Name: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve (Video) | JoVE Journal
Uploaded: 2013-03-18T12:00:00.000+00:00
Duration: 11 min 27 s
Description: Learn selective tracing of auditory fibers in avian embryos using microdissection and fluorescent dye injection into the acoustic ganglion. This technique enables high-resolution visualization of eighth nerve development in early chick embryos.

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5 minutes

The authors wish to thank Dr. Candace Hsieh for suggestions and assistance with imaging techniques and Dr. Doris Wu for expertise on chick inner ear anatomy during early embryogenesis. This work was supported by NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796, and DOE GAANN P200A120165.

1. Prepare the Following Dissection Tools and Reagents
<ul>
	<li>Artificial cerebrospinal fluid (aCSF; 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 20 mM NaHCO3, 3 mM HEPES, 10 mM Glucose, 2 mM CaCl2, 1.3 mM MgSO4) continuously infused with 95% O2 / 5% CO2 at room temperature. For infusion, fill to 2/3 a 500 ml wide-mouth Nalgene jar with a hole drilled in the lid. Tank will be attached by tubing to a glass stem bubbler, which penetrates the aCSF through the hole in the jar lid. Adjust pressure to obtain a constant stream of bubbles that reach about 1/3 of the liquid but are not forceful enough to cause splashing over the rim. Submerge 5 ml glass vials (up to 6) into the Nalgene jar to separate individual samples from each other and from the gas flow turbulence. ACSF should be oxygenated for at least 20 min prior to any tissue incubation.</li>
	<li>Small silicone dissection dish (35 mm x 10 mm Petri coated using Sylgard Elastomere Kit) with two dissection pins placed into small hexagonal polystyrene (47 mm bottom) weigh boat.</li>
	<li>Two fine-tip forceps, one curved-tip forceps, a 50 ml beaker, and a container for tissue waste.</li>
	<li>Pulled glass micropipette (1.2 OD / 0.9 ID) broken to approximately 20-50 &mu;m opening at the tip and filled with rhodamine dextran amine (RDA, MW = 3,000, Invitrogen) in a 6.25% solution with 0.4% Triton X-100 in PBS. Attach by fine tubing to picospritzer and stabilize to micromanipulator.</li>
</ul>
2. Micro-dissection to Reveal Basilar Papilla at E8 4,5
<ol>
	<li>With curved forceps, cut E8 embryo just below the brainstem, placing head directly onto silicone-coated dissection dish within weigh boat.</li>
	<li>Using dissection pins, position the head with a ventral view so the ear on the side of interest is slightly visible, the head tilted slightly dorsally and pointing 10-30 degrees away from center (Figure 2A). Immediately secure the head with pins. If bilateral tracing is desired, center the head at 0 degrees, or a full ventral view so both sides can be accessed. Similarly, for a left-sided injection, secure the head at -10 to -30 degrees from center so the left side is accessible.</li>
	<li>Using the 50 ml beaker, transfer 30 ml of oxygenated aCSF into the dish, fully submerging the tissue.</li>
	<li>Gently grasp and peel down the lower jaw using fine-tip forceps, then remove the lower eyelid on the side of interest.</li>
	<li>Remove the overlying vascular tissue, including any remaining palate and gullet. These soft tissues should peel away revealing the smooth surface of developing chondrocranium with the distinctive vertebral arteries at its surface. By this point, the characteristic white otoconial mass should be visible.</li>
	<li>Carefully dissect the skin around the external meatus, cartilage of the jaw joint, and the middle ear while leaving the inner ear intact. Use the forceps to gently cut away these cartilaginous structures.</li>
	<li>Remove vertebral arteries and pooling blood using a gentle sweeping motion with the fine forceps tips. Blood can obscure view and coagulation may plug the injection pipette opening.</li>
	<li>The cochlear duct with adjacent sensory epithelium (basilar papilla) and acoustic ganglion lies directly beneath the chondrocranium 6 and can be visualized as an elongated finger-like projection extending from the inner ear ventrally, with the distal-most tip (apex) near the anterior midline 5. There may be a small pooling of blood (red) from the dissection and/or otolithic calcifications (white) at the apex of the cochlear duct (Figure 2B) that can aid in visualization. At the apex is the lagenar macula, a sensory patch thought to be of vestibular function 7,8.</li>
</ol>
3. Selective Labeling of CN VIII Auditory Fibers
<ol>
	<li>Place the injection pipette near the embryo surface at the cochlear duct and basilar papilla region.</li>
	<li>Slowly lower the micropipette to first puncture the cranium. This cranial region is thinner than surrounding areas and requires less force to penetrate. The micropipette should now be inside the cochlear duct. Optional: a small injection of tracing dye can be made here to aid in visualizing the basilar papilla.</li>
	<li>Without repositioning the micropipette, continue lowering until it just breaches the deep border of the cochlear duct (Figure 1). Perform an injection here and repeat in multiple regions along the length of the basilar papilla (Figures 2C, 2D), excluding the most proximal tip where the vestibular lagena is located. With the picospritzer set between 10-30 psi, several injections are made. The volume of injected dye fluid varies, and depends on the extent of labeling desired. Each injection should result in a fluorescent dye-labeled spot of approximately 200 - 400 &mu;m diameter (Figure 2D).</li>
	<li>Once the injection is complete, use forceps to transect the sample above the brainstem and use a transfer pipette to place the caudal portion into a small jar within the container of aCSF continually being perfused with 95% O2 / 5% CO2.</li>
	<li>For each embryo, allow 20-30 min for dye transfer, depending on the distance of central tracing desired.</li>
	<li>Remove tissue and prepare for histological sectioning. In the example shown here, tissue is immersed in 4% paraformaldehyde (in 1X PBS) overnight, 30% sucrose (in 1X PBS) overnight. </li>
</ol>
4. Counterstaining and Analysis
<ol>
	<li>Counterstaining is useful to help orient the observer to anatomical location as well as identify particular regions of interest. An effective counterstain in this preparation is neurofilament immunolabeling (Figures 1, 3B, 3E), which strongly labels axons and allows for demarcation of axonal tracts in the CNS.</li>
	<li>Analysis should be performed throughout the labeled region, as the projections can extend several hundred microns or more along the rostrocaudal axis. Some examples of basic analyses are: targeting error rates, timing and/or changes in divergence of projection patterns. RDA-labeled axons are visualized in the peripheral and central nervous system with high resolution (Figures 3A, 3D).</li>
</ol>

