Patrick R. Hof, MD - US grants

Dopamine is an important neurotransmitter in the central nervous system and its various actions are mediated by several dopamine receptor subtypes. One of these subtypes, designated as the D3 receptor, is expressed in phylogenetically older regions of the brain, including the limbic system. This suggest that D3 receptors play a role in cognition and in the expression of emotive behavioural phenotypes. However, conventional animal studies on the particular function of D3 receptors proved to be difficult because of the high homology and the similar pharmacological characteristics of this receptor subtype with other brain dopamine receptors. To circumvent these difficulties, this proposal lays the groundwork for studies on the specific function of D3 receptors in the whole animal by generating mutant mice that lack functional D3 receptors. This will be achieved by introducing a mutant (non-functional) D3 gene into embryonic stem (ES) cells of mice. Such ES cells are then used to generate chimeric mice that have the ability to transmit the mutant D3 gene to their offspring. Mutant offspring will have a decreased (heterozygous mutants) expression of the D3-receptor protein and such mice can be interbred to generate homozygous mutant mice in which the D3 receptor expression is abolished. Initial studies on such mutant animals aim at testing whether a decreased or abolished D3-receptor protein expression affects the development of brain regions in which D3 receptors are normally expressed. At present, nothing is known about the importance of D3 receptor expression for the normal development of the brain. Thus, comparative anatomical studies of such brain sections obtained at defined developmental stages from mutant and normal (wildtype) mice are proposed. Furthermore, because of the significant anatomical overlap in the expression of D3 receptors and another (very similar) dopamine receptor subtype (named D2), and the potential functional complementarity bet ween these two receptor subtypes, one might expect changes in the expression of D2 receptors to occur in response to a decreased or abolished expression of D3 receptors. Such changes could mask the functional consequences of a "loss of D3 receptor expression" in our mutant animals. Therefore, experiments are proposed that will test whether the expression of D2 receptors differs in wildtype and D3- receptor-mutant animals. The work proposed here will provide a powerful mouse model for future studies on the importance of D3 receptor expression for the expression of certain behavioural phenotypes.

Alzheimer's disease (AD) is characterized by extensive neuronal death in the cerebral cortex. This loss of neurons is correlated with the severe functional decline in cognition and memory observed in AD patients. Neuropathological changes restricted to the hippocampal formation are a consistent reflection of age-related memory impairment, but overt dementia is present only in cases with neocortical involvement. Distinct subpopulations of neocortical neurons undergo severe degeneration in AD, while others are remarkably preserved even at late stages of the disease. Thus, in association neocortical areas a subset of pyramidal neurons are particularly vulnerable in AD, while other neuron classes remain viable throughout the progression of AD. The vulnerable neurons are all characterized by their large size, their extensive dendritic arborization and their relatively high content of neurofilament protein. Further investigations have demonstrated that these neurons are also involved in neurofibrillary tangle (NFT) formation and that there exist age-related shifts in the expression of neurofilament protein and other molecules, such as glutamate receptor subunit proteins (GluRs), that may render a neuron prone to neurodegeneration. However, the degree to which molecular and morphological alterations restricted to identifiable neuronal populations represent reliable thresholds reflecting early degeneration or functional deficits has not yet been determined. This component is designed to analyze quantitatively the molecular and morphologic correlates or functional decline and the progression of neuronal alterations in the superior frontal cortex of AD cases by developing quantitative indices of neurofibrillary degeneration (INDs) based on ratios of stereologic estimates of neurons and NFTs in the superior frontal cortex. We will also determine quantitatively the complement of GluRs in identified sets of corticocortical projections linking the prefrontal cortex to temporal and parietal association areas in the macaque monkey to test the hypothesis that substantial differences exist in the distribution of key GluRs among the neurons of origin of these projections. Based on this prediction, we will investigate whether the neurons at risk in AD exhibit overall low staining intensity for the AMPA subunit GluR2 and that progressive shifts in AMPA and NMDA subunits expression will take place as neurons undergo degenerative changes. We hypothesize that a decrease in GluR2 staining intensity will be concomitant of the appearance of the earliest degenerative neuronal changes in AD, in a selected population of neurons, but that no such association will be observed with NMDAR1. The detailed quantitative data obtained from these studies will provide crucial information on the anatomic and neurochemical determinants of selective neuronal vulnerability in AD.

Mount Sinai School of Medicine (MSSM) seeks funding for an Abilene/Internet2 to better handle the demands of image analysis and processing. A recent National Center for Research Resources (NCRR) grant allowed the institution to upgrade the computer network in the main research building. The wide area network now presents a bottleneck for collaboration with other institutions. The connection, itself, will also support collaborative projects in the following areas:

3-dimensional Morphometric Analysis of Mammalian Neurons: A collaboration between MSSM, investigators from the Courant Institute of Mathematical Sciences (NYU), New York University School of Medicine, SUNY at Stony Brook, and the University of New South Wales, Australia. Researchers at MSSM study the structure-function relationship of neurons in the brain, employing advanced 3D microscopic imaging techniques as well as 3D image processing, rendering, analysis and modeling. CNIC investigators are using confocal and multi-photon microscopes to image (in 3D) entire pyramidal neurons in the cortex at the highest resolution possible. This creates approximately 20-gigabyte data sets for a single neuron. After the data is collected, it is exported to computers, where image stitching, volume rendering, deconvolution and 3D-image analysis is done. This award will greatly facilitate the capabilities of off-site collaborators to access these large datasets as well as manipulate and analyze the data using unique software developed by CNIC investigators and hosted at MSSM.
Neural Mechanisms of Vestibular Function: MSSM researchers and collaborators at Washington University, the University of Utah and NASA-Ames are studying vestibular functions. Electrophysiological and microscopic studies are being performed to determine the basis for regional variations in afferent response dynamics across the vestibular sensory epithelium. 600 Mbytes to 1.2-gigabyte data sets are created using a multi-photo laser. The ability to manipulate the data in 3D via access to volume rendering and 3D image analysis software hosted on MSSM computers will provide invaluable information to off-site collaborators. Furthermore, due to the length of time required to compile the data for any one experiment, it will be greatly beneficial for collaborators to review the data as it is collected (in real time) on the multi-photon microscope. This will avoid any delay in feedback and permit the off-site collaborators to modify experimental conditions.

Data have emerged implicating age-related and amyloid-induced pathology of the cerebral microvasculature as a potential contributing factor to the pathogenesis of Alzheimer's disease (AD). This project will investigate the spatial and temporal linkage between vascular pathology, amyloid and tau accumulation, and neuronal pathology.These putative interactions will be investigated in a mouse model that expresses the "Swedish" double mutation of the amyloid precursor protein (APP), and three distinct groups of human postmortem specimens: 1) neurologically normal elderly cases, 2) cases with mild cognitive impairment and early AD, and 3) centenarian brains. These analyses will focus on the hippocampus and entorhinal cortex as they are the earliest cortical regions to be involved by the degenerative process during brain aging and in AD. Specific Aim I will involve a detailed quantitative analysis of the vasculature of the hippocampus and entorhinal cortex in the three groups of human subjects. We hypothesize that a strong spatial and temporal relationship will exist between the degree of vascular damage and reflections of degeneration. We will employ stereologic probes to develop an accurate quantitative appraisal of the vasculature density in a region- and layer-specific manner. Specific Aim II will involve a comparable analysis in the cerebral cortex of Tg2576 transgenic mice expressing the "Swedish" mutation of APP. We will test the hypothesis that the APP(swe) mice show an age-related pathology of the cortical microvasculature that is similar to that found in AD in humans. Moreover, we propose that the accrued deposition of amyloid in these mice leads to severe changes in the morphologic integrity of the neurons in the vicinity of the deposits. Neuronal morphology will be assessed in a quantitative manner using intracellular injection of hippocampal and neocortical neurons with computerized reconstruction. Specific Aim III will involve ex vivo high resolution magnetic resonance microimaging at 9.4 T (MRM). These MRM data will be related to the morphologic alterations analyzed in Specific Aim II. We hypothesize that there will be an age-dependent loss of volume in hippocampal and entorhinal regions in these transgenic mice and this will correlate with the severity of vascular changes. This project will provide a quantitative assessment of the relative contribution in time and space of age-related amyloid deposition and microvascular damage to neuronal pathology in vulnerable cortical circuits critical for memory and cognition.

Much evidence points to tau- and amyloid-related cellular alterations as major contributing factors to the pathogenesis of Alzheimer's disease (AD). Although numerous studies of neuronal pathology have been carried out in the human brain, they have rarely been performed in a way to assess the effects of the progression of pathological lesions on the morphologic integrity of identified neuronal subpopulations and have generally not been quantitative. This project will investigate the spatial and temporal linkage between abnormally phosphorylated tau and amyloid accumulation, and dendritic atrophy and spine loss in different subtypes of neocortical pyramidal cells characterized by neurochemical and morphologic features. Such putative interactions will be investigated in four groups of human postmortem specimens: 1) neurologically normal elderly cases, 2) cases with mild cognitive impairment and early AD, 3) cases with moderate dementia, and 4) severe AD cases, as well as in a mouse model that expresses only the human tau gene. The early AD cases will emerge as a particularly interesting group of brains as they will permit us to pinpoint the earliest changes in dendritic function in neocortical neurons that have a known risk of enhanced vulnerability to the degenerative process of AD. Based on stereologic analyses from our laboratory, we expect that a small subgroup of large neocortical neurons enriched in nonphosphorylated neurofilaments are the first to contain dendritic lesions that precede the stage at which neurofibrillary tangles (NFT) are forming and dementia becomes evident. These analyses will focus on Brodmann's area 9 in the prefrontal cortex. Area 9 is a severely and early affected neocortical field in AD, which we have extensively characterized in terms of selective neuronal vulnerability. In this project we will expand the regional stereologic assessments of identified neuronal subgroups gathered during the previous funding period by analyzing cellular alterations and their relationships to deposition of amyloid and age-related neuronal pathology at the level of individual neuron morphology. The analyses in mice will permit us to follow the dynamic changes in live animals, obtain very high resolution imaging datasets prior to sectioning these specimens for detailed morphologic analyses, and provide quantitative analyses of neurons potentially at risk of degeneration with a much higher level of resolution than would be achievable in human postmortem materials. Altogether this project will provide a quantitative assessment, in AD cases of different severity, of the relative contribution of age-related neuritic pathology, early stages of amyloid processing, and senile plaques, to the progressive demise of selectively vulnerable neuronal subsets subserving cortical circuits critical for memory and cognition.

Project 1. Project 1. An investigation of oligodendroglia in schizophrenia. Demyelinating diseases have been known to be associated with behavioral changes. Recently, the expression levels of several myelin-related genes have been shown to be consistently decreased in postmortem schizophrenic brains compared to controls. Magnetic transfer imaging (MTI) has also shown consistent reduction in myelin content in schizophrenic brains. Diffusion tensor imaging (DTI), which measures the directionality of white matter tracts, has shown a decrease in anisotropy in the brains of schizophrenic patients, suggesting disruptions in white matter tract coherence and directionality in this disease. These data together make a strong case for oligodendrocyte dysfunction in schizophrenia. The anterior cingulate cortex plays a significant role in motivation, attention, and behavior and, as a component of the limbic system, in affect and memory. It has been clearly implicated in schizophrenia by studies of cytoarchitectural postmortem changes and functional imaging showing hypometabolism in this region in schizophrenia. In this project, we propose a quantitative analysis of possible relationships between oligodendrocytic pathology and abnormalities in cytoarchitecture in the cingulate cortex of postmortem brains from schizophrenic patients and neuropathologic and brain imaging analyses of relevant mice mutants such as the Quaking mouse, as well as genetically modified mice such as MAG, RPTPfi, CNPase, or MAGI knock-outs. Our analyses will use advanced microscopy quantitative approaches including the most rigorous stereologic methods for cell counting, estimators of spatial cellular distribution and cytoarchitectural boundaries, as well as single cell morphology by intracellular loading of fluorescent dyes or gene-gun-based DiOlistics techniques. We also are assessing progression of potential changes in white matter integrity using high field magnetic resonance microscopy in vivo at 9.4 T in the relevant mouse models. The combined analysis of human specimens and relevant mice models within the context of this program offers a superb opportunity to investigate myelin deficits that have a clinical impact and to determine the molecular, developmental, and morphologic characteristics of the neuronal circuits whose alteration is likely to underlie the pathogenesis and clinical manifestations of schizophrenia. the molecular, developmental, and morphologic characteristics of the neuronal circuits whose alteration is likely to underlie the pathogenesis and clinical manifestations of schizophrenia.

