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Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin

BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics....

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Autores principales: Wen, Lei, Wang, Hongxia, Tanimoto, Saki, Egawa, Ryo, Matsuzaka, Yoshiya, Mushiake, Hajime, Ishizuka, Toru, Yawo, Hiromu
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944835/
https://www.ncbi.nlm.nih.gov/pubmed/20886118
http://dx.doi.org/10.1371/journal.pone.0012893
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author Wen, Lei
Wang, Hongxia
Tanimoto, Saki
Egawa, Ryo
Matsuzaka, Yoshiya
Mushiake, Hajime
Ishizuka, Toru
Yawo, Hiromu
author_facet Wen, Lei
Wang, Hongxia
Tanimoto, Saki
Egawa, Ryo
Matsuzaka, Yoshiya
Mushiake, Hajime
Ishizuka, Toru
Yawo, Hiromu
author_sort Wen, Lei
collection PubMed
description BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5–10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5–10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state.
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spelling pubmed-29448352010-09-30 Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin Wen, Lei Wang, Hongxia Tanimoto, Saki Egawa, Ryo Matsuzaka, Yoshiya Mushiake, Hajime Ishizuka, Toru Yawo, Hiromu PLoS One Research Article BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5–10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5–10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state. Public Library of Science 2010-09-23 /pmc/articles/PMC2944835/ /pubmed/20886118 http://dx.doi.org/10.1371/journal.pone.0012893 Text en Wen et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Wen, Lei
Wang, Hongxia
Tanimoto, Saki
Egawa, Ryo
Matsuzaka, Yoshiya
Mushiake, Hajime
Ishizuka, Toru
Yawo, Hiromu
Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin
title Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin
title_full Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin
title_fullStr Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin
title_full_unstemmed Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin
title_short Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin
title_sort opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944835/
https://www.ncbi.nlm.nih.gov/pubmed/20886118
http://dx.doi.org/10.1371/journal.pone.0012893
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