Xiangmin Xu - US grants

[unreadable] DESCRIPTION (provided by applicant): Project Summary: Despite extensive knowledge of the basic blueprint of cortical circuits, detailed knowledge about the connectivity of specific cell types and how they function is still limited. The studies proposed here will investigate the laminar and fine-scale specificities of excitatory and inhibitory synaptic input to identified inhibitory neurons in the cerebral cortex, and will examine in vivo physiology of specific inhibitory cell types and their participation in regulating synchronous and oscillatory cortical activities. Recordings of specific inhibitory cell types can be facilitated by visualization of green fluorescent protein (GFP) expression restricted to known inhibitory neuron types in transgenic mice. Laminar specificity of functional input to specific cell types will be understood by using the technique combining whole cell recordings with scanning laser photostimulation. Furthermore, the fine-scale specificity of connections between pairs of neighboring inhibitory cells or excitatory and inhibitory cells will be revealed by cross-correlation analyses of synaptic responses evoked by photostimulation and recorded simultaneously from the neighboring pairs. In addition, to understand in vivo physiology and function of specific inhibitory cell types, targeted recordings under the guidance of 2- photon imaging will be made from these same cell types in GFP-expressing transgenic mice. We will record spikes from the target cells and measure local field potentials (LFPs) through electrocorticogram (ECoG) recordings. For each inhibitory neuron type, the overall spiking pattern in relation to LFPs and spike- triggered average of LFPs will be assessed to determine whether a correlative relationship exists between spike times and cortical oscillations. Other physiological properties of the recorded cells will also be assessed to further understand the properties of inhibitory neurons and their circuits. Relevance: Studies of the detailed organization of cortical circuits involving specific inhibitory cell types are necessary toward understanding cortical function. Understanding the specific roles of inhibitory cortical neurons has important implications for human health, as these cell types and their activities are involved in the cortical mechanisms that regulate attention and their disruption is implicated in schizophrenia. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a competitive revision application for my NIH Pathway to Independence Award (4 R00DA023700) tilted "Local Connections and In Vivo Physiology of Inhibitory Cortical Neurons", in response to the NIH funding notice (NOT-OD-09-058, NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). Despite extensive knowledge of the basic blueprint of cortical circuits, detailed knowledge about local cortical circuits, the connectivity of specific cell types and how they function is still limited. The studies originally proposed investigate the laminar and fine-scale specificities of excitatory and inhibitory synaptic input to specific types of inhibitory neurons in the cerebral cortex, and examine in vivo physiology of distinct inhibitory cell types and their participation and regulation of cortical activities. We have accomplished the aims of examining the laminar specificity of functional input to distinct cell types by combining whole cell recordings with scanning laser photostimulation in brain slices. We also have progressed toward understanding in vivo physiology of specific inhibitory cell types. Recently, we have developed a technique enabling high-resolution and fast functional imaging in brain slices through a novel combination of voltage sensitive dye imaging and laser scanning photostimulation. This innovation will have broad impacts in the field of cortical circuitry studies, as it facilitates rapid mapping and precise evaluation of cortical organization and function. The innovation will enable the ability to map inhibition output from inhibitory cell types by examining their influence on neuronal population activities evoked by photostimulation and detected by voltage sensitive dye imaging. Given the exciting developments of my original projects, I would like to revise my original aims, and focus on further improving the new technique and extending such technology toward elucidating cortical circuitry. The newly revised Specific Aims are to (1) Refine and improve the high-resolution and fast functional imaging technique in brain slices;(2) Map inhibitory output of specific types of inhibitory neurons using the novel technique;(3) Characterize circuit alterations in dopamine receptor knockout (D2R-/-) mice with the novel technique. This revision requests support to purchase additional equipment and train one minority graduate student. PUBLIC HEALTH RELEVANCE: Studies of the detailed organization of cortical circuits involving specific inhibitory cell types are necessary toward understanding cortical function. These studies have important implications for human health, as these cell types and their activities are involved in many disease models and can mediate the cortical mechanisms related to drug addiction and abuse.

