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Neuroscience

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Published: March 18th, 2013

DOI:

10.3791/50305

1Department of Neurobiology and Behavior, 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.

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.

1. Prepare the Following Dissection Tools and Reagents

  • 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.......

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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),.......

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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.......

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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.

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Comments
Polystyrene Weigh Dish Fisher Scientific 02-202-101
Petri Dish, 35 X 10 mm Fisher Scientific 50820644 Use to make silicone dissection dish
Sylgard Silicone Elastomer Kit World Precision Instruments SYLG184 Coat Petri to make dissection dish
Dissection Pins Various Holds embryo in place during dissection
NaCL Various part of aCSF recipe
KCl Various part of aCSF recipe
KH2PO4 Various part of aCSF recipe
NaHCO3 Various part of aCSF recipe
Glucose Various part of aCSF recipe
CaCl2 Various part of aCSF recipe
MgSO4 Various part of aCSF recipe
Container for aCSF. Suggest translucent wide-mouth Nalgene jar, 500 ml (16 oz) with lid. CPLabSafety QP-PLC-03717 Drill hole opening in top of lid for glass bubling stem to penetrate liquid
Empty 5 ml glass vial or comparable transparent vial American Pharmaceutical Partners, Inc 6332300105 Use during aCSF incubation to keep samples separate from each other and from the bubbling stream
Tank of carbogen (95%O2 / 5%CO2) connected by tubing to bubbler Various Attach by tubing to glass stem bubbler for infusion into aCSF
Glass stem bubbler Various To infuse carbogen into aCSF
Curved-tip forceps World Precision Instruments 501008 To remove embryo head from egg
Two fine-tip forceps World Precision Instruments 501985 For micro-dissection
50 ml Beaker various
Rhodamine Dextran Amine (RDA) Invitrogen various Fluorescent axon tracer
Triton X-100 ICN Biomedicals
Phosphate Buffered Saline, (1X PBS) Various Standard lab reagent
Thin Wall Glass Capillaries, 1.2 OD, .9 ID 4" (100 mm) length World Precision Instruments TW120F-4 Load with RDA. Each capillary makes two glass micropipettes
Needle / Pipette puller David Kopf Instruments Model 720 Settings used: Heat 16.4, Solenoid 2.2
Picospritzer Parker Instrumentation various Attach by fine tubing to glass micropipette
Micromanipulator Narishige various
Dissection microscope with fluorescence Various
4% Paraformaldehyde Various Standard lab reagent
anti-Neurofilament antibody, optional Millipore AB1991 Follow histological protocol recommended by manufacturer
Cryostat and associated materials for sectioning Leica various
Epifluorescent microscope for imaging Zeiss, various

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