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 ...

<ol>
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A., Richmond, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sarco(endo)plasmic+reticulum+calcium+ATPase+SCA-1+regulates+the+Caenorhabditis+elegans+nicotinic+acetylcholine+receptor+ACR-16.">The sarco(endo)plasmic reticulum calcium ATPase SCA-1 regulates the Caenorhabditis elegans nicotinic acetylcholine receptor ACR-16.</a> Cell Calcium. 72, 104-115 (2018).</li><li>Wang, Z., Saifee, O., Nonet, M. L., Salkoff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLO-1+Potassium+Channels+Control+Quantal+Content+of+Neurotransmitter+Release+at+the+C.+elegans+Neuromuscular+Junction.">SLO-1 Potassium Channels Control Quantal Content of Neurotransmitter Release at the C. elegans Neuromuscular Junction.</a> Neuron. 32, 867-881 (2001).</li><li>Abraham, L. S., Oh, H. J., Sancar, F., Richmond, J. E., Kim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Alpha-Catulin+Homologue+Controls+Neuromuscular+Function+through+Localization+of+the+Dystrophin+Complex+and+BK+Channels+in+Caenorhabditis+elegans.">An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans.</a> PLoS Genetics. 6 (8), 1-13 (2010).</li><li>Gourgou, E., Chronis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+induced+oxidative+stress+affects+ASH+neuronal+function+and+behavior+in+C.+elegans.">Chemically induced oxidative stress affects ASH neuronal function and behavior in C. elegans.</a> Scientific Reports. 6, 1-9 (2016).</li><li>Zahratka, J. A., Williams, P. D. E., Summers, P. J., Komuniecki, R. W., Bamber, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+differentially+modulates+Ca2++transients+and+depolarization+in+a+C.+elegans+nociceptor.">Serotonin differentially modulates Ca2+ transients and depolarization in a C. elegans nociceptor.</a> Journal of Neurophysiology. 113 (4), 1041-1050 (2015).</li><li>Kerr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+calcium+transients+in+neurons+and+pharyngeal+muscle+of+C.+elegans.">Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans.</a> Neuron. 26 (3), 583-594 (2000).</li><li>Nekimken, A. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>all-trans 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, sca-1(tm5339); [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</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, slo-1 (eg142); [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</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, [zxIs6{Punc17::chop-2 
			(h134R)::yfp,lin-15(+)}; 
			Pmyo3::RCaMP35]</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...

Watch this Scientific Journal Video about In Vivo Calcium Imaging in C. elegans Body Wall Muscles at JoVE.com

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 ...

