Daytime restriction of feeding prevents illuminated night-induced impairment of metabolism and sleep in diurnal zebra finches.

We investigated whether nocturnal eating was causal to the impairment of metabolism and sleep disruption in diurnal animals exposed to illuminated nights. Adult zebra finches hatched and raised in 12 h light: 12 h darkness (LD) were exposed to 5-lux dim light at night (dLAN, two groups), with a control group maintained on LD. For the next 3 weeks, the food availability to one of the dLAN groups was restricted to the 12 h light period (dLAN -F); the other dLAN (dLAN +F) and LD groups were continued on ad lib feeding. In spite of similar food intakes, dLAN +F condition led to the fat accumulation and weight gain. These birds showed concurrent changes in hepatic expression of genes associated with carbohydrate and lipid metabolism, suggesting an enhanced gluconeogenesis and impaired fatty acids synthesis. Increased sirt1 mRNA levels indicated the activation of molecular mechanisms to counter-balance the metabolic damage under dLAN +F. Furthermore, reduced bout length and total duration of the nocturnal sleep suggested a poorer sleep in dLAN +F condition. Negative sleep effects of dLAN were supported by the lower hypothalamic expression of sleep promoting sik3 and camkii genes, and higher mRNA expression of awake promoting achm3 gene in dLAN +F, compared to the LD condition. Importantly, dLAN-induced negative effects in metabolism and sleep were alleviated in the dLAN -F group. These results suggest the role of timed feeding in alleviating the negative impact of illuminated nights in metabolism and sleep in diurnal zebra finches.

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.

Effects of timed food availability on reproduction and metabolism in zebra finches: Molecular insights into homeostatic adaptation to food-restriction in diurnal vertebrates.

Food availability affects metabolism and reproduction in higher vertebrates including birds. This study tested the idea of adaptive homeostasis to time-restricted feeding (TRF) in diurnal zebra finches by using multiple (behavioral, physiological and molecular) assays. Adult birds were subjected for 1 week or 3 weeks to food restriction for 4 h in the evening (hour 8-12) of the 12 h light-on period, with controls on ad lib feeding. Birds on TRF showed enhanced exploratory behavior and plasma triglycerides levels, but did not show differences from ad lib birds in the overall food intake, body mass, and plasma corticosterone and thyroxine levels. As compared to ad lib feeding, testis size and circulation testosterone were reduced after first but not after third week of TRF. The concomitant change in the mRNA expression of metabolic and reproductive genes was also found after week 1 of TRF. Particularly, TRF birds showed increased expression of genes coding for gonadotropin releasing hormone (GnRH) in hypothalamus, and for receptors of androgen (AR) and estrogen (ER-alpha) in both hypothalamus and testes. However, genes coding for the deiodinases (Dio2, Dio3) and gonadotropin inhibiting hormone (GnIH) showed no difference between feeding conditions in both hypothalamus and testes. Further, increased Sirt1, Fgf10 and Ppar-alpha, and decreased Egr1 expression in the liver suggested TRF-effects on the overall metabolism. Importantly, TRF-effects on gene expressions by week 1 seemed alleviated to a considerable extent by week 3. These results on TRF-induced reproductive and metabolic effects suggest homeostatic adaptation to food-restriction in diurnal vertebrates.

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 brain peptides associated with reproduction and energy homeostasis: Putative roles of gonadotrophin-releasing hormone-II and tyrosine hydroxylase in determining reproductive performance in response to daily food availability times in diurnal zebra finches.

Previous studies have demonstrated 'quality-quantity' trade-offs with daily food availability times in zebra finches. Compared with food access ad lib., zebra finch pairs with restricted food access for 4 hours in the morning produced poor quality offspring, whereas those with the same food access in the evening produced fewer but better quality offspring. The present study investigated whether food-time-dependent differential effects on reproductive performance involved brain peptides associated with reproduction and energy homeostasis in zebra finches. We measured peptide/protein expression of gonadotrophin-releasing hormone (GnRH)-I, GnRH-II, gonadotrophin-inhibitory hormone (GnIH), tyrosine hydroxylase (TH), neuropeptide Y (NPY), cocaine- and amphetamine regulated transcript (CART) and ZENK (a neuronal activation marker) by immunohistochemistry and mRNA expression of genes coding for the type 2 (DIO2) and type 3 (DIO3) deiodinase by a quantitative polymerase chain reaction in male and female zebra finches that were paired and kept under a 12:12 hour light/dark photocycle at 24 ± 2°C temperature for > 12 months with access to food ad lib., or for only 4 hours in the morning or evening. In both sexes, GnRH-I, DIO2 and DIO3 expression did not differ significantly between the three feeding conditions, although levels showed an overall food effect. However, in males, GnIH expression was significantly higher in evening-fed birds compared to ad lib. fed birds. Interestingly, GnRH-II and TH levels were significantly lower in restricted feeding compared to the ad lib. group and, importantly, GnRH-II and TH-immunoreactivity levels were negatively and positively correlated with egg laying latency and reproductive success (offspring/brood/pair), respectively. At the same time, we found no effect on the hypothalamic expression of orexigenic (NPY) and anorexigenic (CART) peptides, or ZENK protein (ie, the neuronal activity marker). These results suggest the involvement of reproductive neuropeptides, with putative roles for GnRH-II and TH, in the food-time-dependent effect on reproductive performance, albeit with subtle sex differences, in diurnal zebra finches, which possess the ability to reproduce year-round, in a manner similar to other continuously breeding vertebrates.

Developmental effects of daily food availability times on song behaviour and neuronal plasticity of song-control system in male zebra finches.

Food availability is a major ecological factor and affects body condition and sexual traits. Here, we investigated whether males' song behaviour, a trait for female mate choice, was sensitive to the food availability period and its timing in songbirds. We manipulated daily food availability to 4 h in the morning or evening, with controls on food ad libitum, and assessed its effects on song behaviour and forebrain song control system in male zebra finches that were held as adult (parent) or offspring (since birth) at 24 ± 2 °C under 12 h daily photoperiod. Food restriction significantly affected both temporal and spectral features of daily song in offspring, not the parent. In offspring, we found reduced mesor (mean 24-h levels), attenuated amplitude (daily maxima relative to mesor) and altered acrophase (estimated time of daily maxima) of 24-h rhythm, and reduced motif length (in morning-fed), per motif unique syllables and an enhanced song pitch (in evening-fed). There was also a positive correlation of motif length with cheek patch and plasma testosterone levels, and of per motif syllables with cheek patch and daily activity levels in offspring. Among main song controlling forebrain nuclei, LMAN and HVC were reduced in size, and Area X and HVC showed decreased neuronal recruitment in offspring on food restrictions. These results demonstrate the importance of daily food availability and its timing in determining males' sexual signals, and support growing evidence that among vertebrates well-fed males contain reproductive traits that females use for its mate choice.

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.