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 intraperitoneal injections. Mice are decapitated after the animal no longer responds to painful stimuli, such as a toe pinch.
1. Extraction of Temporal Bones
<ol>
	<li>Decapitate the mouse immediately postmortem with surgical scissors (17 cm long) and cut the skull bone with scissors from the posterior aspect forward along the center line of the skull after exposing the skull bone by pulling the skin anteriorly.</li>
	<li>Remove the brain tissue using forceps and manually remove the temporal bones with the thumb and index finger for mice three months of age or older. Use small surgical scissors (11 cm long) to cut the temporal bone of mice younger than three months old.</li>
	<li>Put the temporal bone into a Petri dish (30 mm in diameter) containing ice-cold fresh 4% paraformaldehyde (PFA) solution dissolved in phosphate-buffered saline (PBS), pH 7.4. 
	NOTE: Prepare fresh 4% PFA solution and adjust the pH to 7.4 just before fixing the temporal bones, because improper pH balancing of the PFA solution will decrease the quality of the immunolabeling.</li>
</ol>
2. Fixation and Perfusion
<ol>
	<li>Remove the stapes from the oval window and puncture the round window membrane with size #5 forceps. Under a stereo dissection microscope make a small hole into the apex of the cochlea using a 27 G needle connected to a 1 mL syringe.</li>
	<li>Gently and slowly perfuse the cochlea with 4% PFA solution via the round and oval windows until the solution washes out of the small hole at the apex. Transfer the cochlea (one or two cochleae per vial) to 20 mL volume scintillation vials containing 10 mL of 4% PFA solution.</li>
	<li>Gently agitate the scintillation vials at room temperature (RT) for 2 h and leave overnight in a refrigerator (4 &#176;C) on a rotator. 
	NOTE: The time of fixation can be adjusted depending on the antibody used. For example, limiting the fixation to 1.5 h will allow for successful immunolabeling of post synaptic terminals when using the GluA 2 antibody.</li>
</ol>
3. Decalcification of the Temporal Bone
<ol>
	<li>Remove the PFA solution and wash the cochleae with fresh PBS 3x for 5 min each.</li>
	<li>Add 20 mL of 4% ethylenediaminetetraacetic acid disodium salt (EDTA) solution (pH 7.4) to the scintillation vials and keep in a refrigerator for 48&#8211;72 h on a rotator with gentle agitation. Change the EDTA solution daily until the cochleae are decalcified. Check if the cochleae are decalcified by touching the bony vestibular portion with forceps to assess for elasticity or simply cut a small piece from the edge of the vestibular portion. If such cut results in a crushed piece, the cochlea is not decalcified. 
	NOTE: The EDTA solution may interfere with the immunoreaction of primary antibodies depending on the solution concentration. Prepare 4% EDTA in PBS and adjust the pH to 7.4. For consistent decalcification of temporal bones, fresh EDTA solution is used.</li>
	<li>Change the solution to PBS for microdissection after decalcification is complete. 
	NOTE: If the cochleae are not sufficiently decalcified, dissection of the cochleae cannot be performed.</li>
</ol>
4. Microdissection of the Cochlear Epithelium
NOTE: Once decalcification is complete, microdissection of cochlear epithelium for immunolabeling needs to be performed as soon as possible. In a clean 30 mm Petri dish containing PBS, the inner ear is oriented in the following manner: in reference to the Petri dish's top (lid) and bottom, the cochlear round and oval windows face the top. In reference to dissector, the cochlear portion is oriented toward the front (away from dissector) and vestibular portion toward the back (near dissector). The following describes the steps in detail.
<ol>
	<li>Hold the vestibular portion of the temporal bone with forceps and cut the apical turn with the scalpel at a 45&#176; angle as indicated by the red line (Figure 1a).</li>
	<li>Cut vertically along the faint line between the round window and the oval window, as indicated by the red line (Figure 1b) to separate the cochlea from the vestibular portion (Figure 1c). 
	NOTE: This cochlear portion contains the middle, basal, and hook portions of the epithelium.</li>
	<li>Place the cochlear portion with the basal turn toward the bottom and the middle turn toward the top of the Petri dish and cut the bony capsule and lateral wall of the middle turn toward the end from which the apical section was removed, as indicated by the red line (Figure 1d,e).</li>
	<li>Continue cutting to completely separate the middle portion from the basal and hook regions (Figure 1f,g). 
	NOTE: The hook region of the cochlea is the end of the cochlear epithelium corresponding to sensitivity to tones 48 kHz and higher in mice.</li>
	<li>Put the basal and hook portion with the basilar membrane site oriented toward the bottom of the dish and vertically cut the modiolus off the hook region to remove the modiolus as indicated by the vertical red line (Figure 1g). 
	NOTE: The modiolus is the conical-shaped central axis of the cochlea that consists of spongy bone and cochlear nerve as well as the spiral ganglion. It may be preferable to perform this cut with scissors.</li>
	<li>Cut the basal and hook regions as the other red line indicates in Figure 1g to separate the hook portion (Figure 1h). 
	NOTE: The cochlea has now been separated into apical, middle, basal, and hook portions. The next steps will be the final dissection of each of the individual turns, using the middle turn as an example.</li>
	<li>Cut away the relatively large portions of bony capsule and lateral wall tissue of the middle turn as indicated by the red line (Figure 1i). 
	NOTE: The lateral wall of the cochlear duct is formed by the spiral ligament and the stria vascularis.</li>
	<li>Hold the lateral wall with forceps to align the bony capsule and lateral wall with the bottom of the Petri dish and cut these tissues from the basilar membrane side. Then flatten the specimen to orient the sensory hair cell surface side up. Trim away the rest of the bony capsule and lateral wall (Figure 1j).</li>