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<li>Maklad, A., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+vestibular+afferent+projections+into+the+hindbrain+and+their+central+targets%5BTitle%5D)+AND+%22Brain+Research+Bulletin%22%5BJournal%5D)">Development of vestibular afferent projections into the hindbrain and their central targets.</a> Brain Research Bulletin. 60, 497-510 (2003).</li>
<li>Rubel, E. W., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auditory+system+development%3A+primary+auditory+neurons+and+their+targets%5BTitle%5D)+AND+%22Annual+Review+of+Neuroscience%22%5BJournal%5D)">Auditory system development: primary auditory neurons and their targets.</a> Annual Review of Neuroscience. 25, 51-101 (2002).</li>
<li>Satoh, T., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+analysis+of+inner+ear+cells+using+genomic+tags+for+clonal+identification%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Lineage analysis of inner ear cells using genomic tags for clonal identification.</a> Methods Mol. Biol. 493, 47-63 (2009).</li>
<li>Bok, J., Chang, W., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterning+and+morphogenesis+of+the+vertebrate+inner+ear%5BTitle%5D)+AND+%22The+International+Journal+of+Developmental+Biology%22%5BJournal%5D)">Patterning and morphogenesis of the vertebrate inner ear.</a> The International Journal of Developmental Biology. 51, 521-533 (2007).</li>
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<li>Fekete, D. M., Campero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axon+guidance+in+the+inner+ear%5BTitle%5D)+AND+%22The+International+Journal+of+Developmental+Biology%22%5BJournal%5D)">Axon guidance in the inner ear.</a> The International Journal of Developmental Biology. 51, 549-556 (2007).</li>
<li>Momose-Sato, Y., Glover, J. C., Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+functional+synaptic+connections+in+the+auditory+system+visualized+with+optical+recording%3A+afferent-evoked+activity+is+present+from+early+stages%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Development of functional synaptic connections in the auditory system visualized with optical recording: afferent-evoked activity is present from early stages.</a> Journal of Neurophysiology. 96, 1949-1962 (2006).</li>
<li>Marrs, G. S., Spirou, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+assembly+of+auditory+circuits%3A+spiral+ganglion+and+brainstem%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Embryonic assembly of auditory circuits: spiral ganglion and brainstem.</a> The Journal of Physiology. 590, 2391-2408 (2012).</li>
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<li>Huang, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brn3a+is+a+transcriptional+regulator+of+soma+size%2C+target+field+innervation+and+axon+pathfinding+of+inner+ear+sensory+neurons%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons.</a> Development. 128, 2421-2432 (2001).</li>
<li>Jones, J. M., Warchol, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+Gata3+transcription+factor+in+the+acoustic+ganglion+of+the+developing+avian+inner+ear%5BTitle%5D)+AND+%22The+Journal+of+Comparative+Neurology%22%5BJournal%5D)">Expression of the Gata3 transcription factor in the acoustic ganglion of the developing avian inner ear.</a> The Journal of Comparative Neurology. 516, 507-518 (2009).</li>
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No conflicts of interest are declared.

