Fred Turek - US grants

The annual change in daylength is the major environmental factor regulating the timing of reproduction in many seasonally breeding mammals. The overall objective of the proposed research is to elucidate the neural and endocrine events by which light alters the hypothalamic- pituitary-gonadal axis. Although it is well established that a circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN) plays a central role in measuring the length of the day, little is known about the functional relationship between the operating characteristics of this clock and the photoperiodic reproductive response. In addition, the neuropeptides/neurotransmitters within the circadian clock system responsible for mediating the effects of light on reproduction have not been identified. Recent research efforts have provided novel experimental approaches for altering the circadian clock and/or the photoperiodic response, offering unique opportunities for addressing these issues. The proposed studies will exploit these advances to delineate the mechanisms relating circadian time-keeping to the photoperiodic reproductive response. Three separate experimental strategies involving three different rodent species will be utilized; each species has been chosen because of the advantages it offers in addressing specific questions about the functional relationship between the circadian and reproductive systems. These experiments will involve male rodents, and the circadian rhythm of locomotor activity will be used as a marker of the state of the circadian clock. Serum LH, FSH and testosterone levels, as well as testicular size, will be monitored to assay the state of the reproductive system. In one series of experiments, the period of the circadian clock of golden hamsters will be altered following pharmacological manipulation, during genetic selection or naturally during senescence, to test the hypothesis that changes in circadian period will alter the critical daylength necessary for the stimulation of neuroendocrine-gonadal activity. In a second series of experiments, the photoperiodic reproductive response of Djungarian hamsters will be altered by either genetic or environmental means, to determine if the circadian clock has also been altered. Finally, the role of vasopressin and/or serotonin in he seasonal reproductive response will be determined by examining the photoperiodic response in the Brattleboro rat which is unable to produce vasopressin, or in golden hamsters with lesions of the serotoninergic input to the SCN. It is anticipated that these studies will further delineate the mechanisms responsible for the control of fertility and offer new insights into the effects of light on the reproductive and circadian systems. the completion of these experiments should also provide a better understanding of the physiological and ecological importance of aging for the photoperiodic reproductive response, and of the role played by genetic factors in this response. These studies are anticipated to lead to novel approaches for manipulating seasonal rhythms through pharmacological and non-pharmacological interventions. Such interventions could be important for regulating the seasonal reproductive cycle of economically useful animals as well as for the treatment of various mood disorders that have been associated with seasonal rhythms in humans.

Many living systems including humans express physiological and behavioral rhythms that vary with a 24-hour period. These rhythms are regulated by an internal circadian clock that is synchronized to the environmental light-dark cycle. Efforts to elucidate the physiological mechanisms underlying the generation of circadian rhythms in mammal have focused on the suprachiasmatic nucleus (SCN) of the hypothalamus as a site of a circadian pacemaker. However, little information exists concerning the cellular and molecular mechanisms responsible for the generation of circadian rhythms in the SCN. Our long term objectives are to understand the cellular and biochemical events that underlie circadian rhythms in mammals. The mechanisms that generate circadian rhythms will be studied in vivo using microinjection of pharmacological agents directly into the SCN of hamsters as a model for investigating the role of protein synthesis in a mammalian circadian pacemaker. The site of action of protein synthesis inhibitors on the circadian oscillator will be localized by microinjection of these agents into specific brain regions. We will test the hypothesis that protein synthesis inhibitors alter circadian rhythmicity by a direct action on SCN cells. The phase dependence of the effects of protein synthesis inhibitors in the SCN will be determined. The pharmacological specificity of protein synthesis inhibitors will be defined by examining the effectiveness of other inhibitors that have different molecular mechanisms of action, inactive analogues, and other agents that control for the known side effects of these inhibitors. Finally, the correlation between the amount of protein synthesis inhibition and the magnitude of perturbation of the circadian pacemaker will be studied. Taken together, these experiments would establish that protein synthesis is required for the generation of mammalian circadian oscillations and provide a foundation for future work to identify the proteins and elucidate their function. Although there is a fairly extensive literature in the field of circadian rhythms on the effects of protein synthesis inhibitors, there are no studies in a vertebrate preparation. The elucidation of the role of protein synthesis should aid ultimately in the search for the molecular components of the circadian clock. An understanding of the biological basis of circadian rhythms in mammals may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunction such as sleep disorders, mental health and endocrine abnormalities.

This proposal is a request for partial support of the Second Meeting of the Society for Research on Biological Rhythms at Amelia Island Plantation, Florida on May 9-13, 1990. The conference will cover a number of topics in the area of rhythms including neural and cellular oscillators, pulsatile and ultradian rhythms, circadian rhythms and annual cycles. The levels of research interests range from molecular genetics, biochemistry, and cell biology to physiology, neuroscience, and clinical applications. This broad coverage of rhythmic phenomena in living systems is a reflection of the goals of the Society which are to promote the advancement of scientific research in all aspects of biological rhythms, to disseminate research results concerning biological rhythms to the general public, to develop and enhance the education and training of students and researchers in this field, and to foster interdisciplinary communication. The organization of the conference will be similar to the First Meeting of the Society which was held at Wild Dunes, South Carolina on May 11-14, 1988, except that the meeting will be extended to three full days in 1990. The meeting will have symposia, workshops, oral presentations and poster presentations. there will be six major symposia on topics of broad appeal featuring invited speakers with two symposia held each morning for three days. The symposia are scheduled to cover the following topics: 1) Mutations to study molecular mechanisms of rhythms; 2) Shift work; 3) Cellular models of circadian rhythms; 4) Mechanisms of photoperiodism; 5) Interval and developmental timing; and 6) Mammalian pacemakers: is the SCN the only one? Approximately twelve workshops will be held on various specialized topics. Approximately twelve slide presentation sessions will be held from submitted abstracts and poster presentation sessions will be held from submitted abstracts. The first meeting of the Society was a great success with almost 300 participants attending. With the continued growth of the Society with over 300 members from sixteen countries, we expect the second meeting to exceed the attendance of the first meeting.

Although a variety of stimuli have been identified which can alter the circadian clock system of young animals, little is known about how aging alters the response of the clock to external and internal environmental stimuli. Preliminary data in the golden hamster demonstrate that the response of the circadian clock system to the primary external environmental synchronizing agent, light, is dramatically altered in old age (i.e. > 18 months of age). In addition, with advanced age the circadian clock in this species appears to become unresponsive to feedback signals about the activity-rest state. The overall objectives of the proposed studies are, 1) to determine in detail how the response of the circadian clock system to light or changes in the activity-rest state is altered in old age, and 2) to determine the physiological basis for age- related changes in the response of the circadian clock to environmental stimuli. To examine the effects of aging on the response of the circadian system to various stimuli, the circadian rhythm of locomotor activity in golden hamsters will be used as a marker rhythm for the state of the circadian clock located in the suprachiasmatic nucleus (SCN) that underlies most behavioral and physiological rhythms in this species. With respect to age- related changes in the response of the clock to light, a series of experiments will be carried out to determine 1) the extent to which aging alters both the phase-shifting and entraining effects of light, and 2) whether age-related alterations in the response to light are due to changes in the reception of photic information or to changes at the level of the SCN. Whether age-related changes in the response of the clock to light are associated with an alteration in light-induced protein synthesis within the SCN will also be determined. With respect to age-related changes in the response of the clock to signals about the activity-rest state, a series of experiments will be carried out to determine 1) if the circadian clock of old animals is completely insensitive to the phase shifting effects of various stimuli that induce acute changes in the activity state, as well as phase shifts in the circadian clock of young animals, and 2) the age at which the circadian clock becomes totally or partially insensitive to the phase-shifting effects of feedback signals about the activity-rest state of the animal. In view of recent findings indicating that norepinephrine may be involved in mediating the effects of stimulated activity on the circadian clock, a series of experiments will be carried out to determine if age-related changes in central noradrenergic activity underlie the loss of responsiveness to such stimuli with advanced age, and whether the response of the circadian clock to such stimuli can be restored by increasing central adrenergic activity in old animals. In addition to providing new insight into how the response of the circadian clock system to external and internal stimuli is altered with age, these findings may lead to new clinical approaches for treating disorders in the elderly that are associated with a disruption of circadian rhythmicity.

Many organisms, including humans, express physiological and behavioral rhythms that vary with a 24-hour period. These rhythms are regulated by an internal circadian clock that is synchronized to the physical environment by external stimuli. Efforts to elucidate the physiological mechanisms underlying the generation of circadian rhythms in mammals have focused on the suprachiasmatic nucleus (SCN) of the hypothalamus as a site of a circadian pacemaker. However, little information exists concerning the cellular and molecular mechanisms responsible for the generation of neural circadian rhythms in the SCN. Recent findings have demonstrated that inhibition of protein synthesis can lead to phase shifts in the circadian clock of mammals and can alter the response of the clock to the phase- shifting effects of light. The experiments outlined in this proposal will use in vivo and in vitro approaches to explore in detail the role of protein synthesis in the generation of circadian rhythms and in mediating the effects of both photic and non-photic stimuli on the mammalian circadian clock. In in vivo studies using the golden hamster, protein synthesis inhibitors will be used to determine when protein synthesis is required for light to be able to induce phase shifts in the circadian clock. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate out proteins in SCN tissue of hamsters injected with 35-S- methionine, proteins produced at specific times of the circadian day and/or in response to various stimuli that can induce phase shifts in the circadian clock (e.g., pulses of light or darkness, exposure to a novel running wheel or treatment with short-acting benzodiazepines) will be identified. These studies should identify proteins that 1) express circadian oscillations, 2) are produced in response to light or other stimuli that can induce phase shifts in the circadian clock and 3) are actually associated with the phase shifts induced by these stimuli. In addition, a new in vitro system involving dispersed rat SCN cells in culture has demonstrated that SCN cells can produce a circadian rhythm in vasopressin release in vitro. This system will be utilized to identify specific proteins that are produced by SCN cells in culture and to identify common proteins that are produced by the SCN under in vitro and in vivo conditions. Taken together, the results from the proposed studies should provide new information on the role of protein synthesis in the entrainment and generation of circadian rhythms in mammals and should ultimately lead to an understanding of the molecular components of the mammalian circadian clock. An understanding of the biological basis of circadian rhythms in animals may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunction that have been observed in sleep disorders, mental illness and/or endocrine abnormalities.

The first (and only) single gene mutation in a mammalian circadian system has recently been discovered in the golden hamster, this mutation alters the period of the circadian clock (the endogenous period under free- running conditions is reduced from the normal 24 hrs to about 20 hrs) and the response of the clock to the phase shifting effects of light and activity-inducing stimuli. These effects on the circadian clock are associated with ,.about a 50% decrease in the lifespan of the mutant hamsters. The overall objectives of the proposed studies are to determine 1) if decreased longevity in tau mutant hamsters is due to genetic or environmental factors, 2) if age-related changes in the circadian clock system occur at an earlier age in tau mutant animals, 3) what effect the tau mutation has on the timing of age-related changes in the circadian clock system, and 4) if longevity is reduced in the tau mutant animal because of the mutation's effects on the circadian clock itself. To reach these objectives, the circadian rhythm of locomotor activity will be used as a marker rhythm for the state of the circadian clock located in the suprachiasmatic nucleus (SCN) that underlies most behavioral and physiological rhythms in mammals. In one series of experiments, male and female wild-type and tau mutant hamsters will be exposed to either constant light (LL) or light-dark (LD) cycles in the two circadian ranges (i.e. 20 or 24-hr LD cycles) to determine if the decreased lifespan in the mutant animals is due to an intrinsic genetic effect on longevity or to the inability of the circadian clock of mutant animals to entrain normally to the standard 24-hr light cycle. In a second series of experiments, the effects of age on 1) the period of the clock, 2) the phase relationship of the activity rhythm to entraining light-dark cycles, 3) the rate of reentrainment following phase shifts in the light-dark cycle, and 4) the response of the circadian clock to light pulses or to a variety of activity-inducing stimuli will be determined in wild-type and tau mutant hamsters. The results of these studies will establish whether a mutation in the circadian system has an effect on normal age-related changes in the circadian clock system, as well as whether such changes occur at an earlier age in the mutant hamsters that corresponds in time with the decreased lifespan of the animals. In a third series of experiments, the effects of destruction of the circadian clock in the SCN on longevity will be established in order to determine if changes in the clock itself are responsible for the reduced lifespan of the mutant animals. A final series of experiments involving the transplantation of fetal SCN tissue from both wild-type and mutant hamsters to animals of the same or different genotype will be carried out to determine the role of transplanted SCN tissue in the restoration of circadian function in old hamsters. In view of the central role played by the circadian clock in the regulation of diverse biological systems, age-related changes in the clock can be expected to have a major impact on the health of the organism. A variety of circadian abnormalities have been found in elderly humans, including those suffering from Alzheimer's disease and disturbances of the sleep-wake cycle. Studies on how circadian mutations affect age-related changes in the circadian clock and whether circadian disturbances induced by mutations affect aging itself, may provide new information on the underlying physiological bases for age-related dysfunction within the circadian system and how altered clock function may contribute to the aging process.

Many organisms, including humans, express physiological and behavioral rhythms that vary with a 24-hour period. These rhythms are regulated by an internal circadian clock that is synchronized to the physical environment by external stimuli. Efforts to elucidate the physiological mechanisms underlying the generation of circadian rhythms in mammals have focused on the suprachiasmatic nucleus (SCN) of the hypothalamus as a site of a circadian pacemaker. However, little information exists concerning the cellular and molecular mechanisms responsible for the generation of neural circadian rhythms in the SCN. Recent findings have demonstrated that inhibition of protein synthesis can lead to phase shifts in the circadian clock of mammals and can alter the response of the clock to the phase- shifting effects of light. The experiments outlined in this proposal will use in vivo and in vitro approaches to explore in detail the role of protein synthesis in the generation of circadian rhythms and in mediating the effects of both photic and non-photic stimuli on the mammalian circadian clock. In in vivo studies using the golden hamster, protein synthesis inhibitors will be used to determine when protein synthesis is required for light to be able to induce phase shifts in the circadian clock. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate out proteins in SCN tissue of hamsters injected with 35-S- methionine, proteins produced at specific times of the circadian day and/or in response to various stimuli that can induce phase shifts in the circadian clock (e.g., pulses of light or darkness, exposure to a novel running wheel or treatment with short-acting benzodiazepines) will be identified. These studies should identify proteins that 1) express circadian oscillations, 2) are produced in response to light or other stimuli that can induce phase shifts in the circadian clock and 3) are actually associated with the phase shifts induced by these stimuli. In addition, a new in vitro system involving dispersed rat SCN cells in culture has demonstrated that SCN cells can produce a circadian rhythm in vasopressin release in vitro. This system will be utilized to identify specific proteins that are produced by SCN cells in culture and to identify common proteins that are produced by the SCN under in vitro and in vivo conditions. Taken together, the results from the proposed studies should provide new information on the role of protein synthesis in the entrainment and generation of circadian rhythms in mammals and should ultimately lead to an understanding of the molecular components of the mammalian circadian clock. An understanding of the biological basis of circadian rhythms in animals may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunction that have been observed in sleep disorders, mental illness and/or endocrine abnormalities.

Although a variety of stimuli have been identified which can alter the circadian clock system of young animals, little is known about how aging alters the response of the clock to external or internal environmental stimuli. Preliminary data in the golden hamster demonstrate that the response of the circadian clock system to light and feedback signals from the activity-rest cycle are dramatically altered in old age (i.e. >15 months of age). The overall objectives of the proposed studies are: 1) to more fully delineate the effects of age on the circadian clock system, 2) to determine the role of monoamines in age-related changes in the characteristics and response of the circadian clock to environmental stimuli, and 3) to determine the extent to which circadian properties can be restored in advanced age. To examine the effects of aging on the response of the circadian system to various stimuli, the circadian rhythm of locomotor activity in golden hamsters will be used in all studies as a marker rhythm for the state of the circadian clock located in the suprachiasmatic nucleus (SCN) that underlies most behavioral and physiological rhythms in this species. One series of studies will examine the effects of age on 1) entrainment to various light-dark cycles, 2) the response to various activity-inducing stimuli, and 3) the feedback effects of endogenous activity on the clock. In examining the role of monoamines in age-related changes in the circadian system, the effects of age on monoaminergic inputs and neurotransmission in the region of the SCN and intergeniculate leaflet (IGL) of the thalamus will be determined using immunohistochemical and in vivo microdialysis techniques. The hypothesis that age-related changes in the circadian clock system can be reversed by pharmacologically-induced changes in brain monoaminergic neurotransmission will also be tested. Preliminary studies indicate that transplantation of fetal SCN tissue into the brains of old animals can reverse age-related changes in the response of the clock to some environmental stimuli, and further studies will be carried out to determine the extent of this restored function. In addition, the hypothesis that age-related changes in the circadian clock can be prevented or reversed by increasing the strength of the entraining light- dark cycle will also be tested. The rhythms of drinking behavior and body temperature will also be monitored in those studies that seek to reverse the effects of aging on the circadian clock system. It is anticipated that the elucidation of the physiological basis for age-related changes in the responsiveness of the clock, and the mechanisms which underlie the ability of various experimental manipulations to restore normal circadian function to old animals, will lead to new insight into the mechanisms by which environmental stimuli alter clock function. Furthermore, determining how the response of the circadian clock system to external and internal stimuli is altered with age, may lead to new clinical approaches for treating disorders in the elderly that are associated with a disruption of circadian rhythmicity.

