This protocol provides two methods to mobilize C.Elegans to perform in vivo calcium imaging of body wall muscles. This method can help answer questions related to calcium homeostasis and calcium handling, and an accessible synapse in genetically tractable organism. This technique can provide a better understanding of the mechanisms regulating muscle calcium homeostasis, and can, therefore, be important in identifying conditions or molecular pathways that impact excitation contraction coupling through the misregulation of calcium cycling.
These methods can provide insight into areas of research including, but not limited too, neurobiology, developmental biology, and cell biology. These techniques could be adapted to study a variety of cell types and C.elegans, other related nematodes as well as filsafat larvae. Visual demonstration of this method is critical so the experimenter can understand the dissection technique, and be able to accurately assess calcium transients from properly immobilized animals.
Start by setting up the microscope for fluorescent imaging. Use a scientific CMOS camera capable of full frame imaging at 100 hertz in order to track rapid changes in calcium levels. Control camera acquisition and LED fluorescent excitation with a micromanager software running in ImageJ.
Use an opensource electronic platform plug-in which connects to an external opensource microcontroller board to manage the external timing pulses that control fluorescent excitations. To control the timing logic, activate the micromanager acquisition protocol in the software according to manufacturers instructions. Use two LEDs to stimulate channel rhodopsin with blue light and record RCaMP changes.
Activate channel rhodopsin with am LED with the peak admission wavelength of 470 nanometers and a bandpass filter, and excite RCaMP with an LED with a peak admission wavelength of 594 nanometers and a bandpass filter. Co-illuminate both LEDs and transmit the light to the same optical path using a dichroic beam combiner. Control the timing of the LED illumination with a solid state switches controlled by TTL signals according to manuscript directions, and set the LED intensity with the current controlled load noise linear power supplies, then program the blue light simulation protocol into a simulator.
In this experiment turn on the blue light simulation after two seconds of capturing RCaMP fluorescence only and use a train of five, two millisecond blue light pulses with 50 millisecond intervals to activate channel rhodopsin. If using the dissection preparation, perform C.elegans dissection in low light. Place the animals in the dissection dish with the silicone coated coverslip base that is filled with a one millimole of calcium extracellular solution prepared according to manuscript directions.
Glue down the animals using the liquid topical skin adhesive with blue coloring along the dorsal side of the worm. And make a lateral cuticle incision along the glue and worm interface using glass needles. Use a mouth pipette to clear the internal viscera from the worm cavity, then glue down the cuticle flap of the animal to expose the ventral medial body wall muscles for imaging.
If using the nano-bead preparation, make a molten 5%Agra solution using distilled water to a final volume of 100 milliliters. Use a Pasteur pipette to place a drop of molten-Agra solution onto a glass slide and immediately place a second glass slide over the top using gently pressure to create an even Agra's pad. Remove the top slide and at approximately four microliters of polystyrene nano-beads to the middle of the pad.
In low light add four to six C.elegans into the nano-beads solution, making sure that the animals do not lay on top of each other, and carefully place a coverslip on top. When when ready to image, place the prepared slide or dissection dish on the microscope and find and focus on a worm using 10 times magnification and dim bright field illumination. Switch to 60 times magnification in RCaMP fluorescent excitation to identify a ventrum medial body wall muscle that is anterior to the vulva and in the correct vocal plane.
Then change the image pathway from the eyepiece to the camera by pulling out the toggle and clicking live within the data acquisition software. Click the ORI button in the data acquisition software, and create a box around the muscle that is being focused on. On the stimulator, switch on the previously programmed blue light stimulation pathway, then click Acquire on the imaging software to capture the image.
When C.elegans are grown on all-trans retinal, successfully incorporating retinal and activating the channel rhodopsin, a calcium transient can be evoked. If animals are not exposed to all-trans retinal no muscle calcium transient is triggered. This protocol was used to investigate loss of function sca-1 mutants, which impact the sarcoplasmic reticulum calcium pump encoded by the sca-1 gene.
But, the dissection preparation increases in baseline levels of RCaMP fluorescence were observed in the mutant compared to the control, suggesting that the mutation needs the elevated levels of resting cytoplasmic calcium. There was a significant decrease in peak calcium levels in the sca-1 mutants as compared to the control. However, no change was seen in the rised peak time or the half decay time.
Importantly, similar results can be observed using the less technically challenging nano-bead preparation. Loss of function slo-mutants, which disrupt the calcium activated big potassium channels were evaluated in order to investigate mutants that indirectly impact calcium handling in C.elegans. When baseline levels of RCaMP were measured the mutants displayed increased fluorescence compared to the controls.
When evaluating the kinetics of the calcium transient no changes in peak calcium levels were seen. The slo-1(eg142)mutants, however, exhibited a significantly increased rise to peak time. The most important step to remember when completing the procedure is to ready experimental animals in all-trans retinal.
Without this step the channel rhodopsin will be inactive, preventing light induced calcium transients from being triggered. Following this procedure, electrophysiology and behavioral analysis can be done to evaluate the functional impact of any observed changes in calcium homeostasis. Using the immobilization methods outlined here, paves the way for further exploration of calcium dynamics and demonstrates that comparable results can be observed in response to optogenetic neuronal stimulation and capture of muscle calcium transients using the far less challenging bead immobilization technique.
