Name: cochlear surface preparation in the adult mouse
Uploaded: 2019-11-06T16:00:00
Description: Watch this Scientific Journal Video about Cochlear Surface Preparation in the Adult Mouse at JoVE.com

The research project described was supported by grant R01 DC009222 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. This work was conducted in the WR Building at MUSC in renovated space supported by grant C06 RR014516. Animals were housed in MUSC CRI animal facilities supported by grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources. The authors thank Dr. Jochen Schacht for his valuable comments and Andra Talaska for proofreading of the manuscript.

All research protocols involving male adult CBA/J mice at the ages of 10&#8211;12 weeks and C57BL/6J mice at the ages of 6&#8211;8 weeks were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina (MUSC). Animal care was under the supervision of the Division of Laboratory Animal Resources at MUSC.
NOTE: For the procedures presented below, mice are anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via intrape...

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Cochlear microdissection of whole-mount surface preparations in combination with immunolabeling provides a basic tool for investigation of inner ear pathologies and molecular mechanisms. This modified adult mouse cochlear dissection method using the cell and tissue adhesive simplifies this difficult procedure.
Although this modified cochlear surface preparation method is relatively easy and accessible, it still requires practice in order to achieve proficiency. To make the correct cuts, the di...

The cochlea is dedicated to the detection of sound and responsible for hearing. The cochlear duct is coiled in a spiral shape in the bony labyrinth and holds the auditory sensory end organ, the organ of Corti (OC). The OC rests on the basilar membrane, making up the cochlear epithelium, with a length of about 5.7 mm when uncoiled in adult CBA/CaJ mice1,2. Because the OC is tonotopically organized with high frequencies detected in the base and low frequencies in the apex, the cochlear epithelium is often divided into three parts for analytical comparisons: the apical, middle, and basal turns corresponding to low, middle, and high frequency detection, respectively. In addition to an array of supporting cells, the OC is composed of one row of inner hair cells (IHCs) located medially and three rows of outer hair cells (OHCs) located laterally with respect to the cochlear spiral.
Correct auditory processing depends on the integrity of the sensory hair cells in the cochlea. Damage to or loss of sensory hair cells is a common pathological feature of acquired hearing loss, caused by numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process3. Additionally, the integrity and function of the inner hair cell/auditory nerve synapses can be impaired by mild insults4. With the availability of molecular and genetic information and manipulation of genes by knockout and knock-in techniques, mice have been widely used in hearing science. Although the adult mouse cochlea is minuscule and the cochlear epithelium is surrounded by a bony capsule resulting in technically difficult microdissections, surface preparations of the epithelium in combination with immunolabeling or immunohistochemistry and confocal imagery have been broadly used for investigation of cochlear pathologies, including losses of ribbon synapses and hair cells, changes in levels of proteins in sensory hair cells and supporting cells, and hair cell regeneration. Cochlear surface preparations have also been used to determine the pattern of expression of reporter genes (i.e., GFP) and confirm successful transduction and identify transduced cell types. These techniques have been previously used for the study of molecular mechanisms underlying noise-induced hearing loss using adult CBA/J mice5,6,7,8,9.
Unlike immunohistochemistry using paraffin sections or cryosections to obtain small cross-sectional portions of the cochlea that contain three outer hair cells (OHCs) and one inner hair cell (IHC) on each section, cochlear surface preparations allow visualization of the entire length of the OC for counting sensory hair cells and ribbon synapses and immunolabeling of sensory hair cells corresponding to specific functional frequencies. Table 1 shows the mapping of hearing frequencies as a function of distance along the length of the cochlear spiral in adult CBA/J mouse according to studies from Muller1 and Viberg and Canlon1,2. Cochlear surface preparations have been widely used for investigation of cochlear pathologies4,5,6,7,8,9,10,11,12,13,14,15. The whole-mount cochlear dissection method was originally described in a book edited by Hans Engstrom in 196616. This technique was subsequently refined and adapted to a variety of species as described in the literature by a number of scientists in hearing science10,11,12,13,15,17 and by the Eaton-Peabody Laboratories at Massachusetts Eye and Ear18. Recently, another cochlear dissection method was reported by Montgomery et al.19. Microdissection of the cochlea is an essential and critical step for cochlear surface preparations. However, dissecting mouse cochleae is a technical challenge and requires considerable practice. Here, a modified cochlear surface preparation method is presented for use in adult mouse cochleae. This method requires decalcification and use of cell and tissue adhesive (i.e., Cell-Tak) to adhere the pieces of cochlear epithelium to 10 mm round coverslips for immunolabeling. Cell and tissue adhesive has been widely used for immunohistochemistry20. This modified cochlear microdissection method is relatively simple compared to those previously reported18,19.

<table>
	<tbody>
		<tr>
			<td>10-mm Rund Coverslips</td>
			<td>Microscopy products for science and industry</td>
			<td>260367</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-mouse IgG2</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Goat Anti-mouse IgG1</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21125</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Goat Anti-rabbit IgG (H+L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboard Micro Slide Trays</td>
			<td>Fisher Scientific</td>
			<td>12-587-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>BD Biosciences</td>
			<td>354240</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST fine science tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Disodium Salt</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-gel with Tris Buffer</td>
			<td>Electron Microscopy Sciences</td>
			<td>17985-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Four-well Cell Culture Dishes</td>
			<td>Greiner Bio-One</td>
			<td>627170</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-myosin VIIa Antibody</td>
			<td>Proteus Biosciences</td>
			<td>25-6790</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CtBP2 Antibody</td>
			<td>BD Biosciences</td>
			<td>#612044</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Glu2R Antibody</td>
			<td>Millipore</td>
			<td>MAB397</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>31872</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>BP665-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td>100491-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Surface preparations of the cochlear epithelium, in combination with immunolabeling and confocal imaging, have been used broadly in hearing science for the investigation of cochlear pathologies, such as quantification of ribbon synapses, sensory hair cells, and protein expression in sensory hair cells5,6,7,8. Although the dissection of adult mouse cochleae for surface preparations is not simple...

Watch this Scientific Journal Video about Cochlear Surface Preparation in the Adult Mouse at JoVE.com

Cochlear Surface Preparation in the Adult Mouse

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 ...

