Optical Recording of Electrical Activity of Cortical Layer 2/3 Pyramidal Neurons Using A Genetically-Encoded Voltage Probe

Voltage-Sensitive Fluorescent Protein 2.3, VSFP2.3, is a genetically-encoded probe of membrane voltage using fluorescence resonance energy transfer (FRET) between a pair of cyan (CFP) and yellow (YFP) fluorescent proteins to convert voltage-activated motions of a voltage sensor domain from Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) into a differential voltage dependent fluorescence signal. To evaluate the utility of VSFP2.3 as a probe of electrical activity of neurons in intact brain tissue, we performed targeted whole cell current clamp and simultaneous optical recordings from L2/3 pyramidal cells of the mouse cerebral cortex in acute brain slices. VSFP2.3 expression in neocortical cells was achieved by in-utero electroporation into the cortical ventricular zone at embryonic age 15.5 of a plasmid vector containing VSFP2.3 under the CAG hybrid promoter. This procedure resulted in strong VSFP2.3 fluorescence at postnatal age (up to day 30 tested) from a restricted cortical area, mostly within somato-sensory cortex, with the fluorescence originating from a clustered population of pyramidal neurons with cell bodies in layer 2/3. Electric current injection into VSFP2.3-positive cells (postnatal day 16-22) revealed an optical response signal to sub-threshold slow depolarization of the somatic membrane that could be resolved in single trials. While the optical signal in response to fast action potentials was noisy in single trials, S/N above two was obtained by event-triggered averaging over a few (5-10) action potentials. We also tested for the optical response to synaptically evoked EPSPs which were reliably detected at near threshold amplitudes in single trials. Our results provide the first demonstration of an optical readout of neuronal activity at cellular resolution using a genetically-targetable voltage probe in intact brain tissue in-vitro.