Tomoko Yoshikawa

Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization.

The phases of central (SCN) and peripheral circadian oscillators are held in specific relationships under LD cycles but, in the absence of external rhythmic input, may damp or drift out of phase with each other. Rats exposed to prolonged constant light become behaviorally arrhythmic, perhaps as a consequence of dissociation of phases among SCN cells. The authors asked whether individual central and peripheral circadian oscillators were rhythmic in LL-treated arrhythmic rats and, if rhythmic, what were the phase relationships between them. The authors prepared SCN, pineal gland, pituitary, and cornea cultures from transgenic Period1-luciferaserats whose body temperature and locomotor activity were arrhythmic and from several groups of rhythmic rats held in LD, DD, and short-term LL. The authors measured mPer1gene expression by recording light output with sensitive photomultipliers. Most of the cultures from all groups displayed circadian rhythms. This could reflect persistent rhythmicity in vivo prior to culture or, alternatively, rhythmicity that may have been initiated by the culture procedure. To test this, the authors cultured tissues at 2 different times 12 h apart and asked whether phase of the rhythm was related to culture time. The pineal, pituitary, and SCN cultures showed partial or complete dependence of phase on culture time, while peak phases of the cornea cultures were independent of culture time in rhythmic rats and were randomly distributed regardless of culture time in arrhythmic animals. These results suggest that in behaviorally arrhythmic rats, oscillators in the pineal, pituitary, and SCN had been arrhythmic or severely damped in vivo, while the cornea oscillator was free running. The peak phases of the SCN cultures were particularly sensitive to some aspect of the culture procedure since rhythmicity of SCN cultures from robustly rhythmic LD-entrained rats was strongly influenced when the procedure was carried out at any time except the 2nd half of the day.

Circadian organization is governed by extra-SCN pacemakers.

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.

Ontogeny of Circadian Organization in the Rat

The mammalian circadian system is orchestrated by a master pacemaker in the brain, but many peripheral tissues also contain independent or quasi-independent circadian oscillators. The adaptive significance of clocks in these structures must lie, in large part, in the phase relationships between the constituent oscillators and their micro- and macroenvironments. To examine the relationship between postnatal development, which is dependent on endogenous programs and maternal/environmental influences, and the phase of circadian oscillators, the authors assessed the circadian phase of pineal, liver, lung, adrenal, and thyroid tissues cultured from Period 1-luciferase (Per1-luc ) rat pups of various postnatal ages. The liver, thyroid, and pineal were rhythmic at birth, but the phases of their Per1-luc expression rhythms shifted remarkably during development. To determine if the timing of the phase shift in each tissue could be the result of changing environmental conditions, the behavior of pups and their mothers was monitored. The circadian phase of the liver shifted from the day to night around postnatal day (P) 22 as the pups nursed less during the light and instead ate solid food during the dark. Furthermore, the phase of Per1-luc expression in liver cultures from nursing neonates could be shifted experimentally from the day to the night by allowing pups access to the dam only during the dark. Peak Per1-luc expression also shifted from midday to early night in thyroid cultures at about P20, concurrent with the shift in eating times. The phase of Per1-luc expression in the pineal gland shifted from day to night coincident with its sympathetic innervation at around P5. Per1-luc expression was rhythmic in adrenal cultures and peaked around the time of lights-off throughout development; however, the amplitude of the rhythm increased at P25. Lung cultures were completely arrhythmic until P12 when the pups began to leave the nest. Taken together, the data suggest that the molecular machinery that generates circadian oscillations matures at different rates in different tissues and that the phase of at least some peripheral organs is malleable and may shift as the organs function changes during development.

Retinal pathways influence temporal niche

In mammals, light input from the retina entrains central circadian oscillators located in the suprachiasmatic nuclei (SCN). The phase of circadian activity rhythms with respect to the external light:dark cycle is reversed in diurnal and nocturnal species, although the phase of SCN rhythms relative to the light cycle remains unchanged. Neural mechanisms downstream from the SCN are therefore believed to determine diurnality or nocturnality. Here, we report a switch from nocturnal to diurnal entrainment of circadian activity rhythms in double-knockout mice lacking the inner-retinal photopigment melanopsin (OPN4) and RPE65, a key protein used in retinal chromophore recycling. These mice retained only a small amount of rod function. The change in entrainment phase of Rpe65−/−;Opn4−/− mice was accompanied by a reversal of the rhythm of clock gene expression in the SCN and a reversal in acute masking effects of both light and darkness on activity, suggesting that the nocturnal to diurnal switch is due to a change in the neural response to light upstream from the SCN. A switch from nocturnal to diurnal activity rhythms was also found in wild-type mice transferred from standard intensity light:dark cycles to light:dark cycles in which the intensity of the light phase was reduced to scotopic levels. These results reveal a novel mechanism by which changes in retinal input can mediate acute temporal-niche switching.

Suprachiasmatic nucleus: cellular clocks and networks.

The suprachiasmatic nucleus (SCN), the master circadian clock of mammals, is composed of multiple circadian oscillator neurons. Most of them exhibit significant circadian rhythms in their clock gene expression and spontaneous firing when cultured in dispersed cells, as well as in an organotypic slice. The distribution of periods depends on the SCN tissue organization, suggesting that cell-to-cell interaction is important for synchronization of the constituent oscillator cells. This cell-to-cell interaction involves both synaptic interactions and humoral mediators. Cellular oscillators form at least three separate but mutually coupled regional pacemakers, and two of them are involved in the photoperiodic regulation of behavioral rhythms in mice. Coupling of cellular oscillators in the SCN tissue compensates for the dysfunction due to clock gene mutations, on the one hand, and desynchronization within and between the regional pacemakers that suppresses the coherent rhythm expression from the SCN, on the other hand. The multioscillator pacemaker structure of the SCN is advantageous for responding to a wide range of environmental challenges without losing coherent rhythm outputs.

A circadian egg timer gates ovulation

Summary Since the pioneering work of Everett and Sawyer, the idea that pituitary gonadotrophins provide the critical timing cue for ovulation has remained unquestioned [1]. It is widely accepted that the timing of ovulation depends entirely on the timing of luteinizing hormone (LH) secretion, itself driven by neuroendocrine releasing factors controlled by the circadian clock in the suprachiasmatic nucleus (SCN) [2,3]. As a consequence, there has been little investigation of a role for the ovary in this process. However, we and others have demonstrated the presence of endogenous circadian clocks in the rat ovary [4–6]. Here we describe a circadian rhythm of ovarian sensitivity to LH that determines the ovulatory response to gonadotrophins. It is plausible that the circadian clock in the ovary may set the responsiveness of the ovarian follicle to the LH surge. Our results significantly alter the classic view that gonadotrophins provide the only timing cue for ovulation. They suggest that the ovary itself plays a major role in the process and provide a new perspective that will inform future research on infertility and ovarian physiology.

CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIα weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

Spatiotemporal profiles of arginine vasopressin transcription in cultured suprachiasmatic nucleus

Arginine vasopressin (AVP), a major neuropeptide in the suprachiasmatic nucleus (SCN), is postulated to mediate the output of the circadian oscillation. Mice carrying a reporter gene of AVP transcription (AVPELuc) were produced by knocking‐in a cDNA of Emerald‐luciferase (ELuc) in the translational initiation site. Homozygous mice did not survive beyond postnatal day 7. Using the heterozygous (AVPELuc/+) mice, a bioluminescence reporter system was developed that enabled to monitor AVP transcription through AVP‐ELuc measurement in real time for more than 10 cycles in the cultured brain slice. AVPELuc/+ mice showed circadian behaviour rhythms and light responsiveness indistinguishable from those of the wild‐type. Robust circadian rhythms in AVP‐ELuc were detected in the cultured SCN slice at a single cell as well as tissue levels. The circadian rhythm of the whole SCN slice was stable, with the peak at the mid‐light phase of a light–dark cycle, while that of a single cell was more variable. By comparison, rhythmicity in the paraventricular nucleus and supraoptic nucleus in the hypothalamus was unstable and damped rapidly. Spatiotemporal profiles of AVP expression at the pixel level revealed significant circadian rhythms in the entire area of AVP‐positive cells in the SCN, and at least two clusters that showed different circadian oscillations. Contour analysis of bioluminescence intensity in a cell‐like region demonstrated the radiation area was almost identical to the cell size. This newly developed reporter system for AVP gene expression is a useful tool for the study of circadian rhythms.

Daily exposure to cold phase-shifts the circadian clock of neonatal rats in vivo

Maternal rhythms entrain the prenatal and neonatal circadian clock in the suprachiasmatic nucleus (SCN) before light entrainment is established. However, the responsible time cues for maternal entrainment are not identified. To examine the role of cyclic changes of ambient temperature in maternal entrainment, blind neonatal rats carrying a clock gene (Per2) bioluminescence reporter were exposed to either of three ambient temperatures (10, 20 or 30 °C) during 6‐h maternal separation in the early light phase. Cold exposure was performed from postnatal day 1 (P1) to P5. On P6, the SCN was harvested and cultured for photometric monitoring of the circadian rhythm in Per2 expression. Here we demonstrate that the daily cold exposure phase‐delayed the circadian Per2 expression rhythms at P6 in a temperature‐dependent manner. Exposure to 10 °C produced the largest phase‐shift of 12.7 h, and exposure to 20 and 30 °C yielded moderate shifts of 4.1 and 4.5 h, respectively. There was no significant difference in the phase‐shifts between the latter two temperatures, indicating that ambient temperature is not the sole factor for the phase‐shift. Behavioral rhythms that developed after weaning reflected the phase‐shift of clock gene expression rhythm in the SCN. These findings indicate that a daily exposure to an ambient temperature of 10 °C during the neonatal period is capable of resetting the circadian clock in the SCN, but other factors yet unidentified are also involved in maternal entrainment.

Localization of photoperiod responsive circadian oscillators in the mouse suprachiasmatic nucleus

The circadian pacemaker in the suprachiasmatic nucleus (SCN) yields photoperiodic response to transfer seasonal information to physiology and behavior. To identify the precise location involved in photoperiodic response in the SCN, we analyzed circadian Period1 and PERIOD2 rhythms in horizontally sectioned SCN of mice exposed to a long or short day. Statistical analyses of bioluminescence images with respective luciferase reporters on pixel level enabled us to identify the distinct localization of three oscillating regions; a large open-ring-shape region, the region at the posterior end and a sharply demarcated oval region at the center of the SCN. The first two regions are the respective sites for the so-called evening and morning oscillators, and the third region is possibly a site for mediating photic signals to the former oscillators. In these regions, there are two classes of oscillating cells in which Per1 and Per2 could play differential roles in photoperiodic responses.