CRYPTOCHROME mediates behavioral executive choice in response to UV light

Abstract

Drosophila melanogaster CRYPTOCHROME (CRY) mediates behavioral and electrophysiological responses to blue light coded by circadian and arousal neurons. However, spectroscopic and biochemical assays of heterologously expressed CRY suggest that CRY may mediate functional responses to UV-A (ultraviolet A) light as well. To determine the relative contributions of distinct phototransduction systems, we tested mutants lacking CRY and mutants with disrupted opsin-based phototransduction for behavioral and electrophysiological responses to UV light. CRY and opsin-based external photoreceptor systems cooperate for UV light-evoked acute responses. CRY mediates behavioral avoidance responses related to executive choice, consistent with its expression in central brain neurons.

Keywords: Drosophila; UV; cryptochrome; neural decision making; phototransduction.

Fig. 1.

l-LNv electrophysiological response to UV light is attenuated in flies lacking CRY-based phototransduction. (A–D) Representative trace for control l-LNv UV light response (A) (365 nm, 640 µW/cm², violet bar; lights off, <0.01 µW/cm², black bar; the gap in the x axis removes <1 s, wherein a noise transient is caused by manual opening of the shutter to expose the prep to light) vs. representative traces for cry^−/− (B), hk^−/− (C), and “cry rescue” (D) flies (pdf-GAL4–driven LNv UAS-CRY expression in a cry^−/− background). (E–G) Dose–response quantification of l-LNv firing frequency (FF) response (FF on/FF off) to UV light at low (20 µW/cm²), intermediate (150 µW/cm²), and high (640 µW/cm²) intensities. (E) Electrophysiological response of control flies increase with increasing intensities of UV light (1.19 ± 0.04, n = 17, low; 1.33 ± 0.07, n = 15, intermediate; 1.77 ± 0.12, n = 15, high intensity). The significantly attenuated UV light responses of cry^−/− (1.04 ± 0.02, n = 17, P = 0.01, low; 1.17 ± 0.06, n = 15, P = 0.129, intermediate; 1.35 ± 0.07, n = 15, P = 0.005 vs. control, high intensity) and hk^−/− (0.99 ± 0.04, n = 15, P = 0.002, low; 1.13 ± 0.03, n = 14, P = 0.049, intermediate; 1.37 ± 0.07, n = 26, P = 0.008 vs. control, high intensity) flies do not differ from each other (P = 0.622, low; P = 0.879, intermediate; P = 0.978, high intensity). (F) Dose–response quantification of FF for control vs. cry^−/− and cry rescue flies. Full rescue is achieved at low (1.18 ± 0.03, n = 15, P = 0.99 vs. control) and intermediate intensities (1.24 ± 0.03, n = 15; P = 0.14 vs. control), but is incomplete at high-intensity UV light (1.45 ± 0.05, n = 15, P = 0.03 vs. control and P = 0.68 vs. cry^−/−). (G) Dose–response quantification of FF for control vs. hk^−/− and pdf-GAL4–driven rescue of WT-HK (UAS-HK-WT) or of redox sensor-disabled point mutant HK-D260N (UAS-HK-D260N), both in hk^−/− genetic background. WT-HK rescue flies also achieve rescue at low (1.21 ± 0.03, n = 16, P = 0.97 vs. control) and intermediate (1.26 ± 0.03, n = 16, P = 0.732 vs. control) intensities, but not at high intensity (1.34 ± 0.04, n = 16, P = 0.001 vs. control, and P = 0.99 vs. hk^−/−). The redox sensor-disabled point mutant HK-D260N fails to rescue the light response at all UV light intensities (1.03 ± 0.04, n = 13, P = 0.033, low; 1.09 ±0.03, n = 15, P = 0.004, intermediate; 1.19 ± 0.05, n = 11, P ≤ 0.001 vs. control, high intensity; P ≥ 0.417 vs. hk^−/− all intensities). (H) Representative trace for glass^60j (gl^60j) mutant l-LNv UV light response. (I) Representative trace for gl^60j- cry^−/− double-mutant l-LNv UV light response. (J) Dose–response quantification FF for gl^60j and gl^60j- cry^−/− double-mutant flies. gl^60j flies response do not significantly differ from control (1.20 ± 0.06, n = 18, P = 0.87, low; 1.19 ± 0.05, n = 14, P = 0.15, intermediate; 1.54 ± 0.07, n = 16, P = 0.098 vs. control, high intensity). gl^60j- cry^−/− double mutant has significantly attenuated UV response compared with control (1.01 ± 0.03, n = 20, P = 0.002, low; 1.08 ± 0.04, n = 32, P = 0.003, intermediate; 1.26 ± 0.05, n = 28, P ≤ 0.001 vs. control, high intensity) and do not differ from cry^−/− response (P = 0.499, low; P = 0.252, intermediate; P = 0.157 vs. cry^−/−, high intensity). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S1.

l-LNv electrophysiological UV light response is not attenuated in sevenless flies. Dose–response quantification of l-LNv firing frequency (FF) response (FF on/FF off) to UV light at low (20 µW/cm²), intermediate (150 µW/cm²), and high (640 µW/cm²) intensities. l-LNv electrophysiological UV response of sevenless flies do not significantly differ from that of control (1.23 ± 0.06, n = 9, P = 0.547, low; 1.37 ± 0.05, n = 9, P = 0.661, intermediate; 1.72 ± 0.14, n = 9, P = 0.816 vs. control, high intensity).

Fig. 2.

Drosophila acute arousal response to UV light is CRY-dependent. (A) Representative averaged double-plotted actogram of n = 32 flies given three 5-min light pulses (365 nm, 3 mW/cm²) during three consecutive nights. Flies respond acutely to light pulses, but remain entrained to the LD 12:12 environmental cues. (B) Percentage of sleeping flies that awaken during the light pulse for UV (365 nm, 3 mW/cm²) and orange light (595 nm, 7 mW/cm²). Compared with control flies (0.72 ± 0.03, n = 384 flies for UV), a significantly lower percentage of cry^−/− flies (0.61 ± 0.04, n = 192 flies, P = 0.039 vs. control) awaken in response to UV light pulse. Percentage of sleeping cry^−/− flies that awaken during orange light pulse does not differ from percentage of sleeping control flies that awaken (0.32 ± 0.03, n = 224 flies for control; 0.38 ± 0.04, n = 128 flies, for cry^−/−; P = 0.264 cry^−/− vs. control). Both norpA^P24 and gl^60j flies have a significantly lower percentage of flies that awaken in response to UV light pulses (0.54 ± 0.03, n = 192 flies, P ≤ 0.001 for norpA^P24 vs. control; 0.09 ± 0.02, n = 64 flies, P ≤ 0.001 for gl^60j vs. control). A significantly higher percentage of norpA^P24 flies awaken (0.56 ± 0.03, n = 160 flies, P ≤ 0.001 for vs. control), whereas a significantly lower percentage of gl^60j flies awaken in response to orange light pulses (0.13 ± 0.02, n = 64 flies, P ≤ 0.001 vs. control). (C–F) Time course of activity of awake flies during and after UV (C and E) or orange (D and F) light pulse. Each point on the graph represents a bin of 5 min, with the first bin collected during the pulse. (C) During the UV light pulse, control flies show a dramatic increase in arousal activity (activity/baseline is 2.98 ± 0.23, n = 384 flies), whereas cry^−/− flies remain relatively inactive (1.35 ± 0.12, n = 192 flies, P ≤ 0.001 vs. control), only responding after the pulse (cry^−/− vs. control, P > 0.213 for all bins, after the light pulse). (D) cry^−/− and control fly activities do not differ during and after the orange light pulse (n = 128 flies, cry^−/− vs. control, n = 224 flies, P > 0.064 for all bins). (E) Activity of awake norpA^P24 flies does not differ from that of control flies (n = 192 flies, norpA^P24 vs. control, n = 384 flies, P > 0.174 for all bins). gl^60j flies show a significantly lower arousal response both during and after the UV light pulse (n = 64 flies, gl^60j vs. control, P < 0.010 for bins during and 5–20 min after the light pulse). (F) gl^60j flies show a significantly lower arousal response during and after orange light pulse (n = 64 flies, gl^60j vs. control, n = 224 flies, P < 0.018 for bins during and 5–25 min after the light pulse). norpA^P24 flies have significantly higher activity than control flies during the orange light pulse (n = 160 flies, norpA^P24 vs. control, P ≤ 0.001), but do not differ in activity after the orange light pulse (norpA^P24 vs. control, P < 0.332 for all bins, after the light pulse). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S2.

Drosophila head and eye cuticles transmit UV light. The proportion of 365-nm UV light transmitted through cuticle tissues was calculated as the amount of UV light transmitted through either eye (n = 10) or head (n = 7) cuticle tissues divided by baseline measurements of transmittance through a droplet of PBS. ***P < 0.001.

Fig. S3.

Drosophila CRY-mediated acute arousal response to UV light is not attenuated in mutants lacking trpA1. (A) Percentage of sleeping flies that awakened during the 5-min light pulse for UV (365 nm, 3 mW/cm²) and orange (595 nm, 7 mW/cm²) light. Percent of sleeping trpA1¹ flies that awaken in response to UV (0.72 ± 0.03, n = 192 flies, P = 0.928 vs. control) or orange (0.32 ± 0.02, n = 192 flies, P = 0.919 vs. control) light does not differ from that of control flies. (B) Time course of activity of awake control and trpA1¹ flies during and after UV light pulse does not differ from each other (all P > 0.481 vs. control). (C) Time course of activity of awake control and trpA1¹ flies during and after orange light pulse does not differ from each other (all P > 0.115 vs. control).

Fig. 3.

Drosophila positive phototaxis behavior toward UV-light is attenuated in mutants lacking CRY- and in mutants lacking external photoreceptors. (A) A DAM2 Drosophila Activity Monitor (32 channels with dual infrared beams; Trikinetics) was mounted to the front of the light-tight chamber holding a population of 40 flies and sealed with a glass cover on the outer face. (B) Average phototaxis activity counts per min toward a very low-intensity UV light pulse (365 nm, 3 µW/cm², five exposures of 5-min light; indicated by violet arrows) followed by 55 min of darkness starting at circadian time (CT) 21 to CT 3 for control (nine experimental repeats, n = 40 flies per experiment), cry^−/− (three experimental repeats, n = 40 flies per experiment), norpA^P24 (four experimental repeats, n = 40 flies per experiment), and gl^60j flies (four experimental repeats, n = 40 flies per experiment). (C) Average phototaxis activity counts per min toward five 5-min orange light pulses (595 nm, 3 µW/cm², indicated by orange arrows) followed by 55 min of darkness starting at CT 21 to CT 3 for control (four experimental repeats, n = 40 flies per experiment), cry^−/− (five experimental repeats, n = 40 flies per experiment), norpA^P24 (three experimental repeats, n = 40 flies per experiment), and gl^60j flies (six experimental repeats, n = 40 flies per experiment). (D–G) Average phototaxis activity in 5-min bins relative to the UV (D and F) or orange (E and G) light pulses averaged from B and C. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S4.

Drosophila-positive phototaxis behavior toward UV light is not attenuated in mutants lacking trpA1. (A) Average phototaxis activity counts per min toward a very low-intensity, five 5-min UV light pulses (365 nm, 3 µW/cm²; indicated by violet arrows), followed by 55 min of darkness starting at CT 21 to 3 for control (nine experimental repeats, n = 40 flies per experiment) and trpA1¹ flies (four experimental repeats, n = 40 flies per experiment). (B) Average phototaxis activity counts per min toward a very low, five 5-min orange light pulse (595 nm, 3 µW/cm²; indicated by orange arrows), followed by 55 min of darkness starting at CT 21 to 3 for control (four experimental repeats, n = 40 flies per experiment) and trpA1¹ flies (three experimental repeats, n = 40 flies per experiment). (C and D) Average phototaxis activity in 5-min bins relative to the UV (C) or orange (D) light pulses averaged from A and B. **P < 0.01.

Fig. 4.

CRY-based phototransduction contributes to UV light avoidance behavior in Drosophila. (A) Diagram of the “light choice” apparatus. Standard Trikinetics Drosophila activity monitors were modified to fit behavior tubes of 2× length, which have food and air hole on both sides of the tube. Flies are first entrained in standard 12:12 white light LD without any cover. The 12:12 white light LD is then replaced by 12:12 UV light LD. (B) Half of the monitor is then covered with cardboard to provide flies a choice between UV light-exposed (400 µW/cm²) and shaded environments. UV light is turned on only during the entrained daytime (ZT0-12). (C–E) Preference for UV-exposed vs. shaded environment is measured by percent of activity in each environment over total amount of activity for each ZT. Gray shade indicates shaded environment preference (light avoidance), and violet-shade indicates UV environment preference. (C) cry^−/− flies have a significant defect UV light avoidance behavior at all times of the day compared with control flies and prefer the UV environment over the shaded (cry^−/−, n = 78 vs. control, n = 76, all P < 0.05). (D) Similarly, hk^−/− flies also have a significant defect in UV light avoidance behavior at all times of the day compared with control flies and prefer the UV environment over the shaded (hk^−/−, n = 77 vs. control, all P < 0.05). (E) Mutant flies lacking all opsin-based external photoreceptors (gl^60j) show significantly less UV avoidance compared with control flies only during the midday, ZT1-6 (gl^60j, n = 76 vs. control, all P < 0.05). (F) Average percent activity in UV-exposed environment during the day vs. night. cry^−/− and hk^−/− flies have significantly higher activity in the UV-exposed environment during the day compared with control flies (P < 0.05). Daytime percent activity in UV-exposed environment of gl^60j flies does not significantly differ from control. Percent activities in UV-exposed environment for cry^−/−, hk^−/−, and gl^60j flies during the nighttime do not differ from control. *P < 0.05.

Fig. S5.

CRY-based phototransduction mediates Drosophila choice of light environment. Average activity count over ZT time in the UV-exposed environment (365 nm, 400 µW/cm²; violet bars) vs. the shaded environment (black bars) is shown. The UV light was on during the daytime (ZT0–12) and off during the nighttime (ZT12–24; shaded gray on the graphs). (A) Control flies prefer the shaded environment over the UV-exposed one during the midday (n = 76). (B and C) cry^−/− flies (n = 78) (B) and hk^−/− flies (C) both lack the preference for shaded environment in the midday and prefer the UV-exposed environment at other times of the day (n = 77). (D) gl^60j flies choose the shaded environment over the UV-exposed one during the midday (n = 76), similar to control flies. *P < 0.05; **P < 0.01; ***P < 0.001.