Imaging chromosome dynamics in live larvae

Chromatin dynamics constitute a fundamental component of genome regulation and cell function36. In order to visualize and quantify chromosome dynamics in live zebrafish, the zebrafish telomeric repeat binding factor a (terfa) was cloned, and the telomere marker EGFP-Terfa was expressed in zebrafish neurons, resulting in a nucleus-specific punctum pattern (Fig. 1a–e). To verify that EGFP-Terfa marks chromosomes, the human telomere marker uas:dsRED-TRF137 was co-injected with either uas:EGFP-Terfa or the DNA binding-site-deleted construct uas:EGFP-Terfa del into one-cell-stage tg(HuC:Gal4) embryos. While zebrafish and human telomeric markers co-localized (Fig. 1f–i), deletion of the Terfa DNA binding site resulted in non-specific protein aggregates in the nucleus (Fig. 1j–l). To further validate that the puncta mark chromosomes, the zebrafish centromere protein a (cenpa) was cloned, and the EGFP-Cenpa was used as a centromere marker. Two-color imaging showed that EGFP-Cenpa and dsRED-TRF1 puncta were expressed adjacently on the chromosome, but not co-localized, as expected from telomeric and centromeric markers (Fig. 1m–p). To continuously image chromosome dynamics in all neurons of live fish, a stable tg(uas:EGFP-Terfa) transgenic line was generated and crossed with tg(HuC:Gal4) zebrafish (Fig. 1q, r). Neurons in the telencephalon (Te), rhombencephalon (Rh), spinal cord (SC), and habenula (Hb), of 6-day post-fertilization (dpf) larvae were imaged during 9.5 min. Single-particle tracking (SPT) analysis38 was used to detect and quantify the motility of telomere trajectories (Supplementary Movie 1, Fig. 1s, t). While telomeres in the Rh, SC, and Hb neurons showed a similar volume of motion of 0.0069 ± 0.0002, 0.0066 ± 0.0003, and 0.0063 ± 0.0004 µm3, respectively, telomeres in Te neurons showed increased volume of motion (0.01 ± 0.0004 µm3, Supplementary Fig. 1a). Calculation of the mean square displacement (MSD) for single trajectories of all Te and Rh neurons demonstrated anomalous subdiffusion of the puncta (Fig. 1u), which is typical to chromosome diffusion38. Indeed, analysis of both telomeric and centromeric markers that are expressed in the same SC neurons (Fig. 1m–p, Supplementary Movie 2) showed similar dynamics (Supplementary Fig. 1b), verifying that the puncta mark chromosomes. Altogether, these results transfer in vitro chromatin experiments to whole organisms, and demonstrate the capability of monitoring chromosome dynamics in live larvae.

Fig. 1 Imaging single chromosome dynamics in live larvae. a The DNA constructs were used to express telomere, centromere, and truncated telomere markers. b–e One-plane view of a representative neuron that expresses cellular tagRFP (magenta) and nuclear EGFP-Terfa (yellow) telomeric markers in live larvae. f–i Co-localization of zebrafish (f) and human (g) telomeric markers in the nucleus of a zebrafish neuron. j–l Co-expression of truncated zebrafish (j) and human (k) telomeric markers. m–p Co-expression of centromere (m) and telomere (n) markers. Dorsal (q) and lateral (spinal cord, r) views of 6-dpf tg(HuC:Gal4)/tg(uas:EGFP-Terfa) larvae (Te telencephalon, Rh rhombencephalon, SC spinal cord). s Live time-lapse imaging of a single neuron nucleus shows the movement of chromosomes measured over 9.5 min. Dashed box shows high magnification of a single trajectory. t 3D single-particle tracking (SPT) shows the volume of motion of chromosomes in a single nucleus. u In all Te and Rh neurons, the time-averaged mean square displacement (MSD) for telomeres trajectory (Te: n = 340 chromosomes, Rh: n = 475 chromosomes). Lines represent the means, and the shaded area represents the standard deviation (SD). Scale bar = 1 µm Full size image

Sleep increases chromosome dynamics in neurons

In the diurnal zebrafish larvae, sleep is regulated by circadian and homeostatic processes and is defined as at least 1 min of immobility accompanied by an increased arousal threshold15,39. Continuous video tracking of behavior showed that, as is the case in WT larvae40, 6 dpf tg(HuC:Gal4)/tg(uas:EGFP-Terfa) larvae sleep more during the night, under a 14 h light/10 h dark to constant darkness (LD to DD) cycle (day: 8.6 ± 0.7 min/h; night: 28.05 ± 1.2 min/h; subjective day: 17.8 ± 1.01 min/h; Fig. 2a). Moreover, analysis of larval activity specifically during the wake bouts showed that the average time of activity is reduced during the night compared to the day (day: 5.6 ± 0.25 s/min; night: 1.6 ± 0.06 s/min, Supplementary Fig. 2). The behavioral immobility and reduced sensory input during sleep may favor behavioral-state-dependent cellular processes in neurons. Alterations in chromatin structure can affect a variety of nuclear processes, including genome stability, transcription, DNA repair, chromosome segregation, and condensation24,37. To test whether chromosome dynamics change between day and night, single nuclei were imaged in Te and Rh neurons of 6 dpf tg(HuC:Gal4)/tg(uas:EGFP-Terfa) larvae during the day [zeitgeber time 4 (ZT4)], night (ZT18), and the following subjective day (ZT4, Fig. 2a). Rh and Te neurons were selected because these regions regulate locomotor activity and cognition, and because these neurons exhibit standard and high levels of chromosome dynamics, respectively (Supplementary Fig. 1a). Remarkably, the time-lapse imaging of telomere markers showed that chromosome dynamics increased by approximately two-fold during nighttime sleep in both brain regions (average volume of motion, day: Rh—0.006 ± 0.0006 µm3, Te—0.01 ± 0.0008 µm3; night: Rh—0.012 ± 0.002 µm3, Te—0.023 ± 0.003 µm3; subjective day: Rh—0.006 ± 0.0005 µm3, Te—0.011 ± 0.001 µm3, Fig. 2b–e, Supplementary Movie 3, 4). Using centromeric markers, similar changes in chromosome dynamics were visualized in SC neurons (day: 0.0047 ± 0.0004 µm3; night: 0.0078 ± 0.0006 µm3, Fig. 2f, Supplementary Movie 2). In order to differentiate between sleep and circadian effect, the 6 dpf tg(HuC:Gal4)/tg(uas:EGFP-Terfa) larvae were sleep-deprived for 4 h during the night, and behavioral sleep rebound was observed during the following subjective day (ZT4; LD to DD: 12.4 ± 0.8 min/h; SD: 27.5 ± 1.3 min/h, Fig. 2a). Immediately following SD, chromosome dynamics were reduced by approximately two-fold (Rh—0.0062 ± 0.0005 µm3, Te—0.01 ± 0.0009 µm3, Fig. 2d, e) compared with the levels observed during the night in the control group, and were similar to the levels observed during the day. On the following day, after 10 h of recovery, when the sleep-deprived larvae demonstrated sleep rebound (Fig. 2a), chromosome dynamics increased by approximately two-fold (Rh—0.011 ± 0.0008 µm3, Te—0.02 ± 0.002 µm3, Fig. 2d, e) in the sleep-deprived larvae, and reached the levels observed during nighttime sleep in the sibling control larvae. These results show that sleep increases chromosome dynamics in a homeostatic-dependent manner.

Fig. 2 Sleep increases chromosome dynamics in neurons. a Recording of sleep was performed in 6 dpf control or sleep-deprived larvae under an 14 h light/10 h dark cycle following by constant darkness (LD to DD, control: n = 119 larvae, sleep deprivation (SD): n = 96 larvae). b, c The area scanned by all chromosomes from all imaged Te neurons during 9.5 min (day: n = 26 cells; night: n = 29 cells). Color was coded according to the levels of volume of motion. d–f Volume of chromosome dynamics over 9.5 min per cell (EGFP-Terfa in d and e, EGFP-Cenpa in f). d Te neurons, ctrl: day (n = 26 cells), night (n = 29 cells), subjective day (n = 25 cells). SD: day (n = 26 cells), night (n = 35 cells), subjective day (n = 25 cells). P = 1.3 × 10−7, F = 17.54, degrees of freedom = 2. e Rh neurons, ctrl: day (n = 23 cells), night (n = 30 cells), subjective day (n = 35 cells). SD: day (n = 27 cells), night (n = 25 cells), subjective day (n = 26 cells). P = 7 × 10−6, F = 12.82, degrees of freedom = 2, determined by two-way ANOVA followed by Tukey test. f SC neurons: day (n = 33 cells), night (n = 33 cells). *P = 0.0001, determined by two-tailed t-test: two samples assuming unequal variance. Red crosses indicate outliers. g, h Monitoring sleep and chromosome dynamics under melatonin (MT) treatment. Blue background represents time under treatment. g Pretreated and EtOH/MT-treated larvae (EtOH: n = 48; MT: n = 48 larvae). P = 7.4 × 10−4, F = 14.3, degrees of freedom = 3, determined by two-way ANOVA followed by Tukey test. Values are presented as means ± SEM. h Pre-treated cells (n = 22) and MT-treated cells (n = 25). *P = 7 × 10−6, determined by two-tailed t-test: two samples assuming unequal variance. i Te neurons, aanat2 +/+ : day (n = 16 cells), night (n = 21 cells). aanat2−/−: day (n = 14 cells), night (n = 32 cells). P = 1.7 × 10−3, F = 12.26, degrees of freedom = 1. j Rh neurons, aanat2+/+ : day (n = 24 cells), night (n = 24 cells). aanat2−/−: day (n = 19 cells), night (n = 30 cells). P = 7.6 × 10−4, F = 10.42, degrees of freedom = 1, determined by two-way ANOVA followed by Tukey test. Boxplots: black diamond represents the mean, boxes indicate the median and the 25th-to-75th percentiles, whiskers extend to the most extreme data points. Letters or asterisks indicate significant differences. ZT zeitgeber time Full size image

To further validate the regulation of chromosome dynamics by the sleep state, the tg(HuC:Gal4)/tg(uas:EGFP-Terfa) larvae were treated during the day with melatonin, which is a strong sleep-promoting hormone in the diurnal zebrafish14,41. Sleep time and chromosome dynamics were monitored prior to and during melatonin treatment. As expected, while ethanol (EtOH) administration did not affect sleep time, under 3 h of melatonin treatment, sleep increased in melatonin-treated larvae (prior to melatonin treatment: 7.7 ± 1.5 min/h; 3 h following melatonin treatment: 30.7 ± 2.4 min/h, Fig. 2g). In accordance, melatonin-derived sleep increases chromosome dynamics (prior to melatonin treatment: 0.006 ± 0.0005 µm3; during melatonin treatment: 0.013 ± 0.0013 µm3, Fig. 2h). Thus, sleep is sufficient to increase chromosome dynamics. In order to understand if sleep is not only sufficient but also necessary to increase chromosome dynamics, we crossed the tg(HuC:Gal4)/tg(uas:EGFP-Terfa) zebrafish with arylalkylamine N-acetyltransferase-242 mutant zebrafish (aanat2−/−), which lack melatonin signaling. The aanat2−/− larvae exhibit reduced sleep time during the night, although their intrinsic molecular circadian clock is intact43. Imaging single neurons during day and night revealed that while chromosome dynamics increased in aanat2+/+ larvae during the night (day: Rh—0.006 ± 0.0006 µm3, Te—0.01 ± 0.001 µm3; night: Rh—0.009 ± 0.0009 µm3, Te—0.014 ± 0.0008 µm3, Fig. 2i, j), as was the case in WT larvae (Fig. 2d, e), it was reduced during the night in aanat2−/− (night: Rh—0.006 ± 0.0006 µm3, Te—0.009 ± 0.0009 µm3) compared with aanat2+/+ larvae. Thus, although the molecular circadian clock is intact, chromosome dynamics were similar in both day and night, which is in accordance with the reduced nighttime sleep in aanat2−/− larvae (Fig. 2i, j). These results show that chromosome dynamics in neurons are regulated by the behavioral sleep/wake state.

Sleep-dependent changes in chromosome dynamics may not be specific to neurons. To test whether these changes are also present in other cell types, we monitored chromosome dynamics in peripheral endothelial and Schwann cells. Chromosome dynamics were imaged during day and night in endothelial and Schwann cells using tg(fli:Gal4)/tg(uas:EGFP-Terfa) and mbp:Gal4-injected tg(uas:EGFP-Terfa) 6 dpf larvae, respectively (Supplementary Fig. 3a, b). Chromosome dynamics did not differ between day and night in both cell types (Supplementary Fig. 3c). These results show that sleep-dependent changes in chromosome dynamics that were observed in neurons, do not occur in endothelial or Schwann cells.

Sleep reduces DSBs that are accumulated during wakefulness

What is the physiological benefit of sleep-dependent chromosome dynamics? Since the genome can be hit by dozens of DSBs per day33,44, we speculated that sleep and increased chromosome dynamics are essential for the recovery from wakefulness-derived DNA damage. To test our hypothesis, the levels of DSBs and chromosome dynamics were monitored in Rh neurons during the 24-h sleep/wake cycle. The γH2AX marker, which is activated as part of the DNA damage response system45, was used to quantify DSBs during day and night in single cells (Fig. 3a–e). Whole head staining showed increased localization of γH2AX in the Te compared to other brain areas, such as the Rh (Fig. 3a). This DSB enrichment is correlated with the increased neuronal activity detected in the Te (Supplementary Fig. 4). In the 24-h experiment, we quantified DSBs in the Rh because it better represents the distribution of γH2AX in the entire CNS. During the day, the number of DSBs consistently increased, and peaked 1 h before darkness (15.1 ± 0.46 γH2AX foci, Fig. 3d). During the night, the number of DSBs dramatically decreased, reached the minimum levels at ZT19 (5.4 ± 0.24 γH2AX foci, Fig. 3d), and remained low until the beginning of the day. Concurrently, during the day, chromosome dynamics remained at similar low levels (ZT1: 0.0063 ± 0.0007; ZT5: 0.0067 ± 0.0005; ZT9: 0.007 ± 0.0008; ZT13: 0.0065 ± 0.0005 µm3, Fig. 3d). In contrast, following 1 h of darkness, chromosome dynamics increased by two-fold, and the high levels were maintained during the entire night (ZT15: 0.014 ± 0.0016; ZT19: 0.012 ± 0.0018; ZT23: 0.01 ± 0.0011 µm3, Fig. 3d). These results show that while chromosome dynamics keep constant low levels, DSB number accumulates during the day. During the night, following robust increase in chromosome dynamics, the number of DSBs was gradually reduced, showing that chromosome dynamics correlate with efficient reduction of DSBs during the night.

Fig. 3 Chromosome dynamics are essential for reducing the number of accumulating DSBs. a–c Dorsal view of 6 dpf larvae stained with γH2AX. a Arrows indicate the telencephalon (Te) and rhombencephalon (Rh). Dashed box showing the Rh represents the area analyzed in d and e. Representative images from the Rh region during day (b) and night (c) are shown. Dashed circle indicates a single neuron. d The number of DSBs (P = 1 × 10−16, F = 118, degrees of freedom = 7) and chromosome dynamics (*P = 8 × 10−7, F = 7, degrees of freedom = 7) in single nuclei over 24 h, in 14 h light/10 h dark cycle. Determined by one-way ANOVA followed by a Tukey test. Lower case letters indicate significant changes between γH2AX groups. Asterisks indicate significant changes between chromosome dynamics groups. White and black horizontal bars represent light and dark periods, respectively. e Numbers of DSBs (dot plot, means ± SEM). Ctrl: day (n = 83 cells), night (n = 131 cells), subjective day (n = 105 cells). SD: day (n = 86 cells), night (n = 170 cells), subjective day (n = 135 cells). P = 1 × 10−6, F = 62.04, degrees of freedom = 2, determined by two-way ANOVA followed by a Tukey test. White, black, and gray rectangles represent day, night, and subjective day, respectively. Dotted white line represents the sleep deprivation (SD) period. f–h Co-expression of Lap2β-EGFP (f) and human telomeric markers (g) in SC-neuronal nucleus. i Volume of chromosome dynamics in control (Lap2β−, day: n = 25; night: n = 21 cells) and Lap2β-overexpressing cells (Lap2β+, day: n = 25; night: n = 19 cells). *P = 0.026, F = 5.1, degrees of freedom = 1, determined by two-way ANOVA followed by a Tukey test. j–l Representative images of double immunohistochemistry using α-γH2AX (magenta) and α-EGFP (green) in single SC neurons during day (j) and night (k, l). Dashed circle indicates a single nucleus. m The number of DSBs (dot plot, means ± SEM) in control (Lap2β−, day: n = 177; night: n = 177 cells) and Lap2β-overexpressing cells (Lap2β+, day: n = 17; night: n = 49 cells). *P = 1 × 10−6, F = 18.8, degrees of freedom = 1, determined by two-way ANOVA followed by a Tukey test. Zeitgeber time (ZT4)-day, ZT18-night. Boxplots: black diamond represents the mean, boxes indicate the median and the 25th-to-75th percentiles, whiskers extend to the most extreme data points. Letters or asterisks indicate significant differences. Scale bar = 1 µm Full size image

In order to distinguish between circadian and sleep effect on DSB levels, we quantified the number of DSBs in sleep-deprived larvae. In the control group, the number of γH2AX foci per cell in the Rh neurons decreased during the night and increased back to daytime levels on the following subjective day (day: 9.3 ± 0.61; night: 6.1 ± 0.36, subjective day: 10.16 ± 0.46, Fig. 3e). In contrast, following SD, the γH2AX foci number increased during the night (11.9 ± 0.37), and then, post-sleep recovery, was reduced on the following subjective day (6.76 ± 0.44, Fig. 3e). These experiments show that DSBs increased during wakefulness and decreased during sleep in neurons.

In order to test whether the amount of DSBs changed between day and night in other cell types, we performed immunostaining assays using both anti-γH2AX and anti-EGFP in tg(fli:EGFP) and tg(mbp:EGFP) 6 dpf larvae (Supplementary Fig. 3d-i). In contrast to the day/night changes observed in neurons, the number of DSBs was constantly low during day and night in endothelial and Schwann cells (Supplementary Fig. 3j), suggesting that wakefulness and sleep do not affect the levels of DSBs in these cells.

Chromosome dynamics are necessary to reduce DSBs

In order to causally link chromosome dynamics and the efficient reduction in DNA damage, we manipulated chromosome dynamics by overexpressing the zebrafish Lamina-associated polypeptide 2 (Lap2β) in specific neurons. This protein physically interacts with lamins and anchors chromatin to the nuclear lamina in mammals and zebrafish46,47. In order to monitor chromosome dynamics and DSBs in Lap2β-overexpressing and control neurons, pT2-uas:Lap2β-EGFP and pT2-uas:dsRED-TRF1 constructs were co-injected into tg(HuC:Gal4) embryos, and neurons that express either both Lap2β-EGFP and dsRED-TRF1 (Fig. 3f–h) or only dsRED-TRF1 in the SC were analyzed during day and night in 6 dpf larvae. As expected, chromosome dynamics increased during the night in the control dsRED-TRF1-expressing neurons (day: 0.006 ± 0.0004; night: 0.009 ± 0.001 µm3, Fig. 3i). In contrast, overexpression of Lap2β inhibited chromosome dynamics, specifically during the night, and the levels were similar during both day and night (day: 0.005 ± 0.0005; night: 0.005 ± 0.0007 µm3, Fig. 3i). Thus, the overexpression of Lap2β impedes the sleep-dependent increase of chromosome dynamics. Next, we measured the number of γH2AX foci in the control and in Lap2β-overexpressing neurons. As was found in the Rh (Fig. 3b–e), the number of γH2AX foci per neuron in the SC decreased by approximately 30% during the night (day: 5.1 ± 0.18; night: 3.5 ± 0.14, Fig. 3j, k, m). Remarkably, the number of γH2AX foci increased by 120% in Lap2β-overexpressing neurons compared with the control neurons during the night (7.95 ± 0.63 γH2AX foci, Fig. 3l, m). These results show that genetic inhibition of chromosome dynamics increases the number of DSBs specifically during nighttime sleep. Furthermore, these results suggest that sleep-dependent increase in chromosome dynamics is necessary to reduce DNA damage.

Neuronal activity can reduce chromosome dynamics

Which cellular processes induce DSBs during wakefulness, and how do they affect chromosome dynamics? In mammals, neuronal activity promotes the formation of DSBs34,35. In zebrafish, we found sleep-dependent changes in the levels of chromosome dynamics and DSBs in neurons (Figs. 2 and 3) but not in two other non-excitable cell types (Supplementary Fig. 3), and showed correlation between increased spontaneous neuronal activity and enriched expression of γH2AX in the Te (Fig. 3a, Supplementary Fig. 4). Therefore, since chromosome dynamics is required to reduce the number of DSBs we rationalized that the intensity and frequency of neuronal activity should affect chromosome dynamics within individual neurons. To study activity-dependent chromosome dynamics, a tg(uas:RCaMP1b) transgenic fish was generated and crossed with the tg(HuC:Gal4)/tg(uas:EGFP-Terfa) line. Then, spontaneous chromosome dynamics and neuronal activity were simultaneously imaged in one plane in single neurons within the Rh during day and night (Fig. 4a–c). The results showed moderate negative correlation between neuronal activity and chromosome dynamics (Fig. 4d–g). While the average frequency of single cell activity was reduced (day: 0.138 ± 0.022 Hz; night: 0.079 ± 0.015 Hz, Fig. 4d–f), the average chromosome dynamics increased during the night in Rh neurons (day: 0.02 ± 0.002 µm2; night: 0.027 ± 0.002 µm2, Fig. 4e, g). Taking into account that neurons in the Rh and SC regulate tail movement and locomotor activity48, which are markedly reduced during sleep, these results show that, at the single-cell level, chromosome dynamics increased in resting cells during the night.

Fig. 4 Neuronal excitation reduces chromosome dynamics in the Rh and SC. a–c Simultaneous 2D imaging of neuronal activity (magenta) and chromosome dynamics (green) in the Rh neurons. d Raster plot of ΔF/F of 27 and 26 RCaMP1b-expressing single cells during day and night, respectively. Grayscale: ΔF/F amplitude. e A moderate negative correlation between chromosome dynamics and neuronal activity during day and night (R Day = −0.62, R Night = −0.64, R Total = −0.64), determined by Pearson correlation coefficient. The average single-cell activity (f, *P activity = 0.035) and chromosome dynamics (g, *P dynamics = 0.03) during day and night. Determined by two-tailed t-test: two samples assuming unequal variance. h–l Optogenetic stimulation of neuronal activity reduces chromosome dynamics. h–j The neurons expressed both dsRED-TRF1 and ChR2-YFP (arrow) or only dsRED-TRF1 (arrowhead). k, l Chromosome dynamics before and following the light stimuli (ChR2−, n = 9 cells; ChR2+, n = 10 cells). *P = 0.031, determined by two-tailed t-test: two-paired samples for means. Average change is marked by red line. m–o Inhibition of neuronal activity during the night does not affect chromosome dynamics. m Representative raster plots of ΔF/F in the Rh of 6 dpf tg(HuC:GCaMP5) larvae under DMSO (n = 6 larvae) or BAPTA-AM (n = 6 larvae) treatments. Grayscale: ΔF/F amplitude. Bottom: histogram of the Ca2+ transients of all cells. n Average spontaneous neuronal activity in single neurons of the Rh under either DMSO (n = 6 larvae) or BAPTA-AM (n = 6 larvae) treatment (ZT23). *P = 2 × 10−4, t = 2.3, degrees of freedom = 7, determined by two-tailed t-test: two samples assuming unequal variance. o Volume of chromosome dynamics during nighttime (ZT23) in DMSO (n = 13 cells) and BAPTA-AM (n = 11 cells)-treated larvae. Values are presented as boxplots and means (black diamonds) or as dot plots and means ± SEM. Boxplots indicate the median and the 25th-to-75th percentiles. The whiskers extend to the most extreme data points. ZT zeitgeber time. Scale bar = 2 µm Full size image

In order to causally test the effect of neuronal activity on chromosome dynamics, we used optogenetics and transiently expressed the neural-activating cation channel channelrhodopsin-2 (ChR2-YFP)49 and the telomere marker dsRED-TRF1 in neurons of the SC (Fig. 4h–j), which showed similar chromosome dynamics and spontaneous neuronal activity as in Rh neurons (Supplementary Fig. 1a, Supplementary Fig. 4). Chromosome dynamics were quantified in single cells that did or did not express the ChR2-YFP before and following blue light stimulation during daytime wakefulness (Fig. 4h–j). While chromosome dynamics did not change post-stimulation in dsRED-TRF1-expressing cells (Fig. 4k), they were reduced in stimulated dsRED-TRF1+ChR2-YFP-expressing cells (pre-stimulation: 0.0073 ± 0.001 µm3; post-stimulation: 0.005 ± 0.0007 µm3, Fig. 4l). Thus, neuronal activity reduces chromosome dynamics in a single active cell under spontaneous conditions and optogenetic manipulation.

Neuronal activity could affect chromosome dynamics via the formation of DSBs. In order to uncouple neuronal activity from DNA damage, neuronal activity was inhibited specifically during the night, when the DSB level is low. The Ca+2 chelator BAPTA-AM, which lowers intracellular Ca+2 levels, was used. To validate the efficiency of BAPTA-AM in Rh neurons, we quantified spontaneous neuronal activity in BAPTA-AM-treated and control 6 dpf tg(HuC:GCaMP5) larvae. As expected, the average activity of a single Rh neuron was reduced by two-fold in BAPTA-AM-treated larvae compared with the control group (DMSO: 0.08 ± 0.002 Hz; BAPTA-AM: 0.04 ± 0.004 Hz, Fig. 4m, n). In these neurons, chromosome dynamics were monitored at the end of the night (ZT23), when the DNA damage is low (Fig. 3d). Inhibition of neuronal activity did not affect chromosome dynamics, which remain high in both BAPTA-AM-treated and control larvae (DMSO—0.0084 ± 0.001, BAPTA-AM—0.008 ± 0.001 µm3, Fig. 4o). These results indicate that neuronal activity is not an essential regulator of chromosome dynamics; however, it can reduce chromosome dynamics, possibly via the induction of DSBs.

Sleep and chromosome dynamics increased following induction of DSBs

Neuronal activity is only one of several processes that can induce DSBs during wakefulness. The causes for DNA damage are diverse and include intrinsic and extrinsic factors33. In order to simulate the effect of these different factors, we used etoposide (ETO), which induces DSBs45, and the number of γH2AX foci, chromosome dynamics, and sleep time were monitored during the day (Fig. 5a–d). As expected, after 2 h under ETO treatment (ZT2–4), the γH2AX foci number increased (DMSO: 10.44 ± 0.37; ETO: 14.01 ± 0.47, Fig. 5b). Furthermore, sleep time (Fig. 5c) and chromosome dynamics (Fig. 5d) did not change under ETO treatment. However, 1 h following ETO withdrawal, sleep time increased in ETO-treated larvae (DMSO: 6.95 ± 1.1 min/h; ETO: 13.28 ± 1.8 min/h, Fig. 5c), while DSB levels remained high (DMSO: 10.87 ± 0.35; ETO: 13.7 ± 0.46 γH2AX foci, Fig. 5b) and chromosome dynamics remained low (DMSO: 0.0065 ± 0.0004; ETO: 0.0094 ± 0.001 µm3, Fig. 5d). Notably, following 2 h of recovery from the ETO treatment, sleep time remained high (DMSO: 6.71 ± 1 min/h; ETO: 11.4 ± 1.8 min/h, Fig. 5c), and chromosome dynamics increased by approximately two-fold (DMSO: 0.0062 ± 0.0005; ETO: 0.015 ± 0.0017 µm3, Fig. 5d), accompanied by a reduction of the number of DSBs (9.65 ± 0.42 γH2AX foci, Fig. 5b). In order to examine how the formation of DSBs will affect sleep time and chromosome dynamics during the night, we exposed the larvae to ETO for 2 h (ZT16–18). Similar to the results obtained during the day, under ETO treatment, the γH2AX foci number increased by 60% (DMSO: 5.4 ± 0.33; ETO: 8.66 ± 0.4, Fig. 5e) and sleep time did not change (Fig. 5f). However, chromosome dynamics decreased by approximately two-fold (DMSO: 0.0126 ± 0.003; ETO: 0.0049 ± 0.0004 µm3, Fig. 5g). These results suggest that while chromosome dynamics are low during the formation of DSBs, the accumulation of DSBs during wakefulness triggers sleep, which increases chromosome dynamics and eventually reduces the number of DSBs.