Neurons and molecules involved in noxious light sensation in Caenorhabditis elegans

Abstract

Ultraviolet (UV) light is dangerous to unpigmented organisms, inducing photodamage of cells and DNA. The transparent nematode Caenorhabditis elegans detects light and exhibits negative phototaxis in order to evade sunlight. UV absorption is detected by the photosensor protein LITE-1 that also responds to reactive oxygen species. We investigated which neurons express LITE-1 and act as noxious photosensors and how they transmit this sensation to the nervous system to evoke escape behavior. We identified the interneuron AVG as a main focus of LITE-1 function in mediating the noxious light-evoked escape behavior, with minor roles of the interneuron PVT, the sensory ASK neurons, and touch receptor neurons. AVG is activated by blue light, and also its optogenetic stimulation causes escape behavior, while its optogenetic inhibition reduced escape. Signaling from AVG involves chemical neurotransmission, likely directly to premotor interneurons, and to other cells, by extrasynaptic signaling through the neuropeptide NLP-10. NLP-10 signaling is somewhat required for the acute response, yet is more important for maintaining responsiveness to repeated noxious stimuli. The source of NLP-10 in this context is largely AVG, yet also other cells contribute, possibly ASK. We uncover entry points of sensory information to neuronal circuits mediating noxious UV/blue light responses.

Keywords: Ca2+ imaging; LITE-1; WormBase; behavioral analysis; negative phototaxis; neuropeptide; optogenetics.

Graphical Abstract

Fig. 1.

LITE-1 is required mainly in AVG and in ASK and TRNs as secondary site of action for noxious light-evoked escape behavior. a) Mean crawling speed (±SEM) of animals before (0–299 s), during (300–330 s), and after (331–600 s) blue light illumination. Tested strains were wild type, lite-1(ce314), and lite-1 mutants expressing LITE-1 solely in neurons AVG, PVT, ASK, and in TRNs, or simultaneously in AVG, PVT, and ASK. Speed was normalized to the mean crawling speed from 270 to 299 s. Illumination is indicated by the shaded regions behind the graphs. N = 20, 15, 14, 14, 7, 4, and 4 experiments, with n = 20–32 animals each, respectively. b) Close-up of 270–360 s, including the illumination period. c) Comparison of speed among the indicated strains during the illumination period. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. One-way ANOVA with Tukey test (*P < 0.05; ****P < 0.0001). Gray asterisks indicate significant difference to wild type, others as indicated. Note that in a comparison of the time windows of 305–320 s, the triple rescue strain was not different to wild type. Full statistical analysis can be found in Supplementary Data File 1. d) Comparison of speeds between strains during the first and last 10 s of illumination. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. Two-way ANOVA with Tukey test. Gray and orange asterisks: Significant differences to wild type and lite-1, respectively (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). e) Time until maximum speed is reached during illumination period. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. One-way ANOVA with Tukey test (ns, not significant). f) Time until maximum speed is reached after stimulus onset. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. One-way ANOVA with Tukey test (****P < 0.0001).

Fig. 2.

The AVG neuron is activated by blue light and releases a signal by chemical neurotransmission. a) Exemplary images of RCaMP signal in AVG (cell body and axon, anterior is left) before (top) and right after (bottom, outlined box) blue light application. Scale bars: 50 µm. b) Mean (±SEM) change of fluorescence intensity of RCaMP in AVG before (20–29 s), during (30–39 s), and after (40–50 s) application of blue light pulses (indicated by bars). n = 20 animals for wild type and n = 19 for lite-1. F₀ was calculated during seconds 20–29. c) Mean RCaMP signal intensity during blue light application of individual animals. Median (thick line) and 25/75 quartiles (dotted or thin lines). Unpaired t-test (****P < 0.0001). d) Schematic illustration of illumination of the AVG soma (right) with blue light, restricted to the soma. AVG soma (filled circle) was placed in the center of the field of view. Animals were exposed to yellow light continuously (shaded box). Blue light was solely presented to the soma (blue), using a video projector. BioRender license LB281CGZH2. e) Mean (±SEM) change of fluorescence intensity of RCaMP in AVG before (5–9 s), during (10–19 s), and after (20–30 s) application of blue light (indicated by blue bar); n = 12. F₀ was calculated during seconds 5–9. f) Mean RCaMP signal intensity of the AVG process in individual animals, before and during blue light application. Median (thick line) and 25/75 quartiles (dotted or thin lines). Paired t-test (ns, not significant). g) Mean crawling speed (±SEM) of animals before (0–299 s), during (300–330 s), and after (331–600 s) blue light illumination. Tested strains were wild type and animals expressing optoSynC (optogenetic tool for immobilization of synaptic vesicles) in AVG. Speed was normalized to the mean crawling speed from 270 to 299 s. Illumination indicated by blue shade. N = 6 experiments with 27–38 animals each. h) Close-up of locomotion speed 30 s before, during, and after illumination. i) Comparison of crawling speed during illumination. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. Unpaired, 2-tailed t-test (P = 0.0052).

