A high-conductance chemo-optogenetic system based on the vertebrate channel Trpa1b

Optogenetics is a powerful research approach that allows localized optical modulation of selected cells within an animal via the expression of genetically encoded photo-excitable ion channels. Commonly used optogenetic techniques rely on the expression of microbial opsin variants, which have many excellent features but suffer from various degrees of blue spectral overlap and limited channel conductance. Here, we expand the optogenetics toolbox in the form of a tunable, high-conductance vertebrate cation channel, zTrpa1b, coupled with photo-activated channel ligands, such as optovin and 4g6. Our results demonstrate that zTrpa1b/ligand pairing offers high light sensitivity, millisecond-scale response latency in vivo, as well as adjustable channel off latency. Exogenous in vivo expression of zTrpa1b in sensory neurons allowed subcellular photo-activation, enabling light-dependent motor control. zTrpa1b/ligand was also suitable for cardiomyocyte pacing, as shown in experiments performed on zebrafish hearts in vivo as well as in human stem cell-derived cardiomyocytes in vitro. Therefore, zTrpa1b/optovin represents a novel tool for flexible, high-conductance optogenetics.


Supplemental Methods
Photomotor response (PMR) assay For Fig. 1a-e and Fig. 3, Zebrabox (ViewPoint Behavior Technology) with the built in Zebralab quantification program was used. Four 3 dpf larvae in 300 µL volume were placed in individual wells of a square flat-bottom 96-well plate (7701-1651; Whatman UNIPLATE). Light flashes were programed as: 10 s no light, 1 s 100% light, 10 s no light, 1s 100% light and 10 s no light. All PMR experiments were conducted at room temperature. Mean behavioral excitation score is calculated by taking the 75 th percentile of the motion index from 1 s -3 s following the light stimulus. To calculate the off latency, five consecutive data points at a 30 ms interval were first combined and then averaged. The time point where the last motion index at the value 15 was calculated as the off latency. The value of 15 is the lowest value that allows us to detect above background motion activity. Light intensity was measured using a hand-held laser meter (LaserCheck, Coherent 1098293).
For Fig. 1f -h, experiments were performed on WT larvae at 3 dpf using an inverted compound microscope (AxioObserver A1; Zeiss) equipped with an EMCCD camera (C9100; Hamamatsu), a violet LED light source (415 nm) with a CW 310 mW maximum output power (BLS-LCS-0415-03-22; Mightex) which is controlled by a BioLED light source control module (BLS-SA02-US; Mightex) and a pulse master multi-channel stimulator (A30; World Precision Instruments). Schott longpass absorption glass (RG610; Chroma) was added in the transmitted light path to eliminate unwanted excitation. A dichroic mirror (T510LPXRXT; Chroma) is used for appropriate excitation of optovin and bright field acquisition. The software MetaMorph (Molecular Devices) was used to control the execution of TTL signals and camera capture using the stream acquisition with trigger function. Larvae were individually placed in 100 µL of media in a single well of a 96-well clear bottom black microplate (07-200-567; Fisher Scientific). A NA 0.25 5x objective was used. Photo-activation was made with a pulse of 30 ms 415 nm light in the mid-trunk region of zebrafish larvae. Bright field time-lapse was captured for 500 frames at ~103 frames per second (FPS). A light pulse was applied at frame 20. Percentage responding was quantified based on whether there was any motion in the entire acquisition period. Response time is the duration from the beginning of the light pulse to the first movement of the larvae.
For Fig. 2b, the corresponding construct was injected in trpa1b mutant embryos as described previously. At 2 dpf, larvae with one or more Rohon-Beard neurons expressing the transgene were picked and treated with 10 µM optovin for 1 hour. The experimental setup previously described for Fig. 1f -h was used.
For Fig. 2c, similar to the experimental setup used for Fig. 2b, trpa1b mutant larvae were injected with the ngn1:zTrpa1b-2A-EGFP construct and screened for mosaic Rohon-beard neuron expression at 2 dpf. Larvae were incubated with 10 µM optovin for 1 h before experiment. Larvae were then briefly anesthetized with 0.02% MS222 before being mounted laterally in 1% low melting agarose. The mounting protocol has been previously described 1 . Larvae were than incubated in E3 without MS222 before the photo-stimulation experiment. Photoactivation was performed on a confocal microscope (Zeiss Observer Z1) using a NA 1.2 40x water immersion objective with the bleaching function in the Zen Black 2009 (Zeiss) software. A 405 nm laser in line scan mode at 80% power was used to activate a 40.5 µm x 5.7 µm rectangular region for approximately 4 s. Time-lapse confocal single plane images were acquired before and after photoactivation was performed. SI S1. Chemical synthesis information of compound 11, 17 and 19.

S3. Heart rate of
Video S1. Photomoter response of trpa1b-/-mutants with transient, mosaic expression of zebrafish Trpa1b or Trpa1a in Rohon-Beard neurons using the neurogenin1 (ngn1) promoter. Experiments were performed at 2.5 dpf. Larvae were pretreated with 10 µM optovin. Video S4. Optogenetic pacing of zebrafish hearts in vivo -optovin. Ventral views of a zebrafish heart at 2 dpf with different photo-activation frequencies.
Photo-activation was performed at the atrium of the heart of a transgenic trpa1b-/-mutant larva expressing zTrpa1b in cardiomyocytes. The larva was pretreated with 10 µM optovin and microinjected with ivabradine into the pericardial space to inhibit pacemaker activity. The stimulation frequency and output heartbeat frequency are indicated on the individual panels (Simulation frequency / Heartbeat frequency).
Video S5. Optogenetic pacing of zebrafish hearts in vivo -4g6. Ventral views of a zebrafish heart at 2 dpf with different photo-activation frequencies.
Photo-activation was performed at the atrium of the heart of a transgenic trpa1b-/-mutant larva expressing zTrpa1b in cardiomyocytes. The larva was pretreated with10 µM 4g6 and microinjected with ivabradine into the pericardial space to inhibit pacemaker activity. The stimulation frequency and output heartbeat frequency were indicated on the individual panels (Simulation frequency / Heartbeat frequency).