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The development of efficient genetically encoded indicators and actuators has opened up the possibility of reading and manipulating neuronal activity in living tissues with light. To achieve precise and reconfigurable targeting of large numbers of neurons with single-cell resolution within arbitrary volumes, different groups have recently developed all-optical strategies based on two-photon excitation and spatio-temporal shaping of ultrashort laser pulses. However, such techniques are often complex to set up and typically operate at a single wavelength only. To address these issues, we have developed a novel optical approach that uses a fiber bundle and a spatial light modulator to achieve simple and dual-color two-photon light patterning in three dimensions. By leveraging the core-to-core temporal delay and the wavelength-independent divergence characteristics of fiber bundles, we have demonstrated the capacity to generate high-resolution excitation spots in a 3D region with two distinct laser wavelengths simultaneously, offering a suitable and simple alternative for precise multicolor cell targeting.
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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.
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Glándulas Endocrinas , Holografía , Animales , Ratones , Interneuronas , Calcio , Neuronas GABAérgicasRESUMEN
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.
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Holografía , Pez Cebra , Ratones , Animales , Neuronas/fisiología , Estimulación Luminosa/métodos , Holografía/métodos , Fotones , Optogenética/métodos , LuzRESUMEN
Two-photon light-targeting optogenetics allows controlling selected subsets of neurons with near single-cell resolution and high temporal precision. To push forward this approach, we recently proposed a fast light-targeting strategy (FLiT) to rapidly scan multiple holograms tiled on a spatial light modulator (SLM). This allowed generating sub-ms timely-controlled switch of light patterns enabling to reduce the power budget for multi-target excitation and increase the temporal precision for relative spike tuning in a circuit. Here, we modified the optical design of FLiT by including a de-scan unit (deFLiT) to keep the holographic illumination centered at the middle of the objective pupil independently of the position of the tiled hologram on the SLM. This enables enlarging the number of usable holograms and reaching extended on-axis excitation volumes, and therefore increasing even further the power gain and temporal precision of conventional FLiT.
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Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Dendritas/metabolismo , Endocitosis , Endosomas/metabolismo , Células HeLa , Hipocampo/citología , Humanos , Ratones , Optogenética , Fosforilación , Multimerización de Proteína , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Receptor trkB/metabolismo , Proteína S6 RibosómicaRESUMEN
Computer-generated holography enables efficient light pattern generation through phase-only wavefront modulation. While perfect patterning usually requires control over both phase and amplitude, iterative Fourier transform algorithms (IFTA) can achieve phase-only approximations which maximize light efficiency at the cost of uniformity. The phase being unconstrained in the output plane, it can vary abruptly in some regions leading to destructive interferences. Among such structures phase vortices are the most common. Here we demonstrate theoretically, numerically and experimentally, a novel approach for eliminating phase vortices by spatially filtering the phase input to the IFTA, combining it with phase-based complex amplitude control at the spatial light modulator (SLM) plane to generate smooth shapes. The experimental implementation is achieved performing complex amplitude modulation with a phase-only SLM. This proposed experimental scheme offers a continuous and centered field of excitation. Lastly, we characterize achievable trade-offs between pattern uniformity, diffraction efficiency, and axial confinement.
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Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor electrical subthreshold events as they travel from synapses on distal dendrites and summate at particular locations to initiate action potentials. It is now possible to carry out these measurements at the scale of individual dendritic spines using voltage imaging. In these measurements, the voltage-sensitive probes can be thought of as transmembrane voltmeters with a linear scale, which directly monitor electrical signals. Grinvald et al. were important early contributors to the methodology of voltage imaging, and they pioneered some of its significant results. We combined voltage imaging and glutamate uncaging using computer-generated holography. The results demonstrated that patterned illumination, by reducing the surface area of illuminated membrane, reduces photodynamic damage. Additionally, region-specific illumination practically eliminated the contamination of optical signals from individual spines by the scattered light from the parent dendrite. Finally, patterned illumination allowed one-photon uncaging of glutamate on multiple spines to be carried out in parallel with voltage imaging from the parent dendrite and neighboring spines.
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Computer Generated Holography achieves patterned illumination at the sample plane through phase modulation of the laser beam at the objective back aperture. This is obtained by using liquid crystal-based spatial light modulators (LC-SLMs), which modulate the spatial phase of the incident laser beam. A variety of algorithms is employed to calculate the phase modulation masks addressed to the LC-SLM. These algorithms range from simple gratings-and-lenses to generate multiple diffraction-limited spots, to iterative Fourier-transform algorithms capable of generating arbitrary illumination shapes perfectly tailored on the base of the target contour. Applications for holographic light patterning include multi-trap optical tweezers, patterned voltage imaging and optical control of neuronal excitation using uncaging or optogenetics. These past implementations of computer generated holography used binary input profile to generate binary light distribution at the sample plane. Here we demonstrate that using graded input sources, enables generating intensity graded light patterns and extend the range of application of holographic light illumination. At first, we use intensity-graded holograms to compensate for LC-SLM position dependent diffraction efficiency or sample fluorescence inhomogeneity. Finally we show that intensity-graded holography can be used to equalize photo evoked currents from cells expressing different levels of chanelrhodopsin2 (ChR2), one of the most commonly used optogenetics light gated channels, taking into account the non-linear dependence of channel opening on incident light.
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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.
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Interneuronas/ultraestructura , Neocórtex/citología , Células-Madre Neurales/ultraestructura , Oligodendroglía/ultraestructura , Corteza Somatosensorial/citología , Potenciales de Acción/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Diferenciación Celular , Expresión Génica , Genes Reporteros , Interneuronas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Microtomía , Neocórtex/crecimiento & desarrollo , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Oligodendroglía/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Corteza Somatosensorial/crecimiento & desarrollo , Corteza Somatosensorial/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica , Técnicas de Cultivo de Tejidos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Correlating patterned neuronal activity to defined animal behaviors is a core goal in neuroscience. Optogenetics is one large step toward achieving this goal, yet optical methods to control neural activity in behaving rodents have so far been limited to perturbing all light-sensitive neurons in a large volume. Here we demonstrate an all-optical method for precise spatial control and recording of neuronal activity in anesthetized and awake freely behaving mice. Photoactivation patterns targeted to multiple neuronal somata, produced with computer-generated holography, were transmitted to the mouse brain using a micro-objective-coupled fiber bundle. Fluorescence imaging through the same device, via epifluorescence, structured illumination, or scanless multipoint confocal microscopy, allowed imaging of neurons and recording of neuronal activity. The fiberscope was tested in mice coexpressing ChR2-tdTomato and GCaMP5-G in cerebellar interneurons, delivering near-cellular resolution photoactivation in freely behaving mice.
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Holografía/métodos , Fibras Ópticas , Imagen Óptica/métodos , Optogenética/métodos , Animales , Conducta Animal/fisiología , Cerebelo/fisiología , Neuroimagen Funcional/instrumentación , Neuroimagen Funcional/métodos , Ratones , Neuronas/fisiología , Imagen Óptica/instrumentación , Estimulación Luminosa/métodosRESUMEN
The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.
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Encéfalo/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuronas/metabolismo , Algoritmos , Animales , Velocidad del Flujo Sanguíneo , Encéfalo/irrigación sanguínea , Encéfalo/citología , Calcio/metabolismo , Señalización del Calcio , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Cristales Líquidos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Modelos Estadísticos , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Pixilated spatial light modulators are efficient devices to shape the wavefront of a laser beam or to perform Fourier optical filtering. When conjugated with the back focal plane of a microscope objective, they allow an efficient redistribution of laser light energy. These intensity patterns are usually polluted by undesired spots so-called ghosts and zero-orders whose intensities depend on displayed patterns. In this work, we propose a model to account for these discrepancies and demonstrate the possibility to efficiently reduce the intensity of the zero-order up to 95%, the intensity of the ghost up to 96% and increase diffraction efficiency up to 44%. Our model suggests physical cross-talk between pixels and thus, filtering of addressed high spatial frequencies. The method implementation relies on simple preliminary characterization of the SLM and can be computed a priori with any phase profile. The performance of this method is demonstrated employing a Hamamatsu LCoS SLM X10468-02 with two-photon excitation of fluorescent Rhodamine layers.
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Luz , Cristales Líquidos/química , Óptica y Fotónica , Silicio/química , Fluorescencia , Imagenología Tridimensional , Lentes , Neuronas/citologíaRESUMEN
Digital holography is an emerging technology that can generate complex light patterns for controlling the excitability of neurons and neural circuits. The strengths of this technique include a high efficiency with which available light can be effectively utilized and the ability to deliver highly focused light to multiple locations simultaneously. Here we demonstrate another strength of digital holography: the ability to generate instantaneous three-dimensional light patterns. This capability is demonstrated with the photolysis of caged glutamate on the dendritic arbor of hippocampal neurons, to study the nature of the integration of inputs arriving on multiple dendritic branches.
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Dendritas/fisiología , Holografía/métodos , Estimulación Luminosa/instrumentación , Algoritmos , Espinas Dendríticas/fisiología , Ácido Glutámico/química , Ácido Glutámico/efectos de la radiación , Hipocampo/citología , Hipocampo/efectos de la radiación , Luz , Vías Nerviosas/fisiología , Neuronas/fisiología , Fotólisis , Programas InformáticosRESUMEN
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.
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Corteza Cerebral/fisiología , Estimulación Luminosa/métodos , Fotones , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Channelrhodopsins , Proteínas Fluorescentes Verdes/genética , Humanos , Luz , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Microscopía de Contraste de Fase , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-ClampRESUMEN
Temporal focusing of ultrashort pulses has been shown to enable wide-field depth-resolved two-photon fluorescence microscopy. In this process, an entire plane in the sample is selectively excited by introduction of geometrical dispersion to an ultrashort pulse. Many applications, such as multiphoton lithography, uncaging or region-of-interest imaging, require, however, illumination patterns which significantly differ from homogeneous excitation of an entire plane in the sample. Here we consider the effects of such spatial modulation of a temporally focused excitation pattern on both the generated excitation pattern and on its axial confinement. The transition in the axial response between line illumination and wide-field illumination is characterized both theoretically and experimentally. For 2D patterning, we show that in the case of amplitude-only modulation the axial response is generally similar to that of wide-field illumination, while for phase-and-amplitude modulation the axial response slightly deteriorates when the phase variation is rapid, a regime which is shown to be relevant to excitation by beams shaped using spatial light modulators. Finally, general guidelines for the use of spatially modulated temporally focused excitation are presented.
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Algoritmos , Interpretación de Imagen Asistida por Computador/métodos , Iluminación/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Modelos Teóricos , Simulación por Computador , Luz , Dispersión de RadiaciónRESUMEN
Multiphoton excitation by temporally focused pulses can be combined with spatial Fourier-transform pulse shaping techniques to enhance spatial control of the excitation volume. Here we propose and demonstrate an optical system for the generation of such spatiotemporally engineered light pulses using a combination of spatial control by a two-dimensional reconfigurable light modulator, with a dispersive optical setup for temporal focusing. We show that although the properties of a holographic beam significantly differ from those of plane-wave illumination used in previous temporal focusing realizations, this leads only to a slightly reduced axial resolution. We show that the system can provide scanningless, arbitrarily shaped, depth resolved excitation patterns that offer new perspectives for multiphoton photoactivation and optical lithography applications.
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We report on a simple yet powerful implementation of objective-type total internal reflection fluorescence (TIRF) and highly inclined and laminated optical sheet (HILO, a type of dark-field) illumination. Instead of focusing the illuminating laser beam to a single spot close to the edge of the microscope objective, we are scanning during the acquisition of a fluorescence image the focused spot in a circular orbit, thereby illuminating the sample from various directions. We measure parameters relevant for quantitative image analysis during fluorescence image acquisition by capturing an image of the excitation light distribution in an equivalent objective backfocal plane (BFP). Operating at scan rates above 1 MHz, our programmable light engine allows directional averaging by circular spinning the spot even for sub-millisecond exposure times. We show that restoring the symmetry of TIRF/HILO illumination reduces scattering and produces an evenly lit field-of-view that affords on-line analysis of evanescnt-field excited fluorescence without pre-processing. Utilizing crossed acousto-optical deflectors, our device generates arbitrary intensity profiles in BFP, permitting variable-angle, multi-color illumination, or objective lenses to be rapidly exchanged.