DESCRIPTION (provided by applicant): The hippocampus contains diverse types of GABAergic inhibitory neurons. Previous work suggests that different subtypes of these inhibitory neurons have distinct functions, but the rules for their regulation of the hippocampal circuit remain to be determined. We hypothesize that functional differences between inhibitory neurons result from distinct circuit connections of different types of inhibitory neurons. The goal of the proposed studies is to map local and long-range direct synaptic connections to major inhibitory neuronal types in CA1 of the mouse hippocampus. We hypothesize that specific types of inhibitory neurons selectively receive local and distant excitatory synaptic inputs from different brain regions, and that each of these inputs to specific inhibitory neurons differentially contributes to their inhibitory regulation of hippocampal target neurons. Specifically, (1) we aim to identify local excitatory connections to specific types of inhibitory hippocampal neurons by laser scanning photostimulation (LSPS). We have combined LSPS with whole-cell recordings from inhibitory neurons in living brain slices to map intrahippocampal sources of excitatory input to the most numerous inhibitory cell types including parvalbumin-expressing (PV+) basket cells, cholecystokinin- expressing (CCK+) basket cells, axo-axonic cells, and somatostatin-expressing (SOM+) oriens-lacunosum moleculare (O-LM) cells. We will test the hypothesis that axo-axonic cells, PV+ and CCK+ basket cells receive differential strength of excitatory input from CA3 vs. CA1 to support their feedforward and feedback inhibition of CA1 network activity. We will also test the hypothesis that O-LM cells only receive excitation from excitatory pyramidal cells in CA1 and strictly perform local feedback inhibition. (2) We aim to identify long-range synaptic connections to selected groups of inhibitory hippocampal neurons with a novel rabies-based tracing system and optogenetic stimulation. We will use knock-in mouse lines that express Cre (Cre recombinase) in selected groups of inhibitory neurons (e.g., PV-Cre, SOM-Cre or CCK-Cre) to limit rabies infection and monosynaptic retrograde tracing to each selected cell group in the intact brain. The rabies tracing will be followed by channelrhodopsin-assisted circuit mapping to functionally characterize the specificity of distant connections to identified cell type within each targeted Cre-expressing cell group. We will test the hypotheses that PV+ inhibitory cells, but not SOM+ inhibitory cells, receive strong distant connections from entorhinal cortex and the medial septum, and that the CCK+ cell group has strong direct synaptic connections with the amygdala. These studies should establish the operational rules of the major classes of inhibitory neurons within the hippocampal circuit.

? DESCRIPTION (provided by applicant): Inhibitory neurons are key regulators of cortical operations. Their dysfunction has been implicated as a major factor in many brain disorders. While recent studies indicate physiological and functional differences between specific types of inhibitory neurons, neural circuit mechanisms that give rise to these differences in cortical regions underlying cognition and executive function are not well understood. We focus our studies of inhibitory neuron circuit organization and function in the prelimbic area of medial prefrontal cortex (mPFC). This region is highly relevant to schizophrenia, autism, attention deficit disorders and others. The guiding hypothesis for this proposal is that the distinct connectivity of each type of inhibitory neurons differentially governs computationally distinct neural signal transformations in the mPFC, and that circuit connectivity differences between these cell types can be mapped to determine their specific roles in regulation of cortical network dynamics and behavioral output. Our proposed experiments will focus on the three major, non- overlapping inhibitory cell types or groups (parvalbumin-expressing, somatostatin-expressing and vasoactive intestinal peptide-expressing interneurons). A new Cre-dependent, genetically modified rabies-based tracing system will be used to map monosynaptic global circuit connections in the intact brain to these selected inhibitory neurons. To complement the anatomical rabies tracing, physiological input characterization will be accomplished by laser scanning photostimulation and channelrhodopsin (ChR2)-assisted circuit mapping. These studies will allow mapping of both local and long-range functional inputs to identified subtypes within each targeted cell group in brain slice preparations. Building on assessing input connections, we will map local functional outputs of these major inhibitory neuronal groups. Computational and behavioral analysis of the input and output circuit connections of specific inhibitory neuron types will be applied to understand how they regulate mPFC network oscillations in vivo and how they contribute to mPFC-controlled animal learning. This will be achieved by electrophysiological recordings made in parallel with behavioral performance measures with cell-type specific genetic inactivation. Together, the proposed research will generate new maps of inhibitory neuronal circuit wiring in medial prefrontal cortex, and it will broadly illuminate how inhibitory neuronal circuits regulate normal and maladaptive behaviors linked to neuropsychiatric and neurological diseases.

