Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

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 waves^1,2. Because these stimuli influence homeostasis, development, and reproduction, animals must modify their behaviors to cope with them^3,4,5. 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 (

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.......

Materials

Name	Company	Catalog Number	Comments
Data anaylsis software
DualViewImaging.nb	author		For analysis of acquired data
Mathematica12	Wolfram		For running data anaysis software DualViewImaging
Escherichia coli and C. elegans strains
E. coli OP50	Caenorhabditis Genetics Center	OP50	Food for C. elegans. Uracil auxotroph. E. coli B.
lite-1(ce314) strain	Caenorhabditis Genetics Center	KG1180	Light-insensitive mutant
lite-1(ce314) strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoter	author	ST12	lite-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 vibrometer	Polytec Japan	IVS-500	For quantifying  frequency and displacement generated by the accoustic transducer
Mouse macro system
Assay.txt	Author		Script for temporally and specially controlling mouse cursol in Windows
HiMacroEx	Vector	https://www.vector.co.jp/download/file/winnt/util/fh667310.html	Free download software for controling mouse cursor based on a script
Nematode growth media plate
Agar purified, powder	Nakarai tesque	01162-15	For preparation of NGM plates
Bacto pepton	Becton Dickinson	211677	For preparation of NGM plates
Calcium chloride	Wako	036-00485	For preparation of NGM plates
Cholesterol	Wako	034-03002	For preparation of NGM plates
di-Photassium hydrogenphosphate	Nakarai tesque	28727-95	For preparation of NGM plates
LB broth, Lennox	Nakarai tesque	20066-95	For culture of E. coli OP50
Magnesium sulfate anhydrous	TGI	M1890	For preparation of NGM plates
Potassium Dihydrogenphosphate	Nakarai tesque	28720-65	For preparation of NGM plates
Sodium Chloride	Nakarai tesque	31320-05	For preparation of NGM plates
Petri dishes (60 mm)	Nunc	150270	For preparation of NGM plates
Nonlocalized vibration device
Amplifier	LEPY	LP-A7USB	For stimulation with controllable vibration
Acoustic transducer	MinebeaMitsumi	LVC25	For stimulation with controllable vibration
WaveGene Ver. 1.5	Thrive	http://efu.jp.net/soft/wg/down_wg.html	Free download software for controling vibration property
Noninvasive calcium imaging
2-Channel benchtop 3-phase brushless DC servo controller	Thorlabs	BBD202	Compatible controller for MLS203-1 stages
479/585 nm BrightLine dual-band bandpass filter	Semrock	FF01-479/585-25	For acquisition of two channel images (GCaMP and TagRFP)
505/606 nm BrightLine dual-edge standard epi-fluorescence dichroic beamsplitter	Semrock	FF505/606-Di01-25x36	For acquisition of two channel images (GCaMP and TagRFP)
512/25 nm BrightLine single-band bandpass filter	Semrock	FF01-512/25-25	For acquisition of two channel images (GCaMP and TagRFP)
630/92 nm BrightLine single-band bandpass filter	Semrock	FF01-630/92-25	For acquisition of two channel images (GCaMP and TagRFP)
Computer	Dell	Precision T7600	Windows7 with Intel Xeon CPU ES-2630 and 8 GB of RAM
High-speed x-y motorized stage	Thorlabs	MLS203-1	Fast XY scannning stage
Image splitting optics	Hamamatsu photonics	A12801-01	For acquisition of two channel images (GCaMP and TagRFP) generated by W-VIEW GEMINI Image spliting optics
LED light source	CoolLED	pE-4000	For generating 470 nm and 560 nm excitation light
Microscope	Olympus	MVX10
sCMOS camera	Andor	Zyla
x 2 Objective lens	Olympus	MVPLAPO2XC	Working distance 20 mm and numerical aperture 0.5
Plasmid
pKDK66 plasmid	author	pKDK66	Co-injection marker
pTAK83 plasmid	author	pTAK83	Plasmid for expression of TagRFP under the control of  the flp-18 promoter
pTAK144 plasmid	author	pTAK144	Plasmid for expression of NLS-GCaMP5G-NLS under the control of  the flp-18 promoter
Tracking software
homingback.vi	author		SubVi file for tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage
LabVIEW	National instruments		For running tracking software
Zyla Control ver.2.6CI.vi	author		For tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage