Andrea Meredith - US grants

Circadian patterning of neuronal activity and behavior

Understanding how information is encoded in the nervous system is essential to understanding how animal behavior is generated. However, few model systems have well-characterized neural correlates that directly correspond to specific behavioral outputs. One exception is the generation of innate circadian rhythmicity. Daily behavioral rhythms (~ 24 hrs) are a universal trait of animals, vital for adaptation to the environment and overall fitness. In mammals, the principal circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. To understand how circadian time is encoded in the SCN and translated into circadian behavioral outputs, this project investigates how the daily pattern of neuronal activity in regulated. Using transgenic manipulation of ion channel expression and function, the consequences of fundamentally altering the daily rhythm of neural activity in the SCN will be investigated by electrophysiology and behavioral studies. Alteration of ion channel activity is predicted to change the expression of circadian behaviors. This project is expected to advance knowledge in how the brain controls innate circadian behavior, but more broadly, provides a novel approach for understanding the direct links between neural coding and the resultant behavioral effects.

Students involved in this research will learn an integrative approach to biological function, from molecular-cell-circuit-system/behavior. In particular, training in electrophysiology, a discipline traditionally without adequate representation of women and minorities, is the cornerstone of the research project. The lab draws upon the inner-city Baltimore area both for student involvement and dissemination of major findings through local public outlets such as the Baltimore Sun newspaper and Maryland Science Center.

? DESCRIPTION (provided by applicant): Circadian rhythmicity is a fundamental aspect of human physiology. Disruption of circadian rhythm is a significant health burden, impacting sleep, cardiovascular, metabolic and psychiatric disorders. Understanding how circadian modulation of the body's physiology contributes to human health and disease requires a basic understanding of how circadian rhythms are generated and expressed. The overall goal of the proposed research is to identify the basic mechanisms of that encode circadian rhythmicity in the suprachiasmatic nucleus (SCN), the brain's clock. The SCN circuit undergoes synchronized daily oscillations in action potential (AP) firing, and circadian behavioral and physiological characteristics are established by this SCN circuit rhythm. Daily modulation of ion channel activity is a critical basis for generating the circadian rhythm in neuronal activity in the SCN. Oe such ion channel, the BK calcium- and voltage-activated potassium channel (Kcnma1) is a central regulator of circadian rhythm. Daily modulation of BK current magnitude drives SCN circuit rhythmicity, and loss of BK channel function disrupts circadian behavioral and physiological rhythms. The day-night difference in BK current level is mediated by the ?2 subunit, which causes inactivation of BK channels during the day. The circadian variation in BK current properties based on ?2 function is unique, but it is not clear how ?2 regulates AP activiy to shape the rhythms in SCN neuronal activity. The proposed studies test the hypothesis that ?2-mediated inactivation is the critical property required for the BK channel's dynamic role in SCN AP firing, and that this process is central to circadian behavior. This hypothesis will be investigated using electrophysiological recordings of BK currents and APs in acute SCN brain slices, providing data to correlate changes in AP waveforms with inactivating properties of the ?2 subunit. Furthermore, the consequences for inactivation of BK currents that stem from circadian changes in BK's calcium source will be investigated. Lastly, the impact of a human epilepsy-linked mutation and other single nucleotide polymorphisms (SNPs) that vary BK/?2 current properties will be tested and the relevance to SCN and behavioral rhythmicity will be determined. The outcome of this project will be an understanding of how the daily regulation of BK currents governs SCN excitability, providing new physiological and translational insight into the mechanisms that influence human BK channel activity.

DESCRIPTION (provided by applicant): The BK large conductance Ca+2-dependent K+ channel is potent regulator of urinary bladder smooth muscle (UBSM) contractility. Kcnma1-/- knockout mice, lacking the BK channel, exhibit increased UBSM tone, hyperactive contractions, and unstable bladder pressure. This bladder overactivity leads to urinary incontinence in Kcnma1-/- mice during the sleep period, suggesting that these mice are a novel rodent model for nocturia, a disorder characterized by excessive urination during the sleep period. The goal of this proposal is to determine how BK channels are involved in the daily regulation of voiding and develop a mechanistic explanation of the poorly understood processes in the lower urinary tract that govern the normal day-night (circadian) patterning of urine voiding. Consistent with the goals of the R21 mechanism, we propose a high- risk, but high-gain, hypothesis geared towards revealing a completely new understanding of nocturia that is based on the derangement of circadian rhythmicity. Using a multidisciplinary approach that combines urodynamic measurements with transgenic analysis, molecular biology, and electrophysiology, the role of the BK channel in the daily regulation of bladder function will be addressed. The first specific aim of the proposed work is to determine how bladder function is different between day and night in urinary bladder smooth muscle by contractile studies, recording of BK currents by electrophysiology, and analysis of protein expression. The second specific aim will determine the mechanism of a day-night change in bladder function by cystometry in Kcnma1-/- and other transgenic mouse lines, addressing the interaction of the bladder and central brain pathways. These studies have the potential to identify a fundamentally new mechanism and novel level of control for bladder function. The proposed studies are highly significant for elucidating critical mechanisms to target for the treatment of nocturia in human patients. PUBLIC HEALTH RELEVANCE: Nocturia, excessive urination at night, is a common and often persistent disorder affecting >50% of people in some age groups and significantly decreasing quality of life. The goal of the proposed research is to develop a mechanistic explanation of the poorly understood processes that govern the normal day-night (circadian) pattern of urine voiding and that may go awry in nocturia.

This proposal requests funds to support an international symposium on ?Genetic and Animal Models of Ion Channel Function in Physiology and Disease,? the 70th Annual Symposium of the Society of General Physiologists (SGP). The meeting will be held on September 7 ? 11, 2016, at the Marine Biological Laboratory in Woods Hole, MA. The SGP annual symposium has an established reputation as the leading meeting for physiologists, cell biologists, and biophysicists spanning across all career stages and professional arenas. Each year the meeting topic is unique, chosen to highlight emerging topics of interest. The 2016 conference will bring together 150 ? 200 scientists and trainees who use genetic manipulation to elucidate the molecular, cellular, and integrated physiology of ion channels in innovative animal models. This is an exciting time in the field, as researchers combine structural information, cellular imaging, and electrophysiology with genome, transcriptome, and transgenic technologies to uncover the fundamental ion channel-based processes that govern physiology and pathophysiology. A core theme for this meeting is the focus on new directions in ion channel physiology stemming from combining human genetics, genome engineering, imaging, and stem cells. Dr. Jeff Noebels from Baylor University will deliver the Society?s ?Friends of Physiology? keynote lecture, highlighting the future of interplay between genetics and animal models in investigating the mechanisms of human disease. SGP symposia are large enough to provide detailed and in depth analyses of a focused area of research, while being small enough to maximize individual discussions and foster collaborative interactions between students, postdoctoral fellows, new investigators, and established leaders within the field. The Marine Biological Laboratory campus is an idyllic venue that affords participants an intimate environment and ample formal and informal opportunities to share scientific insights. An ambitious program has been developed for this meeting, organized by recognized leaders in ion channel physiology and transgenics, Drs. Andrea Meredith (University of Maryland) and Mark Nelson (University of Vermont). The symposium will focus on five central themes: (1) Innovative animal models of human channelopathies, (2) Identification of novel physiological roles for ion channels using animal models, (3) Alterations in ion channel function in established disease models, (4) Use of induced pluripotent stem cell (iPSC) and patient-derived stem cell models, and (5) Imaging physiological function and excitability in animal models. These topics represent an integration of ion channel physiology from molecular to whole organism levels with a special emphasis on human disease- associated mutations. The Society of General Physiologists provides excellent administrative support to ensure the success of the meeting. The overarching goal of the meeting is to inspire young investigators and to communicate new and significant ideas essential to the advancement of human health and treatment of disease.

Deciphering neural coding will require deconstructing the complex and intertwined signaling mechanisms that drive cellular excitability, synaptic plasticity, and circuit dynamics in the brain. This fundamental objective has been extremely challenging because unraveling the temporal and spatial interactions of multiple signaling pathways requires coordinated observation of multiple networks within individual cells and multiple neurons within intact circuits. Large gaps in knowledge remain because our current tools for tracking the dynamics of molecular activity are poorly suited for investigating more than one reporter at a time. Here, we propose to tackle this constraint through development of a novel methodology for simultaneous optical imaging of multiple quantitative FRET biosensors within single neurons, using FLuorescence Anisotropy Reporters (FLAREs). Numerous FLAREs targeting canonical signaling pathways, including calcium, cAMP, and the MAPK cascade, have been constructed in several colors allowing simultaneous imaging of up to three sensors in a single preparation, either in the same or complimentary pathways. We propose three aims to validate and further develop this technology to tailor it for studying cells and circuitry in acute and cultured slices from the mouse brain during neural coding. We will first adapt an optical sectioning microscopy method that is highly advantageous for fluorescence polarization imaging, known as dual-inverted Selective Plane Illumination Microscopy (diSPIM), for FLARE imaging. We will also expand the FLARE palette to include key regulators of synaptic function (Rac, CaMKII) and membrane excitability (voltage). Construction of the FLARE-SPIM instrument will enable proof of principle studies on two high-value neuronal circuits. First, pushing the limits of subcellular spatial resolution, FLARE-SPIM imaging will be performed on key signaling molecules in single dendritic spines in acute hippocampal brain slices during induction of long-term potentiation. Second, pushing the limits of cellular temporal resolution, we will track the rhythmic fluctuations of voltage, calcium, PKA and ERK activities during circadian oscillations of neuronal activity exhibited in organotypically-cultured suprachiasmatic nucleus brain slices. Together, these studies will lay the foundation for systematic exploration of neuromodulation within cells and neuronal circuitry, providing critical and unprecedented new insights for the spatial and temporal interactions between signaling pathways. Through collaboration with other Brain Initiative groups working on similar problems, this foundational work will be scalable to add suites of sensors that visualize nodes of coordinated cellular activity and reveal and measure the complexity of neural coding within intact brain circuits.

Project Summary: The Training Program in Integrative Membrane Biology (TPIMB) is designed to train pre-doctoral students in the biology, biophysics, and physiology of biological membranes. Now in its 4th decade, the TPIMB continues to lead the effort in interdisciplinary training at the interface of molecular, cell, and systems biology at the University of Maryland School of Medicine (UMSOM), by focusing on the role of the cell membrane and intracellular membranes in mediating and integrating the functions of cells and their interactions with the environment. The program is guided by the idea that studies of membrane biophysics and physiology, and of membrane-based signaling cascades, can provide unique insights into the biology of cells and tissues, in both healthy and diseased states. The faculty of the TPIMB, numbering 39 individuals, are well-funded, highly interactive, and devoted to mentoring students studying a broad range of subjects related to membrane biology. Mentors are selected for their interests, extramural support, and commitment to mentoring. Trainees are selected based on their interests, graduate course grades, recommendations, and previous research experience. Requirements are a core course on Mechanisms in Biomedical Sciences, as well as a year-long class in research ethics, both of which are common to all laboratory research-based graduate education at the UMSOM. Trainees are also required to take a series of specialized upper level courses in membrane biochemistry, biophysics, links between membrane defects and human diseases, biostatistics, hypothesis testing and experimental design. They may also take electives in cellular and systems physiology, pharmacology, or neuroscience. Trainees participate regularly in student-oriented activities, such as a student seminar series, monthly get-togethers, and an annual retreat. The UMSOM supports this program generously through contributions to faculty salary, additional stipends for students, and funds for the TPIMB's regular activities. Current NIH/NIGMS funding supports 7 trainees, but this leaves many students following our curriculum and taking part in our activities without direct support. We enjoy a strong partnership with other NIGMS-supported programs at the UMSOM including the Medical Scientist Training Program and Meyerhoff Graduate Fellows Program, a nationally recognized program that supports underrepresented minorities in the sciences. The strength of our graduate training efforts, successes in recruitment, and the stable federal funding at the UMSOM suggest that continued funding for our program is well justified. With continued support, the faculty and students in the TPIMB can continue to spearhead the effort to integrate training in membrane, cell and systems biology at the UMSOM.