The overall goal of this procedure is to establish an acute tissue slice preparation of the mouse RO nasal organ or VNO in order to perform calcium imaging experiments on subpopulations and or individual neurons in living tissue. This is accomplished by first extracting the VNO from the mouse's nose, and then micro dissecting it from its cartilaginous capsule under a dissecting microscope in cold A CSF. Next, the isolated VNO is positioned vertically in an agar block and acute coronal tissue slices are cut using a microtome to verify the tissue integrity.
A few slices are observed after immunohistochemistry under a confocal microscope. Other slices are loaded with a calcium sensitive dye and then perfused with putative pheromonal ligands and neuronal activation is observed using calcium imaging. This method can help resolve key issues in the olfactory field.
For example, hundreds of pheromone receptors are expressed in the romo nasal organ, but very few of them have identified ligands. Pheromone receptors are very difficult to express in a heterologous system. We needed to develop an efficient in vivo assay to assign specific pheromones to a specific receptors.
We can use tact mice where a specific subpopulation of neuron expressed unknown receptors and can be identified by the expression of GFP. We could also use this assay to pharmacologically investigate ion channels and enzymes involved in Fairmont transduction cascade demonstrating the procedure. Today will be Julian and Fabian one, two researchers from my laboratory Before starting the dissection.
Prepare fresh, cold, artificial cerebral spinal fluid, A CSF saturated with oxy carb by combining all the ingredients except the calcium chloride. Saturate the solution by directly bubbling it with oxy carb. Then add the calcium chloride and adjust with double distilled water to the desired volume.
Keep the A CSF solution on ice until use. Following euthanasia and decapitation of an adult mouse, prepare a Petri dish with cold and continuously oxy, carbonated A CSF. After removing the lower jaw, place the mouse head in the Petri dish.
Position the mouse head under the dissecting microscope in order to visualize the palette. Next, make an incision horizontally in the upper part of the palate with micro scissors and then remove the palate membrane with micro dissecting forceps. Hydrate and clean the exposed cavity with A CSF.
Cut the upper and lower part of the nasal septum. Then delicately extract the nasal septum containing the VNO and place it directly in clean A CSF vertically. Separate the two parts of the VNO using micro dissecting forceps.
Identify the cartilaginous capsule of the VNO and then delicately remove it, making sure that all the cartilaginous pieces are removed. The isolated VNO, the cartilaginous pieces and the nons dissected VNO are indicated here year first, prepare 3%low melting auger and then assemble the workspace with the necessary tools. Fill the embedding mold with auger and make sure that the temperature is below 41 degrees Celsius before submerging the dissected VNO Position the tissue vertically in order to obtain coronal slices.
Place the embedding mold on ice for one minute until the auger solidifies. After one minute, remove the auger block, cut the agar into a parametal shape, and then glue it to the microtome support using the microtome cut coronal VNO slices at a speed of 0.5 millimeters per second, an amplitude of 0.7 millimeters and a thickness of 100 micrometers in cold. A CSF.
Collect the tissue slices with a brush and place them in a tissue culture plate filled with cold A CSF in order to verify the structural integrity of the collected tissue slices can be fixed and then immuno stain for cytoskeletal components or other proteins of interest. Here, immuno staining against acetylated tubulin in purple demonstrates the structural integrity of the tissue slice. Moreover, the observation of a specific neuronal population where a particular pheromone receptor has been tagged by GFP shows the conserved neuronal morphology neurons with small oval cell bodies and long dendrites reaching the surface of the lumen ending with microvilli can be observed working in a dark room.
Incubate VNO slices in a loading solution of cold A CSF with seven micromolar URA 2:00 AM and 0.1%onic for one hour at 37 degrees celsius after an hour. Keep the loaded slices on ice with oxy carbonation until use. Use a paint brush to place the loaded slides in a perfusion chamber containing A CSF and secure the slides with a tissue slice anchor.
Place the perfusion chamber on the microscope stage of a calcium imaging setup and continuously perfuse with oxy carbonated A CSF at room temperature. Observe the loaded VNO slices using the fluorescence wavelengths corresponding to the dye, which is three 40 to three 80 nanometers for four O 2:00 AM.Use the appropriate software to record intracellular calcium ratio changes. A perfusion system is necessary to induce chemo stimulation.
Here, a coronal slice of vno tissue from a mouse where the subpopulation of neurons expressing the V one RB two pheromone receptor also expresses GFP is immuno stained against the acetylated tubulin protein shown in purple. Higher power views of the slice demonstrate the conservation of the tissue integrity. Neurons with long dendrites are shown reaching the surface of the lumen and expression of the structural protein can be seen in the dendritic knobs and microvilli for calcium imaging.
FU loaded tissue slices are first visualized using Hoffman phase contrast to locate the field of interest starting from the blood vessel, the first white arrow, the observer slowly progresses towards the lumen, the second white arrow and the neuro epithelial region. The oval cell body of the RO nasal neuron. The third white arrow can be identified by its long and thin dite.
The fourth white arrow extending into the lumen, the neuronal dendritic knob. Fifth white arrow bears microvilli in contact with pheromones RA loaded cells here shown at three 80 nanometers are then sequentially illuminated at three 40 and three 80 nanometers, and this fluorescent wavelength ratio is graft corresponding to the intracellular calcium changes in the selected neurons, black, red and blue circles. A TP is used to determine the viability of the neurons.
Notice the range of calcium responses exhibited across the three selected neurons and the fact that neuron number two red circle does not respond to a TP and therefore will be discarded from further analysis. Chemo stimulation is then achieved using both urine and a mix of mouse pheromones looking in the depth of the tissue slice. A subpopulation of neurons expressing both GFP and the V one RRB two receptor can be localized.
Here, a neuron expressing both GFP and the V one RB two receptor is shown alongside with a non V one RB two expressing neuron. Both cells are loaded with URA 2:00 AM and the V one RB two expressing neuron shows a calcium spike in response to perfusion of the receptor's ligand two, he known. Interestingly, the non V one RB two expressing neuron also responds to two hep known demonstrating the complexity of pheromone coating in the VNO.
After watching this video, you should have a good understanding of how to extract and microdisect the mouse for mar cell organ in order to perform acute tissue slice preparation. This protocol is also adaptable for physiological investigation using alternative calcium dyes and microscope setups. Good luck with your experiment.
