Jennifer Luebke - US grants

The mammalian cerebral cortex is the vastly complex and enigmatic
neural structure responsible for cognitive abilities such as reasoning,
imagining and perceiving. A very useful model system for investigation of
the poorly understood cellular mechanisms underlying cortical function is
the rat somatosensory cortex, which contains discrete columns or "barrels"
of vertically-aligned, input-specific cells that process sensory
information from a single whisker. Barrel cortex has six layers (I-VI),
each comprised of cells with characteristic electrophysiological,
morphological and/or connectional properties. The functional integrity of
input-specific barrels is dependent upon a balance of inhibitory and
excitatory interactions of local inhibitory cells (interneurons) and
excitatory output cells (pyramidal cells) between and within layers. In
recent years, progress has been made in detailing the characteristics and
interactions of cells in layers II-VI of cerebral cortex, but little is
known of the properties of Layer I (LI) neurons and virtually nothing is
known of the role(s) they play in the microcircuitry of cortical columns.
This is a very significant gap, because LI is ubiquitous throughout the
cerebral cortex, and, unlike other cortical layers, contains a cellular
population comprised almost exclusively of inhibitory interneurons. LI
cells are strategically positioned to exert profound inhibitory influences
on pyramidal output cells, which receive many intra- and extracortical
inputs on their complex processes in LI. The goal of the proposed studies
is to gain an understanding of the role of LI neurons in functional columns
of cerebral cortex using the rat barrel cortex as a model system. The
overall hypothesis is that LI interneurons can be grouped into distinct
classes that play important roles in maintaining input-specific sensory
integration within and between barrels, through distinct inhibitory actions
on LII/III pyramidal cells and on other LI neurons. Physiological
recordings with intracellular biocytin filling of LI interneurons and
LII/III pyramidal cells in in vitro slices of rat barrel cortex will be
employed in experiments designed to test this hypothesis. Data from these
studies will yield vital information on the role of distinct classes of
interneurons in the cortical circuits responsible for integration of
sensory information.

DESCRIPTION (provided by applicant): Working memory, which is essential for abilities such as abstract thinking, problem solving, and cognitive flexibility, is significantly impaired with normal aging in a large proportion of humans and non-human primates. While it is known that these abilities are mediated in large part by pyramidal cells of the prefrontal cortex (PFC), the specific neural substrates of age-related decline in PFC function are not known. Given our aging population, this is a highly relevant question that will be directly addressed in an innovative manner by the proposed studies, which employ a unique experimental model- the behaviorally characterized rhesus monkey. Information during working memory tasks is encoded in a temporally dynamic and specific manner by the action potential (AP) firing rates of layer 3 pyramidal cells in the PFC. Preliminary studies demonstrate that there is a significant age-related increase in the firing rates of these cells in vitro, and their firing rates are significantly associated with cognitive performance. The overall goal of this proposal is to comprehensively and simultaneously examine, within individual layer 3 pyramidal cells, age-related alterations in interrelated cellular properties (ionic currents, synaptic responses, morphology), which likely contribute to functionally significant alterations in firing rate. Aged and young rhesus monkeys will be assessed on a battery of cognitive tasks, enabling determination of degree of cognitive impairment. Whole-cell patch-clamp recordings and Lucifer Yellow (LY) filling of layer 3 pyramidal cells in PFC slices prepared from these monkeys will then be employed in three highly integrated Specific Aims. In Aim 1, intrinsic membrane and AP firing properties will be assessed with current-clamp recordings; subsequently, voltage-clamp analyses of currents that influence the temporal pattern of AP firing, (lc, IAHP, sIAHP and lh), will be used to test the specific hypothesis that age-related changes in the properties of these currents lead to increased firing rates. In Aim 2, voltage-clamp recordings will assess age-related changes in glutamatergic and GABAergic postsynaptic currents in these cells, testing the specific hypothesis that changes in synaptic transmission are related to altered signaling. Aim 3 experiments will explore age-related changes in the detailed dendritic architecture and dendritic spines of LY-filled layer 3 pyramidal cells, testing the specific hypothesis that alterations in morphological structure underlie changes in the signaling properties of these cells. Data from each of these aims will be cross correlated and also correlated with cognitive performance scores within the aged group of monkeys. This multi-faceted approach will provide unique information on the effects of normal aging on diverse but interrelated cellular properties within individual neurons that play a critical role in the execution of working memory tasks. Such information is vital to the development of a detailed understanding of the cellular mechanisms of cognitive decline, and a prerequisite for the future development of therapeutic interventions.

DESCRIPTION (provided by applicant): Excitatory neurons of the cerebral cortex are generated prenatally from a diverse group of neural precursors, some of which have only been identified recently. Radial glia stem cells (RGCs) generate neurons directly and at least three separate lineages of Intermediate Neural Precursor Cells (IPCs), which are themselves produced from RGC, also produce neurons. Intriguingly, neurons within each cortical lamina are derived from these different parent cells. The reason why the neocortex requires so many individual precursor cell types, and whether the diverse ancestry of neurons within each layer plays a functional role in neocortical circuitry, has not been established. The numbers of IPCs are thought to be abnormal in several developmental disabilities, including Fragile X and Down's syndromes. In this project, we identify specific lineages of neocortical pyramidal neurons with novel genetic fate mapping tools and determine their structural, functional and connectional characteristics with patch clamp electrophysiology and high resolution 3D imaging. Our preliminary data indicate that neurons from individual precursor lineages, even within the same lamina, are imparted with specific functional properties and that the multiple IPC groups therefore directly underlie neuron and circuit complexity in the neocortex. Morphological and electrophysiological parameters will be quantitatively examined using a multidisciplinary approach and a two-laboratory collaboration.

? 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.

Normal aging in primates often leads to impaired cognitive function, particularly in working memory (WM), which begins to decline in middle-age. Cognitive changes correlate with structural and functional changes to neurons and white matter pathways in the prefrontal cortex (PFC), a brain area that is a key player in WM. However, we currently lack a mechanistic understanding of how the changes at the single-cell and pathway level impact network function and thus WM performance. Moreover, the prefrontal cortex is only one node in a distributed network of brain regions that contributes to WM, and aging also alters these other ?particularly fronto-parietal and visual? areas and long-range inter-areal connections. The central goal of this project is to advance our understanding of the computational and neural mechanisms underlying WM as well as the age- related changes to this executive function in the rhesus monkey model of normal aging. Specifically, we will test the hypothesis that WM arises through coordinated interaction of visual and fronto-parietal brain regions, and that aging-related decline in WM results from changes to both local circuit dynamics and inter-area communication. Our interdisciplinary approach will combine psychophysical, anatomical and physiological experiments with theory and computational modeling, taking advantage of the complementary expertise of the collaborating laboratories. The proposed research has the following specific aims: Aim 1: Identify aging effects on individual neurons, white matter pathways and resting state fMRI activity in fronto-parietal and visual cortices. Aim 2: Develop a multi-area computational neural network model in which WM function emerges from interacting distributed circuits. Aim 3: Model-based interpretation and experimental validation of the neuronal mechanisms underlying age-related WM decline.

The higher order dorsolateral prefrontal cortex (LPFC) and the limbic anterior cingulate cortex (ACC) are key areas in the frontal neural network that mediates executive cognitive functions, which often decline during normal aging. There is strong evidence that layer 3 (L3) pyramidal cells in these higher-order areas are selectively vulnerable during normal aging in the primate, especially compared to those in sensory cortices such as the primary visual cortex (V1). Indeed, extensive morphologic, electrophysiological and structural age-related changes are present in LPFC but not V1 L3 pyramidal cells in the rhesus monkey. However, the mechanisms underlying these area-specific vulnerabilities, and whether LPFC and ACC exhibit similar vulnerability to aging, is not known. The overall hypothesis of this proposal is that LPFC and ACC L3 pyramidal cells share transcriptomic and phenotypic profiles that are highly distinct from V1 neurons, and that underlie selective vulnerability of these frontal areas to age-related synaptic dysfunction and hyperexcitability. The cognitive status of young and aged rhesus monkeys will be assessed on a battery of behavioral as part of other projects. Single-cell Patch-Seq transcriptomic assessment of physiologically characterized L3 pyramidal cells in acute slices of LPFC, ACC and V1 prepared from these monkeys will then be performed. Transcriptomic findings will be validated with RNAscope in situ hybridization and immunohistochemical assessment of proteins on/in biocytin filled, morphologically characterized neurons. This project has two aims: 1) assessment of the transcriptomic profiles of physiologically-characterized pyramidal cells in young vs. aged LPFC, ACC and V1. We will use whole-cell patch-clamp recordings to quantify over 30 physiological variables in L3 pyramidal cells and then harvest these cells for Patch-Seq to determine their transcriptomic profiles. 2) assessment of the morphology and protein expression of pyramidal cells in young vs. aged LPFC, ACC and V1. We will characterize protein expression on a separate subset of non-harvested but biocytin-filled morphologically characterized cells and thus validate Aim 1 gene expression findings. Data on specific age-related genetic changes in expression of ion channels and synaptic markers in individual L3 neurons will be related to age-related changes in genes for oxidative stress, inflammation, and neurodegeneration such as caspase 3 and TNF?. The project will reveal mechanisms underlying differential age-related neuronal dysfunction and mechanisms that can compensate for changes to restore cellular function, and thus has broad implications for therapeutic strategies to reduce cognitive decline during normal aging. This study will form the basis of future studies to investigate relationships and co-dependence of age-related cellular changes in a variety of cell types, laminae and cortical areas during aging that can be correlated with cognitive performance in rhesus monkeys.

Abstract: The objective of the proposed research is to understand the diverse lamina-specific neurons, and connections between the dorsolateral prefrontal cortex (DLPFC) and the rostral aspect of the dorsal premotor cortex (PMdr) during decision-making. Decision-making refers to our ability to choose and perform appropriate actions based on sensory cues and context to achieve behavioral goals. Pressing the brakes to stop the car in response to a red light or choosing what dress to wear are common decisions that we make in our everyday life. Disrupted activity in brain areas such as the DLPFC and PMdr contribute to the impairments in decision-making observed in mental illness. Our past work and other research has provided some insight into the involvement of DLPFC and PMdr in decision-making and that these areas are strongly interconnected. However, we currently do not understand 1) the relationship between biophysical properties and morphological structure, and in vivo decision-related activity of neurons in different layers of these brain areas, and 2) whether the connections between DLPFC and PMdr are feedforward, feedback or lateral (both feedforward and feedback). We address these open questions by using a multimodal approach that combines in vivo neurophysiology in DLPFC and PMdr of behaving monkeys, decoding and granger causality analysis, optical stimulation of DLPFC inputs to PMdr, tract tracing experiments and in vitro single neuron electrophysiology and morphometry in slices from the same subjects. Our first aim uses laminar multi-contact electrodes to investigate neuronal responses across layers of PMdr, and DLPFC while monkeys perform a novel decision-making task that separates perceptual decisions from action selection. We will investigate if in vivo differences are related to differences in biophysical and morphological properties of these neurons with in vitro whole-cell patch-clamp recordings of lamina-specific neurons in PMdr slices. In Aim 2, we examine the granger causality between the local field potentials recorded simultaneously in DLPFC and PMdr to understand whether DLPFC sends a feedforward driving input or a modulating feedback input. We combine these in vivo experiments with anatomical tracing experiments in DLPFC to understand the bidirectional laminar pattern of DLPFC and PMdr connections. In Aim 3, we will inject an opsin in DLPFC and stimulate the anterograde fibers in PMdr in vivo to causally investigate whether the pattern of activity induced in PMdr by stimulation of DLPFC is consistent with feedforward, feedback, or lateral connections. To obtain a more detailed understanding of the pattern of inputs from DLPFC to PMdr, we will investigate in vitro synaptic responses of these PMdr neurons in layers 3 and 5 to optical stimulation of afferent DLPFC fibers and localize the morphological compartments of PMdr neurons to which DLPFC afferent fibers provide inputs. Impact: This project will elucidate the in vivo and in vitro laminar dynamics within and interactions between two critical, clinically relevant brain areas. Such data is a prerequisite for future development of circuit- level therapeutics for mental illness and brain machine interfaces for recovery following brain injury.

Normal aging in primates often leads to impaired cognitive function, particularly in working memory, which begins to decline in middle-age. Our group and others have established that age-related cognitive impairment is not due to overt death of neurons but rather is associated with a constellation of sublethal changes to neurons particularly in layer (L3), such as spine, synapse and myelin loss and consequent alterations to synaptic and intrinsic electrophysiological properties. Importantly these structural and functional changes have been abundantly observed with aging in neurons and white matter pathways in the prefrontal cortex (PFC), a brain area that is a key player in working memory. By contrast, the properties of primary visual cortex (V1) pyramidal neurons are largely spared during normal aging. We currently lack a mechanistic understanding of why pyramidal neurons in these two brain areas are differentially vulnerable in normal aging or how age- related changes at the single-cell and pathway level in PFC impact network function and thus working memory performance. 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 key differences in both the intrinsic properties and the neuropil context of neurons in the two areas, and a greater susceptibility of neurons in LPFC to increases in oxidative stress and inflammation. We propose a novel experimental approach -multiplexed immunohistochemistry combined with high resolution structural analyses of physiologically characterized individual neurons- to compare the properties of individual LPFC and V1 pyramidal neurons in the context of their surrounding neuropil in young and aged rhesus monkeys. These monkeys will also have been assessed for cognitive status, pathway integrity, and CSF pro-inflammatory cytokine levels as part of other existing NIH- funded projects. This project has two aims: 1) To assess the morphological properties of physiologically characterized L3 pyramidal neurons in LPFC and V1 of young and aged monkeys. We will assess dendritic topology and the number and density of dendritic spine subtypes and correlate these data with existing data on 30 different physiological properties of these same cells. 2) To characterize the normative properties and effects of aging on the same L3 pyramidal neurons studied in Aim 1 in the context of the neuropil. We will perform in situ immunofluorescence multiplexing of ~20 protein targets on the same tissue sample to determine the molecular phenotype of biocytin-filled layer 3 pyramidal neurons. A major outcome of this project will be the ability to quantitatively specify those parameters that differ between L3 pyramidal neurons in two highly distinct brain areas and which combination of parameters best predict cognitive impairment in aging. This study will form the basis of future series of larger studies to investigate relationships and co-dependence of age-related cellular changes in a variety of cell types, laminae and cortical areas during normal aging that can be correlated with cognitive performance in rhesus monkeys.