Il sistema olfattivo come modello per studiare gli schemi di crescita assonale e Morfologia<em> In Vivo</em

The overall goal of this procedure is to label olfactory sensory neurons in vivo to visualize axonal morphology and development. This is accomplished by first poring fluoro four coupled dextran into single olfactory sensory neurons. The second step is to wait 24 hours to allow the dye to spread into the axonal processes of the sensory neurons.

Next, a tadpole is anesthetized and a three dimensional image stack of the olfactory bulb is acquired using multi photon microscopy. The final step is to repeatedly examine the labeled neuron in the same animal after specified time intervals. Ultimately, reconstruction of cellular morphology allows the visualization of changes in axonal morphology.

This method can help answer key questions in the field of neuroscience, such as how axons grow towards the target regions in the fore brain and how synapsis are formed. Before the experiment. Ensure that the electroporation setup consists of a stereo microscope with a large working distance and is equipped with fluorescent illumination and filter sets for the dye.

Used for electroporation use either a dedicated single cell electroporation or a generic square pulse generator attached to an oscilloscope. Connect the electroporation outputs to a pipette holder and a bath electrode. Ensure both the pipette holder and the bath electrode contain silver wires coated with a thin layer of silver chloride.

Next, mount the pipette holder on a micro manipulator to allow precise positioning. After that, fabricate the micro pipettes for electroporation with bo silicate glass capillaries with an internal filament. Set the parameters of the micro pipette polar to produce a longer shank and a smaller tip opening micro pipette with high pipette resistance for single cell electroporation.

Then measure the pipette resistance directly with the dedicated single cell electroporated. In this procedure, dissolve fluoro four coupled dextran in the frog ringer at a concentration of three millimolar. Divide this stock solution into small aliquots and store the one for future use in the freezer.

Next, backfill the micro pipette with one to five microliters of dextran solution. Carefully flick the micro pipette to remove residual air bubbles from the pipette tip. Then mount the micro pipette in the pipette holder.

Make sure that the silver wire inside the pipette is in contact with the dye solution. Now, place a small piece of tissue in a Petri dish and cover it with a small amount of water containing 0.02%trica. Next, anesthetize a pre metamorphic tadpole in the water containing 0.02%trica.

Confirm proper anesthesia by touching it and making sure that it is non-responsive. Then carefully transfer the tadpole to the tissue covered Petri dish. Ensure that the electrode is in contact with the wet tissue, and position the micro pipette tip close to the olfactory organ of the tadpole using a micro manipulator.

After that, penetrate the skin covering the olfactory organ. With the pipette tip cautiously advance the tip into the centrally located sensory neuron layer of the main olfactory epithelium or MRO nasal epithelium. Now trigger the positive square voltage pulses in order to transfer the dye into the sensory neurons by applying a single voltage pole or trains of multiple pulses.

Then visualize the successful dye extrusion and electroporation using a fluorescence stereo microscope. The dye spreads quickly into the cell body and dendrites after a successful electroporation. After repeating the procedures for the second olfactory organ of the tadpole, transfer the tadpole into a beaker filled with fresh water for recovery, and allow the electroporated dye to spread to the sensory neurons and the olfactory bulb.

At least 24 hours after electroporation, anesthetize the tadpole in the water containing 0.02%trica. Next, carefully transfer the tadpole to an imaging chamber. Cut a small rectangle out from a strip of param.

Then cover the tadpole with the param, leaving the anterior tail cephalon through the cutout window. Fix the param with needles on the dish without injuring the tadpole, and make sure the tadpole is submerged in sufficient water containing 0.02%Trica. Subsequently mount the imaging chamber on the stage of an upright multi photon microscope or a confocal microscope.

Acquire a three dimensional stack of images of the olfactory bulb and ensure that the imaging procedure does not exceed 10 to 15 minutes. After that, return the tadpole to normal water in a separate tank and prevent it from light exposure. In this image, four deck strands coupled to different fluoro fours were electroporated at four distant locations of the olfactory organ, lateral indicated by green, intermediate indicated by yellow medial indicated by red, and the vomer nasal organ indicated by orange.

This allows the visualization of the accessory olfactory bulb and the three main projection fields of the main olfactory bulb. This image shows three dimensional reconstructions of different axonal growth patterns of single olfactory sensory neurons superimposed on the structure of the olfactory bulb, and here is an example of a single olfactory sensory neuron axon projecting into the olfactory bulb and forming tufted arborization in spherical glomerular. As seen here in Opus Levu, these axons bifurcate regularly before connecting to one, two, or multiple gmer.

This is a representative example of an individual olfactory sensory neuron repeatedly investigated by in vivo time lapse imaging showing continuous changes in axonal morphology. After watching this video, you should have a good understanding of how to label olfactory sensory neurons via electroporation and how to monitor their development using in vivo TimeLapse imaging.