Axel Nimmerjahn, Ph.D. - US grants

DESCRIPTION (Provided by the applicant) Abstract: Microglia are key players in most if not all central nervous system (CNS) disorders including neuropathic pain, Rett syndrome, amyotrophic lateral sclerosis and Nasu-Hakola disease. Recent evidence suggests that these cells also participate in regulation of neurogenesis, circuit remodeling and synaptic plasticity during normal postnatal development, influences they may continue to exert in the adult brain. Despite these vital roles in normal physiology and pathology, we know virtually nothing about these cells' signaling, how it is influenced by local environment and disease, and how it influences other cells and their dynamics in the normal brain. This dearth of knowledge is largely due the inability of in vitro approaches to accurately recapitulate the brain's environment to which microglia are highly sensitive and adaptable and a lack of proper tools to monitor and manipulate microglial cells in vivo. To begin to overcome these limitations we have developed a set of fluorescence staining and genetic manipulation, imaging and data analysis tools for the study of cellular dynamics in the normal brain. These tools have allowed us to reveal, for example, novel forms of astrocytic and neuronal excitation in behaving mice and microglia's surveillance behavior in the healthy adult brain. Building on these approaches we now propose to develop a new set of tools for minimally invasive monitoring and manipulation of microglia in both superficial and deep brain structures. This will allow us to deliver unprecedented insight into these cell's functional dynamics and interactions. Our research therefore has broad implications for our view of microglia, their beneficial and detrimental roles in the healthy and diseased brain and more generally for our ability to uncover complex cell-cell interactions in the normal brain. Public Health Relevance: Microglial cells are involved in onset, progression and/or resolution of essentially all brain pathologies. However, little is known about their properties in the intact brain. We propose to fill this essential gap in our knowledge through development and application of novel research tools.

DESCRIPTION (provided by applicant): Deciphering the relationship between animal behavior and cellular activity in the central nervous system (CNS) is perhaps one of the greatest challenges in neuroscience research today. Traditionally, electrophysiological approaches have been used to sparsely sample from electrically excitable cells of freely moving animals. This has led to the discovery of important behaviorally related phenomena such as place, grid, and head-direction cells in the brain and central pattern generator (CPG) neurons in the spinal cord. Optical imaging in combination with new labeling approaches now allows minimally invasive and comprehensive sampling from dense networks of electrically and chemically excitable cells, such as neurons and glial cells. Imaging in head- restrained mobile mice and with miniaturized head-borne microscopes, for example, has led to the discovery of unanticipated forms of behaviorally related neuronal and glial cell excitation in cortical and hippocampal microcircuits. Long wavelength two- and three-photon excitation now enables imaging in brain regions previously accessible only by invasive endoscopic methods. In contrast, imaging in the spinal cord, the primary neurological link between the brain and other parts of the body, is limited to superficial dorsal regions in anesthetized animals. Because anesthesia precludes animal behavior and alters cellular activity, and because essential central pattern generator components are located in deep tissue regions key aspects of spinal cord physiology have remained elusive. Additionally, because current imaging approaches are limited to either the spinal cord or brain, little is known about how the communication between these CNS regions contributes to behavior. Overcoming such critical barriers in the study of CNS function and dysfunction requires development and application of new tools and approaches. As part of this application new tools and approaches for minimally invasive optical recordings from spinal cord microcircuits during animal behavior, from presently inaccessible deep spinal cord regions, and from anatomically connected brain-spinal cord networks will be developed. The rationale for the proposed research is that once these barriers have been overcome new and unanticipated insight into spinal cord physiology and pathology will be gained. Three specific aims will be pursued: 1) Enable study of spinal cord microcircuits in behaving mice through development of restraint and freely moving imaging approaches; 2) Enable minimally invasive study of deep spinal cord regions in live mice through development of adaptive infrared imaging approaches; and 3) Enable minimally invasive study of spinal cord-brain communication in live mice through development of parallel imaging approaches. Together, the proposed research contribution is significant because it will provide new and unanticipated insight into how defined cell types and their activity patterns relate to spinal cord physiology, brain-spinal cord communication, and animal behavior. It is innovative because it will provide a unique set of tools and approaches with groundbreaking possibilities in multiple areas of science.

