A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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 calmo....
1. Microscope setup
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
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 neurons22,23 or to induce calcium waves in pharyngeal muscles24. Another group utilized .......
The authors thank Dr. Alexander Gottschalk for ZX1659, the RCaMP and channelrhodopsin containing worm strain, Dr. Hongkyun Kim for the slo-1(eg142) worm strain, and the National Bioresource Project for the sca-1(tm5339) worm strain.
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved