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

Wide-field imaging from brain slices stained with a voltage-sensitive dye (VSD) and simultaneously loaded with a ${\mathrm{Ca}}^{2+}$ indicator allows investigating neuronal excitability and synaptic transmission at a multi-cellular scale. So far, achieving this 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 ${\mathrm{Ca}}^{2+}$ indicators to record signals elicited by electrical stimulation in hippocampal slices.

Transversal mouse hippocampal slices were stained with EF-630. ${\mathrm{Ca}}^{2+}$ 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 the hippocampal areas of $\sim 750\times 250{\mu m}^2$ at $1\text{-}\mu m$ pixel resolution.

After assessing all ${\mathrm{Ca}}^{2+}$ indicators, we selected Calbryte520 for achieving $>30\text{-}\min$ stable recordings in combination with EF-630. Action potentials and related ${\mathrm{Ca}}^{2+}$ transients were sequentially 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 unprecedented optical measurements of both electrical and ${\mathrm{Ca}}^{2+}$ transients in brain slices, providing unique information on neuronal excitability and network activity.