Circadian rhythm and response to an acute stressor of urinary corticosterone, testosterone, and creatinine in adult male mice

In small laboratory species, steroid measures can be obtained more frequently and less invasively from urine than blood. Insofar as urinary levels reflect systemic levels, they could provide advantages particularly for measurement of glucocorticoids, whose blood levels react rapidly to handling and stress. In Experiment 1, urinary samples were collected from male mice every second hour over a 14:10 h light:dark cycle. Samples were analyzed via enzyme immunoassay for corticosterone, testosterone, and creatinine. Corticosterone had peak concentrations 1 h after light offset and a trough 1 h after light onset. Testosterone showed peak concentrations 5-7 h after light onset and lowest concentrations during the dark phase of the cycle. Creatinine showed some variation over the light-dark cycle, but steroid measures showed similar trends with and without adjustment for creatinine. In Experiment 2, mice were stressed via an injection at times close to the determined peak and trough levels of corticosterone. In urinary samples taken 90 min after injection, corticosterone was significantly higher in injected animals at both times relative to levels in control animals, but testosterone was unaffected by injection stress. In Experiment 3, serum and urine samples were collected from mice every sixth hour across the diurnal cycle. Corticosterone peaked in urine and serum immediately after light offset, and urinary measures predicted those in serum. These data indicate that urinary corticosterone reflects systemic levels in mice, document circadian variation in urinary testosterone, and indicate that circadian variation in creatinine is minimal, but potentially relevant in stressed animals.     

The influence of phase shift in the light-dark cycle on humoral immune responses of mice to sheep red blood cells and polyvinylpyrrolidone

The influence of a phase shift in the light-dark cycle on humoral immune responses against sheep red blood cells (SRBC), a thymus-dependent antigen, and against polyvinylpyrrolidone (PVP), a thymus-independent antigen, was studied by using 180 BALB/c mice and 150 C3H/HeN mice. Significant suppression of the immune response to SRBC and the number of splenic plaque-forming cells (PFC) and hemagglutination (HA) titers was observed on days 5 and 6 after inversion of the light-dark cycle. On the other hand, the number of splenic PFC and HA titers in the blood against PVP were minimally suppressed by the phase shift in C3H/HeN mice, except for distortion of the rhythmicity. Corticosterone levels in the blood on days 5 and 6 after inversion were higher than those under a normal lighting regimen. The appearance of the high corticosterone level in the blood after the inversion almost concurred with the suppression of the immune response to SRBC. A decrease of the proportion of splenic T cells was also observed on day 6 after the inversion. These results show that a phase shift in the light-dark cycle provokes suppression of the immune response to SRBC, possibly through an increase of secretion of corticosterone after light-dark inversion, which induces a decrease of the proportion of T lymphocytes in the spleen.     

Neuromedin U is involved in nociceptive reflexes and adaptation to environmental stimuli in mice

Following our recent observations of inactivity and slowed movement in neuromedin U knockout (NMU KO) mice, we compared nociceptive reflexes and environmental adaptation in NMU KO and wild-type mice. Hot plate and formalin tests revealed that reflexes to heat and pain were significantly decreased in NMU KO mice. Conversely, intracerebroventricular injection of NMU into wild-type mice stimulated nociceptive reflexes in a dose-dependent manner. After NMU injection, increased c-Fos expression was observed in a wide range of locations in hypothalamus, brainstem, and spinal cord. NMU mRNA expression increased in the spinal cord, but not in the hypothalamus, 2 and 4 h after formalin injection. When their light-dark cycle was advanced or retarded by 5 h, NMU KO mice required a longer time to re-entrain into the new light-dark cycle than did wild-type mice. Wild-type mice displayed increased blood pressure after their environmental temperature was changed from 23 to 37 degrees C, whereas no such increase was observed in NMU KO mice. Blood corticosterone levels were significantly increased after 10 min of immobilization stress in wild-type mice, but not in NMU KO mice. These results suggest that endogenous NMU may be involved in reflexes and adaptation to environmental stimuli.     

