Could Chronic Hypothermia in a Human Affect the Clock System?

AbstractHibernation-like episodes would be particularly interesting for clinical and spatial use if they could be observed and induced in humans. As animal hibernation differs from hypothermia with its control by a temperature-dependent clock, we undertook to find evidence that human hypothermia might affect the circadian clock system. We revisited Siffre's 1962 abyss experiment. Deprived of temporal information and showing signs of chronic hypothermia, Siffre underestimated his stay underground by 22 d. We show that the temperature-dependent clock equation for classical hibernators accurately predicts Siffre's subjective times, and we list potential conditions to be further explored for inducing hibernation-like bouts in humans.

Implicating a Temperature-Dependent Clock in the Regulation of Torpor Bout Duration in Classic Hibernation.

Syrian hamsters may present 2 types of torpor when exposed to ambient temperatures in the winter season, from 8°C to 22°C (short photoperiod). The first is daily torpor, which is controlled by the master circadian clock of the body, located in the SCN. In this paper, we show that daily torpor bout duration is unchanged over the 8°C to 22°C temperature range, as predicted from the thermal compensation of circadian clocks. These findings contrast with the second type of torpor: multi-day torpor or classic hibernation. In multi-day torpor, bout duration increases as temperature decreases, following Arrhenius thermodynamics. We found no evidence of hysteresis from metabolic inhibition and the process was thus reversible. As a confirmation, at any temperature, the arousal from multi-day torpor occurred at about the same subjective time given by this temperature-dependent clock. The temperature-dependent clock controls the reduced torpor metabolic rate while providing a reversible recovery of circadian synchronization on return to euthermy.

Circadian clocks in rat skin and dermal fibroblasts: differential effects of aging, temperature and melatonin.

As a peripheral tissue localized at the interface between internal and external environments, skin performs functions which are critical for the preservation of body homeostasis, in coordination with environmental changes. Some of these functions undergo daily variations, such as temperature or water loss, suggesting the presence of time-keeping mechanisms. Rhythmic functions are controlled by a network of circadian oscillators present virtually in every cell and coordinated by the central clock located in the suprachiasmatic nuclei. At the molecular level, circadian rhythms are generated by conserved transcriptional-translational feedback loops involving several clock genes, among which Per1 and Per2 play a central role. Here we characterize clock activity in skin of the transgenic Per1-luciferase rat during postnatal development and adulthood, by real-time recording of bioluminescence in explants and primary dermal fibroblasts, and report marked transformation in circadian properties, from early life to aging. Using primary dermal fibroblast cultures we provide evidence that melatonin treatment phase dependently increases the amplitude of circadian oscillations and that ambient temperature impacts on their period, with slight overcompensation. Together, these findings demonstrate that skin contains a self-sustained circadian clock undergoing age-dependent changes. Dermal fibroblasts, one of the major skin cell types, also exhibit robust, yet specific, circadian rhythmicity which can be fine-tuned by both internal (melatonin) and external (temperature) factors.

Circadian organization of the rodent retina involves strongly coupled, layer-specific oscillators.

Rhythmic physiology is central to retinal function and survival and adapts vision to daily light intensity changes. Mammalian retina rhythmically releases melatonin when cultured under constant conditions, and the occurrence of clock gene [e.g., Period (Per)] expression has been shown for most cellular layers. However, contribution of the distinct layers to genesis of circadian rhythms within the retina is still debated. To characterize their endogenous oscillatory capacity and their communication at the whole-tissue level, we used a vibratome-based method to isolate individual or paired retina cellular layers from the mPer2(Luc) mouse and Per1-luciferase (Per1-Luc) rat, and real-time recorded bioluminescence. We report that each layer of the mouse retina harbors a self-sustained oscillator whose period is significantly longer (∼ 26 hours) than in whole-retina explants (∼ 22.9 hours), indicating that the period is correlated with the degree of coupling. Accordingly, the maximal period (∼ 29 hours) is reached upon complete enzymatic dissociation of the retina. By using pharmacological approaches, we demonstrate that connection between retina oscillators involves gap junctions but only minor contribution from the main retina neurochemicals. Taken together with results from Per1-Luc rats, these data show that mammalian retina consists of a network of layer-specific oscillators whose period is determined by their connectivity.

Rat retina shows robust circadian expression of clock and clock output genes in explant culture.

PURPOSE: Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. METHODS: Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. RESULTS: Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. CONCLUSIONS: Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.

The evolution of mammalian hibernation: lessons from comparative acid-base physiology.

The conquest of land has endowed air-breathers with the capability to utilize ventilation not only to acquire oxygen but also to control blood and intracellular acid-base state. Hypercapnic acidosis (resulting from ventilatory control and/or behavioral choice), thus, has become a universal component of hypometabolic states in air-breathers, with inhibitory and/or protective roles. Here, special emphasis is placed on the understanding of alterations of acid-base state associated with changes in temperature. Hypercapnic acidosis in connection with hypometabolism has been found in a variety of air-breathing clades, from snails to mammals through lungfish, amphibians, and reptiles. The discovery of the plesiomorphic character of mammalian hibernation has made the transfer to hibernation biology of the experience gained in the application of hypercapnic acidosis (the so-called "pH-stat" procedure) relevant to acid-base control in clinical artificial hypothermia. This paves the way for mutual benefits from such reciprocal exchange of information between hibernation biology and clinical applications.

Oxygen-induced retinopathy induces short-term glial stress and long-term impairment of photoentrainment in mice.

BACKGROUND: Retinopathy of prematurity is a serious potentially blinding disease of pre-term infants. There is extensive vascular remodeling and tissue stress, but data concerning alterations in retinal neurons and glia, and long-term functional sequelae are still incomplete. METHODS: ROP was induced using the oxygen-induced retinopathy (OIR) mouse model. Postnatal day 7 (P7) 129SVE mice were exposed to hyperoxia (75 ± 0.5 % oxygen) for 5 days, and then returned to normoxia to induce OIR. Exposed animals were euthanized at 5 (P17-OIR) and 14 days (P26-OIR) after return to normal air, together with corresponding age-matched control mice (P17-C and P26-C respectively) raised only in room air. Their retinas were examined by immunohistochemistry using a battery of antibodies against key glial and neuronal proteins. A further group of OIR mice and controls were examined at 10 weeks of age for their ability to re-entrain to changing 12 h light/12 h dark cycles, assayed by wheel-running actimetry. In this protocol, animals were subjected to three successive conditions of 300 lux, 15 lux and 1 lux ambient light intensity coupled with 6 hours of jetlag. Animals were euthanized at 4 months of age and used in immunoblotting for rhodopsin. RESULTS: Compared to P17-C, immunohistochemical staining of P17-OIR sections showed up-regulation of stress-related and glutamate-regulatory proteins in astrocytes and Müller glial cells. In contrast, glial phenotypic expression in P26-OIR retinas largely resembled that in P26-C. There was no loss in total retinal ganglion cells (RGC) at either P17-OIR or P26-OIR compared to corresponding controls, whereas intrinsically photosensitive RGC showed significant decreases, with 375 ± 13/field in P26-OIR compared to 443 ± 30/field in P26-C (p < 0.05). Wheel actimetry performed on control and OIR-treated mice at 4 months demonstrated that animals raised in hyperoxic conditions had impaired photoentrainment at low illuminance of 1 lux, as well as significantly reduced levels of rhodopsin compared to age-matched controls. CONCLUSIONS: OIR leads to transient up-regulation of retinal glial proteins involved in metabolism, and partial degeneration of intrinsically photosensitive RGC and rod photoreceptors. OIR affects circadian photo-entrainment at low illuminance values, possibly by affecting the rod pathway and/or intrinsically photosensitive RGC input to the circadian clock. This study hence shows that retinopathy of prematurity affects light-regulated circadian behavior in an animal model, and may induce similar problems in humans.

Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius).

In mammals the light-dark (LD) cycle is known to be the major cue to synchronize the circadian clock. In arid and desert areas, the camel (Camelus dromedarius) is exposed to extreme environmental conditions. Since wide oscillations of ambient temperature (Ta) are a major factor in this environment, we wondered whether cyclic Ta fluctuations might contribute to synchronization of circadian rhythms. The rhythm of body temperature (Tb) was selected as output of the circadian clock. After having verified that Tb is synchronized by the LD and free runs in continuous darkness (DD), we submitted the animals to daily cycles of Ta in LL and in DD. In both cases, the Tb rhythm was entrained to the cycle of Ta. On a 12-h phase shift of the Ta cycle, the mean phase shift of the Tb cycle ranged from a few hours in LD (1 h by cosinor, 4 h from curve peaks) to 7-8 h in LL and 12 h in DD. These results may reflect either true synchronization of the central clock by Ta daily cycles or possibly a passive effect of Ta on Tb. To resolve the ambiguity, melatonin rhythmicity was used as another output of the clock. In DD melatonin rhythms were also entrained by the Ta cycle, proving that the daily Ta cycle is able to entrain the circadian clock of the camel similar to photoperiod. By contrast, in the presence of a LD cycle the rhythm of melatonin was modified by the Ta cycle in only 2 (or 3) of 7 camels: in these specific conditions a systematic effect of Ta on the clock could not be evidenced. In conclusion, depending on the experimental conditions (DD vs. LD), the daily Ta cycle can either act as a zeitgeber or not.

Human skin keratinocytes, melanocytes, and fibroblasts contain distinct circadian clock machineries.

Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.

Floppy eyelid syndrome is associated with obstructive sleep apnoea: a prospective study on 127 patients.

A few investigations have raised the question of a possible relationship between obstructive sleep apnoea syndrome (OSAS) and floppy eyelid syndrome (FES). FES is an easily inverted floppy eyelid with papillary conjunctivis, and is a subset of the general pathology, lax eyelid syndrome. The aim of the current study is to determine whether OSAS severity is associated with FES. One hundred and 27 consecutive subjects (aged 25-75 years) referred to the Strasbourg University Sleep Clinic with suspicion of OSAS were included. All patients underwent overnight ambulatory respiratory polygraphy, comprehensive ophthalmological examination and completed standard sleep questionnaires. OSAS severity was defined based on the patient's obstructive apnoea-hypopnoea index (AHI). As expected, age, body mass index (BMI) and the proportion of males increased with OSAS severity. FES was observed in 15.8% of the subjects without OSAS, 25.8% of the total OSAS population and the frequency was significantly increased (40%) in patients with severe OSAS (AHI > 30 h(-1)). A significant correlation between OSAS severity and FES was found after adjustment for age, sex and BMI, using a principal component analysis (PCA). The multivariate analysis included clinical, polygraphic and comorbidity data and was followed by logistic regressions for the main components extracted from the PCA. In summary, our findings show an association between OSAS severity and FES and suggest that severe OSAS might be an independent risk factor for FES. These two disorders may share common biological determinants, such as tissue elasticity. Finally, clinicians should be aware of this association so that underlying OSAS or FES can be detected.

Is the torpor-arousal cycle of hibernation controlled by a non-temperature-compensated circadian clock?

During the hibernation season, mammalian hibernators alternate between prolonged bouts of torpor with a reduced body temperature (Tb) and short arousals with a return to euthermy. Evidence is presented here to show that this metabolic-and also physiological and neuroanatomical-rhythm is controlled by a clock, the torpor-arousal (TA) clock. The temperature dependence of torpor bout duration in 3 species of Spermophilus (published data) may be described by assuming that the TA clock is a circadian clock (probably not the suprachiasmatic clock) that has lost its temperature compensation. This loss might result either from a permanent deletion, or more likely from a seasonal epigenetic control at the level of the clock gene machinery. This hypothesis was verified over the full Tb range on published data from 5 other species (a monotreme, a marsupial, and 3 placental mammals). In a hibernation season, instantaneous subjective time of the putative TA clock was summated over each torpor bout. For each animal, torpor bout length (TBL) was accurately predicted as a constant fraction of a subjective day, for actual durations in astronomical time varying between 4 and 13 to 20 days. The resulting temperature dependence of the interval between arousals predicts that energy expenditure over the hibernation season will be minimal when Tb is as low as possible without eliciting cold thermogenesis.

Phase shift of the circannual reproductive rhythm in European hamsters by 2 days of long photoperiod.

OBJECTIVES AND DESIGN: In European hamsters a circannual clock drives the seasonal changes in the reproductive state. Its resetting by photoperiod is clearly phase dependent. In mid subjective winter a 1-month pulse of long photoperiod (LP) advances the onset of the reproductive phase of animals maintained in constant short photoperiod (SP) by up to 1.5 months. The present study investigated whether shorter pulses, i.e. 8, 4 or 2 days LP-pulses are still effective to phase shift the circannual rhythm. MAIN FINDINGS: All pulses induced gonadal development after a similar time relative to the offset of the pulse and earlier than in the control group. Thus, they all shared a similar effectiveness. CONCLUSIONS: In European hamsters a very brief LP-pulse can phase shift the reproductive rhythm but its strength is not determined by its duration at least not in the tested range.

Circannual phase response curves to short and long photoperiod in the European hamster.

This study investigated in male European hamsters (Cricetus cricetus ) whether entrainment of circannual rhythms follows the principles of the nonparametric entrainment model. In 2 experiments the times of the year when long (LP) or short photoperiod (SP) are able to synchronize the reproductive cycle were determined, by recording phase response curves (PRCs). A total of 28 groups of 10 hamsters were synchronized by SP, before being subjected to 2 converse experiments: a) 14 groups were transferred to constant LP, only interrupted by SP for 1 month (SP-pulse), the pulse being increasingly delayed between groups by 2 weeks or 1 month steps; and b) the remaining 14 groups stayed in constant SP interrupted by LP for 1 month (LP-pulse) at different phases of the cycle. In a 3rd experiment 5 groups of 10 European hamsters were subjected to constant LP interrupted by 1-month SP-pulses in regular non-365-day zeitgeber intervals (circannual T-cycles) differing between groups (c). The reproductive state was checked every 2 or 4 weeks. The PRCs revealed that an SP-pulse had a very strong phase-resetting capability of -180 degrees to at least +81 degrees in subjective summer (a). During subjective winter when the animals hibernate, a SP-pulse had only weak effectiveness (a) whereas an LP-pulse advanced the circannual clock by up to +41 degrees (b). In the latter conditions a further advance of up to +156 degrees was achieved by the decrease in photoperiod at the return to SP conditions, which terminated the reproductive phase already after 4 to 5 weeks. In different circannual T-cycles the animals entrained for at least 2 cycles (c). In conclusion, 1) the circannual rhythm of European hamsters can be entrained by one photoperiodic signal per cycle, 2) the decrease in photoperiod is most important for its synchronization, and 3) as in circadian clocks the resetting of circannual clocks follows the principles of the nonparametric entrainment model.

Asymmetric control of short day response in European hamsters.

The European hamster (Cricetus cricetus) is a circannual species in which the synchronization of the circannual cycle to the natural year occurs during 2 annual phases of sensitivity. Around the summer solstice, the animals are sensitive to a shortening of photoperiod. During this sensitive phase, pronounced changes in circadian output parameters are observed, indicating a different functional state of the circadian system. This special state is assumed to be necessary to develop the extreme sensitivity to short day length in European hamsters during this phase. In natural conditions, the animals are able to recognize the shortening of photoperiod already in mid-July, when the photoperiod is reduced only by 30 min. To investigate the short-day response in sensitive European hamsters on the basis of the 2-coupled oscillator model of Pittendrigh and Daan (1976), daily activity and the reproductive state of European hamsters were recorded after an asymmetrical reduction of photoperiod from long (LD 16:08) to short (LD 08:16) photoperiods. The activity pattern of the animals showed an immediate response to the short photoperiod at the day of transfer when the night was extended only into the evening, but there was a significant delay in the response time when the night was extended into the morning. Thus, the evening oscillator E is more important in inducing the photoperiodic response than the morning oscillator M. Moreover, the broad intragroup variation in the latter conditions strongly suggests that the changes in the activity pattern were endogenously induced and that the animals were not able to recognize a lengthening of the night into the morning. Gonadal regression started in both groups 3 weeks after the change in the activity pattern, indicating that this process is initiated when the circadian system has received the short-day signal either through changes in photoperiod or through the circannual clock.

Timed hypocaloric feeding and melatonin synchronize the suprachiasmatic clockwork in rats, but with opposite timing of behavioral output.

Temporal organization of the molecular clockwork and behavioral output were investigated in nocturnal rats housed in constant darkness and synchronized to nonphotic cues (daily normocaloric or hypocaloric feeding and melatonin infusion) or light (light-dark cycle and daily 1-h light exposure). Clock gene (Per1, Per2 and Bmal1) and clock-controlled gene (Vasopressin) expression in the suprachiasmatic nuclei was assessed over 24 h. Light and exogenous melatonin synchronized the molecular clock, signaling, respectively, 'daytime' and 'nighttime', without affecting temporal organization of behavioral output (rest/activity rhythm). By contrast, synchronization to hypocaloric feeding led to a striking temporal change between gene expression in the suprachiasmatic clock and waveform of locomotor activity rhythm, rats then becoming active during the subjective day (diurnal-like temporal organization). When the time of feeding coincided with activity offset, normocaloric feeding also synchronized the locomotor activity rhythm with no apparent switch in temporal organization. Peak of Per2 expression in the piriform cortex occurred between the beginning and the middle of the activity/feeding period, depending on the synchronizer. These data demonstrate that even though the suprachiasmatic clockwork can be synchronized to nonphotic cues, hypocaloric feeding likely acts downstream from clock gene oscillations in the suprachiasmatic nuclei to yield a stable yet opposite organization of the rest/activity cycle.

Daily variations of blood glucose, acid-base state and PCO2 in rats: effect of light exposure.

The suprachiasmatic nuclei (SCN) of the hypothalamus are the site of the main circadian clock in mammals. Synchronization of the SCN to light is achieved by direct retinal inputs. The present study performed in rats transferred to constant darkness shows that blood glucose, pH and PCO2 display significant diurnal changes when measurements were made during the subjective day, the early subjective night or the late subjective night. The effects of a 30-min light exposure (100 lx) on these metabolic parameters at each of these circadian times were assessed. Regardless of the circadian time, light induced an increase in blood glucose, but did not affect plasma pH and PCO2. This study suggests that blood glucose, PCO2 and acid-base state are under circadian control, most likely mediated by the SCN, while the hyperglycemic response to light seems not to be gated by a circadian clock and may thus involve retinal inputs to non-SCN retino-recipient areas.

Light exposure during daytime modulates expression of Per1 and Per2 clock genes in the suprachiasmatic nuclei of mice.

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master circadian clock in mammals. Nocturnal light pulses that reset the circadian clock also lead to rapid increases in levels of Per1 and Per2 mRNA in the SCN, suggesting that these genes are involved in the synchronization to light. During the day, when light has no phase-shifting effects in nocturnal rodents, the consequences of light exposure for Per expression have been less thoroughly studied. Therefore, the effects of light exposure during the day were assessed on Per1 and Per2 mRNA in the SCN of mice. Expression of Per1 and Per2 was generally increased by 30-min light pulses during the subjective day, with more pronounced effects in the morning. One exception was noted for a transient decrease in Per2 expression after a short light pulse applied at midday. Prolonged light exposure (up to 3 hr) starting at midday markedly increased Per2 expression but not that of Per1. Moreover, the amplitude of the daily variations of both Per and the duration of Per1 peak was increased in mice exposed to a light-dark cycle compared with those transferred to constant darkness. Finally, the amplitude of the daily variations of both Per and the basal level of Per1 were increased in mice under a light-dark cycle compared with animals synchronized to a skeleton photoperiod (i.e., with daily dawn and dusk 1-hr exposures to light). Taken together, the results indicate that prolonged light exposure during daytime positively modulates daily levels of Per1 and Per2 mRNA in the SCN of mice.

In the rat, exogenous melatonin increases the amplitude of pineal melatonin secretion by a direct action on the circadian clock.

The effect of exogenous melatonin on pineal melatonin synthesis was studied in the rat in vivo. Daily melatonin profiles were measured by transpineal microdialysis over 4 consecutive days in rats maintained on a 12-h light : 12-h dark schedule (LD 12 : 12). Curve-fitting was used to determine the amplitude of the peak of melatonin production, and the times of its onset (IT50) and offset (DT50). A subcutaneous injection of melatonin (1 mg/kg) at the onset of darkness (ZT12) induced an advance of IT50 on the second day after the treatment, in 50% of the animals kept in LD. When the animals were switched to constant darkness, the treatment caused no detectable advance of IT50, while 70% of individuals showed a significant delay in DT50 2 days after the injection. Locally infusing the drug by reverse microdialysis into the suprachiasmatic nuclei (SCN) failed to enhance the shift in melatonin onset. Following subcutaneous melatonin injection, a significant increase ( approximately 100%) in melatonin peak amplitude was observed. This increase persisted over 2 days and occurred only when the melatonin was applied at ZT12, but not at ZT6, 17 or 22. The effect was also observed when the drug was infused directly into the SCN, but not into the pineal. Thus, the SCN are the target site for the effect of exogenous melatonin on the amplitude of the endogenous melatonin rhythm, with a similar window of sensitivity as its phase-shifting effect on the pacemaker.

Circadian tryptophan hydroxylase levels and serotonin release in the suprachiasmatic nucleus of the rat.

Serotonin (5-HT) plays an important role in the regulation of the time-keeping system in rodents. In the present study, we have investigated the interplay between the rhythms of 5-HT synthesis and release in the suprachiasmatic nuclei (SCN) of the rat. The quantitative distribution of tryptophan hydroxylase (TpH) protein was used as an index of 5-HT synthesis, in perikarya and terminals areas. In the raphe medianus, the maximal levels of TpH was reached in the early daytime period, followed by a decrease before the onset of darkness. Conversely, in the axon terminals of the SCN the highest levels of TpH were found before the onset of the dark-period. Furthermore, TpH amount in SCN displays variations depending on the anatomical area of the SCN. Extracellular 5-HT peaked at the beginning of the night, as evidenced by in vivo microdialysis in the SCN. The 5-HT metabolite, 5-HIAA, presented a similar pattern, but the acrophase occurred in the middle of the dark period. These results suggest that TpH is transported from the soma to the nerve terminals in which 5-HT is synthesized during daytime. This would fill the intracellular stores of 5-HT to provide for its nocturnal release.