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Neuroscience

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

Published: October 4th, 2018

DOI:

10.3791/58347

1Department of Physiology and Cell Biology, School of Medicine, University of Nevada, 2Department of Pharmacology, Larner College of Medicine, University of Vermont

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

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of 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.

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....

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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 e.......

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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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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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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.).

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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

  1. Sanes, J. R., Lichtman, J. W. Development of the vertebrate neuromuscular junction. Annual Review of Neuroscience. 22, 389-442 (1999).
  2. Darabid, H., Perez-Gonzalez, A. P., Robitaille, R. Neuromuscular synaptogenesis: coordinating partners with multiple functions. Nature Reviews Neuroscience. 15 (11), 703-718 (2014).
  3. Fatt, P., Katz, B. An analysis of the end-plate potential recorded with an intracellular electrode. Journal of Physiology. 115 (3), 320-370 (1951).
  4. Todd, K. J., Robitaille, R. Purinergic modulation of synaptic signalling at the neuromuscular junction. Pflugers Archive. 452 (5), 608-614 (2006).
  5. Hennig, G. W., et al. 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. Frontiers of Cellular Neuroscience. 9, 436 (2015).
  6. Heredia, D. J., Schubert, D., Maligireddy, S., Hennig, G. W., Gould, T. W. A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology. Frontiers of Cellular Neuroscience. 10, 276 (2016).
  7. Johnson, B. R., Hauptman, S. A., Bonow, R. H. Construction of a simple suction electrode for extracellular recording and stimulation. Journal of Undergraduate Neuroscience Education. 6 (1), A21-A26 (2007).
  8. Hong, S. J., Chang, C. C. Use of geographutoxin II (mu-conotoxin) for the study of neuromuscular transmission in mouse. British Journal of Pharmacology. 97 (3), 934-940 (1989).
  9. Heredia, D. J., Feng, C. Y., Hennig, G. W., Renden, R. B., Gould, T. W. Activity-induced Ca2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y1 receptors and regulates muscle fatigue. Elife. 7, e30839 (2018).
  10. Cho, J. H., et al. The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators. ACS Chemical Biology. 12 (4), 1066-1074 (2017).

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