The optogenetic (r)evolution

Optogenetics is a rapidly evolving field of technology that allows optical control of genetically targeted biological systems at high temporal and spatial resolution. By heterologous expression of light-sensitive microbial membrane proteins, opsins, cell type-specific depolarization or silencing can be optically induced on a millisecond time scale. What started in a petri dish is applicable today to more complex systems, ranging from the dissection of brain circuitries in vitro to behavioral analyses in freely moving animals. Persistent technical improvement has focused on the identification of new opsins, suitable for optogenetic purposes and genetic engineering of existing ones. Optical stimulation can be combined with various readouts defined by the desired resolution of the experimental setup. Although recent developments in optogenetics have largely focused on neuroscience it has lately been extended to other targets, including stem cell research and regenerative medicine. Further development of optogenetic approaches will not only highly increase our insight into health and disease states but might also pave the way for a future use in therapeutic applications.     

Precise spatiotemporal control of optogenetic activation using an acousto-optic device

Light activation and inactivation of neurons by optogenetic techniques has emerged as an important tool for studying neural circuit function. To achieve a high resolution, new methods are being developed to selectively manipulate the activity of individual neurons. Here, we report that the combination of an acousto-optic device (AOD) and single-photon laser was used to achieve rapid and precise spatiotemporal control of light stimulation at multiple points in a neural circuit with millisecond time resolution. The performance of this system in activating ChIEF expressed on HEK 293 cells as well as cultured neurons was first evaluated, and the laser stimulation patterns were optimized. Next, the spatiotemporally selective manipulation of multiple neurons was achieved in a precise manner. Finally, we demonstrated the versatility of this high-resolution method in dissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo. Taken together, our results show that the combination of AOD-assisted laser stimulation and optogenetic tools provides a flexible solution for manipulating neuronal activity at high efficiency and with high temporal precision.     

Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.     

Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals

Optogenetic control of the peripheral nervous system (PNS) would enable novel studies of motor control, somatosensory transduction, and pain processing. Such control requires the development of methods to deliver opsins and light to targeted sub-populations of neurons within peripheral nerves. We report here methods to deliver opsins and light to targeted peripheral neurons and robust optogenetic modulation of motor neuron activity in freely moving, non-transgenic mammals. We show that intramuscular injection of adeno-associated virus serotype 6 enables expression of channelrhodopsin (ChR2) in motor neurons innervating the injected muscle. Illumination of nerves containing mixed populations of axons from these targeted neurons and from neurons innervating other muscles produces ChR2-mediated optogenetic activation restricted to the injected muscle. We demonstrate that an implanted optical nerve cuff is well-tolerated, delivers light to the sciatic nerve, and optically stimulates muscle in freely moving rats. These methods can be broadly applied to study PNS disorders and lay the groundwork for future therapeutic application of optogenetics.     

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain''s global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.     

Algal photoreceptors: in vivo functions and potential applications

Many algae, particularly microalgae, possess a sophisticated light-sensing system including photoreceptors and light-modulated signaling pathways to sense environmental information and secure the survival in a rapidly changing environment. Over the last couple of years, the multifaceted world of algal photobiology has enriched our understanding of the light absorption mechanisms and in vivo function of photoreceptors. Moreover, specific light-sensitive modules have already paved the way for the development of optogenetic tools to generate light switches for precise and spatial control of signaling pathways in individual cells and even in complex biological systems.     

A high-throughput method to deliver targeted optogenetic stimulation to moving C. elegans populations

We present a high-throughput optogenetic illumination system capable of simultaneous closed-loop light delivery to specified targets in populations of moving Caenorhabditis elegans. The instrument addresses three technical challenges: It delivers targeted illumination to specified regions of the animal’s body such as its head or tail; it automatically delivers stimuli triggered upon the animal’s behavior; and it achieves high throughput by targeting many animals simultaneously. The instrument was used to optogenetically probe the animal’s behavioral response to competing mechanosensory stimuli in the the anterior and posterior gentle touch receptor neurons. Responses to more than 43,418 stimulus events from a range of anterior–posterior intensity combinations were measured. The animal’s probability of sprinting forward in response to a mechanosensory stimulus depended on both the anterior and posterior stimulation intensity, while the probability of reversing depended primarily on the anterior stimulation intensity. We also probed the animal’s response to mechanosensory stimulation during the onset of turning, a relatively rare behavioral event, by delivering stimuli automatically when the animal began to turn. Using this closed-loop approach, over 9,700 stimulus events were delivered during turning onset at a rate of 9.2 events per worm hour, a greater than 25-fold increase in throughput compared to previous investigations. These measurements validate with greater statistical power previous findings that turning acts to gate mechanosensory evoked reversals. Compared to previous approaches, the current system offers targeted optogenetic stimulation to specific body regions or behaviors with many fold increases in throughput to better constrain quantitative models of sensorimotor processing.

This study resents a new targeted illumination method for the nematode Caenorhabditis elegans, allowing delivery of optogenetic stimulation to specific body parts of many animals at once, automatically triggered by the animals’ behavior.     

Optogenetic control of cell function using engineered photoreceptors

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light‐actuated tools allow manipulation of molecular events with ultra‐fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub‐micrometre precision within cells. While light‐actuated chemicals such as photolabile ‘caged’ compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever‐growing optogenetic toolkit.     

Identification of Optogenetically Activated Striatal Medium Spiny Neurons by Npas4 Expression

Optogenetics is a powerful neuromodulatory tool with many unique advantages to explore functions of neuronal circuits in physiology and diseases. Yet, interpretation of cellular and behavioral responses following in vivo optogenetic manipulation of brain activities in experimental animals often necessitates identification of photoactivated neurons with high spatial resolution. Although tracing expression of immediate early genes (IEGs) provides a convenient approach, neuronal activation is not always followed by specific induction of widely used neuronal activity markers like c-fos, Egr1 and Arc. In this study we performed unilateral optogenetic stimulation of the striatum in freely moving transgenic mice that expressed a channelrhodopsin-2 (ChR2) variant ChR2(C128S) in striatal medium spiny neurons (MSNs). We found that in vivo blue light stimulation significantly altered electrophysiological activity of striatal neurons and animal behaviors. To identify photoactivated neurons we then analyzed IEG expression patterns using in situ hybridization. Upon light illumination an induction of c-fos was not apparent whereas another neuronal IEG Npas4 was robustly induced in MSNs ipsilaterally. Our results demonstrate that tracing Npas4 mRNA expression following in vivo optogenetic modulation can be an effective tool for reliable and sensitive identification of activated MSNs in the mouse striatum.     

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered,light-switchable anti-CRISPR protein

Optogenetic control of CRISPR–Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.     

An Integrated In Vitro Imaging Platform for Characterizing Filarial Parasite Behavior within a Multicellular Microenvironment

Lymphatic Filariasis, a Neglected Tropical Disease, is caused by thread-like parasitic worms, including B. malayi, which migrate to the human lymphatic system following transmission. The parasites reside in collecting lymphatic vessels and lymph nodes for years, often resulting in lymphedema, elephantiasis or hydrocele. The mechanisms driving worm migration and retention within the lymphatics are currently unknown. We have developed an integrated in vitro imaging platform capable of quantifying B. malayi migration and behavior in a multicellular microenvironment relevant to the initial site of worm injection by incorporating the worm in a Polydimethylsiloxane (PDMS) microchannel in the presence of human dermal lymphatic endothelial cells (LECs) and human dermal fibroblasts (HDFs). The platform utilizes a motorized controllable microscope with CO₂ and temperature regulation to allow for worm tracking experiments with high resolution over large length and time scales. Using post-acquisition algorithms, we quantified four parameters: 1) speed, 2) thrashing intensity, 3) percentage of time spent in a given cell region and 4) persistence ratio. We demonstrated the utility of our system by quantifying these parameters for L3 B. malayi in the presence of LECs and HDFs. Speed and thrashing increased in the presence of both cell types and were altered within minutes upon exposure to the anthelmintic drug, tetramisole. The worms displayed no targeted migration towards either cell type for the time course of this study (3 hours). When cells were not present in the chamber, worm thrashing correlated directly with worm speed. However, this correlation was lost in the presence of cells. The described platform provides the ability to further study B. malayi migration and behavior.     

光遗传学在细菌生产调控中的应用进展

合成生物学的发展使得人们可以根据需求对微生物进行改造,作为“工厂”高效地合成催化所需物质,并通过添加化学诱导物的方式对生命过程进行调控。然而,化学诱导的潜在毒性以及不可逆性等限制其应用。光遗传学技术利用特定波长的光信号实现对细胞生命过程的调控,具有特异性、可逆性、高时空分辨率等特点。近年来,人们对不同来源的光敏蛋白进行改造,开发出各种不同波长、不同效应的光遗传元件用于基因回路的构建,进而实现对细菌蛋白合成、代谢过程的调控。光遗传技术在人与细菌之间搭起了实时的信号沟通桥梁,实现更为精准的物质生产调控:(1)通过光控治疗因子的合成分泌进行药物递送;(2)通过代谢通路的控制提高目的产物的催化效率;(3)通过光诱导控制生物活材料的形成。随着探索的深入,更小体积、更多波长、更高效率的光遗传元件将被开发出来,实现多输入的细菌生命活动调控。     

Broad-Band Activatable White-Opsin

Currently, the use of optogenetic sensitization of retinal cells combined with activation/inhibition has the potential to be an alternative to retinal implants that would require electrodes inside every single neuron for high visual resolution. However, clinical translation of optogenetic activation for restoration of vision suffers from the drawback that the narrow spectral sensitivity of an opsin requires active stimulation by a blue laser or a light emitting diode with much higher intensities than ambient light. In order to allow an ambient light-based stimulation paradigm, we report the development of a ‘white-opsin’ that has broad spectral excitability in the visible spectrum. The cells sensitized with white-opsin showed excitability at an order of magnitude higher with white light compared to using only narrow-band light components. Further, cells sensitized with white-opsin produced a photocurrent that was five times higher than Channelrhodopsin-2 under similar photo-excitation conditions. The use of fast white-opsin may allow opsin-sensitized neurons in a degenerated retina to exhibit a higher sensitivity to ambient white light. This property, therefore, significantly lowers the activation threshold in contrast to conventional approaches that use intense narrow-band opsins and light to activate cellular stimulation.     

Bimodal Activation of Different Neuron Classes with the Spectrally Red-Shifted Channelrhodopsin Chimera C1V1 in Caenorhabditis elegans

The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or –inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1′s) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540–580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.     

High-throughput single-cell manipulation in brain tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.     

De novo spatiotemporal modelling of cell-type signatures in the developmental human heart using graph convolutional neural networks

With the emergence of high throughput single cell techniques, the understanding of the molecular and cellular diversity of mammalian organs have rapidly increased. In order to understand the spatial organization of this diversity, single cell data is often integrated with spatial data to create probabilistic cell maps. However, targeted cell typing approaches relying on existing single cell data achieve incomplete and biased maps that could mask the true diversity present in a tissue slide. Here we applied a de novo technique to spatially resolve and characterize cellular diversity of in situ sequencing data during human heart development. We obtained and made accessible well defined spatial cell-type maps of fetal hearts from 4.5 to 9 post conception weeks, not biased by probabilistic cell typing approaches. With our analysis, we could characterize previously unreported molecular diversity within cardiomyocytes and epicardial cells and identified their characteristic expression signatures, comparing them with specific subpopulations found in single cell RNA sequencing datasets. We further characterized the differentiation trajectories of epicardial cells, identifying a clear spatial component on it. All in all, our study provides a novel technique for conducting de novo spatial-temporal analyses in developmental tissue samples and a useful resource for online exploration of cell-type differentiation during heart development at sub-cellular image resolution.     

Optogenetic investigation of neural circuits in vivo

The recent development of light-activated optogenetic probes allows for the identification and manipulation of specific neural populations and their connections in awake animals with unprecedented spatial and temporal precision. This review describes the use of optogenetic tools to investigate neurons and neural circuits in vivo. We describe the current panel of optogenetic probes, methods of targeting these probes to specific cell types in the nervous system, and strategies of photostimulating cells in awake, behaving animals. Finally, we survey the application of optogenetic tools to studying functional neuroanatomy, behavior and the etiology and treatment of various neurological disorders.     

Illuminating neural circuits and behaviour in Caenorhabditis elegans with optogenetics

The development of optogenetics, a family of methods for using light to control neural activity via light-sensitive proteins, has provided a powerful new set of tools for neurobiology. These techniques have been particularly fruitful for dissecting neural circuits and behaviour in the compact and transparent roundworm Caenorhabditis elegans. Researchers have used optogenetic reagents to manipulate numerous excitable cell types in the worm, from sensory neurons, to interneurons, to motor neurons and muscles. Here, we show how optogenetics applied to this transparent roundworm has contributed to our understanding of neural circuits.     

Reshaping the optical dimension in optogenetics

Optogenetics has been revolutionizing circuit neuroscience in the last few years. Optical methods combined with genetics and molecular techniques have provided new tools for stimulation of neurons, which hold great promise to provide a solution to the circuit mapping problem and more generally provide us with the ability to artificially control the natural stimulus space. Nevertheless, until very recently almost all applications of optogenetics have been based on relatively simple optical schemes mainly used for inducing population activity in neuronal assembles. In this context, alternative optical schemes that enhance the spatial or temporal resolution of excitation and allow for flexible and arbitrary generation of light patterns have all synergetic impact on the development of new optogenetic actuators. In the following we discuss and compare the main new optical techniques that have become available in the recent years. Their respective strengths and limitations as well as their application to different biological contexts are illustrated.     

Lights,cytoskeleton, action: Optogenetic control of cell dynamics

Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein–protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.