The physiological aspects of experience-dependent critical period plasticity has been extensively studied starting with the pioneering studies of Hubel and Wiesel in the 1960s. However the molecular mechanisms that translate sensory deprivation into functional changes in circuit connections remain poorly understood. Neuregulin-1 (NRG1) signaling through its tyrosine kinase receptor ErbB4 is essential for the normal development of the nervous system, and has been linked to neuropsychiatric disorders such as schizophrenia. NRG1 is widely expressed in excitatory neurons, inhibitory interneurons and glial cells in the visual cortex, while ErbB4 expression is largely restricted to parvalbumin-expressing (PV) neurons. We discovered recently that NRG1/ErbB4 signaling in PV neurons is critical for the initiation of critical period visual cortical plasticity by controlling excitatory synaptic inputs onto PV neurons and thus PV-cell mediated cortical inhibition that occurs following visual deprivation. Building on the strong premise from the literature, this discovery and our data showing that NRG1 effects depend on specific neuronal types and are modulated further by deprivation duration, we propose to provide a detailed analysis of NRG1 signaling actions implicated in visual cortical plasticity at the cellular and circuit levels. We hypothesize that NRG1 signaling critically regulates functional circuit connections of PV inhibitory interneurons during short and prolonged visual deprivation that underlies the initiation and establishment of visual critical period cortical plasticity. We also hypothesize that manipulation of ErbB4 signaling in PV neurons is sufficient to extend the ocular dominance plasticity after the closure of the critical period. To test our hypotheses, in Aim1, we will use our established cell-type specific mRNA expression analysis and neurochemical immunostaining to map cellular NRG1 expression in normal and deprived cortex, and determine whether non-PV cell types contribute to the source of PV neuron NRG1. In Aim 2, we will combine ex vivo functional circuit mapping and in vivo 2-photon calcium imaging to test whether NRG1/ErbB4 signaling is required for maintenance of PV neuron excitatory inputs in normal cortex and for restoration of their excitatory inputs in deprived cortex. In Aim 3, we will use pharmacological and genetic approaches to manipulate ErbB4 signaling in PV neurons to extend and attempt to re-open the critical period window of cortical plasticity. Together the proposed research will advance our understanding of molecular mechanisms underlying visual cortical plasticity, and help to develop new therapeutic approaches to treat amblyopia and other neurodevelopmental disorders.

Project Summary / Abstract The bed nucleus of the stria terminalis (BNST) plays an important role in fear and stress, and has been implicated in anxiety disorders including posttraumatic stress disorder (PTSD). The BNST contains a collection of sub-nuclei delineated by cytoarchitecture, molecular and neurochemical features. Dorsal and ventral subregions in anterior BNST contain substantial populations of neurons that express the stress-associated neuropeptide, corticotropin-releasing hormone (CRH) known to modulate BNST-related anxiety-like behaviors. Most of what we know about intrinsic and extrinsic BNST circuit connections comes from classic anatomical studies. These studies tend to have low spatial and cell type resolution. Further, there is little work on the functional synaptic connections within the BNST. Thus many aspects of the circuit organization are not well understood. We propose to use recent technological advancements in genetic cell targeting, molecular and viral tracing, and functional circuit mapping to determine the synaptic circuit organization of specific BNST neuron types with a focus on CRH+ neurons. We hypothesize that CRH-expressing neurons differentially govern neural signal input/output transformations in anxiolytic and anxiogenic promoting subregions of the BNST, and that distinct circuit connectivity differences between these cell types can be mapped to determine their specific roles in regulating stress behavior responses. To test our hypothesis, we will first map global and local circuit connections to CRH+ neuronal types in putative anxiolytic and anxiogenic subregions (Aim 1). New genetically modified rabies-based tracing will be used to map monosynaptic global circuit connections in the intact brain to these selected BNST neurons. To functionally verify the results of the anatomical rabies tracing studies, physiological input characterization of local and long-range connections will be determined by laser scanning photostimulation and channelrhodopsin (ChR2)-assisted circuit mapping. Following input mapping, we will map anatomical and functional output projections of CRH+ neuronal types using anterograde directed viral tracing and ChR2-assisted circuit mapping (Aim 2). This will establish CRH+ projection targets in hypothalamic regions related to the regulation of hypothalamus-pituitary-adrenal (HPA) axis activity. In addition, behavioral analysis of the function of CRH+ neuron types will test how these cell types in specific BNST subregions are integrated in larger functional networks and how they contribute to control of stress responses (Aim 3). This will be achieved by specific optogenetic activation in parallel with physiological and behavioral measures. Together, these studies will generate new maps of CRH+ neuronal circuit wiring in anxiogenic and anxiolytic subregions of the BNST. This research will advance our understanding of BNST-centered neural mechanisms to better understand and treat pathological anxiety disorders.

PROJECT SUMMARY/ABSTRACT The visual system exhibits a heightened sensitivity to the quality visual experience during an interval late in development termed the critical period. Discordant vision during the critical period is the cause of amblyopia, a prevalent visual disorder in children. Treatment of amblyopia is most effective in children before the close of the critical period. Subsequently, the flexibility with brain circuitry diminishes in adulthood and effective therapy is more difficult. In a mouse model of amblyopia, disrupting normal vision by closing one eye for only a few days (monocular deprivation, MD) during the critical period, but not thereafter, also perturbs the normal binocularity of neurons in visual cortex and decreases visual acuity. Yet how these adaptive changes, or plasticity, first emerge within neurons that form the circuits in visual cortex is poorly understood. Likewise, how plasticity propagates from the first neurons to adapt to other neurons connected to these neurons by synapses is unclear. The short duration of the critical period in mice is one factor impeding the study of how the greater plasticity confined to the critical period contributes to the induction as well as recovery from amblyopia. The nogo-66 receptor gene (ngr1) is required to close the critical period. In ngr1 mutant mice, plasticity during the critical period is normal, but it is retained in adult mice. Importantly, ngr1 mutant mice spontaneously recover visual acuity in this model of amblyopia. In the proposed research, we take advantage of this extended critical period in ngr1 mice to investigate what is unique about plasticity during the critical period that promotes recovery from amblyopia. We compare how MD alters the function and connectivity of populations of neurons in visual cortex with a combination of sophisticated repeated in vivo calcium imaging and laser-scanning photostimulation synaptic mapping. We will begin to unravel how plasticity within visual cortex proceeds during abnormal vision (MD), as well as how this plasticity is restricted to the critical period with these experiments. In addition to improving understanding of how experience-dependent plasticity changes the function of brain circuits, these studies may reveal new avenues for developing therapeutic approaches to treat amblyopia and perhaps other neurodevelopmental disorders that result from maladaptive developmental plasticity.

Project Summary / Abstract The bed nucleus of the stria terminalis (BNST) plays a central role in the normal adaptive response to stress, and has been implicated in general anxiety disorders, posttraumatic stress disorder (PTSD) and stress-induced drug abuse. Most existing knowledge about intrinsic and extrinsic BNST circuit connections is derived from classic anatomical studies which do not reveal functional synaptic connections. Due to previous technical limitations, many aspects of the intrinsic circuitry and local circuit organization of synaptic inputs in the BNST are not well understood. We propose to apply recent technological advancements in genetic cell targeting and photostimulation-based circuit mapping to build a high resolution functional map of the synaptic circuit organization of specific BNST neuron types. We hypothesize that strong local inhibitory connections are a major organizational principle in the anterior dorsal BNST, and that the relative balance of local BNST inhibitory synaptic connections underlies functional interactions between the anxiolytic and anxiogenic promoting subregions. To test this hypothesis, in Specific Aim1, we will use laser scanning photostimulation to map local synaptic input connections to three non-overlapping neuron types in the anterior dorsal BNST, including non- GABAergic CaMKII? expressing neurons, GABAergic somatostatin-expressing (SOM) and corticotropin- releasing hormone expressing (CRH) neurons. In Specific Aim 2, we will complement and extend laser scanning photostimulation studies with optogenetic stimulation-based circuit mapping to analyze inputs from specific neuronal subsets within the local BNST circuits. If successfully implemented, the proposed studies will lead to important progress toward understanding cell-type based local BNST circuit organization and function.

Project Summary / Abstract Encoding of environmental location and navigational behavior in mammals involves large ensembles of specific neuron types across multiple interacting brain regions. ?Place cell? and ?grid cell? mapping of spatial location in the CA1 region of hippocampus and medial entorhinal cortex (EC), respectively, is thought to be fed forward to associative cortical brain regions including the posterior parietal cortex (PPC) and retrosplenial cortex (RSP) to map conjunctions of egocentric and external spatial relationships. This notion implies that the hippocampal-neocortical pathway involves a gradual transformation of spatial cognition to action along with encoding of specific route information at intermediate processing stages. While the characterization of this hippocampal feedforward output to the neocortical system has been conceptually useful for our understanding of spatial navigation processes, it is now time to consider the role of the largely unexplored ?top-down? neocortical inputs from RSP to the hippocampus. The subiculum (SUB) is an under-investigated brain structure well positioned to mediate circuit interactions between the hippocampal and neocortical systems. Based on our recent discoveries, we hypothesize that specific subsets of SUB neurons receive significant direct ?top-down? inputs from RSP and that these inputs yield specialized SUB encoding of multiple spatial relationships including the axis of travel, boundary vectors, and route sub-spaces. These SUB neurons are expected to overlap with the population of CA1-projecting SUB neurons that exert direct feedback regulation of hippocampus-associated spatial mapping and learning. We propose to study the synaptic circuit organization and functional implications of this ?top-down? pathway from RSP cortex, to SUB, to hippocampal CA1, using recent technological advancements. To test the hypothesis, in Aim 1, we will map brain-wide circuit input connections of CA1-projecting SUB neurons and compare these to EC-projecting, and RSP-projecting SUB neurons using new viral tracing and optogenetic stimulation mapping. A combinatorial viral and genetic strategy will be used to selectively label projection-specific SUB neurons for circuit studies and physiological characterization. In Aims 2 and 3, we will link circuit connection mapping to neurophysiological function and behavior. Tetrode recordings and in vivo GCaMP6-based calcium imaging of CA1 at single-cell resolution in freely moving animals will resolve how RSP inputs and projection-specific SUB neurons modulate CA1 place cell activities and how they contribute to spatial learning and navigation. The studies will be conducted in conjunction with behavioral analyses addressing how animals learn object-place associations and routes through environments having multiple interconnected pathways. Genetically targeted neuronal inactivation will be used to establish the causality of circuit connections and function. The proposed studies are aligned with the specified goals of Targeted Brain Circuits Projects, and will contribute to a mechanistic understanding of how dynamic patterns of specific SUB neural activity are transformed into spatial navigation and cognition.

Project Summary The development of trans-synaptic viral tracers is an important component of the BRAIN Initiative. At present, the lack of viral-based anterograde monosynaptic tracing tools with high signal strength and low toxicity is a gap in neuroscience. Herpes simplex virus (HSV) type 1 strain 129 (H129) is the most promising viral tool for anterograde neuronal tracing. However, current versions of genetically modified H129 viruses are limited by high virulence and toxicity, weak label signals that require immunostaining for detection, and time-dependent spread across multiple synapses. There is also a concern of the directional specificity of anterograde propagation of H129 recombinants, as they may propagate retrogradely. Investigators in the field have been working actively to develop improved versions of anterograde viral tracers, but progress has been limited. We have formed a strong interdisciplinary collaborative team composed of virologists and systems neuroscientists to develop anterograde monosynaptic recombinant H129 tracers with high signal strength and little or no toxicity for multi-species neural circuit analysis. Our published work and preliminary data establish the feasibility and key methodologies for the proposed research. We will capitalize on our established bacterial artificial chromosome (BAC) based system for rapid generation of recombinant H129 vectors and precise control of the H129 payload. We have a sound plan to reduce viral toxicity, enhance label signals and generate variants carrying different functional payloads. Our overall goal is to create a new set of safe, effective and validated anterograde-directed viral vectors that allow efficient labeling in monosynaptic projection targets of specific neuron types. These new tools will have a broad impact by enabling optical imaging, physiological recording, and activity manipulation of defined anterograde projection networks. For rapid resource sharing, we will create a service platform through the UCI Center for Virus Research to disseminate the new molecular tools to the neuroscience community.

The transplantation of embryonic inhibitory neurons has recently emerged as a promising avenue for cell-based brain repair. Previously, we and others have shown that transplanted inhibitory neurons restore juvenile plasticity to the circuits of adult visual cortex. In this study, we set out to elucidate the mechanisms for transplant-induced cortical plasticity. We hypothesize that transplanted inhibitory neurons reactivate plasticity by creating a new, disinhibitory microcircuit in recipient adult visual cortex. First, we will map out the effects of transplantation on the inter- and intralaminar balance of excitation and inhibition. Next, we will compare the cell-type specific effects of brief monocular deprivation on visual activity in host cortex. Lastly, we will test whether transplanted neurons make long-range connections with appropriate circuits. To evaluate these hypotheses, this proposal will take advantage of recent advances in optogenetic dissection of inhibitory circuits in brain slice, multiphoton imaging of defined cortical cell types using genetically encoded calcium indicators, chemical-genetic tools for testing the mechanisms of transplant- induced plasticity and viral tracing and whole brain clearing to identify the brain-wide connections onto transplanted cells. If successful, the proposed studies shed light on the mechanisms of transplant-induced cortical reorganization. These studies are also likely to give insight into the normal developmental regulation of cortical plasticity by inhibitory cells.

Project Summary / Abstract Alzheimer?s disease (AD) is the most common cause of progressive dementia (memory and cognitive loss) in older adults. Presently, more than 5.5 million Americans may have dementia caused by AD. There is no cure for this debilitating condition. It is increasingly critical that we develop better early diagnostic tools and new treatment strategies for this neurodegenerative disease. Previous gene expression studies using brain tissue and cross-sectional design identify genes whose expression correlates with AD progression. Gene expression is regulated by the cell?s epigenome comprising of DNA methylation, histone modification and non- coding RNAs. We propose to characterize the epigenome of key cell types in neural circuits responsible for learning and memory. Our goal is to determine how the epigenome shapes hippocampal circuit activity and behaviors during AD progression, using the latest single cell genomic technologies coupled with functional circuit mapping and behavioral analysis. We will use two AD mouse models that recapitulate neuropathological features and functional defects observed in human Alzheimer?s. Our guiding hypothesis is that AD neurodegeneration causes significant alterations in the epigenome of cells, including maladaptive changes in accessible chromatin landscape and gene expression programs in disease relevant cell types. This in turn causes defects in specific neural circuit functionality during AD pathogenesis. In Aim 1, we will generate a comprehensive epigenome- and transcription-based cell atlas for hippocampal CA1 and subiculum, and identify epigenomic changes that accompany AD progression in each cell type in AD model mice and age-matched control mice. Single nucleus ATAC-seq (snATAC-seq), single nucleus RNA-seq (snRNA-seq) and the newly developed Methyl-HI in single cells for joint mapping of DNA methylation and chromatin contacts will be key approaches. The proposed work will allow for creation of the first single cell multi-omics atlas of the hippocampal circuits, and will allow us to track the epigenomic changes exhibited by multiple specific cell populations at different AD-like neurodegeneration stages. In Aims 2 and 3, we will investigate the cell subtype specific epigenomic and gene expression basis of neural circuit activities and related memory behaviors in AD model mice of middle age. We will measure epigenomic and behavioral changes in response to genetically targeted ontogenetic hippocampal circuit manipulation and histone deacetylase inhibition. Further, we will determine the beneficial effects of simple behavioral interventions via physical exercise on AD-related epigenomic signatures in Aim 3. Together, our proposed research will provide a new framework to study the molecular underpinnings of neural circuit activities affected during the course of AD pathogenesis. It will also lead to the identification of new therapeutic targets and molecular biomarkers for early detection and better treatment of AD.

Project Summary / Abstract Dementia and age-related cognitive decline is an escalating major health concern in the United States. Approximately 20% of the US population will be 65 or older by year 2030, and roughly 8 million of these individuals are expected to suffer from Alzheimer?s disease (AD). We propose to examine detailed AD-related neural circuit mechanisms that will be critical for developing new AD treatment strategies by use of two complementary AD mouse models, which share many features of human AD. Our guiding hypothesis is that AD-related neurodegeneration causes maladaptive changes of memory circuit connections and neural ensemble activities in the hippocampus. We discovered recently in the mouse that non-canonical subicular back- projections to hippocampal CA1 underlie object-place learning, a prominent impairment in AD. This circuit has been recently identified in human brain. We will test our hypothesis that significant impairments in bidirectional information processing between hippocampal CA1 and the subiculum (SUB) develop over time during AD progression. In Aim 1, we will determine the effect of AD-like neurodegeneration on local and global circuit connections to hippocampal CA1 and SUB excitatory neurons. We will map and compare circuit input connections and output projections of excitatory CA1 and SUB neurons in adult control, and AD-like mice using retrograde monosynaptic rabies tracing and anterograde monosynaptic herpes simplex virus (HSV) tracing. Further, we will perform experiments in postmortem human hippocampus of aged-matched control and AD patients to map the SUB-CA1 pathway in human brains and understand detailed changes of this brain circuit in AD patients. In Aim 2, we will test the hypothesis that neurodegeneration in AD-like mice degrades object- location memory encoded by hippocampal CA1 and SUB excitatory neurons. To map neuronal activity to behavioral performance, we will use in vivo miniature microscopic imaging to examine and compare spatial representations of CA1 excitatory neurons and SUB excitatory neurons during open-field exploration, track- based route-running and object-location memory tasks. Thus, we can longitudinally track progressive AD-like functional defects. In Aim 3, we will determine whether spatial memory can be rescued by patterned stimulation of the non-canonical SUB-CA1 back-projection in the AD model mice. Human literature and our preliminary data show high relevance of our proposed research for Alzheimer?s disease. Together, the proposed research will advance our understanding of specific neural mechanisms underlying AD etiology and help to identify new therapeutic targets in humans.

Project Summary AAV-based gene therapy requires the development of safe, efficient, and target-specific vectors. AAV-mediated gene therapy for peripheral tissues (blood and skeletal muscle) has made great strides, focused mainly on gene replacement for loss of function diseases. However, there has been little research on dominantly inherited CNS disorders that are caused by toxic gene products. We have assembled a world class, multi-disciplinary academic research team supported by our industrial partners to develop innovative AAV-based gene knockdown and replacement treatments for rare genetic diseases including Spinocerebellar Ataxia Type 7 (SCA7) and valosin- containing protein (VCP) multisystem proteinopathy. We respond to the RFA Project Objectives and propose three Specific Aims. In Aim 1, we will design and manufacture new AAV vectors with improved critical quality attributes (safety, efficacy, target specificity) for gene therapy. We propose innovative neuron specific gene delivery, temporal control of gene expression and reduced immune responses in the CNS. We have constructed AAV vectors that express EGFP and mRNA barcodes for improved screening to support our proposed AAV treatments. In Aim 2, we will develop advanced quantitative analytics by combining next-generation sequencing and bar-coded AAVs for efficient assessment of in vivo gene delivery in the mouse model. We will screen and compare AAV-mediated gene expression with different capsid variants using different promoters / enhancers. Single-cell RNAseq also will be used to assess immunological responses of selected AAV vectors by different administration routes. In Aim 3, to effectively treat the SCA7 and VCP diseases, we will develop novel AAV vectors that simultaneously knockdown toxic gene products while replacing normal gene products that are codon optimized to be unaffected by knockdown. This proposed combinatorial treatment will establish a proof-of- concept for many other dominant inherited diseases, where loss of normal allele expression due to non-specific silencing causes its own problems. We have well developed models of the SCA7 and VCP diseases for Aim 3 studies. Our team has great expertise in developing, manufacturing and applying AAV vectors in basic research and preclinical application. Our published work and preliminary data establish the feasibility and key methodologies for the proposed research. Critically, we have an established viral production facility and distribution platform operating out of our UCI Center for Neural Circuit Mapping that supports viral reagent design, validation, and manufacturing.

