Light triggers a network switch between circadian morning and evening oscillators controlling behaviour during daily temperature cycles

Abstract

Proper timing of rhythmic locomotor behavior is the consequence of integrating environmental conditions and internal time dictated by the circadian clock. Rhythmic environmental input like daily light and temperature changes (called Zeitgeber) reset the molecular clock and entrain it to the environmental time zone the organism lives in. Furthermore, depending on the absolute temperature or light intensity, flies exhibit their main locomotor activity at different times of day, i.e., environmental input not only entrains the circadian clock but also determines the phase of a certain behavior. To understand how the brain clock can distinguish between (or integrate) an entraining Zeitgeber and environmental effects on activity phase, we attempted to entrain the clock with a Zeitgeber different from the environmental input used for phasing the behavior. 150 clock neurons in the Drosophila melanogaster brain control different aspects of the daily activity rhythms and are organized in various clusters. During regular 12 h light: 12 h dark cycles at constant mild temperature (LD 25°C, LD being the Zeitgeber), so called morning oscillator (MO) neurons control the increase of locomotor activity just before lights-on, while evening oscillator (EO) neurons regulate the activity increase at the end of the day, a few hours before lights-off. Here, using 12 h: 12 h 25°C:16°C temperature cycles as Zeitgeber, we attempted to look at the impact of light on phasing locomotor behavior. While in constant light and 25°C:16°C temperature cycles (LLTC), flies show an unimodal locomotor activity peak in the evening, during the same temperature cycle, but in the absence of light (DDTC), the phase of the activity peak is shifted to the morning. Here, we show that the EO is necessary for synchronized behavior in LLTC but not for entraining the molecular clock of the other clock neuronal groups, while the MO controls synchronized morning activity in DDTC. Interestingly, our data suggest that the influence of the EO on the synchronization increases depending on the length of the photoperiod (constant light vs 12 h of light). Hence, our results show that effects of different environmental cues on clock entrainment and activity phase can be separated, allowing to decipher their integration by the circadian clock.

Fig 1. MO and EO circadian clocks are required for synchronization to temperature cycles in constant light.

Male flies were synchronized to LD 25°C for two days, before being exposed to LL 25°C for three days, followed by temperature cycles in LL (LLTC 25°C:16°C), which were delayed by 5-h compared to the initial LD cycle. A) Double plotted average actograms of one representative experiment. N: UAS-cyc^DN/+ = 19, Mai179>cyc^DN = 21, cry[19]>cyc^DN = 22. Conditions are indicated to the left. White areas indicate lights on and 25°C, grey areas lights off and 25°C during LD, and blue areas lights-on and 16°C during LLTC. Cartoon on the right shows clock neurons expressing Mai179 and cry[19] drivers. B-C) Median of normalized locomotor activity during the last day of LD (left) and the 6^th day of LLTC (right). White bars represent lights-on, black bar lights off in LD (left), yellow and blue bars indicate thermophase (25°C) and cryophase (16°C) in LL, respectively (right). N: UAS-cyc^DN/+ = 59, Mai179 > cyc^DN = 55, Mai179-Gal4/+ = 59. C) N: UAS-cyc^DN/+ = 36, cry[19] > cyc^DN = 44, cry[19]-Gal4/+ = 47. D-E) Box plots showing the slope of the evening peak on the last day of LD (D) and the 6^th day of LLTC (E). The slope is calculated as follows: (Act_max−Act_ZTmin) / (ZT_max−ZT_min), with ZT_min being the last time point of the minimum median value and ZT_max the first time point with the maximum value before startle response to Zeitgeber change. N: UAS-cyc^DN/+ = 95 (B+C). For other genotypes see B-C. D) UAS-cyc^DN/+: ZT_min8.5, cry[19]-Gal4/+: ZT_min9.5, Mai179>cyc^DN, Mai179-Gal4/+, cry[19]>cyc^DN: ZT_min9. E) UAS-cyc^DN/+ and cry[19]>cyc^DN: ZT_min7, Mai179>cyc^DN: ZT_min7.5, Mai179-Gal4/+: ZT_min6.5, cry[19]-Gal4/+: ZT_min5.5. UAS-cyc^DN/+: ZT_max11.5, Mai179>cyc^DN: ZT_max11.5 and cry[19]>cyc^DN: ZT_max11, Mai179-Gal4/+: ZT_max10, and cry[19]-Gal4/+: ZT_max9. Statistical test: Kruskal wallis [57]. *: p<0.05, **:p<0.005, ***:p<0.001. In the box plots, the lowest line indicates the first interquartile, the central line the median, the upper line the third interquartile, the cross the average, and the whiskers indicate the minimum and maximum, except for outliers.

Fig 2. Blocking clock function in Mai179-Gal4 cells does not prevent temperature synchronization of PER expression in other clock neurons.

PER immunostaining on the 6^th day of LLTC6. A) PER signals in the LNd at ZT0 and ZT12 in controls (UAS-cyc^DN/+) and Mai179 >cyc^DN brains. Scale bar: 10μm. B) Average relative PER levels in the LNd. Only the 3 LNd (presumably CRY^-) were visible and quantified in Mai179 >cyc^DN brains, while all 6 LNd were quantified in the controls. C, D) Average relative PER levels in the other Lateral (C) and Dorsal Neurons (D).

Fig 3. Circadian clock disruption in EO neurons prevents synchronized behavior in temperature cycles during constant light.

