Daytime light spectrum affects photoperiodic induction of vernal response in obligate spring migrants.

It is not well understood how the spectral composition (wavelength) of daylight that varies considerably during the day and seasons affects photoperiodic responses in a seasonal species. Here, we investigated the molecular underpinnings of wavelength-dependent photoperiodic induction in migratory redheaded buntings transferred to 13 h long days in neutral (white), 460 nm (blue), 500 nm (green) or 620 nm (red) wavelength that were compared with one another, and to short day controls for indices of the migratory (body fattening and weight gain, and Zugunruhe) and reproductive (testicular maturation) responses. Buntings showed wavelength-dependent photoperiodic response, with delayed Zugunruhe and slower testis maturation under 620 nm red light. Post-mortem comparison of gene expressions further revealed wavelength-dependence of the photoperiodic molecular response. Whereas there were higher retinal expressions of opn2 (rhodopsin) and opn5 (neuropsin) genes in red daylight, and of rhodopsin-like opsin (rh2) gene in green daylight, the hypothalamic opn2 mRNA levels were higher in blue daylight. Similarly, we found in birds under blue daylight an increased hypothalamic expression of genes involved in the photoperiodic induction (thyroid stimulating hormone subunit beta, tshb; eye absent 3, eya3; deiodinase type 2, dio2) and associated neural responses such as the calcium signaling (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, atp2a2), dopamine biosynthesis (tyrosine hydroxylase, th) and neurogenesis (brain-derived neurotrophic factor, bdnf). These results demonstrate transcriptional changes in parallel to responses associated with migration and reproduction in buntings, and suggest a role of daylight spectrum in photoperiodic induction of the vernal response in obligate spring avian migrants.

Melatonin and curcumin reestablish disturbed circadian gene expressions and restore locomotion ability and eclosion behavior in Drosophila model of Huntington's disease.

Deficit in locomotion (motor) ability and disturbance of the circadian behavior and sleep-wake pattern characterize Huntington's disease (HD). Here, we examined the disturbance of circadian timing with the progression of HD pathogenesis, and tested the efficacy of melatonin and curcumin in preventing the motor deficit and disturbed eclosion behavior in the Drosophila model of HD. To examine circadian timing, we assayed mRNA expression of genes of the transcriptional feedback (TF) loop that generates the near 24-h rhythmicity. We performed qPCR of the Period, Timeless, Clock, Cycle, Clockwork, and Cryptochrome genes in transgenic fly heads from elav-Gal4 (pan neuronal) and PDF-Gal4 (PDF-specific neurons) driver lines through the progression of HD disease post-eclosion, from day 1 to its terminal stage on day 13. Cycle was arrhythmic from day 1, but Period and Timeless became arrhythmic on day 13 of the HD pathogenesis in elav, but not PDF, neurons. Twenty-four-hour mRNA rhythms showed alteration in the waveform properties (mesor and amplitude, not acrophase), but not in the persistence, in both elav-Gal4 and PDF-Gal4 HD flies; however, disturbance of the clock gene rhythm was delayed in PDF-Gal4 flies. To assess the preventive effects on HD pathogenesis, flies of both driver lines were provided with melatonin (50, 100, or 150 µg) or curcumin (10 µM) in the diet commencing from the larval stage. Both melatonin (100 µg) and curcumin reestablished the 24-h pattern in mRNA expression of Period and Timeless to normal (control) levels, and significantly improved both locomotion ability and eclosion behavior of HD flies. We suggest that the disturbance of circadian timekeeping progressively accelerated HD pathogenesis, possibly via modulation of the transcriptional state that resulted in the modification of the Huntington gene. These findings suggest melatonin and curcumin might be potential therapeutic agents for the treatment of HD in humans, although this needs specific investigation.

Light at night affects gut microbial community and negatively impacts host physiology in diurnal animals: Evidence from captive zebra finches.

The gastrointestinal tract (GIT) hosts a large number of diverse microorganisms, with mutualistic interactions with the host. Here, in two separate experiments, we investigated whether light at night (LAN) would affect GIT microbiota and, in turn, the host physiology in diurnal zebra finches (Taeniopygia guttata). Experiment I assessed the effects of no-night (LL) and dimly illuminated night (dim light at night, dLAN) on fecal microbiota diversity and host physiology of birds born and raised under 12 h photoperiod (LD; 12 h light: 12 h darkness). Under LL and dLAN, compared to LD, we found a significant increase in the body mass, subcutaneous fat deposition and hepatic accumulation of lipids. Although we found no difference in total 24 h food consumption, LL/ dLAN birds ate also at night, suggesting LAN-induced alteration in daily feeding times. Concurrently, there were marked differences in amplicon sequence and bacterial species richness between LD and LAN, with notable decline in Lactobacillus richness in birds under LL and dLAN. We attributed declined Lactobacillus population as causal (at least partially) to negative effects on the host metabolism. Therefore, in experiment II with similar protocol, birds under LL and dLAN were fed on diet with or without Lactobacillus rhamnosus GG (LGG) supplement. Clearly, LGG supplement ameliorated LL- and dLAN-induced negative effects in zebra finches. These results demonstrate adverse effects of unnatural lighting on GIT bacterial diversity and host physiology, and suggest the role of GIT microbiota in the maintenance of metabolic homeostasis in response to LAN environment in diurnal animals.

Developmental effects of constant light on circadian behaviour and gene expressions in zebra finches: Insights into mechanisms of metabolic adaptation to aperiodic environment in diurnal animals.

A most crucial feature of biological adaptation is the maintenance of a close temporal relationship of behaviour and physiology with prevailing 24-h light-dark environment, which is rapidly changing with increasing nighttime illumination. This study investigated developmental effects of the loss of night on circadian behaviour, metabolism and gene expressions in diurnal zebra finches born and raised under LL, with controls on 12L:12D. Birds under LD were entrained, and showed normal body mass and a significant 24-h rhythm in both activity-rest pattern and mRNA expression of candidate genes that we measured. But, under LL, birds gained weight and accumulated lipid in the liver. Intriguingly, at the end of the experiment, the majority (4/5th) of birds under LL were rhythmic in activity despite arrhythmic expression in the hypothalamus of c-Fos (neuronal activity), Rhodopsin and Mel1-a genes (light perception), and clock genes (Bmal1, Per2 and Rev-erb ß). In peripheral tissues, LL induced variable clock gene expressions. Whereas 24-h mRNA rhythm was abolished for Bmal1 in both liver and gut, it persisted for Per2 and Rev-erb ß in liver, and for Per2 in gut. Further, we found under LL, the loss of 24-h rhythm in hepatic expression of Fasn and Cd36/Fat (biosynthesis and its uptake), and gut expression of Sglt1, Glut5, Cd36 and Pept1 (nutrient absorption) genes. As compared to LD, baseline mRNA levels of Fasn and Cd36 genes were attenuated under LL. Among major transporter genes, Sglt1 (glucose) and Cd36 (fat) genes were arrhythmic, while Glut5 (glucose) and Pept1 (protein) genes were rhythmic but with phase differences under LL, compared to LD. These results demonstrate dissociation of circadian behaviour from clock gene rhythms, and provide molecular insights into possible mechanisms at different levels (behaviour and physiology) that diurnal animals might employ in order to adapt to an emerging overly illuminated-night urban environment.

Sleep in unnatural times: illuminated night negatively affects sleep and associated hypothalamic gene expressions in diurnal zebra finches.

We investigated the effects of exposure at ecologically relevant levels of dim light at night (dLAN) on sleep and the 24 h hypothalamic expression pattern of genes involved in the circadian timing (per2, bmal1, reverb-ß, cry1, ror-α, clock) and sleep regulatory pathways (cytokines: tlr4, tnf-α, il-1ß, nos; Ca2+-dependent pathway: camk2, sik3, nr3a; cholinergic receptor, achm3) in diurnal female zebra finches. Birds were exposed to 12 h light (150 lux) coupled with 12 h of absolute darkness or of 5 lux dim light for three weeks. dLAN fragmented the nocturnal sleep in reduced bouts, and caused sleep loss as evidenced by reduced plasma oxalate levels. Under dLAN, the 24 h rhythm of per2, but not bmal1 or reverb-ß, showed a reduced amplitude and altered peak expression time; however, clock, ror-α and cry1 expressions showed an abolition of the 24 h rhythm. Decreased tlr4, il-1ß and nos, and the lack of diurnal difference in achm3 messenger RNA levels suggested an attenuated inhibition of the arousal system (hence, awake state promotion) under dLAN. Similarly, changes in camk2, sik3 and nr3a expressions suggested dLAN-effects on Ca2+-dependent sleep-inducing pathways. These results demonstrate dLAN-induced negative effects on sleep and associated hypothalamic molecular pathways, and provide insights into health risks of illuminated night exposures to diurnal animals.

Changes in the expression of genes involved in DNA methylation and histone modification in response to daily food availability times in zebra finches: epigenetic implications.

We hypothesised that daily food availability times serve as an 'epigenetic' factor and affect reproductive physiology in continuously reproducing species. This we tested by measuring mRNA expression of genes coding for enzymes involved in DNA methylation-demethylation (dnmt, tet) and histone modification (hat1, hdac) in the hypothalamus, liver and gonads of male and female zebra finches that were paired for a year under 12 h light:12 h dark conditions with food availability restricted to 4 h in the morning (morning FA group) or evening (evening FA group), with controls provided with food ad libitum The overall hypothalamic and hepatic expression patterns of hat1 and hdac were similar but those of dnmt and tet were different between males and females. Irrespective of the timing of food availability, both hat1 and hdac mRNA levels were increased in the hypothalamus, but not in the liver, in which hat1 mRNA levels were increased in the morning FA group. While hypothalamic tet levels were higher in evening FA males, hepatic tet levels were higher in morning FA birds (tet1, only males). Gonadal expression levels similarly varied and showed sex differences. Histone-modifying genes did not show food availability effects, except for elevated testicular hdac3 levels. Similarly, testicular dnmt3b and tet2 mRNA levels were increased and decreased in morning and evening FA groups, respectively, whereas ovarian dnmt1 and tet2 levels were reduced in morning FA and tet1 levels were reduced in evening FA groups. The present results suggest that an enforced daily feeding schedule in the long term could serve as a conditioning environment that shapes overall hypothalamic regulation, and liver and gonadal function at the epigenetic level in diurnal vertebrates.

Illuminated night alters behaviour and negatively affects physiology and metabolism in diurnal zebra finches.

Light at night (LAN) negatively impacts the behaviour and physiology; however, very little is known about molecular correlates of LAN-induced effects in diurnal animals. Here, we assessed LAN-induced effects on behaviour and physiology, and examined molecular changes in the liver of diurnal zebra finches (Taeniopygia guttata). Birds were exposed to dim LAN (dLAN: 12L = 150 lux: 12D = 5 lux), with controls on 12L (150 lux): 12D (0 lux). dLAN altered daily activity-rest and eating patterns, induced nocturnal eating and caused body fattening and weight gain, and reduced nocturnal melatonin levels. Concomitant increased nighttime glucose levels, decreased daytime thyroxine and triglycerides levels, and hepatic lipid accumulation suggested the impairment of metabolism under dLAN. Transcriptional assays evidenced dLAN-induced negative effects on metabolism in the liver, the site of metabolic homeostasis. Particularly, increased g6pc and foxo1 mRNA expressions suggested an enhanced gluconeogenesis, while increased egr1 and star expressions suggested enhanced cholesterol biosynthesis and lipid metabolism, respectively. Similarly, overexpressed sirt1 indicated protection from the metabolic damage due to elevated gluconeogenesis and cholesterol biosynthesis under dLAN. However, no effect on genes involved in lipogenesis (fasn) and insulin signalling pathway (socs3 and insig1) might indicate for the post transcriptional/post translational modification effects or the involvement of other genetic pathways in LAN-induced effects. We also found daily rhythm in the hepatic expression of selected clock and clock-controlled genes (per2, bmal1 and reverb-beta), with an elevated mesor and amplitude of per2 oscillation, suggesting a role of per2 in the liver metabolism. These results demonstrate dLAN-induced negative effects on the behaviour and physiology, and provide molecular insights into metabolic risks of the exposure to illuminated nights to diurnal animals including humans in an urban setting.