Name: Subcellular Imaging of Neuronal Calcium Handling In Vivo (Video) | JoVE Journal
Uploaded: 2023-03-17T12:30:00.000+00:00
Duration: 7 min 14 s
Description: Learn subcellular calcium imaging in Caenorhabditis elegans neurons using genetically encoded indicators and high-resolution microscopy. This method enables precise measurement of compartmentalized calcium dynamics in vivo.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the National Institutes of Health (NIH) (R01- NS115947 awarded to F. Hoerndli). We would also like to thank Dr. Attila Stetak for the pAS1 plasmid.

1. Creating transgenic strains
<ol>
	<li>Using a cloning method of choice8,9, clone expression vectors to contain the Pflp-18 or Prig-3 promoter (for AVA-specific signal in the ventral nerve cord), followed by the Ca2+ indicator of choice, and then a 3&#39; UTR (see the discussion for more information)10. A list of plasmids and their sources can be found in Supplemental Table 1.</li>
	<li>Follow the established protocol for the creation of transgenic strains via the microinjection of a DNA mix (&#60;100 ng/&#956;L; see Supplemental Table 1 for the concentrations of each plasmid) containing the Ca2+ indicator transgene (~20 ng/&#956;L) and the rescue DNA lin-15+ (selection marker) into the gonad of a 1 day old adult C. elegans10 (genotype: lin-15(n765ts); phenotype: multi-vulva)10,11. 
	NOTE. Maintain lin-15(n765ts) parents at 15 &#176;C to suppress temperature-sensitive mutation.</li>
	<li>Maintain the injected parents at 20 &#176;C until the F1 progeny reach adulthood to allow for transgenic selection using the absence of the multi-vulva phenotype.</li>
	<li>Clonally passage13 adult F1 progeny that do not have the multi-vulva phenotype as this indicates the expression of the extrachromosomal array containing the Ca2+ indicator11.</li>
	<li>Assess the expression of the Ca2+ indicator in the F2 or F3 progeny that do not have the multi-vulva phenotype by mounting and imaging 1 day old adults as described later in section 3. 
	NOTE. A relatively high expression of the indicator is required for the rapid acquisition of images as described here (see the discussion for more detail).</li>
	<li>Perform experiments on subsequent generations of the transgenic strains with an optimal expression of the Ca2+ indicator (see the discussion for more detail), maintaining the strains under standard conditions (20 &#176;C on NGM plates)12.</li>
</ol>
2. Optical setup
<ol>
	<li>Use a microscope capable of long-term time-lapse imaging. 
	NOTE. The representative data were acquired using an inverted spinning disk confocal microscope equipped with 488 nm and 561 nm excitation lasers.</li>
	<li>Use a low-magnification objective (i.e., 10x/0.40) for the crude localization of the worm.</li>
	<li>To achieve subcellular resolution, switch to and localize the neurons using a high-magnification objective. 
	NOTE. A 100x/1.40 NA oil objective was used to acquire the representative data in this study.</li>
	<li>Acquire the images using an ultra-sensitive camera capable of rapid image acquisition (&#62;50 fps).</li>
	<li>Use a standard emission filter for the Ca2+ indicator of choice (i.e., a 525 nm &#177; 50 nm emission filter for GCaMP6f and mitoGCaMP).</li>
	<li>Add a Z-drift corrector (ZDC) for the acquisition of image streams longer than 10 s, as the desiccation of the agar pad during the imaging session causes the neurite to drift out of focus within several seconds. 
	NOTE. If a microscopy set-up does not allow for ZDC, or if long (&#62;20 min) imaging periods are desired, then apply a border of silicon grease or melted petroleum jelly around the agar pad to slow the desiccation.</li>
</ol>
3. Preparation of worms for imaging
<ol>
	<li>Preparing the agar pads
	<ol>
		<li>Make 3 mL of 10% agar by dissolving molecular grade agar in M912 in a 13 mm x 100 mm glass culture tube and microwaving for several seconds (see Table of Materials). 
		NOTE. Molten agar can be kept on a heat block for up to 1 h or can be made fresh each time agar pads are needed.</li>
		<li>Place a microscope slide between two additional slides that each have two layers of laboratory tape (see Figure 1A-i).</li>
		<li>Cut the tip of a 1,000 &#956;L pipette tip (without a filter), and use it to pipette a small drop of agar onto the center coverslip (Figure 1A-i).</li>
		<li>Flatten the agar by pressing another slide down on top of the agar (Figure 1A-ii).</li>
		<li>After cooling, cut the agar into a small disc using the opening of a 13 mm x 100 mm glass culture&#160;tube (Figure 1A-iii), and then remove the surrounding agar.</li>
	</ol>
	</li>
	<li>Preparing the worm-rolling solution
	<ol>
		<li>Dissolve muscimol powder in M9 to create a 30 mM stock. Separate into 50 &#956;L aliquots, and store at 4 &#176;C.</li>
