The authors thank Dr. Alexander Gottschalk for ZX1659, the RCaMP and channelrhodopsin containing worm strain, Dr. Hongkyun Kim for the slo-1(eg142) worm strain, and the National Bioresource Project for the sca-1(tm5339) worm strain.

1. Microscope setup
<ol>
	<li>Use a compound microscope with fluorescence capabilities. For this study, data were collected on an upright microscope (Table of Materials) fitted with LEDs for excitation.</li>
	<li>In order to properly visualize fluorescence changes in body wall muscles, use a high magnification objective.
	<ol>
		<li>For dissected preparations, use a 60x NA 1.0 water immersion objective (Table of Materials).</li>
		<li>For preparations using nanobeads, use a 60x NA 1.4 oil immersion objective (Table of Materials). This magnification ensures sufficient resolution of muscles onto the camera sensor.</li>
	</ol>
	</li>
	<li>Use a high sensitivity camera attached to the microscope to capture images at a high frame rate in order to track rapid changes in calcium levels. For this study, image with an sCMOS system (Table of Materials) capable of full frame imaging at 100 Hz, as rise times for the Ca2+ signals can be as rapid as tens of milliseconds.</li>
	<li>Control camera acquisition and LED fluorescence excitation with a micro-manager software running in ImageJ10 according to the manufacturer&#39;s instructions (Table of Materials).</li>
	<li>Use an open-source electronic platform plugin, which connects an external open-source microcontroller board (Table of Materials), to manage the external timing pulses to control the fluorescence excitation. 
	NOTE: Digital outs are available from digital pins 8 to 13 providing output bits 0 to 5. These can be addressed as base 2 values of 1, 10, 100, etc. Serial port settings are indicated in Table 1 and available from the micro-manager website (Table of Materials). Firmware for the microcontroller board is similarly available at this website.</li>
	<li>To control the timing logic, activate the micromanager acquisition protocol in the software according to the manufacturer&#39;s instructions (Table of Materials). 
	NOTE: This simultaneously initiates a preset camera frame duration and the number of frames to be activated, as well as the logical shutter and preset amber LED for fluorescence excitation. In this case, the amber LED solid state switch is controlled by microcontroller bit 1 output through pin 9. The camera frames out TTL (back plate out 3 connector) triggers a stimulator. This precisely times the activation of the blue light LED for the activation of channelrhodopsin at a set time following the activation of the imaging sequence.</li>
	<li>To stimulate channelrhodopsin with blue light and record RCaMP changes, use two LEDs. Activate channelrhodopsin with an LED with a peak emission wavelength of 470 nm and a bandpass filter (455 to 490 nm) and excite RCaMP with an LED with a peak emission wavelength of 594 nm and a bandpass filter (570 to 600 nm).</li>
	<li>In order to simultaneously activate channelrhodopsin and capture changes in RCaMP fluorescent levels, co-illuminate both LEDs and transmit the light to the same optical path using a dichroic beam combiner.</li>
	<li>Control the timing of LED illumination with solid state switches (Table of Materials) controlled by TTL signals (maximum rated turn-on time, 1 ms, turn-off time 0.3 ms: 0 to 90% turn-on and turn off time of &#60; 100 &#181;s).</li>
	<li>Set the LED intensity with the current controlled low noise linear power supplies (Table of Materials).</li>
	<li>To ensure the precise timing of the blue light LED, activate it directly by TTL pulse to the solid-state relay from a stimulator. Program the blue light stimulation protocol into a stimulator (Table of Materials), which acts as a precise timer from the start of image acquisition to blue LED illumination. In this experiment, turn on blue light stimulation after 2 s of capturing RCaMP fluorescence only and use a train of 5, 2 ms blue light pulses with 50 ms interpulse intervals to activate channelrhodopsin. 
	NOTE: The delay before blue light pulses, the duration of the blue light pulses, the time between pulses, and the number of pulses in the train can all be set at this point and should reflect the specific parameters of experimental interest.</li>
</ol>
2. C. elegans sample preparation and data acquisition
<ol>
	<li>To optically stimulate presynaptic neurons, obtain animals expressing channelrhodopsin in excitatory cholinergic neurons, driven with the unc-17 promoter region, and RCaMP expressed in all body wall muscles driven with the myo-3 promoter region2. 
	NOTE: Only the use of animals with integrated transgenes and robust levels of fluorescence are recommended as the variable expression in the corresponding cell types may affect the reliability of data acquisition.</li>
	<li>Make a working stock of all-trans retinal by diluting the retinal powder (Table of Materials) in ethanol to create a final concentration of 100 mM and store at -20 &#176;C. This working stock will be stable for approximately one year.</li>
	<li>Create a stock of OP50 E. coli, grown in LB media, supplemented with all-trans retinal from the working stock made in step 2.2, at a final concentration of 80 &#181;M. The volume used for the OP50+retinal stock will be dependent on the number of plates necessary for the experiment.</li>
	<li>Seed nematode growth media (NGM) plates with approximately 300 &#181;L of the OP50+retinal stock and allow plates to dry overnight at room temperature in the dark.</li>
	<li>Grow C. elegans strains to the desired age in the dark on OP50+retinal NGM plates at 20 &#176;C. For this experiment, use adult worms.
	<ol>
		<li>For the experiment using the dissected preparation, only use gravid adult worms as performing the dissection on younger, smaller animals is extremely challenging. Leave the animals on the OP50-retinal plates for a minimum of 3 days for an effective channelrhodopsin activation.</li>
	</ol>
	</li>
	<li>If using the dissected preparation8,11 (Figure 1A), perform dissections in low light.
	<ol>
		<li>Place animals in a dissecting dish with a silicone-coated coverslip base that is filled with a 1 mM Ca2+ extracellular solution composed of 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 4 mM MgCl2, 10 mM glucose, 5 mM sucrose, and 15 mM HEPES (pH 7.3, -340 Osm).</li>
		<li>Glue down animals using the liquid topical skin adhesive with blue coloring along the dorsal side of the worm and make a lateral cuticle incision along the glue/worm interface using glass needles.</li>
		<li>Use a mouth pipette to clear the internal viscera from the worm cavity.</li>
		<li>Glue down the cuticle flap of the animal to expose the ventral medial body wall muscles for imaging.</li>
	</ol>
	</li>
	<li>If using the nanobead preparation (Figure 1B)
	<ol>
		<li>Make a molten 5% agarose solution using ddH2O to a final volume of 100 mL.</li>
		<li>Using a Pasteur pipette, place a drop of molten agarose solution onto a glass slide and immediately place a second glass slide over the top, perpendicular to the first, using gentle pressure to create an even agarose pad. Remove the top slide before use.</li>
		<li>Add approximately 4 &#181;L of polystyrene nanobeads (Table of Materials) to the middle of the agarose pad.</li>
		<li>In the low light, pick 4-6 C. elegans into the nanobead solution, making sure animals do not lay on top of each other, and carefully place a coverslip on the top.</li>
	</ol>
	</li>
	<li>Place the prepared slide or dissection dish onto the microscope, and find and focus on a worm using 10x magnification and dim bright field illumination.</li>
	<li>Switch to 60x magnification and RCaMP fluorescence excitation to identify a ventromedial body wall muscle that is anterior to the vulva and in the correct focal plane. 
	NOTE: Muscles anterior to the vulva are selected as they reflect muscles commonly stimulated in electrophysiology experiments.</li>
	<li>Change the image pathway from the eyepiece to the camera by pulling out the toggle and clicking Live within the data acquisition software (Table of Materials). 
	NOTE: Make sure the blue light stimulation pathway is turned off at this point to ensure that the channelrhodopsin does not get activated before capturing images.</li>
	<li>Focus the image within the data acquisition software using the microscope fine focus.</li>
	<li>Once the muscle is clearly in focus, turn off the live image by unclicking the Live button.</li>
	<li>Click the ROI button in the data acquisition software and create a box around the muscle being focused on.</li>
	<li>On the stimulator, switch on the blue light stimulation pathway that has been previously programmed in step 1.10.</li>
	<li>Click Acquire in the imaging software to capture the image through the CMOS camera. To do this, set the exposure time to 10 s with 1,000 frames and 2x binning.</li>
</ol>
3. Data analysis
<ol>
	<li>Open the data file in the imaging software (Table of Materials) and, using the Polygon Tool, outline the muscle of interest. This is the ROI.</li>
	<li>Go to Image | Stacks | Plot Z-axis profile and export the resulting data points into the spreadsheet software (Table of Materials). This function plots the ROI selection mean value versus the time point.</li>
	<li>Move the muscle ROI created with the Polygon tool outside of the animal to get a background fluorescence measurement using the steps outlined in 3.2. Export the resulting data into the spreadsheet workbook.</li>
	<li>In the spreadsheet workbook, subtract the background fluorescence values from the muscle fluorescence values at each time point. This provides the background subtracted fluorescent signal.</li>
	<li>Average the background subtracted fluorescence for the first 2 s of data points. This will give the baseline fluorescence measurement, F.</li>
	<li>Use the baseline fluorescence measurement to calculate the normalized fluorescence level at each time point. To do this, use the equation (&#916;F/F)+1. &#916;F represents (F(t)-F), where F(t) is the fluorescence measurement at any given time point and F is the baseline value. The +1 is added as a y-axis offset.</li>
	<li>Repeat steps 3.2-3.6 for each muscle image that is collected. Using single or multiple muscle cells per image is at the discretion of the researcher. The n of the experiment can be determined by performing a power analysis.</li>
	<li>Use the processed data from steps 3.2-3.6 to make a trace of the fluorescent values in graphing software according to the manufacturer&#39;s instructions (Table of Materials).</li>
	<li>From this trace, measure the kinetics of the calcium transient, such as rise to peak time and half decay time, using the tools provided in the graphing software as per the manufacturer&#39;s instructions.</li>
</ol>

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The authors have nothing to disclose.

GECIs are a powerful tool in C. elegans neurobiology. Previous work has utilized calcium imaging techniques to examine a wide variety of functions in both neurons and muscle cells, including sensory and behavioral responses, with varied methods of stimulation. Some studies have used chemical stimuli to trigger calcium transients in sensory ASH neurons22,23 or to induce calcium waves in pharyngeal muscles24. Another group utilized ...

This paper presents methods for in vivo calcium imaging of C. elegans body wall muscles using optogenetic neuronal stimulation. Pairing a muscle expressed genetically encoded calcium indicator (GECI) with blue light triggered neuronal depolarization and provides a system to clearly observe the evoked postsynaptic calcium transients. This avoids the use of electrical stimulation, allowing a non-invasive analysis of mutants affecting the postsynaptic calcium dynamics.
Single-fluorophore GECIs, such as GCaMP, uses a single fluorescent protein molecule fused to the M13 domain of myosin light chain kinase at its N-terminal end and calmodulin (CaM) at the C-terminus. Upon calcium binding, the CaM domain, which has a high affinity for calcium, undergoes a conformational change inducing a subsequent conformational change in the fluorescent protein leading to an increase in fluorescence intensity1. GCaMP fluorescence is excited at 488 nm, making it unsuitable to be used in conjunction with channelrhodopsin, which has a similar excitation wavelength of 473 nm. Thus, in order to couple calcium measurements with channelrhodopsin stimulation, the green fluorescent protein of GCaMP needs to be replaced with a red fluorescent protein, mRuby (RCaMP). Using the muscle expressed RCaMP, in combination with the cholinergic motor neuron expression of channelrhodopsin, permits studies at the worm neuromuscular junction (NMJ) with simultaneous use of optogenetics and functional imaging within the same animal2.
The use of channelrhodopsin bypasses the need for the electrical stimulation to depolarize the neuromuscular junctions of C. elegans, which can only be achieved in dissected preparations, thus making this technique both easier to employ and more precise when targeting specific tissues. For example, channelrhodopsin has been previously used in C. elegans to reversibly activate specific neurons, leading to either robust activation of excitatory or inhibitory neurons3,4. The use of light-stimulated depolarization also circumvents the issue of neuronal damage due to the direct electrical stimulation. This provides an opportunity to examine the effects of many different stimulation protocols, including sustained and repeated stimulations, on postsynaptic calcium dynamics4.
The transparent nature of C. elegans makes it ideal for the fluorescent imaging functional analysis. However, when stimulating the excitatory acetylcholine neurons at the NMJ, animals are expected to respond with an immediate muscle contraction4, making the immobilization of the worms critical in visualizing discrete calcium changes. Traditionally, pharmacological agents have been used to paralyze the animals. One such drug used is levamisole, a cholinergic acetylcholine receptor agonist5,6,7. Since levamisole leads to the persistent activation of a subtype of excitatory muscle receptors, this reagent is unsuitable for the study of the muscle calcium dynamics. The action of levamisole induces postsynaptic depolarization, elevating the cytosolic calcium, and occluding observations following presynaptic stimulation. To avoid the use of paralyzing drugs, we examined two alternative methods to immobilize C. elegans. Animals were either glued down and then dissected open to expose the body wall muscles, similar to the existing C. elegans NMJ electrophysiology method8, or nanobeads were used to immobilize intact animals9. Both procedures allowed for the reproducible measurements of the resting and evoked muscle calcium transients that were easily quantifiable.
The methods in this paper can be used to measure the baseline cytosolic calcium levels and transients in postsynaptic body wall muscle cells in C. elegans. Examples of data employing the two different immobilization techniques are given. Both techniques take advantage of optogenetics to depolarize the muscle cells without the use of electrical stimulation. These examples demonstrate the feasibility of this method in evaluating mutations that affect postsynaptic calcium handling in the worms and point out the pros and cons of the two immobilization approaches.

<table><tbody><tr><td>all-&lt;em&gt;trans&lt;/em&gt; retinal</td><td>Sigma-Aldrich</td><td>R2500</td><td>Necessary for excitation of channel rhodopsin</td></tr><tr><td>Amber LED</td><td></td><td></td><td>RCaMP illumination</td></tr><tr><td>Arduino UNO</td><td>Mouser</td><td>782-A000066</td><td>Controls fluorescence illumination</td></tr><tr><td>Blue LED</td><td></td><td></td><td>channelrhodopsin illumination</td></tr><tr><td>BX51WI microscope</td><td>Olympus</td><td></td><td>Fixed state compound microscope</td></tr><tr><td>Current controlled low noise linear power supply</td><td>Ametek</td><td>Sorenson</td><td>Controls LED intensity</td></tr><tr><td>Igor Pro</td><td>Wavemetrics</td><td>Wavemetrics.com</td><td>Graphing software</td></tr><tr><td>ImageJ</td><td>NIH</td><td>imagej.nih.gov</td><td>Image processing software</td></tr><tr><td>LUMFLN 60x water NA 1.4</td><td>Olympus</td><td></td><td>Water immersion objective for dissected preparation</td></tr><tr><td>Master-8 Stimulator</td><td>A.M.P.I</td><td></td><td>Master timer for image acquisition and LED illumination</td></tr><tr><td>Micro-Manager</td><td></td><td>micro-manager.org</td><td>Controls camera acquisition and LED excitiation</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td>Spreadsheet software</td></tr><tr><td>pco.edge 4.2 CMOS camera</td><td>pco.</td><td>4.2</td><td>High-speed camera</td></tr><tr><td>PlanApo N 60x oil NA 1.4</td><td>Olympus</td><td></td><td>Oil immersion objective for nanobead preparation</td></tr><tr><td>Polybead microspheres</td><td>Polysciences, Inc.</td><td>00876-15</td><td>For worm immobilization</td></tr><tr><td>solid state switches</td><td>Sensata Technologies</td><td>Crydom CMX100D6</td><td>Controls timing of LED illumination</td></tr><tr><td>Transgenic strain, &lt;em&gt;sca-1(tm5339)&lt;/em&gt;; [&lt;em&gt;zxIs6{Punc17::chop-2&lt;br/&gt; (h134R)::yfp,lin-15(+)};&lt;br/&gt; Pmyo3::RCaMP35&lt;/em&gt;]</td><td>Richmond Lab</td><td>SY1627</td><td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with SERCA mutant allele</td></tr><tr><td>Transgenic strain, &lt;em&gt;slo-1 (eg142)&lt;/em&gt;; [&lt;em&gt;zxIs6{Punc17::chop-2&lt;br/&gt; (h134R)::yfp,lin-15(+)};&lt;br/&gt; Pmyo3::RCaMP35&lt;/em&gt;]</td><td>Richmond Lab</td><td></td><td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line with calcium-activated BK potassium channel mutant allele</td></tr><tr><td>Transgeneic strain, [&lt;em&gt;zxIs6{Punc17::chop-2&lt;br/&gt; (h134R)::yfp,lin-15(+)};&lt;br/&gt; Pmyo3::RCaMP35&lt;/em&gt;]</td><td>Gottschalk Lab</td><td>ZX1659</td><td>Excitatory neuronal channelrhodopsin and body wall muscle RCaMP expressing worm line</td></tr></tbody></table>

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.

This technique evaluated changes in mutants thought to affect the calcium handling or muscle depolarization. Baseline fluorescence levels and fluorescent transients were visualized and resting cytosolic calcium and calcium kinetics within the muscle were evaluated. It is important that the animals were grown on all-trans retinal for at least three days to ensure the successful incorporation of retinal, thereby subsequently activating the channelrhodopsin (Figure 2</st...

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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.

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 ...

in-vivo-calcium-imaging-in-c-elegans-body-wall-muscles

Research

JoVE Journal

Biochemistry

7.6K Views.  University of Illinois at Chicago. Dit protocol biedt twee methoden om C.Elegans te mobiliseren om in vivo calciumbeeldvorming van lichaamswandspieren uit te voeren. Deze methode kan helpen bij het beantwoorden van vragen met betrekking tot calcium homeostase en calcium behandeling, en een toegankelijke synaps in genetisch goed bereikbaar organisme. Deze techniek kan een beter begrip bieden van de mechanismen die spiercalciumhomeostase reguleren en kan daarom belangrijk zijn bij het identificeren van omstandigheden of molecula...

Video: In Vivo Calcium Imaging in C. elegans Body Wall Muscles

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

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.

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.

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

Behavior

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

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 ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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 ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

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

Source: Turek, M. et al. <a href="https://www.jove.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).
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In Vivo Calcium Imaging in C. elegans Body Wall Muscles

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