Fluorescence Imaging of Calcium Dynamics at Neuromuscular Junctions in the Diaphragm of a Transgenic Mouse

Abstract

Source: Heredia, D. J., et. al. Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm. J. Vis. Exp. (2018)

This video demonstrates fluorescence imaging of calcium dynamics at neuromuscular junctions (NMJs) in a transgenic mouse expressing genetically encoded calcium indicators. A NJM is identified by fluorescently tagged acetylcholine receptors in the diaphragm with the phrenic nerve. An image splitter is used to visualize multiple signals simultaneously. An electrical stimulus is delivered, and the change in fluorescence at the NMJ in response to calcium influx is recorded.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of the Diaphragms and Phrenic Nerves from Transgenic Mice

1. Purchase transgenic mice and oligonucleotide primers to genotype 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 with both Cre and conditional GCaMP3/6 alleles—these will henceforth be called double-transgenic mice (e.g., Myf5-Cre, conditional GCaMP3).
      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 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 acetylcholine 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 methods.
   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 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, tighten the dials to clamp it into position, and close the puller door.
   2. Using a P-97 puller, program the following settings: 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 Posatium chloride (KCl).
5. At 10X magnification, lower the electrode into the 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 a 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 Neuromuscular junctions or 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 dual-wavelength imaging.
3. To calculate the maximal fluorescence (Fmax) exhibited by GCaMP3/6-expressing tissue, add 12 µL of 3 M potassium chloride (KCl) to the diaphragm preparations.
   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 so as not to 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 genetically encoded calcium indicators (GECI) or genetically encoded voltage indicators (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.

Materials

List of materials used in this article

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