Studies of the early development of the VIIIth nerve have been limited in part because of the difficulty in identifying embryonic axons arising from multiple distinct ganglia. Several studies have explored the molecular signals guiding auditory and vestibular sensory cell and ganglion cell fates during early development, 5,11,12 but the processes regulating central innervation have yet to be determined. Reports of acoustic ganglion cell projections typically describe peripheral processes to sensory epit...

<table><tbody><tr><td>Polystyrene Weigh Dish</td><td>Fisher Scientific</td><td>02-202-101</td><td></td></tr><tr><td>Petri Dish, 35 X 10 mm</td><td>Fisher Scientific</td><td>50820644</td><td>Use to make silicone dissection dish</td></tr><tr><td>Sylgard Silicone Elastomer Kit</td><td>World Precision Instruments</td><td>SYLG184</td><td>Coat Petri to make dissection dish</td></tr><tr><td>Dissection Pins</td><td>Various</td><td></td><td>Holds embryo in place during dissection</td></tr><tr><td>NaCL</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>KCl</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>Glucose</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>Container for aCSF. Suggest translucent wide-mouth Nalgene jar, 500 ml (16 oz) with lid.</td><td>CPLabSafety</td><td>QP-PLC-03717</td><td>Drill hole opening in top of lid for glass bubling stem to penetrate liquid</td></tr><tr><td>Empty 5 ml glass vial or comparable transparent vial</td><td>American Pharmaceutical Partners, Inc</td><td>6332300105</td><td>Use during aCSF incubation to keep samples separate from each other and from the bubbling stream</td></tr><tr><td>Tank of carbogen (95%O&lt;sub&gt;2&lt;/sub&gt; / 5%CO&lt;sub&gt;2&lt;/sub&gt;) connected by tubing to bubbler</td><td>Various</td><td></td><td>Attach by tubing to glass stem bubbler for infusion into aCSF</td></tr><tr><td>Glass stem bubbler</td><td>Various</td><td></td><td>To infuse carbogen into aCSF</td></tr><tr><td>Curved-tip forceps</td><td>World Precision Instruments</td><td>501008</td><td>To remove embryo head from egg</td></tr><tr><td>Two fine-tip forceps</td><td>World Precision Instruments</td><td>501985</td><td>For micro-dissection</td></tr><tr><td>50 ml Beaker</td><td>various</td><td></td><td></td></tr><tr><td>Rhodamine Dextran Amine (RDA)</td><td>Invitrogen</td><td>various</td><td>Fluorescent axon tracer</td></tr><tr><td>Triton X-100</td><td>ICN Biomedicals</td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline, (1X PBS)</td><td>Various</td><td></td><td>Standard lab reagent</td></tr><tr><td>Thin Wall Glass Capillaries, 1.2 OD, .9 ID 4&quot; (100 mm) length</td><td>World Precision Instruments</td><td>TW120F-4</td><td>Load with RDA. Each capillary makes two glass micropipettes</td></tr><tr><td>Needle / Pipette puller</td><td>David Kopf Instruments</td><td>Model 720</td><td>Settings used: Heat 16.4, Solenoid 2.2</td></tr><tr><td>Picospritzer</td><td>Parker Instrumentation</td><td>various</td><td>Attach by fine tubing to glass micropipette</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>various</td><td></td></tr><tr><td>Dissection microscope with fluorescence</td><td>Various</td><td></td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Various</td><td></td><td>Standard lab reagent</td></tr><tr><td>anti-Neurofilament antibody, optional</td><td>Millipore</td><td>AB1991</td><td>Follow histological protocol recommended by manufacturer</td></tr><tr><td>Cryostat and associated materials for sectioning</td><td>Leica</td><td>various</td><td></td></tr><tr><td>Epifluorescent microscope for imaging</td><td>Zeiss, various</td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