This protocol provides two methods to mobilize C.Elegans to perform in vivo calcium imaging of body wall muscles. This method can help answer questions related to calcium homeostasis and calcium handling, and an accessible synapse in genetically tractable organism. This technique can provide a better understanding of the mechanisms regulating muscle calcium homeostasis, and can, therefore, be important in identifying conditions or molecular pathways that impact excitation contraction coupling through the misregulation of calcium cycling.These methods can provide insight into areas of research including, but not limited too, neurobiology, developmental biology, and cell biology. These techniques could be adapted to study a variety of cell types and C.elegans, other related nematodes as well as filsafat larvae. Visual demonstration of this method is critical so the experimenter can understand the dissection technique, and be able to accurately assess calcium transients from properly immobilized animals.Start by setting up the microscope for fluorescent imaging. Use a scientific CMOS camera capable of full frame imaging at 100 hertz in order to track rapid changes in calcium levels. Control camera acquisition and LED fluorescent excitation with a micromanager software running in ImageJ.Use an opensource electronic platform plug-in which connects to an external opensource microcontroller board to manage the external timing pulses that control fluorescent excitations. To control the timing logic, activate the micromanager acquisition protocol in the software according to manufacturers instructions. Use two LEDs to stimulate channel rhodopsin with blue light and record RCaMP changes.Activate channel rhodopsin with am LED with the peak admission wavelength of 470 nanometers and a bandpass filter, and excite RCaMP with an LED with a peak admission wavelength of 594 nanometers and a bandpass filter. Co-illuminate both LEDs and transmit the light to the same optical path using a dichroic beam combiner. Control the timing of the LED illumination with a solid state switches controlled by TTL signals according to manuscript directions, and set the LED intensity with the current controlled load noise linear power supplies, then program the blue light simulation protocol into a simulator.In this experiment turn on the blue light simulation after two seconds of capturing RCaMP fluorescence only and use a train of five, two millisecond blue light pulses with 50 millisecond intervals to activate channel rhodopsin. If using the dissection preparation, perform C.elegans dissection in low light. Place the animals in the dissection dish with the silicone coated coverslip base that is filled with a one millimole of calcium extracellular solution prepared according to manuscript directions.Glue down the 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 and worm interface using glass needles. Use a mouth pipette to clear the internal viscera from the worm cavity, then glue down the cuticle flap of the animal to expose the ventral medial body wall muscles for imaging.If using the nano-bead preparation, make a molten 5%Agra solution using distilled water to a final volume of 100 milliliters. Use a Pasteur pipette to place a drop of molten-Agra solution onto a glass slide and immediately place a second glass slide over the top using gently pressure to create an even Agra's pad. Remove the top slide and at approximately four microliters of polystyrene nano-beads to the middle of the pad.In low light add four to six C.elegans into the nano-beads solution, making sure that the animals do not lay on top of each other, and carefully place a coverslip on top. When when ready to image, place the prepared slide or dissection dish on the microscope and find and focus on a worm using 10 times magnification and dim bright field illumination. Switch to 60 times magnification in RCaMP fluorescent excitation to identify a ventrum medial body wall muscle that is anterior to the vulva and in the correct vocal plane.Then change the image pathway from the eyepiece to the camera by pulling out the toggle and clicking live within the data acquisition software. Click the ORI button in the data acquisition software, and create a box around the muscle that is being focused on. On the stimulator, switch on the previously programmed blue light stimulation pathway, then click Acquire on the imaging software to capture the image.When C.elegans are grown on all-trans retinal, successfully incorporating retinal and activating the channel rhodopsin, a calcium transient can be evoked. If animals are not exposed to all-trans retinal no muscle calcium transient is triggered. This protocol was used to investigate loss of function sca-1 mutants, which impact the sarcoplasmic reticulum calcium pump encoded by the sca-1 gene.But, the dissection preparation increases in baseline levels of RCaMP fluorescence were observed in the mutant compared to the control, suggesting that the mutation needs the elevated levels of resting cytoplasmic calcium. There was a significant decrease in peak calcium levels in the sca-1 mutants as compared to the control. However, no change was seen in the rised peak time or the half decay time.Importantly, similar results can be observed using the less technically challenging nano-bead preparation. Loss of function slo-mutants, which disrupt the calcium activated big potassium channels were evaluated in order to investigate mutants that indirectly impact calcium handling in C.elegans. When baseline levels of RCaMP were measured the mutants displayed increased fluorescence compared to the controls.When evaluating the kinetics of the calcium transient no changes in peak calcium levels were seen. The slo-1(eg142)mutants, however, exhibited a significantly increased rise to peak time. The most important step to remember when completing the procedure is to ready experimental animals in all-trans retinal.Without this step the channel rhodopsin will be inactive, preventing light induced calcium transients from being triggered. Following this procedure, electrophysiology and behavioral analysis can be done to evaluate the functional impact of any observed changes in calcium homeostasis. Using the immobilization methods outlined here, paves the way for further exploration of calcium dynamics and demonstrates that comparable results can be observed in response to optogenetic neuronal stimulation and capture of muscle calcium transients using the far less challenging bead immobilization technique.

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

Research

JoVE Journal

Biochemistry

7.5K visualizações.  University of Illinois at Chicago. Este protocolo fornece dois métodos para mobilizar C.Elegans para realizar imagens in vivo de cálcio dos músculos da parede corporal. Este método pode ajudar a responder perguntas relacionadas à homeostase de cálcio e manuseio de cálcio, e uma sinapse acessível em organismo geneticamente tratável. Essa técnica pode proporcionar uma melhor compreensão dos mecanismos que regulam a homeostase do cálcio muscular e, portanto, pode ser importante na identificação de condições ou via...