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Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics. This Review discusses temporal focusing microscopy and its applications in neuroscience for imaging and optogenetic activation.

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. Currently, bidirectional control of activity in the same neurons in the same experiment is difficult. Here the authors report a Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES, which they use in a range of organisms including worms, fruit flies, mice and ferrets.

Two-photon excitation with temporally focused pulses can be combined with phase-modulation approaches, such as computer-generated holography and generalized phase contrast, to efficiently distribute light into two-dimensional, axially confined, user-defined shapes. Adding lens-phase modulations to 2D-phase holograms enables remote axial pattern displacement as well as simultaneous pattern generation in multiple distinct planes. However, the axial confinement linearly degrades with lateral shape area in previous reports where axially shifted holographic shapes were not temporally focused. Here we report an optical system using two spatial light modulators to independently control transverse- and axial-target light distribution. This approach enables simultaneous axial translation of single or multiple spatiotemporally focused patterns across the sample volume while achieving the axial confinement of temporal focusing. We use the system's capability to photoconvert tens of Kaede-expressing neurons with single-cell resolution in live zebrafish larvae. Three-dimensional computer-generated holography cannot be implemented with temporal focusing. Here, Hernandez et al . use two spatial light modulators to control transverse- and axial-target light distribution, generating spatiotemporally focused patterns with uniform light distribution throughout the entire volume.

Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables the generation of precise spatiotemporal neuronal activity patterns and thus a broad range of experimental applications, such as high throughput connectivity mapping and probing neural codes for perception. Yet, current holographic approaches limit the resolution for tuning the relative spiking time of distinct cells to a few milliseconds, and the achievable number of targets to 100-200, depending on the working depth. To overcome these limitations and expand the capabilities of single-cell optogenetics, we introduce an ultra-fast sequential light targeting (FLiT) optical configuration based on the rapid switching of a temporally focused beam between holograms at kHz rates. We used FLiT to demonstrate two illumination protocols, termed hybrid- and cyclic-illumination, and achieve sub-millisecond control of sequential neuronal activation and high throughput multicell illumination in vitro (mouse organotypic and acute brain slices) and in vivo (zebrafish larvae and mice), while minimizing light-induced thermal rise. These approaches will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles. Current holographic approaches for neuronal stimulation have limitations in their temporal resolution and the number of targeted neurons. Here, the authors demonstrate an approach for ultra-fast holographic light targeting which, combined with optogenetics, enables sub-millisecond control of sequential neuronal activation and high throughput simultaneous multicell illumination.

In the past 10 years, the use of light has become irreplaceable for the optogenetic study and control of neurons and neural circuits. Optical techniques are however limited by scattering and can only see through a depth of few hundreds µm in living tissues. GRIN lens based micro-endoscopes represent a powerful solution to reach deeper regions. In this work we demonstrate that cutting edge optical methods for the precise photostimulation of multiple neurons in three dimensions can be performed through a GRIN lens. By spatio-temporally shaping a laser beam in the two-photon regime we project several tens of spatially confined targets in a volume of at least 100 × 150 × 300 µm 3 . We then apply such approach to the optogenetic stimulation of multiple neurons simultaneously in vivo in mice. Our work paves the way for an all-optical investigation of neural circuits in previously inaccessible brain areas.

Generalized phase contrast and temporal focusing are combined to shape two-photon excitation patterns that elicit large photocurrents in ChR2-expressing neurons in culture and slices. This method allows precise aiming of the stimulating light at single neuronal processes, neurons or groups of neurons and can elicit simultaneous excitation of multiple cells using optogenetics. Light-gated ion channels and pumps have made it possible to probe intact neural circuits by manipulating the activity of groups of genetically similar neurons. What is needed now is a method for precisely aiming the stimulating light at single neuronal processes, neurons or groups of neurons. We developed a method that combines generalized phase contrast with temporal focusing (TF-GPC) to shape two-photon excitation for this purpose. The illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups of cells. The TF-GPC two-photon excitation patterns generated large photocurrents in Channelrhodopsin-2–expressing cultured cells and neurons and in mouse acute cortical slices. The amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and, thereby, be used to trigger single action potentials or trains of action potentials.

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na+ channels.

