Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

Constant light and pinealectomy disrupt daily rhythm in song production and negatively impact reproductive performance in zebra finches.

We assessed the circadian clock control of singing and reproductive performance in zebra finches. Experiment 1 examined changes in body mass, testis size, and plasma corticosterone and testosterone levels in male birds exposed to constant light (LL, 100 lx) and constant darkness (DD, 0.5 lx), with controls on 12L:12D (L = 100 lx, D = 0.5 lx). There was a significant increase in the body mass and testis size under LL and a decrease in testis size under the DD. Using a similar design, experiment 2 assessed the persistence of the circadian rhythm in singing along with activity-rest pattern in cohort I birds that were entrained to 12L:12D and subsequently released in DD or LL, and in cohort II birds that were entrained to 12L:12D and following pinealectomy were released in DD. Both activity and singing patterns were synchronized with the light phase under 12L:12D, free-ran with a circadian period under DD, and were arrhythmic under the LL. There was an overall decreased and increased effect on singing under DD and LL, respectively, albeit with differences in various song parameters. The pinealectomy disrupted both activity and singing rhythms but did not affect singing or the overall song features. Pinealectomized bird pairs also exhibited a significant reduction in their nest-building and breeding efforts, resulting in a compromised reproductive performance. These results suggest a circadian clock control of singing and more importantly demonstrate a role of the pineal clock in breeding behaviors, leading to a compromised reproductive performance in diurnal zebra finches.

Born without night: the consequence of the no-night environment on reproductive performance in diurnal zebra finches.

We investigated the consequence of no-night environment (constant light, LL) on reproductive performance in zebra finches in the parent (P) and subsequent (F1) generation. As a measure of the overall effects on metabolic reproductive health, we monitored daily activity behaviour, recorded song and cheek patch size in males, and measured body size and hormone levels. As compared with controls under 12 h light:12 h darkness (12 h:12 h LD), both P and F1 pairs showed a compromised reproductive success, as evidenced by fewer fledglings and fewer viable offspring with longer fledging durations, and increased offspring mortality with three successive clutches under LL. The overall negative effect of the no-night environment was increased in the F1 generation. As compared with P pairs, F1 pairs had more failed nesting and breeding attempts, took longer to initiate reproduction, incubated fewer eggs, produced fewer viable offspring with longer fledging duration, and showed increased offspring mortality. Consistent with negative reproductive effects, P males showed significant changes in the motif duration and other spectral features of song, and both F1 and F2 males copied poorly the song of their parent under LL. Plasma corticosterone and sex hormone (testosterone in males and oestradiol in females) levels were significantly lower under LL. Daily plasma melatonin rhythm persisted but with a reduced amplitude under LL. These results demonstrate the importance of night in reproduction in a continuously breeding diurnal species, and give insight into the possible impact on physiology of animals whose surrounding environment is consistently losing the darkness of night.

Illuminated night alters hippocampal gene expressions and induces depressive-like responses in diurnal corvids.

Artificial light at night induces circadian disruptions and causes cognitive impairment and mood disorders; yet very little is known about the neural and molecular correlates of these effects in diurnal animals. We manipulated the night environment and examined cellular and molecular changes in hippocampus, the brain region involved in cognition and mood, of Indian house crows (Corvus splendens) exposed to 12 hr light (150 lux): 12 hr darkness (0 lux). Diurnal corvids are an ideal model species with cognitive abilities at par with mammals. Dim light (6 lux) at night (dLAN) altered daily activity:rest pattern, reduced sleep, and induced depressive-like responses (decreased eating and self-grooming, self-mutilation, and reduced novel object exploration); return to an absolute dark night reversed these negative effects. dLAN suppressed nocturnal melatonin levels; however, diurnal corticosterone levels were unaffected. Concomitant reduction of immunoreactivity for DCX and BDNF suggested dLAN-induced suppression of hippocampal neurogenesis and compromised neuronal health. dLAN also negatively influenced hippocampal expression of genes associated with depressive-like responses (bdnf, il-1ß, tnfr1, nr4a2), but not of those associated with neuronal plasticity (egr1, creb, syngap, syn2, grin2a, grin2b), cellular oxidative stress (gst, sod3, cat1) and neuronal death (caspase2, caspase3, foxo3). Furthermore, we envisaged the role of BDNF and showed epigenetic modification of bdnf gene by decreased histone H3 acetylation and increased hdac4 expression under dLAN. These results demonstrate transcriptional and epigenetic bases of dLAN-induced negative effects in diurnal crows, and provide insights into the risks of exposure to illuminated nights to animals including humans in an urban setting.

Constant light environment suppresses maturation and reduces complexity of new born neuron processes in the hippocampus and caudal nidopallium of a diurnal corvid: Implication for impairment of the learning and cognitive performance.

Periodic day-night environment shapes the temporal pattern in the behaviour and physiology (e.g. 24-h activity-rest and sleep-wake cycles) and the advanced brain function, such as learning, memory and decision making. In a previous study, we showed the abolition of 24-h rhythm in the activity-rest pattern, and an attenuated cognitive performance in diurnal Indian house crows (Corvus splendens) under constant light (no-night; LL) environment. Present study extended this, and investigated LL-induced effects on the neurogenesis (birth, maturation and neurite complexity of new born neurons) in the hippocampus and caudal nidopallium, the brain regions directly associated with learning and cognition in birds. We performed immunohistochemistry of doublecortin (DCX; a neurogenesis marker) and tyrosine hydroxylase (TH, a key enzyme of the dopamine biosynthesis) in the brain section containing hippocampus or caudal nidopallium of Indian house crows exposed for 2 weeks to LL, with controls maintained under 12L:12D. As expected, crows showed arrhythmicity with a significantly reduced rest period in the 24-h activity-rest pattern, and a decreased cognitive performance when tested for the spatial and pattern association learning tasks under LL. Importantly, there was a significant decrease in DCX-immunoreactive (ir) cells and, as shown by Sholl analysis, in the complexity of DCX-ir neurites in both, the hippocampus and caudal nidopallium of crows under LL, as compared to those under 12L:12D. The anatomical proximity of DCX-ir neurons with TH-ir fibers suggested a functional association of the new born hippocampal and caudal nidopallial neurons with the learning, and perhaps cognition in Indian house crows. These results give insights into possible impact of the loss of night on brain health and functions in an emerging ecosystem in which other diurnal species including humans may be inadvertently exposed to an illuminated night, such as in an overly lighted metropolitan urban habitat.

A single incidental dark pulse during daytime attenuated food anticipatory behavior.

Using an open-source operant feeding device (FED3), we measured food-seeking nose poking behavior in mice. When the mice were exposed to 4 h restricted feeding at night, all mice exhibited robust food anticipatory nose poking starting ~4 h before scheduled mealtime. When the light-dark cycle was advanced by 6 h, mice exhibited two distinct bouts of anticipatory poking, one corresponding to actual mealtime which continued at the same time of day, and one corresponding to predicted mealtime which shifted parallel with the light-dark cycle. Likewise, two similar bouts of food-seeking behavior appeared when the light-dark cycle was delayed for 9 h. These data suggest that food anticipatory behavior is encoded to a circadian oscillator that entrains to the light-dark cycle. Two weeks after advancing the light-dark cycle, mice incidentally received a 3.5 h dark pulse in the middle of the day. This single dark pulse had a negligible effect on running wheel behavior but caused a temporary attenuation of both food anticipatory poking and pellet intake. These results suggest that the circadian oscillator controlling food anticipatory poking is sensitive to light disruption and that proper food anticipation is critical for sufficient food intake during temporally restricted feeding.

Protocol to study circadian food-anticipatory poking in mice using the feeding experimentation device version 3.

Food-anticipatory nose poking is a unique food-seeking behavior driven by the food-entrainable oscillator. Here, we present a protocol to record a novel food-seeking nose poking behavior in mice under temporally restricted feeding followed by food deprivation using the open-source feeding experimentation device version 3 (FED3). We describe steps for setting up the FED3 and cage, training, and habituation. We then detail procedures for setting up the schedule for time-restricted feeding and food deprivation and for generating ethograms from FED3 data. For complete details on the use and execution of this protocol, please refer to Ehichioya et al.1.

Administration of blue light in the morning and no blue-ray light in the evening improves the circadian functions of non-24-hour shift workers.

In modern 24-hour society, various round-the-clock services have entailed shift work, resulting in non-24-hour schedules. However, the extent of behavioral and physiological alterations by non-24-hour schedules remains unclear, and particularly, effective interventions to restore the circadian functions of non-24-hour shift workers are rarely explored. In this study, we investigate the effects of a simulated non-24-hour military shift work schedule on daily rhythms and sleep, and establish an intervention measure to restore the circadian functions of non-24-hour shift workers. The three stages of experiments were conducted. The stage-one experiment was to establish a comprehensive evaluation index of the circadian rhythms and sleep for all 60 participants by analyzing wristwatch-recorded physiological parameters and sleep. The stage-two experiment evaluated the effects of an intervention strategy on physiological rhythms and sleep. The stage-three experiment was to examine the participants' physiological and behavioral disturbances under the simulated non-24-hour military shift work schedule and their improvements by the optimal lighting apparatus. We found that wristwatch-recorded physiological parameters display robust rhythmicity, and the phases of systolic blood pressures and heart rates can be used as reliable estimators for the human body time. The simulated non-24-hour military shift work schedule significantly disrupts the daily rhythms of oxygen saturation levels, blood pressures, heart rates, and reduces sleep quality. Administration of blue light in the morning and no blue-ray light in the evening improves the amplitude and synchronization of daily rhythms of the non-24-hour participants. These findings demonstrate the harmful consequences of the non-24-hour shift work schedule and provide a non-invasive strategy to improve the well-being and work efficiency of the non-24-hour shift population.

A time memory engram embedded in a light-entrainable circadian clock.

A longstanding mystery in chronobiology is the location and molecular mechanism of the food-entrainable oscillator (FEO).1,2,3 The FEO is an enigmatic circadian pacemaker that controls food anticipatory activity (FAA). The FEO is implicated as a circadian oscillator that entrains to feeding time. However, the rhythmic properties of the FEO remain a mystery in part due to technical limitations in distinguishing FAA from locomotor activity controlled by the primary circadian pacemaker in the suprachiasmatic nucleus (SCN). To overcome this limitation, we used the Feeding Experimentation Device version 3 (FED3) to measure food-seeking, nose-poking behavior. When food availability was limited to 4 h at night, mice exhibited strong anticipatory nose-poking behavior prior to mealtime. When food availability was moved to the daytime, mice quickly expressed daytime anticipatory nose pokes without displaying transients. Unexpectedly, the mice also maintained nighttime anticipatory nose pokes, even though food pellets were no longer dispensed at night. We next tested if food anticipation was directly encoded on a light-entrainable oscillator by shifting the light-dark cycle without changing mealtime. Anticipatory behavior shifted in parallel with the light-dark cycle, although meal timing was unchanged. Next, we tested whether encoding meal timing for anticipatory nose pokes required a functional SCN by studying Period 1/2/3 triple knockout mice with disabled SCN. Food anticipatory nose poking of Period knockout mice shifted in parallel with the light-dark cycle independent of a functional SCN clock. Our data suggest that food anticipation time is embedded in a novel, extra-SCN light-entrainable oscillator.

Gut microbiota depletion minimally affects the daily voluntary wheel running activity and food anticipatory activity in female and male C57BL/6J mice.

Emerging evidence has highlighted that the gut microbiota plays a critical role in the regulation of various aspects of mammalian physiology and behavior, including circadian rhythms. Circadian rhythms are fundamental behavioral and physiological processes that are governed by circadian pacemakers in the brain. Since mice are nocturnal, voluntary wheel running activity mostly occurs at night. This nocturnal wheel-running activity is driven by the primary circadian pacemaker located in the suprachiasmatic nucleus (SCN). Food anticipatory activity (FAA) is the increased bout of locomotor activity that precedes the scheduled short duration of a daily meal. FAA is controlled by the food-entrainable oscillator (FEO) located outside of the SCN. Several studies have shown that germ-free mice and mice with gut microbiota depletion altered those circadian behavioral rhythms. Therefore, this study was designed to test if the gut microbiota is involved in voluntary wheel running activity and FAA expression. To deplete gut microbiota, C57BL/6J wildtype mice were administered an antibiotic cocktail via their drinking water throughout the experiment. The effect of antibiotic cocktail treatment on wheel running activity rhythm in both female and male mice was not detectable with the sample size in our current study. Then mice were exposed to timed restricted feeding during the day. Both female and male mice treated with antibiotics exhibited normal FAA which was comparable with the FAA observed in the control group. Those results suggest that gut microbiota depletion has minimum effect on both circadian behavioral rhythms controlled by the SCN and FEO respectively. Our result contradicts recently published studies that reported significantly higher FAA levels in germ-free mice compared to their control counterparts and gut microbiota depletion significantly reduced voluntary activity by 50%.

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro or in cells that overexpress protein, the physiological relevance of LLPS is unclear. PERIOD proteins are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Here we show that when transgene was stably expressed, PER2 formed nuclear phosphorylation-dependent LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins is a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian studies.

Genetics and functional significance of the understudied methamphetamine sensitive circadian oscillator (MASCO).

The last 50 years have witnessed extraordinary discoveries in the field of circadian rhythms. However, there are still several mysteries that remain. One of these chronobiological mysteries is the circadian rhythm that is revealed by administration of stimulant drugs to rodents. Herein we describe the discovery of this circadian rhythm and its underlying oscillator, which is frequently called the methamphetamine-sensitive circadian oscillator, or MASCO. This oscillator is distinct from canonical circadian oscillators because it controls robust activity rhythms independently of the suprachiasmatic nucleus and circadian genes are not essential for its timekeeping. We discuss these fundamental properties of MASCO and integrate studies of strain, sex, and circadian gene mutations on MASCO. The anatomical loci of MASCO are not known, so it has not been possible thus far to discover its novel molecular timekeeping mechanism or its functional significance. However, studies in mutant mice suggest that genetic approaches can be used to identify the neural network involved in the rhythm generation of MASCO. We also discuss parallels between human and rodent studies that support our working hypothesis that a function of MASCO may be to regulate sleep-wake cycles.

Dissociation of circadian activity and singing behavior from gene expression rhythms in the hypothalamus, song control nuclei and cerebellum in diurnal zebra finches.

Under periodic day-night environment, most circadian functions maintain a close phase relationship relative to each other, suggesting a common circadian pacemaker control of different overt rhythms. In birds, this seems highly unlikely, given multioscillatory nature of the circadian pacemaker and downstream generation of several circadian behaviors. We hypothesized the dissociation of overt rhythms from circadian gene oscillations, if the two were loosely coupled, under an aperiodic light condition. We tested this in daily rhythms in singing, activity and clock gene expressions in adult male zebra finches (Taeniopygia guttata) that were born and raised under the constant light (LL; 24L:0D), with controls on an LD cycle (12L: 12D). Particularly, we monitored daily pattern of singing and activity behavior, and measured 24 h mRNA expression of immediate early gene (c-Fos), clock genes (Bmal1, Per2 and Rev-erb ß) and epigenetic marker genes (Dnmt3b and Tet2) in the hypothalamus, and of clock genes and genes coding for the aromatase (Arom), androgen receptor (Ar) and dopamine receptor (Drd2) in the song control nuclei (Area X and HVC) and cerebellum (motor control region). We found persistence of daily rhythms in activity and singing in all birds under LD, but in only 70% (14/20) birds under LL; thus, both behaviors were arrhythmic in 30% (6/20) birds) under LL. The overall song quality was also declined under LL. The clock genes showed daily rhythms in the hypothalamus, song control nuclei (except Per2 in Area X) and cerebellum under LD, although with differences in peak expression times; however, there was loss of rhythmicity in clock genes (except Bmal1 in Area X and HVC) under LL. We also found daily Ar mRNA rhythm in the Area X and cerebellum under LD. These results demonstrate for the first time the persistence of clock gene oscillations in the song control brain regions and show the dissociation of circadian behavior from genetic oscillations in relation to an imposed light environment.

Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions in response to cognitive performance in Indian house crows exposed to abrupt light environment.

Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment.