Estimation of a presynaptic calcium level is a key task in studying synaptic transmission. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The main advantage of the presented loading technique is that the staining has been performed only for the cells of interest.
Our method for recording calcium intransience provides extremely good spatial-temporal resolution. The presented registration method can be helpful to neurophysiologists who need to register fast periodic processes using a confocal microscope. The most difficult step in the loading technique is holding the nerve inside the pipette.
The diameter of the pipette must be perfectly fit, so make some several ones. Knowing all the steps in the protocol are complicated and demonstrating each of them will help a lot. Begin by fixing the levator auris longus muscle tissue in a slightly stretched manner in the elastomer coated Petri dish with the fine stainless steel pins and add Ringer's solution to the dish until the muscle is fully covered.
Next, use a micropipette puller to prepare a micropipette with a fine sharp tip for the intracellular recordings. Use capillaries without internal filaments. After scoring the taper with an abrasive, break off the micropipette tip, leaving a tip open to about 100 micrometers in diameter.
Then, fire polish the tip until the internal diameter shrinks from 80 micrometers to 12 micrometers. When done, attach a silicone tube to one side and a syringe without a needle to the other side of the filling pipette. Under a stereo microscope, find where the nerve trunk turns into separate nerve branches.
Place the filling pipette on the Petri dish using wax and then move the pipette tip until it stands above the nerve. Cut the nerve close to the muscle fiber with fine scissors, leaving a small piece of the nerve stump, about one millimeter long. Then, aspirate the nerve stump with some Ringer's solution into the tip of the filling pipette, without pinching the nerve.
Once done, remove the silicone tube from the filling pipette. Later, use a syringe with a long filament to draw the dye loading solution. Then, insert the filament tip with the loading solution into the filling pipette and release the mixture onto the nerve stump, followed by incubating the nerve preparation at room temperature in the dark for 30 minutes.
After the incubation, rinse the nerve preparation with fresh Ringer's solution before treating it with 50 milliliters of Ringer's solution in a glass beaker at 25 degrees Celsius for two hours. For the confocal microscopy, mount the nerve preparation into a silicon elastomer coated experimental chamber and fix it, slightly stretched, with a set of steel microneedles. Then, rinse the preparation extensively with the Ringer's solution.
Install a suction electrode on the chamber. After mounting the preparation chamber onto the microscope stage, place the inlet and outlet fittings into the chamber. Turn on a gravity flow driven system to remove the excess solution from the preparation.
Once done, plug the stimulating suction electrode into an electric stimulator and ensure that muscular contractions occur after stimuli. Then, fill up the perfusion system with the Ringer's solution containing 10 micromolar d-tubocurarine and switch on the perfusion suction pump to start perfusion. In the laser scanning confocal microscope, or LSCM software, choose electrophysiology and acquisition mode to set imaging parameters.
In the job menu settings, select the trigger settings to trigger the stimulator with the microscope sync pulse and set the trigger out on frame field to the OUT 1 channel. Turn the scanning mode to XYT at a frequency of scanning of 1, 400 hertz. The zoom factor should be 6.1 with the pinhole fully open.
Ensure that sequential, transpassing, bidirectional X mode is on, then, set the minimum time to form a frame at 52 milliseconds and collect frames in a raw video at 20 frames. Set excitation wavelength of the argon laser at 488 nanometers with 8%of output power. When the parameters are set, press the live mode button to get a live preview of nerve terminals loaded with the dye.
In the live mode, search for the region of interest, or ROI, to obtain the best focus. Then, shift the delay on the stimulator by two milliseconds less relative to the previous value and run the data acquisition software to acquire 26 sequences by shifting each sequence two milliseconds from the previous one. To process the captured videos, run Leica Application Suite X, or LAS X software, and open the project created.
Then, click on export and save as to save frames in TIF format in the destination folder. Next, run the ImageJ software and click on file, import, and image sequence. In the open image sequence window, choose the destination folder and open the first frame.
From the sequence options, go to the starting image field and set the frame number to one for the first frame. Then, in the increment field, set the value equal to the number of frames in the initial signal recording before clicking okay. To save the generated file of stitched first frames in a separate folder, click on the file, save, and folder tabs in order.
Repeat the procedures for 19 frames by setting the corresponding frame number in the starting image field. When all the images are obtained, click on file, import, and image sequence to select one in the starting image and increment fields. The final video with all the frames stitched together at increased temporal resolution can be seen.
Then, save the video file in TIF format. To analyze the stitched video, click on image, stacks, tools, and stack sorter. Then, go to analysis, tools, and ROI manager.
Drag and drop the TIF file saved previously into the ImageJ window and then expand the image for a better view. To improve the image visualization, click on image, adjust, to turn brightness contrast to auto. Set the background close to the nerve terminal by drawing ROI and adding value to the ROI manager.
Then, calculate the background by clicking on more and multi measure. Copy mean values to paste to the spreadsheet program. Then, calculate the average value.
Next, subtract the calculated average value from the stacks by clicking on process, main, and subtract. Enter the obtained value and draw ROI around a nerve terminal via a polygon line before adding it to the ROI manager. Measure the transient intensity of the nerve terminal by clicking more and multi measure tabs.
Then, copy the mean intensity values and paste them to the spreadsheet program to calculate the average offset of signals. Divide the signal values by the average offset value. From the obtained value, subtract one and then multiply by 100%followed by plotting a calcium transient graph to determine the amplitude.
After loading the nerve preparation with a dye, most of the synapse is located close to the nerve stump, indicated a sufficient level of fluorescence. After the image processing, calcium transience with the desired spatial and temporal resolution were obtained. One of the most important steps in this protocol is that the nerve stump must be placed in the dye containing solution in the first few minutes after cutting the nerve.
This method can be used to load various dyes and drugs into motor neurons. Protocol of registration can be useful for assessing calcium in dendrites and spines in CNS preparations. Our technique allows to register a calcium signal with good spatial and temporal resolution.
Studying calcium transience is a powerful tool in research regulation of a neurotransmitter release and synaptic plasticity.
