Diverse forms of life have evolved 24-hour or circadian timekeeping systems serving to coordinate internal biological events with the daily solar cycle. The generation of circadian rhythms by this timekeeping system ensures that internal processes occur at the appropriate time of day or night in relation to the environmental cycle and to other functionally-affiliated events. For mammals, endogenous oscillations in gene expression are a prevalent feature of oscillatory cells residing in the suprachiasmatic nucleus (SCN) and non-SCN tissues. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain the intrinsic rhythm-generating properties of the SCN, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared to that found in the rat SCN in vivo. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations for 2 cycles. Many (35%) of these rhythmicallyregulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. To screen for output signals that may distinguish oscillatory cells in the mammalian SCN from peripheral-type oscillators, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN in vivo. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited rhythmic fluctuations in the expression of the core clock genes and 323 (2.6%) functionally diverse transcripts. Overlap in rhythmically expressed transcripts among these different oscillator models was limited to the clock genes and four genes that function in metabolism or transcription. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (Nos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of antisense oligonucleotide-mediated inhibition of Nos2 suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. Thus, our comparative analysis of circadian gene expression in SCN and non-SCN cells has important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.

Thesis (Ph.D.)--East Carolina University, 2009. / Presented to the faculty of the Department of Physiology. Advisor: Jian Ding. Title from PDF t.p. (viewed June 12, 2010). Includes bibliographical references.

Thesis (Ph.D.)--Kent State University, 2009. / Title from PDF t.p. (viewed Apr. 15, 2010). Advisor: J. David Glass. Keywords: Suprachiasmatic nucleus; serotonin; nonphotic; arginine vasopressin; hamster. Includes bibliographical references (p. 91-111).

The mammalian brain contains a regulatory center in the diencephalic region known as the hypothalamus that plays a critical role in physiological homeostasis, and contains a variety of centers for behavioral drives, such as hunger and thirst. Located deep within the hypothalamus is the suprachiasmatic nucleus (SCN), or the master biological clock, that organizes rhythmic physiology and behavior, such that critical events take place at the most appropriate time of the day or night and in the most appropriate temporal, phase relationships. Cell-to-cell communication is essential for conveying inputs to and outputs from the SCN. The goal of the present study was to use an immortalized neural cell line (SCN2.2), derived from the presumptive anlage of the rat suprachiasmatic nucleus, as an in vitro model system to study intercellular communication among SCN cells. I tested whether the pineal neurohormone melatonin could modulate cell-to-cell signaling, via both dye coupling (gap junctional communication) and calcium waves (ATP-dependent gliotransmission). I also tested whether extracellular ATP could influence the spread of calcium waves in SCN2.2 cells. Lastly, the ability of extracellular ATP to modulate SCN physiological responses to melatonin in SCN2.2 cells was examined. I show that melatonin at a physiological concentration (nM) reduced dye coupling (gap junctional communication) in SCN2.2 cells, as determined by a scrape loading procedure employing the fluorescent dye lucifer yellow. Melatonin caused a significant reduction in the spread of calcium waves in cycling SCN2.2 cultures as determined by ratiometric calcium imaging with Fura-2 AM, a calcium sensitive indicator dye. This reduction was greatest when an endogenous circadian rhythm in extracellular ATP accumulation, determined by luciferase assay, was at its trough or lowest extracellular concentration. In addition, melatonin and ATP interacted in the regulation of gliotransmission (calcium waves), and this interaction was also specific to particular phases of the endogenous SCN physiological rhythmicity. Thus, I have established that a complex interaction exists between established melatonin signaling pathways and this newly discovered ATP accumulation rhythm, with the mechanisms underlying this relationship linked to endogenous cycling of SCN cellular physiology.

No description available.

Suprachiasmatic nuclei (SCN) is the master circadian pacemaker that generates coordinated rhythms and drives oscillations in other peripheral tissues. Extracellular vesicles (exosomes) have been implicated in cell-to-cell communication and the regulation of circadian clock. However, mammalian clock-derived exosomes have not been characterized. This thesis examine the contents of exosome released from SCN2.2 cells in vitro using a combination of proteomics, next-generation sequencing, and bioinformatic analyses. SCN2.2 cells-derived exosomes, that carry unique microRNAs and proteins, could be taken up by fibroblast cells in vitro. Interestingly, several unique microRNAs and proteins found in SCN2.2 cells-derived exosomes have shown circadian rhythmicity in other cells. In addition, differential expressed microRNAs secreted by SCN cells were also observed outside of exosomes. Taken together, these studies demonstrate that exosomes, containing small RNAs, RNAs and proteins, are released from SCN2.2 cells and likely have a biological role in circadian regulation of metabolism in downstream cells.

