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Peter Hegemann - One of the best experts on this subject based on the ideXlab platform.

  • The form and functIon of Channelrhodopsin
    Science (New York N.Y.), 2017
    Co-Authors: Karl Deisseroth, Peter Hegemann
    Abstract:

    BACKGROUND Channelrhodopsins (ChRs) are naturally occurring Light-Gated Ion Channels that are important for allowing motile algal cells to find suitable light levels. In neuroscience, ChRs have become broadly significant for helping to enable the control of specific circuit elements with light (i.e., optogenetics). Research into how sensatIon, cognitIon, and behavior arise from neuronal activity dynamics has been enabled by the expressIon of ChRs, and other members of the microbial opsin family, in specific cells or in specific connectIons within nervous systems of behaving animals. Both the unique Light-Gated Channels themselves and opportunities for their biological applicatIon have been under intense investigatIon. The resulting studies of atomic-scale structure-functIon relatIonships have led not only to sophisticated understanding of the underlying chemical processes governing these unique seven-transmembrane Channels from the plant kingdom, but also (via optogenetics) to the discovery of fundamental neural circuit principles underlying adaptive and maladaptive behavior in animals. ADVANCES The atomic-scale understanding of Light-Gated Ion Channel functIon has spanned the key processes of activatIon/deactivatIon gating, light adaptatIon, color tuning, and Ion selectivity. A ChR crystal structure–derived, molecular dynamics–calculated pore snapshot (top left panel of the figure) summarizes the wide scope of biophysical and biochemical discoveries. Molecular modeling and redesign have created multiple modes of coupling between delivered photons and spikes in an approach that has illuminated basic principles of protein functIon and also created new tools for optogenetics. In the top right panel of the figure, the top trace shows a photon-spike transductIon mode arising from the ChETA mutatIon, which results in high-speed, high-fidelity single blue flash–single spike coupling. The second trace shows red photon-spike transductIon arising from a redshifted ChR found in nature and then engineered for stronger, more redshifted performance (C1V1). The third trace shows bistable excitatIon photon-spike logic, in which step-functIon opsin (SFO) mutatIons were introduced to create stalled photocycles, allowing stable excitatIon without continuous light delivery. The bottom trace shows bistable inhibitIon photon-spike logic; ChRs that are normally catIon-conducting, and are therefore excitatory in neural systems, were converted to anIon-conducting (inhibitory) ChRs by replacing negatively charged pore residues, followed by SFO mutatIons for bistability. The C1V1 and SFO designs together allowed us to determine that the medial prefrontal neocortex modulates interactIons between two distant subcortical structures to control reward-mediating physiology and behavior (clarityresourcecenter.org/ofMRI.html; www.optogenetics.org). OUTLOOK The ChR Light-Gated pore will continue to be studied for its own elegant properties, which are paradigmatic among Ion Channels because Light-Gated systems allow structure-functIon analysis on the femtosecond time scale. Meanwhile, psychiatry has already yielded some of its deepest mysteries to ChR pore structural insights, including in exploratIons of clinically relevant behavioral states such as anhedonia. Many more opportunities for ChRs in basic neuroscience remain untapped, with the potential for precisIon redesign to achieve new applicatIons and new roles integrated with other advanced technologies.

  • conversIon of a light driven proton pump into a light gated Ion Channel
    Scientific Reports, 2015
    Co-Authors: A Vogt, Suneel Kateriya, Satoshi P. Tsunoda, Yanan Guo, Marcus Elstner, Peter Hegemann
    Abstract:

    Interest in microbial rhodopsins with Ion pumping activity has been revitalized in the context of optogenetics, where light-driven Ion pumps are used for cell hyperpolarizatIon and voltage sensing. We identified an opsin-encoding gene (CsR) in the genome of the arctic alga Coccomyxa subellipsoidea C-169 that can produce large photocurrents in Xenopus oocytes. We used this property to analyze the functIon of individual residues in proton pumping. ModificatIon of the highly conserved proton shuttling residue R83 or its interactIon partner Y57 strongly reduced pumping power. Moreover, this mutatIon converted CsR at moderate electrochemical load into an operatIonal proton Channel with inward or outward rectificatIon depending on the amino acid substitutIon. Together with molecular dynamics simulatIons, these data demonstrate that CsR-R83 and its interacting partner Y57 in conjunctIon with water molecules forms a proton shuttle that blocks passive proton flux during the dark-state but promotes proton movement uphill upon illuminatIon.

  • color tuned Channelrhodopsins for multiwavelength optogenetics
    Journal of Biological Chemistry, 2012
    Co-Authors: Matthias Prigge, Jonas Wietek, Satoshi P. Tsunoda, Franziska Schneider, Carrie Shilyansky, Karl Deisseroth, Peter Hegemann
    Abstract:

    Channelrhodopsin-2 is a Light-Gated Ion Channel and a major tool of optogenetics. It is used to control neuronal activity via blue light. Here we describe the constructIon of color-tuned high efficiency Channelrhodopsins (ChRs), based on chimeras of Chlamydomonas Channelrhodopsin-1 and Volvox Channelrhodopsin-1. These variants show superb expressIon and plasma membrane integratIon, resulting in 3-fold larger photocurrents in HEK cells compared with Channelrhodopsin-2. Further molecular engineering gave rise to chimeric variants with absorptIon maxima ranging from 526 to 545 nm, dovetailing well with maxima of Channelrhodopsin-2 derivatives ranging from 461 to 492 nm. AdditIonal kinetic fine-tuning led to derivatives in which the lifetimes of the open state range from 19 ms to 5 s. Finally, combining green- with blue-absorbing variants allowed independent activatIon of two distinct neural cell populatIons at 560 and 405 nm. This novel panel of Channelrhodopsin variants may serve as an important toolkit element for dual-color cell stimulatIon in neural circuits.

  • Fast, repetitive light-activatIon of CaV3.2 using Channelrhodopsin 2.
    Channels (Austin Tex.), 2010
    Co-Authors: Matthias Prigge, Anton Rösler, Peter Hegemann
    Abstract:

    Channelrhodopsin-2 (ChR2) is a Light-Gated Ion Channel that is successfully used in neurosciences to depolarize cells with blue light. In this regard control of membrane voltage with light opens new perspectives for the characterizatIon of Ion Channels and the search for inhibitors or modulators. Here, we report a control of membrane potential with ChR2 and the potassium Channel mTrek for the purpose of screening for Ion Channel specific drugs. To verify principle we have chosen the voltage gated calcium Channel Ca(V)3.2 as potential drug target. For this purpose we transfected the ChR2 gene into a HEK293T-cell line that permanently expresses Ca(V)3.2 and the K-Channel mTrek. The resting potential was adjusted with low concentratIon of extracellular potassium Ions whereas transient depolarizatIon was achieved by activatIon of ChR2 with short pulses of blue light. Calcium Ion influx through Ca(V)3.2 was monitored by observing fura-2 fluorescence. This approach allowed a repetitive activatIon of Ca(V)3.2. The Ca(2+) influx was specifically blocked by the inhibitor mibefradil. Since this assay is genetically-encoded, it may be employed for a variety of voltage-gated calcium Channels and should be applicable to multi-well reader formats for high-throughput screening.

  • Chlamyrhodopsin represents a new type of sensory photoreceptor.
    The EMBO journal, 1995
    Co-Authors: W Deininger, P Kröger, U Hegemann, F Lottspeich, Peter Hegemann
    Abstract:

    In order to find optimal light conditIons for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transductIon chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparatIon of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot regIon of whole cells during growth and cell divisIon. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might functIon as a Light-Gated Ion Channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolutIon.

Alipasha Vaziri - One of the best experts on this subject based on the ideXlab platform.

  • two photon single cell optogenetic control of neuronal activity by sculpted light
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Bertalan K Andrasfalvy, Boris V Zemelman, Jianyong Tang, Alipasha Vaziri
    Abstract:

    Recent advances in optogenetic techniques have generated new tools for controlling neuronal activity, with a wide range of neuroscience applicatIons. The most commonly used approach has been the optical activatIon of the Light-Gated Ion Channel Channelrhodopsin-2 (ChR2). However, targeted single-cell-level optogenetic activatIon with temporal precessIons comparable to the spike timing remained challenging. Here we report fast (≤1 ms), selective, and targeted control of neuronal activity with single-cell resolutIon in hippocampal slices. Using temporally focused laser pulses (TEFO) for which the axial beam profile can be controlled independently of its lateral distributIon, large numbers of Channels on individual neurons can be excited simultaneously, leading to strong (up to 15 mV) and fast (≤1 ms) depolarizatIons. Furthermore, we demonstrated selective activatIon of cellular compartments, such as dendrites and large presynaptic terminals, at depths up to 150 μm. The demonstrated spatiotemporal resolutIon and the selectivity provided by TEFO allow manipulatIon of neuronal activity, with a large number of applicatIons in studies of neuronal microcircuit functIon in vitro and in vivo.

  • Two-Photon Optogenetic Control of Neuronal Activity with Single Synapse PrecisIon by Sculpted Light
    Biophysical Journal, 2010
    Co-Authors: Alipasha Vaziri
    Abstract:

    Recent advances in optogenetic techniques have provided new tools for controlling neuronal activity, opening up the way to a range of studies in neuroscience. The most widely used approach has been the optical activatIon of the genetically expressed Light-Gated Ion Channel Channelrhodopsin-2 (ChR2) to initiate populatIon activity in neuronal circuits. However, single cell resolutIon of optogenetic activatIon has remained challenging. This is because neither single-photon nor conventIonal two-photon excitatIon provides the necessary combinatIon of high spatial selectivity and the simultaneous stimulatIon of a sufficiently large membrane area necessary to induce fast and significant depolarizatIons by ChR2 in a single neuron.The presented work reports on two-photon excitatIon of ChR2 allowing the generatIon of fast and large ChR2-mediated currents in single cells with high spatial and temporal resolutIon by using temporally focused beams. It is demonstrated that this technique efficiently induces strong depolarizatIon and reliable actIon potential firing in single ChR2-expressing neurons in rat and mouse hippocampal slices. It is further shown that subcellular compartments such as dendrites and large presynaptic terminals can be activated by the TF-2P technique. The superb spatial and temporal resolutIon provided by this technique allows so far unattainable precisIon for fine manipulatIon of neuronal activity to study and control the functIon of neuronal microcircuits in vitro and in vivo.

Tsuneo Urisu - One of the best experts on this subject based on the ideXlab platform.

  • INCUBATIon TYPE PLANAR PATCH CLAMP AS A NEW POTENTIAL TECHNOLOGY FOR DEVELOPING NEURONAL NETWORK HIGH THROUGHPUT SCREENING DEVICES
    Biomedical Engineering: Applications Basis and Communications, 2016
    Co-Authors: Tsuneo Urisu
    Abstract:

    Ion-Channel current recordings based on an incubatIon type planar patch clamp were first reported in 2008, using HEK293 cells expressed with TRPV1 Channels and capsaicin as ligand molecules. At first the success probability (number of devices which normally worked/total number of devices fabricated) was extremely low (several %). Several years later, we have succeeded in significantly decreasing the base line noise by using a salt-bridge-type Ag/AgCl electrode and successfully demonstrated the applicatIon of an incubatIon type planar patch clamp to ligand gated Ion-Channel biosensors and light gated Ion-Channel biosensors using HEK293 cells expressed with ChRWR. Furthermore, a spontaneous Ion-Channel current from a neuronal network was successfully observed by using a planar patch clamp chip, on which the neuronal network was occasIonally formed with a soma of a neuron on a micro through-hole. Although the neuronal network was not controlled, this success shows the high potential of realizing a high throu...

