The overall goal of this procedure is to monitor stimulus induced calcium responses, both in GFP tagged neurons, spatially, and temporally in brain slices using a non-green fluorescent calcium indicator dye. This is accomplished by first extracting the brain of a transgenic mouse expressing GFP in specific cells. The second step is to prepare tissue slices with a microtome.
Next, the calcium indicator dye loading solution is pipetted onto the slices to enable the dye to be taken up by the brain cells. The final step is to perform a timestamp recording of fluorescent cells using confocal microscopy. Ultimately, calcium responses in GFP tagged neurons are shown using multicolor calcium imaging analysis.
The main advantage of this technique over existing methods of confocal microscopy is that we can image calcium in green fluorescent GFP tech neurons using a redshifted fluorescent calcium indicator die. The huge stroke shift of the fewer red permits us to do multicolor analysis of the red fluorescence in combination with the green GFP using a single excitation wavelengths. We first had the idea for this method when we were confronted with the problem of monitoring calcium signals in neurons that we only could identify using a genetic fluorescent tech, which was the green fluorescence protein.
I will now demonstrate a various critical steps of this procedure. Begin the procedure by pouring the heated agar solution into a square Petri dish to a height of one centimeter after cooling and hardening the 4%agar gel is cut into blocks of one cubic centimeter and placed in a bottle for storage. After decapitating the mouse cut the scalp with a single edge razor blade in the central sagittal direction from the frontal bone to the external occipital protuberance.
Subsequently further open the cut scalp first in the rostral lateral direction. Then in the ventral direction. Then make two lateral and one rostral cut.
In the foramen magnum with small spring scissor carefully cleave off the cranium. At this moment, the occipital inter parietal and parietal bones should be detached. If the dura matter is still present, carefully remove it with a pair of forceps to prevent damage to the brain.
Next, remove the squamal bone, the anterior ethmoidal and the frontal foramen. Now the brain can be easily removed with an inverted micro spoon spatula and the cranial nerves can be severed. The brain should land on its ventral side on a cut resistant surface with the ventral surface of the brain down.
Use a single edge razor blade to remove the cerebellum. This straight cut surface will be the basis for mounting the brain on a plate to enable slicing of coronal brain sections. Next, glue the brain with the cut surface on the base plate of the microtome using a small amount of the fast curing high performance cyanoacrylate adhesive super glue.
Subsequently glue a one cubic centimeter agar gel block at the ventral side of the brain onto the base plate of the microtome to support and fix the brain while slicing. After the bond has dried, put the plate into the bath of the microtome containing six degrees Celsius cold oxygenated extracellular solution. Then cut the brain into slices of 300 microns thickness with low slicing velocity, the coronal brain slices are collected until the whole brain has been sectioned.
Now prepare a 20%onic F1 27 in DMSO solution by adding DMSO solution to onic F1 27 powder. Next, dissolve it by directly sonic the solution for two minutes. After that, add five microliters of 20%onic F1 27 solution to a tube of 50 micrograms of cell permeable furred am.
Mix the solution with a pipette. Then add 45 microliters of extracellular solution to the mixture and vortex it. Briefly, add an additional 325 microliters of extracellular solution and sonicate the tube for another three minutes.
After sonication, add 1.156 milliliters of oxygenated extracellular solution to obtain the final calcium indicator D loading solution. Transfer the coronal brain slices to a six well cell culture plate filled with oxygenated extracellular solution up to six slices per well. Then aspirate the oxygenated extracellular solution from the chambers being careful not to damage the brain slices.
Next pipette 750 microliters of the calcium indicator D loading solution directly to each. Well, the brain slices should be covered by the solution containing rore am. After that, incubate the slices in the oxygen carbon dioxide cell culture incubator for 45 to 60 minutes at 37 degrees Celsius.
At the end of the incubation, replace the calcium indicator DI loading solution with fresh oxygenated extracellular solution to prevent overloading the cells. To monitor the stimulus induced changes in the fluorescent signal, transfer one of the fure red loaded brain slices to a recording chamber and position it as desired. After mounting the chamber on the confocal microscope setup, perfuse the slice for 10 minutes with oxygenated extracellular solution to remove any surplus of extracellular calcium indicator dye.
After exiting the recording chamber, the PERFUSE eight is collected in a vacuum bottle. Then look at the slice through the microscope at low magnification. Make a note of the orientation of the slice and find the area of interest in the slice in our case the hypothalamic area in the brain.
Afterward, change to high magnification. Find the cell of interest in the slice by collecting GFP images and simultaneously checking the fluorescence intensity of the URA red signal. Adjust the laser power to a value that allows measurements in the change of URA red fluorescence intensity, and prevents bleaching of the two fluorophores.
Next, start to acquire images at the rates between 0.5 to two hertz to collect the fure and GFP signals. This figure shows the confocal image of the GN, RHR Tau GFP neurons in a coronal brain slice. This is the relatively uniform fluorescence signal observed after loading the brain slice with fure am and this is the merged image showing the neurons loaded with the calcium indicator dye.
Here are examples of the somatic stimulus induced calcium responses from individual GFP tagged neurons in different hypothalamic brain areas. The distinct calcium signals between the GN RHR expressing neurons of different hypothalamic nuclei can be compared using the area under the curve as an estimate for the total change in calcium in a given cell during the same period Once mastered. This technique can be done in less than 90 minutes, including the preparation of the calcium indicator dye, the loading and the incubation of the slices if it is performed properly.
