Combining membrane potential and calcium imaging in brain slices using the voltage-sensitive dye ElectroFluor630 and the calcium indicator Calbryte520

Wide-field imaging from brain slices stained with a voltage sensitive dye (VSD) and simultaneously loaded with a Ca 2+ indicator allows investigating neuronal excitability and synaptic transmission at multi-cellular scale. So far, achieving this type of combined imaging has been limited by experimental constraints. We assessed the ability of the red-IR emitting VSD ElectroFluor630 (EF-630) to be combined with blue-excitable green-emitting Ca 2+ indicators to record signals elicited by electrical stimulation in hippocampal slices. Transversal mouse hippocampal slices were stained with EF-630. Ca 2+ indicators, either Fluo-4, Fluo-8, Cal520 or Calbryte520, were loaded using their AM-ester forms. Fluorescence, during stimulation of the CA3 region was imaged at 5 kHz from hippocampal areas of ∼750X250 µm 2 at 1 µm pixel resolution. After assessing all Ca 2+ indicators, we selected Calbryte520 for achieving >30 minutes stable recordings in combination with EF-630. Action potentials and related Ca 2+ transients were detected in the CA3 stimulated area whereas synaptic signals were observed in the CA1 region. On these signals, we tested the pharmacological blockade of either action potentials or glutamatergic synaptic potentials. We report novel optical measurements of both electrical and Ca 2+ transients in brain slices, providing unique information on neuronal excitability and network activity.