Data have emerged implicating age-related and amyloid-induced pathology of the cerebral microvasculature as a potential contributing factor to the pathogenesis of Alzheimer's disease (AD). This project will investigate the spatial and temporal linkage between vascular pathology, amyloid and tau accumulation, and neuronal pathology.These putative interactions will be investigated in a mouse model that expresses the "Swedish" double mutation of the amyloid precursor protein (APP), and three distinct groups of human postmortem specimens: 1) neurologically normal elderly cases, 2) cases with mild cognitive impairment and early AD, and 3) centenarian brains. These analyses will focus on the hippocampus and entorhinal cortex as they are the earliest cortical regions to be involved by the degenerative process during brain aging and in AD. Specific Aim I will involve a detailed quantitative analysis of the vasculature of the hippocampus and entorhinal cortex in the three groups of human subjects. We hypothesize that a strong spatial and temporal relationship will exist between the degree of vascular damage and reflections of degeneration. We will employ stereologic probes to develop an accurate quantitative appraisal of the vasculature density in a region- and layer-specific manner. Specific Aim II will involve a comparable analysis in the cerebral cortex of Tg2576 transgenic mice expressing the "Swedish" mutation of APP. We will test the hypothesis that the APP(swe) mice show an age-related pathology of the cortical microvasculature that is similar to that found in AD in humans. Moreover, we propose that the accrued deposition of amyloid in these mice leads to severe changes in the morphologic integrity of the neurons in the vicinity of the deposits. Neuronal morphology will be assessed in a quantitative manner using intracellular injection of hippocampal and neocortical neurons with computerized reconstruction. Specific Aim III will involve ex vivo high resolution magnetic resonance microimaging at 9.4 T (MRM). These MRM data will be related to the morphologic alterations analyzed in Specific Aim II. We hypothesize that there will be an age-dependent loss of volume in hippocampal and entorhinal regions in these transgenic mice and this will correlate with the severity of vascular changes. This project will provide a quantitative assessment of the relative contribution in time and space of age-related amyloid deposition and microvascular damage to neuronal pathology in vulnerable cortical circuits critical for memory and cognition.

DESCRIPTION (provided by applicant): Cognitive impairment in normal aging and neurodegenerative disease is accompanied by altered morphologies on multiple scales: from the fine-grained geometry of individual spines to the global topologies of multi-neuron and vasculature networks that are distorted by space-occupying histopathologic lesions. A mechanistic understanding of the role of these structural changes in producing the observed cognitive deficits requires accurate 3D representations of neuronal morphology, and realistic biophysical modeling that can directly relate structural changes on multiple scales to altered neuronal firing patterns. To date however, no tools capable of resolving, digitizing and analyzing neuronal morphology on both local and global scales, and in true 3D, have been available. The central goal of this project is development of an automated analysis system for digitization, 3D reconstruction and geometric analysis of detailed and accurate neuronal morphology, capable of handling morphologic details on scales spanning local spine geometry through complex tree topology to the gross spatial arrangement of multi-neuron networks. As a specific example we will analyze morphologic changes in a Tg2576 mouse model of Alzheimer's disease (AD), in which amyloid deposition, altered cortical microvasculature and neural abnormalities provide easily identifiable examples of pathologic lesions. Four Specific Aims will address this broad objective: (1) To develop a semi-automated system for 3D tree extraction and spine analysis from laser scanning microscopy (LSM) imaged data, with sub-voxel resolution for accurate neuronal morphometry at the finest scales;(2) to image and digitize in 3D individual neurons, multineuron and vasculature networks, and senile plaques from human and Tg2576 mouse models of AD;(3) to develop tools for global analysis of spatially complex cellular structures in 3D;(4) to distribute and maintain all software, and develop a database-driven web repository for distribution of digitized neurons and networks. By providing true 3D morphometry of complex neural structures on multiple scales, the tools developed in this study will enable future multiscale biophysical modeling studies capable of testing hypothesized mechanisms by which altered dendritic structure, spine geometry and network branching patterns in normal aging and neurodegenerative disease determine pathologies of working memory and cognitive function. Such studies will provide crucial insight into general mechanisms of memory induction and maintenance that underlie normal cognitive function, its dysfunction in diseased states, and potential mechanisms for its restoration.

