Bright daylight produces negative effects on affective and cognitive outcomes in nocturnal rats.

The daily light/dark cycle affects animals' learning, memory, and cognition. Exposure to insufficient daylight illumination negatively impacts emotion and cognition, leading to seasonal affective disorder characterized by depression, anxiety, low motivation, and cognitive impairment in diurnal animals. However, how this affects memory, learning, and cognition in nocturnal rodents is largely unknown. Here, we studied the effect of daytime light illuminance on memory, learning, cognition, and expression of mRNA levels in the hippocampus, thalamus, and cortex, the higher-order learning centers. Two experiments were performed. In experiment one, rats were exposed to 12 L:12D (12 h light and 12 h dark) with a 10, 100, or 1000 lx daytime light illuminance. After 30 days, various behavioral tests (novel object recognition test, hole board test, elevated plus maze test, radial arm maze, and passive avoidance test) were performed. In experiment 2, rats since birth were raised either under constant bright light (250 lx; LL) or a daily light-dark cycle (12 L:12D). After four months, behavioral tests (novel object recognition test, hole board test, elevated plus maze test, radial arm maze, passive avoidance test, Morris water maze, and Y-maze tests) were performed. At the end of experiments, rats were sampled, and mRNA expression of Brain-Derived Neurotrophic Factor (Bdnf), Tyrosine kinase (Trk), microRNA132 (miR132), Neurogranin (Ng), Growth Associated Protein 43 (Gap-43), cAMP Response Element-Binding Protein (Crebp), Glycogen synthase kinase-3ß (Gsk3ß), and Tumour necrosis factor-α (Tnf-α) were measured in the hippocampus, cortex, and thalamus of individual rats. Our results show that exposure to bright daylight (100 and 1000 lx; experiment 1) or constant light (experiment 2) compromises memory, learning, and cognition. Suppressed expression levels of these mRNA were also observed in the hypothalamus, cortex, and thalamus. These results suggest that light affects differently to different groups of animals.

Restraint stress-induced effects on learning, memory, cognition, and expression of transcripts in different brain regions of mice.

BACKGROUND: Stress is one of the prevalent factors influencing cognition. Several studies examined the effect of mild or chronic stress on cognition. However, most of these studies are limited to a few behavioral tests or the expression of selected RNA/proteins markers in a selected brain region. METHODS: This study examined the effect of restraint stress on learning, memory, cognition, and expression of transcripts in key learning centers. Male mice were divided into three groups (n = 6/group)-control group, stress group (adult stressed group; S), and F1 group (parental stressed group). Stress group mice were subjected to physical restraint stress for 2 h before light offset for 2 weeks. The F1 group comprised adult male mice born of stressed parents. All animals were subjected to different tests and were sacrificed at the end. Transcription levels of Brain-Derived Neurotrophic Factor (Bdnf), Tyrosine kinase (TrkB), Growth Associated Protein 43 (Gap-43), Neurogranin (Ng), cAMP Response Element-Binding Protein (Creb), Glycogen synthase kinase-3ß (Gsk3ß), Interleukine-1 (IL-1) and Tumour necrosis factor-α (Tnf-α) were studied. RESULTS: Results show that both adult and parental stress negatively affect learning, memory and cognition, as reflected by taking longer time to achieve the task or showing reduced exploratory behavior. Expression of Bdnf, TrkB, Gsk3ß and Ng was downregulated, while IL-1 and Tnf-α were upregulated in the brain's cortex, thalamus, and hippocampus region of stressed mice. These effects seem to be relatively less severe in the offspring of stressed parents. CONCLUSIONS: The findings suggest that physical restraint stress can alter learning, memory, cognition, and expression of transcripts in key learning centers of brain.

Seasonality in tropical birds.

The survival of offspring depends on environmental conditions. Many organisms have evolved with seasonality, characterized as initiation-termination-reinitiation of several physiological processes (i.e., body fattening, molt, plumage coloration, reproduction, etc). It is an adaptation for the survival of many species. Predominantly seasonal breeders use photoperiod as the most reliable environmental cue to adapt to seasonal changes but supplementary factors like temperature and food are synergistically involved in seasonal processes. Studies from diverse vertebrate systems have contributed to understanding the mechanism involved in seasonal reproduction at the molecular and endocrine levels. Long-day induced thyrotropin (thyroid-stimulating hormone) released from the pars tuberalis of the pituitary gland triggers local thyroid hormone activation within the mediobasal hypothalamus. This locally produced thyroid hormone, T3, regulates seasonal gonadotropin-releasing hormone secretion. Most of the bird species studied are seasonal in reproduction and linked behavior and, therefore, need to adjust reproductive decisions to environmental fluctuations. Reproductive strategies of the temperate zone breeders are well-documented, but less is known about tropical birds' reproduction and factors stimulating the annual breeding strategies. Here, we address seasonality in tropical birds with reference to seasonal reproduction and the various environmental factors influencing seasonal breeding.

Molecular expression of clock genes in central and peripheral tissues of white-rumped munia (Lonchura striata).

To synchronize with the fluctuating environment, organisms have evolved an endogenous time tracking mechanism referred to as the biological clock(s). This clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks have been identified in the hypothalamus, the pineal and the retina which interact and generate circadian time at a functional level. However, there is a limited knowledge of molecular clockwork and integration between central and peripheral clocks in birds. Therefore, we studied the daily expression of clock genes (Bmal1, Clock, Per2, Cry1, Npas2, Rev-Erbα, E4bp4, Pparα, Hlf and Tef) in three central circadian clocks (hypothalamus, pineal and retina), other brain areas (cerebellum, optic tectum and telencephalon) and in the peripheral tissues (liver, intestine, muscle and blood) of white-rumped munia. Adult birds were exposed to equinox photoperiod (12 L:12D) for 2 weeks and were then sampled (N = 5 per time point) at six-time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21). Daily expressions of clock genes were studied using qPCR. We observed daily variations and tissue-specific expression patterns for clock genes. These results are consistent with the autoregulatory circadian feedback loop proposed for the mammalian system and thus suggest a conserved tissue-level circadian time generation in white-rumped munia.

Molecular and epigenetic regulation of seasonal reproduction in Terai tree frog (Polypedates teraiensis).

Seasonal breeders predominantly use photoperiod as the predictable environmental cue to time their reproduction. Terai tree frogs are long-day seasonal breeders, but the molecular mechanism is unknown. We tested the role of different photoperiodic conditions on expression levels of candidate genes involved in seasonal reproduction and epigenetic regulation. Four experiments were performed. In experiment 1, frogs were exposed to long (LD: 16L:8D) or short photoperiod (SD: 8L:16D). In experiment 2, animals were procured at four different phases of breeding, i.e., during April (emergence just after hibernation), June (breeding phase), August (post-breeding), and October (just before hibernation). In experiments 3 and 4, frogs were exposed to equinox photoperiod but different (10, 100, or 500 lx) light intensities (exp. 3) or wavelength (red: 640 nm, green: 540 nm, blue: 450 nm or white; exp. 4). After 2 weeks, animals were euthanized, and their brain was harvested. mRNA levels of transcripts involved in photoperiodic transduction (Eya3 and Opn5), reproduction (Tshß, GnRH, Dio2, and Dio3), and epigenetics regulation (Dnmt1, Dnmt3a, Hdac1, Hdac3, and Tet2) were measured. Results show that LD promotes the upregulation of Eya3, Opn5, Tshß, GnRH, and Dio2. Differential expression of Opn5 during LD and SD suggests its involvement in light perception. Dio3 levels were upregulated in SD (exp.1) and during the post-breeding phase (exp. 2). These results employ the limited role of light intensity and spectrum in reproduction. This is the first study showing molecular machinery involved in the amphibian system's seasonal reproduction and epigenetic regulation.

Urban environment alter the timing of progression of testicular recrudescence in tree sparrow (Passer montanus).

Urbanization is a rapidly growing phenomenon that affects wildlife. Laboratory studies show the effects of night light on the physiology of the organisms. Limited studies have been conducted on birds in their natural habitat. Here, we studied the effects of the urban environment on reproduction-linked phenomenon and molecules involved in the regulation of seasonal breeding. Birds (N=5/time/site) were procured from urban and rural sites at specific times, i.e., in March (stimulatory phase), June (reproductive phase), September (refractory phase), and December (sensitive phase) of 2018. Immediately after procurement, birds were brought to the laboratory. Bodyweight, bill color, molt in body feathers, and testes size were recorded. The next day, all the birds were sacrificed in the middle of the day. Blood was collected and serum was used for ELISA of corticosterone, triiodothyronine (T3), and thyroxine (T4). mRNA levels of thyroid-stimulating hormone-ß (Tshß), type 2 deiodinase (Dio2), type 3 deiodinase (Dio3), gonadotropin-releasing hormone (GnRh), and gonadotropin inhibitory hormone (GnIh) were measured in hypothalamic tissue. Urban birds showed higher levels of corticosterone during the stimulatory phase. There was a delay in the initiation of testicular growth in urban birds and it was supported by reduced levels of T3 in blood plasma and relatively lower transcription of Dio2 and GnRH mRNA in urban birds. Our findings suggest that the urban environment delays the timing of reproduction in birds and could be the consequence of local environmental conditions.

High temperature attenuates testicular responses in tree sparrow (Passer montanus).

The majority of birds use environmental cues to time their reproduction. Photoperiod is the most dominant cue, but other environmental factors may play a critical role in successful reproduction. Some previous studies show the effect of temperature on the timing of nest building and reproduction. Here we tested if the temperature can modulate the reproductive responses of tree sparrows. Three experiments were performed on adult male birds. In experiment 1, birds (n = 5/group) were exposed to either high (30 ± 2 °C) or low temperature (20 ± 2 °C). Change in body mass, bill color, and testes volume was recorded every 30 days. In experiment 2, a similar temperature protocol was followed, but birds were euthanized after 30 days. In experiment 3, birds were first exposed to SD (8L:16D) for 30 days but either with high (30 ± 2 °C) or low temperature (20 ± 2 °C). After 30 days, birds were exposed to LD (14L:10D), but half of the high-temperature birds were moved to low temperature, and half of the low-temperature birds were moved to high temperature. After 30 days, all birds were euthanized. In experiment 2 and 3 immediately after euthanization birds, blood samples were collected, serum was used for hormone assay. mRNA levels of thyroid-stimulating hormone-ß (Tshß), type 2 deiodinase (Dio2), type 3 deiodinase (Dio3), gonadotropin-releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) were measured in hypothalamic tissue. Results from experiment 1 show that high temperature attenuates the testicular responses and accelerates the timing of regression. Experiment 2 shows that on day 30, testicular responses are similar, but reproductive genes express differentially in two groups. Experiment 3 shows that exposure to high temperatures during the photosensitive stage affects the testicular response at the poststimulatory state. Together, these findings suggest that high temperature modulates reproductive responses of tree sparrow.

Season dependent effects of urban environment on circadian clock of tree sparrow (Passer montanus).

Great efforts have been made recently to understand the effect(s) of urban environments on the circadian and seasonal physiology of wild animals, but the mechanisms involved remain largely unknown. Most laboratory studies and a few studies on animals in the wild suggest alterations occur in the physiological functions of organisms in urban habitats. Here, we addressed the effects of the interaction of seasons and urban environments on clock gene expression in three tissues of tree sparrows (Passer montanus). Tree sparrows (N = 30 per site per time of year) were procured from rural and urban habitats during periods corresponding to their three physiological states, i.e., June (longest photoperiod; reproductive phase), September (equinox photoperiod; refractory phase), and December (shortest photoperiod; sensitive phase). Birds (N = 5 per time per site per month) were sampled at six time points; ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0 = sunrise time) and clock gene expression in the hypothalamus, pineal gland, and retina was studied. Our results show that there is persistence of the circadian clock in both rural and urban birds throughout the year. In urban birds Bmal1, Npas2, Per2, and Cry1 acrophases were advanced, compared to rural birds, while Clock acrophase was delayed, depending on the tissue and time of year. This difference could be because of changes in the availability, duration, and intensity of sunlight during different times of the year and/or differential photoreceptor sensitivities, differential physiological states, or a combination of all these factors. These important results reveal, for the first time in any species, season-dependent effects of an urban environment on the molecular machinery of the circadian clock.

Hypothalamus but not liver retains daily expression of clock genes during hibernation in terai tree frog (Polypedates teraiensis).

Circadian clock(s) allow an organism to be in synchrony with the surrounding environment and perform daily and seasonal physiological processes, including hibernation, migration, and reproduction. To cope with adverse environmental conditions, organisms have evolved various strategies. Insects undergo diapause, while some higher animals either migrate or hibernate/aestivate during unfavorable environmental conditions. Hibernation is an energy conservation strategy used to cope with adverse environmental conditions. Limited knowledge is available on the physiology of hibernation in non-mammalian vertebrates. Some studies suggest that metabolism is altered during amphibian hibernation, but nothing is known about the circadian clock. In the present study, we investigated daily oscillation of clock genes in the brain and liver of the terai tree frog (Polypedates teraiensis) during two annual phases of life: breeding and hibernation. Adult male terai tree frogs were procured from their natural habitat on the Mizoram University campus (23°N 92°E) during their breeding and hibernation phases. Body mass and testes weight were recorded. Animals were sacrificed at six time points: ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0, zeitgeber time 0, indicates the sunrise time at the respective time of the year; N = 5 frogs per time point). Quantitative real-time polymerase chain reaction (qPCR) was performed for clock genes (Bmal1, Clock, Per2, and Cry2) in the hypothalamus and liver. Our results showed that body and testes weights decreased during hibernation. Further, the hypothalamus retained daily clock gene oscillations during breeding and hibernation. However, the liver lost this daily oscillation during hibernation. The maintained rhythm in hypothalamus in contrast to other hibernating animals might be the result of the fact that these animals hibernate at a higher temperature and might be more alert. As the animals have no food intake during their hibernation season which might be the reason the animals loose their rhythm in liver clock genes. These results suggest that retaining daily clock gene oscillations in the hypothalamic clock could be important for internal time tracking and post-hibernation emergence.

Effect of urban environment on pineal machinery and clock genes expression of tree sparrow (Passer montanus).

Increasing urbanisation is altering the physiology of wild animals and the mechanisms involved are largely unknown. We hypothesised that altering the physiology of urban organisms is due to the effect of extra light at night on the circadian clock by modulating the expression of pineal machinery and clock genes. Two experiments were performed. In Experiment 1, immediately after being procured from their respective sites (urban and rural sites), birds were released individually in LLdim light conditions. Circadian rhythm period, activity duration, and total activity count were calculated and did not differ between urban and rural birds. In Experiment 2, birds (from urban and rural habitats) were sampled at six time points at regular 4-h intervals, beginning 1 h after sunrise. We measured daily variations in plasma melatonin levels. We also analysed the expression levels of Aanat, Mel1A and Mel1B as an indicator of melatonin biosynthesis and action machinery. Clock and clock-controlled genes (Bmal1, Clock, Per2, Per3, Cry1 and Npas2) were studied in the hypothalamus, the pineal gland, and retina to investigate the effects of urban habitats on the circadian clock. Our results show that there is a lower expression of Aanat in the pineal gland and relatively low plasma melatonin levels in urban birds. Further, clock genes are also differentially expressed in all three central tissues of urban birds. We propose that alterations in the melatonin biosynthesis machinery and the expression of clock genes could result in miscalculations in the internal timing of the organism, with environmental timings leading to altered physiology in urban wild animals.

Isolation, characterization, and therapeutic activity of bergenin from marlberry (Ardisia colorata Roxb.) leaf on diabetic testicular complications in Wistar albino rats.

Bergenin is one of the phytochemical constituents in marlberry (Ardisia colorata Roxb.) having antioxidant, anti-diabetic, and anti-inflammatory properties. A. colorata has been used as an herbal medicine in Southeast Asia particularly in Northeast India to treat diabetes. Bergenin was isolated from methanol extract of A. colorata leaf (MEACL) by column chromatography and TLC profiling. Characterization and structural validation of bergenin were performed by spectroscopic analyses. A LC-ESI-MS/MS method was developed for the quantitation of bergenin and validated as per the guidelines of FDA and EMA. The validated method was successfully utilized to quantify bergenin concentration in MEACL samples. Therapeutic efficacy of bergenin was investigated on streptozotocin-induced diabetic rats by following standard protocols. Bergenin supplementation significantly improved the physiological and metabolic processes and in turn reverses diabetic testicular dysfunction via increasing serum testosterone concentrations and expression pattern of PCNA, improving histopathological and histomorphometric manifestations, modulating spermatogenic events and germ cell proliferation, restoring sperm quality, reducing sperm DNA damage, and balancing the antioxidant enzymes levels. Hence, A. colorata leaf is one of the alternate rich resources of bergenin and could be used as a therapeutic agent for diabetic testicular complications.

Daily expression of clock genes in central and peripheral tissues of tree sparrow (Passer montanus).

Almost all organisms live in a fluctuating environment. To achieve synchrony with the fluctuating environment, organisms have evolved with time-tracking mechanism commonly known as biological clocks. This circadian clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks reside within the nervous tissues in the hypothalamus, pineal and retina, which interact with each other and produce circadian time at a functional level. There is limited knowledge available of the molecular clockwork, and of integration between central and peripheral clocks in birds. Here, we studied daily expression of canonical clock genes (Bmal1, Clock, Per2, Per3, Cry1 and Cry2) and clock-controlled gene (Npas2) in all three central tissues (hypothalamus, pineal and retina) and in peripheral tissues (liver, intestine and muscle). Wild caught adult male tree sparrows were exposed to equinox photoperiod (12L:12D) for 2 weeks and after that birds were sacrificed (N = 5 per time point) at six time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21; ZT0 is lights on). Daily expression of clock genes was studied using qPCR. Bmal1, Clock, Per2, Per3, Cry1, Cry2 and Npas2 showed daily oscillation in all tissues except Cry2 in hypothalamus, pineal and intestine. We observed tissue-specific expression pattern for all clock and clock-controlled genes. Bmal1 transcripts expressed during early phase of night. Clock acrophase was observed during middle or late day time in the central clock while during early-to-middle phase of night in peripheral tissues. Npas2 expression pattern was similar to Bmal1. Per genes peaked either late at night or early during day time. However, Cry genes were peaked either at late day time (Cry1in retina, liver and intestine; Cry2 in liver and intestine) or at early night phase (Cry1 in hypothalamus, pineal and muscle; Cry2 in hypothalamus, pineal, retina and muscle). Our results are consistent with the autoregulatory circadian feedback loop, and suggest a conserved tissue-level circadian time generation in tree sparrows. Change in peak expression timing of these genes in different tissues implicates tissue-specific contribution of individual clock genes in the circadian time generation.

Seasonality in terai tree frog (Polypedates teraiensis): Role of light and temperature in regulation of seasonal breeding.

Seasonality is represented as an initiation-termination-reinitiation of any physiological processes. Photoperiod is the most predictable environmental cue which organism use to time their daily physiology and seasonal functions. In natural light environmental conditions, day and night components change in terms of duration, intensity and spectrum of light available. In many vertebrate species, daytime light intensity and spectrum of light play a critical role in gonadal recrudescence-regression cycle. In tropical conditions, many amphibian species show a clear relationship between reproduction and seasonal distribution of rainfall. In temperate conditions, reproduction is usually centered during spring and summer seasons when environmental conditions are favorable. Poikilotherms are highly sensitive to change in environmental temperature and their physiology and metabolic activities depend on environmental temperature. How environmental factors (light and temperature) influence, the reproduction of terai tree frog (Polypedates teraiensis) is not known. We hypothesized that light acts as a proximate factor but the temperature is the ultimate factor for reproduction of terai tree frog. Three experiments were performed. In experiment one, we studied the annual reproductive cycle under natural environmental conditions. Beginning from the month of March till October 2016, we procured adult frogs (male and female) in the middle of each month. Monthly observations were recorded for body weight and gonadal weight and GSI was calculated. We found the annual change in body mass, gonadal weight and GSI in both male and female with high gonadal weight and GSI values from March to June. These results suggest that terai tree frog is seasonal breeders, and their breeding corresponds with long days. In experiment two we addressed the effects of light duration, light intensity and light spectrum on gonadal growth regression cycle. We observed that long days promote gonadal growth regression cycle. However, after achieving critical daylength there is no additive effect of light duration. Further, light intensity and spectrum have limited role in gonadal growth regression cycle of this species. In experiment three we tested the role of temperature on body weight and testicular growth under stimulatory photoperiod (12 L:12D). Group one was exposed to high temperature (34 ±â€¯2 °C), while group two was exposed to low temperature (22 ±â€¯2 °C). We found that low temperature promotes testicular recrudescence under laboratory conditions. Altogether our study suggests both photoperiod and temperature are involved in the regulation of seasonal breeding in tree frog. Findings from the above study could be used for captive breeding of amphibians and may be helpful in amphibian conservation programmes.