How does the brain develop differently to support nocturnality in some mammals, but diurnality in others? To answer this question, one might look to the suprachiasmatic nucleus (SCN), the pacemaker of the mammalian brain, which is required for all circadian biological and behavioral rhythms. Light arriving at the retina entrains the SCN to the daily light-dark cycle via the retinohypothalamic tract (RHT). However, in all mammals studied thus far, whether nocturnal or diurnal, the SCN exhibits a rhythm of increased activity during the day and decreased activity at night. Therefore, structures downstream of the SCN are likely to determine whether a species is nocturnal or diurnal. From an evolutionary perspective, nocturnality appears to be the primitive condition in mammals, with diurnality having reemerged independently in some lineages. However, it is unclear what mechanisms underlie the development of one or the other circadian phase preference. In adult Norway rats (Rattus norvegicus), which are nocturnal, the RHT also projects to the ventral subparaventricular zone (vSPVZ), an adjacent region that expresses an in-phase pattern of SCN-vSPVZ neuronal activity (in other words, activity in the SCN and vSPVZ increase and decrease together). In contrast, in adult Nile grass rats (Arvicanthis niloticus), a diurnal species that is closely related to Norway rats, an anti-phase pattern of SCN-vSPVZ neuronal activity is expressed (in other words, activity in the SCN increases as activity in the vSPVZ decreases, and vice versa). We hypothesized that these species differences in activity pattern result in part from a weak or absent RHT-to-vSPVZ projection in grass rats. Using a developmental comparative approach, we assessed differences in behavior, hypothalamic activity, and RHT and SCN connectivity to the vSPVZ between these two species. We report that a robust retina-to-vSPVZ projection develops in Norway rats around the end of the second postnatal week when nocturnal wakefulness and the in-phase pattern of SCN-vSPVZ activity emerge. In grass rats, however, such a projection does not develop and the emergence of the anti-phase SCN-vSPVZ activity pattern during the second postnatal week is accompanied by increased diurnal wakefulness. When considered within the context of previously published reports on RHT projections in a variety of other nocturnal and diurnal species, our current findings suggest that how and when the retina connects to the hypothalamus differentially shapes brain and behavior to produce animals that occupy opposing temporal niches.

Circadian clocks are responsible for daily rhythms in gastrointestinal function which are vital for normal digestive rhythms and health. The present study examines the roles of the circadian pacemaker, the suprachiasmatic nuclei (SCN), and the sympathetic nervous system in regulation of circadian gastrointestinal rhythms in Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6- hydroxydopamine has no effect on feeding and locomotor rhythmicity, but attenuates stool output rhythms. Again, restricted feeding reestablishes these rhythms. Ex vivo, intestinal tissue from mPer2LUC knockin mice expresses circadian rhythms of luciferase bioluminescence. 6-hydroxydopamine has little effect upon these rhythms, but timed administration of β−adrenergic agonist isoproterenol causes a phase-dependent phase shift in PERIOD2 expression rhythms. Collectively, the data suggest the SCN are required to maintain feeding, locomotor and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the gastrointestinal system.

Molecular, cellular, and physiological processes within an organism are set to occur at specific times throughout the day. The timing of these processes is under control of a biological clock. Nearly all organisms on Earth have biological clocks, ranging from unicellular bacteria and fungi to multicellular plants, insects, reptiles, fish, birds, and mammals. The biological clock is an endogenous time-keeping mechanism that generates the onset of many processes and coordinates the phases of processes over 24 hours. While the biological clock allows these organisms to maintain roughly 24-hour, or circadian, timing in daily processes, many organisms have the ability to set their clocks, or entrain them, to changes in light. In mammals, the suprachiasmatic nucleus (SCN) is the master biological clock that entrains daily physiological and behavioral rhythms to the appropriate times of day and night. The SCN is located in the hypothalamus and contains thousands of neurons and glia that function in coordinating system-level physiological rhythms that are entrained to environmental light cues. Many of these neurons and glia are individual circadian oscillators, and the cellular mechanisms that couple them into ensemble oscillations are emerging. Adenosine triphosphate (ATP) is a transmitter involved in local communication among astrocytes and between astrocytes and neurons. ATP released from astrocytes may play a role in SCN cellular communication and synchrony. Extracellular ATP accumulated rhythmically in the rat SCN in vivo, and ATP released from rat SCN astrocytes in vitro was rhythmic, with a periodicity near 24 hours. ATP released from mouse SCN astrocytes was circadian, and disruption of the molecular clock abolished rhythmic extracellular ATP accumulation. SCN astrocyte cultures with disrupted molecular clocks also had marked reductions in total ATP accumulation compared to SCN astrocyte cultures with functional biological clocks. Furthermore, ATP-induced calcium transients were rhythmic, and this rhythmic purinergic sensitivity was abolished in clock mutant astrocytes. Pharmacological blockade of purinergic signaling, with antagonists of both the P2X7 and P2Y1 receptors, led to a gradual reduction in the amplitude of coordinated ATP accumulation over three days. These purinergic receptor antagonists, as expected, led to a reduction in calcium responses of SCN astrocytes to ATP and led to a dampening of clock gene expression rhythms as determined by PER2::LUC bioluminescence reporting in SCN astrocytes. These data demonstrate that astrocytes of the mammalian SCN rhythmically release ATP and are rhythmically sensitive to ATP in a manner dependent on their intrinsic molecular clock. Ensemble rhythmicity of SCN astrocytes is, in turn, dependent on that rhythmic purinergic signaling via both P2X and P2Y classes of ATP receptors. These results are indicative of a functional role for ATP accumulation within the SCN, with astrocytes releasing ATP every 24 hours for continual signaling onto astrocytes and neurons to maintain daily coordinated synchrony of the clocks in these cells.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.