This study utilizes fluorescence microscopy to visualize neuromuscular junctions in transgenic mice. It focuses on the dynamics of calcium influx in response to electrical stimulation, providing insights into neuromuscular signaling.
Begin with a fluorescence microscope setup containing an immobilized diaphragm with phrenic nerves from a transgenic mouse.
The diaphragm's neuromuscular junction consists of a motor neuron, a muscle fiber, and Schwann cells containing acetylcholine receptors and calcium channels.
These cells have fluorescently labeled acetylcholine receptors and express intracellular calcium indicators.
An image splitter attached to the setup enables simultaneous visualization of both fluorescent signals.
The phrenic nerve is positioned in a suction electrode to deliver electrical stimulus.
Under the fluorescence microscope, visualize acetylcholine receptors to identify a neuromuscular junction.
To set a maximum baseline fluorescence, add a potassium chloride solution to depolarize the cell membrane temporarily. This triggers intracellular calcium influx, causing an increase in the indicators' fluorescence.
Adjust the brightness settings to prevent oversaturation of the fluorescence signal.
After repolarization, apply electrical stimulations to trigger calcium influx into the cell and record the change in intracellular fluorescence at a high frame rate.
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 3 is expressed in muscle cells.
If desired, set up the image splitter with bandpass filters and 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 1 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 subtype, together with the static fluorescently labeled alpha-bungarotoxin neuromuscular junction signal.
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