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 molecular grade agar in M9 and microwave for several seconds. To make the agar pads, first prepare two slides by adding two layers of laboratory tape. Then place a microscope slide between the two slides. Cut the tip of a 1,000 microliter pipette tip and use it to pipette a small drop of agar onto the center of the coverslip.

Flatten the agar by pressing another slide down on top of the agar. After cooling, cut the agar into a small disk using the opening of a 10 milliliter tube and then remove the surrounding agar. Next, prepare the worm rolling solution by dissolving muscimol powder in M9 to create a 30 millimolar stock. Dilute the stock in a one to one ratio with polystyrene beads to make the rolling solution.

To position the worm for imaging, first place 1.6 microliters of the rolling solution onto the center of the agar pad. Then, using a preferred worm pick, transfer a worm of the desired age into the rolling solution on the agar pad. Wait for about five minutes for the muscimol to reduce the worm movement, and then drop a 22 by 22 millimeter coverslip on top of the agar pad.

For imaging neurites in the ventral nerve cord, roll the worm by slightly sliding the coverslip. To mount the worm on the microscope, place a drop of immersion oil onto the coverslip. Find the worm using a low magnification objective in bright field. Then switch to 100X objective and locate the AVA neurite using the illumination of the GCaMP or mito-GCaMP with the 488 nanometer imaging laser.