Biological and Medicinal Chemistry

Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue

Abstract

Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemical synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically-encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer (PeT) triggered RhoVR voltage-sensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethyleneglycol (PEG) linker (RhoVR-Halos). When applied to cells, RhoVR-Halos selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halos maintain high voltage sensitivities—up to 34% ΔF/F per 100 mV—and fast response times typical of untargeted RhoVRs, while gaining the selectivity typical of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca2+ indicators like GCaMP to provide simultaneous voltage and Ca2+ imaging. In brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read out of action potentials from labeled cortical neurons in rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically-encodable systems, permitting imaging modalities inaccessible to either technique individually.

Content

Thumbnail image of 01 EWM RhoVR Halo Formatted.pdf

Supplementary material

Thumbnail image of 02 EWM RhoVR Halo Supporting Information.pdf
02 EWM RhoVR Halo Supporting Information