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 forward movement to a backward escape response when they encounter nonlocalized vibration⁶. The neural circuit underlying this behavior is composed primarily of five sensory neurons, four pairs of interneurons, and several types of motor neurons^7,8. Additionally, worms habituate to such mechanical stimuli after spaced training involving repeated stimulation^9,10,11. Therefore, this simple behavioral response constitutes an ideal system to investigate neural mechanisms underlying both nonlocalized vibration-evoked behavior and memory. A protocol for calcium imaging in freely behaving worms under the influence of nonlocalized vibrations is illustrated. Compared with previously reported systems, this system is simple in that it does not require an additional camera for tracking; however, it allows us to change the frequency, displacement, and duration of nonlocalized vibration. Because activation of the AVA interneurons induces the backward escape response, worms co-expressing GCaMP, a calcium indicator, and TagRFP, a calcium-insensitive fluorescent protein, under the control of an AVA-specific promoter were used as an example (see Table of Materials for details). The protocol demonstrates the activation of AVA neurons as a worm switches from forward to backward movement. This protocol facilitates understanding the neural circuit mechanism underlying mechanosensory behavior.

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 (Table of Materials).

2. Hardware setup

1. Prepare a microscope equipped with a piezoelectric device for stimulation¹³, a 2x objective lens, a high-speed sCMOS camera, image splitting optics, an x-y motorized stage, an LED light source for excitation, and a computer (Figure 1). Essential specifications of the sCMOS camera include 2560 x 2160 active pixels, 6.5 µm in size, with a 10-tap camera link as an interface option. Key specifications of the X-Y motorized stage include 110 mm x 75 mm travel range, 250 mm/s max velocity, 2,000 mm/s² max acceleration, and 0.25 µm unidirectional repeatability.
2. Turn on the computer and the x-y motorized stage controller.
3. Turn on the amplifier and set the Volume adjuster to 10 and the Bass and Treble adjusters to 8.
4. To excite GCaMP and TagRFP, turn on the 488 nm and 560 nm lights of the LED light source. Set the gauges of 470 nm and 550 nm of the control pod in the light source to 5% so that the intensity of the LED light is suitable for imaging.
   ​NOTE: At this LED intensity, photobleaching of GCaMP and TagRFP fluorescence does not occur during recording.

3. Software setup for calcium imaging

1. Download and install three software packages in Windows: mouse macro software, software for controlling vibration property, software for running tracking software, and software for data analysis (Table of Materials).
2. Double click on the tracking software file.
3. Push the Run button and wait for 5 min to stabilize the software (Figure 2A).
4. Provide the information on exposure time and binning. 0.033 s exposure time with 2 x 2 binning ensures smooth image acquisition (Figure 2B).
   NOTE: Only every 10^th image acquired is displayed in order to reduce the memory load.
5. Provide the information for image acquisition (Figure 2C). For example, when 500 images are recorded twice with an interval of 10 s, enter 1,000 in the Images Total box, 500 in the Images box, and 10 in the Interval (s) box.
6. Turn on the Acquisition button (Figure 2D).
7. Split the fluorescence image using image splitting optics, and the two images (GCaMP channel, 500-525 nm (Figure 2E); TagRFP channel, 584-676 nm (Figure 2F)) are projected onto two halves of the sCMOS camera. Calibrate the coordinates of the GCaMP and TagRFP channels (Figure 2G). Save the software settings.
8. Set the directory to output the data file (Figure 2H).
9. Turn off the Acquisition button (Figure 2D).
10. Double click on mouse macro system software (Figure 3A) and set the vibration properties (Figure 3B). Read the Assay.txt file with the mouse macro system so that the mouse cursor is controlled based on the coordinates and the schedule in that file (Figure 3A). Set the coordinates of the mouse cursor (magenta squares in Figure 3A) and wait time until stimulation after starting the recording (magenta in Figure 3A). Set the frequency and the amplitude values in the software for vibration control (Figure 3B).

4. Preparation of worms for calcium imaging

1. Transfer a single worm expressing both GCaMP and TagRFP from the incubated plate to a new fresh NGM plate on which E. coli OP50 has been streaked.
2. Attach the NGM plate to the piezoelectric acoustic transducer using transparent adhesive tape (Figure 4). Do not press the plate onto the actuator because this alters the vibration frequency.

5. Calcium imaging under the control of specific vibration properties

1. Turn on the Acquisition button (Figure 2D).
2. Find the worm under bright light and excitation light (488 nm and 560 nm).
3. Set the zoom value of microscopy to 2.5x. The field of view is 1.1 mm x 1.1 mm with a resolution of 2.6 µm/pixel.
4. Turn off the bright light and turn on the Homing button (Figure 2I) to track the fluorescent spot of a worm. This homing button starts the movement of the X-Y stage to keep the maximum intensity region of the worm in the middle of the field of view in the TagRFP image.
   NOTE: In terms of the flp-18 promoter, the fluorescent intensity of the AVA neurons is strongest and those from other neurons are faint or not detected. Therefore, the low magnification system is not required for tracking and the stage moves throughout the recording.
5. Ensure that the values of intensity bars in Figure 2E and 2F are approximately 1,000. If they are not, fine-tune the 470 nm and 550 nm gauges of the control pod in the light source.
6. Press the Run button in the mouse macro system to allow mouse cursor control according to a schedule described in Figure 3A. This starts to record using the tracking software and stimulate by wave gene software while tracking the worm.
7. Check whether the output BMP file is appropriately created.

6. Data analysis

1. Create a folder on the desktop and name it CalciumImaging.
2. Create a folder inside the CalciumImaging folder and name it CIResult, for the output result files.
3. Double click on the DualViewImaging.nb file written in data analysis software for Windows.
4. Insert the file path to the CIResult folder (e.g., DirectoryName[/Users/Sugi0911/Desktop/CalciumImaging/CIResult/test.rtf]) (Figure 5).
5. Insert the file path to the folder including the BMP file (e.g., SetDirectory[/Users/Sugi0911/Desktop/XXX/]) (Figure 5), in which XXX denotes the folder name including BMP files.
6. Push the Shift and Return keys simultaneously to start an automatic analysis. This analysis uses the value of a maximum intensity region of field of view in the TagRFP image (See the legend of Figure 6 for detailed calculation procedure in the program).
7. Check whether the following output files are appropriately created in the CIResult folder.
   XXX-GCaMP.tif (image file illustrating traces of GCaMP intensities for all images)
   XXX-GCaMP.xls (spreadsheet file listing GCaMP intensities in all images)
   XXX-TagRFP.tif (image file illustrating traces of intensities of TagRFP for all images)
   XXX-TagRFP.xls (spreadsheet file listing TagRFP intensities in all images)
   XXX-Ratio.tif (image file illustrating traces of the ratio value for all images)
   XXX-Ratio.xls (spreadsheet file listing the ratio value in all images)

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...

Materials

List of materials used in this article

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