Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain

Abstract

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 ...

Kilohertz two-photon fluorescence microscopy imaging of neural activityin vivo

“…3d ). These characteristic time constants are consistent with those measured in an independent work [12].…”

“…Finally and most importantly, we imaged neurons expressing genetically encoded voltage indicator ASAP3 [12] in V1 of head-fixed awake mice. Among all voltage indicators, the ASAP family are currently the only ones that monitor voltage signal in brain slice or in vivo with 2PFM, albeit within very restricted field of views [12–14]. As an inverse sensor, ASAP3 reports membrane depolarizations and action potentials as downward deflections in fluorescence.…”

High and asymmetric somato-dendritic coupling of V1 layer 5 neurons independent of visual stimulation and locomotion

“…Coupling could in422 principle also be observed with lower frequency back propagating action potentials, provided423 they are paired with tuft inputs(Larkum, 2013; Manita et al, 2015). Further experiments are424 needed to resolve these potential mechanisms in vivo, for example using voltage-sensitive425 dyes(Chavarha et al, 2018; Roome and Kuhn, 2018;Adam et al, 2019) or dendritic 426 electrophysiological recordings (Moore et al, 2017), that would provide the temporal 427 resolution to resolve the different types of dendritic events.428 Notably, both apical tuft signals and the somato-dendritic coupling may differ 429 between different subtypes of layer 5 pyramidal neurons. It is known that at least two main430 types exist: intratelencephalic neurons which connect cortical areas, and pyramidal tract 431 neurons which project to multiple subcortical areas (Harris and Shepherd, 2015; Gerfen, 432 Economo and Chandrashekar, 2018).…”

Advances in Engineering and Application of Optogenetic Indicators for Neuroscience

“…Therefore, optical voltage sensors are an ideal method to noninvasively measure electrical activity at single cell resolution. However, despite recent progress in development of new voltage sensitive probes [60,65,[80][81][82], voltage imaging still remains technically more challenging than calcium imaging. One of the major challenges is associated with the sub-millisecond timescale of membrane potential changes during neuronal activity.…”