Li Zhang - US grants

musculoskeletal system; skeletal system; computers; biomedical resource; biological products;

DESCRIPTION (provided by applicant): Most physiological studies in the primary auditory cortex (Al) have focused on neural spike output. However, to understand the processing and computation performed by auditory cortical neurons, it is necessary to examine the synaptic mechanisms underlying cortical response properties, i.e. to correlate the synaptic inputs of cortical cells with their outputs under various sound stimuli. In our pilot studies, we have developed techniques of in vivo whole-cell recording from auditory cortical neurons, as to establish a fundamental understanding of the synaptic connection basis for cortical responses. Here, I propose to systematically characterize the synaptic inputs, in terms of both excitatory and inhibitory inputs, underlying the frequency tuning and the supra-/sub-threshold structure of the frequency-intensity tonal receptive fields (TRFs) of single A1 neurons. In Aim 1, I will address how the tonal inputs are represented by a single Al neuron. I will determine the spatial relationship between supra- and sub-threshold TRFs of single A1 neurons, by recording tone-evoked membrane potential responses with in vivo whole-cell current-clamp method, and also characterize the change of subthreshold TRFs with the characteristic frequencies (CFs) of A1 neurons. In Aim 2, I will determine the role of spectrotemporal interaction between excitatory and inhibitory synaptic inputs in shaping the frequency tuning and TRFs of Al neurons. TRFs of pure excitatory and inhibitory synaptic inputs will be derived by using in vivo-whole-cell voltage-clamp recording. In particular, I will determine the role of the cortical inhibition in shaping the frequency tuning and TRFs. In Aim 3, I will characterize the contributions of thalamocortical and intracortical components to the excitatory synaptic TRFs of single A1 neurons by exploiting pharmacological approaches to silent the intracortical connections. With the understanding of the origins of the excitatory inputs, a basic model of synaptic input circuits underlying the TRFs of A1 neurons will be constructed. As a starting point, this project will specifically target the histologically determined excitatory pyramidal neurons in the input layers (layer 3-4) of adult rat A1. This study will be a direct extension of our pilot studies, and will generate information essential for understanding the cortical mechanisms underlying sound processing and representation in the auditory cortex. Taken together, the application of whole-cell recording technique in these studies will provide unique opportunities to address the fundamental issues concerning the mechanisms underlying auditory cortical responses, and are also likely to yield new level of information to the understanding of physiology and pathology of the auditory cortex.

An essential step to achieving an understanding of auditory cortical function is determining how information contained in sensory inputs is represented and processed in different individual cortical neurons. Recently, several laboratories have successfully applied a "blind" in vivo whole-cell voltage-clamp recording technique to cortical neurons. This technique can resolve sensory-driven excitatory and inhibitory synaptic inputs onto the recorded neuron, making it possible to construct a synaptic connectivity model to predict the neuron's response under arbitrary sensory stimulation. Although this technique can be combined with post hoc histological methods to reconstruct the morphology of recorded cells, its "blind" nature largely limits its potential in examining various cell types in the cortex, especially those that are small in size or spatially sparse; the "blind" patch-clamp recording technique will normally result in a biased sampling of pyramidal neurons in the cortex. In this exploratory project, we will study a new technique for revealing functional synaptic inputs made onto different types of individual cortical neurons [unreadable] two-photon imaging guided whole-cell (TPGWC) recording [unreadable] in which fluorescence-labeled neurons are visualized by two-photon imaging and specifically targeted for patch recording. This technique has largely benefited from recent developments in mouse genetics in the labeling of specific cell types with fluorescence proteins, such as green fluorescence protein (GFP), whose expression is controlled by cell-type specific promoters. Initially, we plan to apply this recording technique to GFP-labeled GABAergic interneurons in the input layers (L3/4) of the mouse primary auditory cortex (A1), and aim at addressing two fundamental questions: a) What subthreshold/spike TRF properties are possessed by Al inhibitory neurons? b) How do cortical excitatory and inhibitory synaptic inputs determine subthreshold/spike TRF structure of A1 inhibitory neurons?

DESCRIPTION (provided by applicant): Understanding the structure of cortical synaptic circuits is key to comprehending information representation and processing in the auditory cortex. However, due to technical limitations, the general structure of cortical synaptic circuits, and how this structure determines cortical function, remains largely unknown. As a first step to addressing this issue, in this project, we will investigate the patterns of excitatory and inhibitory synaptic inputs underlying the functional responses of individual cortical neurons and reveal the synaptic mechanisms determining or shaping these response properties. In the auditory cortex, patterns of synaptic inputs can be largely reflected by their frequency-intensity tonal receptive fields (TRFs). These patterns represent basic structural properties of synaptic input circuitry underlying the functioning of individual cortical neurons. Using an in vivo whole-cell recording technique, we will determine the spectrotemporal pattern of synaptic inputs for both excitatory and inhibitory neurons in the input layers of the adult rat auditory cortex. We will dissect the thalamocortical components of excitatory inputs by pharmacologically silencing the cortex. The cell type of recorded neurons will be determined by their spiking and morphological properties. We will determine excitatory and inhibitory synaptic mechanisms for the frequency/ intensity tuning of cortical pyramidal neurons by revealing the patterns of excitatory and inhibitory synaptic inputs with in vivo whole-cell voltage-clamp recording techniques. We will explicate the contribution of thalamocortical excitaotry inputs to the response properties of cortical neurons by developing a novel pharmacological approach to effectively and specifically silence the cortex. Finally, by distinguishing cortical inhibitory neurons according to histology and physiology, we will determine response properties of cortical GABAergic interneurons, and their underlying synaptic mechanisms.

[unreadable] DESCRIPTION (provided by applicant): Occlusion of the middle cerebral artery elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. Thrombolysis with tissue plasminogen activator (tPA) promotes adverse vascular events that limit the therapeutic window of stroke to three hours. Advanced age exacerbates vascular dysfunction after stroke which limits the utilization of tPA. Proteasome inhibitors enhance endothelial nitric oxide synthase (eNOS) expression and improve endothelial function. Our preliminary studies demonstrate that treatment with a proteasome inhibitor, VELCADE, an agent in clinical use for the treatment of cancer, effectively reduces cerebral infarction, and concomitantly reduces secondary thrombosis and microvascular permeability in young rats. In addition, treatment with VELCADE in combination with tPA extends the therapeutic window to at least 6 hours after stroke without increasing hemorrhagic transformation. However, stroke is a major cause of death and disability in the elderly. To mimic clinical situation, we propose to investigate the effect of VECLADE on aged rats. In Aim 1, we hypothesize that treatment with VELCADE dose dependently reduces infarct volume and neurological functional deficit in aged rats after stroke. Optimal doses of VELCADE extend the therapeutic window for stroke. In Aim 2, we will investigate the effects of combination treatment with VELCADE and tPA on cerebral infarction, neurological function, thrombolysis, microvascular thrombus formation, vascular patency and integrity in aged rats after embolic stroke. By reducing the adverse vascular events, VELCADE amplifies the thrombolytic effect of tPA, and permits a reduction in the effective therapeutic dose of tPA. In Aim 3, using eNOS knockout mice and NOS inhibitors, we will examine the mechanisms that underlie the beneficial effects of VELCADE alone or in combination with tPA in the treatment of stroke. We propose that eNOS mediates the neuroprotective effect of VELCADE by down-regulation of pro- coagulation genes and matrix metalloproteinases (MMPs), which provoke thrombosis, and BBB damage. VELCADE counteracts the detrimental effects of delayed administration of tPA on vascular function and consequently improves microcirculation and vascular integrity. Our study may provide fundamental insights into the mechanisms underlying beneficial effects of VELCADE and combination of VELCADE and tPA in embolic stroke, and may lead to a novel treatment strategy for stroke. PUBLIC HEALTH RELEVANCE: Stroke elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. As the only FDA approved drug for the treatment of acute stroke, tissue plasminogen activator (tPA) potentiates adverse vascular events that limit the therapeutic window of stroke to three hours. Advanced age exacerbates vascular dysfunction after stroke which limits the utilization of tPA. Proteasome inhibitors enhance endothelial nitric oxide synthase (eNOS, an important regulator of vascular homeostasis) expression. Treatment with a potent proteasome inhibitor, VELCADE, an agent in clinical use for the treatment of cancer, effectively reduces the development of adverse vascular events, and concomitantly reduces cerebral infarction. Therefore, in the current application, we propose to investigate the neuroprotective effects of VELCADE alone and incombination with tPA and the mechanisms underlying the beneficial effects in aged rats after embolic stroke. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Single-walled carbon nanotubes are molecular wires that exhibit interesting structural, electrochemical, and optical properties. The near-infrared optical absorption properties of polymer- and biomaterial-functionalized single-walled carbon nanotubes have attracted particular attention for optical nanobiosensors. Deoxyribonucleic acids (DNAs) play important physiological and pathological roles in living organisms. The unique structural properties of single-walled carbon nanotubes make them extremely useful as a carrier of macromolecular enzymes and DNAs. Optical properties of DNA-functionalized single-walled carbon nanotubes are sensitive to many chemical and biological materials such as hydrogen peroxide, glucose, protein, oxygen, ammonia, and ascorbic acid. This provides us with useful approaches to develop optical biomedical sensing applications. Hydrogen peroxide is one of the main products for many enzyme-catalyzed chemical reactions. The over accumulation of hydrogen peroxide in living systems is related to aging and severe diseases. It has been found that the linear optical absorption properties of DNA[unreadable]functionalized carbon nanotubes in the near-infrared range significantly depend on the concentration of hydrogen peroxide. This proposed program concentrates on nonlinear optical absorption properties of double-stranded DNA-functionalized carbon nanotubes. We will use a nanosecond pulsed near-infrared laser system and employ the open aperture Z-san technique to characterize nonlinear optical absorption coefficient of the dispersions of double-stranded DNA-functionalized carbon nanotubes under different concentrations of hydrogen peroxide to determine the relation between the nonlinear optical absorption coefficient and the concentration of hydrogen peroxide. This research can be extended from the dispersions to the thin films of double-stranded DNA-functionalized carbon nanotubes during the academic year. The results will reinforce the potential of double-stranded DNA-functionalized carbon nanotubes for use in optical biosensing.

DESCRIPTION (provided by applicant): Inhibitory synaptic circuits play important roles in shaping cortical processing. Our understanding of the functional engagement of inhibitory circuits composed of different inhibitory cell types however remains poor. The recent development of molecular and genetic tools in the mouse, in combination with the innovative techniques of in vivo electrophysiology, has now made it possible to systematically dissect synaptic circuitry underlying specific cortical functions. In this project, we will integrate multile approaches to investigate the synaptic, in particular inhibitory circuitry mechanisms underlying auditory processing in the mouse primary auditory cortex (A1). In the first part, we will apply in vivo cell-attached and whole-cell recordings to investigate synaptic mechanisms for specific laminar processing in A1, a direct extension of the previously funded project. Second, we will combine in vivo two-photon imaging and patch-clamp recordings and utilize optogenetic methods to dissect functional roles of different types of inhibitory neuron. Finally, with high-quality whole-cell recordings in awake behaving mice, we will investigate cortical synaptic circuitry mechanisms for auditory processing functions in awake cortex, and their modulation by different behavioral states.

? DESCRIPTION (provided by applicant): Type II diabetes mellitus (T2DM) is a common metabolic disease and an established risk factor for cognitive dysfunction in the elderly population. However, the pathological mechanisms that underpin the development and progression of DM-related deficits remain unclear. Recent investigations1-4 have altered the traditional model of cerebrospinal fluid (CSF) hydrodynamics. The brain lacks specialized organ-wide anatomic structure to facilitate lymphatic clearance although the brain has complex architecture and high metabolic activity. However, a newly identified glymphatic system has been shown to modulate the CSF-interstitial (ISF) exchange, which facilitates clearance of interstitial solute from the brain parenchyma. Impairment of the glymphatic system is involved with the development of neurodegenerative conditions, including Alzheimers disease and sleep disorders, etc2, 4. Although the impact of the glymphatic system is being investigated in Alzheimers disease and sleep disorders, with promising results, there are no reported data related to diabetes and the glymphatic system. Using a model of T2DM in middle-aged rats and noninvasive MRI methodologies, our preliminary data indicate that compared with age-matched non-DM rats, the T2DM rats reduced clearance rate of interstitial Gd-DTPA agent from brain parenchyma by approximately 84 % and increased clearance time by 4.2 time of Non-DM rats in the hippocampus, leading to accumulation of Gd-DTPA agent in these regions and consequently high MRI signal intensity in T1 weighted MRI (T1WI). In parallel, ex-vivo confocal imaging analysis revealed that in Non-DM rats, the concentration of interstitial Texas Red-conjugated dextran (TR-3, MW 3kD) reached a plateau in the brain interstitium approximately 3h after injecting TR-3 into the cisterna magna and after that TR-3 began to clear and was almost completely cleared from brain parenchyma at 6h after the injection, whereas in middle-age T2DM rats TR-3 accumulated in the hippocampal interstitium with time and exhibited strong fluorescent signals at 6h after the injection. These ex-vivo data are consistent with in vivo MRI findings, indicating that T2DM impairs the glymphatic clearance of interstitial solutes in the brain. In addition, T2DM rats exhibited microvascular thrombosis and blood brain barrier (BBB) leakage in the hippocampus in immunofluorescent analysis and also showed spatial learning deficits compared to Non-DM rats. Based on our novel preliminary data, we will employ MRI and 3D confocal microscopy to evaluate for