Dual polarity voltage imaging reveals subthreshold dynamics and concurrent spiking patterns of multiple neuron-types

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between distinct, targeted cell populations. However, current indicator families lack the requisite sensitivity for in vivo multipopulation imaging. We describe high-performance green and red sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants, pAce and pAceR. Our indicators enable 0.4-1 kHz voltage recordings from >50 neurons per field-of-view in awake mice and [~]30-min continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered behavioral state-dependent interactions between distinct neocortical subclasses, as well as contributions to hippocampal field potentials from non-overlapping projection neuronal ensembles. By combining three mutually compatible indicators, we demonstrate concurrent triple-population voltage imaging. Our approach will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution. One Sentence SummaryA new suite of voltage sensors enables simultaneous cellular-resolution activity imaging from multiple, targeted neuron-types in awake animals.