Mary E. Harrington - US grants

Circadian rhythms are endogenous physiological and behavioral rhythms with a period of approximately 24 hours. Difficulty shifting circadian rhythms has been hypothesized to underlie the impaired performance and discomfort induced in humans by either crossing several time zones or working shift schedules. The mammalian circadian system may be thought of as consisting of three parts; a pacemaker, input pathways which can phase shift and entrain the pacemaker, and output pathways by which the pacemaker can influence the rest of the brain. Many type of evidence indicate that the cells of the hypothalamic suprachiasmatic nucleus (SCN) constitute the major pacemaker. The experiments that I am proposing are designed to answer several specific questions. The research will focus on a single input pathway to the suprachiasmatic nuclei from cells in the lateral geniculate nucleus (the geniculo-hypothalamic tract or GHT), and a single output pathway from the suprachiasmatic nucleus to the hypothalamic subparaventricular area. My research will answer three questions about the GHT of the hamster. 1) What are the responses of SCN cells to a peptide contained in GHT terminals? 2) How do visual responses of GHT neurons compare with those of SCN neurons? 3) How does GHT input affect visual responses of SCN neurons? Other experiments will address two questions about the projection of SCN neurons to the subparaventricular area. 1) What are the neuropharmacological characteristics of this projection? 2) How does this output pathway function in the control of circadian rhythms? In the studies proposed here, I will use golden hamsters, either in lesion-behavior studies with wheeling-running activity measured, or in electrophysiological preparations (both in vivo and in vitro). The golden hamster is an appropriate animal model for the study of mammalian circadian rhythms, in large part because the hamster exhibits very precise behavioral rhythms in wheel-running activity. A wealth of previous literature on formal and physiological aspects of hamster circadian systems and the many parallels among the hamster circadian system and circadian systems of other mammals, including that of humans, help to make the hamster particularly useful.

Mary E. Harrington, 0234203, RUI: Circadian rhythms in NPY and Y5 receptor deficient mice.

Biological rhythms are reset each day by external signals to maintain a 24 hour internal cycle. The light:dark cycle is one very important cue for synchronizing these circadian rhythms. Responses to light are mediated through a direct retinal input to the circadian clock, an input that can be modified by other neural inputs. One important modulating neurochemical is neuropeptide Y (NPY). Interactions between NPY and light resetting provide a point of flexibility for adjustments of light entrainment relative to ecological or internal factors.
The present research will examine the role of NPY in circadian clock phase resetting by light, making use of mice with deletion of either the NPY gene or the gene for an important NPY receptor, the NPY Y5 receptor. Wheel-running activity rhythms of these mice will be studied and it is expected that NPY- and NPY Y5 receptor-deficient mice will show decreased stability and reliability of entrainment to a light:dark cycle. Mice studied under constant darkness will reveal effects these neurochemicals have on circadian rhythm generation and expression.
The mammalian circadian pacemaker is located in the hypothalamic suprachiasmatic nuclei (SCN). Suprachiasmatic nucleus cells maintain a self-sustained oscillation in culture, allowing measures of phase-shifting behavior from an isolated clock. To determine if the Y5 receptor mediates the effects of NPY on light phase shifts, mice with gene deletions will be compared with controls for the effect of NPY applied to the circadian clock in the SCN after a phase shift. It is expected that the Y5 receptor deficient mouse will not show evidence for NPY blocking light-induced phase shifts, providing definitive proof of the importance of this receptor in mediating the effect of NPY on circadian clock resetting.
This work relates to the everyday situation of maintaining synchronization with the external 24 h light:dark cycle. Expanding our understanding of the role of NPY in the circadian system will allow better knowledge of the neural basis of flexibility as we adapt to changing environmental cycles. This research will be conducted at an undergraduate college for women, with promising undergraduate women and minorities taking leadership roles in conducting the studies.

A combination of computational and neurobiological methods will be used to explore the mechanisms which generate endogenous daily (circadian) rhythms in a mammal. The master circadian pacemaker, which resides within the suprachiasmatic nucleus of the hypothalamus (SCN), is comprised of thousands of cell-autonomous oscillators. One objective of this work is to establish a brain slice preparation in which a luciferase reporter allows us to track oscillations in the expression of critical clock genes in real time. The researchers will focus on identified transcriptional-translational feedback loops of genes whose expression is critical to circadian rhythmicity. Molecular methods will be used to selectively neutralize the expression of specific genes, and to examine the effects of such manipulations on the pacemaker and subordinate oscillators. In particular, the relationship between the master oscillator and its damping slaves in the subparaventricular zone will be investigated; the later comprises the major output of the SCN through which the central clock regulates much of the physiology and behavior of the rest of the organism.

The intellectual merit of this work is centered upon coordination of the computational modeling with neurobiological and molecular approaches. It will extend to interactions among distributed oscillators. The broader impact of this work includes its application to understanding the health-related conditions arising from jet lag and shift work. Furthermore, this research will be carried out both at the University of Massachusetts at Amherst and at Smith College, the nation's largest liberal arts college for women. Undergraduate and graduate students will have opportunities to conduct hands-on experiments using state-of-the art equipment, working with mentors who are active researchers. This collaborative efforts will offer opportunities for women and under-represented minorities to gain training at the cutting edge of mathematics, science and engineering. The instructional efforts will be integrated with the newly formed Picker Engineering Program at Smith College. Regular meetings will be held, at which students and faculty review the current literature on biological clocks. The collaborative and interdisciplinary nature of this research draws together students and faculty from diverse backgrounds including Neuroscience, Biology, Computer Science, and Engineering.

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Harrington

Dr. Mary E. Harrington, Smith College, along with her counterparts, Dr. Diego Golombek, Universidad Nacional de Quilmes, and Dr. Mario Guido, University of Cordoba, are organizing a workshop on Chronobiology, to be held in Buenos Aires, Argentina, October 25 - 28. The goals of this activity are to identify common research priorities for collaboration in the field, and to introduce students to current research questions and technologies. Their workshop will precede the 8th Latin American Symposium on Chronobiology and will take advantage of this large gathering of the world experts in the field.

This satellite workshop will lay the groundwork as an introduction to the field, directed primarily at students who are new to the topic. This is an opportunity for students to network among the senior researchers in this exciting new field of science. This is a relatively new field of research (circadian biology) and it impacts a broad range of biological systems.

[unreadable] DESCRIPTION (provided by applicant): Arousal and sleep deprivation can regulate circadian rhythms in multiple ways. One extremely important effect is the ability of these non-photic, higher level inputs to antagonize the synchronizing effects of light, as shown, for example, in studies where non-photic stimuli can block photic phase shift responses. We recently discovered that blocking the non-photic neuropeptide Y (NPY) input pathway to the suprachiasmatic nuclei (SCN) can potentiate photic phase shifting, suggesting that this pathway tonically suppresses the effects of light, and pharmacological treatments can release the animal from these inhibitory effects. We know that non-photic inputs to the circadian clock are represented by both NPY and serotonergic pathways. Remarkably, blocking both NPY and serotonergic inputs potentiates photic effects to an even greater degree, such that a 5 min light pulse is able to induce a 7 h phase shift of circadian rhythms of a treated hamster, as compared to a circa-1 h phase shift in an untreated hamster. This grant will build upon these findings of greatly potentiated light-induced phase shifts when the arousal-related NPY and serotonergic inputs are pharmacologically blocked. We will test these hypotheses: Hypothesis 1: NPY Y5 and serotonin 5HT1A receptors regulate the phase shifting effects of light at multiple phases throughout the subjective night. Hypothesis 2: Non-input inputs do not need to coincide with light, but can alter the response to light when presented within 1 h of light onset or offset. Hypothesis 3: NPY and 5HT inputs alter light-induction of per1 and per2 gene expression. [unreadable] The capability of NPY and serotonin antagonists to potentiate and agonists to attenuate the effects of light on the circadian system could be a potentially useful tool in clinical research. In cases of entrainment disorders, such as delayed or advanced sleep phase syndrome, potentiating the effects of light at one time while blocking effects of light at another phase might be an approach to correct the phase of entrainment. Symptoms of jet lag might be shortened in duration if resetting effects of light in the new time zone are potentiated. Shift workers might benefit from blocking the effects of light at night. The progress of cancer is slower in patients with enhanced circadian rhythmicity, a benefit that might accrue from better circadian entrainment [unreadable] [unreadable]

Circadian or daily rhythms sculpt our responses to the environment. How flexible is the body system that controls our daily rhythms? Clearly circadian rhythms are strongly influenced by light:dark signals, and seasonal changes in day length. We will research the plasticity, or ability to change, in the mammalian circadian system. Circadian rhythms can be measured over many cycles in cultured tissues, such as the suprachiasmatic nuclei (SCN) in the brain, the master pacemaker for the circadian system in mammals, and also in several peripheral tissues, such as the heart, liver, or lung. Circadian rhythms in SCN and peripheral tissues can be measured by light emissions, or bioluminescence, from cultured tissues of a transgenic mouse, the mPer2Luc knockin mice. This project examines responses to altered duration of light changes in period or cycle length after altered length light:dark cycles and will determine the effects of exercise and thus sleep time. These studies will provide answers to fundamental questions about circadian rhythms in mammals. The project will determine if environmental changes alter the behavior of peripheral organs and the SCN when they are isolated in tissue culture and will determine if peripheral organs can also express plasticity of period induced by syncrhonization to varied light:dark-cycles. Finally, the project will deternine if exercise can slow the rate of re-synchronization of SCN and peripheral organs. These experiments will clarify the complex relationship between the environment, the SCN master pacemaker, and the slave oscillators in peripheral tissues. These studies will be conducted at an undergraduate college for women, with promising undergraduate women, many from under-represented minority groups, taking leadership roles. Students will be guided by the PI as they conduct research during the academic year and through summer internships. Participation in an active research lab will provide talented women encouragement and training as they begin scientific careers.

DESCRIPTION (provided by applicant): Circadian or daily rhythms modulate physiological responses. Robust daily rhythms are predictive of improved prognosis for cancer patients, independent of performance status measures. Disruption of circadian rhythms is associated with poor sleep quality and negative mood, fatigue, and reduced quality of life. The ability to care for a patient at home is often lost when the patient no longer sleeps during the night. The mechanism by which tumors suppress circadian rhythms and impair quality of life is unknown. We hypothesize that cytokine release induced by tumors may act directly in the neural system driving circadian rhythms and this action may induce fatigue and circadian rhythm disruption. Previous studies have shown that central administration of the cytokines TGF-1 and neuregulin-1 and systemic administration of IFN-1 can disrupt behavioral rhythms in hamsters and mice. As an animal model of tumor-induced disruption, we will determine if peripheral administration of TGF-1 and neuregulin-1 can similarly disrupt locomotor activity rhythms in mice. We will determine if cytokines can suppress the rhythms expressed by isolated suprachiasmatic nucleus (SCN), immune system organs, thymus and spleen, and other tissues (lung and mammary gland). Further experiments will assess if erlotinib or gefitinib, blockers of epidermal growth factor receptor (EGFR) tyrosine kinase activation used for chemotherapy, can improve circadian rhythm regularity and amplitude following disruption by TGF-1. These experiments are designed to mimic clinical data showing such effects in patients. We will determine with animals the optimal timing for administration of erlotinib or gefitinib to maximize the potential benefit to the circadian system. This research will increase our understanding of the biological mechanism by which tumor growth can impact the circadian system. We will describe effects of circadian clock output suppression on immune system function and on rhythms endogenous to select organs. Our studies will clarify if such effects could be mediated by cytokines, if action is peripheral or central, and if clinical reports of improved quality of life following administration of specific cytokine receptor blockers might be attributable to improved circadian regularity. This research will apply directly to clinical use of EGFR blockers, by investigating a rationale for optimal timing of these compounds. Cancer patients often suffer from disrupted circadian rhythms resulting in poor sleep quality, negative mood, fatigue and reduced quality of life. Tumors induce cytokine release, which may be responsible for circadian disruption and subsequent fatigue. By blocking the action of cytokines with specific compounds, we hope to clarify the downstream effects of cytokines on the immune and circadian systems. Clinical reports of improved quality of life after administration of cytokine blockers might be attributable to improved circadian rhythms.

Daily or circadian rhythms shape both our daily sleep-wake cycles but also our internal physiology. These rhythms are driven by circadian clocks in many cells and tissues within our bodies, and are synchronized by a pacemaker in the brain, the suprachiasmatic nucleus or SCN. This lab has a long-term goal to better understand the resetting dynamics of all the circadian clocks within the body. The objective of this project is to determine the properties of the circadian clock within the liver, and, in particular, how liver cells communicate with each other to keep a consistent time of day. This information will help build a more accurate model of the multi-oscillator circadian system. The central hypothesis is that the liver is a coupled circadian oscillator, and that liver cells are functionally coupled leads to several predictions that can be tested using luminometry and bioluminescence imaging in cultured cells. The first three aims will determine the mechanisms by which the cells of the liver, hepatocytes, communicate information about circadian period and phase. The final aim is to demonstrate that the liver can maintain a rhythm in the absence of input from the SCN. These experiments will greatly enhance the understanding of how a functional, multi-level circadian system is assembled from clocks in millions of single cells, and how this system reacts when confronted with disruptions such as frequent jet lag or rotating shift work. These studies will be conducted at an undergraduate college for women, with promising undergraduate women, many from under-represented minority groups, taking leadership roles in conducting the studies. Participation in an active research lab will provide talented women encouragement and training as they begin scientific careers.

DESCRIPTION (provided by applicant): Animal models have been essential in allowing progress in our understanding of other subjectively- defined symptoms such as anxiety and depression, but currently researchers have no widely accepted animal model for the study of fatigue. This grant will develop a new animal model of fatigue based on interventions expected to decrease circadian amplitude. The long-term goal is to understand the role of the circadian system in the physiological and neural basis for fatigue. The objective in the current application is to validate an animal model of fatigue, building on our preliminary studies that demonstrate selective behavioral effects of chronic proinflammatory cytokines. The central hypothesis is that reduction of circadian outputs induces behavioral changes that can be validated as indicating fatigue in rodents. Our rationale for pursuing this line of research is the recognition that progress in understanding the neurobiology of fatigue will be facilitated by detailing the role of the circadian system as an important modulator of the neural and physiological pathways stimulating arousal and activity. The expected outcomes will be a novel animal model of fatigue. Specific Aim 1: Develop an animal model of fatigue based on chronic low-grade inflammation. Experiments will test the working hypothesis, based on preliminary data, that chronic pro-inflammatory cytokine IL-12 administration will dramatically decrease more effortful tunnel burrowing and wheel-running activity but will have little effect on general locomotor activity. To characterize this model as one related to aging, this experiment will demonstrate differential effects of chronic pro-inflammatory cytokines on fatigue depending on the age or gender of the animal. Specific Aim 2: Determine if our animal model of fatigue involves disruption of circadian clock output. Experiments will test the working hypothesis that treatment with pro-inflammatory cytokines will lead to circadian disruption measured in mice housed under constant darkness, interacting with the age and gender of the animal. To assess this treatment some experiments will use mice with the Per2Luc transgene and will assess circadian desynchrony by in vitro measures of bioluminescence. What may make the proposed project particularly innovative is the application of the PI's expertise in chronobiology to the question of the neural basis of fatigue. This contribution is significant because it will open the study of fatigue to laboratory animal researchers and chronobiologists, enabling studies of fatigue at levels such as mapping neural circuits, modulating neurotransmitters, or targeting genetic manipulations. 1

Project Summary Circadian rhythm disruption, as experienced in shift work, nocturnal light exposure, and aging, is a serious risk factor for disease, including cancer, diabetes, obesity, and cardiovascular and neurodegenerative diseases. As we learn more about the circadian system, it is becoming clear that these disruptions arise not only from shifts in the clocks of individual organs, but also from disruptions of the complex interactions among the clocks of different organs. To study these interactions at a system level, we have developed a method to measure circadian rhythms in specific organs of living animals. We will apply this method to address a fundamental unresolved question in the application of chronobiology to health: By what method can we optimize entrainment of the entire circadian system to shifted light signals? Using our method of in vivo, molecular, organ-specific detection of rhythm in behaving animals, we will search for new methods of resetting the clock that maintain phase alignment among clock components. In parallel, we will continue to develop new approaches for the study of circadian rhythm dynamics in vivo. Our first aim is to measure the entrainment dynamics of liver and brain suprachiasmatic nuclei (SCN) in response to an advanced light cycle. We will then determine if timed food availability can serve to shift the liver faster so that is does not lag behind the shift of the SCN. Undergraduate students at the largest US women's college, Smith College, will conduct this research. Through a multifaceted program with documented success, we will recruit 1st-year students from under-represented minority groups and engage them in hands-on scientific research, with engaged mentorship. Student research teams will include majors with strong quantitative training (e.g., statistics and data science, engineering) and results will be shared via our lab site on the Open Science Framework. Students will be involved in all aspects of the research and will present results at conferences and contribute to publications.