Sleep/Wake Disruption in a Mouse Model of BLOC-1 Deficiency.
ABSTRACT: Mice lacking a functional Biogenesis of Lysosome-related Organelles Complex 1 (BLOC-1), such as those of the pallid line, display cognitive and behavioural impairments reminiscent of those presented by individuals with intellectual and developmental disabilities. Although disturbances in the sleep/wake cycle are commonly lamented by these individuals, the underlying mechanisms, including the possible role of the circadian timing system, are still unknown. In this paper, we have explored sleep/circadian malfunctions and underlying mechanisms in BLOC-1-deficient pallid mice. These mutants exhibited less sleep behaviour in the beginning of the resting phase than wild-type mice with a more broken sleeping pattern in normal light-dark conditions. Furthermore, the strength of the activity rhythms in the mutants were reduced with significantly more fragmentation and lower precision than in age-matched controls. These symptoms were accompanied by an abnormal preference for the open arm in the elevated plus maze in the day and poor performance in the novel object recognition at night. At the level of the central circadian clock (the suprachiasmatic nucleus, SCN), loss of BLOC-1 caused subtle morphological changes including a larger SCN and increased expression of the relative levels of the clock gene Per2 product during the day but did not affect the neuronal activity rhythms. In the hippocampus, the pallid mice presented with anomalies in the cytoarchitecture of the Dentate Gyrus granule cells, but not in CA1 pyramidal neurones, along with altered PER2 protein levels as well as reduced pCREB/tCREB ratio during the day. Our findings suggest that lack of BLOC-1 in mice disrupts the sleep/wake cycle and performance in behavioural tests associated with specific alterations in cytoarchitecture and protein expression.
Project description:We have previously established that CAST/EiJ (CAST) mice differ from normal mice, such as C57BL/6J (B6), in the timing of wheel-running onset relative to light/dark cycles. These mice provide an animal model for studies of the genetic and neurobiological basis for circadian phase misalignment in humans. Neither differences in endogenous circadian period nor the shape of the photic phase response curve explain the difference in the timing of activity onset between CAST and B6 mice, suggesting a mechanism downstream of the circadian clock. Here, we further test the hypothesis that the two strains differ with respect to circadian oscillations at the molecular level.Sleep/wake cycles were examined and rhythms of Period1 (Per1) and Period2 (Per2) expression were measured in the cerebral cortex, suprachiasmatic nucleus (SCN), and other hypothalamic regions.Basic sleep and molecular research laboratory.Male mice of the B6 and CAST inbred strains.None.Sleep/wake cycles were advanced by approximately 4 h in CAST mice relative to B6 mice. This was paralleled by phase-advanced rhythms of Per1 and Per2 expression, as measured byin situ hybridization, in the cerebral cortex of CAST relative to B6. By contrast, the timing of circadian oscillations and the photic induction ofPer1 and Per2 expression in the SCN were unaffected by strain.The advanced phase of wheel running and sleep/wake cycles in CAST mice relative to B6 mice is apparently not associated with differences in molecular oscillations in the SCN clock itself, but most likely in mechanisms downstream of the SCN clock. CAST mice may therefore provide a model system to investigate circadian downstream mechanisms underlying unusual patterns of entrainment to the ambient photoperiod.Jiang P; Franklin KM; Duncan MJ; O'Hara BF; Wisor JP. Distinct phase relationships between suprachiasmatic molecular rhythms, cerebral cortex molecular rhythms, and behavioral rhythms in early runner (CAST/EiJ) and nocturnal (C57BL/6J) mice. SLEEP 2012;35(10):1385-1394.
Project description:In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates daily rhythms including sleep-wake, hormone release, and gene expression. The cells of the SCN must synchronize to each other to drive these circadian rhythms in the rest of the body. The ontogeny of circadian cycling and intercellular coupling in the SCN remains poorly understood. Recent in vitro studies have recorded circadian rhythms from the whole embryonic SCN. Here, we tracked the onset and precision of rhythms in PERIOD2 (PER2), a clock protein, within the SCN isolated from embryonic and postnatal mice of undetermined sex. We found that a few SCN cells developed circadian periodicity in PER2 by 14.5 d after mating (E14.5) with no evidence for daily cycling on E13.5. On E15.5, the fraction of competent oscillators increased dramatically corresponding with stabilization of their circadian periods. The cells of the SCN harvested at E15.5 expressed sustained, synchronous daily rhythms. By postnatal day 2 (P2), SCN oscillators displayed the daily, dorsal-ventral phase wave in clock gene expression typical of the adult SCN. Strikingly, vasoactive intestinal polypeptide (VIP), a neuropeptide critical for synchrony in the adult SCN, and its receptor, VPAC2R, reached detectable levels after birth and after the onset of circadian synchrony. Antagonists of GABA or VIP signaling or action potentials did not disrupt circadian synchrony in the E15.5 SCN. We conclude that endogenous daily rhythms in the fetal SCN begin with few noisy oscillators on E14.5, followed by widespread oscillations that rapidly synchronize on E15.5 by an unknown mechanism.SIGNIFICANCE STATEMENT We recorded the onset of PER2 circadian oscillations during embryonic development in the mouse SCN. When isolated at E13.5, the anlagen of the SCN expresses high, arrhythmic PER2. In contrast, a few cells show noisy circadian rhythms in the isolated E14.5 SCN and most show reliable, self-sustained, synchronized rhythms in the E15.5 SCN. Strikingly, this synchrony at E15.5 appears before expression of VIP or its receptor and persists in the presence of blockers of VIP, GABA or neuronal firing. Finally, the dorsal-ventral phase wave of PER2 typical of the adult SCN appears ∼P2, indicating that multiple signals may mediate circadian synchrony during the ontogeny of the SCN.
Project description:In mammals, the master circadian clock is located in the suprachiasmatic nucleus (SCN), where most neurons show circadian rhythms of intracellular Ca2+ levels. However, the origin of these Ca2+ rhythms remains largely unknown. In this study, we successfully monitored the intracellular circadian Ca2+ rhythms together with the circadian PER2 and firing rhythms in a single SCN slice ex vivo, which enabled us to explore the origins. The phase relation between the circadian PER2 and Ca2+ rhythms, but not between the circadian PER2 and firing rhythms, was significantly altered in Cry1/Cry2 double knockout mice, which display a loss of intercellular synchronization in the SCN. In addition, in Cry1/Cry2 double knockout mice, circadian Ca2+ rhythms were abolished in the dorsolateral SCN, but were maintained in the majority of the ventromedial SCN. These findings indicate that intracellular circadian Ca2+ rhythms are composed of an exogenous and endogenous component involving PER2 expression.
Project description:Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.
Project description:Self-sustaining oscillations are essential for diverse physiological functions such as the cell cycle, insulin secretion and circadian rhythms. Synthetic oscillators using biochemical feedback circuits have been generated in cell culture. These synthetic systems provide important insight into design principles for biological oscillators, but have limited similarity to physiological pathways. Here we report the generation of an artificial, mammalian circadian clock in vivo, capable of generating robust, tunable circadian rhythms. In mice deficient in Per1 and Per2 genes (thus lacking circadian rhythms), we artificially generate PER2 rhythms and restore circadian sleep/wake cycles with an inducible Per2 transgene. Our artificial clock is tunable as the period and phase of the rhythms can be modulated predictably. This feature, and other design principles of our work, might enhance the study and treatment of circadian dysfunction and broader aspects of physiology involving biological oscillators.
Project description:<h4>Study objectives</h4>That sleep deprivation increases the brain expression of various clock genes has been well documented. Based on these and other findings we hypothesized that clock genes not only underlie circadian rhythm generation but are also implicated in sleep homeostasis. However, long time lags have been reported between the changes in the clock gene messenger RNA levels and their encoded proteins. It is therefore crucial to establish whether also protein levels increase within the time frame known to activate a homeostatic sleep response. We report on the central and peripheral effects of sleep deprivation on PERIOD-2 (PER2) protein both in intact and suprachiasmatic nuclei-lesioned mice.<h4>Design</h4>In vivo and in situ PER2 imaging during baseline, sleep deprivation, and recovery.<h4>Settings</h4>Mouse sleep-recording facility.<h4>Participants</h4>Per2::Luciferase knock-in mice.<h4>Interventions</h4>N/A.<h4>Measurements and results</h4>Six-hour sleep deprivation increased PER2 not only in the brain but also in liver and kidney. Remarkably, the effects in the liver outlasted those observed in the brain. Within the brain the increase in PER2 concerned the cerebral cortex mainly, while leaving suprachiasmatic nuclei (SCN) levels unaffected. Against expectation, sleep deprivation did not increase PER2 in the brain of arrhythmic SCN-lesioned mice because of higher PER2 levels in baseline. In contrast, liver PER2 levels did increase in these mice similar to the sham and partially lesioned controls.<h4>Conclusions</h4>Our results stress the importance of considering both sleep-wake dependent and circadian processes when quantifying clock-gene levels. Because sleep deprivation alters PERIOD-2 in the brain as well as in the periphery, it is tempting to speculate that clock genes constitute a common pathway mediating the shared and well-known adverse effects of both chronic sleep loss and disrupted circadian rhythmicity on metabolic health.
Project description:Circadian clocks drive daily rhythms in virtually all organisms. In mammals, the suprachiasmatic nucleus (SCN) is recognized as the master clock that synchronizes central and peripheral oscillators to evoke circadian rhythms of diverse physiology and behavior. How the timing information is transmitted from the SCN clock to generate overt circadian rhythms is essentially unknown. Prokineticin 2 (PK2), a clock-controlled gene that encodes a secreted protein, has been indicated as a candidate SCN clock output signal that regulates circadian locomotor rhythm. Here we report the generation and analysis of PK2-null mice. The reduction of locomotor rhythms in PK2-null mice was apparent in both hybrid and inbred genetic backgrounds. PK2-null mice also displayed significantly reduced rhythmicity for a variety of other physiological and behavioral parameters, including sleep-wake cycle, body temperature, circulating glucocorticoid and glucose levels, as well as the expression of peripheral clock genes. In addition, PK2-null mice showed accelerated acquisition of food anticipatory activity during a daytime food restriction. We conclude that PK2, acting as a SCN output factor, is important for the maintenance of robust circadian rhythms.
Project description:Following general anaesthesia (GA), patients frequently experience sleep disruption and fatigue, which has been hypothesized to result at least in part by GA affecting the circadian clock. Here, we provide the first comprehensive time-dependent analysis of the effects of the commonly administered inhalational anaesthetic, isoflurane, on the murine circadian clock, by analysing its effects on (a) behavioural locomotor rhythms and (b) PER2::LUC expression in the suprachiasmatic nuclei (SCN) of the mouse brain. Behavioural phase shifts elicited by exposure of mice (n = 80) to six hours of GA (2% isoflurane) were determined by recording wheel-running rhythms in constant conditions (DD). Phase shifts in PER2::LUC expression were determined by recording bioluminescence in organotypic SCN slices (n = 38) prior to and following GA exposure (2% isoflurane). Full phase response curves for the effects of GA on behaviour and PER2::LUC rhythms were constructed, which show that the effects of GA are highly time-dependent. Shifts in SCN PER2 expression were much larger than those of behaviour (c. 0.7 h behaviour vs. 7.5 h PER2::LUC). We discuss the implications of this work for understanding how GA affects the clock, and how it may inform the development of chronotherapeutic strategies to reduce GA-induced phase-shifting in patients.
Project description:The hypothalamic suprachiasmatic nucleus (SCN), which in mammals serves as the master circadian pacemaker by synchronizing autonomous clocks in peripheral tissues, is composed of coupled single-cell oscillators that are driven by interlocking positive/negative transcriptional/translational feedback loops. Several studies have suggested that heme, a common prosthetic group that is synthesized and degraded in a circadian manner in the SCN, may modulate the function of several feedback loop components, including the REV-ERB nuclear receptors and PERIOD2 (PER2). We found that ferric heme (hemin, 3-100 microM) dose-dependently and reversibly damped luminescence rhythms in SCN explants from mice expressing a PER2::LUCIFERASE (PER2::LUC) fusion protein. Inhibitors of heme oxygenases (HOs, which degrade heme to biliverdin, carbon monoxide, and iron) mimicked heme's effects on PER2 rhythms. In contrast, heme and HO inhibition did not damp luminescence rhythms in thymus and esophagus explants and had only a small effect on PER2::LUC damping in spleen explants, suggesting that heme's effects are tissue-specific. Analysis of the effects of heme's degradation products on SCN PER2::LUC rhythms indicated that they probably were not responsible for heme's effects on rhythms. The heme synthesis inhibitor N-methylprotoporphyrinIX (NMP) lengthened the circadian period of SCN PER2::LUC rhythms by about an hour. These data are consistent with an important role for heme in the circadian system.
Project description:Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders.