Final Report Summary - MULTIGEVOS (The other side of optogenetics: multicolored genetically encoded hybrid voltage sensors (GEVOS) for ultrafast membrane potential measurements in cortical microcircuits) Optical voltage sensing using genetically expressed probes is highly desirable for large scale recordings of neuronal activity. The presently available genetically encodable Ca2+ indicators (GECI) are not well-suited for accurate detection of single action potentials (APs) and are unable to record membrane hyperpolarizations or depolarizations below AP threshold. The genetically encodable voltage indicators (GEVI) currently in use also have several drawbacks including slow response, low fluorescence, or excessive bleaching. The hybrid voltage sensing approach uses a genetically encoded fluorophore targeted to the membrane and a small quencher molecule that moves in the membrane in a voltage-dependent manner to quench or unquench the fluorophore. The most widely used quencher is dipycrilamine (DPA). However, this molecule presents several drawbacks including its explosive properties, large capacitive load on the membrane, and a narrow absorption spectrum. We have set out to search for molecules to replace DPA in the hybrid voltage sensing approach. After testing several compounds, we identified an inorganic dye (D3) with an absorption spectrum comparable to that of DPA. In hybrid voltage sensing experiments, D3 outperformed DPA in every aspect studied. Whereas DPA significantly increased membrane capacitance at a concentration of 3-5 μM, as previously reported, D3 at 20 μM did not produce significant changes in resting membrane potential, input resistance, membrane capacitance, AP threshold or width of AP at half amplitude. When optically measuring the voltage signal with 10 μM D3 in cultured mouse or rat cortical neurons expressing membrane-targeted GFP, both hyperpolarizations and depolarizations below AP threshold could be recorded with a significantly more linear voltage response than that given by DPA. Optical recordings sampled at >1,300 s-1 accurately reflected the shapes and amplitudes of APs elicited in the cultured neurons by depolarizing steps in whole-cell current-clamp recordings. One of the possible advantages of D3 was observed in an optical recording lasting over 60 min after the dye has been washed out from the media surrounding the cells. This indicates that D3 remains in the membrane in the vicinity of the fluorophore for a significant amount of time even after washout. This property will most likely enable its future use in vivo. Our findings indicate that the hybrid voltage sensor method with compounds other than DPA may hold great promise for the GEVI approach, possibly even for fluorophores spanning over a wide range of emission wavelengths.
