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 &#34;Science of Soft Robot&#34; project (Grant no. JP18H05474), the PRIME from Japan Agency for Medical Research and Development (grant number 19gm6110022h001), and the Shimadzu foundation.

1. Cultivation of worms until calcium imaging
<ol>
	<li>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.</li>
	<li>Incubate this NGM plate for 4 days at 20 &#176;C in an incubator (<st...

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The authors have no conflicts of interest to disclose.

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...

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 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 neurons7,8. Additionally, worms habituate to such mechanical stimuli after spaced training involving repeated stimulation9,10,11. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Data anaylsis software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DualViewImaging.nb</td>
			<td>author</td>
			<td></td>
			<td>For analysis of acquired data</td>
		</tr>
		<tr>
			<td>Mathematica12</td>
			<td>Wolfram</td>
			<td></td>
			<td>For running data anaysis software DualViewImaging</td>
		</tr>
		<tr>
			<td>Escherichia coli and C. elegans strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td>Food for C. elegans. Uracil auxotroph. E. coli B.</td>
		</tr>
		<tr>
			<td>lite-1(ce314) strain</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>KG1180</td>
			<td>Light-insensitive mutant</td>
		</tr>
		<tr>
			<td>lite-1(ce314) strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoter</td>
			<td>author</td>
			<td>ST12</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Laser Doppler vibrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lase Doppler vibrometer</td>
			<td>Polytec Japan</td>
			<td>IVS-500</td>
			<td>For quantifying&#160; frequency and displacement generated by the accoustic transducer</td>
		</tr>
		<tr>
			<td>Mouse macro system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assay.txt</td>
			<td>Author</td>
			<td></td>
			<td>Script for temporally and specially controlling mouse cursol in Windows</td>
		</tr>
		<tr>
			<td>HiMacroEx</td>
			<td>Vector</td>
			<td>https://www.vector.co.jp/download/file/winnt/util/fh667310.html</td>
			<td>Free download software for controling mouse cursor based on a script</td>
		</tr>
		<tr>
			<td>Nematode growth media plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar purified, powder</td>
			<td>Nakarai tesque</td>
			<td>01162-15</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Bacto pepton</td>
			<td>Becton Dickinson</td>
			<td>211677</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Wako</td>
			<td>036-00485</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Wako</td>
			<td>034-03002</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>di-Photassium hydrogenphosphate</td>
			<td>Nakarai tesque</td>
			<td>28727-95</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>LB broth, Lennox</td>
			<td>Nakarai tesque</td>
			<td>20066-95</td>
			<td>For culture of E. coli OP50</td>
		</tr>
		<tr>
			<td>Magnesium sulfate anhydrous</td>
			<td>TGI</td>
			<td>M1890</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogenphosphate</td>
			<td>Nakarai tesque</td>
			<td>28720-65</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Nakarai tesque</td>
			<td>31320-05</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Petri dishes (60 mm)</td>
			<td>Nunc</td>
			<td>150270</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Nonlocalized vibration device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>LEPY</td>
			<td>LP-A7USB</td>
			<td>For stimulation with controllable vibration</td>
		</tr>
		<tr>
			<td>Acoustic transducer</td>
			<td>MinebeaMitsumi</td>
			<td>LVC25</td>
			<td>For stimulation with controllable vibration</td>
		</tr>
		<tr>
			<td>WaveGene Ver. 1.5</td>
			<td>Thrive</td>
			<td>http://efu.jp.net/soft/wg/down_wg.html</td>
			<td>Free download software for controling vibration property</td>
		</tr>
		<tr>
			<td>Noninvasive calcium imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Channel benchtop 3-phase brushless DC servo controller</td>
			<td>Thorlabs</td>
			<td>BBD202</td>
			<td>Compatible controller for MLS203-1 stages</td>
		</tr>
		<tr>
			<td>479/585 nm BrightLine dual-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-479/585-25</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>505/606 nm BrightLine dual-edge standard epi-fluorescence dichroic beamsplitter</td>
			<td>Semrock</td>
			<td>FF505/606-Di01-25x36</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>512/25 nm BrightLine single-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-512/25-25</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>630/92 nm BrightLine single-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-630/92-25</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Precision T7600</td>
			<td>Windows7 with Intel Xeon CPU ES-2630 and 8 GB of RAM</td>
		</tr>
		<tr>
			<td>High-speed x-y motorized stage</td>
			<td>Thorlabs</td>
			<td>MLS203-1</td>
			<td>Fast XY scannning stage</td>
		</tr>
		<tr>
			<td>Image splitting optics</td>
			<td>Hamamatsu photonics</td>
			<td>A12801-01</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP) generated by W-VIEW GEMINI Image spliting optics</td>
		</tr>
		<tr>
			<td>LED light source</td>
			<td>CoolLED</td>
			<td>pE-4000</td>
			<td>For generating 470 nm and 560 nm excitation light</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Zyla</td>
			<td></td>
		</tr>
		<tr>
			<td>x 2 Objective lens</td>
			<td>Olympus</td>
			<td>MVPLAPO2XC</td>
			<td>Working distance 20 mm and numerical aperture 0.5</td>
		</tr>
		<tr>
			<td>Plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pKDK66 plasmid</td>
			<td>author</td>
			<td>pKDK66</td>
			<td>Co-injection marker</td>
		</tr>
		<tr>
			<td>pTAK83 plasmid</td>
			<td>author</td>
			<td>pTAK83</td>
			<td>Plasmid for expression of TagRFP under the control of&#160; the flp-18 promoter</td>
		</tr>
		<tr>
			<td>pTAK144 plasmid</td>
			<td>author</td>
			<td>pTAK144</td>
			<td>Plasmid for expression of NLS-GCaMP5G-NLS under the control of&#160; the flp-18 promoter</td>
		</tr>
		<tr>
			<td>Tracking software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>homingback.vi</td>
			<td>author</td>
			<td></td>
			<td>SubVi file for tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National instruments</td>
			<td></td>
			<td>For running tracking software</td>
		</tr>
		<tr>
			<td>Zyla Control ver.2.6CI.vi</td>
			<td>author</td>
			<td></td>
			<td>For tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage</td>
		</tr>
	</tbody>
</table>

calcium imaging in freely behaving caenorhabditis elegans with well-controlled, nonlocalized vibration

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.

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...

Watch this Scientific Journal Video about Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration at JoVE.com

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

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.

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

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

Our system allows researchers to evoke non localized vibrations with well-controlled properties at non-scaled displacement and to quantify calcium current during responses of the irritants to the vibrations. As a main advantage of this technique, we can noninvasively investigate the neural mechanisms, underlying mechanisms of behavior. Prepare a new nematode growth medium or NGM plate on which Escherichia coli OP50 is streaked in a square pattern using a cell spreader so that the worm spends most of the time in the bacteria during calcium imaging.Four days before a calcium imaging experiment, transfer two adult ST12 worms onto the plate, and place the plate in a 20 degree Celsius incubator for four days. Perform calcium imaging using a microscope equipped with an acoustic transducer device for stimulation, a 2x objective lens, a high speed CMOS camera, image-splitting optics. Turn on the precision computer in the X Y motorized stage controller.Then turn on the amplifier and set the volume adjuster to 10 in the base and treble adjusters to eight. To excite GCaMP and tag RFP, turn on the 488 and 560 nanometer lights of the LED light source. Set the 470 and 550 nanometer gauges of the control pod in the light source to 5%so that the intensity of the LED light is suitable for imaging.Download and install the following software packages and windows. Mouse macro software, software for vibration control, software for running tracking, and software for data analysis. Double click on the tracking software file, then set the exposure time to 0.033 seconds, and binning to two by two to ensure smooth image acquisition.Click the run button and wait for five minutes to stabilize the software. Input the information for image acquisition. For example, to record 500 images without an interval, enter 500 in the images total box, 500 in the images box, and zero in the intervals box.After turning on the acquisition button, split the fluorescence image using image splitting optics, projecting the GCaMP channel and tag RFP channel onto two halves of the CMOS camera. Calibrate the coordinates of the GCaMP and tag RFP channels and save the settings. Then set the directory to output the data file and turn off the acquisition.Read the assay. text file with the mouse macro system so that the mouse cursor is controlled based on the coordinates and the schedule in that file and double click on the software file for vibration control. Set the coordinates of the mouse cursor in wait time until stimulation after starting the recording, then set the frequency and the amplitude values in the software for vibration control.Transfer a single worm expressing both GCaMP and tag RFP from the incubated plate to a fresh NGM plate where E coli OP50 has been streaked. Attach the NGM plate to the acoustic transducer using transparent adhesive tape. For calcium imaging, turn on the acquisition button and find the worm at 2.5x magnification.Set the field of view as 1.1 by 1.1 millimeter with a resolution of 2.6 microns per pixel. Turn off the bright light and click on the homing button to track the fluorescent spot of a worm, which initiates 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 tag RFP image. Ensure that the values of intensity are approximately 1000.If not, fine tune the 470 and 550 nanometer gauges of the control pod in the light source. Press the run button in the mouse macro system to allow mouse cursor control, and verify that the output BMP file is appropriately created. For data analysis, create a folder on the desktop and name it Calcium Imaging"then create a folder inside the Calcium Imaging folder and name it CI result for the output result files.Double click on the dual view imaging. MB file written in the data analysis software, and insert the file paths to the CI result folder in the folder with the BMP file. Push the shift and return keys simultaneously to start an automatic analysis, which uses the value of a maximum intensity region in the tag RFP image.Finally, check whether the output files are appropriately created in the CI result folder. In the representative analysis, GCaMP and tag RFP channel data was obtained as a series of images. Displacement of the Petri dish induced by non-localized vibration system was also quantified.The displacement can be controlled by setting the amplitude value in the software for vibration control, and the volume adjuster and the amplifier. Whereas the frequency can be regulated by setting the frequency value in the software. A transient calcium response of AVA neurons was observed upon stimulation with vibrations having a frequency of 630 Hertz and a displacement of approximately 4.5 micrometers per second, indicating that the Ava neuron was activated during a worm's backward response to the non localized stimulation.In our procedure, we can quantify only a single neuron. However, this system can also be combined with a biometric imaging method to noninvasively quantify multiple neurons or even the whole brain.

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Research

JoVE Journal

Behavior

2.6K visualizações.  Hiroshima University. Nosso sistema permite que os pesquisadores evoquem vibrações não localizadas com propriedades bem controladas em deslocamento não dimensionado e quantifiquem a corrente de cálcio durante as respostas dos irritantes às vibrações. Como principal vantagem dessa técnica, não podemos investigar invasivamente os mecanismos neurais, mecanismos subjacentes ao comportamento. Prepare um novo meio de crescimento de nematoide ou placa de NGM na qual Escherichia coli OP50 é listrado em um padr?...