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ONLINE SUPPLEMENTARY MATERIAL
Ultrafast optogenetic control
Lisa A. Gunaydin, Ofer Yizhar, André Berndt, Vikaas S. Sohal,
Karl Deisseroth and Peter Hegemann
Correspondence and requests for materials should be addressed to
P.H ([email protected]) or K.D. ([email protected]).
Nature Neuroscience: doi:10.1038/nn.2495
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Rec
over
y (%
)
100
75
50
25
Time (s)0 20 40 60
2s
ΔI1ΔI2
WTE123A
a
b
c
d
WT ChR2
τ in : 55.5 ms
0.4 sτoff : 9.8 ms
E123A
τ in : 14.7 ms
τoff : 5.5 ms
20 ms
5 ns
Wavelength (nm)
Act
ivity
(a.u
.)
1.0
0.5
0.0450 475 500 525 550 575
WTE123A
WT 9.2 msE123A 4.0 ms
τ off
Supplementary Figure 1. Characterization of an alternative fast mutant: E123A.
Nature Neuroscience: doi:10.1038/nn.2495
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Supplementary Figure 1:
Characterization of an alternative fast mutant: E123A.
(a) Voltage clamp recordings of WT ChR2 and E123A in Xenopus oocytes under stimulation
with a 1 s light pulse (blue bars, 500 nm ± 25, 50 mW/cm2) at -100 mV and 100 mM NaCl in the
external solution (pH 7.5). τ-values characterize the mono-exponential transition from peak to
stationary currents (τin) and current kinetics (τoff) after the light was switched off. τoff = 9.8 ± 1.3
ms for WT (n = 10) and τoff = 6.2 ± 1.3 ms for E123A (n = 11; p < 0.005). Vertical black bars: 10
nA. (b) Recovery of peak current in WT ChR2 and E123A upon stimulation with a second light
pulse after a variable dark period, recorded in oocytes. Inset shows the peak recovery of WT
ChR2 after 2s in darkness at -75 mV in standard solution (100 mM NaCl, pH 7.5). The ratio of
ΔI2 to ΔI1 yields the recovery in percent. Values for WT ChR2 (black circles) and E123A (pink
circles) are plotted versus the dark interval between the two flashes; data points were fit by a
mono-exponential decay (colored lines). Peak recovery was τ = 10.7 ± 0.8 s for WT (n = 15) and
τ = 0.9 ± 0.2 s for E123A (n = 4; p < 0.005). (c) Oocyte current recordings; excitation delivered
with a 5 nanosecond laser flash (470 nm, 0.5 mW/cm2, blue arrow) at -100 mV. Photocurrent
decay was fitted mono-exponentially with τ-values shown for WT ChR2 (black) and E123A
(pink), and traces were normalized to highlight differences in decay kinetics. Off kinetics were τ
= 9.2 ± 1.3 ms for WT (n = 9) and 4.0 ± 0.2 ms for E123A (n = 11; p < 0.005). Flash-to-peak
Nature Neuroscience: doi:10.1038/nn.2495
current times were tp = 2.4 ± 0.3 ms for WT (n = 9) and tp = 0.8 ± 0.1 ms for E123A (n = 11; p <
0.005). Vertical bars correspond to 2.5 nA (WT: black; E123A: pink). (d) The action spectrum
of E123A (pink, n = 3) is shown in comparison with spectrum of WT ChR2 (black, n = 6).
Current amplitudes were measured at the different wavelengths, normalized to respective
maximum values, and corrected for fluctuations of flash intensity.
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% s
ucce
ssfu
l spi
kes
Pulse-train interval1 2 3 4 1 2 3 4
1 2 3 4
Supplementary Figure 2. Temporal stationarity during extended trains.
WT ChR2ChETA
Nature Neuroscience: doi:10.1038/nn.2495
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Supplementary Figure 2:
Temporal stationarity during extended trains.
The percentage of successful spikes is plotted over time for WT ChR2 and ChETA, across a
range of light pulse frequency and light pulse width. Note the deterioration over time of WT
ChR2 performance in following light pulses. In contrast, the performance of ChETA (in addition
to showing improved efficacy at high frequencies for >1 ms pulse width) was temporally
stationary, in that performance in response to later light flashes was indistinguishable from that
for earlier light flashes in the train, across all conditions. Trains of 40 light pulses were delivered,
and spike responses were grouped into four intervals corresponding to successive blocks of 10
light pulses (interval 1 = light pulses 1-10, interval 2 = light pulses 11-20, interval 3 = light
pulses 21-30, interval 4 = light pulses 31-40).
Nature Neuroscience: doi:10.1038/nn.2495
ChETA_Suppl_Material_ 1-7-10ChETA_Suppl_Fig1_1-7-10ChETA_Suppl_Fig2_12-30-09ChETA_Suppl_Material_ 1-7-10