		<li>Thaw a new aliquot of 30 mM muscimol every 3-5 days (and store at 20 &#176;C while in use).</li>
		<li>Dilute a 30 mM muscimol stock in a 1:1 ratio with polystyrene beads to make the rolling solution.</li>
	</ol>
	</li>
	<li>Positioning a worm for imaging
	<ol>
		<li>Place 1.6 &#956;L of the rolling solution onto the center of the agar pad. 
		NOTE: The amount of liquid should be adjusted for imaging younger (less) or older animals (more).</li>
		<li>Using the preferred worm pick (i.e., a glass or platinum wire pick)13, transfer a worm of the desired age without the multi-vulva phenotype11 into the rolling solution on the agar pad (Figure 1A-iv). 
		NOTE. The representative data are from 1 day old hermaphrodites (GCaMP6f strain = FJH185; mitoGCaMP strain; FJH597; see Supplemental Table 1), which can be identified by the presence of only one row of eggs. See the discussion for more detail on working with worms of different ages.</li>
		<li>Wait for ~5 min for the muscimol to reduce the worm movement, and then drop a 22 mm x 22 mm coverslip on top of the agar pad, physically restricting the worm movement.</li>
		<li>For imaging neurites in the ventral nerve cord, roll the worm into the orientation shown in Figure 1B,C by lightly sliding the coverslip. 
		NOTE. To visualize the ventral nerve cord, position the worm with the head upward, and roll the worm until the intestine is on the right side of the proximal portion and the left for the distal portion of the worm (from the viewer&#39;s perspective). Invert this orientation (Figure 1C) for imaging neurites in the dorsal nerve cord.</li>
	</ol>
	</li>
	<li>Mounting the worm on a microscope
	<ol>
		<li>Place a drop of immersion oil onto the coverslip before mounting it onto the microscope stage.</li>
		<li>Find the worm using a low-magnification objective (10x) objective in brightfield.</li>
		<li>Switch to the 100x objective, and adjust the focus.</li>
		<li>Locate the AVA neurite using the illumination of the GCaMP or mitoGCaMP with the 488 nm imaging laser. 
		NOTE: Use small adjustments to prevent squishing the worm or pulling off the coverslip.</li>
	</ol>
	</li>
</ol>
4. Acquisition of high-resolution image streams
<ol>
	<li>In vivo GCaMP imaging
	<ol>
		<li>Set the exposure time to 20 ms.</li>
		<li>Adjust the imaging laser and acquisition settings until the basal GCaMP fluorescence is in the mid-range of the camera&#39;s dynamic range14. Refer to the discussion for suggestions on optimizing the imaging parameters using other microscopy setups. 
		&#8203;NOTE. For the optical setup used in this study, set the 488 nm imaging laser to the following output settings: laser power = 50% and attenuation = 1. Modify the additional acquisition settings as follows: EM gain = 200 and pre-amplifier gain = 1.</li>
		<li>After the AVA neurite is located using the GCaMP fluorescence at 100x magnification, set the ZDC (see Table of Materials) to a range of &#177;30 &#956;m, and initiate continuous autofocusing. Manually correct the focal plane as needed before imaging.</li>
		<li>Acquire a stream of images at 100x magnification. Durations as long as 10 min can be achieved with the setup used in this study.</li>
	</ol>
	</li>
	<li>In vivo mitoGCaMP imaging
	<ol>
		<li>Follow steps 4.1.1-4.1.2 as needed to acquire the basal mitoGCaMP fluorescence in the mid-range for the camera&#39;s dynamic range14. 
		NOTE. For the optical set-up used in this study, set the 488 nm imaging laser to the following output settings: laser power = 20% and attenuation = 10. Modify the acquisition settings to the following: EM gain = 100 and pre-amplifier gain = 2.</li>
		<li>After the mitochondria in the AVA neurite are located using the mitoGCaMP fluorescence, set the ZDC as described above in step 3.3.</li>
		<li>Acquire a stream of images at 100x magnification.</li>
	</ol>
	</li>
</ol>
5. Image stream analysis
<ol>
	<li>Open the image stream in an appropriate image software for analyzing the pixel values in an image series (see Table of Materials).</li>
	<li>Using the Polygon Selection tool, define the subcellular region of interest (ROI) containing GCaMP or mitoGCaMP.</li>
	<li>Click on Analyze &#62; Tools &#62; ROI Manager. In the ROI Manager, click Add and then More &#62; Multi Measure. In the Multi Measure window, click OK to automatically collect the average, minimum, and maximum pixel intensities of the ROI defined above for each frame of the image stream for further analysis. 
	NOTE. It is recommended that the average pixel intensity (Favg) is normalized to the minimum intensity (Fmin) for each ROI using the ratio Favg/Fmin to account for fluorescence differences due to positioning in the z-plane. Discard the image streams with worm movement during imaging since this would also affect the fluorescence measurements.</li>
</ol>

