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.     

上转换纳米颗粒介导的无线光遗传学技术的研究进展

光遗传学技术是结合基因工程和光学技术对生物体特定细胞进行精确调控的新兴生物技术,该技术可以特异性地兴奋或抑制靶神经元,成为解析介导特定行为神经环路的强有力的工具.传统技术依赖光纤,对脑组织有损伤且限制了动物的自由活动.新一代上转换纳米颗粒介导的无线光遗传学技术,借助近红外光组织穿透相对深的特性,能够对啮齿类动物脑组织深层核团进行无线调控,克服了传统技术中埋置光纤存在的缺陷.本文总结了上转换纳米颗粒介导的无线光遗传学技术的发展历程及现状,比较分析了这类无线光遗传学技术的优缺点,最后对该技术面临的挑战及未来前景进行了分析和展望.     

Optogenetics: a new enlightenment age for zebrafish neurobiology

Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of \optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.     

The microbial opsin family of optogenetic tools

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.     

Optogenetic manipulation of neural circuitry in vivo

Recent advances in optogenetics have permitted investigations of specific cell types in the nervous system with unprecedented precision and control. This review will discuss the use of optogenetic techniques in the study of mammalian neural circuitry in vivo, as well as practical and theoretical considerations in their application.     

Recent Developments in Optical Neuromodulation Technologies

The emergence of optogenetics technology facilitated widespread applications for interrogation of complex neural networks, such as activation of specific axonal pathways, previously found impossible with electrical stimulation. Consequently, within the short period of its application in neuroscience research, optogenetics has led to findings of significant importance both during normal brain function as well as in disease. Moreover, the optimization of optogenetics for in vivo studies has allowed the control of certain behavioral responses such as motility, reflex, and sensory responses, as well as more complex emotional and cognitive behaviors such as decision-making, reward seeking, and social behavior in freely moving animals. These studies have produced a wide variety of animal models that have resulted in fundamental findings and enhanced our understanding of the neural networks associated with behavior. The increasing number of opsins available for this technique enabled even broader regulation of neuronal activity. These advancements highlight the potential of this technique for future treatment of human diseases. Here, we provide an overview of the recent developments in the field of optogenetics technology that are relevant for a better understanding of several neuropsychiatric and neurodegenerative disorders and may pave the way for future therapeutic interventions.     

Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans

By enabling a tight control of cell excitation, optogenetics is a powerful approach to study the function of neurons and neural circuits. With its transparent body, a fully mapped nervous system, easily quantifiable behaviors and many available genetic tools, Caenorhabditis elegans is an extremely well-suited model to decipher the functioning logic of the nervous system with optogenetics. Our goal was to establish an efficient dual color optogenetic system for the independent excitation of different neurons in C. elegans. We combined two recently discovered channelrhodopsins: the red-light sensitive Chrimson from Chlamydomonas noctigama and the blue-light sensitive CoChR from Chloromonas oogama. Codon-optimized versions of Chrimson and CoChR were designed for C. elegans and expressed in different mechanosensory neurons. Freely moving animals produced robust behavioral responses to light stimuli of specific wavelengths. Since CoChR was five times more sensitive to blue light than the commonly used ChR2, we were able to use low blue light intensities producing no cross-activation of Chrimson. Thanks to these optogenetics tools, we revealed asymmetric cross-habituation effects between the gentle and harsh touch sensory motor pathways. Collectively, our results establish the Chrimson/CoChR pair as a potent tool for bimodal neural excitation in C. elegans and equip this genetic model organism for the next generation of in vivo optogenetic analyses.     

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.     

New era of optogenetics: from the central to peripheral nervous system

Abstract

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.     

Shedding light on current trends in molecular optogenetics

Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid–liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions.     

光敏感通道(Channelrhodopsin-2)——神经回路功能和神经系统疾病研究的新工具

光敏感通道(channelrhodopsin-2,ChR2)是一种受光脉冲控制的具有7次跨膜结构的非选择性阳离子通道蛋白,自1991年从莱茵衣藻中发现后被许多实验室所关注.依据ChR2可以快速形成光电流,使细胞发生去极化反应的电生理特性,ChR2已被广泛应用于神经系统的研究.与传统的神经系统研究方法如电生理技术、神经药理学方法相比,用光脉冲控制带有ChR2的神经元的活动,具有更高的空间选择性和特异性.ChR2作为光基因技术的核心组成部分,对神经科学是一个崭新的应用前景广泛的研究工具.近年来ChR2不仅应用于视觉、躯体感觉、听觉和嗅觉等多条感觉神经回路的形态和功能研究,还被应用于各种临床神经系统疾病的研究.本文总结了目前ChR2在神经系统中的研究进展,并对ChR2未来的应用前景作了进一步展望.     

Form and function in systems neuroscience

'Form follows function' is an architectural philosophy attributed to the great American architect Louis Sullivan, and later taken up by the Bauhaus movement. It stresses that the form of a building should reflect its function. Neuroscientists have used the converse of this dictum to learn the functions of neural circuits, believing that if we study neural architecture, it will lead us to an understanding of how neural systems function. New tools for studying the structure of neural circuits are being developed, so it is important to discuss what the old techniques have taught us about how to derive function from the form of a neural circuit.     

