Baljit S. Khakh - US grants

DESCRIPTION (provided by applicant): The brain contains glial cells, which out number neurons by ten-fold. Long considered to provide only a passive role, increasing evidence now suggests that specialized glial cells, called astrocytes, actively participate in normal brain function through interactions with neurons1. Moreover, astrocyte-neuron interactions are involved in epileptic seizure activity. This is of importance because the mechanisms that give rise to epilepsy are not understood;despite the fact that there are three million epilepsy patients in North America for whom a greater understanding of the mechanisms involved is the only rational way to provide improved disease treatment. Such progress is hampered by the fact that many fundamental and important aspects of astrocyte-neuron interactions remain unclear or experimentally unexplored. In this proposal we seek to determine if astrocytes have an intracellular calcium excitability code, and how this impacts synaptic responses to nearby neurons. We will test the hypothesis that kinetically distinct intracellular calcium waveforms within astrocytes trigger distinct forms of exocytosis, and affect nearby neurons in separable and stereotyped ways. Taken together the results of these experiments will establish the logic of astrocyte-neuron communication, and the basis of signaling between these brain cell types. We will thus discover the mechanisms that go awry in epilepsy. We have three specific aims, Specific Aim 1. Tests the hypothesis that physiological patterns of intracellular calcium transients select between modes of astrocyte exocytosis. Specific Aim 2. Tests the hypothesis that distinct modes of astrocyte exocytosis impact neurons in separable ways. Specific Aim 3. Tests the hypothesis that astrocyte exocytosis affects short and long term changes in synaptic strength. PUBLIC HEALTH RELEVANCE We will determine whether astrocytes undergo exocytosis during intracellular calcium transients, as well as determine the effects on neuronal synaptic transmission and plasticity. In so doing we will establish the basis for understanding the active roles of astrocytes within neuronal networks in general, with specific implications for in vitro models of epileptic seizures. This is important because the mechanisms that give rise to epileptic seizures are incompletely understood, and there is an unmet need for the clinical management of epilepsy.

DESCRIPTION (provided by applicant): Abstract P2X receptors are ATP-gated ion channels that are found in the brain. Once activated these channels open a cation selective pore, leading to depolarization and increased neuronal excitability. We have been developing a non invasive approach to track activation of transmitter-gated P2X cation channels. The method exploits the fact that most transmitter-gated cation channels, including P2X receptors, have appreciable calcium fluxes. We engineered P2X receptors to carry calcium sensors near the inner aspect of the pore, and therefore in a nanodomain. We rigorously tested the method for P2X2 receptors. Within a cell, neuron or network this method allows one to image the location of P2X receptors as well as determine when they are activated, with sensitivity equal to whole-cell patch clamp recording. Additionally, the approach is non invasive and provides micrometer scale spatial information. The data show that a FRET based imaging approach can be used as a general method to track the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in neurons, in real time and in living cells. The approach will help reveal when, where and how different receptors are activated during physiological processes. We have two specific aims with which we seek to exploit and refine our new approach. Specific Aim 1: We will employ in vivo expression of the optical reporters to image and identify the regional expression, location and activation of P2X receptors in distinct neurons of the hippocampus from control and epilepsy prone mice. This is because although ATP is known to regulate hyperexcitability associated with epilepsy its precise role is not yet fully understood, largely because there has been no way to measure P2X receptor activation selectively on neuronal processes. Our approach remedies this shortfall and we will use it to image sites of ATP P2X2 receptor activation on neuronal processes from control and epilepsy prone mice. Together with high resolution electrophysiology our approach will allow us to determine precisely how ATP signaling contributes to increased excitability associated with epilepsy. Overall, we will determine the precise roles that ATP signalling plays in the healthy and epileptic hippocampus. Specific Aim 2: We will generate mice expressing optical reporters for P2X2 receptors. These will be invaluable general tools for the ATP signaling community and specifically will shed light on how P2X receptor signaling contributes to epilepsy. We will design and engineer a new generation of P2X constructs that report receptor activation with faster kinetics and higher spatial sensitivity. This will allow us to image fast millisecond time scale P2X receptor mediated signaling in neuronal processes that are inaccessible to electrophysiological methods. PUBLIC HEALTH RELEVANCE: We will study the mechanisms that determine how ATP signaling and P2X2 receptors contribute to a mouse model of epilepsy. In so doing we will establish the basis for understanding the roles of ATP receptors within neuronal networks in general, as well as the specific roles for P2X2 receptors in epilepsy. This is important because the mechanisms that give rise to epilepsy are incompletely understood, and there is an unmet need for its clinical management in humans.

DESCRIPTION (provided by applicant): Astrocytes play well documented supportive roles in the brain. In addition, emerging roles for astrocytes include signaling to and from neurons, and regulation of local blood flow. Certain astrocyte functions are correlated with, or regulated by, cytosolic calcium transients, which are considered a physiological signal reflecting astrocyte excitability. Astrocytes interact with neurons and blood vessels primarily with their distal processes, but it is currently not possible to measure calcium signals non-invasively in astrocyte processes either in vitro in tissue slice preparations or in vivo. A method for measuring calcium in astrocyte processes in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding astrocyte roles in brain function in the healthy CNS, and would open up new ways to study the impact of reactive astrogliosis, which occurs in response to all forms of injury and disease, on these same CNS functions. To develop such a method, we have modified a genetically encoded calcium sensor called GCaMP2 to carry a membrane tethering domain (called Lck) on its N terminus, thus generating Lck-GCaMP2. Our findings thus far show that Lck-GCaMP2 allows the non-invasive imaging of calcium levels in astrocytes near the membrane and in processes in cell cultures. This level of resolution is possible because the Lck-GCaMP2 is selectively and highly expressed in the plasma membrane, providing micrometer scale spatial information. This approach will help reveal when, where and how astrocytes are activated during physiological and pathophysiological processes. In this proposal we seek to exploit our new approach and provide important new resources for the astrocyte signaling community by generating and characterizing novel transgenic mice that will allow calcium imaging in astrocyte processes in tissue slices and in vivo. We have two specific aims. In Aim 1 we will manufacture gene constructs that target Lck-GCaMP2 to astrocytes and use these constructs to establish founder lines of transgenic mice. In Aim 2 we will characterize and use these Lck- GCaMP2 transgenic mice to study mechanisms and functions of calcium signaling in astrocyte processes in hippocampal slices. We will test two hypotheses: (i) that astrocytes display spatially compartmentalized calcium signaling and (ii) that TGF2 has direct, receptor mediated effects on calcium signaling in the processes of reactive astrocytes, thereby providing a mechanism through which reactive astrogliosis could influence neurons. The work proposed here will provide novel, well characterized optical reporter mice that will allow us and others to measure precisely localized calcium signals in astrocyte somata and processes within intact tissue structures such as brain slices and in vivo. These new reporter mice will be valuable general tools for the astrocyte community by allowing researchers to measure local astrocyte calcium signals in processes that are currently inaccessible to conventional imaging methods. PUBLIC HEALTH RELEVANCE: We will develop mouse models that will allow us and other researchers to monitor and track calcium signaling in astrocytes in vitro and in vivo. The availability of these mice would constitute exceptional tools with which to study the role of astrocytes in the normal healthy brain and in diseases of the nervous system, including epilepsy and neurodegeneration as well as during brain injury and repair.

DESCRIPTION (provided by applicant): Neuropathic pain is a prevalent disorder that accompanies a wide spectrum of diseases including cancer, diabetes, traumatic injuries and AIDS. It is a significant clinical problem because the pain is severe and known analgesics have limited clinical efficacy. One of the important discoveries in pain research in the last few years has been the role of ATP activated P2X4 receptors (P2X4Rs) and microglia-neuron interactions in neuropathic pain. During neuropathic pain P2X4R expression is upregulated in microglia located in the dorsal horn of the pain pathway, and reducing P2X4R function alleviates symptoms of neuropathic pain in rodent models of the disorder. However, many fundamental aspects remain unexplored and there is only a rudimentary understanding of microglial P2X4R properties or the mechanisms involved in their upregulation in neuropathic pain. Progress has been hindered because it is not possible to identify and record from P2X4R expressing cells in tissue slices. A method for identifying P2X4R expressing cells in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding microglial P2X4Rs in the healthy CNS and during disease processes. Here we seek to generate and thoroughly characterize an optical reporter mouse using the red fluorescent protein tdTomato. The availability of this mouse will allow researchers to directly visualize and record from P2X4R expressing cells within tissue slices and thus explore the cellular mechanisms that determine P2X4R upregulation and thus contribute significantly to the understanding of mechanisms and plasticity changes in the pain pathway during neuropathic pain. Within the scope of two aims we will generate and fully characterize the reporter mouse and exploit it to measure P2X4 responses using fluorescence guided patch-clamp recordings from microglia and neurons. In Aim 1 we will generate P2X4R reporter mice expressing tdTomato fluorescent proteins as well as establish and characterize different founder lines of transgenic mice to map the location and identity of P2X4R expressing cells. In Aim 2 we will study P2X4R expressing cells using patch-clamp electrophysiology in tissue slices from reporter mice. We will directly test the hypothesis that P2X4R upregulation is specific to activated microglia in the dorsal horn pain pathway. Overall, our approach will provide novel, well characterized optical reporter mice that will allow us and other researchers to identify and record from P2X4R expressing cells within intact tissue structures such as slices of CNS. These new reporter mice will be valuable general tools for the P2X, microglia and pain research communities.

DESCRIPTION (provided by applicant): Astrocytes are found throughout the brain and play well documented physiological roles. Emerging roles for astrocytes include signaling to and from neurons and regulation of local blood flow. Certain astrocyte functions are correlated with or regulated by cytosolic calcium transients, which are a physiological signal. During the previous grant cycle, we developed and used a membrane tethered genetically encoded calcium indicator called Lck-GCaMP3 with the aim of measuring astrocyte calcium transients. Using Lck-GCaMP3 we made the serendipitous discovery of a novel calcium signal in astrocytes due to transmembrane fluxes mediated by TRPA1 ion channels. Moreover, our ongoing experiments show that TRPA1 mediated calcium fluxes give rise to frequent and highly localized near membrane calcium signals that contribute significantly to the resting calcium levels of astrocytes not only within a single astrocyte, but also in a network of astrocytes and neurons in cell cultures, as well as in acute brain slices. Our preliminary data also show that pharmacological block or genetic deletion of TRPA1 channels reduced inhibitory synapse efficacy onto interneurons and long term synaptic potentiation of Schaffer collateral synapses onto pyramidal neurons. We have three specific aims with which we seek to further extend these findings, test novel hypotheses and evaluate the function of astrocyte TRPA1 mediated calcium signals. In Aim 1 we will study near membrane calcium signals in astrocytes within hippocampal slices. In Aim 2 we will employ a variety of methods to systematically evaluate why blocking astrocyte TRPA1 channels or buffering astrocyte calcium levels below rest reduces inhibitory synapse efficacy onto interneurons, but not pyramidal neurons in the stratum radiatum (s.r.) of the hippocampus. In Aim 3 we will study how long-term potentiation (LTP) is reduced by blocking TRPA1 channels. By completing these experiments we will provide new information on the function of a novel astrocyte calcium signal. This information will contribute significantly to our understanding of astrocytes in neuronal networks and allow us and others to test novel hypotheses on the roles of near membrane Ca2+ signals in astrocyte-neuron signaling, and lay the foundations for determining if TRPA1 channels are valid drug targets in brain disorders that involve astrocytes.

DESCRIPTION (provided by applicant): A central goal of neurobiology is to understand how the brain forms, stores, retrieves and encodes information and how these operations go awry in disease. The focus of this application is astrocytes, which make intimate contacts with neurons throughout the brain. Long considered simply the brain's glue, astrocytes are emerging as important regulators of neuronal function. Deciphering the roles of astrocytes in the brain is considered one of the major open questions in neuroscience. This Pioneer Award application seeks to test the novel hypothesis that an early step in brain disorders involves dysfunction of the very fine termini of astrocytes called branchlets that are known to abut synapses. In this context, we define dysfunction as branchlet withdrawal from synapses or altered branchlet signaling, including trophic support, to synapses. These dysfunctions would alter established astrocyte functions including neurotransmitter clearance, synapse regulation and maintenance. This in turn would alter the timing of synaptic transmission, contribute to excitotoxicity and perhaps trigger synapse removal. By focusing on the striatal microcircuitry, we will test the hypothesis that branchlets represents a hitherto overlooked mechanism in neurological and psychiatric disorders that could be exploited for novel therapeutics.

DESCRIPTION (provided by applicant): The focus of this application is to develop much needed and easy to use optical and genetic tools to permit the study of astrocyte function in physiological compartments for genetically specified and tractable cell populations. Astrocytes interact with neurons via fine specialised distal extensions called peripheral astrocyte processes (PAPs). However, a bottleneck to progress has been lack of methods to monitor calcium signals in PAPs, which is a crucial hurdle to overcome in order to understand diverse astrocyte functions in different parts of the brain. This application is based on advances made in our laboratory that allow us to directly measure calcium signals in PAPs. To develop such a method we modified a genetically encoded calcium indicator (GECI) called GCaMP2 to carry a membrane tethering domain (Lck), thus generating Lck-GCaMP2. Then we improved this ~3-fold to generate Lck-GCaMP3 and expressed this in vivo with adeno associated viruses (AAV). Our recent unpublished findings show that Lck-GCaMP3 allows for non-invasive imaging with spectacular clarity. This is a very exciting innovative breakthrough that for the first time will alow researchers to directly measure physiologically relevant astrocyte signals and functions. Moreover, with recent structure-based refinements we made Lck-GCaMP5G, which is ~3-fold better than Lck-GCaMP3 and ~10-fold better than Lck-GCaMP2. We are now ready to develop novel in vivo tools so that Lck-GCaMP5G can be used by anyone and thus generalise a precise way to study astrocyte function and diversity. In Aim 1 we will generate knock-in mice expressing Lck-GCaMP5G at the Rosa26 locus. In Aim 2 we will generate novel BAC transgenic mice expressing Cre/ERT in genetically specified astrocytes. In Aim 3 we will exploit our novel mice to image astrocyte calcium signals in thalamocortical slices.

? DESCRIPTION (provided by applicant): One of the biggest challenges in neuroscience is to understand how neural circuits in the brain process, encode, store, and retrieve information. Meeting this challenge will require methods to record the activity of intact neural networks in freely behaving animals. Spectacular advances with the development of genetically encoded indicators of neural activity and optogenetic actuators now call for methods to image and manipulate the activity of large populations of identified neurons in freely moving mice over prolonged periods of time. Multi-channel imaging is needed to unequivocally identify individual neurons based on their unique gene expression profiles or projection patterns, and a flexible platform is needed so that the scopes can be easily adapted to address diverse neuroscience questions. Optogenetic capability is needed to draw causal connections between cellular activity patterns and behavior. Finally, currently available technology is limited because it requires the mice to be tethered by wires, limiting their range of behaviors and ability to interact with other animals or their environment. In addition, commercial miniaturized scopes are extremely expensive, and cannot be altered to meet individual end-user needs. Here, we seek to remedy shortfalls in existing technology by developing truly open source next-generation two-channel optogenetics-capable, wireless, miniaturized microscopes for imaging and tracking activity patterns of large neural-cell populations in freely moving mice. We will design, manufacture, optimize and test: a two-channel microscope for imaging two fluorophores (Aim 1), an optogenetics-capable microscope for imaging and optogenetic excitation (Aim 2), a wireless miniaturized microscope (Aim 3), and a microscope with combined two-channel, optogenetics, and wireless capability (Aim 4). All throughout, we will build an online environment for sharing our microscopes with the neuroscience community, lifting barriers for others to build, modify, and implant the microscopes and analyze neural activity data with our software. Our new wearable microscopes will have a transformative impact on neuroscience by permitting for the first time the imaging and manipulation of the activity of hundreds of identified neurons and other cells such as astrocytes in freely behaving animals.

Project description Astrocytes are found throughout the mammalian brain and interact spatially and functionally with neurons, blood vessels and other glia. They serve multiple homeostatic functions and are involved in synapse formation, removal and regulation. One long standing and major open question concerns how astrocytes communicate with other cells such as neurons, microglia and astrocytes. From this perspective, much attention has focussed on extracellular ATP, which is released from neurons, astrocytes and multiple other cells by several mechanisms. Once released, ATP activates a family of ionotropic and metabotropic ATP receptors, and its degradation product activates adenosine receptors. However, it has proven extremely challenging to measure extracellular ATP levels directly and much of our knowledge about ATP signaling in the brain is based on pharmacological and genetic interventions targeting ATP receptors. Hence, the release, concentration, dynamics and spread of ATP in living brain tissue has hardly been explored, despite the fact that it is implicated widely in astrocyte-glial and astrocyte-neuron interactions. Buoyed by significant advances in the design and use of genetically-encoded glutamate sensors, we set out to design and characterise a genetically-encoded sensor for extracellular ATP. In the preliminary data of this application we report an intensity-based ATP-sensing fluorescent reporter (iATPSnFR). iATPSnFR is expressed on cell surfaces and responds to expected extracellular ATP concentrations with an increase in fluorescence intensity of an appropriately attached circularly permuted super folder GFP (cpSFGFP). iATPSnFR can be genetically targeted to specific cell types and imaged with standard epifluoresence and confocal microscopes. The use of iATPsNFR will shed light on the cells releasing ATP and reveal when, where and how astrocytes receive ATP signals during physiological and pathophysiological processes. We expect that iATPSnFR will permit the measurement and tracking of extracellular ATP dynamics directly for the first time. Although we focus on astrocytes, iATPsNFR can be applied to any cell type. In this proposal we have two specific aims with which we seek to develop our new approach and provide important, new, broadly applicable and much needed resources for astrocyte and ATP signalling research.

? DESCRIPTION (provided by applicant): One of the biggest challenges in neuroscience is to understand how neural circuits in the brain process, encode, store, and retrieve information. Meeting this challenge will require methods to record the activity of intact neural networks in freely behaving animals. Spectacular advances with the development of genetically encoded indicators of neural activity and optogenetic actuators now call for methods to image and manipulate the activity of large populations of identified neurons in freely moving mice over prolonged periods of time. Multi-channel imaging is needed to unequivocally identify individual neurons based on their unique gene expression profiles or projection patterns, and a flexible platform is needed so that the scopes can be easily adapted to address diverse neuroscience questions. Optogenetic capability is needed to draw causal connections between cellular activity patterns and behavior. Finally, currently available technology is limited because it requires the mice to be tethered by wires, limiting their range of behaviors and ability to interact with other animals or their environment. In addition, commercial miniaturized scopes are extremely expensive, and cannot be altered to meet individual end-user needs. Here, we seek to remedy shortfalls in existing technology by developing truly open source next-generation two-channel optogenetics-capable, wireless, miniaturized microscopes for imaging and tracking activity patterns of large neural-cell populations in freely moving mice. We will design, manufacture, optimize and test: a two-channel microscope for imaging two fluorophores (Aim 1), an optogenetics-capable microscope for imaging and optogenetic excitation (Aim 2), a wireless miniaturized microscope (Aim 3), and a microscope with combined two-channel, optogenetics, and wireless capability (Aim 4). All throughout, we will build an online environment for sharing our microscopes with the neuroscience community, lifting barriers for others to build, modify, and implant the microscopes and analyze neural activity data with our software. Our new wearable microscopes will have a transformative impact on neuroscience by permitting for the first time the imaging and manipulation of the activity of hundreds of identified neurons and other cells such as astrocytes in freely behaving animals.

SUMMARY Astrocytes are ubiquitous, highly branched cells that tile the entire central nervous system, making contacts with neurons and blood vessels, and serving diverse roles. Established roles include ion homeostasis, neurotransmitter clearance, synapse formation/removal, synaptic modulation and contributions to neurovascular coupling. From several of these perspectives, attention has focussed on astrocyte intracellular Ca2+ signaling as a basis to measure, interrogate and ultimately understand their roles within neural circuits. The focus on Ca2+ is based on knowledge that Ca2+ is a crucial second messenger and on the realisation that astrocytes are not electrically excitable. Thus, astrocyte Ca2+ signaling has been studied in vitro and in vivo for 25 years in a range of physiological settings. Based on these advances, however, a major current challenge is to reduce or otherwise abrogate (i.e. ?silence?) astrocyte calcium signaling and thus evaluate its functions in neural circuits. Such approaches are vital to advance basic brain research and they are of direct relevance to brain diseases in which astrocytes are implicated. As reported in the preliminary data of this application we developed a novel genetic strategy (called CalEx) to largely silence (but not abolish) astrocyte Ca2+ signaling in specific brain areas in vivo in adult mice. Using CalEx, we found compelling evidence for how astrocyte Ca2+ signaling regulates tonic GABAergic inhibition of striatal medium spiny neurons (MSNs) as well as striatum-dependent self- grooming behaviors in vivo. This renewal application will capitalise on these advances by combining state-of-the-art imaging and electrophysiology to test novel hypotheses and evaluate the functions of astrocyte Ca2+ signaling in adult striatal neural circuits in brain slices and in vivo. Aim 1 will evaluate how striatal astrocyte Ca2+ signaling affects neurons and astrocytes. In Aim 2, we will record MSN activity in brain slices and in vivo during silenced astrocyte Ca2+ signaling. Aim 3 will determine how silencing of astrocyte Ca2+ signaling alters gene expression and thus seek to identify molecular pathways that explain the hypothesis-driven experiments of Aims 1 and 2. All of the proposed aims are strongly supported by preliminary data. CalEx was extremely robust, and now permits exploration of a fundamental open question in neuroscience: what is the function of astrocyte Ca2+ signaling in circuits and in vivo? We will address this in a focussed way for the striatum and share our tools freely so others can use them in other brain areas.

SUMMARY In order to understand how the CNS encodes, modifies, stores and retrieves information it is necessary to explore the diverse cell populations that comprise the CNS. There is an emerging consensus that the CNS cannot be satisfactorily understood solely as a collection of circuits1. One significant missing aspect in our collective strategy to comprehensively understand the CNS is the largely unmet need to understand additional cell types such as astrocytes1. Astrocytes represent around 40% of all CNS cells and are found throughout the brain. Their close proximity to neurons has been known for over a century. It is now well established that astrocytes serve vital support roles including buffering of K+ around neurons, clearing neurotransmitters from synapses as well as providing nutrients. Astrocytes may also regulate blood flow to meet demands set by neuronal activity. In addition to these varied supportive roles, increasing evidence suggests that astrocytes regulate neuronal function via synapse formation, synapse removal, and regulation of synaptic function through uptake and release of neuromodulators and neurotransmitters. In addition, astrocytes are proposed to engage in bidirectional communication with neurons in a Ca2+-dependent manner, which in some circumstances involves bidirectional ATP signaling. However, despite progress, experimental studies of astrocytes have lagged behind those of neurons by decades, largely because twentieth century neuroscience was dominated by the emergent field of electrophysiology that provided a precise and valuable way to study electrical activity in neurons and its relationship to neural circuit function and behavior. In contrast, astrocytes do not fire action potentials or display any other type of propagated electrical signals, and thus electrophysiology was ill suited to study these cells. As a result, our understanding of astrocytes, their identity, diversity and dynamics is still in its infancy. We seek to capitalize on recent breakthroughs in our laboratories to advance tools that will allow neuroscientists to study in detail the molecular make-up of astrocytes in different brain areas at multiple levels from gene expression, to proteins (Aim 1), to physiology within neural circuit functions in vivo (Aim 2). We will also provide tools to target astrocytes in a selective and non-invasive manner by gene delivery across the blood-brain-barrier (Aim 3). Our overarching hypothesis is that the availability and open dissemination of new, selective tools to study astrocytes at molecular, cellular and circuit levels of investigation may reveal insights about the CNS as striking and as influential as those revealed by early measurements of electrical signals in neurons. Furthermore, the free dissemination of such tools will catalyze additional advances in the context of physiology and brain disease.

A central goal of neurobiology is to understand how the brain forms, stores, retrieves, modifies and encodes information, and to determine how these operations go awry in neurological and psychiatric diseases. The focus of this application is astrocytes, a type of glia. Long considered simply the brain's glue, astrocytes are emerging as important regulators of neuronal function. Astrocytes are ubiquitous, highly branched cells that tile the entire central nervous system, making contacts with neurons and blood vessels, and serving diverse roles. Established roles include ion homeostasis, neurotransmitter clearance, synapse formation/removal, synaptic modulation and contributions to neurovascular coupling. Deciphering and exploiting the physiological roles of astrocytes in the brain is one of the major open questions in neuroscience. This R35 application seeks to exploit technical and conceptual advances made with R01, R21 and DP1 awards and combine them into a single nimble, long-term research program to systematically explore and comprehensively understand the fundamental biology of astrocytes within adult vertebrate intact neural circuits with the compass-driven goal of exploiting this information for advancing new therapies. In this context, we define dysfunction as astrocyte process withdrawal from synapses or altered astrocyte signaling, including trophic support, to synapses. Such dysfunctions would alter established astrocyte roles including neurotransmitter clearance, synapse regulation and maintenance. This in turn would alter the timing of synaptic transmission and microcircuit function, contribute to excitotoxicity and perhaps trigger synapse removal. By exploiting novel experimental tools and concepts generated as part of DP1 and R21 awards, and by applying them to the exemplar striatal microcircuitry studied as part of successive R01 awards, we will determine how astrocytes regulate intact neural microcircuits in vivo. We will test the overarching hypothesis that astrocytes represent a hitherto largely overlooked mechanism in neural circuit function and in neurological disorders. As part of these efforts, we will utilize, and if necessary develop, state-of-the-art tools for molecular, cellular and circuit levels of evaluation. As described herein, our long-term programmatic goal, therefore, is to deliver pivotal molecular, physiological and mechanistic insights on astrocyte contributions to brain function and disease, laying the groundwork for therapeutic advances to occur. We will continue to share openly our database resources and tools in order to enable additional advances by others. In addition, the research program represents an outstanding opportunity and laboratory environment for training the next generation of scientists and physician-scientists.

Alzheimer?s disease (AD) is a complex age-dependent disorder. It requires multiple approaches to comprehensively understand at a molecular level in order to develop novel diagnostics and disease modifying treatments. Astrocytes and neurons coexist in the brain and both major cell types are known to contribute to AD. The cellular phase of AD is proposed to comprise feedback and feedforward signaling between diverse brain cells as a link between the initial emergence of molecular pathology (abnormal tau and A?) and subsequent disease manifestations. Known glial cell proteins that contribute to this cellular phase are APOE and TREM2, and are associated with significantly increased risk of AD. Moreover, known astrocyte mechanisms include reactivity, which is a complex, non-binary phenomenon with sequelae that depends on context. In the past, most disease related studies have evaluated astrocytes or neurons using assessments of physiology, markers, or with gene expression evaluations. Astrocytes and neurons have not been studied in detail together or with cell-type specific proteomic methods, as proposed here and as requested by the FOA. As a result, despite advances, we have little precise information about the proteomes of astrocytes and neurons during aging in brain areas relevant to AD or in brain regions relevant to specific and defined abnormalities such as seizure activity in AD. Our overarching hypothesis is that astrocytes and neurons display protein dynamics during normal ageing and in mouse models of AD and that these changes reflect signaling between these dominant brain cells during the cellular phase of AD pathogenesis and during aberrant seizure activity and its associated cognitive decline in AD. Aim 1 will characterize cell, brain region, and compartment (plasma membrane versus cytosol) specific proteomic methods for astrocytes and neurons. Aim 2 will determine astrocyte and neuron proteomic dynamics during normal aging in mice. Aim 3 will determine astrocyte and neuron proteomic dynamics during aberrant network activity in AD model mice. Understanding the identities and the extent of cell, brain region, and compartment-specific protein changes for the major brain cell types (astrocytes and neurons) using data-driven unbiased approaches could be foundational and catalytic with regards to new opportunities for translational and mechanistic work.