Cochlear surface preparations allow visualization of the entire lens of the organ of Corti using immunohistochemistry and confocal imaging. It has been broadly used for investigation of specific cochlear pathologies of interest. We will modify the cochlear microdissection method.The main advantage of this technique is the adherence of a piece of cochlear epithelium to 10 millimeter round coverslips for the immunolabeling procedure while avoiding tissue loss during the multiple washing steps. In addition cochlear pathologies, cochlear surface preparation can also be utilized for the assessment expression and hair cell regeneration. Basic microscope skills are required for this technique and visual demonstration of the method can help to ensure that each step is performed correctly.Demonstrating the procedure will be Qiaojun Fang, a graduate student from my laboratory. For temporal bone extraction, immediately post-mortem pull the skin anteriorly to expose the skull bone and use scissors to cut the bone from the posterior aspect forward along the center line of the skull. Use forceps to remove the brain tissue before using the thumb and index finger to manually remove the temporal bones from mice three months of age or older.Place the bones into a 30 millimeter diameter Petri dish containing fresh ice cold 4%PFA and PBS under a dissection microscope and remove the stapes from the oval window. Puncture the round window membrane with number five forceps and use a 27 gauge needle attached to a one milliliter syringe containing fresh 4%PFA to make a small hole into the apex of the cochlea. Gently and slowly perfuse the cochlea with a fixative solution via the round and oval windows until the solution washes out of the small hole at the apex.Then transfer up to two perfused cochleae into individual 20 milliliter scintillation vials containing 10 milliliters of 4%PFA per vial and gently agitate the samples for two hours at room temperature, followed by overnight storage at four degrees Celsius on a rotator. The next morning, wash the cochleae with three five-minute washes with fresh PBS per wash. After the last wash, add 20 milliliters of 4%EDTA to each vial and rotate the samples for 48 hours at four degrees Celsius with gentle agitation.At the end of the incubation, touch the bony vestibular portion of each cochlea with forceps to assess the elasticity of the samples. If the bones are elastic rather than firm, the cochlea have been decalcified. Replace the EDTA for each sample with fresh PBS.For microdissection of the cochlear epithelium, grasp the vestibular portion of the temporal bone with forceps and use a scalpel to cut the apical turn at a 45 degree angle. Cut vertically along the faint line between the round window and the oval window to separate the cochlea from the vestibular portion and orient the cochlear portion with the basal turn toward the bottom and middle turns toward the top of the Petri dish. From this position, cut the bony capsule and lateral wall of the middle turn toward the end from which the apical section was removed and continue cutting to completely separate the middle portion from the basal and hook regions.Placing the basal and hook portion with the basilar membrane site oriented toward the bottom of the dish, vertically cut the medialis off of the hook region to remove the medialis and cut the basal and hook regions to separate the hook portion. To avoid distortion of the tissue, cut the medialis off of the hook region before separation of the basal turn and hook region. Cut away the relatively large portions of bony capsule and lateral wall tissue of the middle turn and holding the lateral wall with forceps to align the bony capsule and lateral wall with the bottom of the Petri dish, cut these tissues from the basilar membrane side.Next, flatten the specimen to orient the sensory hair cell surface side up and trim away the rest of the bony capsule and lateral wall. Then use forceps to remove the tectorial membrane to completely separate the middle region and remove the bony capsule, lateral wall tissue, and tectorial regions of the remaining turns as just demonstrated. When all of the cochlear turns have been dissected, spread 0.5 microliters of cell and tissue adhesive onto the center of one round 10 millimeter coverslip and allow the adhesive to dry for three to five minutes at room temperature.Place the dried coverslips into the Petri dish and stick all four pieces of each sensory epithelium sample onto one coverslip. Then use forceps to grasp one edge of each coverslip to transfer the specimens into individual wells of a four-well cell culture dish for immunolabeling. For immunolabeling of surface cochlear synapse preparations, wash each sensory epithelium three times for five minutes in fresh PBS per wash, followed by the treatment of each sample with two milliliters of 2%non-ionic surfactant for 30 minutes at room temperature on a rotator.At the end of the incubation, aspirate the surfactant solution from each well and block any nonspecific binding with 100 microliters of blocking solution per well for one hour on the rotator with gentle agitation at room temperature. At the end of the blocking incubation, wash the samples three times with PBS as demonstrated under gentle agitation before labeling each epithelium with 100 microliters of the primary antibodies of interest for 24 hours at 37 degrees Celsius protected from light. The next day, wash the samples three times with fresh PBS and gentle agitation per wash and label the specimens with 100 microliters of the appropriate secondary antibodies of interest for two hours at 37 degrees Celsius protected from light.At the end of the incubation, wash the samples with three five-minute washes in fresh PBS per wash before placing each coverslip onto individual glass microscope slides with the samples facing up. Next, carefully add eight microliters of an appropriate mounting medium into the center of each coverslip and use forceps to mount another 10 millimeter coverslip onto each sample. Then seal the sides of each coverslip sandwich with clear nail polish, place the samples into a cardboard slide folder, and store the slides at four degrees Celsius.Although the dissection of adult mouse cochleae for surface preparations is not simple, researchers new to the technique should be able to learn the method after practicing with 10 to 15 ears. Immunolabeling of surface preparations of untreated 10 to 12-week-old CBAJ mice with CTBP2 and GluA2 demonstrates that both presynaptic ribbons and postsynaptic terminals respectively are located below the inner hair cell nuclei and are juxtaposed indicating functional synapses. Immunolabeling for myosin 7A and counterstaining with phalloidin and DAPI reveals the presence of sensory hair cells including the outer hair cells and inner hair cells and their nuclei.Immunolabeling for myosin 7A and counterstaining with phalloidin shows no differences in immunoreactivity or uniformity with or without the use of cell and tissue adhesive. In addition, scanning electron microscopy of surface preparations from untreated six to eight-week-old C57 black 6 mice demonstrate a well-organized V-shaped stereocilia of outer hair cells in three rows of the sample. Remember to fill the cochlear with fixative solution gently and slowly through the round and oval windows until the solution washes out of the small hole at the apex.Before separating the hook region from the basal turn, take care to cut the medialis off of the hook region to facilitate the removal of the medialis.

Research

JoVE Journal

Neuroscience

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

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 ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...