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 (Table of Materials).</li>
</ol>
2. Hardware setup
<ol>
	<li>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 &#181;m 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 &#181;m unidirectional repeatability.</li>
	<li>Turn on the computer and the x-y motorized stage controller.</li>
	<li>Turn on the amplifier and set the Volume adjuster to 10 and the Bass and Treble adjusters to 8.</li>
	<li>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. 
	&#8203;NOTE: At this LED intensity, photobleaching of GCaMP and TagRFP fluorescence does not occur during recording.</li>
</ol>
3. Software setup for calcium imaging
<ol>
	<li>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).</li>
	<li>Double click on the tracking software file.</li>
	<li>Push the Run button and wait for 5 min to stabilize the software (Figure 2A).</li>
	<li>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 10th image acquired is displayed in order to reduce the memory load.</li>
	<li>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.</li>
	<li>Turn on the Acquisition button (Figure 2D).</li>
	<li>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.</li>
	<li>Set the directory to output the data file (Figure 2H).</li>
	<li>Turn off the Acquisition button (Figure 2D).</li>
	<li>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).</li>
</ol>
4. Preparation of worms for calcium imaging
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Calcium imaging under the control of specific vibration properties
<ol>
	<li>Turn on the Acquisition button (Figure 2D).</li>
	<li>Find the worm under bright light and excitation light (488 nm and 560 nm).</li>
	<li>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 &#181;m/pixel.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Check whether the output BMP file is appropriately created.</li>
</ol>
6. Data analysis
<ol>
	<li>Create a folder on the desktop and name it CalciumImaging.</li>
	<li>Create a folder inside the CalciumImaging folder and name it CIResult, for the output result files.</li>
	<li>Double click on the DualViewImaging.nb file written in data analysis software for Windows.</li>
	<li>Insert the file path to the CIResult folder (e.g., DirectoryName[/Users/Sugi0911/Desktop/CalciumImaging/CIResult/test.rtf]) (Figure 5).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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)</li>
</ol>

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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><tbody><tr><td>&lt;strong&gt;Data anaylsis software&lt;/strong&gt;</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>&lt;strong&gt;&lt;em&gt;Escherichia coli&lt;/em&gt; and &lt;em&gt;C. elegans&lt;/em&gt; strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; OP50</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td>Food for &lt;em&gt;C. elegans.&lt;/em&gt; Uracil auxotroph. &lt;em&gt;E. coli&lt;/em&gt; B.</td></tr><tr><td>&lt;em&gt;lite-1(ce314)&lt;/em&gt; strain</td><td>Caenorhabditis Genetics Center</td><td>KG1180</td><td>Light-insensitive mutant</td></tr><tr><td>&lt;em&gt;lite-1(ce314)&lt;/em&gt; strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoter</td><td>author</td><td>ST12</td><td>&lt;em&gt;lite-1(ce314)&lt;/em&gt; mutant carrying the genes expressing NLS-GCaMP5G-NLS (NLS; nuclear localization signal) and TagRFP under the control of the &lt;em&gt;flp&lt;/em&gt;-18 promoter as an extrachoromosomal arrays</td></tr><tr><td>&lt;strong&gt;Laser Doppler vibrometer&lt;/strong&gt;</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&amp;nbsp; frequency and displacement generated by the accoustic transducer</td></tr><tr><td>&lt;strong&gt;Mouse macro system&lt;/strong&gt;</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>&lt;strong&gt;Nematode growth media plate&lt;/strong&gt;</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 &lt;em&gt;E. coli&lt;/em&gt; 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>&lt;strong&gt;Nonlocalized vibration device&lt;/strong&gt;</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>&lt;strong&gt;Noninvasive calcium imaging&lt;/strong&gt;</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>&lt;strong&gt;Plasmid&lt;/strong&gt;</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&amp;nbsp; the &lt;em&gt;flp&lt;/em&gt;-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&amp;nbsp; the &lt;em&gt;flp&lt;/em&gt;-18 promoter</td></tr><tr><td>&lt;strong&gt;Tracking software&lt;/strong&gt;</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...

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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. ...

calcium-imaging-freely-behaving-caenorhabditis-elegans-with-well

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

2.6K Views.  Hiroshima University. 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.

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

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

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.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

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.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Neuroscience

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Developmental Biology

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans

Source: Turek, M. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/52742/agarose-microchambers-for-long-term-calcium-imaging-caenorhabditis">Agarose Microchambers for Long-term Calcium Imaging of&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp.&#160;(2015).
This video introduces the concepts behind calcium imaging and includes an example protocol for time-lapse imaging of worms contained in molded agarose wells.

Source: Turek, M. et al. Agarose Microchambers for Long-term Calcium Imaging of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2015).
This video ...

JoVE Encyclopedia of Experiments

Biology

Caenorhabditis elegans (worm)

Microscopy

Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans

Source: Ravi, B. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56911">Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for calcium imaging in individual neurons of behaving Caenorhabditis elegans using genetically encoded fluorescent reporter proteins. It outlines the steps involved in imaging worm movement and measuring reporter fluorescence ratios from neurons to correlate intracellular calcium levels with neuronal activity and behavioral changes during egg-laying.

1. Strains, Culture Media, and Mounting of Animals

	Grow&#160;C. elegans&#160;worms at 20 &#176;C on standard 60-mm Nematode Growth Medium (NGM) agar ...

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

Summary

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Chapters in this video

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In vivo Neuronal Calcium Imaging in C. elegans

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Imaging Dendritic Spines in Caenorhabditis elegans

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans

Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans