SPECIFIC AIM I: Using rhythms of gene expression reported by a luciferase transgene, we will identify the signals linking brain and peripheral oscillators.

SPECIFIC AIM II: We will evaluate potential countermeasures by exposing our experimental animals to conditions that disrupt the normal circadian phase map and then determining if the potential countermeasures are able to restore it rapidly.

SPECIFIC AIM III: Investigate the effects of chronic low dose methamphetamine in synchronizing disrupted rhythms and evaluate related drugs as potential countermeasures.

(2) THE KEY FINDINGS OF THE PROJECT

We have extended our studies of the methamphetamine-sensitive circadian oscillator (MASCO) and have discovered a sex difference in the effect of methamphetamine on C3H mice, i.e., females are much less responsive than are males. We have now demonstrated that gonadectomy does not alter the circadian response of either males or females to methamphetamine, and thus sex hormones are unlikely to explain the sex difference. In addition, we have been screening circadian mutant mice to determine if there is a role for canonical clock genes in the effect of methamphetamine on free-running period length. All of the arrhythmic mutant mice screened to date have become rhythmic in the presence of methamphetamine. This demonstrates that MASCO is indeed a distinct brain oscillator which may be involved in the pathological responses of the circadian system to the space environment. We have also extended our study of the phase synchronizing effect of methamphetamine on circadian oscillators. We previously demonstrated that ablation of the SCN results in phase desynchrony between internal oscillators and that methamphetamine administration is capable of restoring coherent phase relationships between peripheral tissues. In the past year we have run another group of animals confirming and elaborating on our prior experiments. We have now included two additional tissues (lung and pituitary) in our analysis and compared the effect of methamphetamine to that of food restriction. The results demonstrate that both methamphetamine and food restriction can restore phase synchrony, in at least some tissues, following SCN lesion. This is particularly exciting because programming meal times is a non-invasive and potentially beneficial way to treat circadian disorganization produced by the space environment.

We have finished our studies of the phase-controlling signals to two internal organs, the ovary and the submaxillary gland. One paper has been published from these data and another is in preparation. To summarize these findings: the ovary responds to pituitary hormones and does not require neural signals; the salivary gland responds to both neural signals and to feeding time, if and only if the neural inputs are cut. This variety of responses suggest that internal circadian organization is normally maintained by many different signals that are specific to particular organs or groups of organs and may be hierarchically organized. Understanding and eventually controlling them will be an important but difficult undertaking.

(3) THE IMPACT OF THESE FINDINGS ON THE HYPOTHESES, OBJECTIVES AND SPECIFIC AIMS OF THE ORIGINAL PROPOSAL

Our project is intended to provide some of the basic data that will be needed to minimize the effects of disrupting the circadian rhythms of astronauts and ground-based support personnel during their missions. Such disruption is an inevitable result of the work schedules that these men and women are required to follow. Disruption of circadian organization, which includes but is not limited to sleep deprivation, has repeatedly been shown to affect alertness, cognition, and performance on a variety of tasks. In order to minimize these effects, we have to understand the organization of the system that is being affected. In particular, we have to identify general signals such as meal timing that are capable of maintaining internal synchrony with minimal associated side effects.

• Methylphenidate and, more importantly, rigid feeding schedules may be useful for temporarily stabilizing circadian phase in the face of disrupting environments.

• Sympathetic nervous system agonists and antagonists of several kinds may be used to modify phase relationships among some peripheral circadian oscillators and thus improve the outcomes of chronic therapeutic treatments of cancer and other chronic conditions (i.e., high blood pressure and irregular heart beat).

• The reproductive cycles--in particular their circadian components--of women engaged in activities with disruptive schedules may be regularized by carefully timed application of LH or FSH.

• Our data suggest that shift work schedules in which cycles are repeatedly delayed will be less damaging to the health of workers than those in which these cycles are repeatedly advanced.

• We have shown that disrupted internal circadian organization can be restored by chronic application of low dose methamphetamine and, more importantly, by rigid timing of meals.

• We have demonstrated that the methamphetamine-sensitive circadian oscillator does not rely on the canonical molecular circadian machinery.

(2) THE KEY FINDINGS OF THE PROJECT

We have extended our studies of the methamphetamine-sensitive circadian oscillator (MASCO) and have discovered a sex difference in the effect of methamphetamine on C3H mice, i.e., females are much less responsive than are males. We have initiated experiments to determine if estrogen is protective, and early results suggest that it is. We have explored the effects of methamphetamine on the internal circadian organization of mice bearing lesions of the suprachiasmatic nucleus (SCN). Untreated mice with such lesions have seriously disrupted phase relationships among internal organs. Treatment with chronic low levels of methamphetamine unexpectedly resynchronizes these internal phase relationships in addition to restoring behavioral rhythmicity, with possible benefits to integrated function. We are currently testing the effects of chronic methamphetamine on rhythmicity disrupted by constant light. These surprising results have led us to ask whether programmed feeding, which may act on the same circadian oscillator as methamphetamine, might also restore internal synchrony to arrhythmic mice. Preliminary results suggest that it does so. This is particularly exciting because programming meal times is a non-invasive and potentially beneficial way to treat circadian disorganization produced by the space environment. We have finished our studies of the phase-controlling signals to two internal organs, the ovary and the submaxillary gland, and have submitted two papers for publication. To summarize these findings: the ovary responds to pituitary hormones but not to neural signals; the salivary gland responds to both neural signals and to feeding time, but only if the neural inputs are cut. This variety of responses suggest that internal circadian organization is normally maintained by many different signals that are specific to particular organs or groups of organs and may be hierarchically organized. Understanding and eventually controlling them will be an important but difficult undertaking. (3) THE IMPACT OF THESE FINDINGS ON THE HYPOTHESES, OBJECTIVES AND SPECIFIC AIMS OF THE ORIGINAL PROPOSAL

Our project is intended to provide some of the basic data that will be needed to minimize the effects of disrupting the circadian rhythms of astronauts and ground-based support personnel during their missions. Such disruption is an inevitable result of the work schedules that these men and women are required to follow. Disruption of circadian organization, which includes but is not limited to sleep deprivation, has repeatedly been shown to affect alertness, cognition, and performance on a variety of tasks. In order to minimize these effects, we have to understand the organization of the system that is being affected. In particular, we have to identify general signals such as meal timing that are capable of maintaining internal synchrony with minimal associated side effects.

(4) THE PROPOSED RESEARCH PLAN FOR THE COMING YEAR

During the coming year we will:

• Expand our study of sex difference in response to methamphetamine.

• Extend and confirm our initial results on the effects of methamphetamine and programmed feeding on disrupted circadian systems.

• Use programmed meals as countermeasures under a variety of circumstances that parallel the conditions to which astronauts are exposed.

• We have identified a hierarchy of circadian phase-shifting signals that influence the rhythmicity in salivary glands. Sympathetic neural inputs are dominant, but can be replaced by feeding schedules if the sympathetic nerves are cut.

• We have shown that the circadian phase of the ovary does not respond to neural signals but does respond to pituitary hormones (LH and FSH). We hypothesize that the circadian oscillator in the ovary influences the timing of ovulation. This is a reasonable hypothesis given our findings, but runs counter to the strongly held prevailing idea that the timing of ovulation is exclusively controlled by the pituitary.

• We have shown that disrupted internal circadian organization can be restored by chronic application of low dose methamphetamine and, more importantly, by rigid timing of meals.

(2) THE KEY FINDINGS OF THE PROJECT Using rat and mouse models, we have shown that the circadian system consists of hierarchically dominant oscillators in the central nervous system (CNS), and subordinate, but quasi-independent oscillators in the peripheral organs and tissues. There are at least 2 separate oscillators in the CNS. One, the suprachiasmatic nucleus (SCN) has been extensively studied. The other we have called the methamphetamine-sensitive circadian oscillator (MASCO) because it only manifests control over easily measured circadian end points when the subject is given chronic methamphetamine (or methylphenidate--Ritalin®). The properties of this second oscillator (MASCO) are not well researched, and we have focused some of our attention on it. MASCO can interact with the SCN oscillator, increasing the activity/rest ratio within a single cycle and lengthening the period of the activity rhythms. It is highly sensitive to the action of some drugs, particularly lithium, which, in combination with methamphetamine, lengthens its period from 26 to about 40 hours. We assume that MASCO is somewhere in the brain, but we don't know where. Our attempts to localize MASCO and elucidate its molecular mechanism are supported by a grant from the NIMH. The NSBRI support will be used to further characterize the effects of MASCO on circadian behavior and the physiology of peripheral circadian oscillators. Circadian oscillators in the periphery are kept in adaptive synchrony by signals from the dominant oscillators in the CNS. These signals are likely to be either neural, humeral or both, but at present, we know very little about them. We have begun to study the detailed relationships between central and peripheral oscillators by severing the neural connections between them. So far we have studied the effects of nerve section on ovaries and submaxillary glands with two quite different results. The phase of the circadian clock in the ovary is unaffected by denervation. This, coupled with experiments that show that ovarian phase can be reset by the pituitary hormones LH and FSH, suggest that phase control in the ovary is entirely hormonal. On the other hand, the phase of the oscillator in the submaxillary gland does not respond to food signals as long as its nerve supply is intact, but when the nerves are cut, the time of feeding becomes the only determinant of its phase. This suggests that it responds primarily to sympathetic neural input and secondarily to unknown circulating factors associated with food intake.

(3) THE IMPACT OF THESE FINDINGS ON THE HYPOTHESES, OBJECTIVES AND SPECIFIC AIMS OF THE ORIGINAL PROPOSAL Our project is intended to provide some of the basic data that will be needed to minimize the effects of disrupting the circadian rhythms of astronauts and ground-based support personnel during their missions. Such disruption is an inevitable result of the work schedules that these men and women are required to follow. Disruption of circadian organization, which includes but is not limited to sleep deprivation, has repeatedly been shown to affect alertness, cognition, and performance on a variety of tasks. In order to minimize these effects, we have to understand the organization of the system that is being affected.

(4) THE PROPOSED RESEARCH PLAN FOR THE COMING YEAR During the coming year we will: • Continue to study the behavioral and physiological responses of the circadian system to methamphetamine, methylphenidate, and lithium. • Extend our studies of neural and humeral coupling mechanisms in the circadian system to other peripheral organs. • Begin the study of potential countermeasures that will minimize circadian disruption by measuring their effects on the coupling between the CNS and the periphery.

• We have described the methamphetamine oscillator in a mouse model and discovered the remarkable synergy between the effects of methamphetamine and lithium on free running period. The circadian effects of methamphetamine (or its analogue, Ritalin®) may be useful in stabilizing the circadian system in disruptive environments [J Biol Rhythms 21(3):185-194, 2006].

• We have shown that temporally restricted feeding alters the phase relationship between the circadian rhythms of healthy liver tissue and cancerous tissue from the same liver. This may be useful in designing chronotherapy regimens for treating cancer (International Journal of Cancer 118: 1623-1627, 2006).

• We have shown that advancing phase shifts, but not delaying phase shifts, shorten the lives of aged mice, and that some, but not all, central and peripheral circadian oscillators shift with different trajectories in old vs. young rats (two manuscripts submitted).

• We have made significant progress in defining the roles of neural and hormonal links in maintaining the integrity of the mammalian circadian system.

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