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>

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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 prioritized based on the phenomenon of interest. For subcellular compartments with relatively high basal Ca2+ levels, such as the ER, Ca2+ indicators with low Ca2+ affinity should be considered18. To generate recombinant DNA for the cell-specific expression of a Ca2+ indicator, the preferred cloning technique should be used to insert a cell-specific promoter19,20 followed by the Ca2+ indicator of choice and a 3&#39; UTR. Relatively high expression levels in the AVA neurons can be achieved by constructing a plasmid with the promoters Pflp-18 or Prig-3, a let-858, or unc-54 3&#39; UTR and microinjecting this plasmid at 15-25 ng/&#956;L (see Supplemental Table 1).
The successful microinjection of the plasmids containing the Ca2+ indicator and lin-15+ into the gonad of lin-15(n765ts) mutant worms will yield an F1 progeny that expresses the Ca2+ indicator and are rescued for the multi-vulva phenotype. Only a subset (30%-80%) of the F1 generation will transmit the synthetic DNA to the F2 generation. The expression of the Ca2+ indicator should be assessed in the rescued F2 or F3 progeny on plates with &#60;60% transmission of the synthetic DNA by mounting and imaging the fluorescence in 1 day old adults that are rescued for the multi-vulva phenotype. The ideal expression level is determined based on the properties and location of the Ca2+ indicator, as well as the biological question. If rapid image acquisition is desired, then relatively high expression is ideal. However, an extremely high expression of Ca2+ indicators has been found to cause subcellular mislocalization, as well as non-specific expression in other cells or tissues.
Genetically encoded Ca2+ indicators have been widely employed for in vivo analyses of Ca2+ in C. elegans, as well as in other model organisms. Some of these studies have achieved single-cell resolution monitoring of Ca2+ levels in the neuronal cell body21,22,23,24. Measurements of Ca2+ influx in neuronal sub-compartments have been achieved in vivo in C. elegans as well as vertebrate systems but only by recording the Ca2+ indicator activity in an entire neurite25,26 or populations of dendrites27. Although measuring Ca2+ activity in neuronal cell bodies and dendrites as a whole is reflective of the cell&#39;s spiking activity, it does not allow for the observation of the dynamics of Ca2+ influx (i.e., the direction or rate of propagation) or sub-threshold Ca2+ transients. Few in vivo studies have been able to discern Ca2+ handling in neurons to the spatial and temporal resolution demonstrated here. It should be noted that the temporal dynamics of Ca2+ influx in most C. elegans neurons28 appears to be slower than in vertebrate neurons29, so it is likely that this rapid imaging protocol would be insufficient for capturing similar temporal dynamics in vertebrate systems. However, recent advancements in two-photon microscopy allow for the ultra-fast (120 Hz), subcellular imaging of calcium in individual hippocampal dendrites in freely behaving mice30, which provides promise for achieving similar high-resolution Ca2+ imaging in vertebrates.
It is important to note that this non-ratiometric approach to in vivo Ca2+ imaging requires the use of muscimol, which inhibits the contraction of the body wall muscles, thus limiting the worm movement during imaging31. If there is excessive movement during imaging (consistent movement in more than ~20% of worms), a new rolling solution should be made with a newly thawed aliquot of muscimol, and the time for which a worm bathes in the rolling solution prior to rolling should be extended. Muscimol is a GABA receptor agonist, meaning it inhibits GABAergic neurons, including those that provide input to the AVA neurons. Additionally, many excitatory neurons, including AVA, express low levels of GABA