Summary Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the function of neuronal circuits. The combination of the fast scanning by random-access multiphoton microscopy (RAMP) and the latest two-photon-compatible high-performance fluorescent genetically encoded voltage indicators (GEVIs) has enabled action potential detection in deep layers in in vivo brain. However, neuron connectivity analysis on optically recorded action potentials from multiple neurons in brain tissue has yet to be achieved. With high expression of a two-photon-compatible GEVI, ASAP3, via in utero electroporation and RAMP, we achieved voltage recording of spontaneous activities from multiple neurons in brain slice. We provide evidence for the developmental changes in intralaminar horizontal connections in somatosensory cortex layer 2/3 with a greater sensitivity than calcium imaging. This method thus enables investigation of neuronal network connectivity at the cellular resolution in brain tissue.

Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the structure and function of neuronal circuits. Using fluorescent genetically encoded voltage indicators (GEVI) would be especially desirable, as it would allow cell type-selectivity, longitudinal recordings, and further optical manipulations. By expressing the GEVI ASAP3 via in utero electroporation and rapidly imaging neurons in densely labelled tissues via random-access multi-photon microscopy, we achieve voltage recording of multiple neurons in brain slice with single-trial single-voxel resolution. This approach enables monitoring of subthreshold membrane potential changes and action potentials from multiple locations in soma and dendrites for tens of minutes. By optically recording spontaneous electrical activities in somatosensory cortex neurons, we provide evidence for the development of intralaminar horizontal connections in layer 2/3 with greater sensitivity than calcium imaging. Single-trial optical voltage recordings using ASAP3 thus enables the investigation of network connectivity at cellular resolution.indicators must reside within the plasma membrane to sense changes in transmembrane voltage 6 , the number of responding indicator molecules, and thereby changes in photon flux per voltage transient are significantly limited 7 . In addition to the unfavorable spatial constraint on voltage indicators, the transient nature of voltage also poses a problem. While GECIs can be sampled at slow frame rates of ~30 Hz to detect the long-lasting calcium fluctuations that follow action potentials, sub-millisecond sampling rates are required to detect the transient optical spikes of fast GEVIs in response to action potentials 7 . The higher sampling rate resulting fewer photons collected per frame leads to lower signal-to-noise ratios (SNRs). Excitation power can be increased to boost SNR, but at the price of greater photobleaching and phototoxicity 8 . Given these constraints, to detect fast voltage transients by voltage imaging, GEVIs must have high response amplitudes and fast response kinetics, and be bright and photostable. At the same time, methods to measure GEVI signals must maximize photon flux from the limited area of plasma membrane over the short durations of voltage transients, i.e. be both sensitive and fast.After years of intense engineering efforts, the latest generation of GEVIs now have sufficiently improved performance to be deployed in functional in vivo studies 7,9,10 . For this study we selected a recently reported ASAP3 for its fast kinetics (fast time constant of 0.94 ± 0.06 ms at physiological temperatures) and high responsivity (17.0 ± 0.8% per action potential (AP) in cultured neurons). Most importantly, ASAP3 maintains its responsivity to voltage changes under two-photon excitation, which enabled discrimination of individual spikes in fast-firing neurons in tissue 7 .Although GEVIs have been used to monitor electrical activity in neuronal tissue using widefield singlephoton mic...