High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks

Savitha Sridharan,Marta A Gajowa,Mora B Ogando,Uday K Jagadisan,Lamiae Abdeladim,Masato Sadahiro,Hayley A Bounds,William D Hendricks,Toby S Turney,Ian Tayler,Karthika Gopakumar,Ian Antón Oldenburg,Stephen G Brohawn,Hillel Adesnik

doi:10.1016/j.neuron.2022.01.008

Abstract

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.

Highlights

Microbial opsins, which flux ionic current in response to illumination, have empowered neuroscientists to causally perturb brain circuits, yielding fundamental insight into brain function (Fenno et al, 2011)
Structure-guided design of ultra-potent, ultrafast opsins based on ChroME To engineer an ultra-potent opsin that retains fast kinetics we exploited the opsin ChroME, which is a point mutant of the ultrafast opsin Chronos
Three mutants had notable qualities: a glutamate to aspartate mutation at site 118 in ChroME substantially sped up the kinetics, whereas an isoleucine to alanine at 134 increased peak photocurrents (ChroME: 1.5 ± 0.08 Normalized response Photocurrent (nA); ChroME I134A: 1.7 ± 0.1 nA; Figure 1B)

Summary

Introduction

Microbial opsins, which flux ionic current in response to illumination, have empowered neuroscientists to causally perturb brain circuits, yielding fundamental insight into brain function (Fenno et al, 2011). Many different opsins have been identified or engineered, writing in precise spatiotemporal patterns of neural activity requires opsins that enable the control of large groups of neurons with high temporal fidelity (Mardinly et al, 2018; Marshel et al, 2019; Prakash et al, 2012; Forli et al, 2018). Even one-photon optogenetics applications can benefit owing to thermal constraints during visible light illumination (Owen et al, 2019)

Methods

Results

Conclusion