receptors, so it is likely that the use of muscimol in this protocol decreases the neuronal activity. However, using this protocol, the overall spontaneous activation of AVA is reduced by only ~29% in the presence of muscimol (unpublished data). The activation of other neurons may be more drastically decreased by muscimol, and this can be tested by substituting muscimol for M9 in the rolling solution and conducting imaging in the neuronal cell body at low magnification32. An alternative to using muscimol would be to use a ratiometric Ca2+ indicator33,34, which would allow for fluorescence correction due to worm movement. The downside to this approach is that it requires an optical setup capable of dual-wavelength imaging and limits the ability to use a Ca2+ indicator in combination with many other fluorescent tools.
One major limitation of the microscopy system described is the reduction of emitted light by the spinning disc, which results in a loss of information. This system could be modified to allow for increased capture of emitted light by implementing a disc with larger-diameter pin holes. However, this alteration would decrease the axial (z-plane) resolution and increase the background fluorescence. Another possible microscopy setup for this purpose would be the fast-scanning light sheet microscope, which has been improved in recent years to allow for rapid (up to 50 Hz) imaging in situ and in vivo35.
Assessing the spatiotemporal dynamics of cytoplasmic and mitochondrial Ca2+ will allow for a better understanding of how local Ca2+ transients, and even subthreshold events, may trigger calcium-dependent signaling or modulate mitochondrial biology. This subcellular, in vivo Ca2+ imaging could be particularly useful for the study of synaptic plasticity, activity-dependent changes in neuronal metabolism, and the homeostatic modulation of neuronal excitability. Further investigation of how local, subcellular environments adapt around defined states of neuronal activity (i.e., spatially specific concentrations and lifetime of Ca2+) would provide insight into how Ca2+ dyshomeostasis leads to pathogenic changes in neuronal function. The ability to study this in a living, intact organism is especially useful as it enables longitudinal studies that would shed light on how and why Ca2+ homeostasis changes with natural aging and disease-related neurodegeneration. Applying this method to study aging and neurodegeneration is somewhat limited to the early biological ages in C. elegans (&#60;4 days of adulthood). Indeed, the physical manipulation during mounting and positioning the worms for imaging is difficult after about 5 days of adulthood when the worms become flaccid because of decreased muscle tone and cuticle integrity.
Rapid in vivo imaging of the dynamics and subcellular handling of Ca2+ will be instrumental in our understanding of how neuronal excitability and intracellular signaling are finely regulated spatially and temporally. This protocol could benefit a broad range of research and researchers in many areas due to the diversity of Ca2+-dependent processes and their central role in neuronal function. For instance, the application of this method in healthy neurons would provide insight into why elevated cytoplasmic Ca2+ during aging occurs in conjunction with impairments in synaptic plasticity, inefficient mitochondrial respiration, as well as other dysfunctions36,37.&#160;Additionally, this method could be used to investigate the causes of age-associated Ca2+&#160;dyshomeostasis38.&#160;However, the application of this method for questions related to aging and neurodegeneration is somewhat limited to the early stages of aging (&#60;5 days of adulthood)38. As mentioned above, the imaging of older adult worms (&#62;5 days of adulthood) is difficult using our rolling method, but advances in C. elegans microfluidics show promise for imaging with a similar spatial resolution for up to 12 days of adulthood39,40.

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

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 cord. The rapid acquisition of GCaMP6f fluorescence (50 Hz) enabled the detection of the directional propagation of Ca2+ influx (Figure 2A and Supplemental Video 1). The subcellular quantification of GCaMP6f fluorescence revealed that the onset of Ca2+ influx was delayed in a portion of the neurite just 10 &#956;m away (Figure 2B). Additionally, this protocol allowed for several observations of compartmentalized Ca2+ and subthreshold Ca2+ events. For instance, dendritic spine-like structures found along the AVA neurite were seen to have flashes in GCaMP6f fluorescence that occurred independently of neurite activation and did not lead to a propagation of Ca2+ influx (Figure 2C,D and Supplemental Video 2).
The second protocol aimed to rapidly measure the relative Ca2+ levels in the individual mitochondria in single neurites. Using a worm strain with AVA-specific expression of the mitochondrial matrix-localized Ca2+ indicator mitoGCaMP enabled the rapid detection of changes in mitoGCaMP fluorescence at the level of individual mitochondria within the AVA neurite. This imaging protocol revealed different modes of Ca2+ uptake into the mitochondria, at least in this neuron. First, the results shown in Figure 3A provide an example of the synchronous uptake of a relatively large amount of Ca2+ into a subset of mitochondria. More specifically, these data revealed that Ca2+ uptake into some mitochondria was synchronized (Mito1 and Mito2; Figure 3A,B and Supplemental Video 3), whereas neighboring mitochondria did not appear to take up Ca2+ (Mito3; Figure 3A,B). Similarly, subsets of mitochondria were seen to rapidly uptake and release small amounts of Ca2+. This also appeared to be synchronous (Mito1 and Mito3, Figure 3C,D and Supplemental Video 4), but again only for a subset of mitochondria (Mito 2, Figure 3C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64928/64928fig01.jpg" /> 
Figure 1: Mounting and rolling C. elegans for imaging the ventral nerve cord. (A) An illustration of the steps for preparing the agar pads and picking a single worm into a rolling solution. (B) An example of a worm position prior to it being repositioned for imaging. The red arrow indicates the need for a rightward roll of the worm, which can be achieved by sliding the coverslip to the right (blue arrow). Scale bar = 50 &#956;m. (C) The correct orientation needed for visualizing the ventral nerve cord. Note: The intestine is on the viewer&#39;s right side (using an upright microscope) in the proximal half and is then on the left side in the distal half. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/64928fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64928/64928fig02.jpg" /> 
Figure 2: Rapid acquisition of GCaMP6f fluorescence with subcellular resolution in AVA neurite and spine-like structures. (A) Representative images of GCaMP6f fluorescence at the AVAL and AVAR neurite bifurcation.&#160;Scale bar = 5 &#956;m. (B) Quantification of GCaMP6f fluorescence normalized to the minimum fluorescence (F) for that region (Fmin) for the proximal (orange) and distal (blue) regions defined in the bottom image in panel A. (C) Representative images of GCaMP6f fluorescence at a spine-like projection in the AVA neurite. (D) Normalized GCaMP6f fluorescence in the neurite (orange) and the spine-like projection (blue) from the corresponding regions in the bottom image of panel C. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/64928fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64928/64928fig03.jpg" /> 
Figure 3. Rapid imaging of calcium uptake into individual mitochondria within the AVA neurite. (A,C) Representative images of mitoGCaMP fluorescence over time.&#160;Scale bar = 50&#160;&#956;m. (B) Traces of normalized GCaMP fluorescence (F/Fmin) for the corresponding mitochondria shown in the bottom image of panel A. (D) F/Fmin over 40 s of imaging in the regions highlighted in the bottom image in panel C. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/64928fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video 1: GCaMP6f fluorescence in the AVA neurite. The video was&#160;rendered at 2x speed. Scale bar = 5 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/GCaMP_SingleActivation_02_500_20002XSPEED_best_GreenBlue_SB.avi" target="_blank">Please click here to download this Video.</a>
Supplemental Video 2: GCaMP6f fluorescence in a spine-like projection on the AVA neurite. The video was&#160;rendered at 2x speed. Scale bar = 5 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/GCaMP_SpineActivation_01_350_2350_2XSPEED_best_GreenBlue_SB.avi" target="_blank">Please click here to download this Video.</a>
Supplemental Video 3: MitoGCaMP fluorescence in the AVA neurite-synchronized mitochondrial calcium uptake. The video was&#160;rendered at 4x speed. Scale bar = 5 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/mitoGCaMP_SynchActivation_02_4XSPEED.avi" target="_blank">Please click here to download this Video.</a>
Supplemental Video 4: MitoGCaMP fluorescence in the AVA neurite-rapid uptake and release of calcium by the mitochondria. The video was&#160;rendered at 4x speed. Scale bar = 5 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/mitoGCamP_FlickeringMitos_01_4xSPEED.avi" target="_blank">Please click here to download this Video.</a>
Supplemental Table 1: Genetic reagents and strains used in this study. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64928/Supplemental Table 1-64928R2.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Subcellular Imaging of Neuronal Calcium Handling In Vivo at JoVE.com

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.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

1.2K Views.  Colorado State University College of Veterinary Medicine and Biomedical Sciences. 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.

Video: Subcellular Imaging of Neuronal Calcium Handling In Vivo

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

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 . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

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 .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

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.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

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, ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Developmental Biology

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

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.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

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.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

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

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

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.

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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

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

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Calcium Imaging in Neurons