The components of the VIIIth nerve and the anatomy of the nerve itself are complex and convoluted (Figures 1, 3). By selectively tracing fibers arising from acoustic ganglion cells, segments of the VIIIth nerve as well as primary auditory afferents within the brainstem can be cleanly traced and distinguished from their vestibular counterparts (Figures 2, 3). Likewise, this technique could be used to study peripheral projections of the acoustic ganglion cells (Figure 3G),...

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

The aim of this procedure is to selectively trace auditory fibers from the peripheral nervous system to the central nervous system in an embryo. This is accomplished by first performing a microdose section to reveal the ventral chondro cranium and identify the avian cochlear duct. The second step is to perform a micro injection into the underlying acoustic ganglion cells and their axons.Next, the sample is incubated and oxygenated artificial CSF for 20 minutes to allow for DIA transfer. Finally, a histological preparation and sectioning of the tissue allows for microscopic visualization of the acoustic ganglion central projections. The main advantages of this technique over existing methods are that it enables selective tracing of eighth nerve.Auditory fibers with high resolution allows for rapid dye transfer in live tissue and permits experimentation at an early developmental stage. This method can help answer key questions in the field of hearing research. For example, what are the molecules that guide axon to their central targets?Or how does the circuitry respond to injury or ototoxic exposure? First, add artificial cerebral spinal fluid to a 500 milliliter wide mouth Nalgene jar until the jar is two thirds full. Replace the lid, which has a hole drilled into it onto the jar.Place a stem bubbler attached to a tank of 95%oxygen and 5%carbon dioxide through the hole in the lid and into the A CSF. Turn on the gas and adjust the pressure to obtain a constant stream of bubbles that reach about one third of the liquid, but are not forceful enough to cause splashing over the rim. Submerge up to six five milliliter glass vials into the jar to separate individual samples from one another.And from the gas flow turbulence. Allow the A CSF to oxygenate for at least 20 minutes while the A CSF oxygenates pull glass micro pipettes backfill the micro pipette with a 6.25%rodine Dexter and am immune solution in PBS containing 0.4%Triton XX.Attach the micro pipette to the micro manipulator and break the tip with fine forceps to form a 20 to 50 micron opening. Use fine tubing to attach the stabilized micro manipulator to the pico spritzer.Set the pico spritzer between 10 to 30 PSI to start to complete the rest of the setup. Ensure that there is a small silicon dissection dish with two dissection pins placed into a small hexagonal way. Webo two fine tip forceps.One curve tip forceps, a 50 milliliter beaker and a container for tissue waste in the dissection area. After removing the eh chick embryo from the egg, place the head onto the silicon coated dissection dish. In the whey boat, position the head with a ventral view so that the ear on the side of interest in this case, the right side is visible.Once in the correct position, stabilize the tissue with dissection pins, avoiding the hind brain region. Use the 50 milliliter beaker to transfer 30 milliliters of the oxygenated A CSF into the dish, fully submerging the tissue. Then use fine tipped forceps to gently grasp and peel down the lower jaw.Then remove the lower eyelid on the side of interest. Next, remove the overlying vascular and soft tissues, including any remaining palate and gullet. Once these tissues have been removed, the smooth surface of the ventral cranium and distinctive vertebral arteries should be revealed.Carefully dissect the skin around the external mitos the cartilage of the jaw joint and the middle ear While leaving the inner ear intact. Use the forceps to gently cut away the cartilaginous structures of the jaw and middle ear. Now use a gentle sweeping motion with the fine forceps tips to remove vertebral arteries and pooling blood.This is important as blood can obscure view and coagulation may plug the injection pipette opening. By this point, the characteristic white tial mass should be visible. The arrow in this figure indicates the cochlear duct with adjacent sensory epithelium and acoustic ganglion.It is located directly beneath the chondra cranium, and as seen here can be visualized as an elongated finger-like projection extending from the inner ear ventrally with the apex near the anterior midline to label auditory fibers. First place the injection pipette near the embryo surface of the cochlear duct and baula papular region slowly lower the micro pipette to first puncture the cranium. This region of cranium is thinner than surrounding areas and requires less force to penetrate.The micro pipette should now be inside the cochlear duct. If required. A small injection of tracing dye can be made here to aid in visualizing the basilar per pillar without repositioning the micro pipette continue lowering until it just breaches the deep border