NG2 cells, oligodendrocyte progenitors, receive a major synaptic input from interneurons in the developing neocortex. It is presumed that these precursors integrate cortical networks where they act as sensors of neuronal activity. We show that NG2 cells of the developing somatosensory cortex form a transient and structured synaptic network with interneurons that follows its own rules of connectivity. Fast-spiking interneurons, highly connected to NG2 cells, target proximal subcellular domains containing GABAA receptors with γ2 subunits. Conversely, non-fast-spiking interneurons, poorly connected with these progenitors, target distal sites lacking this subunit. In the network, interneuron-NG2 cell connectivity maps exhibit a local spatial arrangement reflecting innervation only by the nearest interneurons. This microcircuit architecture shows a connectivity peak at PN10, coinciding with a switch to massive oligodendrocyte differentiation. Hence, GABAergic innervation of NG2 cells is temporally and spatially regulated from the subcellular to the network level in coordination with the onset of oligodendrogenesis. Neurons are outnumbered in the brain by cells called glial cells. The brain contains various types of glial cells that perform a range of different jobs, including the supply of nutrients and the removal of dead neurons. The role of glial cells called oligodendrocytes is to produce a material called myelin: this is an electrical insulator that, when wrapped around a neuron, increases the speed at which electrical impulses can travel through the nervous system. Neurons communicate with one another through specialized junctions called synapses, and at one time it was thought that only neurons could form synapses in the brain. However, this view had to be revised when researchers discovered synapses between neurons and glial cells called NG2 cells, which go on to become oligodendrocytes. These neuron-NG2 cell synapses have a lot in common with neuron–neuron synapses, but much less is known about them. Orduz, Maldonado et al. have now examined these synapses in unprecedented detail by analyzing individual synapses between a type of neuron called an interneuron and an NG2 cell in mice aged only a few weeks. Interneurons can be divided into two major classes based on how quickly they fire, and Orduz, Maldonado et al. show that both types of interneuron form synapses with NG2 cells. However, these two types of interneuron establish synapses on different parts of the NG2 cell, and these synapses involve different receptor proteins. Together, the synapses give rise to a local interneuron-NG2 cell network that reaches a peak of activity roughly two weeks after birth, after which the network is disassembled. This period of peak activity is accompanied by a sudden increase in the maturation of NG2 cells into oligodendrocytes. Further experiments are needed to test the possibility that activity in the interneuron-NG2 cell network acts as the trigger for the NG2 cells to turn into oligodendrocytes, which then supply myelin for the developing brain.

Optical means for modulating and monitoring neuronal activity, have provided substantial insights to neurophysiology and toward our understanding of how the brain works. Optogenetic actuators, calcium or voltage imaging probes and other molecular tools, combined with advanced microscopies have allowed an \"all-optical\" readout and modulation of neural circuits. Completion of this remarkable work is evolving toward a three-dimensional (3D) manipulation of neural ensembles at a high spatiotemporal resolution. Recently, original optical methods have been proposed for both activating and monitoring neurons in a 3D space, mainly through optogenetic compounds. Here, we review these methods and anticipate possible combinations among them.

Two-photon voltage imaging has long been heralded as a transformative approach capable of answering many long-standing questions in modern neuroscience. However, exploiting its full potential requires the development of novel imaging approaches well suited to the photophysical properties of genetically encoded voltage indicators. We demonstrate that parallel excitation approaches developed for scanless two-photon photostimulation enable high-SNR two-photon voltage imaging. We use whole-cell patch-clamp electrophysiology to perform a thorough characterization of scanless two-photon voltage imaging using three parallel illumination approaches and lasers with different repetition rates and wavelengths. We demonstrate voltage recordings of high-frequency spike trains and sub-threshold depolarizations from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-Kv. Using a low repetition-rate laser, we perform multi-cell recordings from up to fifteen targets simultaneously. We co-express JEDI-2P-Kv and the channelrhodopsin ChroME-ST and capitalize on their overlapping two-photon absorption spectra to simultaneously evoke and image action potentials using a single laser source. We also demonstrate in vivo scanless two-photon imaging of multiple cells simultaneously up to 250 µm deep in the barrel cortex of head-fixed, anaesthetised mice. Detection of membrane potential changes using voltage indicators typically requires fast imaging rates and highly sensitive imaging methods. Here, the authors introduce scanless two-photon imaging, an approach which enables high signal to noise ratio voltage recordings at kilohertz rates, from multiple neurons simultaneously, both in vitro and in vivo.