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 E136</td>
		</tr>
		<tr>
			<td>Wnt1-Cre mice</td>
			<td>Jax</td>
			<td>#003829</td>
			<td>Drives expression into all&#160; Schwann cells at E13 but not P209</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 neurotransmission6</td>
		</tr>
		<tr>
			<td>CF594-&#945;-BTX</td>
			<td>Biotium</td>
			<td>#00007</td>
			<td>Labels acetylcholine receptor clusters at NMJ</td>
		</tr>
		<tr>
			<td>&#181;-conotoxin GIIIb</td>
			<td>Peptides Int&#39;l</td>
			<td>#CONO20-01000</td>
			<td>Blocks Nav1.4 voltage-dependent sodium channel8</td>
		</tr>
		<tr>
			<td>Silicone Dielectric Gel; aka Sylgard</td>
			<td>Ellswoth Adhesives</td>
			<td># Sil Dielec Gel .9KG&#160;</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&#160;</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&#160;</td>
			<td>Autom8</td>
			<td>MXMScr</td>
			<td>Allows movement of specimen</td>
		</tr>
		<tr>
			<td>Manual micromanipulator&#160;</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td>Holds recording and stimulating electrodes&#160;</td>
		</tr>
		<tr>
			<td>Microelectrode amplifier&#160;</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&#160;</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&#160; stimulation artifacts</td>
		</tr>
		<tr>
			<td>Borosilicate filaments, 1.0 mm outer diameter, 0.5mm internal diameter&#160;</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&#160;</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&#160;</td>
			<td>Pulls and prepares recording electrodes</td>
		</tr>
		<tr>
			<td>1200x1200 pixel, back-illuminated cMOS camera&#160;</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>&#160;Infinity-corrected fluorescent water immersion objectives, W.D. 2mm&#160;</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&#160;</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&#160; (512/25-25 and 630/92-25)&#160;</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&#160;</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 8D5, 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...

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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

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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

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1,2,3,4,5,6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

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 ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

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

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Chapters in this video

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

In vivo Calcium Imaging in Mouse Inferior Olive

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus