Highlights d A PCR-based electroporation screen yielded an improved voltage indicator, ASAP3 d ASAP3 shows larger voltage responses than other fluorescent protein-based sensors d Ultrafast local volume excitation (ULoVE) boosts randomaccess two-photon signals d ASAP3 and ULoVE report subthreshold and spiking potentials in deep brain regions

Understanding information processing in the brain requires us to monitor neural activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope (2PFM) empowered by all-optical laser scanning, we imaged neural activity in vivo at up to 3,000 frames per second and submicron spatial resolution. This ultrafast imaging method enabled monitoring of both supra- and sub-threshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.

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Imaging of transmembrane voltage deep in brain tissue with cellular resolution has the potential to reveal information processing by neuronal circuits in living animals with minimal perturbation. Multi-photon voltage imaging in vivo, however, is currently limited by speed and sensitivity of both indicators and imaging methods. Here, we report the engineering of an improved genetically encoded voltage indicator, ASAP3, which exhibits up to 51% fluorescence responses in the physiological voltage range, sub-millisecond activation kinetics, and full responsivity under two-photon illumination. We also introduce an ultrafast local volume excitation (ULOVE) two-photon scanning method to sample ASAP3 signals in awake mice at kilohertz rates with increased stability and sensitivity. ASAP3 and ULOVE allowed continuous single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution, and with repeated sampling over multiple days. By imaging voltage in visual cortex neurons, we found evidence for cell type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULOVE enable continuous high-speed highresolution imaging of electrical activity in deeply located genetically defined neurons during awake behavior.shaping 7,8 . However, parallel excitation is problematic for imaging, as scattered fluorescence signals from simultaneously excited cells would become intermixed, hindering detection of small responses. Furthermore, as GEVIs reside in the membrane rather than the cytosol, the number of indicator molecules that can be excited in an optical section through a mammalian cell body is typically smaller for GEVIs than for GECIs 4,6 .Given these limitations, the development of GEVIs with larger two-photon responses to electrical events of interest is highly desirable. Currently, the GEVIs with the largest responses to both subthreshold changes and APs are based on two types of voltage-sensing domains: seven-transmembrane helix opsins and four-transemembrane helix voltage-sensing domains (VSDs). Opsin-based GEVIs have been used in vivo with one-photon excitation to report electrical activity of superficially located neurons 9,10 , but their responsivity is severely attenuated under twophoton excitation 4,11 . In contrast, ASAP-family GEVIs, composed of a circularly permuted green fluorescent protein variant inserted within the VSD of G. gallus voltage-sensing phosphatase (Fig. 1a), are fully responsive under twophoton excitation 11 . In particular, ASAP2s demonstrates the largest response per AP of fluorescent protein-based GEVIs, but its kinetics are actually slower than earlier ASAP variants 11 . If ASAP-family kinetics and/or overall responsivity could be improved, then electrical events could be more easily detected by two-photon imaging.Here we report an improved indicator, ASAP3, resulting from novel methods for generation and screening of GEVI libraries in mammalian cells. ASAP3 features the largest responses of fluorescent GEVIs to either steady-state voltages or ...

Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.Electronic supplementary materialThe online version of this article (10.1186/s13041-018-0374-7) contains supplementary material, which is available to authorized users.

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Neuronal activity is routinely recorded in vivo using genetically encoded calcium indicators (GECIs) and 2-photon microscopy, but calcium imaging is poorly sensitive for single voltage spikes under typical population imaging conditions, lacks temporal precision, and does not report subthreshold voltage changes. Genetically encoded voltage indicators (GEVIs) offer better temporal resolution and subthreshold sensitivity, but 2-photon detection of single spikes in vivo using GEVIs has required specialized imaging equipment. Here, we report ASAP4b and ASAP4e, two GEVIs that brighten in response to membrane depolarization, inverting the fluorescence-voltage relationship of previous ASAP-family GEVIs. ASAP4b and ASAP4e feature 180% and 210% fluorescence increases to 100-mV depolarizations, respectively, as well as modestly prolonged deactivation and high photostability. We demonstrate single-trial detection of spikes and oscillations in vivo with standard 1 and 2-photon imaging systems, and confirm improved temporal resolution in comparison to calcium imaging on the same equipment. Thus, ASAP4b and ASAP4e GEVIs extend the uses of existing imaging equipment to include multiunit voltage imaging in vivo.One Sentence SummaryPositively tuned ASAP voltage indicators facilitate imaging of electrical activity in the brain.

Acetylcholine (ACh) is an important neuromodulator in various cognitive functions. However, it is unclear how ACh influences neural circuit dynamics by altering cellular properties. Here, we investigated how ACh influences reverberatory activity in cultured neuronal networks. We found that ACh suppressed the occurrence of evoked reverberation at low to moderate doses, but to a much lesser extent at high doses. Moreover, high doses of ACh caused a longer duration of evoked reverberation, and a higher occurrence of spontaneous activity. With whole-cell recording from single neurons, we found that ACh inhibited excitatory postsynaptic currents (EPSCs) while elevating neuronal firing in a dose-dependent manner. Furthermore, all ACh-induced cellular and network changes were blocked by muscarinic, but not nicotinic receptor antagonists. With computational modeling, we found that simulated changes in EPSCs and the excitability of single cells mimicking the effects of ACh indeed modulated the evoked network reverberation similar to experimental observations. Thus, ACh modulates network dynamics in a biphasic fashion, probably by inhibiting excitatory synaptic transmission and facilitating neuronal excitability through muscarinic signaling pathways.