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

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.

Ultra-parallel label-free optophysiology of neural activity

“…The optical characteristics of each channel could be individually profiled and cumulated to convert optical responses into electrophysiological measurements. Recent studies have precisely calibrated the relationship between the incident optical power on optically-sensitive neurons and the number of action potentials evoked in response ( Sridharan et al., 2022 ). A similar experimental strategy could be adapted to observe the optical response profiles of optically-triggered individual action potentials, without the need for electrodes that reduce the phase stability of the measurements.…”

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

“…Further control has been sought by combining optogenetics with classical electrophysiology (electro-optogenetics) or combining voltage sensors and neuronal activators and/or silencers both based on rhodopsins (all-optical electrophysiology) ( Hochbaum et al, 2014 ; Afshar Saber et al, 2018 ; Sridharan et al, 2022 ). In the latter case, it is important to separate the spectral sensitivities to allow selective control and avoid optical cross-talk.…”

Optogenetic Methods to Investigate Brain Alterations in Preclinical Models

“…From Chronos, a new opsin has been derived, ChroME [ 128 ]; the two molecules share similar action spectra and kinetics, but the latter displays greater photocurrent amplitude; hence, lower laser power and shorter light pulses are required to elicit spikes. Recently, ChroME has been used as a scaffold for the generation of new variants, namely ChroME2f and ChroMe2s, with enhanced photocurrent and fast decay time, respectively [ 129 ].…”