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

Podsumowanie

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

Streszczenie

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.  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.

Wprowadzenie

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 cell^1,2. 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.

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Protokół

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

1. Purchase transgenic mice and oligonucleotide primers to genotype these mice.
   Note: The primers are listed on the “Information” page for each of these mice.
   1. 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.
   2. Genotype the pups and mark the ones that have both Cre and conditional GCaMP3/6 alleles—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.
2. 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—when they are no longer responsive to pinching the tail with a pair of forceps, they are ready for sacrifice.
3. Sacrifice the animal by decapitation with a pair of scissors.
4. Transversely section across the entire animal just below the liver and just above the heart and lungs with iridectomy scissors.
5. 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.
6. Further remove the ribcage and the vertebral column, except for the thin ridge around the diaphragm.
7. Place the diaphragm and the phrenic nerve sample in a microfuge tube with Krebs-Ringer solution with 1 µg/mL 594-αBTX for 10 min in the dark.
   Note: This concentration of 594-αBTX labels ACh receptors (AChRs) without blocking their function (personal observation).

2. Stimulation and Recording of the Muscle Action Potentials

1. 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-αBTX, as well as equilibrates the tissue after dissection.
2. Make a suction electrode according to the established methods⁷.
   1. 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.
   2. 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.
3. Turn off the perfusion and add the muscle-specific myosin inhibitor BHC⁶ or the voltage-gated sodium channel antagonist µ-conotoxin⁸ to a final concentration of 100 µM.
   1. To make 100 µM BHC, pipette 4 µL of 200 mM stock in DMSO and predilute it in 1 mL of Krebs-Ringer solution.
   2. Remove 1 mL of Krebs-Ringer solution from the dish.
   3. 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.
   4. Wait 30 min and then, turn on the perfusion of fresh Krebs-Ringer solution for another 20 - 30 min.
4. Prepare the recording electrode.
   1. 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.
   2. 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.
   3. For embryonic diaphragms, ensure that the resistance is near 60 MΩ, and for older diaphragms, 10 - 20 MΩ. Load the recording electrode with 3 M KCl.
5. At 10X magnification, lower the electrode into muscle, using a second micromanipulator on the opposite side of the stage as a stimulating electrode.
6. Using electrophysiological data acquisition software, wait until the resting membrane potential changes from 0 to -65 mV or below.
7. 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.

3. Imaging of the Fluorescence of the Sample

1. At 20X magnification, locate the endplate band at the center of the muscle by looking for 594-αBTX–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.
2. If desired, set up the image splitter with bandpass filters and a dichroic single-edge filter for the dual-wavelength imaging.
3. In order to calculate the maximal fluorescence (Fmax) exhibited by GCaMP3/6-expressing tissue, add 12 µL of 3 M potassium chloride (KCl) to the diaphragm preparations⁶.
   1. 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.
4. Record at 20 frames per second to not miss any fast events.
5. 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-αBTX 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.
6. 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.

4. Export and Analysis of the Data by a Standard Deviation Map of Fluorescence Intensity (SD_iu16)

1. Record image sequences recorded as 16-bit TIFF stacks and load them into the desired imaging data analysis system for analysis.
2. In the software’s 8d file menu, select Image stack of interest and click to load.
   1. 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.
   2. Hold Shift and click to draw a region of interest (ROI) box in the area identified as the background sample area.
   3. After creating the box, press the space bar to generate a plot of background activity change.
   4. 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.
3. Moving back to the video of interest, scan again to identify the time region where the activity of interest is occurring.
   1. Using the middle mouse button, select this time region in the yellow time box.
   2. 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.
   3. Click on the SD map and then press the ] key 19x to apply the appropriate color heat map.
   4. 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.
   5. Then, press the [ key 19x to return to a grayscale color map.
   6. 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.
   7. Press C to close the density tools while maintaining the threshold settings.
   8. Right-click the SD map and select STM particles and then Find PTCLS.
      Note: This will identify individual cells expressing fluorescent activity.
   9. 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.
   10. While holding Shift, right-click on any one of the now identified particle ROIs on the original video.
   11. 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.
4. For detailed logic underlying these operations, please see the source code file⁹.

