Jack Waters - US grants

[unreadable] DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common form of dementia. AD currently affects approximately one in ten people over 65 years of age and this number is expected to grow to 14 million Americans by 2010. The molecular aberrations that underlie Alzheimer's disease (AD) are well-described, but relatively little is known about the resulting cellular and network changes which lead to the clinical symptoms of AD. Recent studies show that cortical networks are profoundly altered during the early stages of AD, when initial symptoms become apparent. Our long-term objective is to determine the temporal order in which cellular and network defects occur in the cerebral cortex during Alzheimer's disease (AD) and to identify molecular targets through which the earliest changes can be arrested and reversed by therapeutic intervention. During early AD, pyramidal neurons are intact and their cellular properties are largely unchanged, but little is known about the cellular properties of interneurons. Interneurons play a central role in regulating network activity so changes in their properties would have a profound effect on network activity. Furthermore, there is evidence that interneurons are susceptible to degeneration during AD, that loss of interneurons can enhance [unreadable]-amyloid toxicity and that benzodiazepines can both reverse some changes in mouse models of AD and slow progression of AD in humans. Do changes in interneurons lead to dysregulation of cortical networks during AD? We will investigate the possible roles of cortical interneurons in a transgenic mouse model of [unreadable]-amyloid overexpression, investigating both their cellular properties and the associated changes in network function. Specific aim 1: To determine the temporal sequence of cellular changes in interneurons during [unreadable]-amyloid overexpression and compare these changes with those in pyramidal neurons. We will answer this by examining the cellular properties of interneurons and pyramidal neurons in brain slices from [unreadable]-amyloid overexpressing mice and wild-type littermates at different ages, using electrophysiological recording and calcium imaging techniques. Specific aim 2: To describe the changes in cortical networks during [unreadable]-amyloid overexpression and investigate whether these changes are likely to result from an imbalance of inhibition and excitation. We will study network function in anesthetized mice using electrophysiological recording and calcium imaging techniques. These studies will provide critical insight into the possible degeneration of interneurons in AD and the resulting dysregulation of cortical networks. We expect this information to be essential for the successful development of novel therapies for AD. Alzheimer's disease is the most common form of dementia and currently affects approximately one in ten people over 65 years of age. Little is known about the changes in individual neurons and neural networks which lead to the clinical symptoms of Alzheimer's disease. We will study changes in inhibitory interneurons, which are key regulators of neural networks, in a mouse model of Alzheimer's disease. These studies will provide critical insight into the cellular changes that occur in Alzheimer's disease and the resulting degeneration of neural networks. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The dendrites of many neurons contain voltage-gated channels that turn otherwise passive structures into electrically excitable dendrites. Dendritic excitability enables backpropagation of action potentials from the soma into the dendritic tree and the initiation of spikes in the dendrite and these phenomena play important roles in synaptic integration and in synaptic plasticity. Dendritic excitability has been studied extensively in brain slice preparations and to a lesser extent in anesthetized animals, but little is known about dendritic excitability in awake animals. To understand how ongoing synaptic activity affects synaptic integration and plasticity in vivo, in this project we will directly measure dendritic excitability in awake animals using whole-cell recording and two-photon imaging techniques, using the extent of propagation of action potentials into the dendritic tree as a measure of dendritic excitability. Since these are the first studies to use these high-resolution techniques in awake animals, our studies will provide unprecedented information on dendritic function in awake animals.

DESCRIPTION (provided by applicant): The neocortex plays a central role in many higher-order functions such as the interpretation of sensory information, comprehension of language and control of voluntary movements. Many of these processes are shaped by ascending cholinergic drive from neurons in the basal forebrain complex, principally nucleus basalis. Dysfunction of this pathway leads to deficits in many behaviors that involve neocortex and has been tied to clinical conditions such as depression, Parkinson's disease and Alzheimer's disease. Our long-term aim is to understand the cellular and network mechanisms by which ACh from nucleus basalis so profoundly influences cortical function. ACh acts at muscarinic and nicotinic ACh receptors (mAChRs and nAChRs), which are widely expressed in neocortex. Pyramidal neurons in neocortex express both of these receptor types, but the physiological functions of nAChRs in pyramidal neurons are unknown. In this proposal we describe the functions of nAChRs on pyramidal neurons. We directly assess how ACh, released by axons from nucleus basalis, affects pyramidal neurons in motor cortex, using a combination of techniques, including optogenetics, viral tools, genetically-modified mice, immuno-cytochemistry, cellular electrophysiology, two-photon microscopy and electron microscopy. We express channelrhodopsin-2 in cholinergic neurons in nucleus basalis and their axons in neocortex. In preliminary experiments, activation of cholinergic axons depolarized and promoted spiking of layer 5 pyramidal neurons via nAChRs, leading us to hypothesize that ACh facilitates the transfer of information through neocortical networks from ascending excitatory inputs, e.g. from thalamus, to target structures, e.g. motor circuits in the spinal cord. In this proposal we will determine whether the effects of ACh on pyramidal neurons support this hypothesis. We will investigate the mechanisms by which nAChRs affect spiking and the nAChR receptor subunits involved (specific aim 1), determine where in the activated nAChRs are located within the dendritic trees of pyramidal neurons (specific aim 2), and determine whether these postsynaptic nAChRs mediated the effects of ACh in pyramidal neurons in other layers and neocortical areas (specific aim3). Our results will reveal a new mechanism by which ACh modulates the excitability of pyramidal neurons. Our studies will also provide the first evidence, to my knowledge, that ACh has layer-specific effects. The resulting hypothesis has the potential to transform our understanding of the manner in which the cholinergic pathway from nucleus basalis changes network function in neocortex and may therefore have important implications for debilitating conditions such as Parkinson's disease and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: The actions of the neuromodulator acetylcholine in neocortex play central roles many higher-order functions, including arousal, attention and learning and memory, and insufficient acetylcholine in neocortex has been tied to pathophysiological conditions such as Alzheimer's disease. He we study the locations of nicotinic acetylcholine receptors on layer 5 pyramidal neurons in the neocortex and the physiological effects of synaptically-released acetylcholine on these neurons. This study will advance our understanding of the mechanism of action of this essential neuromodulator in the healthy brain, which may also lead to insights into pathological conditions such as Alzheimer's disease.

ABSTRACT Activity-sensitive fluorescent indicators and microscopy have proven valuable tools for measuring neuronal activity, but most forms of cellular microscopy can produce images of neurons only near the brain surface and generally only after removal of tissues overlying the brain surface, such as bone. Many neurons in neocortex are out of reach of cellular microscopy. Here we propose to optimize 3-photon (3P) excitation fluorescence imaging, a form of cellular microscopy, to enable deeper imaging into the brain. Our aims are to acquire cellular information from deep cells in flies, mice, cats and macaques. After optimizing 3P microscopy, we will facilitate the duplication of our microscope and procedures in other neuroscience laboratories. We will provide open-access, detailed information on our microscope and experimental procedures in terminology that?s comprehensible to neuroscientists. In addition, we will work with one or more microscope manufacturers to ensure commercial availability of our microscope design, enabling many neuroscience laboratories to use 3P excitation to image deep into the brain with less invasive preparations, repeatedly over many days, weeks or months. To achieve our broad aims, we will pursue five specific aims: ? Specific aim 1: Further optimize fast 3P excitation. ? Specific aim 2: Record calcium signals from central motor circuits in intact, behaving Drosophila melanogaster. ? Specific aim 3: Study sensory information processing in mouse neocortex and spatial memory in mouse hippocampus. ? Specific aim 4: Study visual information processing in deep layers of cat and macaque cortex. ? Specific aim 5: Share methods and facilitate availability of a commercial fast 3P microscope.

Project Summary Much of our understanding of brain microcirculation comes from studies on arteriolar perfusion. Blood efflux through venules plays an equally important role in determining blood flow through the brain, since all blood entering the brain must exit via venules. The structure and function of cerebral venules can change dramatically during cerebrovascular disease. Preclinical and clinical studies have demonstrated marked alterations in venule tortuosity and vascular wall composition during Alzheimer?s disease and Alzheimer?s disease-related dementias. Compared to arterioles, the slower flow and distinct endothelial biology of venules makes them more susceptible to hemostasis, thrombosis, and immune cell adhesion during disease. Collectively, these factors point to venules as a site of vulnerability in cerebral perfusion that remains highly understudied. This project focuses on principal cortical venules (PCVs), a subset of venules that descend from the brain surface into the deepest layers of cortex and underlying white matter. Although PCVs are less common compared to smaller cortical venules, they extend massive, horizontally projecting branches in deeper tissues, suggesting a critical role in perfusion of deep cortex and adjacent white matter tracts. However, there exists almost no information on the structure, physiology and perfusion territories of PCVs. Cerebral white matter is particularly sensitive to blood flow deficit and degenerates in early stages of Alzheimer?s disease and Alzheimer?s disease-related dementias. Understanding the regulation of perfusion in and near white matter tracts will be critical in understanding the basis of this white matter degeneration. Our central hypothesis is that PCVs are the main drainage system for deep cortical layers and the underlying white matter. In Aim 1, we will test this hypothesis by using emergent deep in vivo two-photon imaging and three-photon imaging to measure how capillary flow is drained in cortical layer 6 and its adjacent white matter tract in the mouse brain, respectively. These activities will be performed in adult (3-9 months) and aged mice (18-24 months) to test a secondary hypothesis that age is associated with deterioration in PCV structure and function. In Aim 2, we will we will quantify the radius of cortical tissue dependent upon PCV drainage by measuring how photothrombotic occlusion of a single PCV affects flow into the cortex through neighboring penetrating arterioles. We will further use histology to assess the volume of hypoxic tissue in gray and white matter created by occlusion of single PCVs. This project is significant because it addresses the understudied topic of venular perfusion in white matter using novel in vivo imaging approaches. It further establishes an experimental foundation needed for future research on venular dysfunction as a mechanism of impaired cerebral blood flow and white matter degeneration in Alzheimer?s disease and Alzheimer?s disease-related dementias.

PROJECT ABSTRACT Recent advances in microscopy permit volume imaging - the near-simultaneous imaging of many neurons in a circuit - with 2-photon (2P) excitation, yielding new insights into the circuits underlying relatively simple behaviors. Volume imaging techniques and 2P excitation have been limited mainly to small animals such as rodents, including studies of mouse cortex. Mouse cortex differs from neocortex in non-human primates and humans and it is unlikely that studies of mouse cortex will be sufficient to understand the role of cortex in humans. In this R34 application, we propose to build a multi-plane 3-photon (3P) fluorescence microscope to perform volume imaging in macaques. Unlike 2P excitation, 3P excitation supports imaging deep into macaque cortex. In a future R01 application, we will use this 3P microscope to image large volumes of macaque primary visual cortex, studying how motion information is pooled across neurons in layer 4B and pairing 3P microscopy with serial-section electron microscopy to study the processing of color information, particularly in layer 4C?. We will pursue four specific aims: - Specific aim 1: Construct and test a multi-plane 3P microscope - Specific aim 2: Measure visual tuning of ~50,000 neurons in layers 1-4 in a macaque. - Specific aim 3: Extend multi-plane 3P imaging through layers 5 and 6 of macaque cortex. - Specific aim 4: Make hardware designs, software and protocols freely available.