<ol><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+calcium+signaling%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neuronal calcium signaling.</a> Neuron. 21 (1), 13-26 (1998).</li>
<li>Brini, M., Calì, T., Ottolini, D., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+calcium+signaling%3A+Function+and+dysfunction%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Neuronal calcium signaling: Function and dysfunction.</a> Cellular and Molecular Life Sciences. 71 (15), 2787-2814 (2014).</li>
<li>Brini, M., Calì, T., Ottolini, D., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+calcium+homeostasis+and+signaling%5BTitle%5D)+AND+%22Metal+Ions+in+Life+Sciences%22%5BJournal%5D)">Intracellular calcium homeostasis and signaling.</a> Metal Ions in Life Sciences. 12, 119-168 (2013).</li>
<li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+inositol+trisphosphate%2Fcalcium+signaling+pathway+in+health+and+disease%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">The inositol trisphosphate/calcium signaling pathway in health and disease.</a> Physiological Reviews. 96 (4), 1261-1296 (2016).</li>
<li>Schrank, S., Barrington, N., Stutzmann, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium-handling+defects+and+neurodegenerative+disease%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectives+in+Biology%22%5BJournal%5D)">Calcium-handling defects and neurodegenerative disease.</a> Cold Spring Harbor Perspectives in Biology. 12 (7), 035212(2020).</li>
<li>Pchitskaya, E., Popugaeva, E., Bezprozvanny, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+signaling+and+molecular+mechanisms+underlying+neurodegenerative+diseases%5BTitle%5D)+AND+%22Cell+Calcium%22%5BJournal%5D)">Calcium signaling and molecular mechanisms underlying neurodegenerative diseases.</a> Cell Calcium. 70, 87-94 (2018).</li>
<li>Jadiya, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+mitochondrial+calcium+efflux+contributes+to+disease+progression+in+models+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Impaired mitochondrial calcium efflux contributes to disease progression in models of Alzheimer's disease.</a> Nature Communications. 10 (1), 3885(2019).</li>
<li>Zeiser, E., Frøkjær-Jensen, C., Jorgensen, E., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MosSCI+and+gateway+compatible+plasmid+toolkit+for+constitutive+and+inducible+expression+of+transgenes+in+the+C.+elegans+germline%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline.</a> PLoS One. 6 (5), 20082(2011).</li>
<li>Zhu, B., Cai, G., Hall, E. O., Freeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-FusionTM+assembly%3A+Seamless+engineering+of+multidomain+fusion+proteins%2C+modular+vectors%2C+and+mutations%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">In-FusionTM assembly: Seamless engineering of multidomain fusion proteins, modular vectors, and mutations.</a> Biotechniques. 43 (3), 354-359 (2007).</li>
<li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transformation+and+Microinjection%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Transformation and Microinjection.</a> WormBook. , (2006).</li>
<li>Giordano-Santini, R., Dupuy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selectable+genetic+markers+for+nematode+transgenesis%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Selectable genetic markers for nematode transgenesis.</a> Cellular and Molecular Life Sciences. 68 (11), 1917-1927 (2011).</li>
<li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+C.+elegans%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Maintenance of C. elegans.</a> WormBook. , (2006).</li>
<li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+C.+elegans%3A+Transferring+worms+grown+on+NGM+plates%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Maintenance of C. elegans: Transferring worms grown on NGM plates.</a> WormBook. , (2006).</li>
<li>Ogama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+beginner%27s+guide+to+improving+image+acquisition+in+fluorescence+microscopy%5BTitle%5D)+AND+%22The+Biochemist%22%5BJournal%5D)">A beginner's guide to improving image acquisition in fluorescence microscopy.</a> The Biochemist. 42 (6), 22-27 (2020).</li>
