Endogenous control of spinule formation in horizontal cells of the teleost retina

Summary The processes of horizontal cells invaginating teleost cone pedicles are studded with small finger-like projections which are present only in the light-adapted state. The aim of this study was to investigate whether the formation and degradation of these so-called spinules, which are thought to function as feed-back synapses onto the cones, is endogenously controlled.Three types of experiment were carried out involving fish entrained to a 12 h light/dark cycle: 1) The number of spinules was determined in goldfish at various times during exposure to either constant darkness (36 h) or constant light (57 h). 2) The time course of spinule formation and degradation in goldfish was investigated following exposure to light or darkness at various times during the light/dark cycle. 3) The time course of flash-induced spinule formation in tench following dark adaptation at noon was compared to that following dark adaptation at midnight.The results of these experiments show that spinule formation and degradation are partially under endogenous control but that they need light for full expression. This endogenous rhythm is reflected in the time courses of spinule formation and breakdown during different phases of the light/dark cycle.     

Influence of circadian disorder on structures and functions of neurons in hippocampus of mice

Objective: This study explored the effect of circadian disorder on structure and function of neurons in hippocampus of mice. Methods: Forty male ICR mice were randomly divided into rhythm disorder group (RDG) and normal rhythm group (NRG). The RDG was treated with 3 h light/5 h dark and 5 h light/3 h dark light–dark (LD) cycle alternately. The normal rhythm group was treated with 12/12 h LD cycle. Electron microscope was used to detect the mouse hippocampal cell ultrastructure. Morris water maze was used to test mice cognitive function. The brain slice electric physiological techniques were used to detect synapses in the hippocampal Long-term potentiation (LTP) effect. Results: The time of through central area of RDG was less than that of NRG. The axoplasm in anterior-posterior membranes of synapses of RDG was dissolved and synaptic vesicles of RDG were decreased. Conclusion: Circadian rhythm disorder induced by irregular light–dark circle can lead to the structural damage of hippocampal neurons and damage the cognitive function in mice.     

Strain dependent response of circadian rhythms during exposure to continuous illumination

Enzymes activities were measured, at three hours intervals, during 30 hours, in various tissues of C57BL/6J and A/J male mice. The measurements, were carried out on mice which were exposed for two, five and twenty one days to continuous illumination. Identical measurements were performed also on mice which were kept in alternating 14 hours light: 10 hours dark. Activity patterns of each group were analysed to test the presence, or absence, of rhythm characteristics. The results of the experiments with C57BL/6J have been previously reported. The comparison of the results, which were obtained from the two strains revealed that under exposure to alternating light: dark conditions all activity patterns exhibited a significant circadian rhythm. Except for one enzyme (thymus GAPD), the times of peak activity (acrophase) were identical for all other examined enzymes, in both strains. On the other hand when the two strains were exposed to continuous illumination they differed in their response to the effect of continuous light. The activity of the same enzyme exhibited different periodicity and/or different acrophase in each of the two strains. This variability reflects the existence of genetic differences, between the strains in the free running behavior of these enzymes' activity rhythms.     

Temporal Variation in Hepatic Superoxide Dismutase Activity in Mice

Circadian variations in superoxide dismutase (SOD) activity were determined in liver homogenates of Balb-C mice that were synchronized under controlled environmental conditions with 12 h light: 12 h dark. The activity of hepatic SOD exhibited a significant circadian rhythm, with a minimum at 01:00 h and maximum at 10:00-13:00 h. It is concluded that fluctuations in hepatic SOD activity render mice more susceptible to the toxic effects of reactive oxygen radicals at particular times of the day.     

The effect of dorsal raphe nucleus (DRN) lesions on the locomotor activity rhythm in mice.

The study employed electrical lesions of dorsal raphe nucleus (DRN) to determine the functional significance of those nuclei in the regulation of wheel-running activity rhythm in mice in light/dark (LD 12:12), constant light (LL), and constant dark (DD) conditions. The wheel-running records showed that raphe nucleus lesions resulted in few days' decrease in common activity and amplitude in LD. The activity phase was not compact but in fragmentary form, especially in DD condition. In some animals an earlier onset of activity after DRN lesion in LD was observed. In LL extension of the rhythm period occurred. Destruction of DRN only slightly modulates the wheel-running circadian rhythm in mice.     

Serotonin increases the phase shift of the circadian locomotor activity rhythm in mice after dark pulses in constant light conditions

Investigations on the effects of the 5-HT agonists and antagonists on the phase of the circadian locomotor activity rhythm of animals kept in constant light conditions (LL) are rare. Therefore the influence of R-(+)-OH-DPAT (5-HT1A receptors agonist) and metergoline (5-HT1/2/7 receptors antagonist) on the phase shift of the locomotor-activity rhythm alone and when combined with dark pulses in mice kept in LL are examined. The results indicate that 8-OH-DPAT administered independently at 12.00CT (Circadian Time) shifted the phase of the circadian rhythm and reinforced the effect of dark pulses on this parameter. 12.00CT was defined arbitrarily as the onset of locomotor activity in constant conditions. Metergoline diminished the phase shifts after dark pulses compared to 8-OH-DPAT. The influence of the serotonin agonist showed that serotonin can reinforce the phase shifting effect of the locomotor activity rhythm after dark pulses in LL condition.     

Different circadian rhythms regulate photoperiodic flowering response and leaf movement in Pharbitis nil (L.) Choisy

The phase shifting effect of red light on both the leaf movement rhythm, and on the rhythm of responsiveness of photoperiodic flower induction towards short light breaks (10 min red light), has been studied in Pharbitis nil, strain Violet, and comparisons between the two rhythms have been made. The phase angle differences between the rhythms after a phase shift with 2 or 6 h of red light given at different times during a long dark period were not constant. The results indicate the involvement of two different clocks controlling leaf movement and photoperiodic flower induction.Abbreviations DD continuous darkness - l:D x:y light/dark cycles with x hours of light and y hours of darkness - PPR rhythm of photoperiodic responsiveness towards light break     

Is the PRC for Dark Pulses in LL a Mirror Image of the PRC for Light Pulses in DD in Mice?

The phase-response curves (PRC) for light pulses in continuous darkness (DD) have been described in many mammals, especially in nocturnal rodents. The PRC for dark pulses in continuous light (LL), however, has been described in a few mammals only, in nocturnal for bat and for hamster and in diurnal for Octodon degus, suggesting that this PRC is mirror imaging the PRC for light pulses. Therefore, the effect of 1-h and 3-h lasting dark pulses on the circadian wheel-running activity rhythm of mice in continuous light was investigated and then the PRC for dark pulses in LL was drawn up. For comparison, the effect of 1-h lasting light pulses on the circadian wheel-running activity rhythm of mice in DD was examined and the PRC for light pulses in DD was constructed. It appeared that the PRC for dark pulses, to a certain degree, represents a mirror image of the PRC for light pulses in mice. However, the advance region of this PRC is longer than that of delay. The mechanism of dark pulses action is discussed.     

Is the PRC for Dark Pulses in LL a Mirror Image of the PRC for Light Pulses in DD in Mice?

The phase-response curves (PRC) for light pulses in continuous darkness (DD) have been described in many mammals, especially in nocturnal rodents. The PRC for dark pulses in continuous light (LL), however, has been described in a few mammals only, in nocturnal for bat and for hamster and in diurnal for Octodon degus, suggesting that this PRC is mirror imaging the PRC for light pulses. Therefore, the effect of 1-h and 3-h lasting dark pulses on the circadian wheel-running activity rhythm of mice in continuous light was investigated and then the PRC for dark pulses in LL was drawn up. For comparison, the effect of 1-h lasting light pulses on the circadian wheel-running activity rhythm of mice in DD was examined and the PRC for light pulses in DD was constructed. It appeared that the PRC for dark pulses, to a certain degree, represents a mirror image of the PRC for light pulses in mice. However, the advance region of this PRC is longer than that of delay. The mechanism of dark pulses action is discussed.     

Chronopharmacokinetics of valproic acid following constant-rate administration in mice.

This study was carried out to investigate the circadian rhythm in the pharmacokinetics of valproic acid (VPA). ICR male mice, housed under a light-dark (12 h:12 h) cycle, were used in these studies. In the constant-rate administration study (536.3 or 1,072.6 micrograms/h), osmotic minipumps were implanted subcutaneously in mice. There was a significant circadian rhythm in plasma VPA concentrations: higher values were obtained in the light phase and lower values were found during the dark phase. A significant circadian rhythm also was shown for clearance (CL) of the drug: lower values were obtained in the light phase and higher values were demonstrated in the dark phase. In the intravenous administration study, VPA (50 mg/kg) was injected into a tail vein of the mice. Mean plasma VPA concentrations were significantly higher in mice injected with the drug at 1700 h than at 0100 h. The CL was higher, the volume of distribution (V) was larger, and the area under the curve (AUC) was smaller (p less than 0.05, respectively) in mice injected with the drug at 0100 h than at 1700 h. As the values of CL and V increased similarly during the dark period, there was no effect on half-life (t 1/2) and obviously on the elimination rate constant (K). These findings indicate that the circadian rhythms of plasma VPA concentrations observed after constant-rate administration are due to those of CL and V. To keep drug concentrations constant, the drug release rate from the osmotic minipump should be controlled according to the rhythm of drug pharmacokinetics.     

Circadian Rhythm of Total Protein Synthesis in the Cytoplasm and Chloroplasts of Gonyaulaxpolyedra

Protein synthesis of Gonyaulax polyedra was analyzed by means of electron microscopic autoradiographs under constant conditions at different times of the 24-hr cycle. Circadian rhythmic changes in the synthesis rate of total protein were determined in the cytoplasm and chloroplasts of growing cells. Three independent series of experiments in constant light showed a maximum of grains per unit area during the 'subjective' dark phase (=phase that corresponds to the dark phase during a 12:12 hr LD cycle) in both compartments. Minimum and maximum grain number are different by a factor of 5-10. The maximum of total protein synthesis coincided with the maximum phase shift by cycloheximide pulses (1) suggesting protein species within the total pool involved in the mechanism of the circadian clock. A similar rhythm of lower amplitude was observed in the mitochondria, but this rhythm cannot with certainty be attributed to these organelles. In a slowly growing culture a rhythm of total protein synthesis was observed that showed a smaller amplitude and a different phasing.     

Effects of acute stress on aggression and the cortisol response in the African sharptooth catfish Clarias gariepinus: differences between day and night

African sharptooth catfish Clarias gariepinus were housed under continuous dim light (1 lx) or 12L:12D (350–0 lx) cycles. The number of skin lesions, as indicator of aggressive acts, and plasma cortisol levels, as indicator of stress‐axis activity, were measured at baseline as well as following a stressor (given in the light or dark phase). Results showed that (1) baseline plasma cortisol levels were not different between photoperiods, (2) the number of baseline skin lesions was highest for C. gariepinus housed under continuous dim light, (3) stressor‐induced peak levels of plasma cortisol were highest in the light phase and (4) the number of skin lesions following a stressor was highest in the dark phase. The higher number of stressor‐related skin lesions in the dark (active) phase suggests increased stressor‐induced aggression while in the active phase. In addition, the data suggest that housing under continuous dim light does not result in higher stress‐axis activity, as measured by baseline levels of cortisol, but does result in more stressor‐induced aggression, as measured by the higher number of skin lesions. The latter may be related to the fact that the continuous dim light photoperiod has twice the number of dark‐phase (active) hours in which stressor‐induced aggression is stronger compared to the 12L:12D photoperiod, which has a light phase in which stressor‐induced aggression is lower.     

Day-night variations in plasma melatonin and arginine vasotocin concentrations in chronically cannulated flounder (Platichthys flesus).

Chronically catheterised, free swimming flounder (Platichthys flesus) have been used in experiments examining the day-night variations in circulating levels of melatonin (Mel) and arginine vasotocin (AVT). Under normal photoperiod (16 h light/8 h dark) serial blood samples taken from individual fish demonstrated a Mel rhythm with daytime levels at 09.00 and 15.00 h (238+/-14 and 179+/-12 fmol x ml(-1), respectively) lower than those at 23.00 h (1920+/-128 fmol x ml(-1)). Maintenance of fish in 24-h light abolished the light/dark Mel rhythm and circulating levels were comparable to those measured during the day in fish under normal photoperiod illumination. In fish maintained under 24 h dark, although a daily rhythm was still apparent, at the time when it would be normally dark, plasma Mel concentration was reduced and at times when it would be normally light, levels were higher than in fish maintained under normal light/dark illumination. Plasma AVT concentrations were higher in fish during the day (4.4+/-0.8 fmol x ml(-1)) than those at night (1.5+/-0.4 fmol x ml(-1)), the opposite to that seen with Mel. During acute study infusion of AVT resulted in reduced levels of plasma Mel, although this did not achieve statistical significance. Infusion of Mel did not alter circulating AVT concentration.     

Circadian rhythm in the number of granulated vesicles in the pinealocytes of mice

Summary Adult, Charles River CD-1, male mice were housed in an environmental control chamber under strict conditions of controlled light (12D/12L) and temperature. The mice were sacrificed at various times throughout the twenty-four hour clock and their pineals prepared routinely for electron microscopy. The number of dense-cored or granulated vesicles present in the polar terminals of pinealocytes were quantitated in thin cross sections through pericapillary areas. A distinct circadian rhythm was observed in the number of granulated vesicles with a three- to four-fold difference between late photoperiod maximum and late dark period minimum. The rhythm was abolished by bilateral superior cervical ganglionectomy. These results are consistent with the hypothesis that the granulated vesicles are synthesized and stored in the pinealocytic cytoplasm during the photoperiod under the tropic influence of norepinephrine, and are released during the dark period when melatonin synthesis is greatest. Melatonin, administered as daily intraperitoneal doses of 50 g over a period of five days, was observed to increase markedly the number of pinealocytic granulated vesicles during the light period, but led during the dark period to a decrease in their numbers to levels below that of diluent-treated controls. It may be that melatonin stimulates the synthesis and/or release of granulated vesicles which represent the packaged form of a major secretory product.Supported in part by N.I.H. Grant No. HD 08795     

Circadian Disorganization Alters Intestinal Microbiota

Intestinal dysbiosis and circadian rhythm disruption are associated with similar diseases including obesity, metabolic syndrome, and inflammatory bowel disease. Despite the overlap, the potential relationship between circadian disorganization and dysbiosis is unknown; thus, in the present study, a model of chronic circadian disruption was used to determine the impact on the intestinal microbiome. Male C57BL/6J mice underwent once weekly phase reversals of the light:dark cycle (i.e., circadian rhythm disrupted mice) to determine the impact of circadian rhythm disruption on the intestinal microbiome and were fed either standard chow or a high-fat, high-sugar diet to determine how diet influences circadian disruption-induced effects on the microbiome. Weekly phase reversals of the light:dark (LD) cycle did not alter the microbiome in mice fed standard chow; however, mice fed a high-fat, high-sugar diet in conjunction with phase shifts in the light:dark cycle had significantly altered microbiota. While it is yet to be established if some of the adverse effects associated with circadian disorganization in humans (e.g., shift workers, travelers moving across time zones, and in individuals with social jet lag) are mediated by dysbiosis, the current study demonstrates that circadian disorganization can impact the intestinal microbiota which may have implications for inflammatory diseases.     

5-HT1B receptor knockout mice exhibit an enhanced response to constant light

Serotonin (5-HT) can act presynaptically at 5-HT1B receptors on retinal terminals in the suprachiasmatic nucleus (SCN) to inhibit glutamate release, thereby modulating the effects of light on circadian behavior. 5-HT1B receptor agonists (1) inhibit light-induced phase shifts of circadian activity rhythms, (2) attenuate light-induced Fos expression in the SCN, and (3) reduce the amplitude of optic nerve-evoked excitatory postsynaptic currents in SCN neurons in vitro. To determine whether functional disruption of the 5-HT1B presynaptic receptors would result in an amplified response of the SCN to light, the period (tau) of the circadian rhythm of wheel-running activity was estimated under several different conditions in 5-HT1B receptor knockout (KO) mice and genetically matched wild-type animals. Under constant light (LL) conditions, the tau of 5-HT1B receptor KO mice was significantly greater than the tau of wild-type mice. A quantitative analysis of the wheel-running activity revealed no differences between wild-type and KO mice in either total activity or the temporal distribution of activity under LL conditions, suggesting that the observed increase in tau was not a function of reduced activity. Under constant dark conditions, the period of the circadian rhythm of wheel-running activity of wild-type and 5-HT1B receptor KO mice was similar. In addition, no differences were noted between wild-type and 5-HT1B receptor KO mice in the rate of reentrainment to a 6 h phase advance in the 12:12 light:dark cycle or in phase shifts in response to a 10 min light pulse presented at circadian time 16. The enhanced response of the SCN circadian clock of the 5-HT1B receptor KO mice to LL conditions is consistent with the hypothesis that the endogenous activation of 5-HT1B presynaptic receptors modulates circadian behavior by attenuating photic input to the SCN.     

Circadian Rhythm of the Liver of Male Rats Pre-Treated with Phenobarbital—ii. Hexobarbital Sleeping Time and Lipids Content in Liver and Serum

The circadian rhythm of hexobarbital sleeping time and lipids content in liver and serum were studied in 226 male Sprague-Dawley rats pretreated daily at 0800-0900 with 70 mg/kg (study 1 or 3) or 50 mg/kg (study 2) phenobarbital (PB) orally for 7 days. Thereafter, eight (study 1) or five (study 2 and 3) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through the following day. The lighting schedule in the colony was 12:12 ± light:dark (light from 0600 to 1800). The hexobarbital sleeping times of PB-pretreated rats were generally shortened compared to the controls and no circadian rhythm was observed. PB-treatment increased slightly the liver content of cholesterol, and significantly that of triglycerides and phospholipids. Liver cholesterol and phospholipids showed circadian rhythms with peaks during the dark phase. No circadian rhythm of liver triglycerides existed. In serum, levels of triglycerides and phospholipids were slightly lowered by PB-treatment, while levels of cholesterol and beta-lipoprotein were not influenced. Serum values did not exhibit circadian rhythms.