Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

This method can be useful to answer key questions in neurobiology fields such as how do presynaptic terminals function in vivo. The main advantage of this set of techniques is that we show complimentary approaches to asses the function of olfactory pathways using Xenopus tadpoles as animal model. Demonstrating this procedure will be myself, Beatrice Terni, and Paolo Pacciolla.

Begin the transection procedure by wetting two pieces of cellulose qualitative filter paper in 0.02%MS2 22 anesthetic solution, and placing them under the dissecting scope. Then pick a tadpole from the tank and immerse it in a dish of anesthetic solution. The tadpole should stop swimming within two to four minutes.

Check for proper anesthetization by the absence of a reaction to mechanical stimuli applied at the tail level using tweezers. Place the anesthetized tadpole on the filter paper under the scope. Position the animal with its dorsal side facing upward so brain structures can be visualized.

For behavioral experiments, use veni scissors to transect both nerves to suppress all odorant information arriving to the olfactory bulb. Load a poled glass pipette with two microliters of calcium green one dextrine solution, and place it in the microinjector. Place an anesthetized tadpole under the dissecting microscope on filter paper, then move the tip of the pipette into the principal cavity of the nasal capsule, and deliver 0.15 to 0.3 microliters of the dye.

Leave the tadpole in place for two to three minutes. Using a Pasteur pipette, drip 0.02%MS2 22 solution onto the more coddle parts of the animal to avoid drying. Transfer the animal to the recovery tank where the tadpole should recover normal swimming within 10 minutes or so.

Observe florescence at the level of the glomerular layer of the olfactory bulb on the day after injection. Prepare an anesthetized tadpole for imaging by first removing the skin above the olfactory bulb. Use veni scissors to make a lateral incision on the tadpole's skin on the edge of the central nervous system at the level of the olfactory bulb.

Avoid extending the cut to the tectum, which can be easily identified by the location of the optic nerve. Keep the animal moist by applying drops of 0.02%MS2 22 solution using a Pasteur pipette then pinch the cut skin using tweezers and pull it over the nervous system. Verify successful removal by the absence of melanocytes above the olfactory bulb.

Place the tadpole into the well of the cell guard coated dish. Position a glass cover slip coated with high vacuum grease to cover the top of the tectum to the end of the tail. Ensure that the olfactory bulb and placodes remain exposed to the extracellular medium.

Fill the pitri dish with Xenopus Ringer's solution containing 100 micromolar tubocurarine to prevent muscle contractions. Place the dish holding the tadpole under an upright microscope. Connect the reservoir containing Xenopus Ringer's solution with the dish using polyethylene tubing for continuous profusion of Xenopus Ringer's solution.

Start perfusing Xenopus Ringer's solution. Maintain the level of the solution in the dish constant throughout the experiment. Continuously evaluate tadpole viability by observing blood circulation through the vessels.

Begin live imaging by using a low magnification objective to visualize the tadpole. Move the micromanipulator axis to place the capillary delivering the odorant solution on the top of one nasal capsule, forming a 90 degree angle with the olfactory nerve. Find the olfactory bulb located ipsilaterally to the nasal capsule.

Switch to a high-magnification water objective with a long working distance. Check the florescence emission by I.Glomerular structures should be obvious. Begin by pipetting 20 milliliters of fresh amino acid solution into an elevated reservoir.

Then take six food deprived tadpoles from their housing tank, and place them in two liters of clean tadpole water to minimize the exposure to odorants. Place a modified six well dish on a white LED transilluminator. Fill each well with 10 milliliters of tadpole water.

Place one tadpole per well, then leave to rest for at least three minutes. Start image acquisition and acquire movies that contain basal, stimulus, and recovery periods. An attractive response can be detected as a movement towards the nozzle delivering the solution of amino acids.

Return animals to their tank after imaging. Tracking tadpole head positions within an area of 35 millimeters by 35 millimeters, or the equivalent size in pixels, allows a quantitative analysis of olfactory-guided behavior. The dotted line represents the proximal area to the odor solution inlet.

Individual plots of tadpole movements are constructed using X Y coordinates obtained by image analysis. The extracted motility plots must faithfully reproduce video images. A region of interest of 8.75 millimeter radius centered on the solution inlet is used to classify the proximity of the animals to the odorant source.

More time spent in the vicinity of the odorant's nozzle indicates positive tropism. Time spent by tadpoles near the nozzle during defined periods, for example, 15 second intervals, allows identification of the ability to detect amino acid solutions. The overall behavior of a population of tadpoles can be obtained by plotting the distribution of individual data.

Positive tropism can be detected when the amino acid solution is prepared at one millimolar or 160 micromolar. Animals do not respond to water application. Following this procedure, other methods like histological techniques or electrophysiology, can be performed to answer additional questions like the alteration of synaptic properties after injury.

After its development, this technique paved the way for researchers in the field of neurobiology to explore the processing of olfactory information in Xenopus tadpole.