This study demonstrates the use of cultured myelinated motor neurons and myotubes to investigate neuromuscular junction activity. The experiment involves live imaging to observe myotube contractions in response to calcium ions and neuromuscular blockers.
Start with a plate containing cultured myelinated motor neurons, muscle-controlling nerve cells, and myotubes, multinucleated muscle cells.
The motor neuron is connected with the myotube through a neuromuscular junction or NMJ, a site for signal transmission.
Add calcium chloride to the well.
Observe under an inverted microscope for live imaging and record a movie.
Calcium ions enter the motor neuron and trigger the release of acetylcholine, or ACh, an excitatory neurotransmitter.
These neurotransmitters bind to ACh receptors on the myotubes, allowing sodium ions to enter and triggering the contraction of the myotubes.
Record the movie. The software then generates a color-coded time-motion graphic for myotube contractions, where red color indicates the fastest speed and blue represents the slowest speed.
Next, introduce a neuromuscular blocker that binds to ACh receptors. This prevents sodium influx and restricts myotube contractions.
Re-record the movie. A decrease in red color indicates a reduction in myotube contractions.
To trigger the myotube contractions, add 25 millimolar calcium chloride to each well of differentiated neuromuscular junction cells. And within one to two minutes of treatment, place the plate on the stage of an inverted microscope. Using live cell microscopy, record a movie of the myotube contraction for 20 seconds. At the end of the recording, add 300 nanograms per milliliter of curare to the culture medium and record the movie for another 20 seconds, before opening the movie file in an appropriate motion vector analysis software program for analysis.
繁體中文
