NATURE METHODS
CORRECTION NOTICE
Nat. Methods 11, 338–346 (2014)
Independent optical excitation of distinct neural populationsNathan C Klapoetke, Yasunobu Murata, Sung Soo Kim, Stefan R Pulver, Amanda Birdsey-Benson, Yong Ku Cho, Tania K Morimoto, Amy S Chuong, Eric J Carpenter, Zhijian Tian, Jun Wang, Yinlong Xie, Zhixiang Yan, Yong Zhang, Brian Y Chow, Barbara Surek, Michael Melkonian, Vivek Jayaraman, Martha Constantine-Paton, Gane Ka-Shu Wong & Edward S BoydenIn the version of this supplementary file originally posted online, CsChrimson was incorrectly labeled as Chrimson. The error has been corrected in this file as of 28 August 2014.
Independent Optical Excitation of Distinct Neural Populations Nathan C Klapoetke1-5, Yasunobu Murata4-5, Sung Soo Kim6, Stefan R. Pulver6, Amanda Birdsey-Benson4-5, Yong Ku Cho1-5, Tania K Morimoto1-5, Amy S Chuong1-5, Eric J Carpenter7, Zhijian Tian8, Jun Wang8, Yinlong Xie8, Zhixiang Yan8, Yong Zhang8, Brian Y Chow9, Barbara Surek10, Michael Melkonian10, Vivek Jayaraman6, Martha Constantine-Paton4-5, Gane Ka-Shu Wong7,8,11, Edward S Boyden1-5 1 The MIT Media Laboratory, Synthetic Neurobiology Group, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 2 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 3 MIT Center for Neurobiological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 4 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 5 MIT McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 6 Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA 7 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada 8 Beijing Genomics Institute-Shenzhen, Shenzhen, China 9 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA 10 Institute of Botany, Cologne Biocenter, University of Cologne, Cologne, Germany 11 Department of Medicine, University of Alberta, Edmonton, Alberta, Canada Co-corresponding authors, Edward S Boyden ([email protected]), Gane KS Wong ([email protected])
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 1 Phylogenetic relation of algal opsins.
Supplementary Figure 2 Sequence alignment.
Supplementary Figure 3 Characterization of channel kinetics of Chronos and Chrimson in HEK293FT cells.
Supplementary Figure 4 Comparison of ion selectivity of Chronos, Chrimson, and ChR2.
Supplementary Figure 5 Opsin screening in cultured neurons.
Supplementary Figure 6 Opsin trafficking in cultured neurons.
Supplementary Figure 7 Inactivation and recovery kinetics.
Supplementary Figure 8 Chronos full inactivation and recovery kinetics.
Supplementary Figure 9 ReaChR and Chrimson comparison in cultured neurons.
Supplementary Figure 10 Green light driven spiking frequency responses in cultured neurons.
Supplementary Figure 11 Electrical versus green light driven spiking fidelity in cultured neurons.
Supplementary Figure 12 Red light driven spiking in cultured neurons.
Supplementary Figure 13 Red and far-red spiking with ChrimsonR in acute cortical slice.
Supplementary Figure 14 Larval motor axons expressing ChR2 fire in response to blue but not red light pulses.
Supplementary Figure 15 Proboscis extension reflex (PER).
Supplementary Figure 16 Optogenetics of freely behaving intact flies.
Supplementary Figure 17 Two-color excitation controls in cultured neurons.
Supplementary Figure 18 Optically evoked post-synaptic currents (PSCs) in acute slice.
Supplementary Figure 19 Optically evoked paired-pulse responses in acute slice.
Supplementary Figure 20 Comparisons of spiking and post-synaptic response timing in acute slice.
Supplementary Figure 21 Retina to superior colliculus projection stimulation with Chronos.
Supplementary Figure 22 Post-synaptic current raw traces.
Supplementary Table 1 Naming convention
Supplementary Table 2 Statistical analysis for opsin comparisons
Supplementary Table 3 Primer sequences
Supplementary Table 4 Solutions used to characterize ion selectivity
Supplementary Video 1 Experimental setup with a visual arena.
Supplementary Video 2 PER of a Gr64f x CsChrimson fly to 720 nm light in darkness.
Supplementary Video 3 Startle response to 720 nm light in darkness
Supplementary Video 4 PER of a Gr64f x CsChrimson fly to 720 nm light in a blue random dot arena.
Supplementary Video 5 Inhibited startle response to 720 nm light in a blue random dot arena.
Supplementary Video 6 Optogenetics in freely behaving intact flies.
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 1 – Phylogenetic relation of algal opsins.
Phylogenetic tree of novel opsins discovered from de novo transcriptomic
sequencing of over 100 algal species. Only full length opsin sequences (i.e. has
seven transmembrane helices) were analyzed. In some cases the transcriptome
sequencing resulted in truncated opsin sequences, and rapid amplification of
cDNA ends (RACE) was additionally performed on the original algal species to
obtain the full length opsin sequence. See Supplementary Table 1 for algal
genus/species names, as well as the nicknames or aliases used in the main text
of the paper (not all of the channelrhodopsins we obtained, were assigned
nicknames or aliases, but instead are referred to just by number). Scale bar is
the number of amino acid substitutions per site.
Nature Methods: doi:10.1038/nmeth.2836
VChR2
gene124alt
gene86
ChR2
gene116
ChR1
VChR1
gene74
gene73
gene125
gene65
gene89
Gene111
gene119
gene62
gene123
gene121
gene85
CaChR1
CyChR1
gene87
gene80
Gene112
DChR1
gene90
gene67
gene122
gene117
gene95
gene120
gene92
gene88
gene93
gene91
gene64
Gene115
gene76
gene75
gene77
gene66
gene68
gene71
gene60
MvChR1
Gene113
Gene114
gene63
gene69
gene96
gene70
gene97
gene107
gene108
gene109
gene84
gene100
gene72
gene79
Halo
gene110
gene104
gene105
gene102
gene103
gene101
gene98
gene106
gene99
Bacteriorhodopsin
Arch
gene78
gene61
gene59
gene118
0.000.050.100.150.200.250.300.35
^
^
^^
^R
RR
RR
R
XX
XX
XX
XX
XX
XO
OO
OO
OO
OX
XO
XO
OO
OX
XX
XX
XX
XX
OX
OO
OO
OO
OO
OO
O
^O
^^
OO
^
OO
OO
OO
X
^^
R
X
O
^
photocurrent in HEK293or cultured neurons
no detectable current in HEK293
previously published
no annotation meansdid not test gene
RACE
Supplementary Figure 1
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 2 – Sequence alignment.
Protein sequence alignment of algal opsins screened in cultured neurons (Fig. 1).
Acidic residues are shown in red, basic residues are shown in blue.
Transmembrane regions are denoted by black bar above alignment based on
C1C2 crystal structure annotation. Schiff base lysine is annotated as *.
Nature Methods: doi:10.1038/nmeth.2836
CoChR
CoChR
CoChR
AgChR
AgChR
AgChR
VChR1
VChR1
VChR1
TsChR
TsChR
TsChR
ChR1
ChR1
ChR1
SdChR
SdChR
SdChR
TcChR
TcChR
TcChR
BsChR2
BsChR2
BsChR2
HdChR
HdChR
HdChR
PsChR1
PsChR1
PsChR1
C1V1_TT_
C1V1_TT_
C1V1_TT_
ChR2
ChR2
ChR2
CsChR
CsChR
CsChR
Chrimson
Chrimson
Chrimson
PsChR2
PsChR2
PsChR2
CbChR1
CbChR1
CbChR1
CnChR2
CnChR2
CnChR2
NsChR
NsChR
NsChR
Chronos
Chronos
Chronos
MvChR1
MvChR1
MvChR1
consensus
consensus
consensus
10 20 30 40 50 60 70 80 90 100 110 120
130 140 150 160 170 180 190 200 210 220 230 240 250
260 270 280 290 300 310 320 330 340 350 360 370 380
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M L G N G S A I V P I D - - Q C F C L A W T D S L G S D T E Q L V A N I L Q W F A F G F S I L I L M F Y A Y Q T W R A
T C G W E E V Y V C C V E L T K V I I E F F H E F D D P S M L Y L A N G H R V Q W L R Y A E W L L T C P V I L I H L S N L T G L K D D Y S K R T M R L L V S D V G T I V W G A T S A M S - T G Y V K V I F F V L G - - - - - - C I Y G A N T F F H A A K V Y I E
S Y - - - - - - - H V V P K G R P R T V V R I M A W L F F L S W G M F P V L F V V G P E G F D A I S V Y G S T I G H T I I D L M S K N C W G L L G H - Y L R V L I H Q H I I I Y G D I R K K T K I N - - - - V A G E E M E V E T M V D Q E D E E T V - - - - -
- - M G - - - - - - - - - - - - - - - - T P D P L L S S - - - - - - - - - - - I P G T D I G L G D W T E Y S N Y Y F L N - - - - - - - - A T N S T H K W V A G P E - D D C F C K A W T F N R G S D E E S V A A F A I A W V V F S L S V L Q L L Y Y A Y A Q W R S
T C G W E E V Y V G I I E L T H I C I A I F R E F D S P A M L Y L S T G N F V V W A R Y A S W L L S C P V I L I H L S N L T G M K G N Y S K R T M A L L V S D I G T I V W G S T S A M S P H N H V K I I F F F L G - - - - - - L V F G L F T F Y A A A K V Y L E
A Y - - - - - - - H T V P K G K C R N I V R F M A W T Y Y V T W A L F P I L F I L G P E G F G H I T Y Y G S S I G H Y V L E I F S K N L W S G T G H - Y L R L K I H E H I I L H G N L T K K T K I N - - - - I A G E P L E V E E Y V E A D D - T D E G V - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M D Y P V A R S - - - - - - - - - - - - - - - L I V R Y P T D L G N G T V C M P R - G Q C Y C E G W L R S R G T S I E K T I A I T L Q W V V F A L S V A C L G W Y A Y Q A W R A
T C G W E E V Y V A L I E M M K S I I E A F H E F D S P A T L W L S S G N G V V W M R Y G E W L L T C P V L L I H L S N L T G L K D D Y S K R T M G L L V S D V G C I V W G A T S A M C - T G W T K I L F F L I S - - - - - - L S Y G M Y T Y F H A A K V Y I E
A F - - - - - - - H T V P K G I C R E L V R V M A W T F F V A W G M F P V L F L L G T E G F G H I S P Y G S A I G H S I L D L I A K N M W G V L G N - Y L R V K I H E H I L L Y G D I R K K Q K I T - - - - I A G Q E M E V E T L V A E E E D D T V K Q S T -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M F A I N P E Y M N E T V L L D - - - - E C T P I Y L D I G P L W E Q V V A R V T Q W F G V I L S L V F L I Y Y I W N T Y K A
T C G W E E L Y V C T V E F C K I I I E L Y F E Y T P P A M I F Q T N G Q V T P W L R Y A E W L L T C P V I L I H L S N I T G L N D D Y S G R T M S L I T S D L G G I C M A V T A A L S - K G W L K A L F F V I G - - - - - - C G Y G A S T F Y N A A C I Y I E
S Y - - - - - - - Y T M P Q G I C R R L V L W M A G V F F T S W F M F P G L F L A G P E G T Q A L S W A G T T I G H T V A D L L S K N A W G M I G H - F L R V E I H K H I I I H G D V R R P V T V K - - - - A L G R Q V S V N C F V D K E E E E E D E R I - -
- M S R R P W L L A L A L A V A L A A G S A G A S T G S - - - - - - - - - - - D A T V P V A T Q D G P D Y V F H R A H E R M L F Q T S Y T L E N N G S V I C I P N N G Q C F C L A W L K S N G T N A E K L A A N I L Q W I T F A L S A L C L M F Y G Y Q T W K S
T C G W E E I Y V A T I E M I K F I I E Y F H E F D E P A V I Y S S N G N K T V W L R Y A E W L L T C P V I L I H L S N L T G L A N D Y N K R T M G L L V S D I G T I V W G T T A A L S - K G Y V R V I F F L M G - - - - - - L C Y G I Y T F F N A A K V Y I E
A Y - - - - - - - H T V P K G I C R D L V R Y L A W L Y F C S W A M F P V L F L L G P E G F G H I N Q F N S A I A H A I L D L A S K N A W S M M G H - F L R V K I H E H I L L Y G D I R K K Q K V N - - - - V A G Q E M E V E T M V H E E D D E T Q K V - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M G G A P A P D A H S A P P G N D S A G G S E Y H A P A G Y Q V N P P Y H P V H G Y E E - - - - Q C S S I Y I Y Y G A L W E Q E T A R G F Q W F A V F L S A L F L A F Y G W H A Y K A
S V G W E E V Y V C S V E L I K V I L E I Y F E F T S P A M L F L Y G G N I T P W L R Y A E W L L T C P V I L I H L S N I T G L S E E Y N K R T M A L L V S D L G T I C M G V T A A L A - T G W V K W L F Y C I G - - - - - - L V Y G T Q T F Y N A G I I Y V E
S Y - - - - - - - Y I M P A G G C K K L V L A M T A V Y Y S S W L M F P G L F I F G P E G M H T L S V A G S T I G H T I A D L L S K N I W G L L G H - F L R I K I H E H I I M Y G D I R R P V S S Q - - - - F L G R K V D V L A F V T E E D K V - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M G W K I N P L Y S D E V A I L E - - - - I C K E N E M V F G P L W E Q K L A R A L Q W F T V I L S A I F L A Y Y V Y S T L R A
T C G W E E L Y V C T V E F T K V V V E V Y L E Y V P P F M I Y Q M N G Q H T P W L R Y M E W L L T C P V I L I H L S N I T G L N D E Y S G R T M S L L T S D L G G I A F A V L S A L A - V G W Q K G L Y F G I G - - - - - - C I Y G A S T F Y H A A C I Y I E
S Y - - - - - - - H T M P A G K C K R L V V A M C A V F F T S W F M F P A L F L A G P E C F D G L T W S G S T I A H T V A D L L S K N I W G L I G H - F L R V G I H E H I L V H G D V R R P I E V T - - - - I F G K E T S L N C F V E N D D E E D D V - - - -
M E A Y A Y P E L L G S A G R S L F A A T V P E - - - - - - - - - - - - - - - - - - - - - - - - - - - - N I S E S T W V D A G Y Q H F W T Q R Q N E T V V C E H Y - - - - T H A S W L I S H G T K A E K T A M I A C Q W F A F G S A V L I L L L Y A W H T W K A
T S G W E E V Y V C C V E L V K V L F E I Y H E I H H P C T L Y L V T G N F I L W L R Y G E W L L T C P V I L I H L S N I T G L K N D Y N K R T M Q L L V S D I G C V V W G V T A A L C - Y D Y K K W I F F C L G - - - - - - L V Y G C N T Y F H A A K V Y I E
G Y - - - - - - - H T V P K G E C R I I V K V M A G V F Y C S W T L F P L L F L L G P E G T G A F S A Y G S T I A H T V A D V L S K Q L W G L L G H - H L R V K I H E H I I I H G N L T V S K K V K - - - - V A G V E V E T Q E M V D S T E E D A V - - - - -
- - - - - - - - - - M S V N L S L W E H G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E D A G Y G H W Y Q G T P N G T L V C S H E - - - - D N I A W L K N K G T D E E M L G A N I C M W M A F A A C L L C L S F Y A Y S T W R A
T C G W E E V Y V C L V E M V K V M I E V F H E N D S P A T L Y L S T G N F I M W I R Y G E W L L S C P V I L I H L S N I T G L Q D Q Y S K R T M Q L L V S D L G T I T M G V T A A L C - G N Y V K W I F F I L G - - - - - - L C Y G V N T Y F H A A K V Y I E
S Y - - - - - - - H I V P K G V C R V C V R V M A W C F F G A W T C Y P L L F V F G P E G L G V L S Y N A S A I G H T I I D I F S K Q V W G F V G H - Y L R I K I H E H I V I H G N L V K P T K V K - - - - V A G M E I D A E E M V E K D E E G A I - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M T T I S E V C G V W A L D N P - - - - - - - E C I E V S G T N D N V K M A Q L C F C M V C V C Q I L F M A S - - - - - Q Y P
K V G W E A I Y L P S C E C F L Y G L A S S G N - - - - G F I Q L Y D G R L I P W A R Y A A W I C T C P S I L L Q I N T I H K C K I S H F N L N T F I V Q A D L I M N I M G V T G A L T T N I A F K W I Y F A I G C I L F I F I V L V V Y D I M T S A A K E W K
A K - - - - - - - G D S K G N L V S T R L I L L R W I F I V S W C V Y P L L W I L S P Q A T C A V S E D V I S V A H F I C D A F A K N M F G F I M W R T L W R D L D G H W D I S R H Y P Q S S Y A K - - - - D G K E E E Q M T A M S Q T D D T E K P H S S Q G
- M S R R P W L L A L A L A V A L A A G S A G A S T G S - - - - - - - - - - - D A T V P V A T Q D G P D Y V F H R A H E R M L F Q T S Y T L E N N G S V I C I P N N G Q C F C L A W L K S N G T N A E K L A A N I L Q W I T F A L S A L C L M F Y G Y Q T W K S
T C G W E T I Y V A T I E M I K F I I E Y F H E F D E P A V I Y S S N G N K T V W L R Y A T W L L T C P V L L I H L S N L T G L K D D Y S K R T M G L L V S D V G C I V W G A T S A M C - T G W T K I L F F L I S - - - - - - L S Y G M Y T Y F H A A K V Y I E
A F - - - - - - - H T V P K G I C R E L V R V M A W T F F V A W G M F P V L F L L G T E G F G H I S P Y G S A I G H S I L D L I A K N M W G V L G N - Y L R V K I H E H I L L Y G D I R K K Q K I T - - - - I A G Q E M E V E T L V A E E E D - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M D Y G G A L S - - - - - - - - - A V G R E L L F V T N P V V V N G S V L V P E D - - Q C Y C A G W I E S R G T N G A Q T A S N V L Q W L A A G F S I L L L M F Y A Y Q T W K S
T C G W E E I Y V C A I E M V K V I L E F F F E F K N P S M L Y L A T G H R V Q W L R Y A E W L L T C P V I L I H L S N L T G L S N D Y S R R T M G L L V S D I G T I V W G A T S A M A - T G Y V K V I F F C L G - - - - - - L C Y G A N T F F H A A K A Y I E
G Y - - - - - - - H T V P K G R C R Q V V T G M A W L F F V S W G M F P I L F I L G P E G F G V L S V Y G S T V G H T I I D L M S K N C W G L L G H - Y L R V L I H E H I L I H G D I R K T T K L N - - - - I G G T E I E V E T L V E D E A E A G A V - - - -
- - M S R L V A A S W L L A L L L C G I T S T T T A S S - - - - - - - - - - - A P A A S S T D G T A A A A V S H Y A M N G F D E L A K G A V V P E D H F V C G P A - D K C Y C S A W L H S H G S K E E K T A F T V M Q W I V F A V C I I S L L F Y A Y Q T W R A
T C G W E E V Y V T I I E L V H V C F G L W H E V D S P C T L Y L S T G N M V L W L R Y A E W L L T C P V I L I H L S N L T G M K N D Y N K R T M A L L V S D V G C I V W G T T A A L S - T D F V K I I F F F L G - - - - - - L L Y G F Y T F Y A A A K I Y I E
A Y - - - - - - - H T V P K G I C R Q L V R L Q A Y D F F F T W S M F P I L F M V G P E G F G K I T A Y S S G I A H E V C D L L S K N L W G L M G H - F I R V K I H E H I L V H G N I T K K T K V N - - - - V A G D M V E L D T Y V D Q D E E H D E G - - - -