<li>Mellem, J. E., Brockie, P. J., Madsen, D. M., Maricq, A. v <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Action+potentials+contribute+to+neuronal+signaling+in+C.+elegans%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Action potentials contribute to neuronal signaling in C. elegans.</a> Nature Neuroscience. 11 (8), 865-867 (2009).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+and+sensitive+GCaMP+calcium+indicators+for+imaging+neural+populations%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Fast and sensitive GCaMP calcium indicators for imaging neural populations.</a> bioRxiv. , (2021).</li>
<li>Kerruth, S., Coates, C., Dürst, C. D., Oertner, T. G., Török, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+kinetic+mechanisms+of+fast-decay+red-fluorescent+genetically+encoded+calcium+indicators%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The kinetic mechanisms of fast-decay red-fluorescent genetically encoded calcium indicators.</a> Journal of Biological Chemistry. 294 (11), 3934-3946 (2019).</li>
<li>de Juan-Sanz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axonal+endoplasmic+reticulum+Ca2%2B+content+controls+release+probability+in+CNS+nerve+terminals%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals.</a> Neuron. 93 (4), 867-881 (2017).</li>
<li>Bhatla, N. C. elegans neural network. , Available from: 
 <a target="_blank" href="http://wormweb.org/neurainet#c=BAG&m=1">http://wormweb.org/neurainet#c=BAG&m=1</a> (2014).</li>
<li>Fung, W., Wexler, L., Heiman, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-type-specific+promoters+for+C.+elegans+glia%5BTitle%5D)+AND+%22Journal+of+Neurogenetics%22%5BJournal%5D)">Cell-type-specific promoters for C. elegans glia.</a> Journal of Neurogenetics. 34 (3-4), 335-346 (2020).</li>
<li>Ali, F., Kwan, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interpreting+in+vivo+calcium+signals+from+neuronal+cell+bodies%2C+axons%2C+and+dendrites%3A+a+review%5BTitle%5D)+AND+%22Neurophotonics%22%5BJournal%5D)">Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review.</a> Neurophotonics. 7 (1), 011402(2019).</li>
<li>Tian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+neural+activity+in+worms%2C+flies+and+mice+with+improved+GCaMP+calcium+indicators%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nature Methods. 6 (12), 875-881 (2009).</li>
<li>Mank, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetically+encoded+calcium+indicator+for+chronic+in+vivo+two-photon+imaging%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">A genetically encoded calcium indicator for chronic in vivo two-photon imaging.</a> Nature Methods. 5 (9), 805-811 (2008).</li>
<li>Birkner, A., Tischbirek, C. H., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+deep+two-photon+calcium+imaging+in+vivo%5BTitle%5D)+AND+%22Cell+Calcium%22%5BJournal%5D)">Improved deep two-photon calcium imaging in vivo.</a> Cell Calcium. 64, 29-35 (2017).</li>
<li>Ryan, K. C., Laboy, J. T., Norman, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deregulation+of+mitochondrial+calcium+handling+due+to+presenilin+loss+disrupts+redox+homeostasis+and+promotes+neuronal+dysfunction%5BTitle%5D)+AND+%22Antioxidants%22%5BJournal%5D)">Deregulation of mitochondrial calcium handling due to presenilin loss disrupts redox homeostasis and promotes neuronal dysfunction.</a> Antioxidants. 11 (9), 1642(2022).</li>
<li>Yang, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcellular+imaging+of+voltage+and+calcium+signals+reveals+neural+processing+in+vivo%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Subcellular imaging of voltage and calcium signals reveals neural processing in vivo.</a> Cell. 166 (1), 245-257 (2016).</li>
<li>Takahashi, N., Oertner, T. G., Hegemann, P., Larkum, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+cortical+dendrites+modulate+perception%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Active cortical dendrites modulate perception.</a> Science. 354 (6319), 1587-1590 (2016).</li>
