Eric L. Bittman - US grants

This study is designed to clarify the reproductive neuroendocrinology of melatonin action by localizing and characterizing its binding in hamster brain. This pineal hormone is responsible for the seasonal modulation of reproduction by daylength in a variety of species including the hamster. Putative receptors for melatonin will be localized and characterized by the following procedures: 1) Autoradiographic analysis of melatonin uptake through In Vivo and In Vitro techniques. This approach will allow as fine a neuroanatomic localization as is currently possible. 2) In Vitro binding studies of specific brain regions and nuclei. This will allow determination of the intracellular distribution of melatonin in target tissues. 3) Studies of the consequences of manipulating photoperiod and reproductive state on melatonin uptake and binding characteristics. This will implicate putative melatonin receptors in physiological responses. Experiments of these types are necessary for future work on mechanisms of photoperiodic modulation of reproduction. These experiments will ultimately provide better understanding of environmental influences on brain-pituitary-gonadal interrelationships. They will exploit the advantages of seasonal breeding as a model of reversible fertility and may lead to improvements in food production and human contraception. Finally, study of the neuroendocrine mechanisms by which daylength drives annual breeding cycles may lead to a better understanding of brain plasticity.

The experiments proposed here will determine the influences of time of day and daylength on the distribution and concentrations of opiate receptors in the brain of the male golden hamster, a photoperiodic seasonal breeder, Autoradiographic methods will be used to determine the quantity and localization of mu, delta, and kappa receptors. Since radioimmunoassays indicate large effects of photoperiod on the concentrations of endogenous opiates and since peptide and receptor distributions are not always matched, we will complement these binding experiments with immunocytochemical studies of the distribution of met- enkephalin, beta-endorphin, and dynorphin in the brains of long and short day hamsters. finally, the functional significance of opiates in seasonal changes in gonadotropin secretion, male sexual behavior, and body weight will be evaluated using brain lesions and the systemic and intracranial application of opiate receptor agonists and antagonists. By manipulating photoperiod and gonadal steroid concentrations, we will determine the role of endogenous opiates in the integration of internally and externally generated signals. These studies will contribute to our understanding of the role of specific neuro-peptides in the coordination of endocrine and behavioral processes, elucidate environmental and hormonal influences on CNS function, uncover mechanisms of reproductive cyclicity which have possible applications to human and animal fertility, expand our knowledge of brain plasticity, and indicate naturally occurring controls over responsiveness to opiates and hormones which may bear implications for syndromes of abuse and addiction.

9319653 Bittman All Multicellular organisms possess an internal clock that insures daily rhythmicity of multiple physiological function even in an aperiodic environment. In higher vertebrates, these endogenous daily or circadian rhythms are driven by the suprachiasmatic nucleus of the hypothalamus. This endogenous oscillator insures that incompatible processes occur at different times, and that metabolic, thermoregulatory, osmoregulatory, immune and reproductive functions and arousal are properly scheduled to meet environmental challenges. Dr. Bittman will elucidate the role of this central pacemaker in the timing of ovulation. Ovulation is triggered by a surge of pituitary luteinizing hormone which results from the discharge in tot he hypothalamic-hypophyseal portal system of gonadotropin releasing hormone, a decapeptide synthesized in cells of the diagonal band, medial septum, preoptic area and anterior hypothalamus. This release of gonadotropin-releasing hormone is ultimately regulated by estrogen. Dr. Bittman will examine the function of specific cell types within the suprachiasmatic nucleus in the regulation of the estrous cycle by determining whether these neurons project directly to cells elsewhere in the brain which in turn control the pituitary gland. Using state-of-the-art neuroanatomical techniques, Dr. Bittman will then determine whether these neurons that receive input from this hypothalamic nucleus contain estrogen receptor or gonadotropin releasing hormone. In addition, he will establish whether the signals from the suprachiasmatic nucleus at specific times of day underly the appropriate timing of ovulation. The results from these studies will help delineate the circadian regulation of neuroendocrine cells which govern integrative processes. This research will provide basic information critical not only to understanding how environmental factors impinge upon the nervous system but may provide the fundamental groundwork towards developme nt of therapeutic approaches to fertility and may have applications to contraception. ***

Contrary to dogma, the adult brain exhibits considerable plasticity. New
cells which are born in particular regions migrate within the forebrain and
differentiate into neurons. The investigator has found that the birth, survival, and/or
differentiation of such cells in adult brains is influenced by both
environmental and hormonal factors. His work will clarify the cellular and
physiological mechanisms which underlie the regulation of new cell
incorporation and address its functional significance.
Dr. Bittman will first determine whether removal of the testes eliminates the
influence of daylength on the number of newborn cells in specific brain
regions, and whether fluctuations in androgen secretion are necessary for
daylength to exert its effects. This work will establish whether daylength
and androgen interact to regulate this phenomenon. The investigator will next establish
whether the influences of the gonads and photoperiod on the incorporation
of new neurons in adulthood are attributable to regulation of cell birth,
programmed cell death, migration of newborn cells, or some combination of
these events. Finally, he will explore the possible functional role of
neurons born in adulthood. This will be accomplished by assessing the
activation of newborn cells in the processing of reproductively relevant
olfactory stimuli.
These experiments will elucidate environmental regulation of brain
plasticity and explore basic mechanisms regulating cell birth, migration,
and death which may prove useful in treatment of damaged or diseased
nervous systems.

DESCRIPTION (adapted from applicant's abstract): The pineal gland hormone melatonin (MEL) controls seasonal breeding by regulating feedback and behavioral actions of gonadal steroid hormones. MEL may also shift the phase of endogenous daily (circadian) rhythms, or modulate entraining effects of light. Both circadian rhythms and seasonal reproduction depend upon the suprachiasmatic nucleus of the hypothalamus (SCN). The high affinity, Gi-coupled mel-1a membrane receptor apparently mediates reproductive and at least some of the circadian effects of MEL. The proposed experiments will give us a more complete understanding of the distribution of mel-1a receptor mRNA and the phenotype of cells which express it in hamsters, and will explore the possible physiological implications of these findings. The first specific aim is to determine whether mel-1a receptor mRNA is expressed in VIPergic and light-responsive mel-1a cells in the SCN of Siberian hamsters, whose response to MEL depends upon integrity of the SCN. The second specific aim will evaluate the possible roles of mel-1a, VIPergic and vasopressinergic cells in outputs of the SCN which might contribute to effects of MEL in photoperiodism and circadian entertainment in Siberian hamsters. We will determine whether cells which are retrogradely labeled from projection fields of the SCN contain mel-1a mRNA. We will determine whether photoperiod or pinealectomy influence the pattern of release of AVP or VIP in SCN terminal fields. The third specific aim will evaluate the functional significance of the patterns of colocalization discovered in the experiments. Agonists or antagonists of AVP or VIP will be applied to projection fields of SCN efferents, and intraventricularly, in order to determine the role of these peptides in photoperiodic effects. If we find that mel-1a receptors are expressed in retinally responsive SCN cells, we will determine whether application of physiological concentrations of MEL to the SCN can influence circadian phase or modulate phase-shifting effects of light in vivo. These experiments will clarify the basic function of the circadian pacemaker and the actions of MEL. This is especially relevant to human health and welfare, since millions of Americans have been self-administering MEL in the belief that it will have a wide range of beneficial effects. Our results will have applications to human pathologies including jet lag, sleep disorders, seasonal depression, and infertility.

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.

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus of the hypothalamus (SCN) contains a master pacemaker that controls a wide variety of circadian rhythms of physiology and behavior. Recent work has provided a thorough understanding of molecular mechanisms by which these oscillations are generated. The rhythmic and light-regulated expression of Period, Bmal1, and Cryptochrome genes are essential to pacemaker function. Surprisingly, these genes are also expressed rhythmically in a variety of peripheral organs, but their sustained oscillations in vivo appear to depend upon integrity of the SCN. The present experiments will focus on the nature of the SCN-dependent signals responsible for the persistence of peripheral oscillations. Such signals may be neural, humoral, or behavioral, and various organs may rely on different signals or a combination of signals. The first two specific aims will focus on the extent of neural control of peripheral oscillations. Specific aim 1 will test further the importance of innervation vs. humoral signals in regulation of circadian rhythms in the periphery through use of Syrian hamsters whose circadian rhythms have been induced to split by long-term exposure to constant light. We will determine whether the asymmetrical haPer1-2 and haBmal1 expression in the SCN of such animals is correlated with asynchrony of expression of the same genes on the left and right sides of the body in bilaterally paired and unpaired peripheral organs. Asymmetrical expression of physiologically important genes in these peripheral organs will also be assessed. Specific aim 2 will extend our findings that SCN lesions eliminate peripheral circadian rhythms in hamsters, and establish whether denervation of testis, spleen or muscle replicates the effects of SCN lesions upon rhythmic haPer1-3 and haBmal1 expression. Specific aim 3 will examine the regulation of peripheral oscillations by non-neural signals by taking advantage of a parabiotic mouse model in which the effects of SCN lesions are reversed by linkage of the blood supply to that of an intact animal. We will extend our characterization of peripheral circadian oscillations in SCN-lesioned, parabiotic mice and determine whether social factors or temperature cues can contribute to this effect. Inasmuch as abnormal phase relationships between the pacemaker and peripheral oscillators appear to contribute to jet lag and may be involved in other pathologies including sleep disorders, seasonal depression, and infertility, our findings will have applications to human health and welfare.

