Chronic exposure to dim artificial light disrupts the daily rhythm in mitochondrial respiration in mouse suprachiasmatic nucleus.

Circadian rhythms of physiology, behavior, and metabolism have an endogenous 24 h period that synchronizes with environmental cycles of light/dark and food availability. Alterations in light cycles are stressful and disrupt such diurnal oscillations. Recently, we witnessed a sudden rise in studies describing the mechanisms behind the interaction between the key characteristics of mitochondrial functions, peripheral clocks, and stress responses. To our knowledge, there is no study in the suprachiasmatic nuclei (SCN) describing the dysregulated mitochondrial bioenergetics under abnormal lighting conditions, which is common in today's modern world. Thus, we aimed to investigate the existence of daily changes in mitochondrial bioenergetics (respiratory control rate, RCR), mitochondrial abundance (mtDNA/nDNA), plasma corticosterone, and to test whether disturbances in the lighting conditions might influence such rhythms. To confirm this, mice were sacrificed, mitochondria were isolated from the suprachiasmatic nuclei in the brain and blood was collected, every 3 h at various time points zeitgeber time/circadian time, (0, 3, 6, 9, 12, 15, 18, 21, and 24 h) under 12:12 h light-dark (LD, 150 lux L: 0 lux D) cycle and chronic artificial dim lighting (LL, 5 lux: 5lux) conditions, of a 24 h period, respectively. Our results demonstrate the existence of robust daily rhythmicity in RCR, mtDNA/nDNA and plasma CORT under a normal LD cycle. However, these rhythms were significantly disrupted and clock genes expressions were dysregulated under chronic dim LL. Furthermore, mitochondrial abundance was significantly reduced during LL compared to their numbers under LD cycle. Our data demonstrate that the circadian clock regulates mitochondrial functions (RCR, number), essential for accomplishing daily energy demands and supply by the SCN neurons. Abnormal light exposure dysregulates mitochondrial functions in the SCN and may alter metabolism, resulting in obesity, diabetes, and other metabolic disorders. Therefore, properly designing lighting conditions in workplaces is essential to mitigate the adverse consequences of light on humans.

Continuous artificial light potentially disrupts central and peripheral reproductive clocks leading to altered uterine physiology and reduced pregnancy success in albino mice.

AIMS: The mechanism behind clock coordination in female reproductive disorders is poorly understood despite the known importance of coordinated and synchronized timing of central and clocks in reproductive organs. We investigated the effect of continuous artificial light (LL) on the central and peripheral reproductive clock gene (Bmal1, Clock, Per1, Per2 and Cry1) and its downstream regulators (Hgf, PR-A and HOXA10) during non-pregnancy and pregnancy phases of female mice. MAIN METHODS: Mice (n = 60) in two sets, were maintained under continuous light (LL) and natural day cycle (LD;12L: 12D) for both non-pregnant and pregnant study. Tissues from hypothalamus-containing SCN, ovary, uterus and serum were collected at different zeitgeber time points (ZT; at 4-h intervals across 24-h periods). KEY FINDINGS: LL exposure desynchronized the expressions of the clock mRNAs (Bmal1, Clock, Per1, Per2 and Cry1) in SCN, ovary, and uterus along with Hgf mRNA rhythm. LL significantly increased the thickness of endometrial tissues. Furthermore, the pregnant study revealed lower serum progesterone level during peri- and post-implantation under LL along with downregulated expression of progesterone receptor (PR) as well as progesterone dependent uterine Homeobox A-10 (Hoxa10) proteins with lowered pregnancy outcomes. SIGNIFICANCE: Our result suggests that LL disrupted the circadian coordination between central and clock genes in reproductive tissue leading to interrupted uterine physiology and altered pregnancy in mice. This led us to propose that duration of light exposure at work-places or home for females is very important in prevention of pregnancy anomalies.

Quantitative Proteomics Reveals Molecular Network Driving Stromal Cell Differentiation: Implications for Corneal Wound Healing.

The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-ß. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGß1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.

Sexual dimorphism in ultradian and 24h rhythms in plasma levels of growth hormone in Indian walking catfish, Clarias batrachus.

Growth hormone (GH), a key regulator of somatic and reproductive growth in vertebrates, has been extensively studied, although primarily in female fish. Despite numerous reports about sex- and species-specific growth patterns in fish, to our knowledge, there is no report about the 24 h rhythm of plasma GH in male fish. Thus, we aimed to investigate temporal variations in plasma GH levels and the existence of any rhythms therein during the reproductively active months of March to August in the male walking catfish, Clarias batrachus. We also aimed to compare the secretory temporal patterns of GH in male-female specimens of C. batrachus to decipher sexual dimorphism in GH secretions in fish. After 14 days of acclimation to the natural environment, male catfish (N = 240 in total) were sorted and randomly divided into eight groups for study at ZT0 (sunrise ~06:00 h), 3, 6, 9, 12, 15, 18, and 21. During each month, physical parameters like duration of photoperiod and water temperature were measured. Male catfish (n = 40/month) in all eight groups were sampled (n = 5/group) at each time point under the natural time-of-year 24 h light-dark (LD) cycle. Male catfish were anesthetized and blood was collected through a caudal puncture, centrifuged, and plasma isolated. Plasma GH was measured using a competitive homologous enzyme-linked immunosorbent assay. Further, testes were removed, weighed, and the gonadosomatic index (GSI) was calculated. A significant effect of time and season (p ˂ 0.05, two-way ANOVA) on plasma GH level was detected. Cosinor analyses verified the existence of statistically significant (p ˂ 0.05) ultradian (12 h) and 24 h rhythms of plasma GH in male C. batrachus, with the higher values of Mesor (time series mean) and amplitude (one-half peak-to-trough difference) of the periodicities from March to July. Mapping of the acrophases (peak times) showed two ultradian and one 24 h acrophase of GH during the early photophase and early scotophase from March to August. Distinct sexual-dimorphism in plasma GH Mesors and acrophases was noticed between male and female catfish. GSI values of male catfish indicate males mature a little earlier than females in terms of size and reproductive activity. The findings that plasma GH show 24 h and seasonal fluctuations in a sex-specific manner collectively demonstrate the importance of considering the effect of biological 24 h and seasonal time and sex on the GH level in regulating the physiology of somatic growth and reproduction in catfish.

Wheel-running activity rhythms and masking responses in the diurnal palm squirrel, Funambulus pennantii.

Several studies have reported activity patterns of various diurnal species from the order Rodentia, in which most of the species are nocturnal. Most of these studies have been performed under controlled laboratory conditions. These studies found that most of these species change their activity patterns when held under laboratory conditions, have a diverse masking response to light, and their activity pattern is influenced by the presence of a running wheel. Squirrels are reported to be strictly diurnal both in the field as well as in laboratory settings, and, therefore, form an interesting species to study to better understand the switch to diurnality. The aim of the current study is to characterize the masking response and temporal organization of wheel-running activity rhythms in the palm squirrel, Funambulus pennantii, under semi-natural (NLD) and controlled laboratory conditions using different lighting schedules. Squirrels were housed individually in a resting cage with running wheel under NLD (n = 10) and squared 12:12 h of light-dark cycle (LD) (n = 20). After stable entrainment under the LD condition, squirrels were divided into two groups. One group was housed under constant darkness (DD) (n = 10) and another group under constant light (LL) (n = 10). Following the stable free-running rhythm under DD and LL, the LD condition was reinforced. The kinetics of the endogenous pacemaker was studied following a 6 h phase advance or delay of LD cycle. Further, palm squirrels were subjected to a 3.5: 3.5 h LD cycle to evaluate the masking response to light and dark. Squirrels demonstrated stable, clear, robust, and strict diurnal activity rhythm during NLD and LD. In DD and LL, F. pennantii free-ran from the phase of the previous LD cycle, and the free-running period was longer in LL than in DD. The percentage of activity during the light phase was significantly higher in NLD and LD (above 96%) compared to activity during the subjective day in the DD and LL conditions (above 91%). The alpha/rho ratio was significantly higher in the LL compared to other lighting schedules. Further, all ten squirrels re-entrained to both 6 h advance and delay shifts within 11 days. In the ultradian cycle, significant positive masking of light was evident in nine of ten squirrels. These results suggest that the: (i) circadian system of F. pennantii is stable and functional under various lighting conditions; (ii) basic temporal organization in activity pattern remained unaltered even in the presence of a running wheel; (iii) diurnality is the inherent trait of F. pennantii, and (iv) behavioral activity rhythms are governed by both the circadian clock and external masking. Thus, palm squirrels can be used as a suitable diurnal model in circadian biology to study the underlying mechanisms of diurnality and effects of different light schedules, wavelengths, and non-photic cues on physiological and behavioral parameters.

Melatonin-induced phase and dose responses in a diurnal mammal, Funambulus pennantii.

Melatonin, an essential pineal hormone, acts as a marker of the circadian clock that regulates biological rhythms in animals. The effects of exogenous melatonin on the circadian system of nocturnal rodents have been extensively studied; however, there is a paucity of studies on the phase-resetting characteristics of melatonin in diurnal rodents. We studied the phase shifting effects of exogenous melatonin as a single melatonin injection (1 mg/kg) at various phases of the circadian cycle on the circadian locomotor activity rhythm in the palm squirrel, Funambulus pennantii. A phase response curve (PRC) was constructed. Adult male squirrels (N = 10) were entrained to a 12:12 h light-dark cycle (LD) in a climate-controlled chronocubicle with food and water provided ad libitum. After stable entrainment, squirrels were transferred to constant dark condition (DD) for free-running. Following stable free run, animals were administered a single dose of melatonin (1 mg/kg in 2% ethanol-phosphate buffered saline (PBS) solution) or vehicle (2% ethanol-PBS solution) at circadian times (CTs) 3 h apart to evoke phase shifts. The phase shifts elicited at various CTs were plotted to generate the PRC. A dose response curve was generated using four doses (0.5, 1, 2 and 4 mg/kg) administered at the CT of maximum phase advance. Melatonin evoked maximum phase advances at CT0 (1.23 ± 0.28 h) and maximum phase delays at CT15 (0.31 ± 0.09 h). In the dose response experiment, maximal phase shifts were evoked with 1 mg/kg. In contrast, no significant shifts were observed in control groups. Our study demonstrates that the precise timing and appropriate dose of melatonin administration is essential to maximize the amelioration of circadian rhythm-related disorders in a diurnal model.

Slow and fast orthodromic and antidromic variants in acute 9-h jet-lagged pygmy field mice.

Biological clocks help organism to adapt temporally to a variety of rhythmic environmental cues. Acute changes in the rhythmicity of entraining cues causes short- to long-term physiological distress in individuals, for example, those occurring during jet-lag after long-haul transmeridial flights, or shift work. Variations in the rate of re-entrainment to a 9 h advanced schedule (simulation of acute Jet-lag/shift work) in the Indian pygmy field mouse, Mus terricolor are reported. Wheel- and lab-acclimated adult male mice were entrained to a 12:12 h light:dark (LD) cycles, followed by a 9 h advance in the LD cycle. In response, these mice either advanced or delayed their activity onsets, with individual variation in the rate and direction. Rapid orthodromic (advancing) re-entrainers exhibited a coincidence of activity onsets with the new dark onset in < = 3 days, while gradually advancing re-entrainers took -9 days or more. Delayers (antidromic) also either re-entrained very rapidly (< = 2 days), or gradually (-9 days). Acrophase measurement confirmed the direction of the transients, which did not depend on the free-running period. Such different patterns might determine the differential survival of individuals under the pressure of re-entrainment schedules seen in jet-lag and shift work.

Phase and period responses to short light pulses in a wild diurnal rodent, Funambulus pennanti.

Photic phase response curves (PRCs) have been extensively studied in many laboratory-bred diurnal and nocturnal rodents. However, comparatively fewer studies have addressed the effects of photic cues on wild diurnal mammals. Hence, we studied the effects of short durations of light pulses on the circadian systems of the diurnal Indian Palm squirrel, Funambulus pennanti. Adult males entrained to a light-dark cycle (12 h-12 h) were transferred to constant darkness (DD). Free-running animals were exposed to brief light pulses (250 lux) of 15 min, 3 circadian hours (CT) apart (CT 0, 3, 6, 9, 12, 15, 18 and 21). Phase shifts evoked at different phases were plotted against CT and a PRC was constructed. F. pennanti exhibited phase-dependent phase shifts at all the CTs studied, and the PRC obtained was of type 1 at the intensity of light used. Phase advances were evoked during the early subjective day and late subjective night, while phase delays occurred during the late subjective day and early subjective night, with maximum phase delay at CT 15 (-2.04 ± 0.23 h), and maximum phase advance at CT 21 (1.88 ± 0.31 h). No dead zone was seen at this resolution. The free-running period of the rhythm was concurrently lengthened (deceleration) during the late subjective day and early subjective night, while period shortening (acceleration) occurred during the late subjective night. The maximum deceleration was noticed at CT 15 (-0.40 ± 0.09 h) and the maximum acceleration at CT 21 (0.39 ± 0.07 h). A significant positive correlation exists between the phase shifts and the period changes (r = 0.684, p = 0.001). The shapes of both the PRC and period response curve (τRC) qualitatively resemble each other. This suggests that the palm squirrel's circadian system is entrained both by phase and period responses to light. Thus, F. pennanti exhibits robust clock-resetting in response to light pulses.