Erik D. Herzog - US grants

DESCRIPTION (provided by applicant): The mammalian suprachiasmatic nucleus (SCN) is required for daily cycles in behavior and physiology. Some other brain regions, including the main olfactory bulb (OB), also express intrinsic circadian rhythms. It is not known how or which cells within these regions synchronize and sustain these rhythms or what role they play in behavior. The neuropeptide vasoactive intestinal polypeptide (VIP) is required for circadian synchrony in the SCN and is found in neurons of other circadian tissues. Little is known about how VIP mediates synchrony within the SCN or if it coordinates rhythms in other brain regions. The proposed studies directly address these issues by taking advantage of long-duration recording technologies--multielectrode arrays and bioluminescent reporters of gene activity--and the unique properties of mice with mutations in genes involved in circadian timekeeping or transgenes that allow conditional manipulation of gene expression and cell viability in specific cell types. Aim 1 tests the hypotheses that VIP entrains daily rhythms in variety of brain regions and cell types. The strategy is to compare VIP-induced changes in acute and circadian firing rate and gene expression patterns in real time of neurons and glia within 3 brain regions. In addition, the role of VIP in the OB and in olfaction will be assayed in vivo. Aim 2 tests the hypotheses that VIP neurons in the SCN are required to entrain and sustain circadian locomotor rhythms and that VIP neurons in the OB are required for rhythms in the OB and in olfactory behavior. This Aim also tests whether clock genes in VIP neurons are essential for their rhythmicity and for these rhythms in behavior. Aim 3 uses correlated firing and gene expression patterns and pharmacology to map functional connectivity and the relative roles of glutamate, nitric oxide and VIP in circadian synchrony within the SCN. The recent discoveries of putative circadian oscillators in many mammalian tissues have led to the hypothesis that the circadian system is hierarchically organized and that disruption of this coordination could underlie various neurological disorders including depression. These experiments will, for the first time, identify the mechanisms that coordinate ensemble daily rhythms in the brain and the distinct roles they play in behavior. Daily rhythms in behavior and mood are driven by circadian clocks in the brain. This project examines the role of specific neurons and signaling molecules in the circadian regulation of neural activity and gene expression in and between brain areas and in locomotor and sensory function.

The discoveries of specific molecules, cells, and brain areas that mediate daily behaviors have made circadian biology one of the fastest growing and most integrative fields in neuroscience. In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus regulate rhythms in physiology and behavior. Recently, our lab and others have also found other circadian oscillators in the brain and body. We are now poised to identify the cells capable of circadian pacemaking, the molecular mechanisms underlying their rhythmicity, and the signaling pathways that synchronize them to coordinate behavior.
This proposal focuses on glia as a system to test the role of specific genes and molecules in circadian rhythm generation and communication. Although glia far outnumber the approximately 10,500 neurons in the SCN, they are seriously understudied. Recent studies on glia have challenged traditional neurobiology. Glia communicate with one another and with neurons through conventional and novel chemical signals. Glia can regulate synapse formation, control synaptic strength and, under specific conditions, differentiate into neurons. Conversely, neural impulse activity regulates a wide range of glial activities, including their proliferation and differentiation. It is not known whether glia are circadian pacemakers, precursor pacemaker cells, targets for pacemakers, or support cells to pacemakers. These features emphasize the importance of studying neural-glial interactions in the circadian system.
Glia will be studied as model circadian oscillators, first as targets for SCN signaling and then as potential modulators of circadian timing. Transgenic mice expressing firefly luciferase under the control of either the Period1 or Period2 promoter will allow real-time observations of core clock mechanisms of different cell types. Co-cultures of SCN explants with pure glia will test when and which SCN cells secrete timing signals that are sufficient to synchronize rhythms in target cells. Replacing the SCN with other brain regions in these co-cultures will test whether the SCN are uniquely capable of imposing periodicity on other cells. Pharmacological blockade of candidate transmitter pathways will test their necessity for this neural-to-glia coordination.
The capacity of glia to modulate neural function will be examined in three ways. Recording bioluminescence from transgenic SCN explants co-cultured with glia of different circadian phenotypes will test whether glia secrete factors that influence SCN periodicity. Long-term, multisite electrical recording from SCN neurons will specifically assess the role of glia in neural rhythmicity. Finally, locomotor recording from SCN-lesioned and -chimeric animals will test the possibility that transplanted glia regulate behavioral rhythmicity.
The proposed research will reveal mechanisms that may coordinate activity across the brain. By examining circadian timing in glia and the modulation of circadian timing by glia, this work makes testable predictions about neuron-glia interactions. The findings will be relevant to daily physiology and intercellular signaling. The impact of this work will be broadened with the generation and distribution of transgenic, circadian mutant mice and the recruitment of new scientists, including those traditionally underrepresented. Students will become involved in the research through hands-on research internships, outreach programs, two undergraduate courses and two graduate courses.

DESCRIPTION (provided by applicant): The mammalian suprachiasmatic nucleus (SCN), required for daily cycles in behavior and physiology. How the cells of the SCN synchronize to coordinate behavior is largely unknown. We have established a collaborative program combining experimental and computational methods to study large numbers of circadian oscillators, their connections, and the real-time kinetics by which they self-synchronize and respond to perturbations in environmental timing cues. To understand circadian regulation within the brain, we must understand the topology and types of interactions between circadian neurons. Aim 1 will monitor the network of SCN oscillators as they synchronize during fetal development, during entrainment, following a phase shift, and after restoration of cell-cell communication in the adult SCN. Using novel wavelet-based time series analyses, we will estimate the strength and direction of individual connections in the SCN. Aim 2 will use graph theory and spatial statistics to quantify network features of the developing and adult SCN. These analyses will define the mechanisms of synchronization during normal development and following environmental perturbations and the relative contributions of local, regional or global coupling which contribute to period precision. Aim 3 will compare the performance of the SCN networks under the four conditions with both deterministic and stochastic model networks. The computational models will investigate the effects of intrinsic noise and cell-cell heterogeneity on circadian synchronization. Revealing how circadian oscillators interact to generate a coherent rhythmic output will have important clinical implications for prevention and treatment of circadian rhythm disruptions, including mood and sleep disorders. PUBLIC HEALTH RELEVANCE: Daily rhythms in behavior, physiology and cognitive performance are driven by circadian clocks in the brain. This project examines the role of network connections and noise in the synchronization of circadian oscillators during normal development and following environmental perturbations using novel modeling, statistical and network analysis tools.

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) is the master circadian pacemaker driving daily rhythms in mammalian physiology and behavior. SCN neurons utilize a transcription/translation feedback loop to generate circadian changes in electrical activity. Although we have known that SCN neurons fire during the day and are silent at night since 1982 and considerable evidence implicates subthreshold K+ conductance(s), the critical K+ conductance(s) have not been identified. In recent studies focused on testing the hypothesis that subthreshold, A-type (IA) voltage-gated K+ (Kv) channels are involved, we found that mice lacking Kv4.2 (Kv4.2-/-) or Kv1.4 (Kv1.4-/-) pore-forming (¿) subunits have markedly shorter circadian periods of locomotor (wheel running) activity than wild-type (WT) mice. Using in vitro extracellular microelectrode recordings, we found that the periods of circadian rhythms in firing are similarly shortened in SCN neurons lacking either Kv4.2 or Kv1.4. Initial experiments here (aim 1) will determine if Kv4.2 and Kv1.4 are the only Kv ¿ subunits contributing to the IA channels that modulate SCN excitability and reveal the effects the combined loss Kv4.2 and Kv1.4 on rhythms in SCN firing and locomotor activity. The goal of aim 2 is to determine if the shorter period of circadian firing in SCN neurons lacking Kv4.2 or Kv1.4 reflects the functioning of IA channels in the synchronization (i.e., network properties) or the cell-autonomous regulation of SCN neuron excitability. This aim will, for the first time, establish whether the critical K+ conductance(s) in different SCN cell types are distinct. A long-standing debate in the field is whether daily changes in membrane potential are required for the generation of circadian rhythms in gene expression. Aim 3 will test directly the hypothesis that Kv4.2- and Kv1.4-encoded IA channel mediated changes in excitability also modulate the period and amplitude of circadian changes in gene expression. Finally, the observation that the cyclic changes in SCN neuron firing and locomotor activity persist (albeit with a shorter period) in the absence of Kv1.4 or Kv4.2 indicates that other K+ conductances regulate the daily oscillations in SCN neuron membrane potentials. In aim 4, we will exploit a novel, high-throughput quantitative Taqman-based RT-PCR based method to quantify the expression levels of multiple K+ channel subunits simultaneously, as a function of circadian time, and to identify the subthreshold K+ conductance(s) that mediates the daily depolarizations and hyperpolarizations in the membrane potentials of SCN neurons. These studies will provide fundamentally important new insights into the roles of specific K+ conductances in regulating/modulating daily rhythms in the excitability of SCN neurons. In addition to guiding further investigations into the molecular, cellular and systemic mechanisms linking daily rhythms in neuronal excitability, gene expression and behavior, these insights will translate to advances in understanding the regulation and dysregulation of circadian rhythms and to the development of novel therapeutic strategies to benefit individuals suffering genetic and environmentally-induced disruptions in circadian rhythms.