[unreadable] DESCRIPTION (provided by applicant): Endogenous daily (circadian) rhythms govern many behavioral and physiological functions. A master pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN) controls these oscillations, but the transcriptional-translational feedback loops that generate them are distributed in cells throughout the body. These peripheral circadian oscillations persist for many cycles, if not indefinitely, in isolated organs after they are placed in culture. The SCN generates neural and endocrine signals in order to regulate circadian oscillations in peripheral tissues, but the way in which these signals act as entraining cues (zeitgebers) and the determinants of the phase taken by peripheral oscillators to the master pacemaker are poorly understood. Although powerful tools are available in the form of transgenic mice, novel approaches are required to determine the behavior, the physiological role, and the importance of the peripheral oscillators. The experiments described in this R21 application will utilize a mouse luciferase reporter construct in order to establish whether the SCN entrains hepatic oscillations, and to determine which specific signals serve as zeitgebers. The roles of the neurotransmitters epinephrine and acetylcholine, as well as hormones including glucocorticoids, insulin and glucagon, will be examined. The influence of fluctuations in glucose concentration will also be tested. A novel-flow through system will be used in which physiological signals (hormones or neurotransmitters) are added to organ culture chambers containing replicate sections of liver taken from transgenic Per2::luc mice. The ability of these signals to regulate period and phase will be established by luminometry, and we will determine whether the criteria of entrainment are met. For the first time for a peripheral oscillator, the range of entrainment will be examined and phase response curves wil be constructed. The health relevance of circadian organization will be evaluated as the impact of entrainment of the hepatic clock to various periods upon secretion of VLDL-cholesterol is determined through assay of the perifusion effluent. Once this luminometry system is established, it may be used to analyze not only entrainment of other hepatic circadian rhythms, but also the clock-like behavior of other organs. These studies will provide important new tools for analysis of the multi-oscillator system that governs normal physiological function and whose malfunction may contribute to disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Fertility depends upon the appropriate timing of functions controlled by specialized neurons of the hypothalamus and preoptic area. The control of ovulation by the suprachiasmatic nucleus (SCN) is one of the best-established physiological functions of the circadian clock. The central question of this proposal is whether circadian oscillators in the SCN are not only necessary, but also sufficient, to explain the timing of the LH surge. Circadian rhythms arise from the operation of well-defined transcriptional-translational feedback loops in which the oscillating expression of core clock genes is critical. These loops underlie circadian rhythms that take place not only in the SCN, but also in other brain regions and the periphery. Neither the extent nor the physiological importance of clock gene expression in specific cell types is well understood. Crosses between mice in which clock genes are floxed and lines bearing appropriate Cre drivers has led to exciting progress in studies of the peripheral organs and the retina, but application of this strategy to understanding the role of circadian rhythms in specific brain functions has not yet been accomplished. Circadian clocks appear to run not only in both GnRH cells which directly regulate the preovulatory LH surge and kisspeptinergic neurons from which they receive input. We will use Cre-Lox technology to determine not only the role of circadian function in each of these cell types, but also the importance for normal function of the coordination of phase and period of master and subordinate oscillators. First, we will selectively eliminate clock function by targeting Bmal1 in neurons that express GnRH or kisspeptin in order to determine whether the appropriate timing of the preovulatory LH surge depends upon local circadian oscillations. Second, we will selectively manipulate the period of master and subordinate oscillators to determine the consequences for the LH surge. Establishment of this approach will open new tools for understanding the mechanisms by which circadian systems coordinate behavior and physiology more generally. Given the pivotal role of neurons that contain such oscillators in a variety of homeostatic and reproductive processes, this research will reveal mechanisms that underlie metabolic diseases and infertility and lead to development of new therapies.

