This work was supported with funds from the National Institutes of Health (NIH) GM103554 and GM110767 to (T.W.G.) and from the National Center for Research Resources 5P20RR018751 and the National Institute of General Medical Sciences 8P20 GM103513 (to G.W.H.).

Animal husbandry and experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the IACUC at the University of Nevada.
1. Preparation of the Diaphragms and Phrenic Nerves from Transgenic Mice
<ol>
	<li>Purchase transgenic mice and oligonucleotide primers to genotype these mice. 
	Note: The primers are listed on the &#8220;Information&#8221; page for each of these mice.
	<ol>
		<li>Breed a 3- to 6-month-old mouse expressing one copy of the appropriate transgenic/knock-in Cre-driver allele and zero copies of the conditional GCaMP3/6 allele with a second mouse of the same age expressing one or two copies of the conditional GCaMP3/6 allele and zero copies of the Cre-driver allele.</li>
		<li>Genotype the pups and mark the ones that have both Cre and conditional GCaMP3/6 alleles&#8212;these will henceforth be called double-transgenic mice (e.g., Myf5-Cre, conditional GCaMP3)6. 
		Note: This way, all data will derive from mice expressing one copy of both Cre and conditional GCaMP3/6 alleles. This is particularly important when adding in other mutant mice (e.g., knockouts) to these crosses.</li>
	</ol>
	</li>
	<li>When the double-transgenic mice are of the appropriate age (e.g., postnatal day 0 or 5 [P0 or P5] or adult), euthanize the mice by decapitating them with scissors (for mice younger than P10) or by placing them in an isoflurane inhalation chamber&#8212;when they are no longer responsive to pinching the tail with a pair of forceps, they are ready for sacrifice.</li>
	<li>Sacrifice the animal by decapitation with a pair of scissors.</li>
	<li>Transversely section across the entire animal just below the liver and just above the heart and lungs with iridectomy scissors.</li>
	<li>Dissect away the liver, the heart, and the lungs, being careful to maintain a length of the phrenic nerve that is sufficiently long to be drawn into a suction electrode (i.e., 1 - 2 cm). 
	Note: The left phrenic nerve can be identified as a white piece of tissue that enters the medial portion of the left diaphragm. It must not be cut when removing the lungs. The right phrenic nerve runs within a piece of fascia that also contains the superior vena cava and is thinner and whiter than the vena cava. Together, they both penetrate the right medial diaphragm.</li>
	<li>Further remove the ribcage and the vertebral column, except for the thin ridge around the diaphragm.</li>
	<li>Place the diaphragm and the phrenic nerve sample in a microfuge tube with Krebs-Ringer solution with 1 &#181;g/mL 594-&#945;BTX for 10 min in the dark. 
	Note: This concentration of 594-&#945;BTX labels ACh receptors (AChRs) without blocking their function (personal observation).</li>
</ol>
2. Stimulation and Recording of the Muscle Action Potentials
<ol>
	<li>Using minutien pins, immobilize the diaphragm by pinning it onto a 6-cm dish coated with silicone dielectric gel and filled with ~8 mL of oxygenated Krebs-Ringer solution and place it onto the microscope stage. Perfuse the diaphragm with more Krebs-Ringer solution (8 mL/min) for 30 min. 
	Note: This rinses the unbound 594-&#945;BTX, as well as equilibrates the tissue after dissection.</li>
	<li>Make a suction electrode according to the established methods7.
	<ol>
		<li>At 4X magnification, using a micromanipulator, move the suction electrode over the left phrenic nerve and apply suction by pulling out the barrel of a 5-mL syringe connected to the tubing that is attached to the suction electrode. 
		Note: When successfully drawn into the suction electrode, the phrenic nerve is taut. Turn on the stimulator and stimulate the phrenic nerve by flipping the manual switch 1x.</li>
		<li>Ensure that the diaphragm contracts in response to the 1-Hz stimulation by visually examining it with brightfield illumination. If not, adjust the voltage by turning the voltage knob incrementally to achieve a supramaximal pulse, which can be verified by a visual examination of muscle contraction. If still not visible, blow out the nerve with the syringe and attempt to draw it in again by applying suction.</li>
	</ol>
	</li>
	<li>Turn off the perfusion and add the muscle-specific myosin inhibitor BHC6 or the voltage-gated sodium channel antagonist &#181;-conotoxin8 to a final concentration of 100 &#181;M.
	<ol>
		<li>To make 100 &#181;M BHC, pipette 4 &#181;L of 200 mM stock in DMSO and predilute it in 1 mL of Krebs-Ringer solution.</li>
		<li>Remove 1 mL of Krebs-Ringer solution from the dish.</li>
		<li>Add the prediluted BHC, to the dish. 
		Note: This predilution helps prevent the induction by undiluted DMSO of a non-transient fluorescent response in GCaMP3-expressing cells.</li>
		<li>Wait 30 min and then, turn on the perfusion of fresh Krebs-Ringer solution for another 20 - 30 min.</li>
	</ol>
	</li>
	<li>Prepare the recording electrode.
	<ol>
		<li>Wearing gloves, place a borosilicate filamented glass with an outer diameter (OD) of 1 mm and an inner diameter (ID) of 0.4 mm into a micropipette puller and tighten the dials to clamp it into position. Close the puller door.</li>
		<li>Using a P-97 puller, program the following setting: heat at 900, pull at 120, velocity at 75, time at 250, pressure at 500, and no additional loops. 
		Note: Resistance (R) is measured using software controls of the amplifier: the data acquisition software confirms resistance by solving the formula V = IR. The software controller passes a known current (I) (typically 1 nA) through the electrode and measures the change in voltage (V), thus enabling us to solve for R.</li>
		<li>For embryonic diaphragms, ensure that the resistance is near 60 M&#8486;, and for older diaphragms, 10 - 20 M&#8486;. Load the recording electrode with 3 M KCl.</li>
	</ol>
	</li>
	<li>At 10X magnification, lower the electrode into muscle, using a second micromanipulator on the opposite side of the stage as a stimulating electrode.</li>
	<li>Using electrophysiological data acquisition software, wait until the resting membrane potential changes from 0 to -65 mV or below.</li>
	<li>Stimulate at 1 Hz and verify the presence of a muscle action potential by checking for a large potential that exhibits a modest overshoot (potential that rises above 0 mV when it starts at -65 mV or below). Do not confuse stimulation artifact with an action potential. 
	Note: Potentials are significantly longer in duration (~5 ms) than stimulation artifacts.</li>
</ol>
3. Imaging of the Fluorescence of the Sample
<ol>
	<li>At 20X magnification, locate the endplate band at the center of the muscle by looking for 594-&#945;BTX&#8211;labeled NMJs under green/yellow light excitation (550 nm). Switch to the blue light excitation (470 nm) to image Ca2+ responses in muscle, motor neuron, or Schwann cells.</li>
	<li>If desired, set up the image splitter with bandpass filters and a dichroic single-edge filter for the dual-wavelength imaging.</li>
	<li>In order to calculate the maximal fluorescence (Fmax) exhibited by GCaMP3/6-expressing tissue, add 12 &#181;L of 3 M potassium chloride (KCl) to the diaphragm preparations6.
	<ol>
		<li>Perform experiments with the brightness bar on the lookup table bar set to 110% of the level at which the GCaMP3/6-expressing tissue exhibits saturation at 20X magnification, without binning in response to KCl.</li>
	</ol>
	</li>
	<li>Record at 20 frames per second to not miss any fast events.</li>
	<li>Stimulate with 1 - 45 s of 20 - 40 Hz of nerve stimulation by delivering a train of impulses using the suction electrode or add pharmacological agonists by bath application or by perfusion and collect dynamic fluorescent Ca2+ responses in one cell subtype together with the static 594-&#945;BTX NMJ signal. 
	Note: If tissue-specific red or far-red GECI or GEVI mice become available for use at the NMJ, they can be used to collect two dynamic signals reflecting two distinct cellular elements at the NMJ.</li>
	<li>When the imaging or electrophysiological experiments are finished because the desired results have been achieved, perfuse water through the perfusion lines and suck water 2x - 3x through the suction electrode to ensure that salts do not build up.</li>
</ol>
4. Export and Analysis of the Data by a Standard Deviation Map of Fluorescence Intensity (SDiu16)
<ol>
	<li>Record image sequences recorded as 16-bit TIFF stacks and load them into the desired imaging data analysis system for analysis.</li>
	<li>In the software&#8217;s 8d file menu, select Image stack of interest and click to load.
	<ol>
		<li>Once the video loads, scan through the time to identify a section that has no cellular fluorescent activity. 
		Note: This region will be used to create a background sample.</li>
		<li>Hold Shift and click to draw a region of interest (ROI) box in the area identified as the background sample area.</li>
		<li>After creating the box, press the space bar to generate a plot of background activity change.</li>
		<li>Right-click the trace and select the assorted option to present the option to Dump ROI as text to make the trace as an xy coordinate text file.</li>
	</ol>
	</li>
	<li>Moving back to the video of interest, scan again to identify the time region where the activity of interest is occurring.
	<ol>
		<li>Using the middle mouse button, select this time region in the yellow time box.</li>
		<li>Right-click on the video and select Stack OPS and then Stat map option 5. 
		Note: This will generate a standard deviation map (SD map) in the left window.</li>
		<li>Click on the SD map and then press the ] key 19x to apply the appropriate color heat map.</li>
		<li>Right-click the SD map and select STM load and save, which will present the option Save stm as tiff to save the SD map.</li>
		<li>Then, press the [ key 19x to return to a grayscale color map.</li>
		<li>Press C and then D to bring up density mapping tools. Using the left mouse button and the center mouse button, adjust the threshold to include all fluorescent activity shown in the SD map.</li>
		<li>Press C to close the density tools while maintaining the threshold settings.</li>
		<li>Right-click the SD map and select STM particles and then Find PTCLS. 
		Note: This will identify individual cells expressing fluorescent activity.</li>
		<li>Right-click the SD map once more and select Create Particle ROIs. 
		Note: This will superimpose the selected cells on the original video of interest.</li>
		<li>While holding Shift, right-click on any one of the now identified particle ROIs on the original video.</li>
		<li>Select ROI Marker and Measure Int in ROI. 
		Note: This will generate individual fluorescent activity plots for each identified ROI in the video of interest. These can be saved by right-clicking any one of these and selecting Assorted, followed by Dump ROI as text.</li>
	</ol>
	</li>
	<li>For detailed logic underlying these operations, please see the source code file9.</li>
</ol>

<ol>
	<li>Sanes, J. R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+vertebrate+neuromuscular+junction.">Development of the vertebrate neuromuscular junction.</a> Annual Review of Neuroscience. 22, 389-442 (1999).</li><li>Darabid, H., Perez-Gonzalez, A. P., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+synaptogenesis:+coordinating+partners+with+multiple+functions.">Neuromuscular synaptogenesis: coordinating partners with multiple functions.</a> Nature Reviews Neuroscience. 15 (11), 703-718 (2014).</li><li>Fatt, P., Katz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+end-plate+potential+recorded+with+an+intracellular+electrode.">An analysis of the end-plate potential recorded with an intracellular electrode.</a> Journal of Physiology. 115 (3), 320-370 (1951).</li><li>Todd, K. J., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+modulation+of+synaptic+signalling+at+the+neuromuscular+junction.">Purinergic modulation of synaptic signalling at the neuromuscular junction.</a> Pflugers Archive. 452 (5), 608-614 (2006).</li><li>Hennig, G. W., 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=Use+of+Genetically+Encoded+Calcium+Indicators+(GECIs)+Combined+with+Advanced+Motion+Tracking+Techniques+to+Examine+the+Behavior+of+Neurons+and+Glia+in+the+Enteric+Nervous+System+of+the+Intact+Murine+Colon.">Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon.</a> Frontiers of Cellular Neuroscience. 9, 436 (2015).</li><li>Heredia, D. J., Schubert, D., Maligireddy, S., Hennig, G. W., Gould, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Striated+Muscle-Specific+Myosin-Blocking+Drug+for+the+Study+of+Neuromuscular+Physiology.">A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology.</a> Frontiers of Cellular Neuroscience. 10, 276 (2016).</li><li>Johnson, B. R., Hauptman, S. A., Bonow, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+simple+suction+electrode+for+extracellular+recording+and+stimulation.">Construction of a simple suction electrode for extracellular recording and stimulation.</a> Journal of Undergraduate Neuroscience Education. 6 (1), A21-A26 (2007).</li><li>Hong, S. J., Chang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+geographutoxin+II+(mu-conotoxin)+for+the+study+of+neuromuscular+transmission+in+mouse.">Use of geographutoxin II (mu-conotoxin) for the study of neuromuscular transmission in mouse.</a> British Journal of Pharmacology. 97 (3), 934-940 (1989).</li><li>Heredia, D. J., Feng, C. Y., Hennig, G. W., Renden, R. B., Gould, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+Ca2++signaling+in+perisynaptic+Schwann+cells+of+the+early+postnatal+mouse+is+mediated+by+P2Y1+receptors+and+regulates+muscle+fatigue.">Activity-induced Ca2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y1 receptors and regulates muscle fatigue.</a> Elife. 7, e30839 (2018).</li><li>Cho, J. H., 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=The+GCaMP-R+Family+of+Genetically+Encoded+Ratiometric+Calcium+Indicators.">The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators.</a> ACS Chemical Biology. 12 (4), 1066-1074 (2017).</li></ol>

The authors have nothing to disclose.

Here we provide some examples of measuring Ca2+ responses in specific cells in intact neuromuscular tissue using GECI-expressing mice. In order to successfully perform these experiments, it is imperative not to injure the phrenic nerve during the dissection. To image Ca2+ responses in Schwann cells at either low or high power (i.e., 20X or 60X), it is necessary to use either BHC or &#181;-conotoxin to block movement. For low-power imaging of Ca2+ responses in muscle cells, it is ...

The NMJ, like all synapses, is composed of three elements: a presynaptic terminal derived from a neuron, a postsynaptic neuron/effector cell, and a perisynaptic glial cell1,2. While the basic aspects of synaptic transmission were first demonstrated at this synapse3, many aspects of this process remain unknown, in part owing to the expression of the same molecules by the distinct cellular elements of this synapse. For example, receptors for both the purine adenine nucleotide ATP and acetylcholine (ACh), which are co-released by motor neurons at the vertebrate NMJ, are expressed by muscle, Schwann cells, and motor neurons, thus complicating the interpretation of any functional effect exerted by these substances (e.g., transmitter release or response, muscle force generation)4. Moreover, although the tripartite components of the NMJ are simple compared to, for example, neurons in the central nervous system which often exhibit multiple synaptic inputs, whether motor neurons, muscle cells, or Schwann cells vary in response to stimuli based on their intrinsic heterogeneity (e.g., embryonic derivation, fiber subtype, morphology) is unclear. In order to address each of these issues, it would be advantageous to simultaneously track the response of many cells within one synaptic element, as well as track, at the same time, such a response in either of the other separate elements. Conventional strategies using chemical dyes to measure calcium signaling cannot achieve these two goals, because bath-applied dye is taken up by multiple cell types after application to tissue, and intracellularly loaded dye can only be used to visualize individual or small cohorts of cells. Here, utilizing transgenic mice expressing GECIs designed to measure cell-specific calcium signaling, together with specific imaging and software tools5, we demonstrate the first of these two overall goals and discuss how the addition of new transgenic tools would help achieve the second. This technique will be useful for anyone interested in tracking calcium dynamics or other cellular signaling events observable through gene-encoded optical sensors in multiple cell populations at the same time.