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Wyniki

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

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Dyskusje

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 µ-conotoxin to block movement. For low-power imaging of Ca²⁺ responses in muscle cells, it is ...

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Materiały

Name	Company	Catalog Number	Comments
Myf5-Cre mice	Jax	#007893	Drives muscle cell expression as early as E136
Wnt1-Cre mice	Jax	#003829	Drives expression into all  Schwann cells at E13 but not P209
Sox10-Cre mice	Jax	#025807	Drives Schwann cell expression at older ages
Conditional GCaMP3 mice	Jax	#029043	Expresses GCaMP3 in cell-specific fashion
Conditional GCaMP6f mice	Jax	#024105	Expresses GCaMP6f in cell-specific fashion
BHC (3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one)	Hit2Lead	#5102862	Blocks skeletal muscle myosin but not neurotransmission6
CF594-α-BTX	Biotium	#00007	Labels acetylcholine receptor clusters at NMJ
µ-conotoxin GIIIb	Peptides Int'l	#CONO20-01000	Blocks Nav1.4 voltage-dependent sodium channel8
Silicone Dielectric Gel; aka Sylgard	Ellswoth Adhesives	# Sil Dielec Gel .9KG	Allows for the immobilization of the diaphragm by minutien pins
Minutien pins (0.1mm diameter)	Fine Science Tools	26002-10	Immobilizes diaphragm onto silicone dielectric gel
Eclipse FN1 upright microscope	Nikon	MBA74100	Allows staging and observation of specimen
Basic Fixed Microscope Platform with Manual XY Microscope Translator	Autom8	MXMScr	Allows movement of specimen
Manual micromanipulator	Narishige	M-152	Holds recording and stimulating electrodes
Microelectrode amplifier	Molecular Devices	Axoclamp 900A	Allows sharp electrode intracellular electrophysiological recording
Microelectrode low-noise data acquisition system	Molecular Devices	Digidata 1550	Allows electrophysiological data acquisition
Microelectrode data analysis system	Molecular Devices	PCLAMP 10 Standard	Performs electrophysiological data analysis
Square wave stimulator	Grass	S48	Stimulates nerve to excite muscle
Stimulus Isolation Unit	Grass	PSIU6	Reduces  stimulation artifacts
Borosilicate filaments, 1.0 mm outer diameter, 0.5mm internal diameter	Sutter	FG-GBF100-50-15	Impales and records nerve-evoked muscle potentials
Borosilicate filaments, 1.5 mm outer diameter, 1.17mm internal diameter	Sutter	BF150-117-15	Lengthened and used for suction electrode
Micropipette Puller	Sutter	P-97	Pulls and prepares recording electrodes
1200x1200 pixel, back-illuminated cMOS camera	Photometrics	Prime 95b	Sensitive camera that allows high-resolution, high-speed imaging
Light Source	Lumencor	Spectra X	Provides illumination from LEDs for fluorescence obsevation
Infinity-corrected fluorescent water immersion objectives, W.D. 2mm	Nikon	CFI60	Provide long working distances for visualization of specimen
Fiber Optic Illuminator with Halogen lamp	Sumita	LS-DWL-N	Provides illumination for brightfield observation
W-View Gemini Image Splitter	Hamamatsu	A12801-01	Projects 1 pair of dual wavelength images separated by a dichroic to single camera
Single-band Bandpass Filters  (512/25-25 and 630/92-25)	SemRock	FF01-512/25-25; FF01-630/92-25	Permits dual band imaging
560 nm Single-Edge Dichroic Beamsplitter	Sem Rock	FF560-FDi01-25x36	Dichroic mirror which separates beams of light to allow dual-wavelength imaging
Imaging data acquisition system	Nikon	NIS Elements - MQS31000	Allows imaging data acquisition
Wavelength control module	Nikon	MQS41220	Module for imaging data acqusiition
Emission splitter hardware module	Nikon	MQS41410	Module for imaging data acqusiition
Imaging data analysis system	NA	Volumetry 8D5, Fiji	Allows analysis of fluorescence intensity and other imaging data