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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 calmo....

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 .......

Representative 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 neurons22,23 or to induce calcium waves in pharyngeal muscles24. Another group utilized .......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
all-trans retinalSigma-AldrichR2500Necessary for excitation of channel rhodopsin
Amber LEDRCaMP illumination
Arduino UNOMouser782-A000066Controls fluorescence illumination
Blue LEDchannelrhodopsin illumination
BX51WI microscopeOlympusFixed state compound microscope
Current controlled low noise linear power supplyAmetekSorensonControls LED intensity
Igor ProWavemetricsWavemetrics.comGraphing software
ImageJNIHimagej.nih.govImage processing software
LUMFLN 60x water NA 1.4OlympusWater immersion objective for dissected preparation
Master-8 StimulatorA.M.P.IMaster timer for image acquisition and LED illumination
Micro-Managermicro-manager.orgControls camera acquisition and LED excitiation
Microsoft ExcelMicrosoftSpreadsheet software
pco.edge 4.2 CMOS camerapco.4.2High-speed camera
PlanApo N 60x oil NA 1.4OlympusOil immersion objective for nanobead preparation
Polybead microspheresPolysciences, Inc.00876-15For worm immobilization
solid state switchesSensata TechnologiesCrydom CMX100D6Controls timing of LED illumination
Transgenic strain, sca-1(tm5339); [zxIs6{Punc17::chop-2
(h134R)::yfp,lin-15(+)};
Pmyo3::RCaMP35
]
Richmond LabSY1627Excitatory 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 LabExcitatory 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 LabZX1659Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line

References

  1. Nakai, J., Ohkura, M., Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nature Biotechnology. 19 (2), 137-141 (2001).
  2. Akerboom, J., et al.

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