Correction: Specific Expression of Channelrhodopsin-2 in Single Neurons of Caenorhabditis elegans (original) (raw)
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PloS one, 2018
In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours ('Quint': ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H1...
High-Throughput Analysis of Behavior Under the Control of Optogenetics inCaenorhabditis elegans
Current protocols in neuroscience, 2018
In this unit, we describe an inexpensive and versatile method for optogenetic stimulation of a large population of genetically engineered Caenorhabditis elegans worms while quantitatively analyzing behavior. A custom light-emitting diode light source is used to deliver blue-light stimuli, causing direct depolarization of neurons expressing the light-gated cation channel Channelrhodopsin-2, which in turn evokes behavioral responses. The behavioral responses are recorded by a high-throughput machine vision-based tracking system, the Multi-Worm Tracker, for detailed analysis. This approach allows researchers to bypass technical obstacles to simultaneously deliver uniform stimuli to a large number of freely behaving animals and investigate the neural underpinnings of behavior.
2021
Optogenetic tools have revolutionized the study of neuronal circuits in Caenorhabditis elegans. The expression of light-sensitive ion channels or pumps under specific promotors allows researchers to modify the behavior of excitable cells. Several optogenetic systems have been developed to spatially and temporally photoactivate light-sensitive actuators in C. elegans. Nevertheless, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, and adjustable optogenetics device for use on different microscopes and worm trackers, called the OptoArm. The OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles and light-color. We demonstrate the OptoArm’s power in a population-based study on contributions of motor circuit cells to age-related motility decline. We find that functional decline of cholinergic neurons mirrors motor decline, while GABAergic neur...
Journal of Neurochemistry, 2011
Photoactivated adenylyl cyclase a (PACa) was originally isolated from the flagellate Euglena gracilis. Following stimulation by blue light it causes a rapid increase in cAMP levels. In the present study, we expressed PACa in cholinergic neurons of Caenorhabditis elegans. Photoactivation led to a rise in swimming frequency, speed of locomotion, and a decrease in the number of backward locomotion episodes. The extent of the light-induced behavioral effects was dependent on the amount of PACa that was expressed. Furthermore, electrophysiological recordings from body wall muscle cells revealed an increase in miniature post-synaptic currents during light stimulation. We conclude that the observed effects were caused by cAMP synthesis because of photoactivation of pre-synaptic PACa which subsequently triggered acetylcholine release at the neuromuscular junction. Our results demonstrate that PACa can be used as an optogenetic tool in C. elegans for straightforward in vivo manipulation of intracellular cAMP levels by light, with good temporal control and high cell specificity. Thus, using PACa allows manipulation of neurotransmitter release and behavior by directly affecting intracellular signaling.
Nature Methods, 2011
Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function, and dysfunction. We used a Bacterial Artificial Chromosome (BAC) transgenic strategy to express Channelrhodopsin2 (ChR2) under the control of cell-type specific promoter elements. We provide a detailed functional characterization of the newly established VGAT-ChR2-EYFP, ChAT-ChR2-EYFP, TPH2-ChR2-EYFP and Pvalb-ChR2-EYFP BAC transgenic mouse lines and demonstrate the utility of these lines for precisely controlling action potential firing of GABAergic, cholinergic, serotonergic, and parvalbumin+ neuron subsets using blue light. This Corresponding author -Guoping Feng (fengg@mit.edu). 9 These authors contributed equally to this work.
Optogenetic Long-Term Manipulation of Behavior and Animal Development
PLoS ONE, 2011
Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used ''slow'' ChR2 variants with mutations in the C128 residue, that exhibit delayed offkinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the ''dauer'' larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.
Optogenetic manipulation of neural circuits and behavior in Drosophila larvae
Nature Protocols, 2012
Optogenetics is a powerful tool that enables spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third larval instar. We have previously shown that, using spatially-defined GAL4 drivers and potent UAS-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies-with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes a week to culture optogenetic animals and about an hour per group for the behavioral assays. Keywords Drosophila; optogenetics; channelrhodopsin-2; behavior; nociception channel has been most successfully utilized. ChR2 is a light-gated cation channel from the green algae, Chlamydomonas reinhardtii 13. Upon photoactivation (with ∼480 nm blue light) ChR2 evokes depolarizing inward current on a millisecond timescale. When expressed Correspondence should be addressed to W.D.T.
Optogenetics: Novel Tools for Controlling Mammalian Cell Functions with Light
International Journal of Photoenergy, 2014
In optogenetics, targeted illumination is used to control the functions of cells expressing exogenous light-activated proteins. Adoption of the optogenetic methods has expanded rapidly in recent years. In this review, we describe the photosensitive channel proteins involved in these methods, describe techniques for their targeting to neurons and other cell types both within and outside the nervous system, and discuss their applications in the field of neuroscience and beyond. We focus especially on the channelrhodopsin protein ChR2, the photosensitive protein most commonly employed in optogenetics. ChR2 has been used by many groups to control neuronal activity, bothin vitroandin vivo, on short time scales and with exquisite anatomical precision. In addition, we describe more recently developed tools such as opsin/G protein-coupled receptor chimeric molecules and a light-activated transgene system. In addition, we discuss the potential significance of optogenetics in the development ...