Feb 11, 2020
Dual Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart during Optogenetic Stimulation of the ICNS
- Pradeep Rajendran1,
- Guy Salama2,
- Ching Zhu1,
- Peter Hanna1
- 1UCLA;
- 2University of Pittsburgh
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna 2020. Dual Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart during Optogenetic Stimulation of the ICNS. protocols.io https://dx.doi.org/10.17504/protocols.io.bcdtis6n
Manuscript citation:
Salama G, Hwang SM. Simultaneous optical mapping of intracellular free calcium and action potentials from langendorff perfused hearts. Curr Protoc Cytom. 2009;Chapter 12(Unit 12):17.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol.
Created: February 11, 2020
Last Modified: February 11, 2020
Protocol Integer ID: 32915
The animal is euthanized by a method approved by IACUC (Institutional Animal Care and Use Committee), the chest opened, the heart and lungs removed, and the aorta quickly cannulated on a Langendorff apparatus to perfuse the coronary vessels. The cannulation step should occur in ice-cold Tyrode's solution (112mM NaCl, 5mM KCl, 25mM NaHCO3, 1mM KH2PO4, 1.2mM MgSO4, 1.8mM CaCl2, 50mM glucose) and completed within 10 minutes of euthanasia.
With a Langendorff apparatus, hearts can be perfused at either constant coronary pressure or constant flow rate. Hearts were immobilized in a custom-built chamber to reduce motion artifact.
Hearts were stained with bolus injections of voltage-sensitive RH237 (30uL of 1mg/mL in DMSO, Thermo Fisher Scientific, S1109) and Ca indicator Rhod-2 AM (30uL of 1mg/ml in DMSO, Biotium, 50024) into the coronary perfusate.
Excitation-contraction uncoupling agents such as blebbistatin were not used.
Light from a 100-W tungsten lamp was collimated, passed through 530 ± 30 nm interference filters, split by a 560 nm dichroic mirror, and focused on the dorsal epicardial surface of the heart for excitation. Emitted fluorescence was collected with tandem camera lenses (50 mm f/1.2 mm, Nikon and 50 mm f/0.95, Navitar) and split with a 600 nm dichroic mirror.
The longer wavelength moiety, containing the Vm signal, was filtered between 610-750 nm and the shorter wavelength moiety, containing the intracellular Ca2+ signal, was filtered between 570-595 nm. The emitted fluorescence signals were recorded using 2 CMOS cameras (SciMedia, MiCAM ULTIMA) with a sampling rate of 2 kHz and 100 x 100 pixels with a 5 x 5 mm field of view. Pixel resolution of the images was 150 x 150 µm2. All stimulations were performed on the dorsal heart at 10 Hz, 10 ms, and 221 mW for 10 s. Data were acquired in 40 s intervals with 15 s collected before and after the stimulation.