nat. methods 11, 338–346 (2014) independent optical ... · nat. methods 11, 338–346 (2014)...

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.


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.


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


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 *.


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 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.


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 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).


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 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.

!


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


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


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.


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


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


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

)


0 5 100

50

100

150CbChR1


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

)


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.

!


5 s

C1V1TT

ChR2

SdChR

CoChR

CsChR

Chrimson

Chronos

wait 30 s in the darkwait 1s in the dark



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.


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 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).

!


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 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


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.


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 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.


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 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.


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 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).


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 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.


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 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.

!


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 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.

!


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 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.


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 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.


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 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.

!


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 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).


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 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).

!


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 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.


a

b c

.

20 pA

20 pA

100 ms

20 ms

20 pA

20 ms



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


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


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


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


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


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


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


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


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


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

!


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.


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.


nat. methods 11, 338–346 (2014) independent optical ... · nat. methods 11, 338–346 (2014)...

Documents