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.


RESULTS
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
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.
Wen
Lei
L
Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan.
Wang
Hongxia
H
Tanimoto
Saki
S
Egawa
Ryo
R
Matsuzaka
Yoshiya
Y
Mushiake
Hajime
H
Ishizuka
Toru
T
Yawo
Hiromu
H
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D002696:Chlamydomonas / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D012243:Rhodopsin