- - - - - M A E L I S S A T R S L F A A G G I N P W P N P Y H H E D M G C G G M T P T G E C F S T E W W C D P S Y G L S D A G Y G Y C F V E A T G G Y L V V G V E - - - - K K Q A W L H S R G T P G E K I G A Q V C Q W I A F S I A I A L L T F Y G F S A W K A
T C G W E E V Y V C C V E V L F V T L E I F K E F S S P A T V Y L S T G N H A Y C L R Y F E W L L S C P V I L I K L S N L S G L K N D Y S K R T M G L I V S C V G M I V F G M A A G L A - T D W L K W L L Y I V S - - - - - - C I Y G G Y M Y F Q A A K C Y V E
A N - - - - - - - H S V P K G H C R M V V K L M A Y A Y F A S W G S Y P I L W A V G P E G L L K L S P Y A N S I G H S I C D I I A K E F W T F L A H - H L R I K I H E H I L I H G D I R K T T K M E - - - - I G G E E V E V E E F V E E E D E D T V - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M T M L E H L E G T M D G W Y A E N D L G Q G A I I A H W V T F F F H M I T T F Y L G Y V S F H S K G P G G K Q P Y F A G Y H E E N N
I G I F V N L F A A I S Y F G K V V S D T H G H N Y Q N V G P F I I G - - - L G N Y R Y A D Y M L T C P L L V M D L - - L F Q L R A P Y K I T C A M L I F A V L M I G A V T N F Y P G D D M K G P A V A W F C F G - - - - - - C F W Y L I A Y I F M A H I V S K
Q Y G R L D Y L A H G T K A E G A L F S L K L A I I T F F A I W V A F P L V W L L S - V G T G V L S N E A A E I C H C I C D V V A K S V Y G F A L A N F R E Q Y D R E L Y G L L N S I G L D G E D V - - - - V Q Q L E K E M Q T N H H K K K S I N S P A V G -
- M A A G L E G L V S S A S R G L H A S I P E N P Y H S D G H H - - L P C G - L T P F G - C M D - D F W C N P E Y G M S Y A G Y T Y C F S E L A F G K L V M V P E - - - - A D A G W L H S H G T Q A E F V A A T A C Q Y T A L S L A L L L L S F Y A Y S A W K A
T C G W E E G Y V C C V E V L F V T L E I S N E F N S P A T L Y L S T G N Y C Y F L R Y G E W L L S C P V I L I H L S N L S G L K N D Y S M R T M R L L V S C I G M L I T G M A G G L G - V G W V K W T L Y F V S - - - - - - C A Y S A Q T Y L Q A A K C Y V E
V Y - - - - - - - A T V P K G Y C R T V V K L M A Y A F F T A W G A Y P I L W A I G P E G L K Y I S G Y S N T I A H T F C D I L A K E I W T F L G H - H L R I K I H E H I L I H G D I R K K V Q V R - - - - V A G E L M N V E E L M E E E G E D T V - - - - -
- - M E P V L G L A S T A V R E L T A G G S G N P Y E S - Y K P P E D P C A - L T P F G - C L T - N F W C D P Q F G L A D A K Y D Y C Y V K A A Y G E L A I V E T - - - - S R L P W L Y S H G S D A E H Q G A L A M Q W M A F A L C I I C L V F Y A Y H S W K A
T T G W E E V Y V C V V E L V K V L L E I Y K E F E S P A S I Y L P T A N A A L W L R Y G E W L L T C P V I L I H L S N I T G L K D D Y N K R T M Q L L V S D I G C V V W G I T A A F S - V G W L K W V F F V L G - - - - - - L L Y G S N T Y F H A A K V Y I E
S Y - - - - - - - H T V P K G H C R L I V R L M A Y C F Y V A W T M Y P I L F I L G P E G L G H M S A Y M S T A L H G V A D M L S K Q I W G L L G H - H L R V K I F E H I L I H G D I R K T T T M Q - - - - V G G Q M V Q V E E M V D E E D E D T I - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - M A D - - - - - - - - - - - F V W Q G A G N G G P S - - - - - - - - - - - - - - A M V S H Y P N G S V L L E S S - G S C Y C E D W Y T S R G N H V E H S L S N A C D W F A F A I S V I F L V Y Y A W A A F N S
S V G W E E I Y V C T V E L I K V S I D Q F L S S N S P C T L Y L S T G N R V L W I R Y G E W L L T C P V I L I H L S N V T G L K D N Y S K R T M A L L V S D I G T I V F G V T S A M C - T G Y P K V I F F I L G - - - - - - C C Y G A N T F F N A A K V Y L E
A H - - - - - - - H T L P K G S C R T L I R L M A Y T Y Y A S W G M F P I L F V L G P E S F G H M N M Y Q S N I A H T V I D L M S K N I W G M L G H - F L R H K I R E H I L I H G D L R T T T T V N - - - - V A G E E M Q V E T M V A A E D A D E T T V - - -
- - - - - - M E T A A T M T H A F I S A V P S A E A T I R - - - - - - - - - - - - - - - - - - - - - - - - - - - - G L L S A A A V V T P A A D A H G E T S N A T T A G A D H G C F P H I N H G T E L Q H K I A V G L Q W F T V I V A I V Q L I F Y G W H S F K A
T T G W E E V Y V C V I E L V K C F I E L F H E V D S P A T V Y Q T N G G A V I W L R Y S M W L L T C P V I L I H L S N L T G L H E E Y S K R T M T I L V T D I G N I V W G I T A A F T - K G P L K I L F F M I G - - - - - - L F Y G V T C F F Q I A K V Y I E
S Y - - - - - - - H T L P K G V C R K I C K I M A Y V F F C S W L M F P V M F I A G H E G L G L I T P Y T S G I G H L I L D L I S K N T W G F L G H - H L R V K I H E H I L I H G D I R K T T T I N - - - - V A G E N M E I E T F V D E E E E G G V - - - - -
- - - - - - M S P P T S P T P D T G H D T P D T G H D T G - - - - - - - - - - - - - - - - - - - - - - - - - - G H G A V E I C F A P C E E D C V T I R Y F V E N D F E G C I P G H F D Q Y S S H G S L H D I V K A A L Y I C M V I S I L Q I L F Y G F Q W W R K
T C G W E V W F V A C I E T S I Y I I A I T S E A D S P F T L Y L T N G Q I S P Q L R Y M E W L M T C P V I L I A L S N I T G M A E E Y N K R T M T L L T S D V C C I V L G M M S A A S - K P R L K G I L Y A V G - - - - - - W A F G A W T Y W T A L Q V Y R D
A H - - - - - - - K A V P K P - L A W Y V R A M G Y V F F T S W L T F P G W F L L G P E G L E V V T G T V S T L M H A C S D L I S K N L W G F M D W - H L R V L V A R H H R K L F K A E E E H A L K K G Q T L E P G M P R S T S F V R G L G D D V E I - - - -
M M L L A S Y H H P C G G . . G C C W . S G . E A . Q W . F . . S . L L . F Y A Y . . W . A
T C G W E E . Y V C . . E . . K V . . E . E F S P A L Y L . . G N . . W L R Y . E W L L T C P V I L I H L S N L T G L K . D Y S K R T M L L V S D . G . I V G . T . A . . G . K . F F . . G C I L F I F L . Y G . T . F A A K V Y I E
. Y G R L D Y L A H T V P K G C R . V . . M A . F F S W M F P . L F . L G P E G . G . S Y . S . I . H . I D L . S K N . W G . L G H L R V K I H E H I L I H G D I R K . . . K G Q T . A G E . E V E . . V . E . E . G
*
Supplementary Figure 2
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 3 – Characterization of channel kinetics of Chronos
and Chrimson in HEK293FT cells.
(a-b) Population data for channel closing rate (tau off) (a) and time to peak (time
to reach 90% of peak photocurrent after the beginning of illumination) (b),
measured with 470 nm illumination and irradiance of 10 mW/mm2, for ChR2,
ChR2 E123A (aka ChETAA), and Chronos, and 590 nm illumination and
irradiance 4.6 mW/mm2 for Chrimson (n = 5 – 8 HEK293FT cells each).
(c) Comparison of channel closing rate (tau off) at various holding potentials for
ChR2 E123A and Chronos, measured with 470 nm illumination, irradiance of 10
mW/mm2. Pulse durations were 2 ms for panels a, c, and 1 s for panel b.
Statistics for panels a and b: ***, P < 0.001, t-test comparing ChR2 E123A
(ChETAA) vs. Chronos. All data are plotted as mean ± s.e.m. throughout.
Nature Methods: doi:10.1038/nmeth.2836
0
1
2
3
4
5
6
7
8
-80 -60 -40 -20 0 20 40 60
Chronos
ChR2 E123A
holding potential (mV)
0
5
10
15
20
25
0
1
2
3
4
5
6
7
8
9
ba c
***
***
chan
nel c
losi
ng ra
te (t
au o
ff) (m
s)
time
to 9
0% o
f pea
k (m
s)
chan
nel c
losi
ng ra
te (t
au o
ff) (m
s)
Supplementary Figure 3
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 4 – Comparison of ion selectivity of Chronos,
Chrimson, and ChR2.
(a-c) Population data for photocurrent density ratios, measured using whole-cell
patch clamp in HEK cells in ion-specific extracellular solutions (see Methods);
using 1 s illumination of 470 nm, irradiance of 10 mW/mm2 for ChR2 and
Chronos, 1s illumination of 590 nm, irradiance of 4.6 mW/mm2 for Chrimson.
Shown is data for ChR2, Chronos, and Chrimson (n = 6 – 10 HEK293FT cells
each). (a) Calcium photocurrent (I_calcium) measured in 90 mM CaCl2, pH 7.4,
vs. sodium photocurrent (I_sodium) measured in 145 mM NaCl, pH
7.4. (b) Proton photocurrent (I_proton) measured in 135 mM NMDG, pH 6.4 vs.
sodium photocurrent (I_sodium). (c) Potassium photocurrent (I_potassium)
measured in 145 mM KCl, pH 7.4 vs. sodium photocurrent (I_sodium).
Nature Methods: doi:10.1038/nmeth.2836
0
0.2
0.4
0.6
0.8
1
1.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
ba c
I_calcium/I_sodium
I_proton/I_sodium
I_potassium/I_sodium
I_calcium/I_sodium I_proton/I_sodium I_potassium/I_sodium
Supplementary Figure 4
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 5 – Opsin screening in cultured neurons.
(a) Representative GFP (left), tdTomato (center) and phase-contrast images
(right) of a tdTomato and opsin-GFP fusion–transfected neuron in culture. The
yellow border indicates the mask boundary used to quantify soma fluorescence.
Scale bars, 10 µm. (b) Comparison of fluorescence and current density across all
opsins screened. Each dot represents the data from a cell expressing one of the
opsins in Fig. 1, pooled over all opsins. Photocurrent was measured for each
opsin with the wavelength nearest its peak using 5 ms pulses (equal photon
fluxes across wavelength; 470 nm, 4.23 mW/mm2; 530 nm, 3.66 mW/mm2; 625
nm, 3.14 mW/mm2). (c) Opsin-GFP and tdTomato fluorescence for each
construct patched and the membrane parameters indicated on the y-axes (n = 3
– 12 cells each). (d) Photocurrent measured at equal photon fluxes using 5 ms
pulses (same irradiances as part a: n = 6 CsChR cells, n = 2 C1V1TT cells, n = 8
Chrimson cells). (e) Action spectra in HEK293FT cells done under the same
conditions as Fig. 1e. Statistics for panels b: *P < 0.05, **P < 0.01 and ***P <
0.001, ANOVA with Dunnett’s post hoc test, with ChR2 as the reference.
!
Nature Methods: doi:10.1038/nmeth.2836
a b
c
d e
ChR
1
ChR
2
C1V
1T
T
VC
hR
1
NsC
hR
SdC
hR
BsC
hR
2
TsC
hR
HdC
hR
TcC
hR
MvC
hR
1
CoC
hR
CsC
hR
Chrim
son
CnC
hR
2
Chro
nos
PsC
hR
1
PsC
hR
2
AgC
hR
CbC
hR
1
0
500
1000
Rm
(M
Ohm
)
ChR
1
ChR
2
C1V
1T
T
VC
hR
1
NsC
hR
SdC
hR
BsC
hR
2
TsC
hR
HdC
hR
TcC
hR
MvC
hR
1
CoC
hR
CsC
hR
Chrim
son
CnC
hR
2
Chro
nos
PsC
hR
1
PsC
hR
2
AgC
hR
CbC
hR
1
0
20
40
60
80
100
Cm
(pF
)
*
ChR
1
ChR
2
C1V
1T
T
VC
hR
1
NsC
hR
SdC
hR
BsC
hR
2
TsC
hR
HdC
hR
TcC
hR
MvC
hR
1
CoC
hR
CsC
hR
Chrim
son
CnC
hR
2
Chro
nos
PsC
hR
1
PsC
hR
2
AgC
hR
CbC
hR
1
-80
-60
-40
-20
0
Resting p
ote
ntial (m
V)
ChR
1
ChR
2
C1V
1T
T
VC
hR
1
NsC
hR
SdC
hR
BsC
hR
2
TsC
hR
HdC
hR
TcC
hR
MvC
hR
1
CoC
hR
CsC
hR
Chrim
son
CnC
hR
2
Chro
nos
PsC
hR
1
PsC
hR
2
AgC
hR
CbC
hR
10
25
50
75
100
Hold
ing c
urr
ent at -6
5 m
V (
|pA
|)
ChR
1
ChR
2
C1V
1T
T
VC
hR
1
NsC
hR
SdC
hR
BsC
hR
2
TsC
hR
HdC
hR
TcC
hR
MvC
hR
1
CoC
hR
CsC
hR
Chrim
son
CnC
hR
2
Chro
nos
PsC
hR
1
PsC
hR
2
AgC
hR
CbC
hR
1
0
200
400
600
800
1000
* **
tdT
om
ato
(A
U/p
ixel)
ChR
1
ChR
2
C1V
1T
T
VC
hR
1
NsC
hR
SdC
hR
BsC
hR
2
TsC
hR
HdC
hR
TcC
hR
MvC
hR
1
CoC
hR
CsC
hR
Chrim
son
CnC
hR
2
Chro
nos
PsC
hR
1
PsC
hR
2
AgC
hR
CbC
hR
1
0
200
400
600
800
1000
****
***
GF
P (
AU
/pix
el)
470 n
m
530 n
m
625 n
m
0
500
1000
1500
Curr
ent (p
A)
470 n
m
530 n
m
625 n
m
0
200
400
600
Curr
ent (p
A)
470 n
m
530 n
m
625 n
m
0
200
400
600
Curr
ent (p
A)
CsChR C1V1TT Chrimson
400 500 600 700
0.0
0.5
1.0 Chrimson
CsChR
C1V1TT
Wavelength (nm)
Norm
alized c
um
ula
tive c
harg
e
Supplementary Figure 5
Ubiquitin tdTomato
0 50 100
0
500
1000
Current density (pA/pF)
GF
P (
AU
/pix
el)
0 50 100
0
500
1000
Current density (pA/pF)
tdT
(A
U/p
ixel)
opsinCaMKII GFP
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 6 – Opsin trafficking in cultured neurons.
Channelrhodopsin trafficking and photocurrent comparison from cultured neuron
screening. Quantitative soma GFP fluorescence versus current measured at the
light power of peak sensitivity (same conditions as Supplementary Fig. 5a) for
each neuron experimented on (indicated by a circle) (left), GFP images for the
median current (middle) and maximum current (right) cells are shown for each
opsin. Scale bar, 10 µm. Note that in the GFP versus current plot, the GFP
intensity values are absolute, and thus can be compared across opsins. However
due to two-log-unit variance in absolute GFP intensity, the brightness and
contrast settings used for the median images are varied across the different
opsins, so the GFP images should not be used to compare brightness across
constructs. That said, the brightness and contrast settings for each opsin's
median and maximum current cells are matched, to illustrate whether higher
expression correlates with increased photocurrent. See Methods for imaging
conditions and Supplementary Table 1 for full genus/species names.
Nature Methods: doi:10.1038/nmeth.2836
0 100 200 300 4000
50
100
150
200TsChR
470 nm current (pA)
GFP
(AU
)
0 500 1000 15000
100
200
300
400
500BsChR2
470 nm current (pA)
GFP
(AU
)
0 1000 20000
200
400
600SdChR
470 nm current (pA)
GFP
(AU
)
0 10 20 30 40 5060
70
80
90
100
110NsChR
470 nm current (pA)
GFP
(AU
)
0 100 200 300 4000
50
100
150
200
250VChR1
530 nm current (pA)G
FP (A
U)
0 250 500 750 10000
50
100
150
200
250C1V1TT
530 nm current (pA)
GFP
(AU
)
0 500 1000 15000
100
200
300
400ChR2
470 nm current (pA)
GFP
(AU
)
0 50 100 150 20060
80
100
120
140ChR1
470 nm current (pA)
GFP
(AU
)Supplementary Figure 6
Nature Methods: doi:10.1038/nmeth.2836
0 1000 2000 30000
200
400
600
800ShChR/Chronos
470 nm current (pA)
GFP
(AU
)
0 500 1000 15000
200
400
600
800
1000
CnChR2
470 nm current (pA)
GFP
(AU
)
0 250 500 750 10000
500
1000
1500CnChR1/Chrimson
625 nm current (pA)
GFP
(AU
)
0 500 1000 1500 20000
500
1000
1500
CsChR
530 nm current (pA)
GFP
(AU
)
0 2000 40000
200
400
600
800
CoChR
470 nm current (pA)G
FP (A
U)
0 5 10160180200220240260280
MvChR1
530 nm current (pA)
GFP
(AU
)
0 200 4000
100
200
300
400TcChR
470 nm current (pA)
GFP
(AU
)
0 100 2000
50
100
150
200HdChR
470 nm current (pA)
GFP
(AU
)
Nature Methods: doi:10.1038/nmeth.2836
0 5 100
50
100
150CbChR1
470 nm current (pA)G
FP (A
U)
0 5 10 15 200
50
100
150
200AgChR
470 nm current (pA)
GFP
(AU
)
0 50 100 150 200 2500
1000
2000
3000
4000PsChR2
530 nm current (pA)
GFP
(AU
)
0 50 100 150 200 2500
500
1000
1500PsChR1
530 nm current (pA)
GFP
(AU
)
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 7 – Inactivation and recovery kinetics.
Normalized traces of photocurrent recovery kinetics, with traces from each
patched cell overlaid, with experiments as performed in Fig. 1h.
!
Nature Methods: doi:10.1038/nmeth.2836
5 s
C1V1TT
ChR2
SdChR
CoChR
CsChR
Chrimson
Chronos
wait 30 s in the darkwait 1s in the dark
Supplementary Figure 7
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 8 – Chronos full inactivation and recovery kinetics.
(a) Peak current recovery ratio vs. waiting time in darkness after 20 s of
illumination (n = 3 cells), followed by a variable period of darkness, and assessed
with a 50 ms pulse. (b) Representative trace showing Chronos inactivation has a
fast and a slow component under continuous illumination.
Nature Methods: doi:10.1038/nmeth.2836
a b
0 100 2000.0
0.2
0.4
0.6
0.8
1.0
Recovery time (s)
Rec
over
y ra
tio
2s
500 pA
20 s 5 s 25 s 30 s 60 s
Supplementary Figure 8
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 9 – ReaChR and Chrimson comparison in cultured
neurons.
Side-by-side comparisons of Chrimson and ReaChR’s spectral sensitivity.
tdTomato was co-expressed with opsin-GFP for unbiased selection of neurons.
(a-d) Raw traces and photocurrent measurements at equal photon fluxes across
wavelengths. All measurements were done first with red, then green, then blue (n
= 5 cells; 5 ms pulse; 470 nm, 4.23 mW/mm2; 530 nm, 3.66 mW/mm2; 625 nm,
3.14 mW/mm2). (e) Short vs. long pulse activation of ReaChR at 625 nm. Raw
traces from a representative cell showing current in response to a 5 ms (left) vs.
1 s (middle) pulse of red light. Additional dashed lines on the 1 s pulse trace
correspond to the current amplitude at the indicated time post-light-onset.
Individual cell data is plotted on the right (n = 4 cells; 5 ms, 3.14 mW/mm2; 1 s, 5
mW/mm2). (f) Turn-on kinetics comparison in response to a 1 s pulse. (n = 4 – 6
cells; 625 nm, 5 mW/mm2).
!
Nature Methods: doi:10.1038/nmeth.2836
ReaChR 625 nm5
ms
1000
ms
0
500
1000
1500
2000
Cur
rent
(pA)
Chrimson
470
nm
530
nm
625
nm
0
500
1000
1500
Cur
rent
(pA)
ReaChR
470
nm
530
nm
625
nm
0
500
1000
1500
Cur
rent
(pA)
625 nm 1000 ms
Chr
imso
n
Rea
ChR
1
10
100
Tim
e to
90%
of p
eak
(ms)
a b
dc
400 pA
500 ms
400 pA
500 ms
470 nm 530 nm 625 nm
470 nm 530 nm 625 nm
e f
200 pA
500 ms
5 ms
20 ms
50 ms
100 ms
0 1000 20000
200
400
600
800
530 nm current (pA)
Tota
l GFP
(AU
)
Chrimson ReaChR
0 1000 20000
200
400
600
800
625 nm current (pA)
Tota
l GFP
(AU
)
Chrimson ReaChRChrimson 5 ms pulse
ReaChR 5 ms pulse
ReaChR 5 ms vs. 1000 ms pulse
Supplementary Figure 9
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 10 – Green light driven spiking frequency responses
in cultured neurons.