Brain functions are executed by intricately coordinated networks of neurons, whose modes of operation are highly sensitive to a constellation of neuromodulators. More specifically, neuromodulators such as dopamine, norepinephrine, serotonin, and acetylcholine exert dramatic control over global brain processes such as arousal, attention, emotion, or cognitive perception. Altered neuromodulator signaling has been linked to neurological and psychiatric disorders such as Parkinson's disease, schizophrenia, depression and addiction. Similarly, opioid neuropeptides play important roles in the modulation of cognition and behavior. While the anatomical structures that produce neuromodulatory signals are well known, little is known about the spatial and temporal evolution of these signals in the innervated brain regions. This is because current measurement techniques, such as microdialysis or cyclic voltammetry, lack the spatial or temporal resolution (and often the molecular specificity) to resolve respective signals. This technical challenge has been a long-standing barrier to our understanding of how neuromodulation alters neural circuit function in order to influence behavior. To address this challenge, this project will develop, validate, and disseminate novel genetically encoded fluorescent proteins for large-scale optical measurement of monoamine neuromodulator and opioid neuropeptide signaling in behaving animals, by bringing together a multi-disciplinary team of investigators with unique and complementary expertise. These sensor proteins have the potential to revolutionize neuroscience in a way similar to genetically encoded indicators for calcium, glutamate, and voltage, which are now in widespread use. Combined with calcium and voltage imaging, neuromodulator sensors will reveal how these systems impinge on cellular and circuit function. In particular, proposed sensors will enable minimally invasive, high-resolution, long-term, and direct measurement of neuromodulator and neuropeptide signaling at synaptic, cellular, and system levels. Sensors for neuromodulatory signaling have remained elusive for a long time. Our team recently developed a first generation of genetically encoded indicators for serotonin (5-HT), norepinephrine (NE), and dopamine (DA) that can report nano- to micromolar concentration changes with high spatial and temporal resolution. Building on this work, the following specific aims are proposed: 1) Optimize and diversify genetically encoded sensors for the monoamines using computational modeling, directed evolution and high-throughput screening; 2) Develop and optimize genetically encoded sensors for opiate neuropeptides using novel protein scaffolds; and 3) Systematically validate the novel neuromodulator and neuropeptide sensors in acute brain slices and behaving animals. Together, this work will provide the neuroscience community with a wide range of well-characterized multi-color indicators for probing the functional role of neuromodulators and neuropeptides in regulating neural circuit function and behavior in both health and disease.

PROJECT SUMMARY A key unresolved question in neuroscience is how different cell types and their activity patterns contribute to sensory processing in the central nervous system. Anatomical and physiological measurements indicate that computations underlying somatosensation are initiated in the dorsal horn of the spinal cord. Genetic, electrophysiological, and circuit-tracing methods have identified a number of neuronal populations involved in this process, as well as their potential contributions. Likewise, histologic, pharmacologic, and genetic studies have revealed important roles for glial cells in the pathogenesis and resolution of aberrant sensations. However, despite these advances, little is known about the dynamic neuronal and glial activity patterns, or the interactions between them, that underlie the moment-to-moment processing of innocuous and noxious stimuli. The recent development of novel two-photon and miniaturized one-photon imaging approaches has enabled stable measurement of cellular calcium excitation in the spinal dorsal horn of behaving animals. These technologies have provided the first insights into how sensory information from mechanoreceptors and nociceptors in the skin activates dorsal horn neurons and astrocytes. Using cutting-edge imaging, optogenetic, and pharmacological approaches, the objective of this proposal is to define how the activity patterns of different types of dorsal horn neurons shape astrocyte calcium excitation, and how astrocyte excitation influences neuronal spiking under physiological and pathophysiological conditions. The rationale for the proposed research is that by uncovering the bi-directional relationship between neuron and astrocyte activity in the spinal dorsal horn, new strategies for pain relief may be developed. Three specific aims will be pursued: 1) Determine how sensory evoked activity patterns in molecularly defined neurons relate to astrocyte calcium excitation in the spinal dorsal horn of behaving animals; 2) Determine how aberrant neuronal activity patterns in preclinical models of pain relate to astrocyte calcium excitation in the spinal dorsal horn of behaving animals; and 3) Determine how targeted manipulation of astrocyte calcium excitation controls aberrant neuronal activity patterns in the spinal dorsal horn of behaving animals. In summary, this work will reveal how molecularly defined neurons encode different sensory stimuli and how their activity patterns relate to astrocyte calcium excitation. These efforts will also reveal how normal activity patterns are altered in two animal models of pain and how pharmacologic and non-pharmacologic interventions targeting astrocytes affect aberrant neuronal activity and sensory processing.

Advanced Biophotonics Core Shared Resource - Project Summary/Abstract The Advanced Biophotonics Core (ABC) provides imaging and analysis instrumentation coupled with technical and collaborative support staff for advanced light and electron microscopy of biological systems. Cancer Center members use the facility for high-throughput imaging assays, high-resolution imaging of live cell and tissue dynamics, super-resolution microscopy, large 3D volume imaging of tissues, electron microscopy analysis of subcellular morphology and protein distribution, and automated computational image processing, visualization, and analysis. The ABC Core is also actively pursuing and developing new cutting-edge imaging and analysis methodologies to better serve the needs of Cancer Center researchers, such as cryo-correlative light and electron microscopy, light-sheet imaging of cleared and expanded tissues, and machine-learning based processing, segmentation, and analysis of light and electron microscope images. The ABC Core is committed to providing Cancer Center members: 1) access to light and electron microscopes, specialized sample preparation reagents and technologies, and computational hardware and software for analysis and visualization, 2) free one-on-one training on all microscopes, as well as image processing and analysis software, 3) consulting and collaborative support for experimental design and implementation of imaging and analysis experiments, 4) sample preparation for electron microscopy, tissue clearing, and expansion microscopy, 5) workshops and demos with advanced microscope and software technologies, 6) weekly open- door imaging boot camp on advanced imaging and image processing techniques, and 7) a monthly Biophotonics scientific seminar series followed by town-hall style discussions with ABC Core staff and users.

Project Summary: Administrative Core The Administrative Core will coordinate all the scientific, regulatory, and external reporting activities of this U19 project. It will provide oversight for all four Research Projects and the Data Science Resource Core to ensure that experiments are coordinated and that proposed schedules for milestones are met. The Administrative Core will support a Program Manager who will help coordinate all scientific activities and regulatory responsi bilities. The Core will also provide for an Administrative Assistant who will support the Project Manager and scientific personnel. The Administrative Core will coordinate meetings between the research teams and the Data Science Resource Core. The Administrative Core will also coordinate scientific leadership meetings between the Principal Investigators, the Project Manager, and any consultants providing support for the project. The Administrative Core will also be responsible for overseeing data sharing between each Research Project to ensure that all generated data are available to all team members.

