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>Groves, A. K., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+sound+in+space:+the+regulation+of+inner+ear+patterning.">Shaping sound in space: the regulation of inner ear patterning.</a> Development. 139, 245-257 (2012).</li><li>Pflieger, J. F., Cabana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vestibular+primary+afferents+and+the+vestibulospinal+projections+in+the+developing+and+adult+opossum,+Monodelphis+domestica.">The vestibular primary afferents and the vestibulospinal projections in the developing and adult opossum, Monodelphis domestica.</a> Anatomy and Embryology. 194, 75-88 (1996).</li><li>Molea, D., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+topography+of+nucleus+magnocellularis+innervation+by+the+cochlear+ganglion.">Timing and topography of nucleus magnocellularis innervation by the cochlear ganglion.</a> The Journal of Comparative Neurology. 466, 577-591 (2003).</li><li>Bissonnette, J. P., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+atlas+of+the+gross+anatomy+of+the+developing+inner+ear+of+the+chicken.">Standard atlas of the gross anatomy of the developing inner ear of the chicken.</a> The Journal of Comparative Neurology. 368, 620-630 (1996).</li><li>Brigande, J. V., Kiernan, A. E., Gao, X., Iten, L. E., Fekete, D. 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A., Haeseler, C., Brix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+patterns+and+spontaneous+activity+of+afferent+fibres+to+the+lagenar+macula+and+apical+basilar+papilla+of+the+chick's+cochlea.">Innervation patterns and spontaneous activity of afferent fibres to the lagenar macula and apical basilar papilla of the chick's cochlea.</a> Hearing Research. 56, 211-226 (1991).</li><li>Code, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferent+neurons+to+the+macular+lagena+in+the+embryonic+chick.">Efferent neurons to the macular lagena in the embryonic chick.</a> Hearing Research. 82, 26-30 (1995).</li><li>Maklad, A., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+vestibular+afferent+projections+into+the+hindbrain+and+their+central+targets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+system+development:+primary+auditory+neurons+and+their+targets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+inner+ear+cells+using+genomic+tags+for+clonal+identification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+and+morphogenesis+of+the+vertebrate+inner+ear.">Patterning and morphogenesis of the vertebrate inner ear.</a> The International Journal of Developmental Biology. 51, 521-533 (2007).</li><li>Appler, J. M., Goodrich, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+the+ear+to+the+brain:+Molecular+mechanisms+of+auditory+circuit+assembly.">Connecting the ear to the brain: Molecular mechanisms of auditory circuit assembly.</a> Progress in Neurobiology. 93, 488-508 (2011).</li><li>Bulankina, A. V., Moser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuit+development+in+the+mammalian+cochlea.">Neural circuit development in the mammalian cochlea.</a> Physiology (Bethesda). 27, 100-112 (2012).</li><li>Fekete, D. M., Campero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+in+the+inner+ear.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+functional+synaptic+connections+in+the+auditory+system+visualized+with+optical+recording:+afferent-evoked+activity+is+present+from+early+stages.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+assembly+of+auditory+circuits:+spiral+ganglion+and+brainstem.">Embryonic assembly of auditory circuits: spiral ganglion and brainstem.</a> The Journal of Physiology. 590, 2391-2408 (2012).</li><li>Milo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+the+function+of+the+transcription+factor+gata3+during+development+of+the+mammalian+inner+ear.">Genomic analysis of the function of the transcription factor gata3 during development of the mammalian inner ear.</a> PloS One. 4, e7144 (2009).</li><li>Fritzsch, B., Eberl, D. F., Beisel, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+bHLH+genes+in+ear+development+and+evolution:+revisiting+a+10-year-old+hypothesis.">The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis.</a> Cellular and Molecular Life Sciences : CMLS. 67, 3089-3099 (2010).</li><li>Jahan, I., Kersigo, J., Pan, N., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurod1+regulates+survival+and+formation+of+connections+in+mouse+ear+and+brain.">Neurod1 regulates survival and formation of connections in mouse ear and brain.</a> Cell and Tissue Research. 341, 95-110 (2010).</li><li>Huang, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brn3a+is+a+transcriptional+regulator+of+soma+size,+target+field+innervation+and+axon+pathfinding+of+inner+ear+sensory+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Gata3+transcription+factor+in+the+acoustic+ganglion+of+the+developing+avian+inner+ear.">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><li>Lu, C. C., Appler, J. M., Houseman, E. A., Goodrich, L. 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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 border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <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>KH2PO4</td>
 <td>Various</td>
 <td></td>
 <td>part of aCSF recipe</td>
 </tr>
 <tr>
 <td>NaHCO3</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>CaCl2</td>
 <td>Various</td>
 <td></td>
 <td>part of aCSF recipe</td>
 </tr>
 <tr>
 <td>MgSO4</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%O2 / 5%CO2) 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" (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

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),...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

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 ...

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9.1K Views.  University of California, Irvine. 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.