Reference - Detail
|Author||Wen L, Wang H, Tanimoto S, Egawa R, Matsuzaka Y, Mushiake H, Ishizuka T, Yawo H.|
|Title||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. 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.
|MeSH||Amino Acid Sequence Animals Cells, Cultured Cerebral Cortex / chemistry Cerebral Cortex / cytology Cerebral Cortex / metabolism Chlamydomonas / genetics Chlamydomonas / metabolism* Electrophysiology HEK293 Cells Humans Kinetics Mice Molecular Sequence Data Neurons / chemistry* Neurons / metabolism Patch-Clamp Techniques Protein Engineering* Protein Structure, Tertiary Rhodopsin / chemistry* Rhodopsin / genetics* Rhodopsin / metabolism|
|DNA material||pCMV-chopGR-Venus (RDB16775) pCMV-ChRGR-Venus (RDB16776)|