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 thr...

<ol>
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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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+pattern+formation+in+the+inner+ear:+do+compartment+boundaries+play+a+role.">Molecular genetics of pattern formation in the inner ear: do compartment boundaries play a role.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 11700-11706 (1073).</li><li>Bellairs, R., Osmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of chick development. , (2005).</li><li>Manley, G. 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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+profiling+of+spiral+ganglion+neurons+reveals+insights+into+auditory+circuit+assembly.">Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 31, 10903-10918 (2011).</li></ol>

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 ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Research

JoVE Journal

Neuroscience

9.1K visualizações.  University of California, Irvine. Aqui descrevemos uma técnica de microdissecção seguido de injeção de corante fluorescente no gânglio acústico de embriões de galinha cedo para rastreamento seletivo das fibras auditivas axônios no nervo e rombencéfalo.