Characterization of second generation voltage-sensitive fluorescent proteins as genetically-encoded probes of membrane voltage of neurons in vitro

Over the last 10 years, our laboratory designed Voltage-Sensitive Fluorescent Proteins (VSFPs) as an optogenetic tool to study neuronal circuit dynamics. In contrast to conventional voltage sensitive dyes, VSFPs allow for a precise control of the expression of such protein probes to selected cell populations, thus avoiding background noise and enabling a rigorous source assignment of optical response signals. The second generation of these probes (VSFP2s) uses the voltage sensor domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) linked at its C-terminal end to a tandem of fluorescent proteins that act as a FRET reporter (Dimitrov et al, 2007). VSFP2.3 and VSFP2.4 are advanced VSFP2 versions containing a tandem of Cerulean/Citrine and Citrine/mKate2, respectively. The present experiments were designed to characterize the functional properties of these probes in cultured neurons and in an acute brain slice preparation. In single pyramidal cells from hippocampal cell cultures transfected with a VSFP2.3 plasmid, we observed ratiometric VSFP2.3 donor and acceptor fluorescence signals in response to an intracellular injection of depolarizing and hyperpolarizing current steps. Using VSFP imaging, we optically recorded spontaneous action potential bursts in both proximal and distal processes. To establish the properties of these VSFP2s in acute brain slices, we performed in-utero electroporation of VSFP2.3/2.4 DNA at embryonic age E15.5, resulting in high expression of VSFP2s in cortical pyramidal cells. Confocal imaging of brain slices from mice (P > 25) revealed bright VSFP fluorescence from plasma membranes of pyramidal cells in layer 2/3 and layer 5 over a large part of the somatosensory cortex. From layer 2/3 pyramidal cell bodies in acute brain slices, we recorded differential VSFP responses to somatic voltage steps and to voltage transients evoked by current pulses or synaptic stimulation in single and averaged trials. Slower membrane potential waveforms like EPSPs were reported at a higher gain than fast action potentials, as expected from the kinetic properties of these probes. Taken together our data validate the function of VSFP2s as optical ratiometric probes for membrane potential in neurons from intact brain tissue with single cell and single trail sensitivity.