Martha Ulbrick Gillette, Ph.D. - US grants

This proposal represents an intent to characterize the physiological substrates which modulate the primary circadian oscillator in the brain of the rat, the neurons of the suprachiasmatic nuclei (SCN). These neurons serve a well-defined and critical role in the generation and entrainment of the daily oscillations of physiological, metabolic and behavioral functions. The in vitro hypothalamic brain slice technique will be used to investigate circadian mechanisms which are difficult to address in the intact animal, but for which the brain slice offers unusual advantage. In our hands, the SCN in vitro sustains a circadian rhythm of firing rate and peptide secretion for up to 38 hr, even when reduced to remove peripheral hypothalamic regions normally included in the slice. Further, we have found that the electrical oscillations can be phase-shifted in vitro in a response curve similar to the intact animals. Thus, our preliminary neurophysiological investigations have shown that not only the 24 hr oscillator but also the phase resetting mechanism is endogenous to the SCN neurons in the brain slice. Preliminary biochemical studies demonstrate circadian changes in the phosphorylation state of specific SCN proteins and suggest that phosphorylation mechanisms may modulate circadian oscillations in the activity of these neurons. We propose to characterize the phase responsiveness of the SCN in vitro to stimuli which reset the oscillations in electrical activity using conventional neurophysiological and organ culture techniques. This precise definition of the phase-response curve will be used in physiological studies to characterize metabolic pathways underlying the electrical oscillations, particularly cyclic nucleotide- and Ca++-dependent protein phosphorylation as well as pathways involving synthesis of new mRNA and protein. Concurrently, biochemical studies will be carried out to further identify changes in phosphoproteins and their regulators during the normal circadian cycle and during phase-shifting. Because the SCN integrates most circadian behaviors and metabolic fluxes, this study has basic relevance to understanding many brain and metabolic dysfunctions, including certain forms of mental illness.

This award provides funds to establish an interdisciplinary site REU program in Neuroscience at the University of Illinois Neural and Behavioral Biology (NBB) Program, a Ph.D. granting interdisciplinary program begun in 1970. The common focus is neuroscience, the interdisciplinary field that seeks to understand the function of nerve cells and systems from the molecular and cellular levels to that of behavior. Faculty in the program that demonstrate an extensive history of involvement of undergraduate in laboratory research have been selected as co- Principal Investigators. Collectively, faculty in NBB have sent about 75 undergraduates who worked with them into scientific careers, including faculty positions at Harvard, Yale, Chicago, Stanford, and Pennsylvania. Even greater numbers have gone on to careers in Medicine and other professional Doctorate-level fields. Neuroscience subfields in which research experience will be offered included behavioral neuroscience, neural development and plasticity, molecular, cellular and genetic neuroscience, neurophysiology, neuroanatomy, computational neuroscience, and cognitive neuroscience.

The suprachiasmatic nuclei (SCN) of the hypothalamus are endogenous oscillators that serve a well-defined, critical role in the generation and entrainment of the daily (circadian) oscillations of physiology, metabolism and behavior of mammals. Our broad research objective is to understand the molecular, cellular and neurophysiological mechanisms by which the SCN keep 24-hr time. The model species studied is the rat. The SCN pacemaker survives intact in the hypothalamic brain slice where it is accessible to experiments aimed at dissecting cellular mechanisms. Our methodological approach combines brain slice culture with neurophysiological techniques that measure the circadian rhythm of the ensemble neuronal activity and whole cell recordings in the slice as well as biochemical analyses that measure cyclic nucleotide levels, enzyme activities and proteins phosphorylated, and immunocytochemical/in situ hybridization techniques that identify and localize molecules of interest. The present proposal develops naturally from our finding that the SCN rhythm can be reset in the brain slice by treatments affecting cAMP-,cGMP- or pertussis toxin-sensitive pathways. Further, even under the constant conditions in the slice chamber, the pacemaker substrates are changing so that the rhythm is reset by cAMP in subjective day, by cGMP during subjective night and by melatonin during the day/night transition. Our specific aims include: A) to more fully explore the role of cAMP/protein kinase A in SCN function (by examining the activity, concentration and phosphorylation state of protein kinase A (PKA), localizing the sites of cAMP and PKA effects, and the involvement of transcription/translation in cAMP stimulation); B) to more fully explore the role of cGMP/protein kinase G at night (by determining the activity, concentration and phosphorylation state of protein kinase G (PKG), localizing cGMP and PKG effect; C) to examine the regulation of cyclic nucleotide phosphodiesterase (by determining the level and regulation of cAMP and cGMP phosphodiesterase activities) and D) to understand the relationship of other second messenger/kinase systems in generating circadian time (by exploring the interactive role of protein kinases in time-keeping). Because the SCN integrates most circadian behaviors and metabolic changes, this study has basic relevance of understanding many brain and metabolic dysfunctions, including sleep disorders and certain forms of mental illness.

Mammalian circadian timekeeping is a property of the suprachiasmatic nuclei (SCN) of the hypothalamus. This biological clock is responsible for the near-24 h endogenous rhythms of activity observable in all mammals. Because we live in an ever-changing environment that varies over the 24 h period of the day, internal synchronization requires daily adjustment of the SCN clock to keep its phase, and thus the animal's, in synchrony with the outside world. The effect of light, the most potent environmental timing signal, upon the clock is a function of the phase of stimulation within the 24 h cycle: Light will delay the clock during early night and advance it during late night, but is without effect during the day. How is this remarkable integration between external and internal time accomplished? Our broad objective is to understand the cellular mechanisms by which light acting through a retinohypothalamic tract (RHT) projection, regulates the SCN clock. Glutamate (GLU) is the putative transmitter of the RHT. From our preliminary studies, we have developed an hypothesis that the pathway through which light can entrain the circadian clock during late night includes: light -> RHT stimulation -> GLU release -> nitric oxide release -> transcription events -> phase shift. The thrusts of this proposal are to elucidate the initial events connecting RHT stimulation to phase shifting of the circadian clock, and to probe the differences in the RHT pathway between early and late night. Based upon our preliminary studies, the proposal has four major aims: 1. To determine whether RHT stimulation in vitro resets the clock in a light-like, GLU-dependent manner. 2. To determine whether nitric oxide synthase activation is part of this pathway. 3. To investigate the involvement of a transcriptional process. 4. To compare the role of these elements between early and late night. The SCN clock survives intact in the hypothalamic brain slice where it is accessible to experiments aimed at dissecting cellular mechanisms. the model species to be studied is the rat. The effect of treatments (afferent stimulation as well as reagents of the putative signal transduction elements applied focally at projection sites, sometimes with antagonists in the bath) upon the phase of the circadian rhythm SCN electrical activity will be determined over 2-3 days in vitro. Mechanisms of phase shifting will be probed using extracellular recording, immunocytochemistry, enzyme assays, western blots and nitric oxide measurements. Because the SCN integrate most circadian behavior, this study is basic to determining the mechanisms responsible for generation and regulation of biological timekeeping. It will have applied relevance to strategies for ameliorating internal desynchronization manifested as disordered sleep, depression, negative affective states and the physiological decline with aging, as well as chronobiological aspects of therapeutic treatment.

DESCRIPTION (provided by applicant): Acetylcholine (ACh) has diverse regulatory roles in the central nervous system. Cholinergic neurons in brainstem and basal forebrain structures send ascending fibers throughout the brain where they may participate in a wide range of functions, including modulation of learning, memory retrieval, mood states, central autonomic control, and the processes regulating sleep/arousal. Mechanisms by which cholinergic signals induce long-lasting changes in neuronal state, and their consequences for behavior, are areas of intense research interest with great importance for human health. We propose to investigate mechanisms of cholinergic signaling via M1 mAChRs (M1.Rs) to behaviorally relevant hypothalamic neurons and immortalized cells from rat, as well as in transgenic mouse models. Imaging of M1.R distribution within the suprachiasmatic nucleus (SCN) reveals substantial receptor concentrations. We have found that M1 subtype-like pharmacological reagents selectively alter the cholinergic response. Preliminary data using transgenic mice and immortalized cell lines support the pharmacology and point to this brain site as an important model in which to evaluate M1.R signaling. We propose to employ techniques ranging from behavioral analyses to extracellular and patch-clamp recordings in brain slices, accompanied by immunocytochemical, biochemical and cell biological approaches, together aimed at uncovering fundamental mechanisms of M1.R signaling. Our specific aims include: 1) To fully evaluate the effect of genetic deletion of the M1.R on the cholinergic response, 2) To assess actions of downstream diffusible messengers, and 3) To define the role of specific PKG isoforms in this M1.R signaling cascade. In addition to making fundamental contributions to understanding muscarinic neuromodulation of CNS neurons, these studies will permit us to evaluate the roles of M1.R-mediated neurotransmission within a defined behavioral axis. This multidisciplinary approach will provide new insights into central cholinergic neurotransmission, muscarinic signal transduction mechanisms, and decision-making processes that form the neural substrates of behavioral change. Signal transduction is a cellular process, and by identifying the roles of specific receptors, second messenger systems and targets, we will be able to understand the causal mechanisms that mediate long-term adjustments in neuronal state. This research has applied relevance for strategies in developing rationally-based therapies for cholinergic disorders, including those altering sleep/arousal, autonomic function, senile dementia, Alzheimer's type (SDAT), Parkinson's disease, Huntingtons chorea and other neuropsychiatric and movement disorders.

protein quantitation /detection; isothiocyanates; biomedical resource; fluoresceins;

Irving R. Epstein (Brandeis University), Rustem F. Ismagilov (University of Chicago), Anna Lin (Duke University) and Jonathan Sweedler (University of Chicago) are jointly supported to study the chemical processes involved in glial-neuronal signaling. The collaborative project includes fabrication of topologically well-defined networks of microchannels to support neuronal and neuron-glial networks, time-resolved imaging of the calcium levels in both neurons and glial cells of rat hippocampus cultures, specific stimulation of spatially and temporally defined glial cells using microfluidic tools, advanced data analysis and modeling.

