All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

Abstract

All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The “Optopatch” constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.

Current approaches to characterize micro- and macroscale circuit mechanisms of Parkinson’s disease in rodent models

“…Thus, fluorescent calcium signals are yet unable to resolve single action potentials (~ 1 ms), but rather serve as a proxy for events of neural activation. Recently, first studies using fluorescent voltage sensors have demonstrated proof-of-principle in rodent models and promise higher temporal resolution in future circuit studies ( Piatkevich et al, 2019 ; Tian et al, 2021 ). On the other hand, electrophysiological amplifiers can detect electrical signals on μs scale (filter frequency up to 20 kHz), exceeding the kinetics of the fastest ion channels and action potentials.…”

Automating the High-Throughput Screening of Protein-Based Optical Indicators and Actuators

“…68 A preprint also recently reported using a similar approach to screen ∼120 000 single cells for voltage indicators with increased brightness and response amplitude, in this case using a green-to-red photoconvertible FP for optical tagging. 69 A key benefit of microscopy-based pooled screening compared with multiwell-based screening is the much higher achievable throughput. For example, multiwell-based screening of 3 million variants, the number of YFPs variants we screened to develop mGold, would require screening four 96-well plates per day for 21 years (assuming one variant per well and screening four plates per day).…”

“…Three million single-cell variants expressed in yeast were screened using SPOTlight, leading to the identification of a bright and photostable yellow fluorescent protein (YFP) variant called mGold that is 4–5-fold more photostable than its predecessor . A preprint also recently reported using a similar approach to screen ∼120 000 single cells for voltage indicators with increased brightness and response amplitude, in this case using a green-to-red photoconvertible FP for optical tagging …”

On the Diverse Functions of Electrical Synapses

“…Extracellular methods, such as multi-electrode arrays, field recordings, and wide-scale imaging of fluorophores overcome this limitation, but do not reveal which neurons are coupled in a network or the strengths or spatial distributions of their coupling. One could hope that cell-specific optogenetics could mitigate the barrier ( Dakin and Li, 2006 ; Qiao and Sanes, 2016 ), and some all-imaging approaches have identified GJ-mediated signals ( Tian et al, 2021 ). In computational studies of neural networks, electrical synapses are most often neglected entirely, which we regard as a vast missed opportunity.…”