Article Figures & Data
Figures
- Figure 1.
Expression of three ChR variants in excitatory neurons of the mouse visual cortex. A, AAVs carrying three opsins were injected into primary visual cortex of wild-type mice; ITR, inverted terminal repeat; WRPE, woodchunk hepatitis B virus post-transcriptional element. B, Spread of viral infection in the cortex. Example image showing GFP expression (green) around the area of a cortical injection of AAV5 carrying the Chronos construct. Magnification: 4×. C, ChR2-GFP, Chronos-GFP, and Chrimson-GFP were robustly expressed in excitatory neurons in cortical layers 2, 3, and 5, as confirmed by DAPI staining (blue). Magnification: 10×. D, Example confocal images showing expression of the three ChR variants (green) in layer 5 pyramidal neurons stained for the neuronal marker NeuN (red). Scale bar: 10 μm. Magnification: 64×. E, Quantification of expression in layers 2/3 (left) and 5 (right) for each opsin.
- Figure 2.
Different ChRs evoke cortical activity with distinct temporal profiles in vivo. A, Raster plots (upper) and histograms (lower) of example MU spike activity during stimulation of excitatory pyramidal neurons with ChR2. The 1.5-s-long interval of light stimulation (10 mW/mm2) is indicated as shaded box. An asterisk indicates the peak firing evoked by the light pulse. B, Same as in A, for Chronos. C, Same as in A, for Chrimson. D, Average PSTH for all recorded sites in ChR2-expressing mice. Red symbols and lines indicate the mean peak time and SEM of the peak time, respectively. Inset shows the initial period if evoked firing in the first 600 ms of light stimulation. E, Same as in D, for Chronos. F, Same as in D, for Chrimson.
- Figure 3.
Amplitude and frequency distribution of evoked spike response varies with optogenetic tool. A, ISIs of spontaneous (black) and evoked (red) MU activity during optogenetic stimulation (10 mW/mm2) in ChR2-expressing cortex. Inset shows an enlarged plot of the initial 200 ms of the evoked spike response. B, Same as in A, for Chronos. C, Same as in A, for Chrimson. Error bars denote SEM. D, Firing rates evoked by ChR2 stimulation over a range of intensities, divided by baseline spontaneous firing immediately before the light pulses. E, Same as in D, for Chronos. F, Same as in D, for Chrimson. Dashed lines indicate linear regression of the data. Error bars denote SEM.
- Figure 4.
Distinct changes in evoked spike patterns during the first 100 ms after light onset. A, ISIs of spontaneous (black) and evoked (red) MU activity during the initial 100 ms of optogenetic stimulation (10 mW/mm2) in ChR2-expressing cortex. B, Same as in A, for Chronos. C, Same as in A, for Chrimson. Error bars denote SEM. D, Firing rates evoked by the initial 100 ms of ChR2 stimulation over a range of intensities, divided by baseline spontaneous firing rates immediately before the light pulses. E, Same as in D, for Chronos. F, Same as in D, for Chrimson. Dashed lines indicate linear regression of the data. Error bars denote SEM.
- Figure 5.
Different ChRs evoke distinct cortical activity profiles in vivo. A, Example traces of cortical LFP activity in response to 1.5 s of 10 mW/mm2 light stimulation of ChR2-expressing pyramidal neurons (upper). Average changes in spectral power density at this site across stimulation trials (lower). B, Same as in A, for Chronos. C, Same as in A, for Chrimson.
- Figure 6.
Distinct recruitment of γ-band activity by different ChRs. A, Normalized power spectra (upper) for spontaneous (blue) and evoked (red) cortical LFP activity in response to ChR2 stimulation, averaged across all stimulation levels, and the ratio between evoked and spontaneous spectra (lower). B, Same as in A, for Chronos. C, Same as in A, for Chrimson. Shaded areas denote ± SEM. D, Change in the relative power in the γ band (30–80 Hz) in response to varying light intensities in cortex expressing ChR2. E, Same as in D, for Chronos. F, Same as in D, for Chrimson. Error bars denote SEM.
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