Recent research suggests that glial cells are actively involved in chemical communication with and between neurons and other glia. Goals of this project include a better understanding of the role of glial cells in individual synapses and in neural networks, elucidation of the interaction between the network topology and the dynamics of individual elements (cells), and the development and evaluation of methods that can be applied to the analysis of complex chemical and biological networks. This project is funded through the Collaborative Research in Chemistry Program (CRC) and provides outstanding opportunities for undergraduate, graduate and postdoctoral students to acquire knowledge and skills in neurochemistry and bioanalytical chemistry.

DESCRIPTION (provided by applicant): Dynamic assembly and disassembly of the actin cytoskeleton underlies diverse cellular processes, including cell division, developmental polarity and intracellular transport. These changes can be local or global, transforming cell state. Extracellular signals mediate experience-induced changes of actin dynamics within synaptic microdomains of neurons. Recent evidence suggests that actin dynamics of the cell body, distinct from those in the synapse, also may be necessary for neurons to sense and respond to extracellular stimuli. We predict that this process contributes to plasticity of behavior by altering transcription. Our overarching goal is to understand how experience signals long-term state changes in neurons that, in turn, change behavior. We hypothesize that glutamatergic neurotransmission changes actin organization, and this is permissive for transcriptional activation. We will test this hypothesis in the suprachiasmatic nucleus (SCN), a brain site with established molecular substrates necessary for temporal organization of behavior. The SCN is a cell-based, ~24-h clock driven by spatial and temporal oscillations that regulate transcription. Specifically, we hypothesize that signaling cascades initiated by glutamate engage the actin cytoskeleton of SCN cells, changing localization of key transcriptional regulators that alter clock state. We will examine the nature and necessity of such changes in actin and their effects on transcriptional activation of clock genes. We will evaluate these mechanisms in rat and mouse models: cell cultures, brain slices and behaving animals. Specific aims will: 1) characterize and localize stimulus-induced changes in actin;2) find the role of actin changes in clock function and behavior, and 3) determine the role of actin changes in regulating transcription. We will use cell biological methods, dynamic imaging, biochemistry, neurobiological measures, and behavioral analyses. The breadth of this systems-based analysis will generate insights into how experience is transformed into long-lasting modification in brain state and behavior. This will enhance the understanding of substrates of long-lasting neural state change, with broad relevance for public health and disease prevention. Dysfunctions in the actin system cause severe neurological disorders, including those of cognition, neurodegeneration, movement and autonomic control. Sleep disorders, learning/memory impairments, drug-addiction and aging will be direct beneficiaries.

How does the brain encode experience so that future behaviors are changed? Altered neural function that long outlasts experience characterizes brain processes from learning to memory modification to resetting the circadian clock, which patterns behavioral changes over the day-night cycle. Underlying mechanisms are not understood. Consensus is emerging that stimulus-induced remodeling of the actin-based cellular architecture redistributes key informational proteins bound there. Which proteins mediate this change? The researcher will use the power of the mammalian circadian clock, where mechanisms, including plasticity, are evolutionarily ancient and conserved, and control of the rhythmic homeostatic patterning of daily behaviors is localized within one brain site, the suprachiasmatic nucleus (SCN). They will employ a cross-disciplinary approach, combining novel analytical chemistry that enables large-scale analysis and identification of actin-binding proteins in local brain regions with neurophysiology and behavior. The researcher will manipulate SCN actin state, comparing effects of natural neural signals on actin-associated proteins with reagents that directly activate or inhibit actin remodeling. This broad approach will permit discovery of protein complexity necessary for circadian neural and behavioral plasticity. Coupled proteomic and functional analyses will provide new insights on how sensory experience is integrated into a long-lasting response spanning molecular, cellular, brain and behavioral levels. Research on this evolutionarily ancient brain system will identify a set of core plasticity elements that may contribute critical emergent properties to all forms of brain plasticity. Thus, findings will impact understanding of fundamental principles of experience-induced brain adaptation. Beyond scientific inquiry, this study will provide training opportunities at the intersection of analytical chemistry and neuroscience for students in the laboratory, as well as outreach to undergraduates, especially minorities under-represented in science.

DESCRIPTION (provided by applicant): Proper wiring of the nervous system requires interplay of intrinsic and extrinsic signals that shape neurite development, architecture and function. Whereas axonal development is relatively well understood, less is known of the forces that shape dendrites, especially the nano-scale filopodia that decorate developing dendritic shafts. What factors influence dendritic filopodia during wiring of brain circuits? Do filopodia contribute to formation of dendritic spines, sites of synaptic information processing and plasticity? We hypothesize that chemical cues in substrate-bound gradients instruct dendrite morphogenesis and maturation via nanometer-scale changes that transform collateral filopodia into spines. We will build upon our recent success in culturing hippocampal neurons from early post-natal rat at very low densities in refined microfluidic environments. Centered in neuroscience, this R21 proposal bridges with materials science to create and exploit complex gradient chemical fields-ones embedding nanometer scale design rules and capable of imprinting the physical environments of neurons in culture with specific immobilized and diffusive factors. These experimental competencies are provided by state-of-the-art microfluidic systems that exploit a variety of physical behaviors to actuate programmed chemo-temporal profiles within the device. Specific aims are to: 1) characterize collateral filopodial behavior in response to 2D surface gradients of bioactive molecules, and 2) build upon these findings to construct 3D gradient environments that encourage filopodial differentiation and enable responses to diffusive stimuli. Models are hippocampal neurons of early post-natal rat and EGFP-actin transgenic mouse. We seek to discover novel insights, solutions and applications that impact mental health, neural repair and restoration of function. The intransigence of brain disorders and damage to treatment is of rising concern as many incurable conditions (schizophrenia, depression, Parkinson's and Alzheimer's disease) have huge economic costs and will increase with the aging of our population. PUBLIC HEALTH RELEVANCE: Nano-scale Processes of Dendrogenesis Proper wiring of the nervous system requires interplay of intrinsic and extrinsic signals that shape neurite development, architecture and function. This proposal seeks to understand the role of nano-scale filopodia in hippocampal dendrogenesis and spine formation by bridging neuroscience with materials science to create and exploit complex gradient chemical fields embedding nanometer-scale design features in nanoliter physical environments. This innovative approach positions us to discover novel insights for normal dendritic spine formation that will offer new strategies, solutions and applications that impact mental health, neural repair and restoration of function, which are of rising concern as many incurable conditions (schizophrenia, depression, Parkinson's and Alzheimer's disease) have huge economic costs and will increase with the aging of our population.

Description (provided by applicant): A central unsolved question in biology is, What coordinates an organism's circadian clocks? Loss of coordination between the central circadian clock in the brain, the suprachiasmatic nucleus (SCN), and circadian clocks in other cells and tissues has been implicated in systems pathologies that lead to sleep disorders. The goal of this discovery proposal is to identify peptides that couple the SCN and [glial] circadian clocks. Studies of peripheral tissues cultured in isolation have revealed that in the SCN's absence, cellular rhythms continue but phase and period properties change in diverse tissues, including brain, liver, lung, muscle, kidney, tail [and spleen]. With decoupling, the various tissue functions lose temporal coherence as well as appropriate alignment to the daily cycle of sleep and wakefulness. Little is known about what couples an organism's circadian clocks, except that diffusible factors are sufficient to entrain many. Discovering coupling factors that communicate time-of-day from SCN to other circadian clocks [in brain and body] has proven difficult. We propose a study applying advanced analytical peptidomic techniques on a micrometer scale coupled with functional determinations of the ability of peptides to restore clock-to-clock coordination, an innovative approach. We aim to: 1) define and characterize induction of SCN-driven synchronization of [glia] rhythms, 2) identify released candidate coupling peptides by peptidomic analysis, 3) determine the necessity/sufficiency of candidate coupling peptides released from the SCN for inducing synchronous rhythms of [glia] clocks, and 4) characterize and evaluate candidate coupling peptides. Successful completion of these aims will poise us for testing coupling in animal models. Loss of synchrony among internal clocks is maladaptive for health and longevity. There are no current approaches to better synchronize or enhance coupling of the internal clocks. Identifying signals that effectively couple circadian rhythms will have major value in treatment of metabolic syndrome, obesity, cardiovascular stress, and physiological decline with aging, all of which manifest with disordered sleep patterns that affect more than 10 million Americans each year. However, to realize therapeutic potential, signals by which the SCN engages other circadian clocks must be identified and placed in temporal context. PUBLIC HEALTH RELEVANCE: This proposal seeks to identify neuropeptides that provide integration of circadian rhythms in body function across the sleep-wake cycle. Loss of coordination between the central circadian clock in the brain, the suprachiasmatic nucleus (SCN), and circadian clocks in other cells and tissues has been implicated in systems pathologies that lead to sleep disorders, and is maladaptive for health and longevity. Identifying signals that effectively couple circadian rhythms throughout the body will have major value in treatment of metabolic syndrome, obesity, cardiovascular stress, and physiological decline with aging, all of which manifest with disordered sleep patterns that affect more than 10 million Americans each year;however, to realize therapeutic potential, signals by which the SCN engages other circadian clocks must be identified and placed in a temporal context.

1040462
Popescu

This proposal is for instrument development of a spatial light interference microscopy facility that will measure samples in both transmission and reflection modes. This quantitative phase imaging instrument will benefit diverse research efforts in the materials and life sciences. In particular, it will enable: (1) non-destructive inspection of nanostructures, semiconductor devices, and new materials such as graphene and carbon/semiconductor nanotubes, (2) observation of the dynamics of live cells and transport in neurons, and (3) exploration of new cancer detection techniques. Current topographic imaging technology severely constrains the size and sheer number of samples that can be measured at high resolution. Thus, the information gathered and new understanding obtained is thereby limited. Numerous new lines of research and opportunities for discovery in fields ranging from medicine and life sciences to semiconductors and material sciences will be enabled once this new form of fast microscopy is made accessible. Further, development of this transformative scientific instrument will provide rich opportunities to broadly integrate research and education.

This goal of this Integrative Graduate Education and Research Training (IGERT) award is to create a graduate training program that will produce the next generation of intellectual leaders in Cellular & Molecular Mechanics and Bio-Nanotechnology. This program represents a highly coordinated and interdisciplinary effort to educate Ph.D. students across the University of Illinois at Urbana-Champaign, University of California at Merced, North Carolina Central University, and partner institutions to tackle the important problems in bionanotechnology spanning the molecular-cellular-tissue scale.

How living cells transduce mechanical signals to functionalities at different length scales, from inside cells to their communication with the extra cellular matrix, presents a scientific grand challenge of our times. Recent advancements in micro/nanotechnology, molecular scale imaging, and computational methodologies will catalyze this quantitative biological revolution at a cellular and molecular scale. Students who have been trained at the intersection of these domains have the potential to revolutionize tissue and regenerative engineering, biological energy harvesting, sensing and actuation, cells-as-a-machine, and synthetic biology, to name a few. Unique training efforts in this program include a two-track educational program to educate engineering and biology students to develop depth and breadth in their area of research, new experimental modules and a summer workshop introducing the IGERT trainees to state-of-the-art equipment and laboratory procedures, an exciting iWORLD program with collaborators around the world that will provide IGERT trainees with international research experiences, and a student leader council that participates in the leadership and drives the management of the IGERT.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

DESCRIPTION (provided by applicant): Spatially defined sub-cellular heterogeneity determines neuron function. Thus, it is not surprising that disease origins can be traced back to the aberrant behavior(s) of dendritic filopodia that wire the brain. Interactions of the myriad filopodia extended by dendrites of individual neurons in their spatial contexts generate the remarkable range of functionalities of the human brain. Even adjacent filopodia encounter distinct local micro-environments and develop individual functionalities. Only by overcoming ensemble averaging of populations and measuring molecular signatures with single- filopodium resolution can we understand the interplay of the diverse intrinsic and extrinsic regulators, and explain the spectrum of neurological functions encompassing healthy and disease states. In particular, there is an unmet need for ways of probing of local regulators of filopodia during the emergence and sculpting of the dendritic arbor. This innovation proposal addresses this need by integrating our expertise in designing and fabricating nanoliter microfluidic environments for ultra-low density neuronal cultures with our expertise in the cell biology of neurons. We propose to use microfluidic device (¿FD) environments and high resolution image analysis to probe changes in localization, activity, and function of specific microRNAs (miRNAs) in developing hippocampal dendrites. miRNAs are short, non-coding RNAs that act as regulators of local protein synthesis, especially during dendrogenesis and local wiring of the nervous system. Our objective is to control the structure and function of individual dendrites within micro-channels of fabricated ¿FDs to isolate individual dendrites. We will use this system to map and influence miR125b functioning in filopodia during their development and in response to glutamatergic stimulation. This novel set of studies will address the need for understanding with high resolution the localization, activation, and function of miR125b during wiring of the hippocampus. This approach will provide new insights on this putative regulator, new tools for studying properties of miRNA control of dendrogenesis in single neurons, and contribute to effective strategies for restoring defects in models of affective dysfunctions, chronic stress, Alzheimer's disease, and autism.

Proposal Number: 1403660
P.I.: Boppart, Stephen A.
Title: Enhanced Optogenetic Control of Neuronal Activity with Tailored Light Stimuli

Non-Technical Explanation

Significance:

This project will investigate how new forms of light may enhance the electrical output and control of neurons that have been genetically modified to be light-sensitive. Optogenetics is a rapidly developing field that uses molecular biology techniques to enable cellular functions to be controlled with light. When neurons (nerve cells) are genetically modified to express a light-activated membrane channel, they can be made to trigger electrical activity when exposed to light. This new level of light-activated control over neuronal activity has yet to be fully exploited, but is offering new opportunities for understanding how neurons and their electrical circuits function within the brain to form thoughts, memories, behaviors, and emotions. In optical science and engineering, advances now make it possible to generate a wide range of new forms of light with customized properties, or what is called tailored light. This research is highly significant and important for the fields of neuroscience and biophotonics. Biophotonics is the science of how light interacts with biological cells and tissues, and neuroscientists seek to understand how the brain and mind work. The new optical sources for generating tailored light in this project will change the way in which we use optogenetics to investigate and understand the function of neurons, neural circuits, and the brain. This project is also highly interdisciplinary, and will provide a unique educational and training opportunity for undergraduate and graduate students to help them solve the complex interdisciplinary problems in engineering and biology in the future. Results from this research will also be integrated into undergraduate and graduate courses in biophotonics, neuroscience, and advanced microscopy. The long-term societal benefits of this research will include raising the public's scientific literacy of how neurons and neural circuits in our brain function, and how new types of light and lasers can be used to probe the complex functions of our brain.