PROJECT SUMMARY Cognitive decline in normal aging is accompanied by morphologic changes on many scales, and in rhesus monkeys, by single cell electrophysiological changes such as altered firing rates and synaptic responses. A subset of 'successful agers' can maintain both normal cognitive function and normal single-cell electrophysiology, suggesting some form of adaptive compensation for morphologic dystrophy at the cellular level. To date, no mechanistic understanding of these cellular changes, nor the inferred compensatory mechanisms, exists. The goal of this unique multidisciplinary project is to develop innovative computational technologies for identifying causal mechanisms underlying the cognitive decline that accompanies aging and neurodegeneration. Based upon these mechanisms, this project will design quantitatively precise strategies for compensating or reversing these changes, to restore a given cellular-level function to normal levels. Three Specific Aims will address this broad objective: (1) To reconstruct the morphology, including dendritic spines, of electrophysiologically characterized young and aged layer 3 (L3) pyramidal cells from the prefrontal cortex of rhesus monkeys that have underwent behavioral testing; (2) To develop morphologically accurate compartment models of young and aged L3 pyramidal cells while developing novel parameter optimization tools; and (3) To predict compensatory mechanisms for restoring normal function in aged or dystrophic neurons using newly-designed sensitivity-analysis techniques. Public dissemination of the 3D morphology and physiology of young and aged neurons from behaviorally characterized primates will provide a unique database for the general neuroscience community to begin to address important cellular and system-level questions. Dissemination of all modeling and analysis software will provide the computational community with efficient tools to apply and extend these techniques. Such studies will generate crucial insight into the cellular bases of aging- and neurodegenerative disease-related changes in cognitive function. The development of novel technologies to predict mechanisms that can compensate for specific morphologic changes to restore a given cellular level function, has far-reaching implications for designing therapeutic interventions for many human diseases.

DESCRIPTION (provided by applicant): In this Lab to Marketplace proposal, we aim to develop the MBF SpineStudio software incorporating the innovative, laboratory-based NeuronStudio software created at the Mount Sinai School of Medicine. Our SpineStudio software will enable automated detection, reconstruction and morphological classification of the structural analysis of dendritic spines. By creating this commercial software, we will improve the existing technology by providing validated, supported, and fully-documented software for automated 3D quantitative dendritic spine analysis. This software will integrate the specific advantages of the NeuronStudio software for a combination of neuron labeling, imaging (including in vivo), and image pre- processing techniques applied by the user without requiring expert knowledge in software programming. The software product will offer automated detection, reconstruction, and separate classification of complex spines (cup-shaped, multi-headed or branched, filopodia). Providing this innovative functionality will help research in important fields such as neurodegenerative diseases, learning, and memory.

DESCRIPTION (provided by applicant): Our central hypothesis is that developmental delay syndromes including autism spectrum disorders lead to alterations in synaptic function in integrative brain regions that result in aberrant behavioral phenotypes. We will explore this hypothesis in a genetically modified rat model. Haploinsufficiency of SHANK3 leads to neurodevelopmental changes that include autism spectrum disorders, attentional disorders, absent or delayed speech, mild to moderate intellectual disability, and motor alterations. The SHANK3 protein forms a key structural part of the postsynaptic density. Because of the closer physiology between rats and humans as compared to mice, rats remain the primary choice of the pharmaceutical industry for studying pharmacokinetic (PK) properties of novel drugs. In addition, rats provide a far more tractable experimental model system for neurobiological, electrophysiological and behavioral studies, and it is of course advantageous, when considering drug development, that the biological assays be done in the same species where the PK studies are carried out. We have used zinc-finger nucleases to develop a genetically engineered rat with a disruption in the full-length rat Shank3 gene. This represents a first-ever genetically modified rat model for ASD and permits us to carry out detail studies in the prefrontal cortex, an area of great importance in autism, not easily studied in mouse models. We propose to carry out a detailed analysis of this model. We plan to test our central hypothesis with the following specific aims: 1) Behavioral assessment of prefrontal function in Shank3-deficient rats; 2) Electrophysiological analysis of prefrontal function in Shank3-deficient rats; and, 3) Neuropathological and neurochemical investigation of prefrontal function in Shank3-deficient rats. 3) The research is innovative, in our opinion, because it will make use of a first-ever rat model of ASD. In addition, it is innovative in the use of state-of-the art approaches to understanding the role of PFC in ASD, a key region not yet studied in detail in ASD model systems. The focus on PFC also allows for studying neuronal pathways that feed into the PFC, including the first-ever behavioral neurophysiological assessment of hippocampal-prefrontal circuitry in a rodent model for ASD. Our approach to high-resolution 3D imaging and analysis of neuronal morphology down to the level of single spine is notably novel. This form of analysis will allow us to identify molecular targets that are affected in Shank3-deficient rats, in particular, te distribution of excitatory receptors and synaptic proteins known to be linked to spine and synapse size and maturity. Finally, our behavioral analyses will make use of novel touchscreen chambers for detailed analysis of PFC function.

? DESCRIPTION (provided by applicant): The proposed program on Research Training in the Neuroscience of Aging will provide predoctoral students in Neuroscience with an integrated training experience in the laboratories of nationally and internationally recognized faculty. The predoctoral training program builds on an exciting, translationally relevant curriculum taught in years one and two of graduate school that has been awarded NIH support through the Jointly Sponsored Predoctoral Early Stage T32 Training Program mechanism (T32 MH087004). The proposed new training program would be the first at Mount Sinai to focus specifically on the neuroscience of aging and age- related neurodegenerative disease (primarily Alzheimer's disease), complementing Mount Sinai's historical concentration and strength in research in these areas. Outstanding training faculty in the proposed program share common research interests in the mechanisms of brain function in health and disease, employing a diversity of experimental approaches and working at levels of analysis ranging from molecular neurobiology to human neuropsychology and neuroimaging. The training program specifically encourages participation of faculty mentors whose research grants directly focus on aging neuroscience research, while not excluding those whose research expertise is critically important for the interdisciplinary training we seek to impart. Through their course work, predoctoral trainees will have received a solid foundation in basic neurobiology and the pathophysiology of neurological and psychiatric disease, as well as biostatistics and the responsible conduct of research. Advanced coursework includes a seminar course on the biology of aging as well as focused elective courses in specific areas of neuroscience. Selection of a research mentor is made in a collaborative environment that actively promotes multidisciplinary, integrative research. Research training will also have a `work in progress' component, to foster important interdisciplinary interactions, hone presentation skills, and improve awareness of ethical issues. Using this approach, the program on Research Training in the Neuroscience of Aging will provide predoctoral students with the guidance and experimental tools, in the laboratories of our training faculty, to launch successful, productive, independent careers in aging research.

