A spinal opsin controls early neural activity and drives a behavioral light response

Abstract

Nonvisual detection of light by the vertebrate hypothalamus, pineal, and retina is known to govern seasonal and circadian behaviors. However, the expression of opsins in multiple other brain structures suggests a more expansive repertoire for light regulation of physiology, behavior, and development. Translucent zebrafish embryos express extraretinal opsins early on, at a time when spontaneous activity in the developing CNS plays a role in neuronal maturation and circuit formation. Though the presence of extraretinal opsins is well documented, the function of direct photoreception by the CNS remains largely unknown. Here, we show that early activity in the zebrafish spinal central pattern generator (CPG) and the earliest locomotory behavior are dramatically inhibited by physiological levels of environmental light. We find that the photosensitivity of this circuit is conferred by vertebrate ancient long opsin A (VALopA), which we show to be a Gα(i)-coupled receptor that is expressed in the neurons of the spinal network. Sustained photoactivation of VALopA not only suppresses spontaneous activity but also alters the maturation of time-locked correlated network patterns. These results uncover a novel role for nonvisual opsins and a mechanism for environmental regulation of spontaneous motor behavior and neural activity in a circuit previously thought to be governed only by intrinsic developmental programs.

Figure 1

Effect of light on frequency of spontaneous coiling behavior. A. Still frames from a movie of a single spontaneous coil in a 27 hpf embryo. B. Trace of detected motion (pixel changes) from video of an individual embryo before (dark) and during (508 nm) illumination; peaks represent individual coiling events. C. Raster plot of coiling events measured simultaneously in 44 dark-adapted 22.5 hpf embryos under dark and light conditions. D. Left, peri-stimulus time histogram of 22.5 hpf embryos from data in (C). Right, histogram of the same fish at 27 hpf. Frequency = mean coils/fish within bins of 2.4 sec. E. Mean baseline coiling frequencies (coils/sec) in the dark (black bars) and under green light (gray bars) over developmental time. Two-tailed paired t test with Bonferroni adjusted p-values, *** p < 0.001; n = 39–75. F. Photo-inhibition [−(Hz_Light−Hz_Dark)/Hz_Dark; coiling measured over 120 sec in each condition] as a function of wavelength. Gray squares are individual responses < 3 s.d. from each mean (black lines). Light power = 133–159 nW/mm²; n = 96. G. Photo-inhibition of coiling frequency by light flashes of indicated durations. Lines indicate normalized mean, bins = 4 sec; s.e.m. in grey; n = 96.

Figure 2

Acute and delayed effects of light on neural activity. A. Left, image of GCaMP5 fluorescence in ventral spinal cord somites 3–8 of a 1020:Gal4; UAS:GCaMP5 embryo. Scale bar = 20 μm. Right, calcium traces from one cell (arrowhead in image) under 2P (black trace, 920 nm) or subsequent period of 1P (gray trace, 488 nm) excitation, following < 5 sec interval. Axes: 100% ΔF/F, 10 sec. B. Left, raster plot of 2P-imaged calcium events in 24 hpf embryos before and after a 5 sec flash (dashed line) with 561 nm light. Measurements from each of 8 fish (horizontal lines delineate individual fish). Right, quantification of calcium event frequency in the 8 fish (grey lines; mean = black line) during a 180 sec period under 2P excitation (baseline) and over 60 sec following the 561 nm light flash (post-stim.). Two-tailed paired t test p < 0.01. C. Frequency of calcium events over 9 hours of development measured under 2P excitation before (pre-stim) and after (post-stim.) a 5 sec 561 nm light flash, as quantified in (B). Two-tailed paired t test, Bonferroni adjusted p-values, *** p < 0.001, ** p < 0.01; n = 21–123 cells from 4–8 fish at each age. D. Photo-inhibition after illumination with a 5 sec light flash at varying wavelengths. 103–110 μW/pixel; n = 16 cells in 8 fish at each wavelength. E. Left, pairwise ipsilateral correlations between cells in 22 hpf embryos following dark or light (508 nm, 13.2 μW/mm²) rearing for 2 hours before 20 hpf. Circle size proportional to event width at half max amplitude (range = 1.8–75.7 sec). Dashed box demarcates non-correlated cells. Right, percentage of non-correlated cells (ipsilateral correlation < 0.1) at 22 hpf. Two-tailed unpaired t test, p = 0.004. n = 6 (dark) and 8 fish (light).

Figure 3

Light inhibition of coiling depends on an extraretinal opsin. A,B. Representative peri-stimulus time histograms of coiling frequency (events/sec; mean across 6 trials; bin = 1 sec) of embryos at 22–25 hpf (A) or 27–31 hpf (B). Zygotes injected with scrambled control morpholino (n = 23 in (A), 34 in (B)) (black) or morpholino against VALopA (n = 24 in (A), 41 in (B)) (gray). C. Left, mean photo-inhibition of scrambled control morpholino (sc; n = 40) and morpholino (mo; n = 55) injected 22–25 hpf embryos. Two-tailed unpaired t test, p < 0.001. Right, frequency distribution of percent photo-inhibition (6 responses per fish) in scrambled control morpholino embryos (black) and morpholino embryos (gray). D. Normalized inhibition or excitation of coiling frequency [−(Hz_Light−Hz_Dark)/Hz_Dark] in first 12 sec after light onset relative to a 120 sec preceding baseline in darkness (n = 39–75). E. Mean photo-inhibition (5 responses per fish) of morpholino-injected embryos mosaically expressing low, intermediate, or high levels of a VALopA rescue construct with a mCerulean marker. One-way ANOVA with Tukey post hoc analysis, * p < 0.05, ** p < 0.01; n = 59 (low), 18 (intermediate), and 11 (high). F. Images of representative embryos expressing low, intermediate, and high levels of mCerulean fluorescence. Scale bar = 200 μm.

Figure 4

VALopA couples to Ga_i/o in Xenopus oocytes. A. Representative voltage clamp currents from an uninjected oocyte (top trace), an oocyte expressing zebrafish VALopA_GFP alone (middle trace), or co-expressing VALopA_GFP with GIRK1_mCherry and GIRK2. Oocytes are initially bathed in ND96, switches to 24 mM K⁺ (high K⁺), and then 5 mM Ba²⁺ in 24 mM K⁺ (Barium). Illumination through a 535/50 nm bandpass filter (~70 mW/mm²) is indicated. B. Summary for the basal (top) and light-evoked (bottom) GIRK currents as in (A). C. Representative currents (protocols as in (A)) from oocytes expressing VALopA_GFP, GIRK1_mCherry and GIRK2 with the catalytic subunit of pertussis toxin, (PTX-S1) and either PTX-sensitive Gα_i3-wt (gray) or PTX-insensitive C351I mutant Gα_i3 (black). D. Summary for the basal (top) and light-evoked (bottom) GIRK currents in oocytes expressing combinations of Gα_i3-wt, Gα_i3-C351I, and PTX. One-way ANOVA with Tukey post hoc analysis * p < 0.05, ** p < 0.01, *** p < 0.001; n is indicated above bars.