<li>Nicoletti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biophysical+modeling+of+C.+elegans+neurons%3A+Single+ion+currents+and+whole-cell+dynamics+of+AWCon+and+RMD%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Biophysical modeling of C. elegans neurons: Single ion currents and whole-cell dynamics of AWCon and RMD.</a> PLoS One. 14 (7), 0218738(2019).</li>
<li>Church, P. J., Stanley, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+L-type+calcium+channel+conductance+with+physiological+levels+of+calcium+in+chick+ciliary+ganglion+neurons%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons.</a> The Journal of Physiology. 496, 59-68 (1996).</li>
<li>O'Hare, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compartment-specific+tuning+of+dendritic+feature+selectivity+by+intracellular+Ca+2%2B+release%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Compartment-specific tuning of dendritic feature selectivity by intracellular Ca 2+ release.</a> Science. 375 (6586), (2022).</li>
<li>Mclntire, S. L., Jorgensen, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+required+for+GABA+function+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genes required for GABA function in Caenorhabditis elegans.</a> Nature. 364 (6435), 334-337 (1993).</li>
<li>Doser, R. L., Amberg, G. C., Hoerndli, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactive+oxygen+species+modulate+activity-dependent+AMPA+receptor+transport+in+C.+elegans%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Reactive oxygen species modulate activity-dependent AMPA receptor transport in C. elegans.</a> The Journal of Neuroscience. 40 (39), 7405-7420 (2020).</li>
<li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+long+Stokes+shift+red+fluorescent+Ca2%2B+indicator+protein+for+two-photon+and+ratiometric+imaging%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.</a> Nature Communications. 5, 5262(2014).</li>
<li>Cho, J. -H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+GCaMP-R+family+of+genetically+encoded+ratiometric+calcium+indicators%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">The GCaMP-R family of genetically encoded ratiometric calcium indicators.</a> ACS Chemical Biology. 12 (4), 1066-1074 (2017).</li>
<li>Smith, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+light+sheet+fluorescence+microscopy+protocol+for+Caenorhabditis+elegans+larvae+and+adults%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults.</a> Frontiers in Cell and Developmental Biology. 10, 1012820(2022).</li>
<li>Müller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+and+calcium+regulation+as+basis+of+neurodegeneration+associated+with+aging%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Mitochondria and calcium regulation as basis of neurodegeneration associated with aging.</a> Frontiers in Neuroscience. 12, 470(2018).</li>
<li>Nikoletopoulou, V., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+homeostasis+in+aging+neurons%5BTitle%5D)+AND+%22Frontiers+in+Genetics%22%5BJournal%5D)">Calcium homeostasis in aging neurons.</a> Frontiers in Genetics. 3, 200(2012).</li>
<li>Chen, C. -H., Chen, Y. -C., Jiang, H. -C., Chen, C. -K., Pan, C. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+aging%3A+Learning+from+C.+elegans%5BTitle%5D)+AND+%22Journal+of+Molecular+Signaling%22%5BJournal%5D)">Neuronal aging: Learning from C. elegans.</a> Journal of Molecular Signaling. 8 (1), 14(2013).</li>
<li>Saberi-Bosari, S., Huayta, J., San-Miguel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidic+platform+for+lifelong+high-resolution+and+high+throughput+imaging+of+subtle+aging+phenotypes+in+C.+elegans%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">A microfluidic platform for lifelong high-resolution and high throughput imaging of subtle aging phenotypes in C. elegans.</a> Lab on a Chip. 18 (20), 3090-3100 (2018).</li>
<li>Sridhar, N., Fajrial, A. K., Doser, R. L., Hoerndli, F. J., Ding, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+acoustic+wave+microfluidics+for+repetitive+and+reversible+temporary+immobilization+of+C.+elegans%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Surface acoustic wave microfluidics for repetitive and reversible temporary immobilization of C. elegans.</a> Lab on a Chip. 22 (24), 4882-4893 (2022).</li>
</ol>

The authors declare no competing interests.

The first consideration when implementing the method described involves the selection of a Ca2+ indicator with ideal characteristics for the given research question. Cytoplasmic Ca2+ indicators typically have a high affinity for Ca2+,&#160;and the sensitivity of these indicators to Ca2+ is inversely related to the kinetics (on/off rate)16,17. This means that either Ca2+ sensitivity or kinetics will need to be...

Calcium ions (Ca2+) are highly versatile carriers of information in many cell types. In neurons, Ca2+ is responsible for the activity-dependent release of neurotransmitters, the regulation of cytoskeletal motility, the fine-tuning of metabolic processes, as well as many other mechanisms required for proper neuronal maintenance and function1,2. To ensure effective intracellular signaling, neurons must maintain low basal Ca2+ levels in their cytoplasm3. This is accomplished by cooperative Ca2+ handling mechanisms, including the uptake of Ca2+ into organelles such as the endoplasmic reticulum (ER) and mitochondria. These processes, in addition to the arrangement of Ca2+-permeable ion channels in the plasma membrane, result in heterogeneous levels of cytoplasmic Ca2+ throughout the neuron.
Ca2+ heterogeneity during rest and neuronal activation allows for the diverse, location-specific regulation of Ca2+-dependent mechanisms1. One example of the concentration-specific effects of Ca2+ is the release of Ca2+ from the ER through inositol 1,4,5-trisphosphate (InsP3) receptors. Low Ca2+ levels in combination with InsP3 are required for the opening of the receptor&#39;s calcium-permeable pore. Alternatively, high Ca2+ levels both directly and indirectly inhibit the receptor4. The importance of Ca2+ homeostasis for proper neuronal function is supported by evidence suggesting that disrupted intracellular Ca2+ handling and signaling is an early step in the pathogenesis of neurodegenerative disorders and natural aging5,6. Specifically, abnormal Ca2+ uptake and release by the ER and mitochondria are linked to the onset of neuronal dysfunction in Alzheimer&#39;s disease, Parkinson&#39;s disease, and Huntington&#39;s disease6,7.
The study of Ca2+ dyshomeostasis during natural aging or neurodegeneration requires the longitudinal observation of Ca2+ levels with subcellular resolution in a living, intact organism in which the native cellular architecture (i.e., the arrangement of synapses and distribution of ion channels) is maintained. To this end, this protocol provides guidance on the use of two readily available, genetically encoded Ca2+ sensors for recording Ca2+ dynamics in vivo with high spatial and temporal resolution. The representative results acquired using&#160;the described method in C. elegans demonstrate how the expression of Ca2+ indicators in the cytoplasm or mitochondrial matrix of single neurons can allow for the rapid acquisition of fluorescent images (up to 50 Hz) that illustrate the Ca2+ dynamics with the additional capability of discerning the Ca2+ levels within single spine-like structures and individual mitochondria.

<table><tbody><tr><td>100x/1.40 Oil objective&amp;nbsp;&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td>&amp;nbsp;UPlanSApo</td></tr><tr><td>10x/0.40 Objective&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td>&amp;nbsp;UPlanSApo</td></tr><tr><td>22 mm x 22 mm Cover glass&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>48366-227&amp;nbsp;</td><td></td></tr><tr><td>Agarose SFR&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>J234-100G&amp;nbsp;</td><td></td></tr><tr><td>Beam homogenizer</td><td>Andor Technologies</td><td></td><td>Borealis upgrade to CSU-X1</td></tr><tr><td>CleanBench laboratory table&amp;nbsp;</td><td>TMC&amp;nbsp;</td><td></td><td>With vibration control</td></tr><tr><td>Disposable culture tubes</td><td>VWR&amp;nbsp;</td><td>47729-572</td><td>&amp;nbsp;13 mm x 100 mm</td></tr><tr><td>Environmental chamber</td><td>Thermo Scientific</td><td>3940</td><td>Set to 20 &amp;deg;C</td></tr><tr><td>Filter wheel or slider</td><td>ASI</td><td></td><td>For 25 mm diameter filters</td></tr><tr><td>FJH 185</td><td>Caenorhabditis Genetics Center&amp;nbsp;</td><td>FJH 185</td><td>Worm strain</td></tr><tr><td>FJH 597</td><td>Caenorhabditis Genetics Center</td><td>FJH 597</td><td>Worm strain</td></tr><tr><td>GFP bandpass emission filter&amp;nbsp;</td><td>Chroma&amp;nbsp;</td><td></td><td>525 &amp;plusmn; 50 nm&amp;nbsp;(25 mm diameter)</td></tr><tr><td>ILE laser combiner&amp;nbsp;</td><td>Andor Technologies&amp;nbsp;</td><td></td><td>4 laser lines&amp;nbsp;</td></tr><tr><td>ILE solid state 488 nm laser</td><td>Andor Technologies&amp;nbsp;</td><td></td><td>50 mW</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>Version 1.52a</td></tr><tr><td>IX83 Spinning disk confocal microscope&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td>With Yokogawa CSU-X1 spinning disc</td></tr><tr><td>iXon Ultra EMCCD camera&amp;nbsp;</td><td>Andor Technologies&amp;nbsp;</td><td></td><td></td></tr><tr><td>Low auto-fluorescence immersion oil&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>Z-81226</td><td></td></tr><tr><td>MetaMorph&amp;nbsp;</td><td>Molecular Devices&amp;nbsp;</td><td></td><td>Version 7.10.1&amp;nbsp;</td></tr><tr><td>Microscope control box</td><td>Olympus</td><td></td><td>IX3-CBH</td></tr><tr><td>Muscimol&amp;nbsp;</td><td>MP Biomedical&amp;nbsp;/ Sigma</td><td>02195336-CF&amp;nbsp;</td><td></td></tr><tr><td>pAS1</td><td>AddGene</td><td>194970</td><td>Plasmid</td></tr><tr><td>pBSKS</td><td>Stratagene</td><td></td><td></td></tr><tr><td>pCT61</td><td></td><td></td><td>Plasmid available from Hoerndli/Maricq lab upon request</td></tr><tr><td>pJM23</td><td></td><td></td><td>Plasmid available from Hoerndli/Maricq lab upon request</td></tr><tr><td>pKK1&amp;nbsp;</td><td>AddGene&amp;nbsp;</td><td>194969</td><td>Plasmid</td></tr><tr><td>Polybead microspheres&amp;nbsp;</td><td>Polysciences Inc.&amp;nbsp;</td><td>00876-15</td><td>&amp;nbsp;0.094 &amp;micro;m</td></tr><tr><td>Stability chamber</td><td>Norlake Scientific</td><td>NSRI241WSW/8H</td><td>Set to 15 &amp;deg;C</td></tr><tr><td>Stage controller</td><td>ASI</td><td></td><td>With filter wheel control</td></tr><tr><td>Standard microscope slide</td><td>Premiere</td><td>9108W-E</td><td>75 mm x 25 mm x 1 mm</td></tr><tr><td>Touch panel controller</td><td>Olympus</td><td></td><td>I3-TPC</td></tr><tr><td>Z-drift corrector&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td>IX3-ZDC2</td></tr></tbody></table>

subcellular imaging of neuronal calcium handling in vivo

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.

These two protocols enable the rapid acquisition of differential Ca2+ levels within the subcellular regions and organelles of individual neurites in vivo with high spatial resolution. The first protocol allows for the measurement of cytoplasmic Ca2+ with high temporal and spatial resolution. This is demonstrated here using the cell-specific expression of GCaMP6f in the glutamatergic AVA command interneurons15, whose neurites run the entire length of the ventral nerve...

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 ...

subcellular-imaging-of-neuronal-calcium-handling-in-vivo

Research

JoVE Journal

Neuroscience

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Functional Calcium Imaging in Developing Cortical Networks

Source: Doser, R., et al. Subcellular Imaging of Neuronal Calcium Handling In Vivo. J. Vis. Exp. (2023)This video demonstrates in vivo imaging of subcellular calcium flux in the ventral nerve cord neurons of transgenic Caenorhabditis elegans strains. Two distinct calcium indicators are employed: a cytoplasmic indicator in one strain and a mitochondrial indicator in another. Neurons are observed under a fluorescence microscope to monitor real-time changes in fluorescence intensity within the...

Imaging Subcellular Calcium Dynamics in Neurons of Caenorhabditis 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.

In vivo Neuronal Calcium Imaging in C. elegans

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca2+ Imaging of the Enteric Nervous System

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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

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

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Learn subcellular calcium imaging in Caenorhabditis elegans neurons using genetically encoded indicators and high-resolution microscopy. This method enables precise measurement of compartmentalized calcium dynamics in vivo.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Explore More Videos