the first time, temporal and spatial profiles of paravascular CSF-ISF exchange throughout the brain during the development of T2DM. We propose to further develop MRI protocol and analysis modeling as an effective means to evaluate the function and status of the glymphatic system (Aim 1). We will then use the optimized MRI protocol and analysis to explore the relationships between impairment of glymphatic system, vascular damage, and functional deficits during develop of DM (Aim 2). Data generated from this application will provide new insights into the progression of DM associated impairment of the glymphatic system.

PROJECT SUMMARY/ABSTRACT Identifying the diversity of cell types in the nervous system will allow for their selective manipulation and reveal their functional contributions in health and disease. However, this is not a trivial undertaking and is hindered by the lack of consensus on which properties to use for classification. Characteristics like anatomical location, connectivity, morphology, molecular profile, and electrophysiological properties have been used as classification systems, but singly, none provide a combined view of all these characteristics. To address this, we propose a multidisciplinary approach that will provide all of this information for each cell type of the mouse hippocampus and subiculum (HPF/SUB). We recently identified all HPF/SUB molecular domains and assembled their connectivity networks using tracing data from the Mouse Connectome Project (www.MouseConnectome.org). Our multiple retrograde tracer injections revealed that the HPF/SUB contain multiple intermixed populations of cells with unique projection targets, suggesting different cell types that could be defined based on their connectional start and end points with anatomic specificity. Therefore, here, we propose to use a quadruple retrograde tracing method to initially classify these neurons based on these connections. Subsequently, a two- step cre-dependent AAV tracing method will determine all outputs of each cell type. To determine their molecular identities, seqFISH, with preselected hippocampal marker genes, will be performed on the tissue from the quadruple retrograde tracing data. Importantly, seqFISH preserves spatial information so that the precise anatomic locations of the tracer-labeled cells and the genes will be retained. Next, rabies injections placed in targets of each HPF/SUB cell type will reveal their morphology. CLARITY and two-photon microscopy will enable morphological assessment in 3D and neuronal reconstructions for further analysis. To examine electrophysiologcial properties, each cell type will be labeled with retrograde tracers for identification purposes and ex vivo cell patch clamping will be performed on the labeled cells. Finally, cre-dependent viral tracing (TRIO) will determine inputs to the different HPF/SUB cells types. With the aid of Expansion Microscopy and two-photon imaging, a combined anterograde/rabies tracing strategy will show precise locations of select inputs to cell types. If successful, this project can be applied to characterize neuronal cell types of the entire brain. All data will be publicly shared. Images from the quadruple retrograde, two-step cre-AAV, and TRIO tracing experiments will be available in the iConnectome Cell Type Viewer. Graphic reconstructions of labeling from these experiments will be compiled and presented within a common neuroanatomic frame through a Cell Type Connectivity Map. The iConnectome Cell Type Morphology Viewer will showcase labeling from the double rabies experiments and provide details like 3D reconstructions and their morphological and electrophysiological properties. Cell type connections will be visualized in an interactive Web Connectivity Matrix. Our in-house informatics pipelines and algorithms will be further developed and optimized to support the proposed features of all viewers. 

Abstract: Diabetes mellitus (DM) is a common metabolic disease in the middle-aged and older population, which is associated with cognitive decline and an increased risk of developing dementia in the elderly. Given the growing size of the aging population and increased prevalence of DM, development of specific interventions to maintain cognitive integrity by counteracting DM induced pathophysiological processes is of major clinical importance. Clinical trials show that the achievement of improved glycemic control may not prevent progression of cognitive impairment. The underlying cause of DM-induced cognitive deficits remains unknown. Exosomes are nanovesicles with a size of 40 to 120 nm in diameter and mediate intercellular communication by transferring proteins, lipids, and genomic materials including mRNAs and microRNAs (miRNAs) between source and target cells. Our preliminary data demonstrated aged-DM rats exhibit substantial cognitive impairment, which is associated with dysfunction of cerebral endothelial cells and neural stem cells. We also found that exosomes derived from dysfunctional cerebral endothelial cells induced by DM communicated with and damaged neural stem cells. More importantly, administration of exosomes isolated from cerebral endothelial cells of healthy young adult brain to aged-DM rats effectively improved cognitive function and minimize DM-induced dysfunction of cerebral endothelial cells and neural stem cells. In this application, we therefore, propose to develop the endothelial exosomes as a mechanism-based therapy for DM-induced cognitive decline in aged population. Our hypotheses are: 1) The cerebral endothelial exosome (CEE) treatment reduces cognitive deficits in the aged-DM rat, 2) The CEE treatment improves cerebral vascular patency and integrity, and promotes neurogenesis and oligodendrogenesis in the aged-DM rat, and 3) Engineered exosomes carrying elevated miR-1 and -146a have enhanced effects on cerebral vascular function, neurogenesis and oligodendrogenesis as well as cognitive function. Aim 1 is to investigate whether the CEE derived from young adult rats improve cognitive function in the aged-DM rat, when the CEE is administered at an early (2 months) or advanced (4 months) stage of DM in aged male and female rats. Aim 2 is to investigate whether the CEE treatment improves cerebral vascular function and enhances neurogenesis and oligodendrogenesis in the aged-DM rat. Cerebral vascular patency and integrity, neurogenesis and oligodendrogenesis will be measured. Aim 3 is to investigate whether treatment of the aged-DM rat with tailored endothelial exosomes carrying elevated miR-1 and miR146a further enhances vascular function, neurogenesis and oligodendrogenesis as well as cognitive function. We will generate the tailored CEE and then administer the tailored CEE to aged-DM rats. These studies are innovative and highly clinically relevant.

Abstract Stroke is one of leading causes of death and disability worldwide, mainly affecting elderly. Tissue plasminogen activator (tPA), the only Food and Drug Administration (FDA) approved treatment, is limited in its use to < 8.5% of stroke patients. Therefore, there is a compelling need to develop new and broader utility therapies for acute ischemic stroke. Vepoloxamer is a well characterized proprietary amphipathic copolymer with rheological properties, which is currently under investigation in a global phase III clinical trial for patients with sickle cell disease. Our preliminary studies demonstrate that administration of Vepoloxamer in combination with tPA 4h after embolic stroke facilitates recanalization and thrombolysis reduces ischemic neuronal damage and improves neurological outcome, but does not increase cerebral hemorrhage in young adult rats. We also found that platelet-derived exosomes contribute to the therapeutic effect of Vepoloxamer on enhanced tPA-thrombolysis. In this application, we propose to investigate effect of Vepoloxamer in combination with tPA on acute stroke and molecular mechanisms underlying the combination therapy on the thrombolysis and neurovascular function in the aged male and female rats. Data generated from this application may provide a novel and potentially useful treatment strategy for patients with acute stroke.