Project Summary: Overall ?What is the function of glial cells in neural centers? The answer is still not known, and it may remain unsolved for many years to come until scientists find direct methods to attack it.? (Ramon y Cajal, 1901). This prophecy turned out to be accurate. Astrocytes, one of the most abundant cell types in the brain, have long been thought of as primarily passive support cells. Over the past two decades, studies indicate that astrocytes play pivotal roles in nervous system development, function, and diseases. However, a major unresolved issue in neuroscience is how astrocytes integrate diverse neuronal signals under healthy conditions, modulate neural circuit structure and function at multiple temporal and spatial scales, and how aberrant excitation and molecular output influences sensorimotor behavior and contributes to disease. The overall goal of this U19 Team-Research BRAIN Circuit Program proposal is to address this fundamental issue by developing a deeper mechanistic understanding of astrocytes? roles in neural circuit operation, complex behaviors, and brain computation theories. Two overarching questions will be addressed: 1) How do astrocytes temporally and spatially integrate molecular signals from the diverse types of local and projection neurons activated during sensorimotor behaviors. 2) How do astrocytes convert this information into functional outputs that modulate neural circuit structure and function at different spatial and temporal scales. A multidisciplinary, comprehensive effort is proposed to address these questions that can only be completed through close collaboration between researchers with unique and complementary expertise. An innovative multi-scale approach integrating functional, anatomical, and genetic analyses with theoretical modeling will be leveraged. This approach involves quantitative behavioral assays, large-scale imaging of cellular and molecular dynamics, targeted cell-type-specific manipulations, high- throughput omic techniques, genetic profiling, protein engineering, machine learning, and computational modeling. By integrating experimental and theoretical approaches, molecular, cellular, and circuit mechanisms will be determined through which astrocytes influence neural circuits and contribute to complex behaviors and brain computation theories. The experimental and data analysis tools developed as part of this project will be invaluable for the broader neuroscience community.

Project Summary: Project 2 - Linking Fast Timescale Neuron-Astrocyte Communication to Neural Circuit Function and Behavior A fundamental yet unresolved question in neuroscience is how non-neuronal cells communicate with the surrounding neurons, influence their function, and potentially affect animal behavior. Astrocytes are in a unique position to modulate neural circuit function. They are ubiquitous in all CNS regions, express receptors for neurotransmitters, neuromodulators, and neuropeptides, extend highly ramified processes that interact with synapses and other CNS elements, and can operate as a syncytium partly due to their gap junctional coupling. These structural and functional properties enable them to modulate synaptic plasticity and neuronal excitability. Indeed, experimental evidence from multiple species and CNS regions now suggests that astrocytes modulate neural circuit function and behavior on both slow and fast timescales. Nevertheless, precisely how astrocytes respond to the composite molecular signals in their environment and how their intricate excitation patterns influence neural circuit function on fast timescales (sub-seconds to minutes) remains unclear. This Project will test the hypothesis that the heterogeneity of astrocyte transients can be understood by the temporal integration of the time-varying molecular signals in their environment. Previous studies have also suggested that astrocytes operate in at least two different modes: 1) Individually, and 2) as a syncytium. Yet, the relevance of these various forms of chemical excitation for neural circuit function remains a mystery. This Project's second hypothesis is that the different activity modes serve distinct physiological roles, enabling astrocytes to influence neural circuits and behavior on different timescales. This Project proposes four major Aims to tackle these issues as part of a team initiative. Aim 1 will determine how molecular signaling by local neurons relates to astrocyte excitation. Aim 2 focuses on elucidating how neuromodulator signaling by projection neurons influences astrocyte activity. Aim 3 will determine how targeted manipulation of astrocyte function (e.g., their ability to detect, temporally integrate, communicate, or respond to extracellular signals) modulates their excitation patterns, neural circuit function, and behavior. Aim 4 will generate a multilayer, multilevel atlas of the investigated neuron-astrocyte circuits. These data will be acquired from a common set of mouse cortical regions involved in sensorimotor processing using a reward-based quantitative behavioral assay. Computational analyses and modeling of this data will be used to identify variables controlling astrocyte excitation, cell-intrinsic parameters constraining this activity, distinct activity modes, and neuronal properties affected by these astrocytic features. Together, the functional and anatomical studies of this Project will a) provide foundational information about how astrocytes (individually or as a syncytium) respond to, integrate, and modulate neural circuit function (Projects 1 and 2); b) guide the development of novel genetically encoded indicators and interventional tools to interrogate neuron-astrocyte circuits in vivo (Projects 2, 3, and 4); c) inform, test, and refine predictive neuron-astrocyte circuit models of sensorimotor processing (Projects 1, 2, and Data Science Resource Core).