Michael Menaker - US grants

Our overall aim is to understand in physiological detail the interaction between the circadian clock that measures the daily light cycle and the reproductive system that is regulated by this light cycle. We will approach this goal by first dissociating the two systems from each other and studying them independently. Although light affects both the clock and the reproductive system, potentially its effects may be experimentally confined to one or the other in at least four different ways: 1) by careful choice of the intensity, duration and wavelength of the light signal itself; 2) by the use of general anesthetics that block its effects on one system but not on the other; 3) by manipulaton of neurotransmitters involved in the neural inputs to one system but not the other; and 4) by exploiting genetic lesions that affect the input pathway to one system but not to the other. We propose experiments that will utilize all four of these general stategies. We hope to obtain unambiguous descriptions of some of the properties of the pathways by which light influences each of the two system and some indication of the extent to which the pathways are independent of each other. We view this study as contributing to increased understanding of a much more general and important problem: How is hypothalamic function regulated by the environment and by other brain structures? The effects of light on the circadian/reproductive axis provide a particularly favorable situation in which to define such interactions experimentally because: 1) light, which can be exceptionally well controlled has profound influences on the reproductive system and is the only important environmental input to the circadian system and 2) much of the neural circuitry is known and the behavior that it controls can be measured with unusual precision. Increased understanding of hypothalamic function is central to the amelioration of many human health problems. Although the relevance of an understanding of circadian organization to problem of sleep, jet lag and other aspects of human performance has been appreciated for some time, its interaction with other brain functions has recently been underlined by the finding that circadian malfunction underlies some kinds of serious affective illness.

Our overall aim is to understand in physiological detail the interaction between the circadian clock that measures the daily light cycle and the reproductive system that is regulated by this light cycle. We will approach this goal by first dissociating the two systems from each other and studying them independently. Although light affects both the clock and the reproductive system, potentially its effects may be experimentally confined to one or the other in at least four different ways: 1) by careful choice of the intensity, duration and wavelength of the light signal itself; 2) by the use of general anesthetics that block its effects on one system but not on the other; 3) by manipulaton of neurotransmitters involved in the neural inputs to one system but not the other; and 4) by exploiting genetic lesions that affect the input pathway to one system but not to the other. We propose experiments that will utilize all four of these general stategies. We hope to obtain unambiguous descriptions of some of the properties of the pathways by which light influences each of the two system and some indication of the extent to which the pathways are independent of each other. We view this study as contributing to increased understanding of a much more general and important problem: How is hypothalamic function regulated by the environment and by other brain structures? The effects of light on the circadian/reproductive axis provide a particularly favorable situation in which to define such interactions experimentally because: 1) light, which can be exceptionally well controlled has profound influences on the reproductive system and is the only important environmental input to the circadian system and 2) much of the neural circuitry is known and the behavior that it controls can be measured with unusual precision. Increased understanding of hypothalamic function is central to the amelioration of many human health problems. Although the relevance of an understanding of circadian organization to problem of sleep, jet lag and other aspects of human performance has been appreciated for some time, its interaction with other brain functions has recently been underlined by the finding that circadian malfunction underlies some kinds of serious affective illness.

Our long-term goals are 1) to understand the mechanism that generates circadian rhythmicity in the retina and 2) to elucidate the role played by rhythmic synthesis of melatonin in retinal function. We will begin by defining optimal culture conditions for studying circadian rhythms of melatonin synthesis in in vitro preparations of isolated neural retinas from rats and mice. We will then measure the activity of the enzymes involved in melatonin synthesis and the breakdown products produced by its catabolism in order to define the proximate source of the circadian rhythmicity. We will determine whether dopamine is also produced rhythmically by cultured retinas. Using mice and rats with genetic defects that affect specific cell types in the retina we will ask which cell types are essential for synthesis and degradation of melatonin (and dopamine) and which are essential for circadian oscillation. We will define the response of the retinal circadian oscillators to light pulses and explore the light input pathway using pharmacological agents that block or mimic the resetting effects of light. Finally we will ask whether melatonin is causally involved in synchronized rhythmic outer segment disc shedding by comparing disc shedding in C3H mice, whose retinas synthesize melatonin rhythmically, with disc shedding in C57 mice whose retinas, like their pineal glands, make no melatonin at all. What we learn about rhythmicity in mammalian retinas in culture is likely to be applicable to pathologies of retinal function in humans, particularly since so many aspects of retinal physiology have been shown to be rhythmic.

DESCRIPTION (provided by applicant): Our overall aim is to elucidate the structure of the mammalian circadian hierarchy. To accomplish this we need to know which organs and tissues contain independent circadian oscillators and how each is coupled to the others and to the environment to control phase and produce adaptive temporal organization. Our guiding hypothesis is that circadian oscillators in the suprachiasmatic nucleus of the hypothalamus (SCN) normally synchronize circadian oscillators in the brain and periphery using a variety of different signals. These include neural impulses, hormonal signals and, unexpectedly, behavioral signals generated by the SCN, modified by the environment, and acting on peripheral oscillators directly through the consequences of the behavior (e.g., feeding cycles entrain the circadian oscillators in the liver). Using transgenic rats in which the mouse Per1 promoter has been linked to a luciferase reporter, we have been able to measure circadian rhythms of light emission from a variety of cultured tissues including the SCN and peripheral tissues such as lung, liver and cornea. We will assess the degree to which the oscillators in peripheral tissues manifest canonical circadian properties such as temperature compensation. We will test the hypothesis that peripheral oscillators are maintained and regulated by a variety of different signals originating in the SCN by lesioning that structure and subsequently culturing peripheral tissues that oscillate when derived from intact animals. We will test the efficacy of several signals that putatively entrain peripheral oscillators (e.g., norepinephrine for pineal, insulin for liver, forced exercise for lung, melatonin and body temperature as systemic signals). Detailed knowledge of the coupling signals involved will eventually enable us to modify the phase of particular oscillators within the system for therapeutic purposes. This information is essential for the rational use of circadian approaches to the management of the performance deficits produced by shift work, insomnia and pathologies with circadian components such as seasonal affective disorder (SAD). Furthermore, it is basic to the design of treatments for many serious conditions (e.g., high blood pressure, cancer) in which responses to therapeutic agents are modulated both positively and negatively by the circadian system.

Circadian clocks, found in virtually all organisms from bacteria to humans, control daily rhythms in physiology and behavior, including the familiar sleep-wake cycle. The day-night cycle synchronizes the circadian clock to the external world. Light is crucial for the proper timing of circadian rhythms, yet organisms live in diverse light habitats, ranging from dark caves to sun-drenched savannahs. How does the circadian clock respond "correctly" to this broad range of light cues? This project tests the hypothesis that circadian clocks have been modified by evolution such that they respond appropriately to light levels normally experienced by a species in its native habitat. To test this idea, the investigators will capture several different species of anoles, small Caribbean lizards living in diverse light conditions. Using automated behavioral recording techniques, they will determine how behavioral activity rhythms change according to the strength of the light-dark cycle. The investigators will also examine the pineal gland, a small organ located in the brain that secretes a hormone called melatonin. The pineal gland is the master circadian clock in lizards, and melatonin production is controlled by light. By measuring the light-induced changes in melatonin production, the investigators can determine the photic (light) sensitivity of the circadian clock. The prediction is that anole species living in the deep forest will be more sensitive to the effects of light on the circadian clock, relative to species living in sunnier habitats. Understanding the evolutionary relationship between the light environment and circadian clocks is important from a conservation standpoint, as "light pollution" from urban settlements is known to disrupt ecosystems. This project will provide research experience for several undergraduate students, in addition to the doctoral training of at least one graduate student.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

With this award to the Biology department at the University of Virginia a bioluminescence/biofluorescence imaging system will be acquired to study molecular signals associated with daily time keeping mechanisms. The instrument will be used for experiments aimed at gaining a better understanding of the internal circadian clocks that allow animals to anticipate daily changes in their environment and to organize a multitude of bodily functions in an optimized daily schedule. The new imaging system optimally visualizes bioluminescent signals produced by clock-controlled expression of luciferase reporter genes in combination with fluorescent signals produced by additional reporter genes that act as markers for specific cell types or biological activities. With the new technology it will be possible to image cell-type specific circadian gene expression at single cell resolution. The proposed experiments will focus on the cellular and network properties of circadian clock function in fruit fly and rodent model systems. These research activities will strongly stimulate the research programs of the (Co-)PIs as well as help in the recruitment and training of researchers and students to the University of Virginia. The instrument and planned research activities will be prominently featured in several undergraduate courses. In addition, existing affiliations will be used to also incorporate training and outreach at the K-12 level. Participating researchers and students will not only be involved in projects at the cutting edge of chronobiological research and imaging technology, but also receive invaluable training in microscopy and data analysis techniques that will be an asset to them throughout their scientific or professional careers.

DESCRIPTION (provided by applicant): We have discovered a new spontaneous circadian mutation in Syrian hamsters, which confers a robust phenotype on the animals' locomotor rhythmicity. The free running period of animals homozygous for the mutation is approximately 28 hours, 4 hours per daily cycle longer than that of wild type animals. Such mutations, most produced in mice by chemical mutagenesis, have been critical in working out what we know thus far about the molecular mechanism that generates cell autonomous circadian rhythmicity in mammals, but our knowledge is far from complete and it is clear that additional unidentified genes must contribute to its function. Furthermore, because hamsters provide many advantages over mice for behavioral and physiological studies, this new mutation provides a unique opportunity for studies of the circadian clock mechanism in mammals. We propose to analyze this new mutation at 3 levels: genetic, behavioral and molecular. Genetic analysis will test our assumption, based on preliminary data, that the mutation is a single, autosomal co-dominant allele. The work in the Menaker laboratory will be focused on the behavioral phenotype and will explore the responses of wild type, heterozygous and homozygous littermates of both sexes to constant darkness, constant light, single light pulses, photoperiods with different light/dark ratis and entraining cycles with different periods, These data will address the role of the mutant gene in the circadian molecular mechanism as well as its impact on the organism's physiology. The Green lab will perform molecular and biochemical analysis of these mutants to determine whether this mutation affects the core intracellular oscillator mechanism or some system-level aspect, such as intercellular coupling. The nature of the mutation will be addressed by sequencing of the known circadian genes, and by analysis of their expression levels. This will also lay important groundwork for the eventual identification of the mutation by whole-genome sequencing. Circadian rhythms modulate much of normal physiology and behavior and circadian disruptions increase vulnerability to a variety of environmental insults. Knowledge of the underlying mechanism is essential to controlling these deleterious effects.