This technique can answer important functional questions about neuronal responses to taste in the geniculate ganglia, a crucial part of the interior chorda tympani taste pathway. This technique can be used to monitor the real-time reactions of multiple individual neurons in a single experimental trial, as cells recorded per animal are significantly higher than typically observed via the electrophysiological method. With the headpost-mounted mouse in the supine position on a heating pad, make a two centimeter midline incision in the skin over the throat, from the sternum to the chin.
And retract the skin and sub-maxillary glands to fully expose the digastric muscles. After locating the seam in the paratracheal musculature, separate the seam with blunt dissection and retract the tissue to open it. Carefully cut an opening in the top of the trachea large enough to fit a piece of polyethylene tubing without cutting more than halfway through the diameter of the trachea and insert the tubing into the trachea toward the lungs.
Reposition the retractors to release the paratracheal musculature and retract the sub maxillary glands. Then, use a small amount of veterinary glue to seal the paratracheal musculature together over the tube. To break open the tympanic bulla, gently tease the desired digastric muscle upward and pull the connective tissue apart.
Make an incision at the anterior end of the muscle, avoiding the blood vessels. And pull back posteriorly until clear of the tympanic bulla. Tilt the head back slightly to lift the tympanic bulla and locate the branch of the carotid artery anterior to the posterior insertion point of the digastric muscle.
Palpate just posterior to this blood vessel for the convex structure of the tympanic bulla and locate a seam in the musculature. Using two sets of fine forceps, blunt dissect at the seam until the bone of the tympanic bulla is visible and use retractors to keep a clear view of the bone. Locate the seam running anterior to posterior on the bulla and use a surgical probe to poke a hole in the bone at the center of the seam, Then, use a set of fine end scissors to cut a circular area in the bone, taking care not to cut blood vessels anterior and posterior to or below the bulla.
To expose the geniculate, locate the cochlea. Anterior to the cochlea is the tensor tympani muscle. Use spring scissors to cut and remove this muscle.
Use the surgical probe to poke a hole in the cochlear promontory and immediately use suction to aspirate any liquid that flows out of the hole. Enlarge the hole in the cochlea, taking care not to damage the blood vessel encircling the cochlea, to the posterior and lateral edge. And tilt the mouse's head forward to locate the hole in the temporal bone beneath the former cochlear structure.
Take note of the ridge anterior to the hole that sits directly over the seventh nerve. And insert a surgical probe into the hole to allow the temporal bone to be carefully lifted to expose the seventh nerve. If the geniculate is not fully visible, gently tilt the animal's head back and attempt to pull up the bone anterior to the nerve.
If the ganglia is still obscured, pull up more bone from beneath, taking care not to place the probe deep beneath the bone, as this may damage the geniculate. To run the tastant panel, use suctioned to remove the liquid from over the geniculate and place the mouse on an absorbent pad under a dissecting microscope. Use the hole left in the bulla, the hole in the temporal bone, and the seventh nerve to locate the geniculate ganglion.
And use the FITCGFP filter on the epifluorescent scope to check for individual GCaMP expressing geniculate ganglion neurons. Place the dispensing needle for one tastant line firmly into the animal's mouth and place a Petri dish below the mouth to catch any fluid. Synchronize the start of video recording with the start of tastant presentation, watching the live feed for responses, drift, and seepage during the recording.
If seepage occurs, suction the liquid until the view of the geniculate is clear and redeliver the tastant. If drift occurs, check that all of the parts of the headpost are firmly tightened. If no response occurs, check that the liquid is flowing and that the microscope and camera are focused on the proper location without anything obscuring the field of view.
When all of the desired experiments are completed, gently ease the retractors and repeat the exposure and tastant analysis on the opposite side of the animal. As observed, taste stimuli applied to the tongue should result in a rapid transient increase in GCaMP fluorescence, causing a noticeable change in brightness among the responding neurons. Analysis of the fluorescence video footage allows the generation of traces corresponding to the changes in fluorescence over the baseline response within individual regions of interest over time.
Changes in the fluorescence intensity above the threshold level is considered a positive response. In addition to damaging the ganglia, it is important to avoid bleeding. If bleeding occurs, wait for the blood to clot before applying saline and suction to remove the blood from the visual field.
Visualization of the geniculate ganglia has allowed researchers to directly measure the responses of neurons to taste-like stimuli and to identify these neurons with tools such as Cre-dependent GCaMP.