Synapses in the spotlight with synthetic optogenetics

Membrane receptors and ion channels respond to various stimuli and relay that information across the plasma membrane by triggering specific and timed processes. These include activation of second messengers, allowing ion permeation, and changing cellular excitability, to name a few. Gaining control over equivalent processes is essential to understand neuronal physiology and pathophysiology. Recently, new optical techniques have emerged proffering new remote means to control various functions of defined neuronal populations by light, dubbed optogenetics. Still, optogenetic tools do not typically address the activity of receptors and channels native to neurons (or of neuronal origin), nor gain access to their signaling mechanisms. A related method—synthetic optogenetics—bridges this gap by endowing light sensitivity to endogenous neuronal receptors and channels by the appending of synthetic, light‐receptive molecules, or photoswitches. This provides the means to photoregulate neuronal receptors and channels and tap into their native signaling mechanisms in select regions of the neurons, such as the synapse. This review discusses the development of synthetic optogenetics as a means to study neuronal receptors and channels remotely, in their natural environment, with unprecedented spatial and temporal precision, and provides an overview of tool design, mode of action, potential clinical applications and insights and achievements gained.     

Development of larval motor circuits in Drosophila

How are functional neural circuits formed during development? Despite recent advances in our understanding of the development of individual neurons, little is known about how complex circuits are assembled to generate specific behaviors. Here, we describe the ways in which Drosophila motor circuits serve as an excellent model system to tackle this problem. We first summarize what has been learned during the past decades on the connectivity and development of component neurons, in particular motor neurons and sensory feedback neurons. We then review recent progress in our understanding of the development of the circuits as well as studies that apply optogenetics and other innovative techniques to dissect the circuit diagram. New approaches using Drosophila as a model system are now making it possible to search for developmental rules that regulate the construction of neural circuits.     

Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila

Here we incorporate recent advances in Drosophila neurogenetics and "optogenetics" into neuroscience laboratory exercises. We used the light-activated ion channel channelrhodopsin-2 (ChR2) and tissue-specific genetic expression techniques to study the neural basis of behavior in Drosophila larvae. We designed and implemented exercises using inexpensive, easy-to-use systems for delivering blue light pulses with fine temporal control. Students first examined the behavioral effects of activating glutamatergic neurons in Drosophila larvae and then recorded excitatory junctional potentials (EJPs) mediated by ChR2 activation at the larval neuromuscular junction (NMJ). Comparison of electrically and light-evoked EJPs demonstrates that the amplitudes and time courses of light-evoked EJPs are not significantly different from those generated by electrical nerve stimulation. These exercises introduce students to new genetic technology for remotely manipulating neural activity, and they simplify the process of recording EJPs at the Drosophila larval NMJ. Relatively little research work has been done using ChR2 in Drosophila, so students have opportunities to test novel hypotheses and make tangible contributions to the scientific record. Qualitative and quantitative assessment of student experiences suggest that these exercises help convey principles of synaptic transmission while also promoting integrative and inquiry-based studies of genetics, cellular physiology, and animal behavior.     

The bright frontiers of microbial metabolic optogenetics

In recent years, light-responsive systems from the field of optogenetics have been applied to several areas of metabolic engineering with remarkable success. By taking advantage of light's high tunability, reversibility, and orthogonality to host endogenous processes, optogenetic systems have enabled unprecedented dynamical controls of microbial fermentations for chemical production, metabolic flux analysis, and population compositions in co-cultures. In this article, we share our opinions on the current state of this new field of metabolic optogenetics.We make the case that it will continue to impact metabolic engineering in increasingly new directions, with the potential to challenge existing paradigms for metabolic pathway and strain optimization as well as bioreactor operation.     

PlexinA2 and semaphorin signaling during cardiac neural crest development.

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.     

Scratching Deer: A Late Prehistoric Campsite In The Green Lakes Valley,Colorado

Abstract

The Scratching Deer site (5 BL 69) is a small, single-component Hog Back Phase campsite inthe upper subalpine forest, western Boulder County, Colorado. The site was occupied 1260 + 95radiocarbon years ago, probably by hunters attracted to the region by stone game-drive systems on a nearby tundra ridgecrest. Although few tools were discarded at the site, analysis ofwaste flakes suggests that large numbers of cutting and scraping tools were resharpened in the occupation area, and that new tools were manufactured, possibly from imported stone that was heat-treated in especially-prepared hearths. Occupation was preceded by an interval of expanded snow cover and accelerated erosion, and was followea by an interval of loess accumulation and redeposition; snowbanks have never again been as persistent on the floor of the Green Lakes valley as they were a few centuries prior to occupation.     

The potential for computer-aided identification in biodiversity research

Species identification lies at the heart of most ecological studies, but it is recognized as a difficult and often frustrating task. Taxonomists have sought to overcome the difficulties of species identification by developing a range of tools and techniques that have increasingly involved the use of computers. We describe recent developments in computer-aided species identification, which have been in four main areas; multi-access keys; hypertext keys; expert systems; and neural networks. We also suggest which technique might be appropriate for a particular taxonomic group.     

From dusk till dawn: one-plasmid systems for light-regulated gene expression

Signaling photoreceptors mediate diverse organismal adaptations in response to light. As light-gated protein switches, signaling photoreceptors provide the basis for optogenetics, a term that refers to the control of organismal physiology and behavior by light. We establish as novel optogenetic tools the plasmids pDusk and pDawn, which employ blue-light photoreceptors to confer light-repressed or light-induced gene expression in Escherichia coli with up to 460-fold induction upon illumination. Key features of these systems are low background activity, high dynamic range, spatial control on the 20-μm scale, independence from exogenous factors, and ease of use. In optogenetic experiments, pDusk and pDawn can be used to specifically perturb individual nodes of signaling networks and interrogate their role. On the preparative scale, pDawn can induce by light the production of recombinant proteins and thus represents a cost-effective and readily automated alternative to conventional induction systems.