Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans

JoVE Encyclopedia of Experiments

Neurowetenschap

0 weergaven • 3:01 min • July 8th, 2025

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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.

For ratiometric calcium imaging, under the Sequence tab, click Start to begin recording, and track the worm with the joystick, keeping the cells and synapses of interest in focus in the center of the field of view. At the moment the fluorescence recording is started, triggers are sent to the waiting brightfield camera and the stage controller, which begin collecting synchronized images and stage position.

Click the Stats button in the histogram window, to show pixel statistics for each channel. Adjust the LED power to ensure a maximum single-pixel mCherry fluorescence at the presynaptic terminus of greater than or equal to 8,000 counts in the red channel, giving approximately 12-bit dynamic range above background. The calcium reporter signals at the presynaptic terminus during resting, or low calcium, should be around 2,500 counts in the green channel.

Record the behavior until one egg-laying active state is reached. Then, save a 10-minute subset containing 6,000 frames before and 6,001 frames after the first egg-laying event. Take care to keep the same subset of brightfield images of worm behavior and time points of the x,y stage position or the precise synchronization of data streams will be lost.

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Laatst bijgewerkt: 15 augustus 2026