(a-b) tdTomato was co-expressed with opsin-GFP for unbiased selection of
neurons to patch. Any neuron that could not drive a train of 1 Hz, 10 pulses, of
green (530nm) light at 5 mW/mm2 with 100% spike probability was excluded from
further spike frequency analysis, since the focus here was on high spike
frequency fidelity. Generally neurons that could not drive spikes at 1Hz (indicated
by red X’s, as depicted in a) had lower GFP intensity than the neurons that could
(indicated by black squares), in b. Each symbol is one cell. (c) Comparison of
current measured at the end of 1 s green light (5 mW/mm2), defined as Imin in the
trace (left). Asterisk indicates escaped spike-like sodium current since TTX was
not used. Jmin is Imin divided by membrane capacitance. Symbols are defined in
the same manner as in a-b. (d-h) Characterization of green light driven spike
frequency responses for cells that passed the 1 Hz spiking test in a-b.
Stimulation train consisted of 40 pulses of green light in all cases (n = 5 – 8 cells
for each opsin). (d) Individual cell spike probability vs. frequencies over
irradiances. Population summary is shown on the right. (e) Population data for
spike fidelity within the 40 pulse stimulation train. (f) Individual cells’ spike
latency, defined as time from light onset to spike peak. (g) Membrane parameter
controls to show that the observed spiking differences were not due to changes
in neural excitability. (h) Comparison of measured and projected current density
to estimate the effective driving force during optical spiking for cells that passed
Nature Methods: doi:10.1038/nmeth.2836
the 1 Hz spiking test in a-c. Measured current density Jmin is the same data set
as panel c. Projected current densities (Jmax, Jmax for 2 ms pulse) are calculated
by multiplying the measured Jmin (from h) by the ratios (Jmax/Jmin or Jmax for 2
ms/Jmin) derived under the same illumination conditions but with TTX blockade
(see Supplementary Fig. 7, for TTX data). “Jmax for 2 ms pulse” refers to the
maximum current density within the 2 ms post-light-onset interval. Statistics for
panels h: *P < 0.05, **P < 0.01 and ***P < 0.001, ANOVA with Dunnett’s post hoc
test, with C1V1TT as the reference.
Nature Methods: doi:10.1038/nmeth.2836
0 20 40 600.0
0.5
1.0
Frequency (Hz)Sp
ike
Prob
abili
ty0 20 40 60
0.0
0.5
1.0
Frequency (Hz)
Spik
e Pr
obab
ility
0 20 40 600.0
0.5
1.0
Frequency (Hz)
Spik
e Pr
obab
ility
0.5 2 50.0
0.5
1.0
Irradiance (mW/mm2)
Spik
e Pr
obab
ility
at 5
Hz
Chr
onos
CsC
hR
C1V
1 TT
0
5
10
15
20
J min
(pA/
pF)
Chr
onos
CsC
hR
C1V
1 TT
0
50
100
150
200
tdT
(AU
/pix
el)
Chr
onos
CsC
hR
C1V
1 TT
0
100
200
300
GFP
(AU
/pix
el)
a b
d
c
100% spiking
optical spike test: 5 mW/mm2 2 ms pulse at 1 Hz
no spiking
25 mV
1 s
*
Imin
0.5 mW/mm2
20 Hz; 5 mW/mm2 40 Hz; 5 mW/mm2 60 Hz; 5 mW/mm2 Spike latency 5 Hz 5 mW/mm2
2 mW/mm2 5 mW/mm2
e f
g
h
Chronos
CsChR
C1V1TT
Chr
onos
CsC
hRC
1V1 T
T
-80
-60
-40
-20
0
Res
ting
Pote
ntia
l (m
V)
Chr
onos
CsC
hRC
1V1 T
T
-80
-60
-40
-20
0
Spik
e Th
resh
old
(mV)
1 2 3 40.0
0.5
1.0
Quartile
Spik
e Pr
obab
ility
1 2 3 40.0
0.5
1.0
Quartile
Spik
e Pr
obab
ility
1 2 3 40.0
0.5
1.0
Quartile
Spik
e Pr
obab
ility
Chr
onos
CsC
hRC
1V1 T
T
0
200
400
600
800
Mem
bran
e re
sist
ance
(MO
hm)
Chr
onos
CsC
hRC
1V1 T
T
0
20
40
60
80
Mem
bran
e ca
paci
tanc
e (p
F)
Chr
onos
CsC
hRC
1V1 T
T
0
10
20
30
40
J min
(pA/
pF)
Chr
onos
CsC
hRC
1V1 T
T
0
10
20
30
40 **
Proj
ecte
d J m
ax (p
A/pF
)
Chr
onos
CsC
hRC
1V1 T
T
0
10
20
30
40
***
*
Proj
ecte
d J m
ax fo
r 2m
s pu
lse
(pA/
pF)
Chr
onos
CsC
hRC
1V1 T
T
0
25
50
75
100
Hol
ding
cur
rent
at -
65 m
V (|p
A|)
0 2 4 6 8 100.01
0.1
1
Mean (ms)
Stan
dard
dev
iatio
n (m
s)
Supplementary Figure 10
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 11 – Electrical versus green light driven spiking
fidelity in cultured neurons.
Additional analysis of optical versus electrical spike probability over various
frequencies at 5 mW/mm2; same dataset as Supplementary Fig. 10d-h.
Electrical stimulation protocol was the same as optical, except the pulse duration
was 5 ms and the input current was varied between 200-800 pA to maximize
spike probability. Plateau potential was measured as the voltage difference
between two horizontal dotted lines as shown in each trace. A single dash to the
left of electrical traces indicates –65 mV. Statistics for b, e, and h: Paired t-test
between electrical and optical spiking were computed at each frequency: *P <
0.05, **P < 0.01 and ***P < 0.001.
Nature Methods: doi:10.1038/nmeth.2836
a b c
d e f
0 20 40 600.0
0.5
1.0
Frequency (Hz)
Spik
e Pr
obab
ility
0 20 40 600.0
0.5
1.0**
Frequency (Hz)
Spik
e Pr
obab
ility
0 20 40 600.0
0.5
1.0** *** ***
Frequency (Hz)
Spik
e Pr
obab
ility
0 20 40 600
10
20
30
40
Frequency (Hz)
Plat
eau
pote
ntia
l (m
V)
0 20 40 600
10
20
30
40
Frequency (Hz)
Plat
eau
pote
ntia
l (m
V)
0 20 40 600
10
20
30
40
Frequency (Hz)
Plat
eau
pote
ntia
l (m
V)
500 ms
50 mV
Electrical 0.5 mW/mm2 5 mW/mm2
Electrical 5 mW/mm22 mW/mm2
Electrical 5 mW/mm22 mW/mm2
g h i
Chr
onos
CsC
hRC
1V1 TT
50 mV
50 mV
Supplementary Figure 11
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 12 – Red light driven spiking in cultured neurons.
(a-d) Red (625 nm) light spike frequency data for Chrimson-expressing neurons
selected based on the presence of tdTomato co-expression (n = 6 cells).
Stimulations were as performed in Fig. 2e (train of 40 pulses, 2 ms pulse
duration). (a) Optical vs. electrical spiking showing Chrimson can reliably drive
spikes only at <20 Hz. (b) Chrimson’s spike fidelity can decrease at higher red
irradiance due to channel inactivation and/or depolarization block. (c-d) Spike
latency, defined as light onset to spike peak, is both a function of irradiance (c)
and Chrimson expression level (d). (e) Action spectrum of a ChrimsonR
expressing HEK293 cell (n = 1 cell). (f) Current measured at the end of 1 s light
pulse (Imin) for Chrimson- or ChrimsonR-expressing cultured neurons. Population
mean and s.e.m. are plotted as black line. (g) Chronos red (625 nm) light
crosstalk characterization (n = 4 cells). Peak voltage crosstalk for 5 ms pulses at
5 Hz and for continuous 500 ms pulse, representative cell (left) and population
average (right). Statistics for a: Paired t-test between electrical and optical
spiking were computed at each frequency: *P < 0.05, **P < 0.01 and ***P < 0.001.
Nature Methods: doi:10.1038/nmeth.2836
a
b c
e f
g
d
400 500 600 7000.0
0.5
1.0
ChrimsonR
Wavelength (nm)
Nor
mal
ized
cum
ulat
ive
char
ge
0 20 40 600.0
0.5
1.0
Electrical
Chrimson
5 mW/mm2
Frequency (Hz)
Spik
e pr
obab
ility
Chrimson 625 nm
5 100.0
0.5
1.0
Irradiance (mW/mm2)
Spik
e pr
obab
ility
at 1
0 H
z
Chrimson 625 nm
5 100
5
10
15
20
end of light pulse
Irradiance (mW/mm2)
Spik
e la
tenc
y at
10
Hz
(ms)
0 5 10 15 200
5
10
15
625 nm 10 mW/mm2
Jmin (pA/pF)
Spik
e la
tenc
y at
10
Hz
(ms)
2 s
0 mV
0 mV
0 mV
50 mV
50 mV
625 nm5 mW/mm2
5 mW/mm2
10 mW/mm2
5 Hz 10 Hz
10 Hz
20 Hz
Electrical
50 mV
50 mV
1 s
625 nm 5 mW/mm2
0
250
500
750
1000
tdTomatoco-transfectionYes No No
I min
(pA)
** *** ***
500 ms
5 ms 5 Hz
5 ms 5 Hz
500 ms
Chronos 625 nm 5 mW/mm2
1 100
5
10
15
20
Red Irradiance (mW mm-2)
Peak
Vol
tage
(mV)
5 mV
5 mV
500 ms
Supplementary Figure 12
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 13 – Red and far-red spiking with ChrimsonR in
acute cortical slice.
Spectral and kinetic characterization of ChrimsonR in slice (n = 4 neurons from
one P60 mouse throughout figure; pCAG-ChrimsonR-tdTomato was
electroporated as specified in Methods). (a) ChrimsonR histology showing layer
2/3 expression (left, scale bar = 50 µm) and individual neuron expression (right,
scale bar = 10 µm). (b) 625 nm spiking fidelity at 20 Hz (5 ms, 40 pulses, 3
sweeps overlaid). (c) Far-red 735 nm spiking with ChrimsonR (5 sweeps overlaid
per condition). Traces from a representative trace from a single neuron (left),
population average (middle), and membrane properties (right).
Nature Methods: doi:10.1038/nmeth.2836
a b
c
Layer 2/3
-80
-60
-40
-20
0
Resting
Potentia
l
Spike T
hresho
ld
Pote
ntia
l (m
V)
100 ms
Electrical 5 ms 20 Hz
625 nm 5 ms 20 Hz
50 mV
ChrimsonR
12 mW/mm25 mW/mm2
735 nm 12 mW/mm2
10 20 40 60 80 1000.0
0.5
1.0
Pulse width (ms)
Spik
e Pr
obab
ility
625 nm 2 mW/mm2 20 Hz
1 2 3 40.0
0.5
1.0
Quartile
Spik
e Pr
obab
ility
25 mV-80 mV
200 ms 10 ms 20 ms 40 ms 80 ms
ChrimsonR 735 nm spiking
Supplementary Figure 13
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 14 – Larval motor axons expressing ChR2 fire in
response to blue but not red light pulses.
(a,b) Intracellular recordings from m6 muscles in 3rd instar larvae expressing
ChR2 in motor neurons. Responses to 470 nm and 617 nm light pulses of
increasing duration are shown. EJPs were only triggered by 16 ms pulses.
Dashes in each panel indicate –50 mV. (c) Probability of light-evoked EJPs after
1, 2, 4, 8, and 16 ms pulses in response to 470nm and 617nm light. As in Fig.
3d. (d) Mean ± s.e.m. number of EJPs evoked in response to light pulses. As in
Fig. 3e. Sample size in each case: n = 6 muscles from 3 animals.
Nature Methods: doi:10.1038/nmeth.2836
A
B
ChR 2 - 617 nm (0.06 mW/mm2)
ChR 2 - 470 nm (0.14 mW/mm2)
Proba
bilityof
evok
edEJP
0.0
0.2
0.4
0.6
0.8
1.0
1 2 4 8 16
Light pulse duration (ms)
Num
berof
EJP
sev
oked
2
1
0 000000 000
1 2 4 8 16
10 mV
100 ms
C
D
Supplementary Figure 14
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 15 – Proboscis extension reflex (PER).
(a) PER score was computed as (P-H)/H, where H is the pixel distance between
the root of antennae and the neck connective (i.e., the head capsule) and P is
the maximum horizontal pixel distance from the center of the head to the tip of
proboscis during 0–2 seconds after initiation of each trial. (b, c) PER of three
different fly groups to 25 light pulses at 470nm (b) and 617nm (c). n = 5 for each
population. (d) PER to light stimulation without CsChrimson expression in sugar
receptors. Data is from the same video recording analyzed for startle response in
Fig. 3g.
!
Nature Methods: doi:10.1038/nmeth.2836
8 12 16 20 240
0.5
1
pulse width (ms)
0.05 0.1 0.2 0.3 0.40
0.5
1
power (mW/mm )
1 2 4 8 160
0.5
1
pulse width (ms)
0.005 0.0075 0.015 0.03 0.060
0.5
1
power (mW/mm )
PE
R score
PE
R score
470nm (40Hz, 25 pulses)
617nm (40Hz, 25 pulses)
H P pUAS-CsChrimson-mVenus in attP18/w-;+/+;pBDPGal4 in attP2
w-;Gr64f-Gal4/UAS-ChR2eYFP line C;Gr64f-Gal4/+
pUAS-CsChrimson-mVenus in attP18/+;+/+;+/+
a
b
c
2
2
16ms
4ms
0.2mW/mm
0.015mW/mm
2
2
d
dark arena dark arena dark arena0
0.5
1
1.5
PER to light stimulation in the absence of
CsChrimson expression (WTB x CsChrimson, n=9)
sco
re
470nm 617nm 720nm
Supplementary Figure 15
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 16 – Optogenetics of freely behaving intact flies.
(a) Hardware configuration of a circular light arena for deep brain stimulation of
freely behaving flies with intact cuticle. Note that the LED array is divided into 4
quadrants (Q1~Q4). (b) Fraction of flies in quadrants 1 and 3 for the
experimental group. If more than 50% of flies are in the illuminated quadrants,
the error bar enters into to the red-shaded zones of the plot. Flies with
CsChrimson expression in PNv-1 neurons avoid illuminated area. See also
Supplementary Video 6. (c) Flies of control group do not avoid illuminated area.
!
Nature Methods: doi:10.1038/nmeth.2836
heat sink
LED array
diffuser
IR absorption film
circular arena with clamps
transparent cover
IR illumination LEDs
recording camera
0.25
0.5
0.75
1
% o
f flie
s in
Q1
and
Q3 VT031497 x CsChrimson
0 30 60 90
0.25
0.5
0.75
1
time (s)
WTB x CsChrimson
a
b
c
% o
f flie
s in
Q1
and
Q3
Q1
Q3
Q2
Q4
Supplementary Figure 16
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 17 – Two-color excitation controls in cultured
neurons.
(a) Current density at end of 1 s light pulse (5 mW/mm2, 470 nm light) Jmin is
defined in the same manner as Supplementary Fig. 10c. This is from the same
dataset as Fig. 4f. (b) Averaged traces of ChR2-, Chronos-, and Chrimson-
expressing neurons at the indicated irradiances of 470 nm light (n = 4 – 7 cells
for each opsin; 1 second pulse; traces are truncated to the first half of
illumination). (c) Chrimson blue current in response to 5 ms or 1 s pulses. Traces
from a representative cell (left) and population average (right) (n = 4 cells). (d)
ChrimsonR blue light voltage crosstalk for individual cells (n = 7 cells). Same
condition as Fig. 4b.
Nature Methods: doi:10.1038/nmeth.2836
0.1 10
10
20
30
Blue Irradiance (mW mm-2)Pe
ak V
olta
ge (m
V)
a
c d
b
0 10 20 300
100
200
300
Jmin (pA/pF)
GFP
(AU
/pix
el)
100 ms
470 nm 0.01 mW/mm2 470 nm 0.1 mW/mm2
0.01 0.1 10
200
400
600
800
Blue Irradiance (mW mm-2)
Cur
rent
(pA)
1000 ms
5 ms
Chrimson
ChR2
Chronos
200 ms
50 pA
1000 ms5 ms
Chrimson 470 nm 0.25 mW/mm2
ChR2 Chronos
ChrimsonR
Supplementary Figure 17
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 18 – Optically evoked post-synaptic currents (PSCs)
in acute slice.
(a) Widefield illumination with 20x objective (1.6 mm diameter spot size at the
focal plane) was used to optically drive spikes in the opsin expressing pre-
synaptic neurons. Both pre- and post-synaptic neurons are in layer 2/3. (b-d)
Either Chronos or Chrimson is expressed in the cortical brain slice; not both. All
stimulations were done at 0.2 Hz. Color and irradiance are as used in Fig. 5g-j
unless otherwise indicated. (b) Chronos PSC from the same neuron shows faster
onset and greater amplitude (potentially with multiple PSC peaks) at higher blue
irradiance. Trials are overlaid for each irradiance. Vertical dashed lines denote
the start and end of 5 ms light pulse for all traces (and also providing a time scale
reference). (c) Multiple traces, not overlaid, showing trial-to-trial variability for
Chrimson PSC at 4 mW/mm2 of red light. (d) Chronos PSC at various holding
voltages shows immediate excitatory PSC followed by strong inhibitory PSC in
response to 1 mW/mm2 of blue light. (e) Red and blue light driven post-synaptic
responses from five different non-expressing neurons downstream of opsin-
expressing neurons, in brain slice now expressing both Chronos and Chrimson.
Here, the dashed lines indicate timing, and the color of the shaded bar within, the
color of light delivered. Black trace is the averaged response, grey traces are
individual trials, throughout this figure.
Nature Methods: doi:10.1038/nmeth.2836
a b d
200 pA
50 ms
-75 m V
-55 m V
-35 m V
-15 m V
c
50 pA20 pA
0.04 mW/mm2
0.06 mW/mm2
0.1 mW/mm2
1.6 mm diameter illumination spot size
20 pA
50 ms
e
Chronosor
Chrimson
ChronosChrimson
Supplementary Figure 18
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 19 – Optically evoked paired-pulse responses in
acute slice.
(a-c) Chrimson and Chronos expressed in separate neurons in the same slice
using triple plasmid in utero electroporation. Patched neurons are post-synaptic
to both Chrimson and Choronos based on optical response to blue and red light.
(a) Paired-pulse responses (PPR) for an exemplar neuron. Blue-blue stimulation
exhibited facilitation, while other paired pulses were linear summation of
individual pulse response. 470 nm, 0.2 mW/mm2; 625 nm, 1 mW/mm2. 5 ms
pulse width and inter-pulse interval 50 ms throughout this figure. Black trace is
the averaged response, grey traces are individual trials, throughout this figure.
(b-c) Blue-blue and red-red responses from the same neuron showed no
differences in the blue paired PSC, while the second red pulse response often
failed at 50 ms.470 nm, 0.37 mW/mm2; 625 nm, 1mW/mm2. (d-g) Chrimson and
Chronos expressed in separate slices. 470nm, 0.3 mW/mm2, 625 nm, 4 mW/mm2
used throughout. (d-e) PPR recorded downstream of Chronos-expressing
neurons. Representative trace (d) and averaged PPR in four different neurons
(e), showing reliable Chronos drive . (f-g) PPR recorded downstream of
Chrimson-expressing neurons. Representative trace (f) and averaged PPR in
four different neurons (g), showing second pulse fails due to kinetic limitation of
Chrimson. An inhibitory PSC (voltage clamped at –55 mV) was recorded from
one neuron, most likely due to mistargeting during in utero electroporation.
!
Nature Methods: doi:10.1038/nmeth.2836
Pulse 1 Pulse 2-100-80-60-40-20
020
EPSC
Pea
k (p
A)
Pulse 1 Pulse 2-10
01020304050
IPSC
Pea
k (p
A)
b c
50 pA
100 ms
10 pA
100 ms
50 pA
100 ms
a
d e
f g
ChronosChrimson
Chronos Pulse 1 Pulse 2-30
-20
-10
0
10
EPSC
Pea
k (p
A)
Pulse 1 Pulse 2-200
-150
-100
-50
0
50
EPSC
Pea
k (p
A)Pulse 1 Pulse 2
-200
-150
-100
-50
0
50
EPSC
Pea
k (p
A)
Pulse 1 Pulse 2-50-40-30-20-10
010
EPSC
Pea
k (p
A)
Blue Blue Blue Red
Red Blue Red Red
Pulse 1 Pulse 2-80
-60
-40
-20
0
20
EPSC
Pea
k (p
A)
Pulse 1 Pulse 2-30
-20
-10
0
10
EPSC
Pea
k (p
A)
Blue Blue Blue Red
Red Blue Red Red
Chrimson
50 pA
50 ms
50 ms
50 pA
Supplementary Figure 19
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 20 – Comparisons of spiking and post-synaptic
response timing in acute slice.
(a) Time-to-spike-peak as a function of irradiance when optically driving
Chrimson neurons in the red and Chronos neurons in the blue; color and pulse
duration as in Fig. 5b-d. Values shown are population average ± standard
deviation. In all subpanels, red squares denote Chrimson, blue circles denote
Chronos (n = 9 cells, 3 mice, Chrimson; n = 11 cells, 4 mice, Chronos). (b) Time-
to-spike-peak for individual Chronos- (0.3 mW/mm2 blue light) and Chrimson-
expressing (5 mW/mm2 red light) neurons. Values are trial mean and standard
deviation (cells patched from 3-4 mice for each opsin). (c) Post-synaptic current
latency (time from beginning of light pulse, to 10% of synaptic current peak) for
individual neurons post-synaptic to Chronos (0.3 mW/mm2 blue) or post-synaptic
to Chrimson (4 mW/mm2 red). Values shown are trial averages and standard
deviation (cells patched from 2-4 mice for each opsin).
Nature Methods: doi:10.1038/nmeth.2836
a b c
0 5 10 15 200
1
2
3
4
time to spike peak mean (ms)
stan
dard
dev
iatio
n (m
s)
0 5 10 15 200
1
2
3
4
PSC onset delay mean (ms)
stan
dard
dev
iatio
n (m
s)
0.1 1 10 1000
5
10
15
Irradiance (mW mm-2)
time
to s
pike
pea
k (m
s)
Supplementary Figure 20
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 21 – Retina to superior colliculus projection
stimulation with Chronos.
Post-synaptic responses recorded in a superior colliculus neuron, downstream of
Chronos expressed in retinal ganglion cells. See methods for retinal virus
delivery details. (a) Diagram of retina projection to superior colliculus (adapted
from Paxinos, G. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates
2nd edn. Academic Press, 2001). (b) Histology of Chronos-GFP axons from
sagittal section of superior colliculus. Scale bar is 100 µm. (c-d) Post-synaptic
current and potential in response to blue light at multiple irradiances (5 pulses, 1
Hz, 5 ms pulse duration in all cases. Traces from a neuron recorded from a P18
mouse).
!
Nature Methods: doi:10.1038/nmeth.2836
a b
c
d
LV
Blue IrradiancemW/mm2 pulse 1 pulse 2 pulse 3 pulse 4 pulse 5
pulse 1 pulse 2 pulse 3 pulse 4 pulse 5
EPSCs recorded downstream of Chronos-expressing axons; 470 nm 5 ms 1 Hz
EPSPs and Spikes recorded downstream of Chronos-expressing axons; 470 nm 5 ms 1 Hz
0.1
0.2
Blue IrradiancemW/mm2
1
2
0.3
0.4
1.0
AAV8-Syn-Chronos-GFP
Chronos-GFP axons projecting from the retinato the superior colliculus, sagittal section at P16
retina
superior colliculus
20 pA
20 pA
20 pA
20 pA
20 pA
20 ms
25 mV
25 mV
20 ms
-60 mV
-60 mV
Supplementary Figure 21
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Figure 22 – Post-synaptic current raw traces.
Unfiltered traces with LED stimulation artifact. Panel (a), (b), and (c) corresponds
to the filtered traces in Fig. 5g, h, and i respectively.
Nature Methods: doi:10.1038/nmeth.2836
a
b c
.
20 pA
20 pA
100 ms
20 ms
20 pA
20 ms
Supplementary Figure 22
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Table 1 – Naming convention !
Gene # NCBI # Alias Genus species
ChR1 Chlamydomonas reinhardtii
ChR2 Chlamydomonas reinhardtii
VChR1 Volvox carteri
MvChR1 Mesostigma viride 60 KF992067 Mesostigma viride 62 KF992054 NsChR Neochlorosarcina sp. 63 KF992043 Monomastix opisthostigma 64 KF992072 SdChR Scherffelia dubia 65 KF992034 BsChR2 Brachiomonas submarina 66 KF992089 TsChR Tetraselmis striata 67 KF992071 Tetraselmis chui 68 KF992081 Tetraselmis chui 69 KF992087 Tetraselmis chui 70 KF992084 Spermatozopsis exsultans 71 KF992066 Pedinomonas minor 72 KF992045 Cyanophora paradoxa 73 KF992053 Stephanosphaera pluvialis 74 KF992059 HdChR Haematococcus droebakensis 75 KF992058 Tetraselmis striata 76 KF992070 Tetraselmis chui 77 KF992057 TcChR Tetraselmis cordiformis 78 KF992042 Pavlova lutheri 79 KF992077 Scherffelia dubia 80 KF992086 BsChR1 Brachiomonas submarina 84 KF992047 Rhodomonas sp. 85 KF992030 Chloromonas reticulata-A 86 KF992041 CoChR Chloromonas oogama 87 KF992078 CsChR Chloromonas subdivisa 88 KF992060 CnChR1/Chrimson Chlamydomonas noctigama 89 KF992073 CnChR2 Chlamydomonas noctigama 90 KF992040 ShChR/Chronos Stigeoclonium helveticum 91 KF992032 Microthamnion kuetzigianum-A 92 KF992049 Chlamydomonas bilatus-A 93 KF992052 Heterochlamydomonas inaequalis 95 KF992076 Chloromonas reticulata-A 96 KF992036 Chlamydomonas noctigama 97 KF992074 PsChR1 Proteomonas sulcata 98 KF992083 Cryptomonas curvata
Nature Methods: doi:10.1038/nmeth.2836
99 KF992085 Chroomonas sp. 100 KF992088 Chroomonas sp. 101 KF992051 Chroomonas sp. 102 KF992090 Chroomonas sp. 103 KF992033 Proteomonas sulcata 104 KF992065 Chroomonas sp. 105 KF992064 Gloeochaete wittrockiana 106 KF992061 Hemiselmis virescens 107 KF992055 Proteomonas sulcata 108 KF992056 PsChR2 Proteomonas sulcata 109 KF992063 Proteomonas sulcata 110 KF992037 Rhodomonas sp. 111 KF992044 Chlamydomonas bilatus-A 112 KF992038 AgChR Asteromonas gracilis-B 113 KF992046 Pyramimonas parkeae 114 KF992035 Monomastix opisthostigma 115 KF992079 Tetraselmis striata 116 KF992075 Lobomonas rostrata 117 KF992031 Lobomonas rostrata 118 KF992050 Stichococcus bacillaris 119 KF992080 Hafniomonas reticulata 120 KF992062 CbChR1 Chlamydomonas bilatus-A 121 KF992039 Hafniomonas reticulata 122 KF992069 Carteria crucifera 123 KF992048 Carteria crucifera 124 KF992068 Volvox aureus 125 KF992082 Phacotus lenticularis
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Table 2 – Statistical Analysis for Figure 1 Figure 1b – 660 nm current C1V1TT used as control group for Dunnett’s post hoc test
Molecule Abs(Dif)-LSD p-Value # of cells mean s.e.m.
AgChR -221 1 3 0 0
BsChR2 -169 1 6 0 0
C1V1TT -166 1 10 22.646 4.12
CbChR1 -169 1 6 0 0
ChR1 -180 1 5 0 0
ChR2 -139 1 11 0 0
Chrimson 489.4 <.0001 11 673.841 119.57
Chronos -149 1 9 1.08 0.61
CnChR2 -180 1 5 0 0
CoChR -143 1 10 0 0
CsChR -184 1 5 3.836 1.46
HdChR -180 1 5 0 0
MvChR1 -264 1 2 0 0
NsChR -180 1 5 0 0
PsChR1 -171 1 6 1.985 1.2
PsChR2 -182 1 5 2.192 1.44
SdChR -160 1 7 0 0
TcChR -180 1 5 0 0
TsChR -196 1 4 0 0
VChR1 -207 1 4 35.13 11.75 Figure 1c – 530 nm current C1V1TT used as control group for Dunnett’s post hoc test
Molecule Abs(Dif)-LSD p-Value # of cells mean s.e.m.
AgChR -138 0.3059 3 0.32 0.16
BsChR2 -154 0.4857 5 108.41 18.68
C1V1TT -371 1 10 408.07 72.59
CbChR1 -82.1 0.1787 4 0 0
ChR1 -188 0.4964 3 50.47 29.79
ChR2 7.531 0.0419 11 38.5 9.73
Chrimson -347 1 10 431.7 96.15
Chronos 204.1 0.0002 8 1005.24 253.54
CnChR2 -213 0.7878 5 167.35 51.07
CoChR 417.3 <.0001 10 1195.9 107.95
CsChR 210.4 0.0005 5 1072.3 213.64
HdChR -152 0.356 3 14.88 3.85
Nature Methods: doi:10.1038/nmeth.2836
MvChR1 -234 0.5428 2 0 0
NsChR -141 0.316 3 3.38 1.69
PsChR1 -116 0.3403 6 96.16 29.81
PsChR2 -165 0.5412 5 119.2 42.73
SdChR -188 0.7707 7 188.22 40.1
TcChR -51.8 0.1227 5 6.07 2.83
TsChR -49.2 0.1174 5 3.38 1.46
VChR1 -205 0.7498 5 159.38 58.38 Figure 1d – 470 nm current ChR2 used as control group for Dunnett’s post hoc test
Molecule Abs(Dif)-LSD p-Value # of cells mean s.e.m.
AgChR -281 0.5733 3 4.76 3.62
BsChR2 -330 0.9825 8 683.24 77.56
C1V1TT -296 0.9554 9 259.56 51.76
CbChR -196 0.3878 4 0 0
ChR1 -211 0.4931 5 66.92 22.74
ChR2 -478 1 12 478.96 115.55
Chrimson -304 0.9773 10 281.87 61.26
CnChR2 -274 0.734 5 828 145.97
CoChR 2274 <.0001 10 3253.58 227.96
CsChR -254 0.6609 5 847.42 186.25
HdChR -287 0.78 5 142.9 30.65
MvChR1 -414 0.7877 2 0 0
NsChR -172 0.3578 5 28.22 4.67
PsChR1 -151 0.322 6 44.74 17.51
PsChR2 -236 0.5912 5 92.53 32.98
SdChR 316.4 0.0001 7 1351.67 184.47
Chronos 222.8 0.0006 9 1217.6 266.72
TcChR -386 0.9837 5 242.45 70.72
TsChR -306 0.8417 9 162.12 66.48
VChR1 -516 0.9577 2 101.41 95.66 Figure 1h – channel turn off kinetics ChR2 used as control group for Dunnett’s post hoc test
Molecule Abs(Dif)-LSD p-Value # of cells mean s.e.m. λ (nm)
BsChR2 -351 0.9996 6 126.4 21.47 470
C1V1TT -374 1 10 37 2.89 530
ChR1 -526 1 4 5.61 1.08 470
ChR2 -378 1 12 14.59 1.39 470
Chrimson -379 1 11 22.4 1.43 625
Nature Methods: doi:10.1038/nmeth.2836
Chronos -429 1 7 3.59 0.21 470
CnChR2 1101 <.0001 5 1608.54 634.85 470
CoChR -369 1 7 86.03 11.63 470
CsChR -487 1 5 8.45 0.71 530
HdChR -485 1 5 21.98 2.03 470
PsChR1 -500 1 4 48.86 4.97 530
PsChR2 -516 1 4 33.22 1.51 530
SdChR -430 1 7 24.99 1.81 470
TcChR -485 1 5 6.96 0.76 470
TsChR -587 1 3 3.83 0.38 470
VChR1 -452 1 4 97.43 15.03 530 Figure 1i – time to 90% of peak ChR2 used as control group for Dunnett’s post hoc test
Molecule Abs(Dif)-LSD p-Value # of cells mean s.e.m. λ (nm)
BsChR2 -0.08 0.0756 4 7.075 0.42696 470
C1V1TT -0.11 0.1051 9 6.76667 0.17341 530
ChR2 -0.93 1 10 5.92 0.25768 470
Chrimson 0.188 0.0143 6 7.18333 0.39951 625
Chronos 2.644 <.0001 8 2.2875 0.28185 470
CoChR 0.351 0.0038 7 4.54286 0.31912 470
CsChR 0.559 0.0011 5 4.22 0.33675 530
SdChR 2.065 <.0001 7 2.82857 0.18988 470
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Table 3 – Primer sequences Primers for generating cDNA library Name Sequence Comment RNA adapter 2 AAUCAUACGACGACCACCGAGAUCAGG 5' mRNA ligation adaptor
DNA adapter 1 AAGCAGTGGTATCAACGCAGACTAC(T)30VN Reverse transcription Primers for algae PCR Name Sequence DNA adapter 2 AATCATACGACGACCACCGAGATCAGG
55 GSP5 GAAACACCTCAATCATCACCTTGACC
55 GSP6 CAGCCTGTCCCGCATCACCAGGAACG
55 GSP8 CGGGCTTACAAGACCAGTACAGCAAG
2565 GSP12 ATCTTGACCTGGAGGTAATGGGCG
232 GSP6 GTGATAATCTTGATTAAGCCGTGGCG
51 GSP2 CGTTCAGCCCCGTTATATTCGATAG
1293 GSP2 AAAAGCAGCCGATGTAGAAGAGGAGC
CCAC19 GSP1 TACGCTACCTCCTTTGTGGTGGAG
CCAC19 GSP2 GTAGTCCACACCACTATCGTCAGCAG
Primer pairs for algae PCR Gene # Algae source Template Primer 1 Primer 2
74 UTEX 55 cDNA DNA adapter 2 55 GSP5
cDNA 55 GSP8 55 GSP6
75 UTEX 2565 cDNA DNA adapter 2 2565 GSP12 76 UTEX 232 cDNA DNA adapter 2 232 GSP6
77 CCAC 51 cDNA DNA adapter 2 51 GSP2 78 UTEX 1293 cDNA DNA adapter 2 1293 GSP2
79 CCAC 19 genomic DNA CCAC19 GSP1 CCAC19 GSP2 UTEX: http://web.biosci.utexas.edu/utex/ CCAC: http://www.ccac.uni-koeln.de
Nature Methods: doi:10.1038/nmeth.2836
Primers for cloning Name Sequence
Cloning P12 GGAGGTGGAAGTGGAAGAGTTCGTGGAGG
Cloning P13 actaggtacctaggcttaCTTATACAGCTCATCCATGCCGTACAG
Cre P1 caaaGAATTCtgagccgccaccATGcccaagaagaagaggaaggtgtccAATTTACTGACCGTACACCAAAATTTGCC
Cre P2 ggacccgccaccactcccgccaccagaATCGCCATCTTCCAGCAGGC
Cre P3 GGCTTCTGGCGTGTGACC
GB1 (dsDNA) CTGTCTCATCATTTTGGCAAAGAATTCCCATAACTTCGTATAAAGTATCCTATACGAAGTTATATCAAAATAGGAAGACCAATGCTTCACCATCGACCCGAATTGCCAAGCATCACCATCGACCCATAACTTCGTATAATGTATGCTATACGAAGTTATACTAGCTAGCGCCGCCACCATGGAAACAGC
GB2 (dsDNA)
GAGCTGTACAAGTAATGAGCGGCCTAGGTACCTAGTATAACTTCGTATAGGATACTTTATACGAAGTTATCATTGGGATTCTTCCTATTTTGATCCAAGCATCACCATCGACCCTCTAGTCCAGATCTCACCATCGACCCATAACTTCGTATAGCATACATTATACGAAGTTATGTCCCTCGAAGAGGTTCGCGGCCGCACTCCTCAGGTGCAG
Gene88 K176R Fwd cccgtgatcctgatcagactgagcaacctgagcg
Gene88 K176R Rev cgctcaggttgctcagtctgatcaggatcacggg
Gene88 P2 ctactaccggtgccgcCACTGTGTCCTCGTCCTCCTCC
Gene88 P27 actagctagcgccgccaccATGGCTGAGCTGATCAGCAGCG
Gene90 P9 gccgccaccATGGAAACAGCCGCCACAATGAC
Gene90 P10 actaggtacctaggccgcgccgccaccATGGAAACAGCCGCCACAATG
GFP P18 ggccgctcattaCTTGTACAGCTCGTCCATGCCGAG
GFP P20 actagctagcTCATTACTTGTACAGCTCGTCCATGC
OE P1 CTGTCTCATCATTTTGGCAAAGAATTCCC
OE P2 CTGCACCTGAGGAGTGCGGCCGCG
OE P13 CAGTGgcggcaccggtagtagcaGTGAGCAAGGGCGAGGAGA
OE P14 actaggtacctaggccgctcattaCTTGTACAGCTCGTCCATGCCG
OE P15 ACTAGCTAGCGCCGCCACCATGG
OE P16 ACTAGGTACCTAGGCCGCTCATTAC
tdTomato P1 ctgcaccggtagtagcaGTGAGTAAGGGCGAGGAAGTGATCAAAG
tdTomato P2 gagtgcggccgctttaCTTATACAGCTCATCCATGCCGTACAGAAAC
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Table 4 – Solutions used to characterize ion selectivity
Solution [Na] (mM)
[K] (mM)
[Ca] (mM) [H] (mM) pH Other (mM)
Intracellular 0 140 0 5.10E-05 7.40 5 EGTA, 2 MgCl2, 10 HEPES
145 mM NaCl 145 5 1 5.10E-05 7.40 10 HEPES, 5 glucose, 2 MgCl2
145 mM KCl 0 145 1 5.10E-05 7.40 10 HEPES, 5 glucose, 2 MgCl2
90 mM CaCl2 0 5 91 5.10E-05 7.40 10 HEPES, 5 glucose, 2 MgCl2
5 mM NaCl 5 5 1 5.10E-04 6.40 135 NMDG, 10 HEPES, 5 glucose, 2 MgCl2
!
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Video 1 – Experimental setup with a visual arena.
The fly was tethered and centered in the visual arena50. In this movie, a flowing
random dot pattern is shown. The visual arena was removed from the setup in
other conditions. Fly behavior was recorded using a camera with 850 nm IR
illuminator.
Supplementary Video 2 – PER of a Gr64f X CsChrimson fly to 720 nm light
in darkness.
A fly with CsChrimson expression in sugar receptors shows PER to deep red
light stimulation.
Supplementary Video 3 – Startle response to 720 nm light in darkness
A control fly without CsChrimson expression shows clear startle response to
deep red light.
Supplementary Video 4 – PER of a Gr64f X CsChrimson fly to 720 nm light
in a blue random dot arena.
PER of a fly with CsChrimson expression in sugar receptors is not affected by
visual distractors.
Nature Methods: doi:10.1038/nmeth.2836
Supplementary Video 5 – Inhibited startle response to 720 nm light in a
blue random dot arena.
The startle response of a control fly without CsChrimson expression is effectively
inhibited.
Supplementary Video 6 – Optogenetics in freely behaving intact flies.
Top: Light-induced CO2 avoidance behavior (VT031497-Gal4 x UAS-
CsChrimson in attP18). Bottom: A control group (WTB x UAS-CsChrimson in
attP18). Circles show raw video images with false color red background
indicating the illuminated quadrants. The effect of light is quantified (see
Methods) and plotted as a single blue line corresponding to the presented
examples and a plot representing the mean of all 9 sessions (±SEM error bars).
Plots will be in red region if more than 50% of flies are in illuminated quadrants.
Replay speed: 4x.
Nature Methods: doi:10.1038/nmeth.2836