Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

Hello my name is Dimitry Samigullin I work in the laboratory of Biophysics of Synaptic Processes of Kazan Institute of Biochemistry and Biophysics, the Russian Academy of Science. In our laboratory we study vast ways of calcium signalization in the Neuromuscular Junction. One of the most approachable methods to measure the synaptic level of calcium is an optical recording.

Here we describe the method of the loading of a calcium sensitive dye, through a nerve stump, in the frog nerve endings. Also, we present the protocol to measure, and analysis of, a vast calcium transient in frog terminals. Using this method it is possible to study the influence of active drugs on the presynaptic level of the calcium during synaptic activity.

Preparing of the dye loading solution. Fluorescein's Calcium Dye Oregon Green BAPTA, comes in 500 microliters via a small amount 500 micrograms of powder. Add 14 microliters of solution, containing HEPES, to the powder.

Vortex loading solution, and spin down to mix thoroughly. Wrap the vial with the loading solution with foil, to avoid exposure of light. Store the loading solution at minus 20 degrees Celsius, in a freezer.

Dye loading procedure. Dissect a cutaneous pectoris muscle from the animal. Mount dissected tissue in a silver lined Petri dish.

Stretch the tissue with fine stainless steel pins. Add Ringer's solution to the Petri dish. Using a razor blade, cut a small piece, around 2 millimeters of the ten microliters pipette tube from the thin end.

Prepare a piece of plasticine for holding the filling pipette on the Petri dish. Connect the filling pipette with a plastic syringe through a silicone tube and adapters made of pipette tubes. Remove temporary Ringer's solution from the Petri dish by a plastic pipette.

Dry preparation with a syringe. That will prevent suction of the solution in the filling pipette. Remove the vial with loading solution from the freezer and allow to thaw at room temperature in a dark place.

With a fine tweezer and scissors, under visual control, with sterile microscope, cut the nerve close to the muscle. Leave around two millimeters of nerve stump. Fix the filling pipette with a tube and the syringe, on the Petri dish using plasticine.

Move it close to the nerve branch. Gently aspirate the nerve stump in the filling pipette. Remove the suction tube from the blunt end of the filling pipette.

Dry the nerve stump in the filling pipette by the syringe. Move up the filling pipette with the nerve stump. Isolate the nerve stump from the outside by Vaseline.

One more time, dry the nerve stump in the filling pipette by the syringe. Draw the zero point five microliters of loading solution using a pipette with a long pipette tip. Gently insert the pipette tip with the loading solution into the filling pipette.

Eject the mixture directly to the nerve stump. Seal the open end of the filling pipette by Vaseline. Add small amount of solution into the Petri dish.

Incubate the preparation at room temperature in a darkened and wet location for five hours. After the incubation procedure, take the preparation, and remove the filling pipette with the loading solution. Place the preparation in a bath and wash it out with Ringer's solution.

Keep the bath with the preparation overnight in a fridge at eight degrees Celsius temperature. Prepare the tissue for microscopy. Take the preparation from the bath.

Mount preparation in a silver lined chamber and slightly stretch the tissue. Rinse the tissue with fresh Ringer's solution. Take the Suction Stimulation Electrode.

Place the Stimulation Suction Electrode in the chamber with its tip close to the cut end of the nerve. Draw the nerve to the electrode. Construction of the electrode is described in Khazokoff's article, a link to the article is in the materials.

Fix the preparation chamber on the microscope stage. Place the temperature probe. Connect inflow tubes in a power cord for Peltier element in the preparation chamber.

Place outflow tube in the preparation chamber. Turn on a perfusion pump. Switch on the term controller unit.

Set the temperature on the controller unit to reach 20 degrees Celsius. Install ultra-violet protection shield. Set up delay duration and the period on the stimulator.

Connect a wire from the suction electrode with the stimulator. Turn it on. Under a low magnification objective, check the muscular contraction by turning the stimulus on.

Fill up the perfusion system with a Ringer's solution with low calcium and high magnesium. Set the perfusion rate of about two milliliters per minute. Replace the objective for 40 times magnification.

