Optical control of zebrafish behavior with halorhodopsin

Abstract

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, enables optical control of membrane potential and reversible silencing of targeted neurons. We generated transgenic zebrafish expressing enhanced NpHR under control of the Gal4/UAS system. Electrophysiological recordings showed that eNpHR stimulation effectively suppressed spiking of single neurons in vivo. Applying light through thin optic fibers positioned above the head of a semi-restrained zebrafish larva enabled us to target groups of neurons and to simultaneously test the effect of their silencing on behavior. The photostimulated volume of the zebrafish brain could be marked by subsequent photoconversion of co-expressed Kaede or Dendra. These techniques were used to localize swim command circuitry to a small hindbrain region, just rostral to the commissura infima Halleri. The kinetics of the hindbrain-generated swim command was investigated by combined and separate photo-activation of NpHR and Channelrhodopsin-2 (ChR2), a light-gated cation channel, in the same neurons. Together this "optogenetic toolkit" allows loss-of-function and gain-of-function analyses of neural circuitry at high spatial and temporal resolution in a behaving vertebrate.

Fig. 1.

Expression of NpHR in zebrafish. (A) Expression pattern of Gal4^s1101t; UAS:Kaede transgenic animals. Dorsal view (i), horizontal optical slice through the hindbrain (ii) and transverse section through the eyes and midbrain (iii) of 5 dpf animals. (B) Expression of NpHR in Gal4^s1101t; UAS:(e)NpHR-XFP animals. (i and ii) NpHR-mCherry and eNpHR-eYFP, respectively. Intracellular blebs (arrows) are labeled in (i). (C) Surface targeting of NpHR. Co-expression of membrane-bound Dendra-kras and NpHR-mCherry in Gal4^s1013t; UAS:Dendra-kras; UAS:NpHR-mCherry animals (i) reveals suboptimal surface targeting. Co-expression of eNpHR-eYFP and membrane-bound mCherry-kras in Gal4^s1013t; UAS:eNpHR-eYFP; UAS:mCherry-kras animals, shows complete surface targeting of eNpHR-eYFP. [Scale bars, 100 μm in (A); 5 μm in (B and C).]

Fig. 2.

Analysis of silencing efficacy in the hindbrain. (A) Schematic of the electrophysiological setup. The optics of a microscope was used for NpHR activation in Fig. 2 B–D (mercury lamp, excitation filter HQ 585/70, beamsplitter 90/10). For Fig. 3, a laser (532 nm) was coupled to an optic fiber to activate NpHR. (B) Unprocessed current trace (top) and corresponding single-unit firing rate histogram (middle) show a cell of a NpHR-mCherry-expressing animal that was silenced during illumination (yellow shaded boxes, 21 mW/mm²). Bottom: A cell from a ChR2-eYFP-expressing animal fired at a higher rate upon stimulation with blue light (blue shaded boxes). (Scale bars, 10 s.) (C) Scatter plot of the firing rate during stimulation (F2) vs. the firing rate without stimulation (F1). NpHR-mCherry cells had a reduced firing rate F2, while cells from non-expressing siblings and blind lakritz/atoh7 mutants clustered around the line of unity (black, F2 = F1). (D) Cumulative probabilities of firing rate ratios F2/F1 (with/without stimulation) in cells from different lines. Cells from NpHR lines were silenced (curve shift to the left) and cells from the ChR2 line were facilitated (shift to the right). The differences between wild-type and eNpHR-eYFP or NpHR-mCherry were highly significant (P < 0.0001, KS test and Ranksum test).

Fig. 3.

Use of fiber optics to control locomotion with NpHR. (A) Release from NpHR activation induced locomotion in head-restrained animals. The tail deflection is plotted over time. (B) The recorded cell showed an above-average firing rate directly after the stimulation (yellow shaded box) was stopped (arrow). (C) Fiber-optic setup for the mapping of the locomotion phenotype. An optic fiber was coupled to a laser and placed over the head-restrained larva with a micromanipulator. (D and E) Spatially defined light application was feasible with optic fibers. (D) The optic fiber (10-μm inner core diameter) was moved across the recorded cell in five steps. False-colored stripes in the image represent the scattered light that was detected by the camera when light was sent through the fiber at positions 3–5. The stripes of scattered light approximately match the theoretical divergence angle of the fiber (8.5°, white solid lines). Dashed lines outline the position of the electrode. pf, pectoral fin. (E) The firing rate ratio F2/F1 of a single cell is plotted for different fiber positions. The cell was only silenced when it was directly illuminated, which demonstrated the precise light application with optic fibers.

Fig. 4.

Mapping of the locomotion phenotype that was induced by rebound from NpHR silencing (A) The probability of observing a forward swim is plotted versus the position of stimulation (200-μm optic fiber) in 3 dpf zebrafish. Mean probabilities of NpHR-mCherry expressors (black) and siblings (gray, only third position) are shown with 95% confidence intervals (Wilson score interval for binomial distributions). (B) The hindbrain region in (A) (asterisk) was mapped in detail with a 50-μm fiber. The probability was maximal in a small area along the midline just caudal to the pectoral fins. A total of 295 trials was analyzed in (A) and (B) (n = 7 animals). (C) The phenotype was mapped in an animal transgenic for Gal4^s1101t; UAS:NpHR-mCherry; UAS:Kaede. The region where the animal responded reliably was illuminated with UV light to photo-convert Kaede. (D) Close-up of the box in (C). A narrow column of tissue was converted, labeling only a few cells in every optical section. The maximal response probability was found in the region of the commissura infima Halleri (CI, dashed lines), which demarcates the border between spinal cord (SC) and hindbrain (HB). The upper right shows the red channel and the lower right the green channel. [Scale bars, 100 μm in (C) and 10 μm in (D).]

Fig. 5.

Kinetics of the rebound-evoked swim command. (A) The NpHR induced forward swimming was blocked by reactivating NpHR. Top: Without reactivation of NpHR, the animal (5 dpf) started to move 267 ± 3.5 ms (SEM) after the light had been turned off and continued to swim for 3.6 ± 0.3 s (SEM). Bottom: When the animal was re-illuminated after 248 ms, the locomotion that had started after 262 ms was blocked at t = 572 ms. (B) The amplitude and duration of the tail contractions depended on the re-illumination time point. Early re-illuminations permitted only smaller amplitudes (see also Movie S4). Images are an average-z-projection of four consecutive minimum-intensity-z-projections. (C) The time difference between the cessation of locomotion and the re-illumination is plotted versus the re-illumination time point. Intervals shorter than 190 ms never permitted tail contractions. Between 190 ms and 250 ms the time difference increased rapidly and approached a maximum value of 263 ± 14 ms (SEM) at 300 ms. Trials in (B) are labeled with an asterisk in (C). (D) In ChR2 expressing animals, the response latency depended on the illumination intensity. Stronger illuminations elicited shorter latencies. Trials from a single 3-dpf animal are plotted.

Fig. 6.

NpHR and ChR2 can be combined and activated separately. (A) Animals transgenic for Gal4^s1101t; UAS:NpHR-mCherry; UAS:ChR2-eYFP were illuminated with red or blue light, or both. Illumination with blue light induced locomotion (i), which could be blocked when red light was added (ii). When red light was followed by blue and red light, no locomotion was induced (iii). In (iv), blue light evoked locomotion was blocked with red light three consecutive times. (B) Experiments in (A) were highly reproducible. The probability of locomotion is plotted for four different genotypes (divided by vertical lines) and four different stimulation protocols. Error bars are 95% confidence intervals for binomial distributions (Wilson score, n = 238 trials in total, n ≥ 2 animals for each genotype). For triple transgenic animals, the blue-evoked locomotion probability was significantly different between trials with (A iii) and without (A i) red illumination (P < 0.0004, z-test for proportions). The inset shows the activation spectra of ChR2 and NpHR (after ref. 12) and the laser lines for ChR2 (488 nm) and NpHR (633 nm).