Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

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), or modified to study projections arising from individual vestibular organs. Because axons are cleanly traced along their entire length, accurate quantitative analyses can be performed distinguishing auditory from vestibular projections during development, including precise timing and patterns of initial innervation, targeting error rates, etc. Additionally, unlike the previous approach of auditory fiber labeling, this approach is performed on non-fixed, living tissue and thus can be used simultaneous to other in vitro experimentation.

Though this technique is suitable for embryos from E6-E9, best results commence at E8 when auditory axons have innervated their primary central targets ³. In contrast, vestibular projections arrive in the brainstem earlier ^9,10 and thus would serve as better candidates for earlier tracing, though technically challenging due to the small size and relative immaturity of the vestibular apparatus at this age. Histological sections should be examined to confirm the location of dye injection, as the apical-most region of the basilar papilla contains a small sensory patch thought to be of vestibular function and if labeled could potentially contaminate the results.

Once the investigator is familiar with the anatomy, three of the most commonly encountered problems are as follows:

• The cochlear duct is filled with tracing dye, but ganglion cell axons are not labeled. This problem likely indicates that the injection was not performed deep to the cochlear duct, where the ganglion cells and nerve fibers reside. This problem may be corrected by ensuring that the injection micropipette first punctures the chondrocranium, then is gently advanced so as to just puncture through the other side of the duct. Alternatively, the injection pipette may have been correctly positioned but delivered insufficient quantities of dye. Increasing injection pressure or number of injections may also overcome this issue.
• The nerve is inadvertently transected as a consequence of excessive pulling on the cartilaginous labyrinth during dissection. This difficulty can be avoided by using fine dissection techniques and limited dissection of the middle ear.
• The injection is too extensive and inadvertently labels other sensory ganglion cells or fibers arising from other nerve components. This problem can be minimized by optimizing injection protocols, and may require reducing the injection pressure or the number of pulses, or shifting the location of injections.

Two examples of potential analyses are:

1. Divergence of projection patterns: determines the extent of labeled axons along a particular axis and how they might be affected following experimentation. Rostrocaudal divergence can be described as:

(Number sections containing labeled axons) x (Thickness of each section) This measure may be corrected for the extent of dye label as a proportion of the basilar papilla, or in counterstained tissue, for the proportion of VIIIth nerve axons labeled. This is a useful tool for analyzing topographic projections, as the labeled axons can be cleanly traced to their terminal fields in the hindbrain. For measurements of tonotopy, dye injections should be limited to the region of interest along the basilar papilla.

2. Targeting errors: determines the frequency of mistargeted axons for every predetermined region of analyzed tissue (can be per section, per embryo, etc.). Perturbations may result in axons following an abnormal projection path or terminating in an inappropriate region. When quantifying errors, numbers should be normalized to account for difference in injection amounts between embryos. Smaller injections are more likely to result in the ability to score single axons. A basic targeting error analysis can be described as:

Figure 1. Coronal view of E6 brainstem for simplification with bilateral acoustic ganglia intact. Schematized red pipette illustrates accurate targeting of acoustic ganglion cells from a dorsolateral entry point. Sections are immunolabeled with anti-neurofilament to highlight axonal projections. Vestibular organs are rostral to sections shown. Region of RDA injection for selective auditory fiber tracing demonstrated with red pipette illustration. VIII = vestibulocochlear nerve, AG = acoustic ganglion, VG = vestibular ganglion. Dorsal is up. Scale bar = 300 μm.

Figure 2. Microdissection and injection of target region. (A) Appropriate position of embryo on dissection dish, ventral view with head tilted dorsally and turned slightly to the side to allow better access to the right side. Right ear is shown and underlying basilar papilla is indicated with a dashed line. Arrowhead in (A) and (B) illustrates initial position of pipette insertion (B) Following dissection, a white otoconial mass is visible, demarcating the apical border of the basilar papilla (arrow). (C,D) First injection should provide an outline of the cochlear duct. (D) Subsequent injections should be made just deep to the cochlear duct and basilar papilla into the acoustic ganglion region. RDA fluorescence is useful in enhancing injection depth perception and each individual injection should create a fluorescent dye-labeled spot of approximately 200 – 400 μm diameter as seen here. Scale bar = 2 mm.

Figure 3. Representative images of selective auditory fiber tracing in an E8 embryo. (A-C) Low power and (D-G) high power 25 μm coronal sections collected from the same embryo at different planes. (A) RDA labeled axons can be traced from injection site (arrowhead), through the cranial nerve (arrow) and caudally toward the primary auditory nuclei targets (double arrowhead). (B) Immunolabeling with anti-neurofilament antibody highlights all axonal projections and the RDA-traced fibers from (A) are within the auditory projection pathway. Overlay of separate (A) and (B) channels shown as a color merge in (C). (D-F) High power image of the same embryo at a more caudal position demonstrates (A) high resolution of fibers at the nerve entry point (arrow) and along the central pathway (double arrowhead). (E) Neurofilament stain of all axons allows demarcation of PNS-CNS as well as putative auditory versus vestibular projections. 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. Overlay of separate (D) and (E) channels shown as a color merge in (F). (G) Histologic examination of the acoustic ganglion reveals location of labeled acoustic ganglion cells from the tracing shown in (A-F) and their peripheral projections (asterisk) are traced retrogradely to the basilar papilla. (H) Illustration of a 700 μm tracing pathway expected with auditory-specific VIII labeling at E8. Injection site (red circle) is caudal to nerve entry point, and central projections travel rostral and caudally once in the hindbrain. Vp = peripheral vestibular, Ap = peripheral auditory, Vc = central vestibular, Ac = central auditory, AG = acoustic ganglion, BP = basilar papilla, LM = lagenar macula, SVG = superior vestibular ganglion, IVG = inferior vestibular ganglion. Scale bar = 300 μm for A-C, 100 μm for D-F, 100 μm in G and 500 μm in H.