Abstract
Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.
Highlights
Optogenetics has greatly advanced our ability to investigate how neural circuits process information and generate behaviour by allowing manipulation of neural activity with high spatio-temporal resolution in genetically-defined neurons (Miesenbock, 2009; Boyden, 2011; Miesenbock, 2011; Adamantidis et al, 2015; Boyden, 2015; Deisseroth, 2015; Deisseroth and Hegemann, 2017)
Two primary experimental requirements should be met to enable controlled and reproducible in vivo optogenetic circuit manipulations: (i) reproducible opsin expression levels, with stable expression systems offering higher reliability and homogeneity than transient ones (Kikuta and Kawakami, 2009; Yizhar et al, 2011; Sjulson et al, 2016), and (ii) calibrated photocurrents recorded in target neurons (Huber et al, 2008; Li et al, 2019)
High levels of expression were achieved in most cases (Figure 1C), with only few opsins showing intracellular puncta suggestive of incomplete trafficking to the plasma membrane (CheRiff and GtACR2) or low expression (Chronos)
Summary
Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. We developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. We performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