? DESCRIPTION (provided by applicant): This application would create a BP-ENDURE program in St. Louis for undergraduate students pursuing training in the neurosciences. The objective of the grant is to provide rigorous and critical training in neuroscience to a diverse cohort of students from three partner institutions (Washington University, the University of Missouri-St. Louis and Harris-Stowe State University). By providing support for 10 funded positions for summer research, this proposal will establish a Pipeline to graduate school. The Pipeline emphasizes sustained training in oral and written science communication, discovery science and outreach experience. Specifically, this proposal will support 10 early-stage trainees annually for up to three years each. Our Pipeline has long-standing commitments to cutting-edge research, to interdisciplinary education, and to providing modern career development. We seek to be a Program that responds to changes in the research environment by helping our students to pursue important and innovative problems and concepts, to adopt new techniques and to communicate effectively with their peers and the general public. The proposal will allow for the addition of three interactive and immersive courses that will appeal to teens and create a community of young scientists who can begin as early as the summer after their freshman year. The curriculum and research environments will remain broad and deep, combining expertise in molecular, cellular and systems-level approaches to the study of neural function and dysfunction. The Program will recruit and retain talented, diverse students through innovative and dedicated coordination with the University and partner schools and be evaluated formally by a Board of Directors and an external assessor. Major new initiatives aimed at accomplishing these goals include: 1) the establishment of a new network of research opportunities for undergraduates interested in the neurosciences, 2) the introduction of three interactive courses (The Teen Brain, Neuroscience Futures, and Skills for a Neuroscientist) to bolster neuroscience fundamentals and a sense of community among the students, 3) enhanced involvement of the undergraduates in the Society for Neuroscience Brain Bee as part of their training in science communication, and 4) refinement of a near peer-mentoring program that has graduate students working with undergraduates and undergraduates working with high school students. These initiatives will ensure our students remain at the forefront of developments in neuroscience research, teaching and outreach.

? DESCRIPTION (provided by applicant): Overview The suprachiasmatic nucleus (SCN) serves as a circadian pacemaker that drives daily rhythms in vertebrate behavior and physiology. It generates robust, self-sustained oscillations that entrain to the local day-night cycle including hormone secretion, metabolism and sleep-wake. In addition, the SCN must adapt to slow seasonal changes in day length and, with travel across time zones, rapid shifts in the 24-h environment. It is an important and fundamental question how the oscillatory systems balance robust rhythmicity with flexible synchronization. Intellectual Merit: We hypothesize that opposing actions of two coupling agents contribute to the robust, yet sensitive, cycling of the network of SCN cells. Vasoactive intestinal peptide (VIP) and ?-aminobutyric acid (GABA) are key neurotransmitters involved in the generation and maintenance of SCN rhythms. Genetic and pharmacological experiments revealed central roles for VIP and its canonical receptor (VPAC2R) and GABA and GABAA receptors in synchronization among circadian cells. We will test the hypothesis that GABA signaling makes SCN timekeeping less precise but more amenable to phase shifting by external stimuli, such as light. We will combine theoretical and experimental tools to study the synergistic actions of VIP and GABA for synchronization among cells and entrainment to the environment. The SCN provides an outstanding model system of coupled oscillators that can be studied in vivo, reduced and simplified to a slice or individual cells in vitro, and modeled in silico. Although it is comprisedof thousands of heterogeneous cells, the SCN offers a unique opportunity to understand the balance between reliable rhythmicity and adaptable synchrony. We have three specific research aims: 1. Data-driven modeling of two competing coupling mechanisms. 2. Experimental studies of GABA blockade and enhancement in wild-type and VIP-deficient mice. 3. Theoretical and experimental optimization of adjustment to shift work via dosing strategies of benzodiazopines. Broader impacts The proposed research includes the development of novel software (the Entrainometer) for analysis of rhythm synchronization to a periodic input. This software will be freely available and useful to a wide range of scientists wanting to reliably measure features of entrainment. The proposal also includes significant outreach, education, and research opportunities. Both Drs. Herzog and Herzel are deeply committed to training young scientists. Dr. Herzog founded the St. Louis Chapter of the Society for Neuroscience through which he runs major outreach activities including NeuroDay (1500 visitors), HealthFest (1500 visitors) and the St. Louis Area Brain Bee (40 competitors, 200 participants). He also runs a 1-week summer course for high school teachers that includes material on circadian rhythms. Both Drs. Herzog and Herzel have taught in and directed the International Chronobiology Summer School for many years (including 2014 in Sapporo, Japan). As the 2014 Program Chair for the Society for Research on Biological Rhythms, Dr. Herzog promoted advocacy to the general public (e.g., a discussion of school start times) and chronobiology education (e.g. how to use chronobiology to excite students about physics, chemistry, math and biology). He continues to share his enthusiasm for rhythms research as the 2014-1016 Chair of Fundraising for SRBR. As part of this collaboration, the PIs will develop Modeling Circadian Clocks, a teaching module, for use by undergraduate and graduate educators around the world.

? DESCRIPTION (provided by applicant): We request partial support for the 2016 Society for Research on Biological Rhythms Conference, to be held near Tampa, Florida, from May 21-25, 2016. This meeting, which attracts 600-700 attendees, will focus on the breadth of topics that represent key research areas in chronobiology, including molecular biology, genetics, cell biology, neurobiology, physiology, metabolism, cancer, aging, immunology, behavior, sleep, mathematical modeling, and applied research. The theme of meeting is the relevance of biological clocks to science and society, which reflects the extent to which circadian clocks affect essentially all aspects of physiology and disease. The meeting will feature 18 symposia of invited speakers, and 12 slide sessions (selected from submitted abstracts) that combine the best of basic clock research with those that translate this information into human applications. The symposium speakers and session chairs are recognized leaders in their fields, and were chosen to represent our breadth and realize our goal of bridging basic and applied circadian clock research. Special attention has been given to cultural and geographical diversity, as well as gender balance. We aim to attract scientists from diverse backgrounds through advertisement of the meeting at minority institutions, and, with NIH support, offers of travel awards to graduate students and postdoctoral fellows prioritizing those from under-represented groups. All meeting abstracts will be made available to the public on the SRBR web site. Training aspects of the meeting are fully developed, and include a highly subscribed, free, one-day training day for students and postdocs, and junior faculty workshops, which precede the main meeting.

An award is made to support the acquisition of a multiphoton confocal microscope to address the research needs of a large, diverse group of researchers in multiple departments at Washington University and in the St. Louis area. The new system will be housed in the Biology Department Imaging Core and will be readily accessible to university and regional users. The new imaging capabilities of this instrument will enable cutting edge multidisciplinary research and promote a well-prepared, diverse STEM workforce including students in underrepresented groups. The microscope will enhance the hands-on training of undergraduates through existing courses, in addition to the many Independent Study undergraduates that already use the Core. Furthermore, the instrument will enable the development of new teaching modules for other undergraduate biology classes where the need to procure data for many students in a short time frame precludes the use of existing instruments. This instrument will also have a large impact on underrepresented students involved in the Science Career Explorations programs, YMCA campus Y program for inner-city school children, and ENDURE program for underrepresented students entering neuroscience graduate school.

The field of live cell imaging has recently enabled researchers to resolve physiological and molecular events in cells deep within tissues with micron- and millisecond-resolution. The new multiphoton system will provide deep, fast and super-resolution imaging technology for the first time to users in the departments of Biology, Physics, and Engineering at Washington University and from area institutions. Initial research uses for the new system will include work on cortical microtubules in plant cells; analysis of biosensors in root cells and pollen dynamics. Other researchers working with animal systems will study neuronal cell populations, tendon fascicles, and a range of other topics. Results from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both training and outreach activities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This application would renew support for the BP-ENDURE program in St. Louis to train undergraduate students underrepresented in the neurosciences. The objective of the grant is to provide rigorous and critical training in neuroscience to a diverse cohort of students from three partner institutions: Washington University, the University of Missouri-St. Louis and Harris-Stowe State University. By supporting 8 new students per year over two years of research, education, and networking experiences, this proposal will establish a Pipeline to graduate school. The Pipeline emphasizes sustained training in oral and written science communication, discovery science and outreach experience. We seek to be a Program that responds to changes in the research environment by helping our students to pursue important and innovative problems and concepts, to adopt new techniques and to communicate effectively with their peers and the general public. The training will create a community of young neuroscientists through three interactive and immersive courses, full-time summer and part-time academic year research in a neuroscience lab matched to the student?s interests and background, and presentations at conferences. The Pipeline provides intensive workshops for mentors. The curriculum and research environments will remain broad and deep, combining expertise in molecular, cellular and systems-level approaches to the study of neural function and dysfunction. Major new initiatives aimed at accomplishing these goals include: 1) creation of a new course at our Partner Institutions to develop the network of future researchers, 2) the introduction of ENDURE-ing Synapses, a course to bolster neuroscience fundamentals, literature reading and presentation skills and a sense of community among the students, 3) mentoring experiences for undergraduates in the Society for Neuroscience Brain Bee as part of their training in science communication, and 4) refinement of a near peer-mentoring program that has graduate students working with undergraduates and undergraduates working with high school students. These initiatives will ensure our students remain at the forefront of developments in neuroscience research, teaching and outreach.

Project Summary This proposal tests the fundamental hypothesis that circadian rhythms in brain tumors provide a temporal therapeutic window during which the efficacy of standard treatments may be substantially optimized. Timed delivery of chemotherapy, or chronotherapy, has an established role in colorectal cancer and leukemia but has never been evaluated in brain tumors. Here we build on exciting preliminary data in which we have demonstrated that the efficacy of Temozolomide (TMZ), an established chemotherapeutic for glioblastoma (GBM) is substantially modulated by the time of day when it is administered. These findings suggest that we may already have the means of significantly improving outcome from this dismal disease. In this proposal we will utilize novel intracranial xenograft models of GBM in which tumor cells have been engineered to serve as reporters of tumor circadian time, and in which recipient mice have been genetically engineered to represent four different patterns of diurnal variations in behavior, or chronotype. We will test in vivo, whether GBM cells maintain stable circadian rhythms, whether these are independent or entrained by the host, whether treatment with TMZ is best optimized to tumor or host circadian rhythm and whether there is therapeutic value in ablating tumor circadian rhythm. Success in these studies will advance our basic understanding of GBM circadian biology and will provide critical information for the translational application of chronotherapy to GBM care.