Diurnal Variation of Plasmatic Melatonin,Corticosterone and Variation of General Activity in Pigeons under Light-Dark Cycle and Constant Light

This work analyzed the diurnal variation of general activity and plasmatic levels of melatonin and corticosterone in pigeons submitted to a 12:00:12:00 h light-dark cycle (lights on at 6:00 a.m.) or to constant light. In both conditions pigeons were observed in 5-min sessions at times 03:00, 06:00, 09:00, 12:00, 15:00, 18:00, 21:00 and 24:00 h during two successive days. Behavior was video taped in the home cages for posterior categorization and quantification. Radioimmunoassays were used to evaluate plasmatic levels of melatonin and corticosterone. Blood samples were obtained at the times of behavioral observation. In the light-dark condition the results showed day-night variation of general activity (p < 0.001) and a robust diurnal rhythm of plasmatic melatonin (p < 0.001). Both of these variations as well as the oscillatory secretion of corticosterone disappeared under constant light condition. The parallel changes in general activity and blunting of melatonin rhythm secretion in constant light condition agree with previous evidences that melatonin may regulate behavioral oscillations in the pigeon. The present data are related to the proposition that the timing system in pigeons may involve neuroendocrine relations characterized by interactions between blood born signalization by melatonin and corticosterone.     

Effect of social isolation on 24-h pattern of stress hormones and leptin in rats

This work analyzes the effect of social isolation of growing male rats on 24-h changes of plasma prolactin, growth hormone, ACTH and leptin, and on plasma and adrenal corticosterone concentrations. At 35 days of life, rats were either individually caged or kept in groups (6-8 animals per cage) under a 12:12 h light/dark schedule (lights on at 08:00 h). A significant arrest of body weight gain regardless of unchanged daily food intake was found in isolated rats after 2 weeks of isolation. On the 4th week, rats were killed at 6 time intervals during a 24-h cycle, beginning at 09:00 h. In isolated rats the 24-h pattern of all parameters tested became distorted, as assessed by Cosinor analysis. When analyzed as a main factor in a factorial analysis of variance, isolation decreased plasma prolactin and growth hormone, increased plasma leptin and corticosterone while decreased adrenal corticosterone. Plasma corticosterone levels correlated significantly with plasma ACTH and with adrenal corticosterone levels in group-caged rats only. These changes can be attributed to an effect of mild stress on the endogenous clock that modulates the circadian hormone release.     

Humoral (immunological) responses in female albino rats during rotating magnetic field exposures

Experiments were designed to evaluate the primary and secondary humoral responses to a rotating magnetic field configuration, which is known to evoke significant biobehavioral changes. Ten days after inoculation with human serum albumin and 10 days before a booster, female rats were exposed to eigher a 0.5 Hz rotating magnetic field (RMF) or to room conditions (control). The lighting schedule was either continuous or involved a light-dark cycle (LD) of 12:12h. A third group of rats served as colony room controls. Group differences of low statistical significance were found when females were exposed to continuous lighting rather than the LD 12:12 light-dark cycle. However, the effects were considered trivial and not sufficient to explain the previously reported biobehavioral changes evoked by this field configuration.     

布氏田鼠社会行为对光周期的适应格局

对实验室内不同光周期条件下的布氏田鼠陌生成体—亚成体间社会行为模式的初步研究显示:不同光周期下成体对亚成体（特别是对雄性亚成体）的行为反应具性二型特征,长光照周期下（light:dark,14L:10D)成体与亚成体同性组合间的争斗较异性组合间的明显,容忍性低,其中成体雄对亚成体雄尤甚。短光照周期下（light:dark,10L:14D）成体雄对亚成体雄、成体雌对亚成体雌及亚成体雄的相容性均较成体雄对亚成体雌的容忍性低,尤成体雌对亚成体雄的排斥表现更为突出,其容忍性也较长光照条件下对应组的低。结果表明,不同光周期条件下布氏田鼠成体与亚成体间的社会行为模式不同。在自然条件下,这些行为模式是其对环境季节变化的一种有效适应,它影响种群中未成年个体存活、补充、扩散重组以及社群结构、种群空间格局季节变化,对该鼠的生存和繁殖有利。     

The photophase light intensity does not affect the scotophase melatonin response in the domestic pig

This study investigated the effects of the photophase light intensity on the scotophase melatonin response. Twelve, 8-month-old crossbred gilts were allocated to three groups of four and housed in temperature- and lighting-controlled climate rooms. The rooms had a light intensity of 40, 200 or 10,000 lx and a light-dark cycle of 12 L:12 D. The gilts were allowed to acclimatize to a new lighting regimen for 1 week before being sampled at 2h intervals for 24h. Following the sampling, pigs were transferred under a different light intensity, allowed to adjust for 1 week and sampled again. The procedure was repeated three times so that all the groups went through all three lighting regimens (light intensities). All the gilts exhibited a clear circadian serum melatonin rhythm under each lighting regimen with high melatonin concentrations occurring during the scotophase. There was no difference in the scotophase melatonin response in terms of mean concentrations or duration of increased melatonin levels within or between the groups under different lighting regimens. There was considerable inter-individual variation in the dark phase melatonin response but the individual profiles were consistent under the different lighting regimens. It is concluded that when a certain threshold light intensity (<40lx) is exceeded, the photophase light intensity has no effect on the scotophase melatonin response. These results imply that extremely high light intensities during the photophase would provide no additional benefits compared with normal comfortable light intensity, if artificial lighting programs were introduced to commercial piggeries in order to reduce seasonal effects on reproduction.     

Role of the pituitary-adrenocortical axis in the control of agonistic behaviour after single and repeated footshock in castrated male mice

Two experiments were conducted to examine the role of the pituitary-adrenocortical axis in the mediation of the effects of single and repeated electric footshock on subsequent agonistic responding in castrated male mice. It was found that (1) preventing corticosterone responses to shock occludes the facilitatory effects of single shock on both aggressive and submissive behaviour and occludes the additional increases in submissive behaviour which normally occur after repeated shock, and (2) blocking pituitary release of ACTH by dexamethasone treatment restores aggressive behaviour after repeated shock, independently of the initial levels of corticosterone and testosterone. These findings suggest that (1) increases in aggressive and submissive behaviour in response to single shock depend on shock-induced increases in corticosterone levels; (2) further increases in submissive behaviour in response to repeated shock require further increases in the corticosterone levels; and (3) inhibition of aggressive behaviour after shock repetition appears to depend on increases in ACTH levels, and occurs independently of the initial corticosterone and testosterone levels.     

Meal timing dominates the lighting regimen as a synchronizer of the eosinophil rhythm in mice.

Mouse eosinophils undergo circadian fluctuation, and the phasing of the rhythm normally is synchronized to the environmental light-dark cycle if food always is available. This study was undertaken to determine whether or not the same rhythm could be synchronized to restricted feeding schedules. It was found that if food is available ad libitum for only short spans (in this case, 4 h during each 24 h period), the rhythm becomes synchronized to the feeding schedule. In addition, restricting food to certain 4 h spans causes the amplitude of the eosinophil rhythm to increase significantly over that of normal, light-dark synchronized animals. Not all rhythmic variables synchronize to restricted feeding schedules. Some remain synchronized to the light-dark cycle; the phasing of others seems to be the result of an interaction between both the light-dark cycle and the feeding schedule. These studies help dispel the popular misconception that all body functions react in the same manner to different synchronizers and emphasize that one must not generalize about the synchronizing effect of feeding or lighting.     

Effect of Pinealectomy on Re-entrainment of Air-gulping Activity in Indian Walking Catfish, Clarias batrachus

The present study was aimed to examine the effect of pinealectomy on re-entrainment of air-gulping activity in Indian walking catfish Clarias batrachus under various LD phases. Live Clarias batrachus of mixed sex (40-50g body weight) were procured from the local market and kept in the stock aquaria under the laboratory conditions for proper acclimation. Following 7 days of acclimation, animals were randomly selected from the stock aquaria and divided into 2 groups. The pineal was surgically removed from one group (P_xG) and the other group was treated as intact control group (IG). Both groups were exposed under LD 12:12 cycle inside the chronocubicles. In every 7 days LD schedule was delayed by 4 hours by lengthening of the light-on time. Both intact and pinealectomized animals are exhibited 24h rhythm in air-gulping behaviour and its entrainability irrespective of phase shifts of LD cycle. However, the phase angle of peak with reference to light on hour gradually decreased from phase 1 to phase 4, irrespective of treatment (intact/pinealectomized). Further, it has been noticed that the occurrence of multifrequency rhythm (24h, 12h and 6h) is being decreased from phase 1 to phase 4. It could be concluded that daily light-dark cycle plays an important role in modulating the rhythmic characteristics of air-breathing activity in Clarias batrachus and that pinealectomy does not modulate the effects of LD cycles on such activity.     

Photic resetting and entrainment in CLOCK-deficient mice

Mice lacking the CLOCK protein have a relatively subtle circadian phenotype, including a slightly shorter period in constant darkness, differences in phase resetting after 4-hour light pulses in the early and late night, and a variably advanced phase angle of entrainment in a light-dark (LD) cycle. The present series of experiments was conducted to more fully characterize the circadian phenotype of Clock(-/-) mice under various lighting conditions. A phase-response curve (PRC) to 4-hour light pulses in free-running mice was conducted; the results confirm that Clock(-/-) mice exhibit very large phase advances after 4-hour light pulses in the late subjective night but have relatively normal responses to light at other phases. The abnormal shape of the PRC to light may explain the tendency of CLOCK-deficient mice to begin activity before lights-out when housed in a 12-hour light:12-hour dark lighting schedule. To assess this relationship further, Clock(-/-) and wild-type control mice were entrained to skeleton lighting cycles (1L:23D and 1L:10D:1L:12D). Comparing entrainment under the 2 types of skeleton photoperiods revealed that exposure to 1-hour light in the morning leads to a phase advance of activity onset (expressed the following afternoon) in Clock(-/-) mice but not in the controls. Constant light typically causes an intensity-dependent increase in circadian period in mice, but this did not occur in CLOCK-deficient mice. The failure of Clock(-/-) mice to respond to the period-lengthening effect of constant light likely results from the increased functional impact of light falling in the phase advance zone of the PRC. Collectively, these experiments reveal that alterations in the response of CLOCK-deficient mice to light in several paradigms are likely due to an imbalance in the shape of the PRC to light.     

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT_1B receptors, decreasing responsiveness to light. Consequently, 5-HT_1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT_1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.     

The effects of feeding and housing on the behaviour of the laboratory rabbit

The effects of housing, feeding time and diet composition on the behaviour of the laboratory rabbit were examined. The animals were caged individually in single or double metal cages with perforated metal floors, metal walls, and bars in the front, or kept as a group in floor pens. The light/dark cycle was 12/12 h with light from 04:00 to 16:00 h and 30 min twilight. One experiment compared feeding equal energy levels of a high energy diet (10.1 MJ/kg) and with a low energy diet (7.0 MJ/kg) at 08:00 h. The second experiment compared feeding the high energy diet at 08:00 h and at 14:00 h. In both studies the behaviour of the rabbits was recorded between 08:00 and 14:00 h and between 16:00 and 22:00 h. Feeding the animals at 14:00 h reduced abnormal behaviour during the dark period compared to feeding at 08:00 h, whereas no difference in behaviour could be detected between feeding a high-energy and a low-energy diet at 08:00 h. Animals in floor pens generally showed less abnormal behaviour than caged animals. The results indicate that the welfare for caged rabbits can be improved by feeding the animals in the afternoon rather than in the morning.     

The effect of various photoperiods on daily oscillations of serum corticosterone and insulin in rats.

The effect of various photoperiods on circadian rhythms of chosen parameters was investigated in laboratory rats. SPF male Wistar rats were adapted for six weeks to artificial light-dark cycles (LD 8:16, 12:12, 16:8). The light was switched on at 07.00 h in all regimens. The rats were killed at 3-hour intervals within 24 h, the serum concentration of corticosterone, insulin, glucose, food and water intake was determined. The external and computative acrophases of corticosterone varied in every photoperiod being dependent on the duration of light, the mesor values decreased in LD 16:8 in comparison with other photoperiods. The external acrophase of insulin was located 4 h after light onset in LD 8:16 and 12:12, in LD 16:8 one hour before light onset. The mesor values were approximately equal in all photoperiods. The circadian rhythms of glucose were similar in all regimens. Circadian variation of food and water consumption culminated at the same time in all regimens, the amount of food consumed in light increased with the light duration. Various photoperiods remarkably influenced circadian oscillations of corticosterone and in part food and water intake which could be considered as photoperiodic traits.     

Effect of Pinealectomy on Re-entrainment of Air-gulping Activity in Indian Walking Catfish,Clarias batrachus

The present study was aimed to examine the effect of pinealectomy on re-entrainment of air-gulping activity in Indian walking catfish Clarias batrachus under various LD phases. Live Clarias batrachus of mixed sex (40-50g body weight) were procured from the local market and kept in the stock aquaria under the laboratory conditions for proper acclimation. Following 7 days of acclimation, animals were randomly selected from the stock aquaria and divided into 2 groups. The pineal was surgically removed from one group (P_xG) and the other group was treated as intact control group (IG). Both groups were exposed under LD 12:12 cycle inside the chronocubicles. In every 7 days LD schedule was delayed by 4 hours by lengthening of the light-on time. Both intact and pinealectomized animals are exhibited 24h rhythm in air-gulping behaviour and its entrainability irrespective of phase shifts of LD cycle. However, the phase angle of peak with reference to light on hour gradually decreased from phase 1 to phase 4, irrespective of treatment (intact/pinealectomized). Further, it has been noticed that the occurrence of multifrequency rhythm (24h, 12h and 6h) is being decreased from phase 1 to phase 4. It could be concluded that daily light-dark cycle plays an important role in modulating the rhythmic characteristics of air-breathing activity in Clarias batrachus and that pinealectomy does not modulate the effects of LD cycles on such activity.     

Effect of photoperiod, injection of pentobarbitone sodium or lesion of the suprachiasmatic nucleus on pre-partum decrease of blood progesterone concentrations or time of birth in the rat

In Sprague-Dawley rats kept under 14L:10D (lights on 05:00-19:00 h), parturition occurred during the light phase on Day 23, and the pre-partum decrease in progesterone concentrations was observed between 07:00 and 15:00 h during the light period on Day 22. When the rats were transferred to reversed light-dark regimen (lights on 17:00-07:00 h) on Day 7, the progesterone decrease and parturition still occurred during the light period on Day 21 and 22-23, respectively. However, when rats were kept in constant darkness from Day 7, parturition occurred independently of the time of day between Day 22 and 24. A gradual decline of progesterone concentrations was randomly observed in individual rats. In Wistar rats kept under the usual light-dark regimen, parturitions were biphasic, occurring during the light periods on Day 22 and 23. The progesterone decrease occurred at the usual time even when the lighting regimen was changed only on the day of the expected progesterone decrease. However, treatment with pentobarbitone sodium at 15:00, 19:00 or 21:00 h, but not at 12:00 or 23:00 h, on Day 21 resulted in a delay of progesterone decrease and of parturition. Complete lesion of the suprachiasmatic nucleus on Day 13 or 14 led to advancement and random distribution of the time of birth. These results suggest that the time of parturition and of pre-partum progesterone decrease may be closely associated with an endogenous circadian system, and a luteolytic factor involving the nervous system may be present during a limited period before parturition.     

Nocturnal activity in a diurnal rodent (Arvicanthis niloticus): the importance of masking

It is known that day-active Nile grass rats, Arvicanthis niloticus, increase the amount of activity in the night relative to that in the day when provided with running wheels. This was confirmed in the present study. Animals without a wheel displayed 69.0% of their general activity in the L phase of a 12:12 h light-dark cycle; animals provided with wheels had only 48.6% of their wheel revolutions in the light. The contribution of direct (masking) responses to light to the increased nocturnality of animals with wheels was examined in two experiments. In experiment 1, masking was tested by exposing the animals to repeated cycles of 30 min of entraining light and 30 min of a different, usually dimmer light, during the L phase of a 12:12 h light-dark cycle. For animals with wheels, there was more running during the 30-min pulses of dim light or darkness than during the 30-min periods of entraining light. In contrast, for animals without wheels, there was more general activity during the 30-min periods of entraining light than during the 30-min pulses of dim light or darkness. In experiment 2, the animals were first exposed to a 12:12 h light-dark cycle and then put on a 1:10:1:12 h LDLD skeleton photoperiod. Animals with wheels increased their running during the subjective day of the skeleton photoperiod compared to that in the actual day of the 12:12 h light-dark cycle. Animals without wheels showed similar levels of general activity during the subjective day of the skeleton photoperiod and the actual day of the 12:12 h cycle. These experiments demonstrate that when Nile rats have running wheels, their increased nocturnal activity is associated with an increased suppression of locomotion in direct response to light. It is possible that changes in masking responses to light may be an essential and integral component of switching between diurnal and nocturnal activity profiles.     

Effect of constant light on prolactin and corticosterone rhythms evaluated using a noninvasive urine sampling protocol in the rat.

Circadian prolactin and corticosterone rhythms are usually investigated in the rat by analysis of plasma hormone profiles. In order to develop a nonstressful methodology for long-term studies, we validated prolactin and corticosterone radioimmunoassays in rat urine samples. Among the criteria of validation, prolactin was identified in urine by Western blot whereas both prolactin and corticosterone levels were undetectable in the urine of hypophysectomized rats. The determination of prolactin and corticosterone levels on serial urine samples showed daily variations in male rats entrained by the light-dark cycle. The acrophases of the 24-hour prolactin and corticosterone profiles were located at 03:26 h and 23:32 h respectively, a delay of 3-4 hours compared with the values of the 24-hour plasma profiles reported in the literature. Corticosterone and prolactin rhythms were abolished or dramatically delayed after 3 weeks of constant illumination. As expected, constant light suppressed the rhythm of 6-sulfatoxymelatonin, the major hepatic metabolite of melatonin. The noninvasive and nonstressful methodology we developed could be of interest for studying the regulation of hormone rhythms and their mutual endocrine interactions in physiological conditions, especially their evolution in the aging process.     

Behavioural and physiological effects of absence of ultraviolet wavelengths on European starlings Sturnus vulgaris

As ultraviolet wavelengths are used in normal avian colour perception, the maintenance of captive birds under artificial lighting (which is normally UV-deficient) may have welfare implications. European starling Sturnus vulgaris juveniles kept in UV-deficient light environments had significantly higher basal plasma corticosterone concentrations than those kept under full spectrum lighting, in the second of two experimental blocks. UV-deficient conditions also led to significant changes in behaviour indicative of escape (less perching and more hanging on the cage and pecking at it). However, the birds from the first block, where the interval between transfer to the experimental set-up from the wild was short (2 days), showed significantly higher basal and maximum plasma corticosterone concentrations than those in the second block and no additional effect of light environment on either corticosterone or behaviour. We hypothesise that this difference between blocks was due to the overriding initial stress of being in captivity swamping any treatment effects. Capture stress had declined in the second set of birds, which entered the experiment after 7–14 days in captivity. Stress effects of UV-deficient lighting appear small relative to the overall impact of captivity, but may nevertheless become apparent after the initial effects of capture subside.