Switch on the Polychrome V.Select emission wavelength and continuous mode illumination. Open the shutter. Under 40 times magnification objective, in fluorescence mode illuminations ensure that the terminals are loaded.

Find the terminal and focus on it. The loaded nerve and well-loaded motor nerve terminals, are represented on the figure. Video capture with Red-Short Neuro CCD Camera.

Run Turbo-SM Software. Select, Change Settings. Input number of frames is 500.

Enter the experiment name. Basic configuration is 1000 frames per second. 80 by 80.

Choose external trigger. Enter pre-trigger time. Minus ten milliseconds.

Enter number of repeats, 20. In Polychrome software, select external trigger illumination mode. Run Clampex software.

Load stimulation protocol. The protocol is available for downloading from our lab page on Kazan Federal University website. Before recording capture a dark frame in Turbo-SM software.

On the trinocular cube select light path exchange levels. 100 percent for camera. Run the stimulation protocol.

Select the region of interest and check the signal. In response to the nerve stimulation we record changing of fluorescence intensity that reflects calcium entry into the nerve ending. Data analysis.

In Turbo-SM software, select file. Then, select, average files. Select files.

Enter name of average file, and average them. Save the average file as fit file. Select, save as fit file.

Enter the file name and save it. Run Image J, and open the instruments below. Analysis, tools, ROI Manager.

Find saved average file in fit format. Drag and drop it to Image J.Zoom the window for a better view. Select a rectangular region of interest over what you believe to be background.

Add it to ROI manager. In ROI manager, select, more.Multi-measure. Load down mean.

Copy data, export to Excel. In Excel, use average function to calculate average value of background for a ratio. Without closing Excel, return to the Image J.Close the window with background calculation without saving.

Use ROI Manager, delete rectangular background drawing. Open instrument process, math, subtract. Enter calculated averaged value of threshold from Excel, and subtract the ground from stacks.

Select a rectangular region of interest around a nerve terminal. Add it to ROI Manager. In ROI Manager, select, more, Multi-measure, load down, mean.

Copy and paste in Excel window. Use Excel instrument plot graph. Select a flat area before starting the signal.

Load the graph with the details from the first values of fluorescence at the rest, to the beginning of the signal. Then determine the numbers of points to average rest fluorescence values. Average these values, then find the average number of the fluorescence at the rest.

Divide signals by fluorescence at the rest. Subtract one and multiply by 100 percent. Plot the signal and calculate the amplitude of calcium transient.

Time parameters of fluorescence signals depend on the speed of the forward and or reverse reactions of calcium-dye-complex formation. The rise time of calcium signal represents dynamics of calcium input. It's diffusion in the terminal, and calcium interaction with dye.

The decay of calcium transient depends on the dye affinity and rate of calcium buffering and its withdrawal. Analysis of calcium transient amplitude can be used to study influence of different active substances on the calcium entry that participate in neurotransmitter release. This loading technique is well suitable for the imaging of changes in cytosomal calcium.

With fluorescent indicators and the both single nerve stimulation, and rhythmic synaptic activity. Analysis of calcium transient amplitude can be used to study the influence of different substances on calcium entry that participates in neurotransmitter release. There are some important points we want to pay attention to.

Incubation procedure includes two steps. Incubation at room temperature, and in a fridge. It is important to control the time incubation with the dye at room temperature.

Depending on the length of the nerve stump, the dye and the temperature in the room, incubation time might vary. If you overexpose the pre-operation terminals and proximal parts, close to the nerve stump, can be overloaded. Due to the long incubation in the fridge, dye is evenly distributed along the nerve endings.

It is essential to place the nerve into the dye loading solution, within several minutes after cutting, to allow the dye to enter into the nerve, since longer time intervals produced ineffective loading, presumably due to receiving closed nerve. To reduce dye compartmentalization it is possible to use the extended form of the dye. It is very important to prevent the long fluorescent illumination of the tissue because it affects its survival.

We use Nomarski optics in the visible light channel to search for the terminals. During the recording, we limit the illuminated view, by the diaphragm. Long duration of loading process can be reduced using shutter stamp of the nerve.

In this case, glass micro-bypass can be used.