Development of a Red Genetically-Encoded Voltage Indicator and its use with Channelrhodopsin for All-Optical Electrophysiology

Abstract

The need for effective genetically-encoded voltage indicators is widely recognized. Several prototypes have been made to date by fusing one or more fluorescent proteins to a voltage sensitive membrane protein (1). However, one factor that has limited the utility of these indicators is poor voltage sensitivity, making it a challenge to express the indicators in neurons and still observe detectable responses (2). In addition, none of the genetically-encoded voltage indicators reported can be combined with channelrhodopsin for optical readout of electrical activity in an all-optical electrophysiology setup. To address these limitations, we set up a directed evolution strategy to screen for both voltage indicator brightness and voltage sensitivity. First, bacterial expression enables us to screen thousands of clones in colonies of E. coli to improve the brightness of the indicator. Second, a medium throughput mammalian electric field stimulation system allows us to test for function of the voltage indicators. This screening has resulted in a family of red voltage indicators (VSDRs). They consist of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase linked to red fluorescent protein mApple. The fluorescence intensity of the VSDRs increases in response to depolarization. We show that the combination of signal size and response speed of VSDRs allows the reliable detection of spontaneous action potentials in cultured mammalian neurons in single trials with widefield fluorescent light microscopy. Critically, we also demonstrate that VSDRs faithfully reports signals from all-optical electrophysiology experiments using channelrhodopsin to depolarize mammalian cells.1. B. J. Baker et al., J. Neurosci. Methods. 2007, 161, 32-38.2. W. Akemann et al., Nat Methods. 7, 2010, 643-649.