Imaging Subcellular Calcium Dynamics in Neurons of Caenorhabditis elegans

Take two separate transgenic Caenorhabditis elegans strains on slides with agar pads. The pads contain a rolling solution with a paralytic agent that minimizes movement.

The worms' ventral nerve cord, or VNC, contains excitatory interneurons that express cytoplasmic calcium indicators in one strain and mitochondrial calcium indicators in the other.

Place a coverslip to roll the worms, orienting the VNC for visualization, then position them under a fluorescence microscope.

Use visible light to locate the worms, then switch to fluorescence mode.

In resting neurons, low intracellular calcium keeps the indicators unbound, resulting in weak fluorescence.

Spontaneous neuronal activity opens voltage-gated calcium channels, allowing calcium influx.

In the strain with cytoplasmic indicators, calcium binds to the indicators, enhancing cytoplasmic fluorescence.

Excess cytoplasmic calcium enters the mitochondria through channels. In the strain with mitochondrial indicators, the calcium-binding increases mitochondrial fluorescence.

Image the neurons in both strains to monitor the subcellular calcium dynamics.

In a 13 by 100 millimeter glass culture tube, prepare 3 milliliters of 10% agar by dissolving