Advances in optical techniques and two-photon (2P) sensitive genetic voltage indicators (GEVIs) enabled in-depth voltage imaging at single-spike and single-cell resolution. These results were achieved using ASAP-type sensors, while rhodopsin-based GEVIs were mainly used with one-photon (1P) illumination. Here, we demonstrate compatibility of rhodopsin-based GEVIs with 2P illumination. We rationally engineer a fully genetically encoded, rhodopsin-based GEVI, just another voltage indicating sensor (Jarvis), and demonstrate its utility under 1P and 2P illumination. We further show 2P usability of the fluorescence resonance energy transfer (FRET)-opsin GEVIs pAce and Voltron2. Comparing 2P scanless with fast 2P scanning illumination revealed that responses are resolved with both approaches, but FRET-opsin GEVIs show improved signal-to-noise ratio (SNR) with low irradiance, inherent to scanless illumination. Utilizing Jarvis and pAce, we establish high-SNR action potential detection at kilohertz imaging rates in mouse hippocampal slices, zebrafish larvae, and the cortex of awake mice, demonstrating high-contrast action potential detection under 2P illumination with rhodopsin-based GEVIs in vitro and in vivo.

Abstract For the brain to compute, electrical signals must propagate over the membranes of individual neurons, connecting synaptic inputs to synaptic outputs 1 . Complex neuronal morphologies coupled with the spatial organization of synaptic inputs and outputs enable diverse voltage transformations that underlie cell-type specific computations 2,3 . However, measuring these transformations in vivo has remained challenging, leaving a crucial gap in our mechanistic understanding of single neuron computation. Here, we develop ASAP7y, a genetically encoded voltage indicator with unprecedented subthreshold sensitivity and expanded excitation compatibility in both mice and flies. We leveraged ASAP7y combined with two-photon random-access microscopy to record sensory stimulus-evoked voltage dynamics with millisecond, subcellular, and subthreshold resolution along the neurites of individual neurons in Drosophila . We found remarkable heterogeneity in voltage propagation across cell-types, delineating a fundamental axis of electrical diversity. Leveraging a nanoscale EM reconstruction of the visual system 4 , we modeled the electrotonic properties of single neurons spanning 717 cell types, revealing how morphology shapes voltage transformations. Finally, we demonstrate that confined voltage propagation creates substrates for local computation, producing subcellular domains with distinct feature selectivity across multiple cell types. These results provide mechanistic insight into how critical single neuron computations arise and reveal parallel processing in single neurons.

ABSTRACTSignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities forin vitroandin vivofluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.AimWe describe the development and applications of T-GECO1 – a high-performance blue-shifted GECI based on theClavularia sp.-derived mTFP1.ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performancein vitrousing one-photon excitation in cultured rat hippocampal neurons,in vivousing one-photon excitation fiber photometry in mice, andex vivousing two-photon Ca2+imaging in hippocampal slices.ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparentKdfor Ca2+is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Parallel light-sculpting methods have been used to perform scanless two-photon photostimulation of multiple neurons simultaneously during all-optical neurophysiology experiments. We demonstrate that scanless two-photon excitation also enables high-resolution, high-contrast, voltage imaging by efficiently exciting fluorescence in a large fraction of the cellular soma. We present a thorough characterisation of scanless two-photon voltage imaging using existing parallel approaches and lasers with different repetition rates. We demonstrate voltage recordings of high frequency spike trains and sub-threshold depolarizations in intact brain tissue from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-kv. Using a low repetition-rate laser, we perform recordings from up to ten neurons simultaneously. Finally, by co-expressing JEDI-2P-kv and the channelrhodopsin ChroME-ST in neurons of hippocampal organotypic slices, we perform single-beam, simultaneous, two-photon voltage imaging and photostimulation. This enables in-situ validation of the precise number and timing of light evoked action potentials and will pave the way for rapid and scalable identification of functional brain connections in intact neural circuits.

AbstractOne of the central goals of neuroscience is to decipher the specific contributions of neural mechanisms to different aspects of sensory perception. Since achieving this goal requires tools capable of precisely perturbing and monitoring neural activity across a multitude of spatiotemporal scales, this aim has inspired the innovation of many optical technologies capable of manipulating and recording neural activity in a minimally invasive manner. The interdisciplinary nature of neurophotonics requires a broad knowledge base in order to successfully develop and apply these technologies, and one of the principal aims of this chapter is to provide some basic but fundamental background information in terms of both physiology and optics in the context of all-optical two-photon neurophysiology experiments. Most of this information is expected to be familiar to readers experienced in either domain, but is presented here with the aim of bridging the divide between disciplines in order to enable physicists and engineers to develop useful optical technologies or for neuroscientists to select appropriate tools and apply them to their maximum potential.The first section of this chapter is dedicated to a brief overview of some basic principles of neural physiology relevant for controlling and recording neuronal activity using light. Then, the selection of appropriate actuators and sensors for manipulating and monitoring particular neural signals is discussed, with particular attention paid to kinetics and sensitivity. Some considerations for minimizing crosstalk in optical neurophysiology experiments are also introduced. Next, an overview of the state-of-the-art optical technologies is provided, including a description of suitable laser sources for two-photon excitation according to particular experimental requirements. Finally, some detailed, technical, information regarding the specific wavefront engineering approaches known as Generalized Phase Contrast (GPC) and temporal focusing is provided.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.

In this study, we explored the complex interactions between platelet-derived growth factor (PDGF) and N-methyl-d-aspartate receptor (NMDAR) and their effect on the excessive proliferation and migration of smooth muscle cells leading to obstructed arteries in pulmonary arterial hypertension (PAH). We report lower expression of glutamate receptor NMDA-type subunit 2B (GluN2B), a subunit composing NMDARs expected to affect cell survival/proliferation of pulmonary artery smooth muscle cells (PASMCs), in PAH patient lungs. PASMC exposure to PDGF-BB stimulated immediate increased levels of phosphorylated Src family kinases (SFKs) together with increased phosphorylated GluN2B (its active form) and cell surface relocalization, suggesting a cross talk between PDGFR-recruited SFKs and NMDAR. Selective inhibition of PDGFR-β or SFKs with imatinib or A-419259, respectively, on one hand, or with specific small-interfering RNAs (siRNAs) on the other hand, aborted PDGF-induced phosphorylation of GluN2B, thus validating the pathway. Selective inhibition of GluN2B using Rö25-6981 and silencing with specific siRNA, in the presence of PDGF-BB, significantly increased both migration and proliferation of PASMCs, thus strengthening the functional importance of the pathway. Together, these results indicate that GluN2B-type NMDAR activation may confer to PASMCs antiproliferative and antimigratory properties. The decreased levels of GluN2B observed in PAH pulmonary arteries could mediate the excessive proliferation of PASMCs, thus contributing to medial hyperplasia and PAH development.

Neurophysiologie tout-optique par excitation biphotonique sans-balayage Afin de comprendre les mécanismes qui sous-tendent les perceptions sensorielles, il est nécessaire, d'une part, d’identifier les neurones qui composent les réseaux concernés et, d'autre part, de déchiffrer les schémas spatiaux et temporels d'activation des différentes cellules en réponse à un stimulus. Les méthodes électrophysiologiques ont permis aux scientifiques d'enregistrer et de manipuler l'activité neuronale avec une grande fidélité, mais elles pâtissent d'une mauvaise résolution spatiale ou d'un rendement très faible. Le développement de l'optogénétique biphotonique au cours des vingt dernières années, ainsi que de nouvelles méthodes optiques complexes, a révolutionné le domaine des neurosciences en permettant de contrôler et d'enregistrer l'activité des populations neuronales avec une résolution cellulaire, dans des expériences dites "tout-optique". Au cours de ma thèse, j'ai travaillé sur la caractérisation biphotonique d'outils pour les deux aspects de la manipulation tout-optique des circuits neuronaux. Tout d'abord, en collaboration avec les laboratoires de Peter Hegemann et de J. Simon Wiegert, j'ai caractérisé une construction composée d'une rhodopsine excitatrice et d'une rhodopsine inhibitrice (appelée BiPOLES) sous excitation biphotonique. En utilisant l'illumination par holographie digitale (CGH), des tranches organotypiques d'hippocampe exprimant la construction et la technique du patch-clamp, j'ai exploré le spectre d'activation à deux photons de la construction et déterminé les longueurs d'onde optimales pour la photo-activation et l'inhibition. J'ai pu montrer que les potentiels d'action pouvaient être photo-évoqués de manière fiable à 1100 nm, et que la lumière de 920 nm permettait d'inhiber les potentiels d'action induits par le courant ou la lumière, via l'inhibition par shunting. La deuxième partie de ma thèse consistait à démontrer que les approches sans balayage pouvaient être utilisées pour l'imagerie de voltage biphotonique dans des échantillons densément marqués. En utilisant des tranches organotypiques d'hippocampe exprimant l'indicateur JEDI-2P et la technique de contraste de phase généralisé (GPC) couplé à l’approche de focalisation temporelle (TF), nous avons pu enregistrer optiquement des potentiels d'action uniques et des trains de potentiels d'action jusqu'à 125 Hz, à des vitesses d'acquisition variant de 500 Hz à 1 kHz. Nous avons également montré que les événements de faible amplitude (jusqu'à 1 mV) pouvaient être détectés en moyennant plusieurs répétitions. Des enregistrements d'activité spontanée de multiples cellules ont pu être réalisés. Enfin, en co-exprimant la rhodopsine ChroME-ST et JEDI-2P, nous avons photo-évoqué des potentiels d'action et les avons enregistrés, ce qui nous a permis de déduire le moment précis du déclenchement des potentiels d’action.