Toward Better Genetically Encoded Sensors of Membrane Potential

Abstract

Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions. Optical signals of membrane potential are useful because membrane potential changes are a direct sign of both synaptic and action potentials. Here we describe recent improvements in the in vitro and in vivo signal size and kinetics of genetically encoded voltage indicators (GEVIs) and discuss their relationship to alternative sensors of neural activity.

Keywords: Ehud Isacoff; Thomas Knopfel; genetically encoded calcium indicators (GECIs); genetically encoded voltage indicators (GEVIs).

Figure 1

Schematic structures of three types of GEVIs. (A). Mosaic sensors combining the voltage sensitive domain of a voltage sensitive phosphatase and a fluorescent protein. (B). Sensors based on the voltage sensitivity of a microbial rhodopsin. (C). Sensors that are a combination of two separate molecules. Figure 1B was modified from [62] ). Figure 1C was modified from [32]

Figure 2

Characterization of ArcLight, a single FP mosaic sensor. (A). The location of the outward facing A227 amino acid. (B). The A227D mutation increased the signal size by a factor of 15. (C). A further increase in signal was found if the linker between the S4 segment and the FP was shortened. (D). The fluorescence change vs membrane potential was sigmoidal with a V_1/2 of −20 mV. Modified from [27].

Figure 3

A simultaneous optical and electrode measurement of an action potential in a cultured hippocampal neuron using QuasAr2, a single chromophore microbial rhodopsin GEVI. Modified from [62].

Figure 4

In vivo individual neuron spikes measured in a layer 2/3 visual cortical neuron of an awake mouse using Ace2N-4AA-mNeon, a microbial rhodopsin FRET quenching GEVI. The spike signals are riding on top of longer lasting depolarizations. Modified from [4].

Figure 5

(A,B) In vivo population measurements using ArcLight, a single FP mosaic GEVI. The mitral/tufted cells were transduced by infection with an AAV1 virus containing ArcLight DNA. (A). A single trial, single glomerulus measurement of the response to the odorant ethyl tiglate presented during four breaths. (B). The overlay of the responses to six repeated trials. There was little change from trial to trial. (C). Comparing ArcLight and GCaMP3 signals from opposite olfactory bulbs in the same preparation in response to odorant presentations lasting one and two breaths. (D). Comparing ArcLight and GCaMP6f. Modified from [12].

Figure 6

The effect of monomeric mutations at the dimerization site of a single FP mosaic GEVI. A. The GEVI TM with 206A-221L-223F which favor dimerization has the largest signal (orange). Individual mutations favoring the monomer at the three sites reduced the signal; double mutations cause a further reduction. B. A schematic model of a TM dimer. Modified from [59].