Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

This technique can be used to advance our understanding of calcium handling in neurons, and how compartmentalized calcium can regulate different mechanisms important for neuronal function in vivo. The main advantage of this technique is that it allows for higher resolution rapid in vivo imaging under physiological conditions in C.elegans that may be combined with other fluorescent tools. This technique could provide insights into mechanism regulating calcium signaling in neurons in many different contexts.

For example, during aging, neuronal injury, and neurodegeneration. Demonstrating this procedure will be Ennis Deihl, research technician. Kaz Knight, a PhD candidate, and Rachel Doser, a postdoctoral researcher from my lab.

Begin by cloning expression vectors that contain a promoter followed by a calcium indicator, and three prime UTR of choice. Create transgenic C.elegans strains by microinjection of a DNA mix containing the rescue DNA Lin-15 plus in the calcium indicator transgene into the gonad of a one-day-old adult C.elegans. Maintain the injected parent worms at 20 degrees Celsius until the F1 progeny reach adulthood.

Select transgenic adults by screening for the absence of the multi-vulva phenotype. That indicates expression of the extra chromosomal array containing the calcium indicator. Clonally passage adult F1 progeny that do not have the multi-vulva phenotype.

Perform experiments on subsequent generations of the transgenic strains with an optimal expression of the calcium indicator. Use a microscope capable of long-term Time-lapse imaging. Locate the worm under a low magnification objective and then switch to a high magnification objective to localize the neurons.

Acquire the images using an ultrasensitive camera capable of rapid image acquisition at more than 50 FPS. Next, use a standard emission filter for the calcium indicator. Add a Z-drift corrector for the acquisition of image streams longer than 10 seconds.

In a 13-by-100-millimeter glass culture tube prepare three 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 1000-microliter pipette tip and use it to pipette a small drop of agar onto the center of the cover slip. Flatten the agar by pressing another slide down on top of the agar. After cooling, cut the agar into a small disc 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 cover slip on top of the agar pad. For imaging neurites in the ventral nerve cord, roll the worm by slightly sliding the cover slip.

To mount the worm on the microscope, place a drop of immersion oil onto the cover slip. Find the worm using a low magnification objective in brightfield. 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.

For in vivo GCaMP imaging, adjust the imaging laser in acquisition settings until the basil GCaMP fluorescence is in the mid range of the dynamic range of the camera and set the exposure time to 20 milliseconds. After the AVA neurite is located using the GCaMP fluorescence at 100 times magnification, set the Z-drift corrector, and initiate continuous autofocusing. Manually correct the focal plane as needed before imaging.

Acquire a stream of images at 100 times magnification. Using this protocol, cytoplasmic calcium was measured with high temporal and spatial resolution. The cell specific expression of GCaMP6F in the glutamatergic AVA command interneurons revealed a directional propagation of calcium influx.

The subcellular quantification of GCaMP6F fluorescence showed that the onset of calcium influx was delayed in a portion of the neurite located only 10 micrometers away. Also, dendritic spine-like structures found along the AVA neurite showed flashes in GCaMP6F fluorescence that occurred independently of neurite activation and did not lead to a propagation of calcium influx. Further, the relative calcium levels were measured in the individual mitochondria in single neurites using a worm strain with a AVA specific expression of the mitochondrial matrix localized calcium indicator mito GCaMP.

The results showed the synchronous uptake of a relatively large amount of calcium into a subset of mitochondria. Specifically, the calcium uptake into some mitochondria was synchronized, whereas neighboring mitochondria did not appear to take up calcium. Similarly, subsets of mitochondria showed rapid uptake and release of small amounts of calcium.

This also appeared to be synchronous but only for a subset of mitochondria. Speed and fine manipulations are essential for positioning animals and retaining neuronal function. Animal health is also paramount.

Even a little starvation is problematic. The hardest part to learn is the rapid orientation of the animals for confocal microscopy without damage. My advice is a lot of practice and good controls.

This protocol could be applied to experiments where calcium is released from other subcellular locations in neurons, other neurons or cells other than neurons in C.elegans. Because this approach in C.elegans leaves animals intact, it is possible to address questions about regulation of subcellular calcium handling in vivo in single neurons of a complete nervous system.