of the cochlear duct.This image shows the ideal position of the pipette in the appropriate region of the acoustic ganglion. Perform an injection at this point and then repeat in multiple regions along the length of the basala papilla. Depending on the extent of the labeling desired and excluding the most proximal tip where the vestibular legina is located.With the pico spritzer set between 10 to 30 PSI, each injection should result in a fluorescent DIA labeled spot of approximately 200 to 400 microns in diameter as seen here. Once the injection is complete, use forceps to transect the sample above the brainstem as illustrated here, and use a transfer pipette to place the cordal portion into one of the small jars within the container of perfused. A CSF.For each embryo allow 20 to 30 minutes for die transfer depending on the distance of central tracing desired. Once the incubation time has elapsed, remove the tissue and prepare for histological sectioning. Here tissue is immersed in 4%paraldehyde in one XPBS overnight, which will then be followed by an overnight dehydration in 30%sucrose in one XPBS.After the tissue is equilibrated overnight in 30%sucrose, it is cryo sectioned coronal. After sectioning, immuno labeling with appropriate antibodies can orient the observer to anatomical location as well as identify particular regions of interest. This image shows the coronal view of E six brainstem with bilateral acoustic ganglia intact.The S schematized red pipette illustrates accurate targeting of acoustic ganglion cells from a dorsal lateral entry point. Sections are immuno labeled with anti neurofilament to highlight axonal projections. The vestibular organs are rostral to the section shown.The vestibular cochlear nerve is labeled as is the acoustic ganglion and vestibular ganglion. The dorsal side is up the scale bar equals 300 microns. The following, a representative low and high power images of selective auditory fiber tracing in an E eight embryo from 25 micron.Coronal sections collected from the same embryo are different planes as seen here. RDA labeled axons can be traced from the injection site indicated by the arrow head through the cranial nerve indicated by the arrow and quarterly towards the primary auditory nuclei targets indicated by the double arrow head, the scale bar equals 300 microns. Immuno labeling with anti neurofilament antibody highlights all axonal projections and the RDA traced fibers from the previous image are within the auditory projection pathway.AP and VP indicate respective auditory and vestibular components peripheral to the nerve entry point. The merged color image shows how the RDA labeled axons fit into the local anatomy. High power image of the same embryo at a more cordal position demonstrates the high resolution of the fibers at the nerve entry point indicated by an arrow and along the central pathway indicated by a double arrow head.Neurofilament stain of all axons allows demarcation of P-N-S-C-N-S as well as putative auditory versus vestibular projections. Again, AP and VP indicate respective auditory and vestibular components peripheral to the nerve entry point, whereas AC and VC indicate respective auditory and vestibular projections central to the nerve entry point. Again, the overlay of separate channels is shown as a color merge.Histologic examination of the acoustic ganglion reveals the location of labeled acoustic ganglion cells from the previous tracing and their peripheral projections indicated by the asterisk, A traced retro greatly to the basilar papilla. Here AG indicates the acoustic ganglion bp, the basala papilla, and lm the Laina macular. The scale bar equals 100 microns.Finally, this illustration shows a 700 micron tracing pathway expected with auditory specific eighth nerve labeling at E eight. The injection site indicated by the red circle is cordal to nerve entry point and central projections travel rostral and quarterly. Once in the hind brain here, the scale bar equals 300 microns.While attempting this procedure, tailor your injections to best fit your analysis criteria. And remember, there will be some variability in the exact number and location of trace axons between samples.

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Research

JoVE Journal

Neuroscience

This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse's brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

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.

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

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

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

In Ovo Electroporation in the Chicken Auditory Brainstem

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

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

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Learn selective tracing of auditory fibers in avian embryos using microdissection and fluorescent dye injection into the acoustic ganglion. This technique enables high-resolution visualization of eighth nerve development in early chick embryos.

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

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