The aim of this procedure is to selectively trace auditory fibers from the peripheral nervous system to the central nervous system in an embryo. This is accomplished by first performing a microdose section to reveal the ventral chondro cranium and identify the avian cochlear duct. The second step is to perform a micro injection into the underlying acoustic ganglion cells and their axons.
Next, the sample is incubated and oxygenated artificial CSF for 20 minutes to allow for DIA transfer. Finally, a histological preparation and sectioning of the tissue allows for microscopic visualization of the acoustic ganglion central projections. The main advantages of this technique over existing methods are that it enables selective tracing of eighth nerve.
Auditory fibers with high resolution allows for rapid dye transfer in live tissue and permits experimentation at an early developmental stage. This method can help answer key questions in the field of hearing research. For example, what are the molecules that guide axon to their central targets?
Or how does the circuitry respond to injury or ototoxic exposure? First, add artificial cerebral spinal fluid to a 500 milliliter wide mouth Nalgene jar until the jar is two thirds full. Replace the lid, which has a hole drilled into it onto the jar.
Place a stem bubbler attached to a tank of 95%oxygen and 5%carbon dioxide through the hole in the lid and into the A CSF. Turn on the gas and adjust the pressure to obtain a constant stream of bubbles that reach about one third of the liquid, but are not forceful enough to cause splashing over the rim. Submerge up to six five milliliter glass vials into the jar to separate individual samples from one another.
And from the gas flow turbulence. Allow the A CSF to oxygenate for at least 20 minutes while the A CSF oxygenates pull glass micro pipettes backfill the micro pipette with a 6.25%rodine Dexter and am immune solution in PBS containing 0.4%Triton XX.Attach the micro pipette to the micro manipulator and break the tip with fine forceps to form a 20 to 50 micron opening. Use fine tubing to attach the stabilized micro manipulator to the pico spritzer.
Set the pico spritzer between 10 to 30 PSI to start to complete the rest of the setup. Ensure that there is a small silicon dissection dish with two dissection pins placed into a small hexagonal way. Webo two fine tip forceps.
One curve tip forceps, a 50 milliliter beaker and a container for tissue waste in the dissection area. After removing the eh chick embryo from the egg, place the head onto the silicon coated dissection dish. In the whey boat, position the head with a ventral view so that the ear on the side of interest in this case, the right side is visible.
Once in the correct position, stabilize the tissue with dissection pins, avoiding the hind brain region. Use the 50 milliliter beaker to transfer 30 milliliters of the oxygenated A CSF into the dish, fully submerging the tissue. Then use fine tipped forceps to gently grasp and peel down the lower jaw.
Then remove the lower eyelid on the side of interest. Next, remove the overlying vascular and soft tissues, including any remaining palate and gullet. Once these tissues have been removed, the smooth surface of the ventral cranium and distinctive vertebral arteries should be revealed.
Carefully dissect the skin around the external mitos the cartilage of the jaw joint and the middle ear While leaving the inner ear intact. Use the forceps to gently cut away the cartilaginous structures of the jaw and middle ear. Now use a gentle sweeping motion with the fine forceps tips to remove vertebral arteries and pooling blood.
This is important as blood can obscure view and coagulation may plug the injection pipette opening. By this point, the characteristic white tial mass should be visible. The arrow in this figure indicates the cochlear duct with adjacent sensory epithelium and acoustic ganglion.
It is located directly beneath the chondra cranium, and as seen here can be visualized as an elongated finger-like projection extending from the inner ear ventrally with the apex near the anterior midline to label auditory fibers. First place the injection pipette near the embryo surface of the cochlear duct and baula papular region slowly lower the micro pipette to first puncture the cranium. This region of cranium is thinner than surrounding areas and requires less force to penetrate.
The micro pipette should now be inside the cochlear duct. If required. A small injection of tracing dye can be made here to aid in visualizing the basilar per pillar without repositioning the micro pipette continue lowering until it just breaches the deep border of the cochlear duct.
This image shows the ideal position of the pipette in the appropriate region of the acoustic ganglion. Perform an injection at this point and then repeat in multiple regions along the length of the basala papilla. Depending on the extent of the labeling desired and excluding the most proximal tip where the vestibular legina is located.
With the pico spritzer set between 10 to 30 PSI, each injection should result in a fluorescent DIA labeled spot of approximately 200 to 400 microns in diameter as seen here. Once the injection is complete, use forceps to transect the sample above the brainstem as illustrated here, and use a transfer pipette to place the cordal portion into one of the small jars within the container of perfused. A CSF.
For each embryo allow 20 to 30 minutes for die transfer depending on the distance of central tracing desired. Once the incubation time has elapsed, remove the tissue and prepare for histological sectioning. Here tissue is immersed in 4%paraldehyde in one XPBS overnight, which will then be followed by an overnight dehydration in 30%sucrose in one XPBS.
After the tissue is equilibrated overnight in 30%sucrose, it is cryo sectioned coronal. After sectioning, immuno labeling with appropriate antibodies can orient the observer to anatomical location as well as identify particular regions of interest. This image shows the coronal view of E six brainstem with bilateral acoustic ganglia intact.
The S schematized red pipette illustrates accurate targeting of acoustic ganglion cells from a dorsal lateral entry point. Sections are immuno labeled with anti neurofilament to highlight axonal projections. The vestibular organs are rostral to the section shown.
The vestibular cochlear nerve is labeled as is the acoustic ganglion and vestibular ganglion. The dorsal side is up the scale bar equals 300 microns. The following, a representative low and high power images of selective auditory fiber tracing in an E eight embryo from 25 micron.
Coronal sections collected from the same embryo are different planes as seen here. RDA labeled axons can be traced from the injection site indicated by the arrow head through the cranial nerve indicated by the arrow and quarterly towards the primary auditory nuclei targets indicated by the double arrow head, the scale bar equals 300 microns. Immuno labeling with anti neurofilament antibody highlights all axonal projections and the RDA traced fibers from the previous image are within the auditory projection pathway.
AP and VP indicate respective auditory and vestibular components peripheral to the nerve entry point. The merged color image shows how the RDA labeled axons fit into the local anatomy. High power image of the same embryo at a more cordal position demonstrates the high resolution of the fibers at the nerve entry point indicated by an arrow and along the central pathway indicated by a double arrow head.
Neurofilament stain of all axons allows demarcation of P-N-S-C-N-S as well as putative auditory versus vestibular projections. Again, 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. Again, the overlay of separate channels is shown as a color merge.
Histologic examination of the acoustic ganglion reveals the location of labeled acoustic ganglion cells from the previous tracing and their peripheral projections indicated by the asterisk, A traced retro greatly to the basilar papilla. Here AG indicates the acoustic ganglion bp, the basala papilla, and lm the Laina macular. The scale bar equals 100 microns.
Finally, this illustration shows a 700 micron tracing pathway expected with auditory specific eighth nerve labeling at E eight. The injection site indicated by the red circle is cordal to nerve entry point and central projections travel rostral and quarterly. Once in the hind brain here, the scale bar equals 300 microns.
While attempting this procedure, tailor your injections to best fit your analysis criteria. And remember, there will be some variability in the exact number and location of trace axons between samples.
