PMID- 30346388
OWN - NLM
STAT- MEDLINE
DCOM- 20190403
LR  - 20191023
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 140
DP  - 2018 Oct 4
TI  - Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of 
      the Mouse Diaphragm.
LID - 10.3791/58347 [doi]
LID - 58347
AB  - 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 (Ca(2+)) 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 Ca(2+), thus permitting 
      the imaging of Ca(2+) 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 Ca(2+) 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.
FAU - Heredia, Dante J
AU  - Heredia DJ
AD  - Department of Physiology and Cell Biology, School of Medicine, University of 
      Nevada.
FAU - Hennig, Grant W
AU  - Hennig GW
AD  - Department of Pharmacology, Larner College of Medicine, University of Vermont.
FAU - Gould, Thomas W
AU  - Gould TW
AD  - Department of Physiology and Cell Biology, School of Medicine, University of 
      Nevada; tgould@medicine.nevada.edu.
LA  - eng
GR  - P20 GM103513/GM/NIGMS NIH HHS/United States
GR  - P20 GM103554/GM/NIGMS NIH HHS/United States
GR  - P20 RR018751/RR/NCRR NIH HHS/United States
GR  - P30 GM110767/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Video-Audio Media
DEP - 20181004
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Recombinant Fusion Proteins)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Calcium/metabolism
MH  - Calcium Signaling/genetics/*physiology
MH  - Diaphragm/*innervation
MH  - Fluorescent Dyes/metabolism
MH  - Mice
MH  - Mice, Transgenic
MH  - Neuromuscular Junction/*metabolism
MH  - Optical Imaging/*methods
MH  - Recombinant Fusion Proteins/genetics/metabolism
PMC - PMC6235423
EDAT- 2018/10/23 06:00
MHDA- 2019/04/04 06:00
PMCR- 2019/10/04
CRDT- 2018/10/23 06:00
PHST- 2018/10/23 06:00 [entrez]
PHST- 2018/10/23 06:00 [pubmed]
PHST- 2019/04/04 06:00 [medline]
PHST- 2019/10/04 00:00 [pmc-release]
AID - 58347 [pii]
AID - 10.3791/58347 [doi]
PST - epublish
SO  - J Vis Exp. 2018 Oct 4;(140):58347. doi: 10.3791/58347.