Project Summary / Abstract Blood vessels, from arteries to capillaries to venules and then to veins, contribute to fundamental physiological processes. However, the vascular responses for repair and restoration of microvascular networks after cortical brain injury are poorly understood, including how neuronal activity influences these processes. A major barrier to research is the poor accessibility of micro-vessels in the brain and associated technical difficulties. To overcome this barrier, we propose to use innovative imaging technologies that we have co-developed to investigate micro-vessel formation and re-growth in response to focal cortical injury. We have used light-weight head-mounted, miniaturized microscopes (?miniscopes?) to dynamically image the vasculature and associated cells with high spatial and temporal resolution. We will use cortical injury models by applying a controlled moderate impact to the mouse motor cortex. Combining in vivo longitudinal miniscope and 2-photon imaging, histological ?vessel painting? and perfusion-weighted magnetic resonance imaging (PWI MRI), we aim to achieve a deeper understanding of microvascular restoration following cortical injury. We will apply targeted optogenetic stimulation of excitatory neurons and specific inhibitory neurons to modulate microvascular repair in early and late phases of vessel re-growth. Our guiding hypothesis is that microvascular restoration and remodeling after cortical injury are regulated by vascularization sequences and cellular processes that are similarly observed in normal vasculogenesis during central nervous system development. In Aim 1, we will identify the time course and spatial pattern of vascular regrowth, and blood flow dynamics after focal cortical injury. Vascular networks and blood flow are visualized with fluorescent-labeled dextrans for in vivo imaging for quantitative measurements. In Aim 2, we will determine the role of endothelial cells in new blood vessel sprouting and the establishment of functional microvascular by imaging Tie2-Cre reporter mice during the first two weeks post- injury. We will also examine the influence of astrocytes and pericytes in vascular re-growth. In Aim 3, we will test the hypothesis that optogenetic stimulation of specific neuron types in a temporally controlled manner facilitates and enhances microvasculature restoration for post-injury repair. We will also examine if and how targeted modulation of neural activities modulate Wnt/ß-catenin and VEGF signaling mechanisms that are critical for micro-vessel re-growth. Behavioral testing will assess the outcomes of the optogenetic treatment. We have strong preliminary data that supports the premise for the proposed research for all aims. The proposed research will advance our understanding of the cellular and molecular mechanisms underlying cortical microvascular restoration and how neural stimulation enhances vascular network formation.

Project Summary/Abstract In response to RFA-MH-20-555, this Instrumentation grant application requests NIMH funds to purchase a customized state-of-the-art light-sheet fluorescence microscope system from the 3i Corporation (Intelligent Imaging Innovations, Inc.). The system has a unique custom-designed stage for imaging large cleared brain samples from mice, rats and non-human primates. This instrumentation will allow for high-speed, three-dimensional imaging of whole rodent brains and large non-human primate and human brain samples at single cell resolution. Our team has the required expertise. We have combined whole brain clearing and light sheet imaging to improve localization and quantification of virally mapped neurons across the entire mouse brain. Available turnkey imaging systems sold by LaVision/Miltenyi and Zeiss are not capable of large brain sample imaging. Our proposed 3i system has large sample imaging capability, superior automated performance and superior analysis capability based on hardware and software improvements developed by the 3i Corporation. Quantitative comparisons of connection patterns will be achieved with our analysis pipeline using automated quantification of brain wide fluorescent labeling, which allows for rapid quantification and analysis. We foresee that this enhanced microscope will allow us to effectively perform 3-D large scale neural circuit mapping in mouse, rat and monkey brain samples to better achieve the goals of our research projects funded by 9 active NIMH grant awards and 14 other NIH grant awards at UCI. This proposed instrument will be located at and managed by the core facility of the newly established UCI Center for Neural Circuit Mapping. We request funds to purchase the customized light-sheet microscope system. The UCI Center for Neural Circuit Mapping will ensure that the instrument is covered under full service contract through the estimated 5 years of instrument lifetime. Institutional commitment of the UCI School of Medicine will cover maintenance and software upgrade needs throughout the instrument lifetime. This new imaging system will significantly enhance data acquisition for our ongoing NIH- funded basic and translational research programs, which address the areas of high priority to NIMH.