	<li>Remove the tectorial membrane using forceps to completely separate the middle region (Figure 1k).</li>
	<li>Repeat steps 4.7&#8722;4.9 for the final dissections of the remaining turns or regions as shown in Figure 1l.</li>
	<li>Prepare a 10 mm round coverslip for adhesion of the sensory epithelium. Use a pipette to hand-spread 0.5 &#181;L of cell and tissue adhesive on the center of the round coverslip. After drying (3&#8211;5 min at RT), put the coverslips in PBS in Petri dishes.</li>
	<li>Stick all four pieces of the sensory epithelium on the 10 mm round coverslip in the Petri dish. Then hold one edge of the coverslip to transfer it to a four-well dish for immunolabeling or immunohistochemistry (Figure 1m). 
	NOTE: Figure 2 illustrates the location of the major cuts.</li>
</ol>
5. Immunolabeling for Cochlear Synapses
NOTE: The protocol for immunolabeling for cochlear synapses followed in this study has been previously described13. Presynaptic ribbons were labeled with a CtBP2 antibody (mouse anti-carboxyl-terminal binding protein 2 IgG1, labeling the B domain of the RIBEYE scaffolding protein). Post synaptic terminals were labeled with GluA2 antibody (mouse anti-glutamate receptor 2 IgG2a, labeling subunits of the AMPA receptor).
<ol>
	<li>Wash the sensory epithelium with PBS 3x for 5 min each wash in a four-well dish and then add 2 mL of 2% nonionic surfactant (i.e., Triton-X 100) into the dish for 30 min at RT on a rotator.</li>
	<li>Remove the nonionic surfactant solution from the dish using a pipette and add 100 &#181;L of blocking solution containing 10% normal goat serum to each well for 1 h on a rotator with gentle agitation at RT.</li>
	<li>Remove the blocking solution from each well, wash with PBS 3x for 5 min each wash under gentle agitation at RT.</li>
	<li>Add 100 &#181;L of the primary antibodies diluted with PBS to each well: mouse anti-GluA2 IgG2a (1:2,000), mouse anti-CtBP2 IgG1 (1:400). Cover each four-well dish with its lid and place into a large humidified container that protects from light. Incubate at 37 &#176;C for 24 h. 
	NOTE: As an option, rabbit anti-myosin VIIa (1:400) for immunolabeling sensory hair cells can be added.</li>
	<li>Wash 3x with PBS for 5 min each wash with gentle agitation at RT.</li>
	<li>Add 100 &#181;L of the secondary antibodies Alexa 594 goat anti-mouse IgG1a (1:1,000), Alexa 488 goat anti-mouse IgG2a (1:1,000) diluted with PBS to each well. Cover each four-well dish with its lid and place into a large humidified container that protects from light. Incubate at 37 &#176;C for 2 h. 
	NOTE: If myosin VIIa is used, add Alexa 350 goat anti-rabbit (1:200).</li>
	<li>Wash 3x with PBS for 5 min. Then, transfer the 10 mm round coverslip onto a slide with the samples on top.</li>
	<li>Carefully add 8 &#181;L of Fluoro-Gel with Tris buffer into the center of the coverslip. Then hold the edge of another 10 mm round coverslip with forceps to mount on top, sandwiching the two coverslips together.</li>
	<li>Use nail polish to seal the slides, put them in a cardboard slide folder, and store in the refrigerator. 
	NOTE: If some Fluoro-Gel leaks out between the coverslips during mounting, clean the edges of the coverslips before sealing the slide with nail polish. Confocal images need to be taken within 7 days.</li>
</ol>

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H., Schacht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+therapeutic+interventions+against+noise-induced+hearing+loss.">Emerging therapeutic interventions against noise-induced hearing loss.</a> Expert Opinion on Investigational Drugs. 26 (1), 85-96 (2017).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+insult+to+injury:+cochlear+nerve+degeneration+after+"temporary"+noise-induced+hearing+loss.">Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss.</a> Journal of Neuroscience. 29 (45), 14077-14085 (2009).</li><li>Chen, F. Q., Zheng, H. W., Hill, K., Sha, S. 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The authors have nothing to disclose.

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-preparation-in-the-adult-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.5K Views.  Medical University of South Carolina. 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.

Video: Cochlear Surface Preparation in the Adult Mouse

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

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 in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

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

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

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

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

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

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

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

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated 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 the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

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 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 ...

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 regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish 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

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

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

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

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 these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

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

Cochlear Surface Preparation in the Adult Mouse

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Chapters in this video

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

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

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

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

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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

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

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies