell stereocilia. We also performed similar experiments at shorter incubation times, and although we observed dextran-TR accumulation in the hair cell body, the signals were weaker and more variable than those at 2 h incubation (Figure 2C).
We next imaged both stereocilia and cell bodies of the hair cells and observed that only those cells that incorporated 3 kDa dextran-TR in their cell body showed fluorescent labeling of their stereocilia (Figure 3A). This relationship between stereocilia and cell body labeling was absent in hair cells from damaged tissue, which also presented unspecific labeling of dextran in several cell types of the sensory epithelium (yellow squares in Figure 2B, and Figure 3B). Importantly, we observed a uniform fluorescence signal along the stereocilia with enrichment at the tips of the shorter stereocilia rows where the MET channel is located18,23 (Figure 3C). Also, we noticed vesicle-like structures in the cell body of the hair cells and the neighboring supporting cells. The vesicle-like pattern of uptake in these cells suggests that dextran-TR can also be taken up by endocytosis (Figure 3C).
The protocol described here also allows for the examination of the uptake of larger dextrans and combinations of different dextrans. Larger dextran of 10 kDa labeled with Texas Red (dextran-TR) or fluorescein (dextran-FITC) also produced a vesicle-like pattern in the cell body of hair cells and supporting cells, in addition to accumulating around the hair cell membrane in a patchy pattern (Figure 4A,B). The 10 kDa dextran-TR also superficially labeled the three stereocilia rows of all the hair cells (Figure 4A, inset), probably due to the negatively charged surface of the hair cell plasma membrane44,45,46. We next examined the uptake of 3 kDa dextran-TR and 10 kDa dextran-FITC simultaneously in the organ of Corti explants. For the 3 kDa dextran-TR, we observed both a diffuse and a vesicle-like pattern. However, the 10 kDa dextran-FITC only displayed a vesicle-like pattern (Figure 4C). These data suggest that dextran uptake occurs by at least two distinct mechanisms that are dependent on the size of the dextran.
We next assessed whether functional MET channels were required for the uptake of 3 kDa dextran-TR. To do this, we tested dextran incorporation in hair cells from the organ of Corti explants in the presence of MET channel blockers (neomycin and amiloride)13,14,47 or the Ca2+ chelator BAPTA, which abolishes MET currents by breaking the tip-links and preventing the gating of the channel25,48,49. In these experiments, the tissue explants were previously incubated for 30 min with neomycin (500 mM), amiloride (150 mM), or with BAPTA (5mM) before the addition of 3 kDa dextran-TR in the presence of the corresponding MET blocker. The presence of BAPTA or the MET channel blockers prevented the stereocilia labeling (Figure 5A) and the uptake of 3 kDa dextran-TR in hair cells (Figure 5B). However, blockade of the MET channel preserved the vesicle-like pattern (Figure 5B), indicating that this pattern of uptake is independent of functional MET channels. Similar results have been observed in hair cells from TMC1/TMC2 double knock-out mice, which lack MET27. Intriguingly, these vesicle-like structures were not observable in the presence of amiloride, which is known to inhibit the Na+-H+ exchanger and thereby inhibit endocytosis50,51. These results indicate that 3kDa dextran-TR enters the hair cells through two different pathways, one common to larger dextrans involving vesicle-like structures and another that depends on functional MET channels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60769/60769fig1.jpg" /> 
Figure 1: Steps of the dissection of a murine organ of Corti. (A) Clean area and materials required for the dissection. (B) Exposed mouse cranium. Forceps are holding the skin, which has folded towards the nose. Black lines indicate the two incisions that are required for the removal of the brain. (C) Incised and opened cranium to allow for removal of the brain with a spatula (on the right). (D) Cranium and temporal bones after brain removal. (E) Excised skull, including the temporal bones (dashed black squares) in a P35 plate covered with media. (F) Excised cochleae. (G) Two dissected organs of Corti on the well of a glass depression plate. (H) Mounted sample on a glass coverslip ready for imaging. <a href="https://www.jove.com/files/ftp_upload/60769/60769fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60769/60769fig2.jpg" /> 
Figure 2: Hair cells uptake 3 kDa dextran-TR. (A) Schematic representation of Texas Red-labeled dextran (dextran-TR) containing six molecules of glucose corresponding to a molecular weight of 1.08 kDa. A succinimidyl ester reaction links a Texas Red molecule (magenta) to a glucose monomer. (B) Representative confocal image showing specific labeling of sensory hair cells (HC) with 3 kDa dextran-TR across the whole organ of Corti from a 6 day-old mouse. The basal (BA), middle (MD), and apical (AP) regions of the organ are indicated. Yellow squares indicate tissue damaged regions. (C) 3 kDa dextran-TR fluorescence after 30, 60, 90, or 120 min incubation with the organ of Corti explants is shown in magenta merged with F-actin in green (top images) and independently in grayscale (bottom images). The image display range was linearly adjusted in each one of the gray images independently for visualization of the 3 kDa dextran-TR fluorescence. Scale bar = 20 mm. This figure has been modified from27. <a href="https://www.jove.com/files/ftp_upload/60769/60769fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60769/60769fig3a.jpg" /> 
Figure 3: Hair cell body and stereocilia labeling with 3 kDa dextran-TR. (A) Confocal images displaying 3 kDa dextran-TR fluorescence (magenta) at the cell body and stereocilia counterstained with phalloidin to label F-actin (green) to visualize hair cell boundaries and stereocilia. The three rows of outer hair cells (OHC) and one row of inner hair cells (IHC) are indicated. Scale bar = 20 mm. (B) Confocal images of a damaged tissue area displaying 3 kDa dextran-TR fluorescence (magenta) at the cell body and stereocilia. Yellow arrows indicate the labeling of non-sensory supporting cells. Scale bar = 20 mm. (C) Closer view of 3 kDa dextran-TR fluorescence at the cell body and stereocilia of outer hair cells (OHC, top) and inner hair cells (IHC, bottom). Scale bar = 5 mm. This figure has been modified from27. <a href="https://www.jove.com/files/ftp_upload/60769/60769fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60769/60769fig4.jpg" /> 
Figure 4: Labeling of larger 10 kDa dextran and a combination of 3 and 10 kDa dextran. (A) Confocal image of hair cells at the hair cell body (top) and stereocilia (bottom) levels incubated with 10 kDa dextran-TR. The dextran fluorescence signal is shown in magenta merged with F-actin in green and independently in grayscale. A couple of IHC are shown at higher magnification in the inset to appreciate the superficial labeling of stereocilia observed with the 10 kDa dextran-TR. Scale bar = 5 mm. (B) Confocal image of hair cells incubated with 10 kDa dextran-FITC represented as in A. (C) Confocal image of hair cells at the hair cell body (top) and stereocilia (bottom) levels incubated simultaneously with 3 kDa dextran-TR and 10 kDa dextran-FITC and imaged using independent channels and depicted separately in gray. In the right panel, F-actin (blue), 10 kDa dextran-FITC (green), and 3 kDa dextran-TR (magenta) are shown together along with phalloidin (blue). Scale bar = 20 mm, and the vesicle-like pattern of uptake is indicated with yellow arrows. This figure has been modified from27. <a href="https://www.jove.com/files/ftp_upload/60769/60769fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60769/60769fig5.jpg" /> 
Figure 5: Functional MET channels are required for 3 kDa dextran-TR uptake. Representative confocal images of hair cells at the stereocilia (A) or cell body (B) level after incubation with 3 kDa dextran-TR in the absence (control) or presence of BAPTA or the MET channel blockers amiloride and neomycin. 3 kDa dextran-TR fluorescence is shown in magenta. Tissue was counterstained with phalloidin to label F-actin for visualization of the hair cell stereocilia and boundaries (green). White arrows point to vesicle-like structures. The three rows of outer hair cells (OHC) and one row of inner hair cells (IHC) are indicated, Scale bar = 20 mm. This figure has been modified from27. <a href="https://www.jove.com/files/ftp_upload/60769/60769fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells at JoVE.com

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

dextran-labeling-uptake-live-functional-murine-cochlear-hair

Universidad Científica del Sur

Research

JoVE Journal

Bioengineering

7.3K Views.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3–10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

Video: Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair 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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetics

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biology

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Neuroscience

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

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 ...

Medicine

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

Summary

Explore More Videos

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

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

Cochlear Surface Preparation in the Adult Mouse

Whole Neonatal Cochlear Explants as an In vitro Model