Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

This method can help answer key questions in the neuromuscular field, such as the role of calcium signaling in muscle or Schwann cells in response to nerve stimulation. The main advantage of this technique is that one can image the activation of specific cell types in response to nerve stimulation. To begin, obtain transgenic mice, and genotype them as detailed in the text protocol.

Following euthanasia, transversely section across the entire animal just below the liver and just above the heart and lungs with iridectomy scissors. Dissect away the liver, the heart, and the lungs. Being careful to maintain a length of the phrenic nerve that is sufficiently long enough to be drawn into a suction electrode.

Further remove the rib cage and the vertebral column, except for the thin ridge around the diaphragm. Place the diaphragm and the phrenic nerve sample in a microfuge tube with Krebs-Ringer solution containing one microgram per milliliter of fluorescently labeled alpha bungarotoxin. Allow the specimen to incubate in the solution for 10 minutes in the dark.

Using minutien pins immobilize the diaphragm by pinning it onto a six centimeter dish coated with silicone dielectric gel and filled with approximately eight milliliters of oxygenated Krebs-Ringer solution. Place the dish on the microscope stage. Then, perfuse the diaphragm with more Krebs-Ringer solution at eight milliliters per minute for 30 minutes.

At four times magnification, using a micromanipulator, move the suction electrode over the left phrenic nerve. Apply the suction by pulling out the barrel of a five milliliter syringe connected to the tubing that is attached to the suction electrode. Ensure that the diaphragm contracts in response to the one hertz stimulation by visually examining it with Brightfield illumination.

If not, adjust the voltage by turning the voltage knob incrementally to achieve a supramaximal pulse which can be verified by visual examination of muscle contraction. If still not visible, blow out the nerve with the syringe and attempt to draw it in again by applying suction. Now, turn off the perfusion to add the muscle-specific myosin inhibitor BHC or the voltage gated sodium channel antagonist mu-conotoxin.

Prepare the BHC pre-dilution by pipetting four microliters of 200 millimolar stock BHC in DMSO in one milliliter of Krebs-Ringer solution. Remove one milliliter of Krebs-Ringer solution from the dish. Then add the pre-diluted BHC to the dish.

After 30 minutes, turn on the perfusion of fresh Krebs-Ringer solution for another 20 to 30 minutes. Wearing gloves, prepare the recording electrode by placing a borosilicate filamented glass with an outer diameter of one millimeter and an inner diameter of 0.4 millimeters into a micropipette puller. Tighten the dials to clamp it into position and close the puller door.

Then program the puller as described in the text protocol. For embryonic diaphragms, measure the resistance using software controls of the amplifier. Ensure that the resistance is near 60 megaohms and for older diaphragms, 10 to 20 megaohms.

Now, load the recording electrode with three molar potassium chloride. At 10 times magnification, lower the electrode into muscle using a second micromanipulator on the opposite side of the stage as a stimulating electrode. Using electrophysiological data acquisition software wait until the resting membrane potential changes from zero to minus 65 millivolts or below.

Stimulate at one hertz and verify the presence of a muscle action potential by checking for a large potential that exhibits a modest overshoot. Do not confuse stimulation artifact with an action potential. At 20 times magnification locate the end plate band at the center of the muscle by looking for fluorescently labeled alpha bungarotoxin neuromuscular junctions under green yellow light excitation.

Switch to blue light excitation to image calcium responses in muscle, motor neuron, or Schwann cells. In this example GCaMP three is expressed in muscle cells. If desired, set up the image splitter with band pass filters and a dichroic single edge filter for the dual wavelength imaging.

Perform experiments with the brightness bar on the lookup table bar set to 110%of the level at which the genetically encoded calcium indicator expressing tissue exhibits saturation at 20 times magnification without binning in response to potassium chloride. Record at 20 frames per second so not to miss any fast events. Stimulate with one to 45 seconds of 20 to 40 hertz of nerve stimulation by delivering a train of impulses using a suction electrode and collect dynamic fluorescent calcium responses in one cell sub-type together with the static fluorescently labeled alpha bungarotoxin neuromuscular junction signal.

Pharmacological agonists can also be added by bath application or by perfusion and the dynamic fluorescent calcium responses can be collected in the same way. When the imaging or electrophysiological experiments are finished because the desired results have been achieved, perfuse water through the perfusion lines and suck water two to three times through the suction electrode to ensure that salts do not build up. A spatial map of Schwann cell calcium responses to phrenic nerve stimulation of a Wnt one GCaMP three mouse diaphragm at post natal day seven show a restriction to terminal parasynaptic Schwann cells at the neuromuscular junction.

The same diaphragm was imaged after labeling with fluorescently labeled alpha bungarotoxin, which bonds to enable acetylcholine receptors of the neuromuscular junction. The same diaphragm was also imaged under Brightfield illumination to guide a recording electrode to the post-synaptic region of the muscle by the neuromuscular junction. Shown here is spatial demarcation of color coded cells or regions of interest taken for analysis of individual calcium transients.

The spatial map of muscle cell calcium responses of a mth 5 GCaMP three mouse diaphragm at P four to phrenic nerve stimulation in the presence of the myosin inhibitor BHC shows a response throughout the entire region of all diaphragm muscle cells. In contrast, stimulation of the same diaphragm in the presence of mu-conotoxin causes a spatially restricted calcium response in the medial region of all diaphragm muscle cells that corresponds to the acetylcholine receptor cluster enriched end plate band. Following this procedure other methods like immuno-histochemistry or immunoblotting can be performed in order to answer additional questions such as, what is the molecular profile of cells identified by electrophysiology or calcium imaging?

This method can provide insight into population responses of Schwann cells and muscle cells to nerve stimulation in normal neonatal mice. It can also be applied to the study of older mice or to the study of mice expressing neuromuscular disease associated proteins.