Optical techniques in optogenetics

2014

The Neuroscience Journal of Shefaye Khatam, 2013

Signal transduction Channelrhodopsin-2 Chlamydomonas reinhardtii All-trans-retinal 13-cis-retinal ChR2 ChR2 (VChR1) Action potential Volvox channelrhodopsin-1 Volvox carteri Step-Function-Opsins (SFOs)

Journal of Visualized Experiments, 2021

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin-has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of lightmediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities. Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Frontiers in Cellular Neuroscience, 2022

Optogenetics, a field concentrating on controlling cellular functions by means of light-activated proteins, has shown tremendous potential in neuroscience. It possesses superior spatiotemporal resolution compared to the surgical, electrical, and pharmacological methods traditionally used in studying brain function. A multitude of optogenetic tools for neuroscience have been created that, for example, enable the control of action potential generation via light-activated ion channels. Other optogenetic proteins have been used in the brain, for example, to control long-term potentiation or to ablate specific subtypes of neurons. In in vivo applications, however, the majority of optogenetic tools are operated with blue, green, or yellow light, which all have limited penetration in biological tissues compared to red light and especially infrared light. This difference is significant, especially considering the size of the rodent brain, a major research model in neuroscience. Our review w...

Cell, 2010

Optogenetic technologies employ light to control biological processes within targeted cells in vivo with high temporal precision. Here, we show that application of molecular trafficking principles can expand the optogenetic repertoire along several long-sought dimensions. Subcellular and transcellular trafficking strategies now permit (1) optical regulation at the far-red/infrared border and extension of optogenetic control across the entire visible spectrum, (2) increased potency of optical inhibition without increased light power requirement (nanoampere-scale chloride-mediated photocurrents that maintain the light sensitivity and reversible, steplike kinetic stability of earlier tools), and (3) generalizable strategies for targeting cells based not only on genetic identity, but also on morphology and tissue topology, to allow versatile targeting when promoters are not known or in genetically intractable organisms. Together, these results illustrate use of cell-biological principles to enable expansion of the versatile fast optogenetic technologies suitable for intact-systems biology and behavior.

Journal of Neuroscience, 2010

This mini-symposium aims to provide an integrated perspective on recent developments in optogenetics. Research in this emerging field combines optical methods with targeted expression of genetically encoded, protein-based probes to achieve experimental manipulation and measurement of neural systems with superior temporal and spatial resolution. The essential components of the optogenetic toolbox consist of two kinds of molecular devices: actuators and reporters, which respectively enable light-mediated control or monitoring of molecular processes. The first generation of genetically encoded calcium reporters, fluorescent proteins, and neural activators has already had a great impact on neuroscience. Now, a second generation of voltage reporters, neural silencers, and functionally extended fluorescent proteins hold great promise for continuing this revolution. In this review, we will evaluate and highlight the limitations of presently available optogenic tools and discuss where these technologies and their applications are headed in the future.

Journal of Experimental Neuroscience, 2017

Several fields in neuroscience have been revolutionized by the advent of optogenetics, a technique that offers the possibility to modulate neuronal physiology in response to light stimulation. This innovative and far-reaching tool provided unprecedented spatial and temporal resolution to explore the activity of neural circuits underlying cognition and behaviour. With an exponential growth in the discovery and synthesis of new photosensitive actuators capable of modulating neuronal networks function, other fields in biology are experiencing a similar re-evolution. Here, we review the various optogenetic toolboxes developed to influence cellular physiology as well as the diverse ways in which these can be engineered to precisely modulate intracellular signalling and transcription. We also explore the processes required to successfully express and stimulate these photo-actuators in vivo before discussing how such tools can enlighten our understanding of neuronal plasticity at the syste...

Journal of Visualized Experiments

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin-has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of lightmediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities. Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

St. Petersburg Polytechnical University Journal: Physics and Mathematics, 2015

The article is devoted to problems of realization and application of optogenetic methods used to identify reasons of various diseases, to monitor the biochemical processes of cell activity and to study various organisms. The problems of delivery, embedding and monitoring the expression of opsin genes into the cell genome of interest have been considered. In the article, the parameters and properties of various opsins and also the main ways of achievement of precise optical control over cell using opsins were presented. The rules for choosing the parameters of a light beam and the features of its putting were pointed out. The characteristic properties of the different measurement technique and recording the experimental quantities were analyzed and given.

2019

Optogenetics is an innovative neuromodulation technique involving the use of light and light-sensitive proteins to control molecular events within a genetically modified cell. The fundamental mechanism behind optogenetics is the deliberate shining of light at light-sensitive cellular membrane proteins which causes some sort of change within a cell. These proteins, called opsins, come in many forms including ion channels, pumps, and G protein-coupled receptors and they are found in a wide range of organisms from vertebrates to prokaryotes [1]. When utilizing optogenetics, researchers must make several considerations including the light source to be used to control the cellular event, the type of cell to be activated by the light, and the tools to be utilized for measuring such cellular activity. We reviewed in detail the mechanism behind optogenetics and the considerations researchers make in employing this technique. We also reviewed outcomes from several studies centered around it ...

Critical Reviews in Biochemistry and Molecular Biology, 2020

Optogenetics has recently gained recognition as a biological technique to control the activity of cells using light stimulation. Many studies have applied optogenetics to cell lines in the central nervous system because it has the potential to elucidate neural circuits, treat neurological diseases and promote nerve regeneration. There have been fewer studies on the application of optogenetics in the peripheral nervous system. This review introduces the basic principles and approaches of optogenetics and summarizes the physiology and mechanism of opsins and how the technology enables bidirectional control of unique cell lines with superior spatial and temporal accuracy. Further, this review explores and discusses the therapeutic potential for the development of optogenetics and its capacity to revolutionize treatment for refractory epilepsy, depression, pain, and other nervous system disorders, with a focus on neural regeneration, especially in the peripheral nervous system. Additionally, this review synthesizes the latest preclinical research on optogenetic stimulation, including studies on non-human primates, summarizes the challenges, and highlights future perspectives. The potential of optogenetic stimulation to optimize therapy for peripheral nerve injuries (PNIs) is also highlighted. Optogenetic technology has already generated exciting, preliminary evidence, supporting its role in applications to several neurological diseases, including PNIs.

2011

Optogenetic technology has shown great promise as a tool to selectively control the electrical activity of neural circuits via optical stimulation. This control is enabled by genetic delivery and expression of light-sensitive proteins to specific cells. Optogenetics research in neuroscience has rapidly expanded and the technology shows the potential for clinical translation in treating neurological disorders including Parkinson's disease and stroke. However, challenges still remain with respect to the clinical application of optogenetic technology. These challenges include developing appropriate gene delivery methods, creating more efficient light-sensitive proteins, and enhancing the specificity of targeting tools. This review will focus on the naturally occurring, lightsensitive opsin proteins and their potential for clinical use. Specifically, various methods for the safe and effective delivery of opsin genes to target cells are examined. By testing the delivery, expression, and stimulation of opsin in different organisms, selective delivery, robust expression, and effective activation of opsin can be optimized. Such testing will naturally focus on primates due to physiological similarities with humans. With further progress, the prospect of clinical translation of optogenetic tools-even beyond neuromodulation with devices such as an optogenetic cardiac pacemaker-may be just around the corner.

Chemical reviews, 2018

Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, a...

Trends in Molecular Medicine, 2011

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. Optogenetics The mammalian brain is composed of billions of neurons interconnected into circuits by trillions of synapses [1]. Some neural systems have been difficult to describe anatomically, such as the exquisitely complicated wiring of the cerebral cortex, while other neural systems are relatively well-characterized, such as the circuits that mediate vision, motor movements, breathing/respiration, and sleep/wake architecture. However, even these systems require functional dissection so that the relative contributions of individual cell types and their synaptic connections can be discerned. Perturbing one element in a neural circuit is especially difficult in vivo, where the complex environment of the brain in an awake, behaving animal imposes obstacles to the stimulation, inhibition, or manipulation of biochemical signaling events in specific cell types. Traditionally, cells and synapses have been manipulated using electrical, physical, pharmacological, and genetic methods (Figure 1) [2]. Although much progress has been made using these classical techniques, considerable drawbacks prevent their use in the study of neural circuits with fine spatial and temporal precision in vivo. Electrical and physical techniques are not spatially precise and can cause stimulation, inhibition, or inactivation of surrounding cells and processes. Pharmacological and genetic methods exhibit improved spatial selectivity but lack temporal resolution at the scale of single action potentials. To overcome these limitations, a new set of tools collectively referred to as "optogenetics" [3] has been developed to precisely stimulate [4-10], inhibit [11-16], or alter biochemical activity [17, 18] in specific cells or their processes with high temporal precision and rapid

Journal of Visualized Experiments, 2015

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing