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In This Article

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

Summary

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

Abstract

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Introduction

Animals are often exposed to nonlocalized mechanical stimuli such as vibrations or acoustic waves1,2. Because these stimuli influence homeostasis, development, and reproduction, animals must modify their behaviors to cope with them3,4,5. However, the neural circuits and mechanisms underlying such behavioral modification are poorly understood.

Mechanosensory behavior in the nematode, Caenorhabditis elegans, is a simple behavioral paradigm, in which worms usually change behavior from....

Protocol

1. Cultivation of worms until calcium imaging

  1. Four days before a calcium imaging experiment, transfer two adult ST12 worms to a new nematode growth medium (NGM) plate (Table of Materials) on which Escherichia coli OP50 are streaked in a square pattern (approximately 4 mm x 4 mm) using a cell spreader so that the worm spends most of the time in the bacteria during calcium imaging12.
  2. Incubate this NGM plate for 4 days at 20 °C in an incubator (

Representative Results

Here, a worm expressing both GCaMP and TagRFP under control of the AVA interneuron-specific promoter is used as an example of calcium imaging in freely behaving C. elegans. GCaMP and TagRFP channel data were obtained as a series of images, some of which are shown in Figure 6 and as a movie (Supplemental Movie 1). The displacement of the Petri plate induced by our nonlocalized vibration system (Figure 7) was also quantified. The displace.......

Discussion

Generally, the quantification of neural activity requires introduction of a probe and/or restraints on animal body movement. However, for studies of mechanosensory behavior, the invasive introduction of a probe and restraints themselves constitute mechanical stimuli. C. elegans provides a system to circumvent these problems, because its features are transparent and because it has a simple, compact neural circuit comprising only 302 neurons. Combining these advantages with the previously developed method of evoki.......

Acknowledgements

We thank the Caenorhabditis Genetics Center for providing the strains used in this study. This publication was supported by JSPS KAKENHI Grant-in-Aid for Scientific research (B) (Grant no. JP18H02483), on Innovative areas "Science of Soft Robot" project (Grant no. JP18H05474), the PRIME from Japan Agency for Medical Research and Development (grant number 19gm6110022h001), and the Shimadzu foundation.

....

Materials

NameCompanyCatalog NumberComments
Data anaylsis software
DualViewImaging.nbauthorFor analysis of acquired data
Mathematica12WolframFor running data anaysis software DualViewImaging
Escherichia coli and C. elegans strains
E. coli OP50Caenorhabditis Genetics CenterOP50Food for C. elegans. Uracil auxotroph. E. coli B.
lite-1(ce314) strainCaenorhabditis Genetics CenterKG1180Light-insensitive mutant
lite-1(ce314) strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoterauthorST12lite-1(ce314) mutant carrying the genes expressing NLS-GCaMP5G-NLS (NLS; nuclear localization signal) and TagRFP under the control of the flp-18 promoter as an extrachoromosomal arrays
Laser Doppler vibrometer
Lase Doppler vibrometerPolytec JapanIVS-500For quantifying  frequency and displacement generated by the accoustic transducer
Mouse macro system
Assay.txtAuthorScript for temporally and specially controlling mouse cursol in Windows
HiMacroExVectorhttps://www.vector.co.jp/download/file/winnt/util/fh667310.htmlFree download software for controling mouse cursor based on a script
Nematode growth media plate
Agar purified, powderNakarai tesque01162-15For preparation of NGM plates
Bacto peptonBecton Dickinson211677For preparation of NGM plates
Calcium chlorideWako036-00485For preparation of NGM plates
CholesterolWako034-03002For preparation of NGM plates
di-Photassium hydrogenphosphateNakarai tesque28727-95For preparation of NGM plates
LB broth, LennoxNakarai tesque20066-95For culture of E. coli OP50
Magnesium sulfate anhydrousTGIM1890For preparation of NGM plates
Potassium DihydrogenphosphateNakarai tesque28720-65For preparation of NGM plates
Sodium ChlorideNakarai tesque31320-05For preparation of NGM plates
Petri dishes (60 mm)Nunc150270For preparation of NGM plates
Nonlocalized vibration device
AmplifierLEPYLP-A7USBFor stimulation with controllable vibration
Acoustic transducerMinebeaMitsumiLVC25For stimulation with controllable vibration
WaveGene Ver. 1.5Thrivehttp://efu.jp.net/soft/wg/down_wg.htmlFree download software for controling vibration property
Noninvasive calcium imaging
2-Channel benchtop 3-phase brushless DC servo controllerThorlabsBBD202Compatible controller for MLS203-1 stages
479/585 nm BrightLine dual-band bandpass filterSemrockFF01-479/585-25For acquisition of two channel images (GCaMP and TagRFP)
505/606 nm BrightLine dual-edge standard epi-fluorescence dichroic beamsplitterSemrockFF505/606-Di01-25x36For acquisition of two channel images (GCaMP and TagRFP)
512/25 nm BrightLine single-band bandpass filterSemrockFF01-512/25-25For acquisition of two channel images (GCaMP and TagRFP)
630/92 nm BrightLine single-band bandpass filterSemrockFF01-630/92-25For acquisition of two channel images (GCaMP and TagRFP)
ComputerDellPrecision T7600Windows7 with Intel Xeon CPU ES-2630 and 8 GB of RAM
High-speed x-y motorized stageThorlabsMLS203-1Fast XY scannning stage
Image splitting opticsHamamatsu photonicsA12801-01For acquisition of two channel images (GCaMP and TagRFP) generated by W-VIEW GEMINI Image spliting optics
LED light sourceCoolLEDpE-4000For generating 470 nm and 560 nm excitation light
MicroscopeOlympusMVX10
sCMOS cameraAndorZyla
x 2 Objective lensOlympusMVPLAPO2XCWorking distance 20 mm and numerical aperture 0.5
Plasmid
pKDK66 plasmidauthorpKDK66Co-injection marker
pTAK83 plasmidauthorpTAK83Plasmid for expression of TagRFP under the control of  the flp-18 promoter
pTAK144 plasmidauthorpTAK144Plasmid for expression of NLS-GCaMP5G-NLS under the control of  the flp-18 promoter
Tracking software
homingback.viauthorSubVi file for tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage
LabVIEWNational instrumentsFor running tracking software
Zyla Control ver.2.6CI.viauthorFor tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage

References

  1. Hill, P. S. M., Wessel, A. Biotremology. Current Biology. 26 (5), 187-191 (2016).
  2. Fettiplace, R., Hackney, C. M. The sensory and motor roles of auditory hair cells. Nature Reviews Neuroscience. 7 (1), 19-29 (2006).
  3. Vogel, V., Sheetz, ....

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