Different categories of fluorescent proteins result in GEVIs with similar characteristics

Abstract

The latest generation of genetically encoded voltage indicators (GEVIs) is significantly advancing our ability to study electrical activity from large numbers of identified neurons. The further refinement of the technology will contribute to our understanding of behavior-evoked information perception, transfer and processing on a cellular level across brain regions. The development of GEVIs relies on synthetic biology which includes rational and random modifications of indicator sequence. One strategy in GEVI design is based on creating chimeras between voltage sensitive protein domains (VSDs) and fluorescent proteins (FPs). However, in this design scenario, the mechanistic details of voltage-induced fluorescence change that would inform rational design and improvements of GEVIs are still largely missing. Here we preformed a systematic study of how nature of the FP and altering the insertion site affects the characteristics of Ciona intestinalis voltage-sensitive phosphatase-based GEVIs. Surprisingly, we found that regardless of vast difference in phylogenesis, biochemical properties, fluorophore structure, sequence and excitation/emission spectra between FPs, the resulting GEVIs exhibit virtually identical decrease in fluorescence intensity in response to depolarization. These results stand in strong contrast to studies demonstrating that small numbers of targeted mutations in the FP sequence cause dramatic changes in both signal size and polarity.

High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations

“…Library production. The production of GEVI site-directed mutagenic libraries was described previously (Platisa et al, 2017(Platisa et al, , 2020. Briefly, the mutagenic PCR reaction was run with a mix of the three forward primers containing three degenerative codons (NDT, VHG, or TGG) and the single reverse primer.…”

“…Functional testing of mutagenic libraries under widefield excitation. The functional screening of GEVI mutants is done as previously described (Platisa et al, 2020;Platisa et al, 2017). In short, a semi-automated screening platform was built around a Nikon Eclipse Ti-E inverted microscope equipped with a Perfect Focus System and a motorized Prior Proscan II stage (Prior Scientific, Inc., USA).…”

Voltage imaging using transgenic mouse lines expressing the GEVI ArcLight in two olfactory cell types

“…Genetically encoded voltage indicators (GEVIs) can report changes in membrane potential from genetically defined cell-types. The membrane localization, and the spatial and temporal complexity of these signals make the design and production of GEVIs challenging [1][2][3][4][5][6]. However, recent advances in GEVI development have resulted in improvements in the sensitivity, speed and signal-to-noise ratio in several GEVIs, allowing for enhanced detection of electrical transients in vivo [7][8][9][10][11][12][13][14][15][16][17].…”