STABLE<EM> IN VIVO</EM> 2-PHOTON VOLTAGE RECORDING OF CORTICAL MICROCIRCUITS USING NEXT-GENERATION ASAP-CLASS GENETICALLY ENCODED VOLTAGE INDICATORS

Understanding how information is represented and processed in the brain requires technologies capable of recording transmembrane potential in defined neuronal populations with high fidelity. However, optical voltage recording in vivo and at depth using 2-photon(2P) microscopy has been limited by the speed, sensitivity and photobleaching of available Genetically Encoded Voltage Indicators (GEVIs).
We have focused on developing the ASAP class of GEVIs, which display fast kinetics and high responsivity under 1P and 2P microscopy. JEDI-2P, an early ASAP derivative, showed improved photostability under 2P excitation and compatibility with deep cortical recordings.
To expand the GEVIs toolbox, refined indicators based on the JEDI-2P backbone were developed and characterized using ULoVE, an Acousto-Optic Deflectors (AODs)-based recording method with kHz sampling rates. We will present JEDI3-sub and JEDI3-hyp, bright-to-dim GEVIs with enhanced sensitivity for subthreshold fluctuations. We will also introduce FORCE1, a novel dim-to-bright GEVI that is photostable and reports spike amplitudes larger than 100% ∆F/F. These indicators were validated in layer 2/3 cortical pyramidal cells and fast-spiking interneurons in head-fixed behaving mice during tens-of-minutes recordings.
Lastly, we demonstrate that the combined high responsivity, photostability and slower repolarization kinetics of FORCE1 enable high-resolution GEVI signal acquisition by imaging at relatively low sampling rates (~250Hz) using an AODs-based method termed diagonal scanning. Notably, we achieved one-hour continuous imaging of 10 neurons in behaving mice, while preserving spike resolution over time.
Taken together, these results validate a GEVI toolbox compatible with long-lasting in vivo recordings and well-suited for studying brain microcircuits under diverse experimental conditions.