Electric potential changes across neuronal membranes form the basis of signaling in the brain. Monitoring these changes is therefore crucial to understanding how neuronal processes underlie both behavior and disease. Voltage-sensitive dyes (VSDs) and genetically encoded voltage indicators (GEVIs) have been developed to allow the monitoring of membrane potential (Vm) through fluorescence microscopy. We recently reported the synthesis of a new class of VSDs, Rhodamine-based Voltage Reporters (RhoVRs), which possess red-shifted excitation and emission profiles and high sensitivity to changes in Vm (up to 47% ΔF/F per 100 mV) at sub-millisecond response times. While effective reporters of Vm in dissociated cultures, VSDs indiscriminately label all cellular membranes, leading to high levels of background fluorescence that make their use in complex tissues difficult. To address this limitation, we have developed genetically targetable RhoVRs using the HaloTag system. HaloTag is a dehalogenase-based labeling system which forms a covalent linkage between a protein tag and a chemical ligand (chloroalkane). By expressing HaloTag extracellularly, then adding RhoVR linked to the HaloTag ligand via a flexible linker, we gained the selectivity inherent to GEVIs while maintaining the favorable photophysics of RhoVRs. A library of RhoVR-HaloTag derivatives was synthesized and screened in dissociated hippocampal neurons in order to minimize off-target labeling and maximize voltage sensitivity. The best performing RhoVR-HaloTag dyes were shown to possess high voltage sensitivities and SNRs better than many recently reported GEVIs. These RhoVR-HaloTags were then used to record Vm changes in acutely prepared mouse brain slice using both 1-photon and 2-photon illumination.

Genetically targeted tetramethylrhodamine voltage reporters

255th National Meeting (2018)

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