Technical Description

Optogenetics is a rapidly expanding field, and one that originated out of the field of neuroscience, where genetic modifications to mammalian neurons enabled photo-activated control of membrane channels to elicit action potentials. While this concept has provided a unique toolkit for exploring neuroscience questions and envisioning new medical science applications, there have been relatively few advances or contributions to optogenetics from the fields of optical science and engineering. This proposal addresses this gap by using advanced optical sources and precise control over the optical properties of the light stimuli to enhance the neural control in optogenetics.

The innovation of this research project is the ability to generate new forms of tailored light, and apply this light as new forms of stimuli to excite, modulate, and control the output of optogenetically-modified neurons in new ways. Conceivably, it is much more practical to modify and control the light stimulus than to genetically modify the biological properties of cells and tissues. As optogenetics advances to in vivo applications, this practical advantage will be even more significant. Therefore, our hypothesis is that by precisely controlling the spectral, temporal, and spatial parameters of novel tailored light stimuli, it is possible to provide enhanced modulation and control of the electrical output activity of optogenetically-modified neurons. To prove our hypothesis, our research plan will be guided by three objectives. First, we will construct an optical stimulus and microscope system to generate these new forms of tailored light. Second, we will optically stimulate and electrically/optically record from cultured hippocampal neurons that have been genetically modified to express Channelrhodopsin-2, a light-gated membrane ion channel, to investigate how tailored light stimuli alters the electrical output and activity from these cells. Third, we will implement an optical feedback system that will measure the optical response of the neurons and adjust the light stimuli parameters to optimize, modulate, and control the electrical output.

The successful outcome of this research project will have far-reaching impact in not only the field of biophotonics, but also in neuroscience and optical science and engineering. Just as optogenetics is expected to make a broad impact in neuroscience, as well as medical science, this research will potentially have an even greater and more rapid impact because it will conceivably be more practical to tailor the light stimulus than to modify the biology to enhance the optogenetic control in the future.

This award is being made jointly by two Programs- (1) Biophotonics, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate), and (2) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate).

Brain plasticity, the adaptability of the nervous system in response to experience, is a modulatory process leading to long-lasting structural and functional changes. Plasticity has been thought to characterize neurons primarily. Preliminary data suggest that glia, cells thought to provide nutritive support for neurons, undergo a new, potentially fundamental form of plasticity: diurnal and light-stimulated morphological dynamics in the central circadian clock, the suprachiasmatic nucleus (SCN). This concept is novel and has not been effectively explored. Knowledge of the patterns and regulators of SCN glial plasticity over the daily cycle will contribute significant new insights on glial cellular dynamics in the brain, an emergent property of an understudied area. Goals of educational and outreach components are to develop and validate effective cross-disciplinary instructional modules involving neuroscience, imaging, and three-dimensional analytic techniques. Emphasis will be on engaging women and groups underrepresented in neuroscience research. Outcomes will include a series of educational materials, presentations, and publications for use with K-12 educators and students and the general public aimed at increasing interest and awareness of recent research findings. This will enhance scientific literacy.

The scientific issues will be addressed using confocal microscopy coupled with computer-based analysis of glial structural complexity and spatial organization in the rat SCN. This study will advance understanding of diurnal modulation of glial plasticity in two ways: 1) providing an unbiased, high-resolution three-dimensional assessment of diurnal patterns of SCN glial plasticity, and 2) generating new insights on signals that alter glial structure and thereby dynamic cell-cell relationships. Assessment of efficacy of the educational and outreach components will be made in partnership with Prof. Lizanne DeStefano, Director of the Illinois STEM Education Initiative and an expert in design and evaluation of innovative STEM education programs. They will employ a robust goal-based evaluation to validate, document, and disseminate this model through publication and presentation.

This award is being made jointly by the Neural Systems Cluster in the Division of Integrative and Organismal Systems and the Instrument Development for Biological Research program (IDBR) in the Division of Biological Infrastructure.

Understanding how the brain enables us to think, act, learn, and remember is challenging. Progress has been impeded by lack of a dynamic picture of interactions and properties that emerge when tiers of interconnected brain cells (neurons) are activated in response to experiences. These interactions cause changes in our behaviors and can affect subsequent activities of these neurons, a process called plasticity. This proposal will develop and use newly created, complementary technologies that will non-invasively control, measure, and analyze brain network dynamics and change in real time. Neuroscientists, engineers, and chemists from the University of Illinois at Urbana-Champaign will work together, each bringing cutting-edge methods to bear on this problem. Approaches include: 1) analyzing slices of brain tissue that maintain dynamic properties in a dish; 2) real-time, label-free imaging of neuron activity by novel optical methods; 3) activating and measuring neuronal activity with flexible, clear electrodes that interface directly with cells; and, 4) measuring and identifying patterns of brain chemicals released by experiences. These approaches will be applied together to better understand the dynamic geography of brain information processing and plasticity. Such comprehensive studies of brain dynamics in space and time have never been done. In the future, these technologies can be applied to many brain regions to advance understanding, broadening their impact. Students will be trained beyond usual disciplines, so that neuroscience, imaging technology, engineering of new materials for electrodes, and high-resolution analysis of neuron-to-neuron signals will be taught and used together. Outcomes will contribute to a workforce trained in new ways to tackle problems beyond current boundaries.

What dynamic interactions and emergent properties of neuronal cells and circuits encode experience and generate changes in complex behaviors? Understanding the temporal and spatial dynamics of signal flow and evolution in multi-tiered neuronal circuits has been elusive. The proposed study will address this gap through transformational research that bridges excellence in fundamental neuroscience with innovative technologies in non-invasive imaging, materials development, and neurochemical analysis. Focus will be on processing of a surrogate sensory signal in the suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, that generates long-term behavioral change. This initiative will enable a pioneering program to develop and integrate novel non-invasive imaging of action potentials assessed by quantitative phase imaging of optical signals, stimulation/sensing by original, transparent, biocompatible electrodes, and chemical analyses of complex peptide-release signatures to understand the spatiotemporal dynamics of information flow in rat SCN circuits. These approaches will be applied together to better understand the dynamic geography of brain information processing and plasticity. Such comprehensive studies of brain dynamics in space and time have not been done previously. In the future, these technologies can be applied to many brain regions to advance understanding, broadening their impact. Students will be trained beyond usual disciplines, so that neuroscience, imaging technology, engineering of new materials for electrodes, and high-resolution analysis of neuron-to-neuron signals will be taught and used together. Outcomes will contribute to a workforce trained in new ways to work beyond current boundaries.

This National Science Foundation Research Traineeship award to the University of Illinois at Urbana-Champaign will address the next frontier in biotechnology: to engineer, and then decipher and harness, the living three-dimensional brain. The program will provide doctoral students with the skills and knowledge base to develop and utilize miniature brain machinery in an effort to understand and regulate brain activities. To achieve the goals of developing cross-disciplinary researchers, trainees will learn diverse fundamentals in biology, mathematics, engineering, and cognitive science, relevant to miniature brain machinery. The training grant anticipates providing a unique and comprehensive training opportunity for sixty (60) PhD students, including thirty four (34) funded trainees. Trainees will be recruited from neuroscience, cell and developmental biology, molecular and integrative physiology, chemistry, chemical and biomolecular engineering, bioengineering, electrical and computer engineering, and psychology. The training program will foster a culture of innovation and translational research, and will produce a new generation of scientists and engineers prepared to tackle major problems in brain studies that can improve the quality of human life.

The research and training program will bridge two dominant, non-overlapping brain research paradigms: i) cognitive and behavioral studies, focused principally on understanding of adaptation, decision-making, psychology, and learning of an individual using bioimaging and computational tools vs. ii) cell and tissue studies, focused on activities of multiple neuronal cells by altering their internal and external microenvironments comprised of biomolecules, extracellular matrix, and external stimuli. The goal of this NRT training program is to unite these two dominant paradigms in brain science studies and bridge the expertise of cell and molecular biologists, physiologists, chemists, nano/micro technologists, and cognitive neuroscientists. This training program prepares students for studies that enable control over the networks producing behavior and thus to study causal relations. The overarching goal of the program is to provide students with an interdisciplinary curriculum grounded in problem-based learning and an immersive research experience that blends techniques from multiple disciplines. A second goal is to increase the participation of women, underrepresented minorities, and students with disabilities in neuroscience, life sciences, chemical sciences, and engineering fields. A third goal is to train students in communication skills with the public. Evaluative studies conducted throughout this research traineeship project will explore the dynamics and efficacy of interdisciplinary collaboration by students in this program. Project outcomes will be a demonstrated, evaluated model for transformative graduate training that is effective in developing broadly trained professionals.

The NSF Research Traineeship (NRT) Program is designed to encourage the development and implementation of bold, new potentially transformative models for STEM graduate education training. The Traineeship Track is dedicated to effective training of STEM graduate students in high priority interdisciplinary research areas, through comprehensive traineeship models that are innovative, evidence-based, and aligned with changing workforce and research needs.

The remarkable functional range of the human brain emerges from interactions of myriad neuronal filopodia that generate specific patterns of connectivity. This neuronal wiring during brain development is shaped by sub-cellular spatial heterogeneity, such as dendritic `hot-spots' and filopodia. Even adjacent filopodia of developing dendrites encounter distinct local micro-environments that alter their fate. Consequently, many disease origins, including altered cognition, can be attributed to aberrant behavior(s) of filopodia during brain wiring. A fundamental, unsolved question is how the local environment modulates developing dendrites and how these interactions control dendritic fate, in normal differentiation and in disease. We hypothesize that local redox state provides crucial context, shaping neurite responses to signals. In particular, there is an unmet need for probing local regulators of filopodia during the sculpting of the dendritic arbor. This proposal addresses this need by integrating our expertise in cell signaling, the neurobiology of redox dynamics, and high-resolution imaging in living cells with our expertise in designing and fabricating nanoliter micro-environments for low density neuronal cultures. We will use microfluidic device (?FD) environments and high-resolution imaging of fluorescent intracellular redox reporters to probe changes in localization, activity, and function of redox signaling in developing hippocampal dendrites. We will use this system to map and influence redox dynamics 1) in isolated, dendritic segments during their development, and 2) in their response to semaphorin 3A, which has a dual nature as an axon repulsion cue and a promoter of dendrite growth. We will gain new insights on how local redox dynamics in early developmental stages and in response to Sema3A stimulation contribute to filopodia/dendritic maturation. These novel studies will address the need for high resolution localization, regulation, and function of redox dynamics in developing dendrites, a topic that has received little attention. This approach will provide fresh insights on this putative regulator, new tools for studying regulation of redox-dynamics during dendrogenesis, and contribute to developing effective strategies for restoring defects in affective disorders, Alzheimer's, schizophrenia, Fragile X syndrome, autism, and chronic stress.

Muscle is a unique tissue that can contract, allowing for movement and essential involuntary actions such as breathing, heart pumping, and digestion in humans and animals. Muscle can also secrete various chemicals, called "myokines," which are involved in proper function of the immune system and brain. Neurons are responsible for transmitting signals between the brain and muscle tissues that control muscle contraction and secretion activity. These signals travel through neurons and then transferred to the muscle through the neuromuscular junction. Many injuries and muscle diseases are due to the loss of connection between neurons and muscle. Therefore, reproducing muscle connected to neurons in vitro (in the lab) would enable better understanding of the role/importance of the neuromuscular junction, and this understanding could lead to better treatments for muscle injuries and diseases. Thus there is an urgent need for creating an in vitro physiologically relevant model of muscle connected with/innervated by neurons. To this end, the investigators aim to engineer and validate muscle that contracts and secretes myokines in response to bioelectrical signals from neurons. The project proposes that these neuron-induced muscular activities depend on size, number, and alignment of the muscle fibers in the engineered muscle. This hypothesis will be studied by co-culturing neuron-forming cells on engineered muscle tissue. The quality of neuron-innervated muscle will be evaluated by monitoring contractions and myokine secretion of muscles in response to a neural impulse. In parallel, the investigators will utilize the research program to train undergraduate and graduate students who are involved in bioengineering-related research. The research program will be incorporated into various outreach activities that aim to attract future young scientists and engineers to the biomedical area. Overall, the project will significantly impact efforts to recreate biologically functional muscle tissues and also educate the next generation of biologists and biomedical engineers in diverse ways.

The project is focused on engineering and validating motor neuron-innervated human skeletal muscle that contracts and, in turn, secrets myokines in response to neurotransmitters (e.g., glutamate). Experiments are designed to test the hypothesis that controlling the expression of acetylcholine receptors on engineered muscle is key to enhancing formation of the neuromuscular junction and that the topology and softness of a matrix on which skeletal myoblasts form muscle fibers modulate acetylcholine expression of muscle fibers. The hypothesis will be examined via three aims. The FIRST Aim is to assess the extent that topology of myoblasts-adhered substrates modulates neural innervation and contraction of muscle in response to neural impulse. Human skeletal myoblast cells will be cultured on grooved substrates with grooves (2 to 100 micrometers in width) patterned onto a collagen conjugated PEDGA gel. Studies are designed to answer to what extent the groove-patterned substrate modulates maturity and expression of acetylcholine receptors of myofibers, modulates the alignment and innervation of motor neurons, and influences the neurotransmitter-respondent muscle contraction. The SECOND Aim is to study the effects of softness of the myoblast-adhered substrate on the neural innervation into muscle. The elastic modulus of the gel will be varied from 5 to 40 kPa. Studies are designed to answer to what extent the gel softness and topology orchestrate maturity of muscle and expression of acetylcholine receptors, modulate neural innervation into muscle fibers, influence the neurotransmitter respondent muscular contraction and if neural innervation is related to the mechanotransduction. The THIRD Aim is to evaluate the extent that neuron-innervated muscle produces myokines in response to a neural impulse. Motor neuron progenitor cells will be plated on the myofibers formed on the grooved substrates and differentiated to motor neurons. The focus will be on analyzing mRNA expression and protein secretion levels that lead to increased secretion of myokines. Studies are designed to answer to what extent the neural impulse increases protein and myokine expression by the innervated muscle, to what extent the innervated muscle increases glucose uptake and fat oxidation in response to the neural impulse and to what extent the neuromuscular junction serves to increase the volume of the innervated muscle. The end result is expected to be a neuron-innervated muscle that will actively express and secrete myokines and, in turn enhance metabolic activity and increase muscle volume over time when the muscle is regularly contracted.

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.