Two-photon voltage imaging of spontaneous activity from multiple neurons reveals network activity in brain tissue

Abstract

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...

Scanless two-photon voltage imaging

“…However, the acquisition rate of conventional 2P-LSM is limited and millisecond transients such as action potentials can only be detected by drastic reduction of the field of view [24][25][26][27] . As a result, several specialised scanning-based techniques have been developed to image neural activity across larger areas at kilohertz rates [28][29][30][31][32][33][34][35] and have yielded spectacular results, such as recording the voltage dynamics of cortical neurons in layer 5 in awake behaving mice. However, these methods are extremely technically demanding, have thus-far been limited to imaging a few cells simultaneously, and…”

Spontaneous and multifaceted ATP release from astrocytes at the scale of hundreds of synapses

“…Pregnant ICR mice were purchased from Japan SLC Inc., and in utero electroporation (IUE) was performed as previously described ( 66 ) with the following modifications. Mice at gestation day 15 were deeply anesthetized with either a mixture of medetomidine (0.3 mg/kg body weight), midazolam (4 mg/kg) and butorphanol (5 mg/kg) or with sodium pentobarbital (50 mg/kg) by intraperitoneal administration.…”

Detecting rapid pan-cortical voltage dynamics in vivo with a brighter and faster voltage indicator

“…1a). We chose to focus on this class of indicators because 34 they have been shown to express efficiently and report voltage in a variety of neuronal types and experimental preparations 24,25,[35][36][37][38] In addition, cpGFP-based sensors have the potential to produce larger responses than FRET-based indicators 34 .…”