  • INCUBATIon TYPE PLANAR PATCH CLAMP AS A NEW POTENTIAL TECHNOLOGY FOR DEVELOPING NEURONAL NETWORK HIGH THROUGHPUT SCREENING DEVICES
    Biomedical Engineering: Applications Basis and Communications, 2016
    Co-Authors: Tsuneo Urisu
    Abstract:

    Ion-Channel current recordings based on an incubatIon type planar patch clamp were first reported in 2008, using HEK293 cells expressed with TRPV1 Channels and capsaicin as ligand molecules. At first the success probability (number of devices which normally worked/total number of devices fabricated) was extremely low (several %). Several years later, we have succeeded in significantly decreasing the base line noise by using a salt-bridge-type Ag/AgCl electrode and successfully demonstrated the applicatIon of an incubatIon type planar patch clamp to ligand gated Ion-Channel biosensors and light gated Ion-Channel biosensors using HEK293 cells expressed with ChRWR. Furthermore, a spontaneous Ion-Channel current from a neuronal network was successfully observed by using a planar patch clamp chip, on which the neuronal network was occasIonally formed with a soma of a neuron on a micro through-hole. Although the neuronal network was not controlled, this success shows the high potential of realizing a high throughput screening device on the basis of Channel current measurements, which contain the most important informatIon on network conditIons.

  • Channelrhodopsin as a Noble Biomaterial Useful for the OperatIon and Performance Test of the IonChannel Devices
    MATERIALS TRANSACTIONS, 2012
    Co-Authors: Hidetaka Uno, Toru Ishizuka, Hiromu Yawo, Zhi-hong Wang, Noriko Takada, Tsuneo Urisu
    Abstract:

    The Light-Gated Ion-Channel protein is expected to be able to improve the performance of IonChannel devices for the multi-point screening and neural network devices based on the planar patch clamp. However, no studies with Light-Gated Ion Channels on a planar patch clamp have been reported. We constructed an incubatIon type of planar patch clamp biosensor using silicon-on-insulator substrates with a 1­2µm micropore in them. Channelrhodopsin 2-expressing C2C12 cells and Channelrhodopsin/wide receiver-expressing HEK293 cells were seeded on the surface of the sensor chip and incubated to almost complete confluence. A laser beam (473nm wavelength) was radiated on the cells adsorbing to the sensor chip’s micropore and the Light-Gated Channel currents of the voltage clamp and actIon potentials of the current clamp modes were observed. Good signal to noise ratios were obtained, although the seal resistance was not so large, 10­30M³. The observed pulse shape of the Ion-Channel currents also qualitatively resembled reported results measured with a pipette patch clamp. These results strongly suggest that the Light-Gated Ion Channel proteins are useful biomaterials for IonChannel devices. [doi:10.2320/matertrans.N-M2012814]

William Guido - One of the best experts on this subject based on the ideXlab platform.

  • Optogenetic StimulatIon of the Corticothalamic Pathway Affects Relay Cells and GABAergic Neurons Differently in the Mouse Visual Thalamus
    2016
    Co-Authors: Chris W. D. Jurgens, Karen A. Bell, Rory A. Mcquiston, William Guido
    Abstract:

    The dorsal lateral geniculate nucleus (dLGN) serves as the primary conduit of retinal informatIon to visual cortex. In additIon to retinal input, dLGN receives a large feedback projectIon from layer VI of visual cortex. Such input modulates thalamic signal transmissIon in different ways that range from gain control to synchronizing network activity in a stimulus-specific manner. However, the mechanisms underlying such modulatIon have been difficult to study, in part because of the complex circuitry and diverse cell types this pathway innervates. To address this and overcome some of the technical limitatIons inherent in studying the corticothalamic (CT) pathway, we adopted a slice preparatIon in which we were able to stimulate CT terminal arbors in the visual thalamus of the mouse with blue light by using an adeno-associated virus to express the Light-Gated Ion Channel, ChIEF, in layer VI neurons. To examine the postsynaptic responses evoked by repetitive CT stimulatIon, we recorded from identified relay cells in dLGN, as well as GFP expressing GABAergic neurons in the thalamic reticular nucleus (TRN) and intrinsic interneurons of dLGN. Relay neurons exhibited large glutamatergic responses that continued to increase in amplitude with each successive stimulus pulse. While excitatory responses were apparent at postnatal day 10, the strong facilitatIon noted in adult was not observed until postnatal day 21. GABAergic neurons in TRN exhibited large initial excitatory responses that quickly plateaued during repetitive stimulatIon, indicating that the degree of facilitatIon was much larger for relay cells than for TRN neurons. The responses of intrinsic interneurons were smaller an

  • Optogenetic stimulatIon of the corticothalamic pathway affects relay cells and GABAergic neurons differently in the mouse visual thalamus.
    PLOS ONE, 2012
    Co-Authors: Chris W. D. Jurgens, Karen A. Bell, A. Rory Mcquiston, William Guido
    Abstract:

    The dorsal lateral geniculate nucleus (dLGN) serves as the primary conduit of retinal informatIon to visual cortex. In additIon to retinal input, dLGN receives a large feedback projectIon from layer VI of visual cortex. Such input modulates thalamic signal transmissIon in different ways that range from gain control to synchronizing network activity in a stimulus-specific manner. However, the mechanisms underlying such modulatIon have been difficult to study, in part because of the complex circuitry and diverse cell types this pathway innervates. To address this and overcome some of the technical limitatIons inherent in studying the corticothalamic (CT) pathway, we adopted a slice preparatIon in which we were able to stimulate CT terminal arbors in the visual thalamus of the mouse with blue light by using an adeno-associated virus to express the Light-Gated Ion Channel, ChIEF, in layer VI neurons. To examine the postsynaptic responses evoked by repetitive CT stimulatIon, we recorded from identified relay cells in dLGN, as well as GFP expressing GABAergic neurons in the thalamic reticular nucleus (TRN) and intrinsic interneurons of dLGN. Relay neurons exhibited large glutamatergic responses that continued to increase in amplitude with each successive stimulus pulse. While excitatory responses were apparent at postnatal day 10, the strong facilitatIon noted in adult was not observed until postnatal day 21. GABAergic neurons in TRN exhibited large initial excitatory responses that quickly plateaued during repetitive stimulatIon, indicating that the degree of facilitatIon was much larger for relay cells than for TRN neurons. The responses of intrinsic interneurons were smaller and took the form of a slow depolarizatIon. These differences in the pattern of excitatIon for different thalamic cell types should help provide a framework for understanding how CT feedback alters the activity of visual thalamic circuitry during sensory processing as well as different behavioral or pathophysiological states.

Sheena A. Josselyn - One of the best experts on this subject based on the ideXlab platform.

  • The past, present and future of Light-Gated Ion Channels and optogenetics.
    eLife, 2018
    Co-Authors: Sheena A. Josselyn
    Abstract:

    The discovery of the mechanisms underlying Light-Gated Ion Channels called Channelrhodospins and the subsequent development of optogenetics illustrates how breakthroughs in science and technology can span multiple levels of scientific inquiry. Our knowledge of how Channelrhodopsins work emerged from research at the microscopic level that investigated the structure and functIon of algal proteins. Optogenetics, on the other hand, exploits the power of Channelrhodospins and similar proteins to investigate phenomena at the supra-macroscopic level, notably the neural circuits involved in animal behavior that may be relevant for understanding neuropsychiatric disease. This article is being published to celebrate Peter Hegemann, Karl Deisseroth and Ed Boyden receiving a 2018 Canada Gairdner InternatIonal Award "for the discovery of Light-Gated Ion Channel mechanisms, and for the discovery of optogenetics, a technology that has revolutIonized neuroscience".