Project Summary / Abstract Age-related cognitive decline is an important concern in the United States, as approximately 20% of the US population is expected to be age 65 or older by year 2030. Understanding the molecular mechansims of brain aging to prolong healthy cognitive function is therefore increasingly important as the population ages and older people remain in the work force. Brain cells exhibit profound and heterogeneous changes during aging at molecular and cellular levels. The simple intervention of physical exercise has emerged as a major positive modulator of cognitive function in aging. In response to RFA-RM-20-005, we have formed an interdisciplinary team with expertise in single-cell genomics, neural circuitry, and aging, to investigate age- and physical activity- related changes of 4D nucleome in post-mortem human brain hippocampus cells across the lifespan with single- cell resolution. We hypothesize that cell-type-specific re-organization of nucleome occurs in the human hippocampal brain region during aging and with physical activity. The changes in nucleome in turn control brain epigenome and transcriptome, modulating neural circuit functionality. The ?Methyl-HiC?, a new approach for joint profiling of DNA methylation and chromatin contacts in single cells, combined with ?Paired-seq?, an ultra- high-throughput method for single-cell joint analysis of open chromatin and transcriptome, will be used to interrogate the chromatin architecture along with DNA methylation, chromatin accessibility and gene expression in the human hippocampus. In Aim 1, we will determine changes in nucleome in major cell types of post-mortem human hippocampus across the life-span with 4 age ranges (20?39, 40?59, 60?79, and 80?99 years old). We will further correlate these changes in nucleome with epigenome and transcriptome in each cell type, to identify vulnerable cell types during aging, and uncover potential gene regulatory programs that could be impacted by aging. In Aim 2, we will determine how physical activity modifies and restores nucleome in specific human hippocampal cell types. We will study two age-matched cognitively?healthy cohorts (70-99 years old) with either high level or low level physical activity, as measured by wearable activity monitors. We will correlate restorative effects on nucleome with epigenome and transcriptome. In Aim 3, we will map how aging and exercise alter nucleome in specific hippocampal cell types with highly controlled quantifiable physical activity in the mouse model, for comparison with human data. These mouse studies allow the exercise variable to be investigated in isolation from effects of other lifestyle factors that can affect hippocampal nucleome, which is not possible with human subjects. The proposed research will help to transform our ability to understand the mechanisms of chromatin organization and function in the context of human brain aging.

Project Summary / Abstract Alzheimer's disease (AD) is the most common cause of human dementia that progressively worsens with age. Sporadic late-onset AD accounts for more than 90 percent of Alzheimer?s cases without clear documented familial history of the disease. However, the vast majority of existing transgenic and knock-in models incorporate disease-causing familial mutations in one or more genes associated with dementias, representing a major limitation. The RFA-AG-21-003 [New/Unconventional Animal Models of Alzheimer?s Disease] highlights the need to develop and characterize naturally occurring ?non-murine models of AD that may represent improved translational potential by better replicating pathological features of the human disease?. We respond to the RFA to apply single cell epigenomic and transcriptomic technologies developed by our team to create cell-type- specific epigenome and transcriptome maps in frontal cortex and hippocampus that are associated with AD-like pathogenesis in two naturally occurring AD animal models: Octodon degus and Canis familiaris. These animals show age-dependent neuropathology and cognitive impairment similar to those observed in human AD, thus they are natural AD models. As both degus and mice are rodents, the studies of long-lived degus will be particularly valuable for a within-mammalian order comparison of which AD gene regulatory pathways are common to spontaneous AD-like features in degus versus different transgenic mouse models. While we generate the resources in alignment with the RFA goals, the proposed research will allow us to develop a comparative analysis to determine conserved epigenetic alterations in the unconventional animal models and bridge our existing databases of mouse models and humans. Maladaptive changes in accessible chromatin accessibility, chromatin organization and gene expression in disease relevant cell types will reveal species- specific and cross-species conserved mechanisms of AD pathogenesis, as well as new targets for AD prevention and treatment. This will provide new insights into the mechanisms of AD pathogenesis in humans. In addition to genome data sharing at the designated NIH depository, resources will be shared and curated at our UCI Center for Neural Circuit Mapping.