Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Summary

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.

Abstract

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.

Protocol

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 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.
• 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.
• Two fine-tip forceps, one curved-tip forceps, a 50 ml beaker, and a container for tissue waste.
• Pulled glass micropipette (1.2 OD / 0.9 ID) broken to approximately 20-50 μm 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.

2. Micro-dissection to Reveal Basilar Papilla at E8 ^4,5

1. With curved forceps, cut E8 embryo just below the brainstem, placing head directly onto silicone-coated dissection dish within weigh boat.
2. 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.
3. Using the 50 ml beaker, transfer 30 ml of oxygenated aCSF into the dish, fully submerging the tissue.
4. Gently grasp and peel down the lower jaw using fine-tip forceps, then remove the lower eyelid on the side of interest.
5. 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.
6. 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.
7. 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.
8. 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.

3. Selective Labeling of CN VIII Auditory Fibers

1. Place the injection pipette near the embryo surface at the cochlear duct and basilar papilla region.
2. 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.
3. 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 μm diameter (Figure 2D).
4. 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.
5. For each embryo, allow 20-30 min for dye transfer, depending on the distance of central tracing desired.
6. 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.

4. Counterstaining and Analysis

1. 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.
2. 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).

Results

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),...

Discussion

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...

Materials

List of materials used in this article

Name	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