Fig. 3.

Optogenetic stimulation of AVG triggers escape behavior. a) Mean crawling speed (±SEM) of animals expressing Chrimson in AVG in wild type (N = 10), lite-1(ce314) (N = 8), and nlp-10(zx29) (N = 8 with 19–35 animals for each strain) was analyzed before, during, and after red light illumination (shaded regions behind the graphs). Crawling speeds were normalized to the respective mean crawling speeds from 270 to 299 s. Animals without exposure to ATR served as a negative controls. b) Close-up of locomotion speed 30 s before, during, and after illumination. c) Statistical analysis of data in a) and b), during red light illumination. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. Two-way ANOVA with Tukey post hoc analysis (ns, not significant; ****P < 0.0001). d and e) As in a) and b), but for wild type or unc-47(e307) animals expressing AVG::Chrimson that were either exposed to ATR or not. N = 6 with 26–31 animals each. f) Time until animals reached their maximum speed during the illumination. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. Mann–Whitney test (P = 0.0022). g) Mean crawling speed (±SEM) of wild type, lite-1, or unc-47 animals before (0–299 s), during (300–330 s), and after (331–600 s) blue light illumination. Crawling speed was normalized to the mean crawling speed from 270 to 299 s. Illumination is indicated by the shaded regions behind the graphs. N = 4 experiments with 22–32 animals each. h) Close-up of locomotion speed 30 s before, during, and after illumination. i) Mean speed during the illumination period. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. One-way ANOVA with Tukey test (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 4.

Expression of NLP-10 in AVG rescues habituation to repetitive blue light stimulation. a) Representative image of NLP-10::mScarlet fluorescence, expressed specifically in AVG. Arrowhead indicates AVG soma; additional signal was seen in coelomocytes. Scale bar indicates 50 µm. b) Fluorescence intensity of mScarlet in nlp-10 and nlp-10; lite-1 mutants in anterior coelomocytes (CC1). Unpaired t-test (ns, not significant). c) Fluorescence intensity of mScarlet in nlp-10 and nlp-10; lite-1 mutants with (second and fourth column) and without (first and third column) repetitive blue light illumination. One-way ANOVA with Tukey post hoc analysis (*P < 0.05; ns, not significant). d) Mean crawling speed (±SEM) of the indicated strains during repetitive blue light stimulation at 300–330, 600–630, 900–930, and 1200–1230 s, as indicated by shaded regions behind the graphs. Crawling speed was normalized to the mean crawling speed from 270 to 299 s. N experiments per strain, as indicated, with 23–41 animals each. e and g) Close-ups of locomotion speed 30 s before, during, and after the first and the fourth illumination period, respectively. f) Comparison of crawling speed during the first (300–330 s) and fourth illumination (1200–1230 s). Median with 25/75 quartiles; whiskers indicate minimum and maximum values. Two-way ANOVA with Tukey post hoc analysis was applied (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Color of asterisks indicates significant differences to the respective strain. Full statistical analyses can be found in Supplementary Data File 1. h) Differences of mean speeds of fourth illumination, deduced by speed levels at first illumination. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. One-way ANOVA with Tukey test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 5.

nlp-10 mutants exhibit reduced sensitivity to repetitive optogenetic activation of AVG. a) Mean crawling speed (±SEM) of wild type and nlp-10(zx29) animals expressing Chrimson in AVG during repetitive red light stimulation at 300–330, 600–630, 900–930, and 1200–1230 s, as indicated by shaded regions behind the graphs. N = 8 experiments with 19 to 27 animals each. b) Difference of speed level upon first vs fourth illumination; mean speed during the fourth illumination was subtracted from mean speed during the first illumination. Median with 25/75 quartiles; whiskers indicate minimum and maximum values. Unpaired, 2-tailed t-test (****P < 0.01). c and d) Close-ups of locomotion speed 30 s before, during, and after the first or the fourth illumination period, respectively.