? DESCRIPTION (provided by applicant): During normal aging in the rhesus monkey, pyramidal cells in the dorsolateral prefrontal cortex (LPFC) undergo significant structural and functional changes that are likely associated with cognitive impairment, while pyramidal cells in the primary visual cortex (V1) are comparatively spared. The overall hypothesis of this project is that selective vulnerability of neurons and associated networks in LPFC compared to V1 during aging is due to a greater susceptibility to increases in oxidative stress, inflammation, and vascular dysfunction in LPFC than in V1. We further hypothesize that intervention with the potent antioxidant and anti-inflammatory polyphenol curcumin will prevent or reduce age-related dysfunction on multiple scales- from the molecular to the behavioral level. This project has three aims: 1) To assess the biomarker and ultrastructural characteristics of V1 and dlPFC neuropil. In situ immunofluorescence multiplexing of ~30 protein biomarkers will be used to determine the molecular phenotype of neurons, glia, vascualture and surrounding neuropil with a GE Global Research MultiOmyxTM platform tailored for use in brain tissue, enabling quantitative, multimarker analyses with high throughput. Using 2D and 3D electron microscopy, inhibitory and excitatory synapses, mitochondria, myelin and axons of neurons as well as microglia and vascular elements will be quantitatively characterized. 2) To characterize the physiological and morphological properties of layer 3 (L3) pyramidal neurons in V1 and dlPFC across the adult lifespan of rhesus monkeys. Using whole-cell patch-clamp recordings we will assess passive membrane properties, AP firing patterns and underlying ionic currents, as well as excitatory and inhibitory postsynaptic currents of L3 pyramidal cells in in vitro slices of PFC and V1. We will then characterize the morphological properties (e.g. dendritic topology, density and detailed morphology of dendritic spines and neurotransmitter receptor and transporter distribution, as well as oxidative stress markers) of these same pyramidal cells using immunohistochemistry and ultra-high resolution confocal laser scanning microscopy. 3) To use computational models of V1 and dlPFC networks and spatial working memory task behavior to make predictions about the functional consequences -at the single neuron, network and behavioral levels- of changes revealed in Aims 1 and 2. Unique to this proposal is the combination of state-of-the art physiological, anatomical and computational approaches together with concurrent behavioral assessment of the aging monkey under control conditions and following therapeutic treatment with curcumin. This project will yield entirely novel and critically needed information on the neural substrates of cognitive decline in the aging primate and provide important insight into the specific mechanisms of action of protective anti-inflammatory and anti-oxidants during normal aging.

During normal aging in the rhesus monkey, pyramidal cells in the dorsolateral prefrontal cortex (LPFC) undergo significant structural and functional changes that are likely associated with cognitive impairment, while pyramidal cells in the primary visual cortex (V1) are comparatively spared. The overall hypothesis of this project is that selective vulnerability of neurons and associated networks in LPFC compared to V1 during aging is due to a greater susceptibility to increases in oxidative stress, inflammation, and vascular dysfunction in LPFC than in V1. We further hypothesize that intervention with the potent antioxidant and anti-inflammatory polyphenol curcumin will prevent or reduce age-related dysfunction on multiple scales- from the molecular to the behavioral level. This project has three aims: 1) To assess the biomarker and ultrastructural characteristics of V1 and dlPFC neuropil. In situ immunofluorescence multiplexing of ~30 protein biomarkers will be used to determine the molecular phenotype of neurons, glia, vasculature and surrounding neuropil with a GE Global Research platform tailored for use in brain tissue, enabling quantitative, multimarker analyses with high throughput. Using 2D and 3D electron microscopy, inhibitory and excitatory synapses, mitochondria, myelin and axons of neurons as well as microglia and vascular elements will be quantitatively characterized. 2) To characterize the physiological and morphological properties of layer 3 (L3) pyramidal neurons and of interneurons in V1 and dlPFC across the adult lifespan of rhesus monkeys. Using whole-cell patch-clamp recordings we will assess passive membrane properties, AP firing patterns and underlying ionic currents, as well as excitatory and inhibitory postsynaptic currents of L3 excitatory and inhibitory neurons in in vitro slices of PFC and V1. We will then characterize the morphological properties (e.g. dendritic topology, density and detailed morphology of dendritic spines and neurotransmitter receptor and transporter distribution, as well as oxidative stress markers) of these same neurons using immunohistochemistry and ultra-high resolution confocal laser scanning microscopy. 3) To use computational models of V1 and dlPFC networks to predict the functional consequences?at the single neuron, network and behavioral levels?of changes revealed in Aims 1 and 2. Simplified models of LPFC and V1 neurons will be incorporated into model networks capable of persistent neural activity, oculomotor spatial working memory, and visual orientation tuning. Unique to this proposal is the combination of state-of-the art anatomical, physiological, and computational approaches together with concurrent behavioral assessment of the aging monkey under control conditions and following therapeutic treatment with curcumin. This project will yield entirely novel and critically needed information on the neural substrates of cognitive decline in the aging primate and provide important insight into the specific mechanisms of action of protective anti-inflammatory and anti-oxidants during normal aging.