Using Optogenetics to Understand Neuronal Mechanisms Underlying Behavior in C. elegans.
ABSTRACT: Approaches for inhibiting and activating neurons are essential for understanding how neurons and neuronal circuits produce behavior. Optogenetics is a recently-developed technique which uses light to manipulate neuronal activity with temporal precision in behaving animals and is widely-used by neuroscience researchers. Optogenetics is also an excellent method to incorporate into an undergraduate neuroscience laboratory module because students can learn to conduct and analyze quantitative behavioral assays, reinforce their understanding of synaptic transmission, and investigate the genetic and neuronal basis of behavior. Here, we describe a module in which students use light to activate serotonergic neurons expressing the light-activated ion channel channelrhodopsin in wildtype and mutant Caenorhabditis elegans, and observe and analyze the effects on the movement behavior of the nematode. The methods described here provide a foundation which students can use to design and conduct additional experiments that may have never been done before. Thus, this laboratory module provides an opportunity for students to learn a state-of-the-art neuroscience technique, think about neuroscience on genetic, cellular and behavioral levels, and to develop an independent research project.
Project description:In recent years optogenetics has rapidly become an essential technique in neuroscience. Its temporal and spatial specificity, combined with efficacy in manipulating neuronal activity, are especially useful in studying the behavior of awake behaving animals. Conventional optogenetics, however, requires the use of lasers and optic fibers, which can place considerable restrictions on behavior. Here we combined a wirelessly controlled interface and small implantable light-emitting diode (LED) that allows flexible and precise placement of light source to illuminate any brain area. We tested this wireless LED system in vivo, in transgenic mice expressing channelrhodopsin-2 in striatonigral neurons expressing D1-like dopamine receptors. In all mice tested, we were able to elicit movements reliably. The frequency of twitches induced by high power stimulation is proportional to the frequency of stimulation. At lower power, contraversive turning was observed. Moreover, the implanted LED remains effective over 50 days after surgery, demonstrating the long-term stability of the light source. Our results show that the wireless LED system can be used to manipulate neural activity chronically in behaving mice without impeding natural movements.
Project description: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.
Project description:A biomedical sciences graduate program needed an introductory class that would develop skills for students interested in a wide variety of disciplines, such as microbiology or cancer biology, and a diverse array of biomedical careers. Faculty created a year-long student-centered course, Scientific Discovery, to serve this need. The course was divided into four modules with progressive skill outcomes. Each module had a focus related to each of the major research areas of the collective faculty: molecular biology, biochemistry, neuroscience, and infectious disease. First-year graduate students enter the program with relevant college-level biology and chemistry coursework but not in-depth content knowledge of any of the focus areas. Each module features a biomedical problem for the students to gain specific content knowledge while developing skills outcomes, such as the ability to conduct scholarly inquiry. In 2015, the theme of the infectious disease module was to create an effective human vaccine to prevent Lyme disease. The module required students to learn fundamental concepts of microbiology and immunology and then apply that knowledge to design their own Lyme disease vaccine. The class culminated with students communicating their creative designs in the form of a "white paper" and a pitch to "potential investors." By the end of the module, students had developed fundamental knowledge, applied that knowledge with great creativity, and met the skills learning outcomes, as evidenced by their ability to conduct scholarly inquiry and apply knowledge gained during this module to a novel problem, as part of their final exam.
Project description:Electrophysiology is a decades-old technique widely used for monitoring activity of individual neurons and local field potentials. Optogenetics has revolutionized neuroscience studies by offering selective and fast control of targeted neurons and neuron populations. The combination of these two techniques is crucial for causal investigation of neural circuits and understanding their functional connectivity. However, electrical artifacts generated by light stimulation interfere with neural recordings and hinder the development of compact closed-loop systems for precise control of neural activity. Here, we demonstrate that transparent graphene micro-electrodes fabricated on a clear polyethylene terephthalate film eliminate the light-induced artifact problem and allow development of a compact battery-powered closed-loop optogenetics system. We extensively investigate light-induced artifacts for graphene electrodes in comparison to metal control electrodes. We then design optical stimulation module using micro-LED chips coupled to optical fibers to deliver light to intended depth for optogenetic stimulation. For artifact-free integration of graphene micro-electrode recordings with optogenetic stimulation, we design and develop a compact closed-loop system and validate it for different frequencies of interest for neural recordings. This compact closed-loop optogenetics system can be used for various applications involving optogenetic stimulation and electrophysiological recordings.
Project description:Optogenetics is a technology that is growing rapidly in neuroscience, establishing itself as a fundamental investigative tool. As this tool is increasingly utilized across the neuroscience community and is one of the primary research techniques being presented at neuroscience conferences and in journals, we believe that it is important that this technology is introduced into the undergraduate neuroscience research laboratory. While there has been a significant body of work concentrated to deploy optogenetics in invertebrate model organisms, little to no work has focused on brining this technology to mammalian model organisms in undergraduate neuroscience laboratories. The establishment of in vivo optogenetics could provide for high-impact independent research projects for upper-level undergraduate students. Here we review the considerations for establishing in vivo optogenetics with the use of rodents in an undergraduate laboratory setting and provide some cost-saving guidelines to assist in making optogenetic technologies financially accessible. We discuss opsin selection, cell-specific opsin expression strategies, species selection, experimental design, selection of light delivery systems, and the construction of implantable optical fibers for the application of in vivo optogenetics in rodents.
Project description:Current neuromodulation techniques such as optogenetics and deep-brain stimulation are transforming basic and translational neuroscience. These two neuromodulation approaches are, however, invasive since surgical implantation of an optical fiber or wire electrode is required. Here, we have invented a non-invasive magnetogenetics that combines the genetic targeting of a magnetoreceptor with remote magnetic stimulation. The non-invasive activation of neurons was achieved by neuronal expression of an exogenous magnetoreceptor, an iron-sulfur cluster assembly protein 1 (Isca1). In HEK-293 cells and cultured hippocampal neurons expressing this magnetoreceptor, application of an external magnetic field resulted in membrane depolarization and calcium influx in a reproducible and reversible manner, as indicated by the ultrasensitive fluorescent calcium indicator GCaMP6s. Moreover, the magnetogenetic control of neuronal activity might be dependent on the direction of the magnetic field and exhibits on-response and off-response patterns for the external magnetic field applied. The activation of this magnetoreceptor can depolarize neurons and elicit trains of action potentials, which can be triggered repetitively with a remote magnetic field in whole-cell patch-clamp recording. In transgenic Caenorhabditis elegans expressing this magnetoreceptor in myo-3-specific muscle cells or mec-4-specific neurons, application of the external magnetic field triggered muscle contraction and withdrawal behavior of the worms, indicative of magnet-dependent activation of muscle cells and touch receptor neurons, respectively. The advantages of magnetogenetics over optogenetics are its exclusive non-invasive, deep penetration, long-term continuous dosing, unlimited accessibility, spatial uniformity and relative safety. Like optogenetics that has gone through decade-long improvements, magnetogenetics, with continuous modification and maturation, will reshape the current landscape of neuromodulation toolboxes and will have a broad range of applications to basic and translational neuroscience as well as other biological sciences. We envision a new age of magnetogenetics is coming.
Project description:Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies.
Project description:Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~?1?mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations.
Project description:A critical technique for understanding how neuronal activity contributes to behavior is determining whether perturbing it changes behavior. The advent of optogenetic techniques allows the immediately reversible alteration of neuronal activity in contrast to chemical approaches lasting minutes to hours. Modification of behavior using optogenetics has had substantial success in rodents but has not been as successful in monkeys. Here, we show how optogenetic inactivation of superior colliculus neurons in awake monkeys leads to clear and repeatable behavioral deficits in the metrics of saccadic eye movements. We used our observations to evaluate principles governing the use of optogenetic techniques in the study of the neuronal bases of behavior in monkeys, particularly how experimental design must address relevant parameters, such as the application of light to subcortical structures, the spread of viral injections, and the extent of neuronal inactivation with light.
Project description:Cell assemblies manipulation by optogenetics is pivotal to advance neuroscience and neuroengineering. In in vivo applications, photostimulation often broadly addresses a population of cells simultaneously, leading to feed-forward and to reverberating responses in recurrent microcircuits. The former arise from direct activation of targets downstream, and are straightforward to interpret. The latter are consequence of feedback connectivity and may reflect a variety of time-scales and complex dynamical properties. We investigated wide-field photostimulation in cortical networks in vitro, employing substrate-integrated microelectrode arrays and long-term cultured neuronal networks. We characterized the effect of brief light pulses, while restricting the expression of channelrhodopsin to principal neurons. We evoked robust reverberating responses, oscillating in the physiological gamma frequency range, and found that such a frequency could be reliably manipulated varying the light pulse duration, not its intensity. By pharmacology, mathematical modelling, and intracellular recordings, we conclude that gamma oscillations likely emerge as in vivo from the excitatory-inhibitory interplay and that, unexpectedly, the light stimuli transiently facilitate excitatory synaptic transmission. Of relevance for in vitro models of (dys)functional cortical microcircuitry and in vivo manipulations of cell assemblies, we give for the first time evidence of network-level consequences of the alteration of synaptic physiology by optogenetics.