A) Immunostaining of a Mai179, Pdf-Gal80>cyc^DN,GFP brain at ZT2 in LD (25°C). GFP: green; PER: red; PDF: blue. Left panel: LNd. The * marks a non-LNd GFP⁺ cell. Scale bar: 10μm. Cartoon depicts clock neurons expressing cyc^DN in Mai179, Pdf-Gal80 flies. B) Average actograms of one representative experiment. N: UAS-cyc^DN,Pdf-Gal80/+ = 14, Mai179, Pdf-Gal80>cyc^DN = 31, Mai179-Gal4/+ = 16. C) Median of the normalized locomotor activity during the last day of LD (left) and the 6^th day of LLTC (right). N: UAS-cyc^DN,Pdf-Gal80/+ = 34, Mai179, Pdf-Gal80>cyc^DN = 48, Mai179-Gal4/+ = 32. D) Box plots showing the slope of the evening peak on the last day of LD (left) and the 6^th day of LLTC (right). Same flies as in (C). For all the genotypes in LD ZT_min = 9.5. In LLTC, UAS-cyc^DN,Pdf-Gal80/+ and Mai179, Pdf-Gal80>cyc^DN: ZT_min7.5 and Mai179-Gal4/+: ZT_min6. UAS-cyc^DN,Pdf-Gal80/+: ZT_max11.5, Mai179, Pdf-Gal80>cyc^DN: ZT_max10.5, and Mai179-GAL4/+: ZT_max9.5.

Fig 4. EO neurons are required for behavioral synchronization to temperature cycles in constant light.

A) Double plotted average actograms of the indicated genotypes. N: UAS-hid,rpr/Y; ls-tim) and cry-Gal4[19]/+ = 20, cry[19]>hid,rpr = 18, UAS-hid,rpr/Y;s-tim/ls-tim = 20, spE-GAL4/+ = 19, spE > hid,rpr = 19. Cartoons indicate clock neurons that remain after hid,rpr-induced ablation. B) Median of normalized locomotor activity during day six of LLTC. Left panel: same flies as in A. N for right panel: UAS-hid,rpr/Y, s-tim/ls-tim = 59, spE>hid,rpr = 55; spE-Gal4/+ = 61. C) Box plots showing the slope of the evening peak on the 6^th day of LLTC. Same flies as in B. C) ZT_min for all the genotypes is ZT9.5, except for spE>hid,rpr (ZT_min = 10). For left group UAS-hid,rpr/+ and cry-Gal4[19]/+: ZT_min4.5, and ZT_min2.5 for cry[19]>hid,rpr. For right group UAS-hid,rpr/+ and spE>hid,rpr: ZT_min5, and spE-Gal4/+: ZT_min7.5. For left group UAS-hid,rpr/+: ZT_max7.5, and cry[19]>hid,rpr and cry[19]-GAL4/+: ZT_max7. For right group ZT8.5 for UAS-hid,rpr/+ and spE>hid,rpr: ZT_max8.5, and spE-Gal4/+: ZT_max11. D) Percentage of flies anticipating (blue) and not anticipating (orange) lights-off in LD (left) or the temperature decrease (right). Same flies as in B.

Fig 5. Clock function in the MO and EO neurons is not required for synchronization to temperature cycles in constant darkness (DDTC).

A) Double plotted average actograms of the indicated genotypes. N: UAS-cyc^DN/+ = 25, cry[19]>cyc^DN = 30; UAS-cyc^DN,pdf-Gal80/+ = 21; cry[19],pdf-Gal80 > cyc^DN = 25. Conditions are indicated to the left. White areas indicate lights on and 25°C, orange areas lights off and 25°C, blue area light off and 16°C. Cartoons to the right indicate clock neurons expressing cyc^DN in the respective genotypes. B-C) Median of normalized locomotor activity during the 6^th day of DDTC (B), and the first two DD days in constant conditions (C). Same flies as in A. The dark grey bars represent the subjective night of DD1, the light grey bars subjective day of DD2.

Fig 6. Environmental cues act as Zeitgeber and set behavioral activity Phase.

The molecular clock in brain clock neurons is entrained by light and temperature. However, these two environmental inputs vary on a day to day basis (e.g., cloudy versus sunny days), and animals change their activity at different times of day in response to the ambient environmental condition. How does the circadian system distinguish between an input that entrains the circadian clock (Zeitgeber) and one that after integration by the clock system, sets the daily activity phase? Here, we demonstrate that the EO determines the behavioral evening peak (Φ) in the presence of light and 25°C, while the circadian clock is entrained with a 25°C:16°C temperature cycle. In contrast, the MO determines the morning peak in constant darkness and 25°C, during the same temperature cycle. We present a model explaining how the environment and the circadian clock shape locomotor activity. On one hand, the on/up and off/down environmental changes that happen once a day entrain the molecular clocks in the system (purple arrow). On the other hand, different environmental input such as light (quality and intensity) and temperature (different levels), here represented by green arrows, are perceived by different oscillators in different manners. Depending on the time of day (the molecular clock status of the system), this will lead to a modification of the network balance and a dominancy of one or several oscillators (blue clocks vs grey ones), resulting in a behavioral activity phase (Φ) according to the internal timing and the current condition. Therefore, to biologically demonstrate this model, we fixed a Zeitgeber (in this study TC 25°C-16°C) and tested how light (here presence/absence) modifies the balance. From this basis, we can now apply more subtle modifications such as the intensity or the quality of light to change the phase of the behavior and use this to understand the principles underlying neuronal network switches.