A technology to record membrane potential from multiple neurons, simultaneously, in behaving animals will have a transformative impact on neuroscience research . Genetically encoded voltage indicators are a promising tool for these purposes, but were so far limited to single-cell recordings with marginal signal to noise ratio (SNR) in vivo [4][5][6] . We developed improved near infrared voltage indicators, high speed microscopes and targeted gene expression schemes which enabled recordings of supra-and subthreshold voltage dynamics from multiple neurons simultaneously in mouse hippocampus, in vivo. The reporters revealed sub-cellular details of back-propagating action potentials, correlations in sub-threshold voltage between multiple cells, and changes in dynamics associated with transitions from resting to locomotion. In combination with optogenetic stimulation, the reporters revealed brain state-dependent changes in neuronal excitability, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behavior. Main textArchaerhodopsin-derived genetically encoded voltage indicators (GEVIs) are excited by red light and emit in the near infrared part of the spectrum, have sub-millisecond response times at physiological temperature, and report neuronal action potentials with a ~40% change in fluorescence brightness [7][8][9] . A transgenic mouse expressing the Arch-based GEVI QuasAr2 and the channelrhodopsin CheRiff (together called 'Optopatch2') enabled optical stimulation and recording in sparsely expressing acute brain slices 4 , but did not attain adequate SNR for voltage imaging in brain in vivo. The primary factors which limited SNR were (a) poor trafficking of QuasAr2 in vivo, leading to intracellular aggregates that contributed to fluorescence background, and (b) high background, due to light scattering, out-of-focus fluorescence, and fluorescence from optically unresolvable neuropil.To address the trafficking challenge, we systematically tested the impacts of trafficking sequences, appended fluorescent proteins, linker sequences, and various point mutations (Methods). We used the Optopatch system 7 to test the SNR for optogenetically induced action potentials first in All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/281618 doi: bioRxiv preprint first posted online Mar. 13, 2018; 2 cultured neurons, and then in acute brain slices (Fig. S1). Incorporation of multiple K ir 2.1 trafficking sequences, a Citrine fluorescent protein fusion (instead of mOrange2), and mutation of a putative ubiquitination site (lysine 171) to arginine, led to a construct that showed high expression and excellent trafficking in vivo, which we called QuasAr3 (Fig. 1). In acute cortical slices, QuasAr2 produced bright intracell...

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