Neuromodulators are essential signaling molecules that regulate many neural processes, including cognition, mood, memory, and sleep, through their influence on brain circuits. Monitoring the release and distribution of neuromodulators in behaving animals is critical for understanding the diverse functions of these molecules. A major impediment to developing this understanding is the lack of tools that can monitor these compounds at the temporal, spatial and concentration scales relevant to these brain processes. Filling this technological gap is one of the most pressing needs in neuroscience research. Our proposal directly bridges this gap by developing a platform of new tools for chronic, non- invasive monitoring of neuromodulators at millisecond, subcellular, and nanomolar resolution. Genetically-encoded fluorescent indicators for calcium and glutamate have transformed investigation of dynamic brain processes in the major model systems, including worms, flies, rodents, and increasingly primates. Building on our prior experience in developing these tools, we now propose to build a new suite of GPCR-activation-based (GRAB) genetically-encoded fluorescent indicators for neuromodulators. Our preliminary data shows we can generate GRABs with >500% fluorescence change and nanomolar affinity in mammalian cells. We propose to further develop and validate these prototypes in cultured neurons, flies, rodent brain slices, anesthetized and behaving mice to maximize their utility. In Aim 1, we will develop GRAB indicators for acetylcholine, serotonin, and norepinephrine by iteratively screening libraries that systematically vary in insertion site, linkers, cpGFP sequence, and FP-GPCR protein surface interface. The dimensions of optimization will be dF/F, membrane surface expression, affinity, and non-disruption of endogenous signaling. Our targeted performance levels are >10x dF/F, nanomolar range affinity and <10 millisecond on-rates in vitro. In Aim 2, performance of top candidate GRAB indicators from the in vitro screen will be validated following long-term expression in drosophila olfactory system, in brain slice, in anesthetized and behaving mouse cortex. Feedback from these experiments will guide iterative optimization in Aim 1. Successful completion of our Aims will yield a suite of powerful molecular constructs, cell-type specific viral tools and technical approaches that will be broadly disseminated to the neuroscience community. The GRAB indicators can be easily integrated with existing mouse models of human mental disorders. Since these probes for neuromodulators are well-suited for a wide range of preparations, and a large number of investigators, they will have a multiplicative impact on our understanding of neural circuit function and dysfunction when combined with other advances supported by the BRAIN Initiative.

ABSTRACT The objective of this application is to investigate glymphatic impairment and cognitive deficits during progression of aging with and without diabetes. Emerging data1-5 indicate that the glymphatic system in the brain mediates the cerebrospinal fluid (CSF)-interstitial (ISF) exchange and solute clearance from the brain parenchyma. However, despite the well-described dysfunction of the glymphatic system in the development of neurodegenerative conditions, there is still no reported study that focuses on the role of the glymphatic system in the development of cognitive impairment during aging and aging with type-2 diabetes (DM). Using non- invasive MRI methodologies to investigate cerebral solute waste clearance in middle-age control and type-2 diabetic (DM) rats, we have found increased impairment of the glymphatic system, as indicated by reduced clearance of interstitial Gd-DTPA in brain parenchyma, primarily in the hippocampus and hypothalamus in DM rats (Fig.2&3)6. In parallel, 3D confocal microscopic analysis of the brain-wide distribution of fluorescent tracers revealed increased delayed clearance of ISF in the hippocampus and hypothalamus from DM rats (Fig.2&3)6. Impairment of the glymphatic system in DM rats was shown to be highly correlated with cognitive deficits as measured by an array of cognitive tests including the Morris Water Maze (MWM) for hippocampal related learning and memory. Importantly, histopathological analysis shows that delayed clearance of interstitial solutes is associated with sporadic cerebral microvascular thrombosis in the hippocampus 2 months after hyperglycemia (15 months from birth), while extensive microvascular thrombosis and para-vascular accumulation of beta- amyloid (A?) are detected at 4 months after induction of hyperglycemia (17 months from birth), suggesting that the impairment of the glymphatic system leads to A? accumulation. Collectively, our preliminary data, for the first time, demonstrate that non-invasive MRI methodologies can detect DM-induced early impairment of the glymphatic system which is highly correlated with hippocampal related dysfunction of learning and memory. Based on our novel preliminary data, we will employ MRI and 3D confocal microscopy to evaluate and quantitatively measure kinetic clearance parameters of the glymphatic system during progression of aging with and without DM (Aim 1). We will then investigate: whether impairment of the glymphatic system predicts cognitive dysfunction, the sensitivity and association between impairment of the glymphatic system, the onset of brain vascular dysfunction, and cognitive deficits during aging with and without DM (Aim 2). Data generated from this application will provide new insights into aging and age-matched DM associated impairment of the glymphatic system and the relationship of the glymphatic system with vascular and cognitive dysfunction.

PROJECT SUMMARY Although great efforts have been dedicated to characterizing neuronal cell types in the brain, systematic studies on the brain-spinal cord connectome and associated spinal neuronal types are lacking. In this project, a team of seven laboratories proposes to use a highly innovative and multidisciplinary approach to systematically characterize neuronal types in the spinal cord based on their anatomy, connectivity, neuronal morphologies, molecular identities, and electrophysiological properties. In Aim 1, we will use a newly developed AAV anterograde transsynaptic tagging method to label spinal cord neurons that receive descending inputs from different brain regions, and use a retrograde viral tracer, AAVretro, to label spinal neurons that project to defined brain regions. These tagged neurons will be imaged in the intact whole spinal cord with a newly developed fast 3D light sheet microscopy technique, and targeted for recording in slice preparations. The axonal collateral patterns, dendritic morphologies, and electrophysiological properties will be compared between different input/output-defined spinal neuron groups. In Aim 2, the gene expression patterns of the tagged neurons will be determined in situ by sequential bar-coded FISH (seqFISH), with candidate marker genes obtained from online resources, or from single-cell sorting and RNA sequencing (Dropseq). In Aim 3, all collected data on connectivity, anatomical cell type distribution map, neuronal morphologies, molecular identities, and electrophysiological properties will be used for classifying spinal neuron types connected with brain, and an open-source data portal will be established which will allow users to search, view, and analyze the multi-modal and integrative cell-type specific data. Together, we aim to construct a comprehensive cell- type atlas of the mouse brain-spinal cord connectome.

Project Summary In this RO1 renewal application, we will explore functional roles of a higher-order thalamic nucleus, the lateral posterior nucleus (LP), in auditory cortical processing. LP is a rodent homologue of the pulvinar nucleus. While previous studies have been mostly focused on involvements of pulvinar in visual functions, there are salient connections between LP/pulvinar and auditory cortex, suggesting potential involvements of LP/pulvinar in auditory information processing as well. However, our knowledge on how LP influences auditory processing in the cortex is lacking. Recently in our preliminary experiments we observed that LP activity could suppress auditory responses in the primary auditory cortex (A1) of awake mice. This has prompted us to propose an extensive investigation into the functional contribution of LP to auditory cortical processing in A1. In Aim 1, we will perform in vivo recordings from individual neurons in supragranular and granular layers of A1 in awake mice and examined their auditory response properties before and after optogenetically inactivation and activation of LP. We will test the hypothesis that LP plays a role in enhancing auditory processing in A1 through a surround-suppression mechanism. In Aim 2, we will specifically manipulate the activity of the LP to A1 axon terminals either optogenetically or chemogenetically, and examine A1 neuron cell types that are innervated by the LP-A1 projection. We will test the hypothesis that the LP modulation of A1 responses is primarily mediated by the LP projection to layer 1 inhibitory neurons. In Aim 3, by manipulating activity of superior colliculus (SC), we will test the hypothesis that a SC-LP-A1 pathway mediates the bottom-up suppressive modulation of A1 responses. In addition, it can also mediate a cross- modality modulation of A1 responses by visual signals. Together, these experiments will enhance our understanding of functional roles of non-primary sensory thalamic nuclei in general.