Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans
Trascrizione
Take transgenic Caenorhabditis elegans worms growing on an agar block.
These worms express a calcium indicator, GCaMP5, and a calcium-insensitive fluorescent protein, mCherry, in neurons that regulate egg-laying behavior.
Mount the worms on an inverted microscope stage.
Visualize GCaMP5 and mCherry fluorescence.
Prior to egg release, the neurons become activated, opening voltage-gated calcium channels, which allow calcium influx.
Intracellular calcium binds to GCaMP5, altering its conformation and increasing fluorescence.
Meanwhile, mCherry fluorescence remains unchanged, enabling tracking of the neuron.
The activated neurons release neurotransmitters that bind to specific muscle receptors, triggering egg release.
After release, the calcium channels close, reducing calcium influx and GCaMP5 fluorescence.
Next, capture brightfield images to observe worm movement.
Reduced movement occurs due to muscle contractions before egg release.
Following egg release, the muscles relax, restoring normal movement.
Measure the GCaMP5-mCherry fluorescence ratio and worm speed to detect egg release events.