<table><tbody><tr><td>Myf5-Cre mice</td><td>Jax</td><td>#007893</td><td>Drives muscle cell expression as early as E13&lt;sup class=&quot;xref&quot;&gt;6&lt;/sup&gt;</td></tr><tr><td>Wnt1-Cre mice</td><td>Jax</td><td>#003829</td><td>Drives expression into all&amp;nbsp; Schwann cells at E13 but not P20&lt;sup class=&quot;xref&quot;&gt;9&lt;/sup&gt;</td></tr><tr><td>Sox10-Cre mice</td><td>Jax</td><td>#025807</td><td>Drives Schwann cell expression at older ages</td></tr><tr><td>Conditional GCaMP3 mice</td><td>Jax</td><td>#029043</td><td>Expresses GCaMP3 in cell-specific fashion</td></tr><tr><td>Conditional GCaMP6f mice</td><td>Jax</td><td>#024105</td><td>Expresses GCaMP6f in cell-specific fashion</td></tr><tr><td>BHC (3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one)</td><td>Hit2Lead</td><td>#5102862</td><td>Blocks skeletal muscle myosin but not neurotransmission&lt;sup class=&quot;xref&quot;&gt;6&lt;/sup&gt;</td></tr><tr><td>CF594-&amp;alpha;-BTX</td><td>Biotium</td><td>#00007</td><td>Labels acetylcholine receptor clusters at NMJ</td></tr><tr><td>&amp;micro;-conotoxin GIIIb</td><td>Peptides Int&#039;l</td><td>#CONO20-01000</td><td>Blocks Nav1.4 voltage-dependent sodium channel&lt;sup class=&quot;xref&quot;&gt;8&lt;/sup&gt;</td></tr><tr><td>Silicone Dielectric Gel; aka Sylgard</td><td>Ellswoth Adhesives</td><td># Sil Dielec Gel .9KG&amp;nbsp;</td><td>Allows for the immobilization of the diaphragm by minutien pins</td></tr><tr><td>Minutien pins (0.1mm diameter)</td><td>Fine Science Tools</td><td>26002-10</td><td>Immobilizes diaphragm onto silicone dielectric gel</td></tr><tr><td>Eclipse FN1 upright microscope&amp;nbsp;</td><td>Nikon</td><td>MBA74100</td><td>Allows staging and observation of specimen</td></tr><tr><td>Basic Fixed Microscope Platform with Manual XY Microscope Translator&amp;nbsp;</td><td>Autom8</td><td>MXMScr</td><td>Allows movement of specimen</td></tr><tr><td>Manual micromanipulator&amp;nbsp;</td><td>Narishige</td><td>M-152</td><td>Holds recording and stimulating electrodes&amp;nbsp;</td></tr><tr><td>Microelectrode amplifier&amp;nbsp;</td><td>Molecular Devices</td><td>Axoclamp 900A</td><td>Allows sharp electrode intracellular electrophysiological recording</td></tr><tr><td>Microelectrode low-noise data acquisition system</td><td>Molecular Devices</td><td>Digidata 1550&amp;nbsp;</td><td>Allows electrophysiological data acquisition</td></tr><tr><td>Microelectrode data analysis system</td><td>Molecular Devices</td><td>PCLAMP 10 Standard</td><td>Performs electrophysiological data analysis</td></tr><tr><td>Square wave stimulator</td><td>Grass</td><td>S48</td><td>Stimulates nerve to excite muscle</td></tr><tr><td>Stimulus Isolation Unit</td><td>Grass</td><td>PSIU6</td><td>Reduces&amp;nbsp; stimulation artifacts</td></tr><tr><td>Borosilicate filaments, 1.0 mm outer diameter, 0.5mm internal diameter&amp;nbsp;</td><td>Sutter</td><td>FG-GBF100-50-15</td><td>Impales and records nerve-evoked muscle potentials</td></tr><tr><td>Borosilicate filaments, 1.5 mm outer diameter, 1.17mm internal diameter&amp;nbsp;</td><td>Sutter</td><td>BF150-117-15</td><td>Lengthened and used for suction electrode</td></tr><tr><td>Micropipette Puller</td><td>Sutter</td><td>P-97&amp;nbsp;</td><td>Pulls and prepares recording electrodes</td></tr><tr><td>1200x1200 pixel, back-illuminated cMOS camera&amp;nbsp;</td><td>Photometrics</td><td>Prime 95b</td><td>Sensitive camera that allows high-resolution, high-speed imaging</td></tr><tr><td>Light Source</td><td>Lumencor</td><td>Spectra X</td><td>Provides illumination from LEDs for fluorescence obsevation</td></tr><tr><td>&amp;nbsp;Infinity-corrected fluorescent water immersion objectives, W.D. 2mm&amp;nbsp;</td><td>Nikon</td><td>CFI60</td><td>Provide long working distances for visualization of specimen</td></tr><tr><td>Fiber Optic Illuminator with Halogen lamp</td><td>Sumita</td><td>LS-DWL-N</td><td>Provides illumination for brightfield observation</td></tr><tr><td>W-View Gemini Image Splitter&amp;nbsp;</td><td>Hamamatsu</td><td>A12801-01</td><td>Projects 1 pair of dual wavelength images separated by a dichroic to single camera</td></tr><tr><td>Single-band Bandpass Filters&amp;nbsp; (512/25-25 and 630/92-25)&amp;nbsp;</td><td>SemRock</td><td>FF01-512/25-25; FF01-630/92-25</td><td>Permits dual band imaging</td></tr><tr><td>560 nm Single-Edge Dichroic Beamsplitter</td><td>Sem Rock</td><td>FF560-FDi01-25x36</td><td>Dichroic mirror which separates beams of light to allow dual-wavelength imaging</td></tr><tr><td>Imaging data acquisition system</td><td>Nikon</td><td>NIS Elements - MQS31000</td><td>Allows imaging data acquisition</td></tr><tr><td>Wavelength control module</td><td>Nikon</td><td>MQS41220</td><td>Module for imaging data acqusiition</td></tr><tr><td>Emission splitter hardware module&amp;nbsp;</td><td>Nikon</td><td>MQS41410</td><td>Module for imaging data acqusiition</td></tr><tr><td>Imaging data analysis system</td><td>NA</td><td>Volumetry 8D&lt;sup class=&quot;xref&quot;&gt;5&lt;/sup&gt;, Fiji</td><td>Allows analysis of fluorescence intensity and other imaging data</td></tr></tbody></table>

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.

Several examples of fluorescence intensity changes, mediated by increases of intracellular Ca2+ within defined cell types of the NMJ, show the utility of this approach. These results are presented as spatial fluorescence intensity maps, which provide the location of responding cells, as well as the intensity of their responses, thus allowing for the evaluation of how many cells respond and how much each cell responds to a particular stimulus. For example, as shown in <strong cl...

Watch this Scientific Journal Video about Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm at JoVE.com

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.

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 ...

ex-vivo-imaging-cell-specific-calcium-signaling-at-tripartite-synapse

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.7K Views.  University of Nevada. Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction. 

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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

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.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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

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

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

Source: Hancock, C. E., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/60288/" target="_blank">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes in vivo calcium imaging in Drosophila neurons using GCaMP. The featured protocol clip shows how to record changes in GCaMP fluorescence from mushroom body neurons during an olfactory conditioning experiment.

Drosophila In Vivo (Drosophila) in vivo (Drosophila) Obrazowanie wapnia: metoda funkcjonalnego obrazowania aktywności neuronalnej

Source: Hancock, C. E., et al. In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster. J. Vis. Exp. (2019).
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Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

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Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

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

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

In vivo Calcium Imaging in Mouse Inferior Olive

Imaging Local Ca²⁺ Signals in Cultured Mammalian Cells

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Drosophila In Vivo (Drosophila) in vivo (Drosophila) Obrazowanie wapnia: metoda funkcjonalnego obrazowania aktywności neuronalnej