The overall objective of the proposed studies is to use experimental genetic approaches that are available in the mouse to identify genetic, and thus molecular, elements that underlie the control of sleep and wakefulness. To reach this objective, two general strategies will be taken. One strategy will involve the use of the recently discovered Click gene which, when mutated, affects the period and expression of the circadian clock underlying the rest-activity and sleep-wake cycles and possibly also the total amount of sleep in mice. Clock represents the first mammalian circadian clock mutation show to affect sleep. Sleep EEG activity will be recorded from mice of three different Click genotypes (wild-type, heterozygotes, and homozygotes) under both entrained and free- running conditions, as well as following periods of sleep deprivation, to determine how this gene regulates both the circadian and homeostatic processes underlying sleep and wakefulness, as well as the interactions between the sleep and circadian systems. The second strategy will involve the use of two different forward genetic approaches to find new genes that are involved in sleep regulation. One approach will use inbred strains of mice to determine the effects of different genetic backgrounds on sleep, and through the use of Quantitative Trait Loci (QTL) analysis identify linkage of chromosomal regions with sleep EEG phenotypes. Isolating chromosomal regions containing candidate sleep regulatory loci on a congenic strain background will allow the effects of each locus to be tested individually and will provide specific regions to be targeted for genetic mapping and gene identification. The second forward genetic strategy will utilize a chemical mutagenesis screen, successfully used to identify Clock, to create mutant animals with an altered phenotypic response of recovery sleep following sleep deprivation. Animals showing an unusual recovery time will be bred and their offspring used to completely characterize the phenotype and genotype of the mutation. Ultimately, positional cloning techniques will be used to identify the mutated genes underlying the homeostatic control of sleep. Determining the molecular mechanisms by which Clock (and its protein product) regulates both the timing and the need for sleep, and the identification of new genes involved in sleep regulation, will provide new information on the genetic and molecular control of sleep. Such information is expected to lead to new treatments for sleep disorders, mental and physical disorders associated with sleep-wake abnormalities, as well as for strategies to influence human fatigue and alertness.

DESCRIPTION (from applicant's abstract): The annual change in day length is the major environmental factor regulating the timing of reproduction in many seasonally breeding mammals. Both the circadian clock located in the hypothalamic suprachiasmatic nucleus and the pineal melatonin rhythm play a central role in relaying information about length of the day to the neuroendocrine-gonadal axis. Despite the fact that a variety of age-related changes in the circadian clock system have now been well documented in mammals, including changes in the response to the entraining effects of the light dark cycle and dampening of the pineal melatonin rhythm, very little is known about how age-related changes in circadian function may impact on the response of seasonally breeding animals to changes in day length. In the proposed studies, golden and Siberian hamsters will be utilized to test various hypotheses regarding 1) the effects of age on the photoperiodic control of neuroendocrine-gonadal activity and the circadian clock system, 2) how aging alters the interactions between the various components of the photoperiodic time measuring system and 3) how day length influences the effects of age on circadian rhythmicity. For all studies the rhythm of locomotor activity will be used as a marker for the state of the circadian clock system. Serum LH, FSH and testosterone levels, as well as testicular size, will be monitored to assay the state of the reproductive system, and in some studies serum melatonin levels will be determined. Various studies are proposed to determine how aging may effect 1) the critical day length for the photoperiodic response and the entrainment to different light-dark cycles, 2) the pattern of melatonin secretion, 3) the response of the neuroendocrine-gonadal axis to melatonin, 4) the sensitivity of the neuroendocrine-gonadal axis to light intensity and 5) feedback relationships in the circadian clock system and in the neuroendocrine-gonadal axis. Other studies will determine if age-related changes in the photoperiodic response can be blocked by stimuli that alter the circadian temporal organization. Taken together, the proposed studies are expected to yield new information about the physiological mechanisms that underlie age related changes in the photoperiodic response. Age related changes in the way hamsters respond to changes in the length of the day offer new avenues for elucidating the physiological and cellular mechanisms that underlie the generation of circadian rhythms as well as the photic control of the hypothalamic-pituitary-gonadal axis. In view of recent demonstrations that the pattern of human endocrine rhythms is also influenced by the length of the day, the results of the proposed studies are expected to increase our understanding of how age-related changes in the human neuroendocrine system may be affected by the seasonal change in day length; such information may also lead to new treatments of mood disorders that have been associated with seasonal rhythms.

One of the most prominent annual rhythms expressed in animals is the photoperiod-dependent seasonal change in reproductive activity. While much progress has been made in understanding the physiological mechanisms underlying the photoperiodic control of reproduction at the system's level, very little is known about the cellular and molecular events within the central nervous system and the hypothalamic-pituitary axis that underlie the transition between the reproductively active and inactive states. The male Siberian hamster is an attractive model for such mechanistic studies for three reasons: 1) pronounced changes in reproductive neuroendocrine function can be induced within 1-2 days of photostimulation, 2) follicle stimulating hormone (FSH) is selectively synthesized and released following photostimulation, and 3) gonadotropin secretion can be induced in males housed in long photoperiod following exposure to a female, but this induction is inhibited in males exposed to short photoperiods. The overall objectives of the proposed project are to elucidate the early cellular and molecular events that underlie the photic induction of increased hypothalamic gonadotropin releasing hormone (GnRH) and pituitary FSH content and to determine how the photoperiod alters the processing of afferent signals in response to exposure to a female that ultimately influences pituitary gonadotropin release. A variety of experimental techniques (i.e., manipulation of hormone and neurotransmitter system by infusion of exogenous hormone or treatment with agonists or antagonists, detection of intracellular changes in mRNA and protein content using ribonuclease protection assays, quantitative rtPCR, in situ hybridization, immunocytochemistry, and radioimmunoassay) will be employed to achieve these objectives. The proposed studies are expected to yield new information about the fundamental mechanisms by which environmental information is integrated to regulate the activity of the hypothalamic-pituitary-gonadal axis and are expected to lead to an improved understanding of fertility and infertility. In view of the increasing evidence that both photoperiod and chemosensory signals can influence human reproduction, the proposed studies are also expected to lead to new insights into the fundamental mechanisms by which human reproduction can be influenced by environmental factors.

The overall objectives of the proposed experiments in this application are to develop the mouse as an animal model for elucidating the genetic and molecular mechanisms for sleep regulation and to tet specific hypotheses regarding the importance of the genetic background and physiological state of both male and female mice in the regulation of sleep as well as the effects of stress on sleep. Stress can have major disruptive effects on sleep as well as the circadian clock, which itself plays a fundamental role in the regulation of sleep. Both genetic and environmental factors contribute to the regulation of sleep as well as to how the sleep and circadian clock systems of any one individual will be affected by stress. In the proposed studies, the effects in mice of acute stress, chronic mild stress, and short- term sleep deprivation on sleep and circadian rhythms will be determined. Mice with genetic differences will be studied. These genetic differences will be polygenic for some studies, or will test the roles of single candidate genes in others. In view of gender differences in both sleep and the response to stress that have been observed in other species, primarily in humans, the proposed studies will include females in different stages of the estrous cycle and after the age-related loss in cyclicity. An additional objective of this proposal is to develop and validate the use of a novel automated sleep analysis system in the mouse. It is anticipated that these studies will contribute to an understanding of the mechanisms whereby stress impacts sleep regulation. The results of these studies also will form a critical basis for use of the tools of mouse genetics to identify genetic and physiological factors which may influence sleep disruption. Such information is likely ultimately to lead to more effective interventions for humans who suffer from sleep disturbances due to stress or related factors, and may lead to new treatments for mental and physical disorders associated with sleep-wage abnormalities.

The overall objectives of the proposed experiments in this application are to develop the mouse as an animal model for elucidating the genetic and molecular mechanisms for sleep regulation and to tet specific hypotheses regarding the importance of the genetic background and physiological state of both male and female mice in the regulation of sleep as well as the effects of stress on sleep. Stress can have major disruptive effects on sleep as well as the circadian clock, which itself plays a fundamental role in the regulation of sleep. Both genetic and environmental factors contribute to the regulation of sleep as well as to how the sleep and circadian clock systems of any one individual will be affected by stress. In the proposed studies, the effects in mice of acute stress, chronic mild stress, and short- term sleep deprivation on sleep and circadian rhythms will be determined. Mice with genetic differences will be studied. These genetic differences will be polygenic for some studies, or will test the roles of single candidate genes in others. In view of gender differences in both sleep and the response to stress that have been observed in other species, primarily in humans, the proposed studies will include females in different stages of the estrous cycle and after the age-related loss in cyclicity. An additional objective of this proposal is to develop and validate the use of a novel automated sleep analysis system in the mouse. It is anticipated that these studies will contribute to an understanding of the mechanisms whereby stress impacts sleep regulation. The results of these studies also will form a critical basis for use of the tools of mouse genetics to identify genetic and physiological factors which may influence sleep disruption. Such information is likely ultimately to lead to more effective interventions for humans who suffer from sleep disturbances due to stress or related factors, and may lead to new treatments for mental and physical disorders associated with sleep-wage abnormalities.

Seeinstructions): Chronic partial sleep restriction (CPSR) is a hallmark of life in modern human society however, only recently have the consequences of CPSR for chronic human illnesses such as obesity and diabetes come to be fully appreciated. Despite the growing awareness of the importance of sleep as a factor in human health and in the development of age-related diseases, few animal models have been developed to systematically determine how aging effects the consequences of CPSR on the sleep-wake regulatory system itself and on other physiological processes, such as energy metabolism and circadian rhythms. During the previous funding period, we developed a model of CPSR that led to the exciting discovery that after only a few days of CPSR, both young and old rats no longer exhibited a homeostatically regulated sleep recovery pattern;they failed to generate increases in NREM EEG delta power and NREM sleep time. In addition, we demonstrated an impact of CPSR on leptin and glucocorticoid concentrations, glucose tolerance, and insulin responsiveness in young rats. These data led us postulate a novel hypothesis that repeated episodes of sleep restriction result in the transition from a homeostatic to an allostatic state of sleep regulation that may lead to adaptive changes in the sleep-wake and other physiological systems to cope with the immediate environmental challenge inducing sleep loss. However, such initially adative changes could have adverse effects if the allostatic response to CPSR is maintained for a prolonged period of time. In the proposed studies, we will systematically examine this working model in two Specific Aims (1) Test the hypothesis that aging accelerates the transition from homeostatis to allostasis during CPSR. Specifically, we will determine if aging impacts the severity and duration of sleep restriction necessary to cause the transition from a homeostatic to an allostatic sleep regulatory state (2) Test the hypothesis that aging alters the effects of CPSR on key hormonal and molecular components of energy metabolism and circadian clock gene expression in the CMS and peripheral tissues, and that the adverse effects of CPSR on metabolism are a consequence and are exacerbated by the transition from a homeostatic to an allostatic sleep regulatory state RELEVANCE (See instructions): The use of an animal model to determine the effects of chronic partial sleep restriction (CPSR) and how aging impacts the response to CPSR, is expected to lead to new insights into the biological mechanisms that link short sleep in humans with cardiometabolic diseases, and new therapeutic strategies for treating age- related metabolic and associated disorders, such as obesity, diabetes, and cardiovascular disease.

DESCRIPTION (provided by applicant): This is a resubmission of a renewal application for continued support of an established T32 Training Grant in Circadian and Sleep Research to train and support predoctoral and postdoctoral students at Northwestern University (NU) and the University of Chicago (U of C). The integration of circadian biology and sleep research in both animal models and humans has been the cornerstone of the relationship between the preceptors on this Training Grant at the U of C and NU for over 20 years, and we have added the term circadian to the title of our Training Program in recognition of the importance of combining these fields in the training of the next generation of young investigators. This Training Program will be led by two established senior investigators under the Multiple Leadership Plan and will involve 13 Primary Training Faculty who have their primary appointments in either basic science or clinical departments. The proposed Training Program will offer predoctoral and postdoctoral students interdisciplinary and multidisciplinary training in a wide range of scientifi disciplines that are highly relevant to understanding the function, regulation and health implications of sleep and circadian rhythmicity. Central to this Program is the training of student in modern basic science, translational research as well as patient oriented research. Multiple research perspectives have fueled for more than a decade the productive interactions and cross-fertilization that have developed between the preceptors in this Program. During the current grant period, the scope of the research activities of the Training Faculty has expanded to include bench to bedside investigations of the impact of sleep-disordered breathing on metabolism and cardiovascular function and we propose to further broaden the program in our renewed program with the addition of six outstanding physician-scientists to the training faculty. As our nation is facing unprecedented epidemics of obesity, diabetes and their cardiovascular consequences, the Training Program proposed in the present application is uniquely positioned to train an interdisciplinary workforce of academic and industry investigators as well as government decision makers to address the roles of sleep disturbances and circadian dysfunction in these public health challenges. A key feature of our Training Program is the inclusion of 10 Collaborating Faculty with additional clinical, scientific and/or educational expertise that greatly enhances our training environment, and conversely, they will benefit from their participation in the program by developing mentoring skills. Our Program will enable trainees to integrate cutting edge approaches and techniques in the areas of genetics, genomics, endocrinology, metabolism, pharmacology, neurobiology, pulmonology, cognitive neuroscience, gerontology and chronobiology into their training in sleep and circadian research. Because the preceptors in this Training Program are actively involved in research at the molecular, cellular, systems, behavioral and epidemiological levels, trainees will be trained in a rich environment of activities that are integrated together for the study of the basic mechanisms of sleep and circadian function at all levels of biological organization.

The integrated goal of this program project application is to apply approaches in both humans (Projects 1 and 2) and experimental male and female mouse models (Project 3) to determine the impact of circadian phase- restricted feeding on age-related disorders of sleep, circadian rhythms, and metabolism. In Project #3, we aim to dissect the molecular mechanisms by which circadian phase-restricted feeding acts as a countermeasure to prevent the age-related decay in circadian robustness, sleep, and metabolic health by exploiting integrated systems level physiological analyses and genome-based approaches. New preliminary data using whole animal calorimetry, feeding monitoring, and RNA analyses support the concept that feeding time plays a key role in the integration of energetics and metabolism throughout life, and that mis-timing in behavioral, energetic, and transcriptional rhythms in relationship to the light cycle represents a hallmark of aging. Our lab and others have also shown that circadian control of NAD+ biosynthesis impacts both core clock function and activity of the sirtuins, key enzymes involved in gene regulation and energetic homeostasis during aging. Yet, a gap remains in understanding whether decay in peripheral clock control of NAD+ biosynthesis and sirtuin function during aging contributes to alterations in circadian behavioral rhythms, sleep, and metabolic homeostasis. We have also recently performed integrated RNA and chromatin immunoprecipitation sequencing (Science, Nov 2015) to reveal cell-type specific functions of the molecular clock in glucose metabolism, and we aim to apply these methodologies to further investigate the global effects of mis-timed feeding on peripheral clock transcription cycles and the impact of such cycles on circadian behavior and sleep homeostasis. We will further perform both chronologic and lifespan-extension analyses to dissect the role of the clock-NAD+-SIRT pathway and application of next-generation sequencing as a discovery platform will uncover how dampened oscillations of clock- controlled metabolic gene regulation contributes to alterations in cell-type specific metabolic dysfunction, and in turn, to alterations in circadian behavior and sleep. Finally, we will also assess whether NAD+ biosynthesis in peripheral tissues is both necessary and sufficient to prevent the age-related decline in sleep and metabolic function. Collectively, results of Project #3 will determine whether circadian phase-restricted feeding is an effective means to attenuate misalignment between oscillations in bioenergetics, metabolic, and transcriptional pathways important in aligning oxidative and reductive phases of physiology with the sleep-wake cycle and will establish new mechanistic insights into feeding time manipulation as an anti-aging intervention in humans.