In Vivo Calcium Imaging in C. elegans Body Wall Muscles

Summary

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

Abstract

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

Introduction

This paper presents methods for in vivo calcium imaging of C. elegans body wall muscles using optogenetic neuronal stimulation. Pairing a muscle expressed genetically encoded calcium indicator (GECI) with blue light triggered neuronal depolarization and provides a system to clearly observe the evoked postsynaptic calcium transients. This avoids the use of electrical stimulation, allowing a non-invasive analysis of mutants affecting the postsynaptic calcium dynamics.

Single-fluorophore GECIs, such as GCaMP, uses a single fluorescent protein molecule fused to the M13 domain of myosin light chain kinase at its N-terminal end and calmodulin (CaM) at the C-terminus. Upon calcium binding, the CaM domain, which has a high affinity for calcium, undergoes a conformational change inducing a subsequent conformational change in the fluorescent protein leading to an increase in fluorescence intensity¹. GCaMP fluorescence is excited at 488 nm, making it unsuitable to be used in conjunction with channelrhodopsin, which has a similar excitation wavelength of 473 nm. Thus, in order to couple calcium measurements with channelrhodopsin stimulation, the green fluorescent protein of GCaMP needs to be replaced with a red fluorescent protein, mRuby (RCaMP). Using the muscle expressed RCaMP, in combination with the cholinergic motor neuron expression of channelrhodopsin, permits studies at the worm neuromuscular junction (NMJ) with simultaneous use of optogenetics and functional imaging within the same animal².

The use of channelrhodopsin bypasses the need for the electrical stimulation to depolarize the neuromuscular junctions of C. elegans, which can only be achieved in dissected preparations, thus making this technique both easier to employ and more precise when targeting specific tissues. For example, channelrhodopsin has been previously used in C. elegans to reversibly activate specific neurons, leading to either robust activation of excitatory or inhibitory neurons^3,4. The use of light-stimulated depolarization also circumvents the issue of neuronal damage due to the direct electrical stimulation. This provides an opportunity to examine the effects of many different stimulation protocols, including sustained and repeated stimulations, on postsynaptic calcium dynamics⁴.

The transparent nature of C. elegans makes it ideal for the fluorescent imaging functional analysis. However, when stimulating the excitatory acetylcholine neurons at the NMJ, animals are expected to respond with an immediate muscle contraction⁴, making the immobilization of the worms critical in visualizing discrete calcium changes. Traditionally, pharmacological agents have been used to paralyze the animals. One such drug used is levamisole, a cholinergic acetylcholine receptor agonist^5,6,7. Since levamisole leads to the persistent activation of a subtype of excitatory muscle receptors, this reagent is unsuitable for the study of the muscle calcium dynamics. The action of levamisole induces postsynaptic depolarization, elevating the cytosolic calcium, and occluding observations following presynaptic stimulation. To avoid the use of paralyzing drugs, we examined two alternative methods to immobilize C. elegans. Animals were either glued down and then dissected open to expose the body wall muscles, similar to the existing C. elegans NMJ electrophysiology method⁸, or nanobeads were used to immobilize intact animals⁹. Both procedures allowed for the reproducible measurements of the resting and evoked muscle calcium transients that were easily quantifiable.

The methods in this paper can be used to measure the baseline cytosolic calcium levels and transients in postsynaptic body wall muscle cells in C. elegans. Examples of data employing the two different immobilization techniques are given. Both techniques take advantage of optogenetics to depolarize the muscle cells without the use of electrical stimulation. These examples demonstrate the feasibility of this method in evaluating mutations that affect postsynaptic calcium handling in the worms and point out the pros and cons of the two immobilization approaches.

Protocol

1. Microscope setup

1. Use a compound microscope with fluorescence capabilities. For this study, data were collected on an upright microscope (Table of Materials) fitted with LEDs for excitation.
2. In order to properly visualize fluorescence changes in body wall muscles, use a high magnification objective.
   1. For dissected preparations, use a 60x NA 1.0 water immersion objective (Table of Materials).
   2. For preparations using nanobeads, use a 60x NA 1.4 oil immersion objective (Table of Materials). This magnification ensures sufficient resolution of muscles onto the camera sensor.
3. Use a high sensitivity camera attached to the microscope to capture images at a high frame rate in order to track rapid changes in calcium levels. For this study, image with an sCMOS system (Table of Materials) capable of full frame imaging at 100 Hz, as rise times for the Ca²⁺ signals can be as rapid as tens of milliseconds.
4. Control camera acquisition and LED fluorescence excitation with a micro-manager software running in ImageJ¹⁰ according to the manufacturer's instructions (Table of Materials).
5. Use an open-source electronic platform plugin, which connects an external open-source microcontroller board (Table of Materials), to manage the external timing pulses to control the fluorescence excitation.
   NOTE: Digital outs are available from digital pins 8 to 13 providing output bits 0 to 5. These can be addressed as base 2 values of 1, 10, 100, etc. Serial port settings are indicated in Table 1 and available from the micro-manager website (Table of Materials). Firmware for the microcontroller board is similarly available at this website.
6. To control the timing logic, activate the micromanager acquisition protocol in the software according to the manufacturer's instructions (Table of Materials).
   NOTE: This simultaneously initiates a preset camera frame duration and the number of frames to be activated, as well as the logical shutter and preset amber LED for fluorescence excitation. In this case, the amber LED solid state switch is controlled by microcontroller bit 1 output through pin 9. The camera frames out TTL (back plate out 3 connector) triggers a stimulator. This precisely times the activation of the blue light LED for the activation of channelrhodopsin at a set time following the activation of the imaging sequence.
7. To stimulate channelrhodopsin with blue light and record RCaMP changes, use two LEDs. Activate channelrhodopsin with an LED with a peak emission wavelength of 470 nm and a bandpass filter (455 to 490 nm) and excite RCaMP with an LED with a peak emission wavelength of 594 nm and a bandpass filter (570 to 600 nm).
8. In order to simultaneously activate channelrhodopsin and capture changes in RCaMP fluorescent levels, co-illuminate both LEDs and transmit the light to the same optical path using a dichroic beam combiner.
9. Control the timing of LED illumination with solid state switches (Table of Materials) controlled by TTL signals (maximum rated turn-on time, 1 ms, turn-off time 0.3 ms: 0 to 90% turn-on and turn off time of < 100 µs).
10. Set the LED intensity with the current controlled low noise linear power supplies (Table of Materials).
11. To ensure the precise timing of the blue light LED, activate it directly by TTL pulse to the solid-state relay from a stimulator. Program the blue light stimulation protocol into a stimulator (Table of Materials), which acts as a precise timer from the start of image acquisition to blue LED illumination. In this experiment, turn on blue light stimulation after 2 s of capturing RCaMP fluorescence only and use a train of 5, 2 ms blue light pulses with 50 ms interpulse intervals to activate channelrhodopsin.
    NOTE: The delay before blue light pulses, the duration of the blue light pulses, the time between pulses, and the number of pulses in the train can all be set at this point and should reflect the specific parameters of experimental interest.

2. C. elegans sample preparation and data acquisition

1. To optically stimulate presynaptic neurons, obtain animals expressing channelrhodopsin in excitatory cholinergic neurons, driven with the unc-17 promoter region, and RCaMP expressed in all body wall muscles driven with the myo-3 promoter region².
   NOTE: Only the use of animals with integrated transgenes and robust levels of fluorescence are recommended as the variable expression in the corresponding cell types may affect the reliability of data acquisition.
2. Make a working stock of all-trans retinal by diluting the retinal powder (Table of Materials) in ethanol to create a final concentration of 100 mM and store at -20 °C. This working stock will be stable for approximately one year.
3. Create a stock of OP50 E. coli, grown in LB media, supplemented with all-trans retinal from the working stock made in step 2.2, at a final concentration of 80 µM. The volume used for the OP50+retinal stock will be dependent on the number of plates necessary for the experiment.
4. Seed nematode growth media (NGM) plates with approximately 300 µL of the OP50+retinal stock and allow plates to dry overnight at room temperature in the dark.
5. Grow C. elegans strains to the desired age in the dark on OP50+retinal NGM plates at 20 °C. For this experiment, use adult worms.
   1. For the experiment using the dissected preparation, only use gravid adult worms as performing the dissection on younger, smaller animals is extremely challenging. Leave the animals on the OP50-retinal plates for a minimum of 3 days for an effective channelrhodopsin activation.
6. If using the dissected preparation^8,11 (Figure 1A), perform dissections in low light.
   1. Place animals in a dissecting dish with a silicone-coated coverslip base that is filled with a 1 mM Ca²⁺ extracellular solution composed of 150 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 4 mM MgCl₂, 10 mM glucose, 5 mM sucrose, and 15 mM HEPES (pH 7.3, -340 Osm).
   2. Glue down 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/worm interface using glass needles.
   3. Use a mouth pipette to clear the internal viscera from the worm cavity.
   4. Glue down the cuticle flap of the animal to expose the ventral medial body wall muscles for imaging.
7. If using the nanobead preparation (Figure 1B)
   1. Make a molten 5% agarose solution using ddH₂O to a final volume of 100 mL.
   2. Using a Pasteur pipette, place a drop of molten agarose solution onto a glass slide and immediately place a second glass slide over the top, perpendicular to the first, using gentle pressure to create an even agarose pad. Remove the top slide before use.
   3. Add approximately 4 µL of polystyrene nanobeads (Table of Materials) to the middle of the agarose pad.
   4. In the low light, pick 4-6 C. elegans into the nanobead solution, making sure animals do not lay on top of each other, and carefully place a coverslip on the top.
8. Place the prepared slide or dissection dish onto the microscope, and find and focus on a worm using 10x magnification and dim bright field illumination.
9. Switch to 60x magnification and RCaMP fluorescence excitation to identify a ventromedial body wall muscle that is anterior to the vulva and in the correct focal plane.
   NOTE: Muscles anterior to the vulva are selected as they reflect muscles commonly stimulated in electrophysiology experiments.
10. Change the image pathway from the eyepiece to the camera by pulling out the toggle and clicking Live within the data acquisition software (Table of Materials).
    NOTE: Make sure the blue light stimulation pathway is turned off at this point to ensure that the channelrhodopsin does not get activated before capturing images.
11. Focus the image within the data acquisition software using the microscope fine focus.
12. Once the muscle is clearly in focus, turn off the live image by unclicking the Live button.
13. Click the ROI button in the data acquisition software and create a box around the muscle being focused on.
14. On the stimulator, switch on the blue light stimulation pathway that has been previously programmed in step 1.10.
15. Click Acquire in the imaging software to capture the image through the CMOS camera. To do this, set the exposure time to 10 s with 1,000 frames and 2x binning.

3. Data analysis

1. Open the data file in the imaging software (Table of Materials) and, using the Polygon Tool, outline the muscle of interest. This is the ROI.
2. Go to Image | Stacks | Plot Z-axis profile and export the resulting data points into the spreadsheet software (Table of Materials). This function plots the ROI selection mean value versus the time point.
3. Move the muscle ROI created with the Polygon tool outside of the animal to get a background fluorescence measurement using the steps outlined in 3.2. Export the resulting data into the spreadsheet workbook.
4. In the spreadsheet workbook, subtract the background fluorescence values from the muscle fluorescence values at each time point. This provides the background subtracted fluorescent signal.
5. Average the background subtracted fluorescence for the first 2 s of data points. This will give the baseline fluorescence measurement, F.
6. Use the baseline fluorescence measurement to calculate the normalized fluorescence level at each time point. To do this, use the equation (ΔF/F)+1. ΔF represents (F(t)-F), where F(t) is the fluorescence measurement at any given time point and F is the baseline value. The +1 is added as a y-axis offset.
7. Repeat steps 3.2-3.6 for each muscle image that is collected. Using single or multiple muscle cells per image is at the discretion of the researcher. The n of the experiment can be determined by performing a power analysis.
8. Use the processed data from steps 3.2-3.6 to make a trace of the fluorescent values in graphing software according to the manufacturer's instructions (Table of Materials).
9. From this trace, measure the kinetics of the calcium transient, such as rise to peak time and half decay time, using the tools provided in the graphing software as per the manufacturer's instructions.

Results

This technique evaluated changes in mutants thought to affect the calcium handling or muscle depolarization. Baseline fluorescence levels and fluorescent transients were visualized and resting cytosolic calcium and calcium kinetics within the muscle were evaluated. It is important that the animals were grown on all-trans retinal for at least three days to ensure the successful incorporation of retinal, thereby subsequently activating the channelrhodopsin (Figure 2

Discussion

GECIs are a powerful tool in C. elegans neurobiology. Previous work has utilized calcium imaging techniques to examine a wide variety of functions in both neurons and muscle cells, including sensory and behavioral responses, with varied methods of stimulation. Some studies have used chemical stimuli to trigger calcium transients in sensory ASH neurons^22,23 or to induce calcium waves in pharyngeal muscles²⁴. Another group utilized ...

Materials

Name	Company	Catalog Number	Comments
all-trans retinal	Sigma-Aldrich	R2500	Necessary for excitation of channel rhodopsin
Amber LED			RCaMP illumination
Arduino UNO	Mouser	782-A000066	Controls fluorescence illumination
Blue LED			channelrhodopsin illumination
BX51WI microscope	Olympus		Fixed state compound microscope
Current controlled low noise linear power supply	Ametek	Sorenson	Controls LED intensity
Igor Pro	Wavemetrics	Wavemetrics.com	Graphing software
ImageJ	NIH	imagej.nih.gov	Image processing software
LUMFLN 60x water NA 1.4	Olympus		Water immersion objective for dissected preparation
Master-8 Stimulator	A.M.P.I		Master timer for image acquisition and LED illumination
Micro-Manager		micro-manager.org	Controls camera acquisition and LED excitiation
Microsoft Excel	Microsoft		Spreadsheet software
pco.edge 4.2 CMOS camera	pco.	4.2	High-speed camera
PlanApo N 60x oil NA 1.4	Olympus		Oil immersion objective for nanobead preparation
Polybead microspheres	Polysciences, Inc.	00876-15	For worm immobilization
solid state switches	Sensata Technologies	Crydom CMX100D6	Controls timing of LED illumination
Transgenic strain, sca-1(tm5339); [zxIs6{Punc17::chop-2 (h134R)::yfp,lin-15(+)}; Pmyo3::RCaMP35]	Richmond Lab	SY1627	Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with SERCA mutant allele
Transgenic strain, slo-1 (eg142); [zxIs6{Punc17::chop-2 (h134R)::yfp,lin-15(+)}; Pmyo3::RCaMP35]	Richmond Lab		Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with calcium-activated BK potassium channel mutant allele
Transgeneic strain, [zxIs6{Punc17::chop-2 (h134R)::yfp,lin-15(+)}; Pmyo3::RCaMP35]	Gottschalk Lab	ZX1659	Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line