Abstract The present research will explore and develop a transgenic approach to producing a bioluminescent reporter strain of Syrian hamster that will be a valuable tool for circadian research. The tau hamster was the first mammalian period mutant to be discovered. Transplantation experiments using tau hamsters established that suprachiasmatic nucleus (SCN) regulates the daily scheduling of physiology and behavior. Discovery that tau is a gain of function of casein kinase 1? was critical to achieving an understanding of the operation of transcriptional-translational feedback loops that generate circadian rhythms, both in the brain's pacemaker and in peripheral oscillators. We recently discovered duper, a new mutation in hamsters which, like tau, speeds up the circadian clock. Duper is not a change in the coding region of casein kinase or any other known clock gene, and the mutation does not affect clock speed in fibroblasts. Thus duper is unlikely to affect the TTFL, but more probably alters coupling relationships within the SCN. Duper causes a striking reduction in jet lag. Our experiments will validate the reporter strain by comparing luminescent traces with results from qRT-PCR. We will then compare the impact of the tau and duper mutations on circadian rhythms in order to determine whether shortening of period through effects on the pacemaker vs alterations in the TTFL have different consequences for organismal function. Although hamsters have provided a valuable model for studies of biological rhythms, their potential as a genetic tool has yet to be realized. We have recently contributed to the first draft of the hamster genome and established methods for making transgenic hamsters. We have employed piggyBac methodology to insert circadian reporters into a hamster cell line. This technology will now enable us to study organismal function. In the first application of the reporter strain, we will determine effects of duper upon the core cell-autonomous feedback loops and upon the coordination of pacemaker function. This will shed light on the mechanisms of circadian desynchrony. Unlike the species in which transgenic reporter strains have thus far been produced, hamsters have a particularly regular, circadian-based estrous cycle and a strong photoperiodic response. Thus development of the hamster reporter strain will make possible future applications to investigate neuroendocrine function. Finally, the importance of the hamster as a disease model for several pathologies including MERS and Ebola, and the relevance of jet lag to the incidence of disease in an era of frequent trans-meridian travel, indicate that the reporter strain will be of widespread utility. Given the importance of circadian organization in behavior and physiology, this research will reveal mechanisms that underlie neurologic diseases, sleep deficiencies and metabolic disorders, and lead to development of new therapies.

Abstract/Project Summary A master pacemaker in the suprachiasmatic nucleus of the hypothalamus coordinates circadian rhythms throughout the body. The phase and period of these oscillations is controlled by environmental signals, principally light. Disruption of circadian organization, such as occurs in shift work and jet lag, compromises health through mechanisms that are poorly understood. We recently discovered duper, a hamster mutation which speeds up the circadian clock and markedly accelerates re-entrainment of behavioral rhythms upon a shift of the light:dark cycle. The duper mutation is not a change in the coding region of casein kinase or any other known clock gene, and seems not to affect the transcriptional- translational feedback loops responsible for cell autonomous oscillations. We will identify the mutant allele by fast homozygosity mapping. We will use qRT-PCR to examine effects of the duper allele upon circadian phase shifts of expression of core clock and clock-controlled genes in heart, liver, kidney, and muscle. Given that transcription of genes critical to metabolic function is controlled by multiple clock proteins or clock-dependent transcription factors, we will use RNA-Seq to reveal the extent of transcriptome-wide phase disruption that occurs within organs during shifts. We will use immunocytochemistry to examine effects of duper on regional SCN function, and factor the contribution of pacemaker vs. peripheral oscillators to the latency of re-entrainment. Finally, we will test the hypothesis that internal desynchronization of circadian rhythms is responsible for aggravation of dilated cardiomyopathy by repeated phase shifts in a commonly used hamster model. Hamsters have proven valuable in studies of biological rhythms, and they offer a unique tool for understanding heart disease. Circadian control of the preovulatory LH surge is well understood in this species and provides a model for control of subordinate oscillators by the hypothalamic pacemaker. Our work will shed light on the mechanisms of circadian desynchrony and allow us to test its role in disease. Given the importance of circadian organization in behavior and physiology, this research will reveal mechanisms that underlie pathologies of heart and lung, as well as changes in liver and kidney that contribute to hypertension and fibrosis. The results will also shed light on causes of sleep deficiencies and metabolic disorders, and are likely to lead to development of new therapies to improve human and animal health.