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Optogenetics brought noninvasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models that receive the full benefit of this technique and provides behavioral studies based on intact individual nervous system. In this chapter, we describe methods to introduce optogenetic genes into zebrafish, and desirable apparatus for photostimulation and motion analysis with an example from our studies.With a compact neural circuit consisting of entirely mapped 302 neurons, Caenorhabditis elegans plays an important role in the development and application of optogenetics. Optogenetics in C. elegans offers the opportunity that drastically changes experimental designs with increasing accessibility for neural activity and various cellular processes, thereby accelerating the studies on the functions of neural circuits and multicellular systems. Combining optogenetics with other approaches such as electrophysiology increases the resolution of elucidation. In particular, technologies like patterned illumination specifically developed in combination with optogenetics provide new tools to interrogate neural functions. In this chapter, we introduce the reasons to use optogenetics in C. elegans, and discuss the technical issues raised, especially for C. elegans by revisiting our chapter in the first edition of this book. Throughout the chapter, we review early and recent milestone works using optogenetics to investigate a variety of biological systems including neural and behavioral regulation.The fruit fly Drosophila melanogaster, an insect 4 mm long, has served as the experimental subject in a wide range of biological research, including neuroscience. In this chapter, we briefly introduce optogenetic applications in Drosophila neuroscience research. First, we describe the development of Drosophila from egg to adult. In fly neuroscience, temperature-controlled perturbation of neural activity, sometimes called "thermogenetics," has been an invaluable tool that predates the advent of optogenetics. After briefly introducing this perturbation technique, we describe the process of generating transgenic flies that express optogenetic probes in a specific group of cells. Transgenic techniques are crucial in the application of optogenetics in Drosophila neuroscience; here we introduce the transposon P-elements, ϕC31 integrase, and CRISPR-Cas9 methods. As for cell-specific gene expression techniques, the binary expression systems utilizing Gal4-UAS, LexA-lexAop, and Q-system are described. We also present a short and basic optogenetic experiment with Drosophila larvae as a practical example. Finally, we review a few recent studies in Drosophila neuroscience that made use of optogenetics. In this overview of fly development, transgenic methods, and applications of optogenetics, we present an introductory background to optogenetics in Drosophila.Spatiotemporal dynamics of cellular proteins, including protein-protein interactions and conformational changes, is essential for understanding cellular functions such as synaptic plasticity, cell motility, and cell division. One of the best ways to understand the mechanisms of signal transduction is to visualize protein activity with high spatiotemporal resolution in living cells within tissues. Optogenetic probes such as fluorescent proteins, in combination with Förster Resonance Energy Transfer (FRET) techniques, enable the measurement of protein-protein interactions and conformational changes in response to signaling events in living cells. Of the various FRET detection systems, two-photon fluorescence lifetime imaging microscopy (2pFLIM) is one of the methods best suited to monitoring FRET in subcellular compartments of living cells located deep within tissues, such as brain slices. This review will introduce the principle of 2pFLIM-FRET and the use of chromoproteins for imaging intracellular protein activities and protein-protein interactions. Also, we will discuss two examples of 2pFLIM-FRET application imaging actin polymerization in synapses of hippocampal neurons in brain sections and detecting small GTPase Cdc42 activity in astrocytes.In this chapter, we introduce a relatively new, emerging method for molecular neuromodulation-bioluminescence-optogenetics. [https://www.selleckchem.com/products/dtnb.html learn more] Bioluminescence-optogenetics is mediated by luminopsin fusion proteins-light-sensing opsins fused to light-emitting luciferases. We describe their structures and working mechanisms and discuss their unique benefits over conventional optogenetics and chemogenetics. We also summarize applications of bioluminescence-optogenetics in various neurological disease models in rodents.There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. link2 In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.The progress in live-cell imaging technologies has revealed diverse dynamic patterns of transcriptional activity in various contexts. The discovery raised a next question of whether the gene expression patterns play causative roles in triggering specific biological events or not. Here, we introduce optogenetic methods that realize optical control of gene expression dynamics in mammalian cells and would be utilized for answering the question, by referring the past, the present, and the future.Cells respond to a wide range of extracellular stimuli, and process the input information through an intracellular signaling system comprised of biochemical and biophysical reactions, including enzymatic and protein-protein interactions. It is essential to understand the molecular mechanisms underlying intracellular signal transduction in order to clarify not only physiological cellular functions but also pathological processes such as tumorigenesis. Fluorescent proteins have revolutionized the field of life science, and brought the study of intracellular signaling to the single-cell and subcellular levels. Much effort has been devoted to developing genetically encoded fluorescent biosensors based on fluorescent proteins, which enable us to visualize the spatiotemporal dynamics of cell signaling. In addition, optogenetic techniques for controlling intracellular signal transduction systems have been developed and applied in recent years by regulating intracellular signaling in a light-dependent manner. Here, we outline the principles of biosensors for probing intracellular signaling and the optogenetic tools for manipulating them.Optogenetic approaches combine the power to allocate optogenetic tools (proteins) to specific cell populations (defined genetically or functionally) and the use of light-based interfaces between biological wetware (cells and tissues) and hardware (controllers and recorders). The optogenetic toolbox contains two main compartments tools to interfere with cellular processes and tools to monitor cellular events. Among the latter are genetically encoded voltage indicators (GEVIs). This chapter outlines the development, current state of the art and prospects of emerging optical GEVI imaging technologies.Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.The cyclic nucleotides cAMP and cGMP are ubiquitous secondary messengers that regulate multiple biological functions including gene expression, differentiation, proliferation, and cell survival. In sensory neurons, cyclic nucleotides are responsible for signal modulation, amplification, and encoding. For spatial and temporal manipulation of cyclic nucleotide dynamics, optogenetics have a great advantage over pharmacological approaches. link3 Enzymerhodopsins are a unique family of microbial rhodopsins. These molecules are made up of a membrane-embedded rhodopsin domain, which binds an all trans-retinal to form a chromophore, and a cytoplasmic water-soluble catalytic domain. To date, three kinds of molecules have been identified from lower eukaryotes such as fungi, algae, and flagellates. Among these, histidine kinase rhodopsin (HKR) is a light-inhibited guanylyl cyclase. Rhodopsin GC (Rh-GC) functions as a light-activated guanylyl cyclase, while rhodopsin PDE (Rh-PDE) functions as a light-activated phosphodiesterase that degrades cAMP and cGMP.