Stephen J Smith, PhD - US grants

The project is aimed at characterizing basic mechanisms of chemical communication between nerve cells. The information obtained should ultimately help in understanding the actions of psychotherapeutically useful drugs, and may possibly provide a basis for rational design of new therapeutic approaches. The work could also provide a better basis for understanding brain mechanisms of alcohol and drug addiction. One focus of our studies is the monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). Serotonin has been implicated in the etiology of clinical depressive disorders and in disturbances of sleep. The experiments will concentrate on regulatory roles of intracellular calcium ion concentration ([Ca++]i) in both presynaptic and postsynaptic mechanisms of synaptic transmission. It is widely believed that [Ca++]i is important in both classes of mechanism, but difficulties in experimental measurement of [Ca++]i in living cells have limited the information available. New methods for measurement of [Ca++]i in isolated cells will be used in conjunction with electrophysiological, pharmacological, and optical means of manipulating and recording cellular activity. Two sets of specific questions will be addressed. The first concerns the role of [Ca++]i in excitation-secretion coupling at presynaptic terminals: (1) What happens to [Ca++]i during excitation? (2) Can kinetics of [Ca++]i explain the kinetics of transmitter secretion? (3) How does [Ca++]i promote secretion? The second set of questions to be addressed concerns the role of [Ca++]i in cellular responsiveness to 5-HT: (1) What happens to [Ca++]i during 5-HT action? (2) How does 5-HT act on [Ca++]i? (3) Does [Ca++]i mediate actions of 5-HT on ion channels? Measurements of [Ca++]i will be carried out by photometry of indicator dyes such as arsenazo III. Cells types to be studied include the ganglion cells of gastropod molluscs, squid giant neurons, and chromaffin cells from bovine (cow) adrenal glands.

This project will address the basic mechanisms of cortical synapse formation. Three specific synapses, two excitatory and one inhibitory, will be studied in organotypic cultures of hippocampal cortex. Brain slices from neonatal rats will be cultured and studied in the living state using time-lapse laser confocal microscopy. Fluorescent stains and ion indicators will be used to visualize cellular structure and activity patterns. Electrophysiological methods will be used to assess and to manipulate synaptic function. Cells examined physiologically will be reidentified after fixation for immunohistochemical and electron microscopic study. The project will begin with a detailed anatomical, immunohistochemical and electrophysiological baseline study of the in vitro development of three synapses: (1) the excitatory mossy fiber synapse onto pyramidal cells in hippocampal area CA3, (2) the excitatory Schaffer collateral fiber synapse onto pyramidal cells in area CA1, and (3) the inhibitory synapse of basket cells onto pyramidal cells in area CA1. The cultured slice system will then be used to pursue four main questions about the physiology and dynamics of synapse formation: 1. How do axonal growth cones navigate in a cortical tissue environment? 2. How do axonal growth cones and immature dendrites make the initial contact leading to synapse formation? 3. What is the sequence and timing of events between initial contact and establishment of a functional synapse? 4. How does electrical activity influence the formation and stability of synapses? Several specific hypotheses regarding each of these questions will be tested. The proposed studies should provide insights into abnormalities of neural development including birth defects, learning disorders and mental retardation. They may also help to suggest medical procedures for prevention or reversal of neurological deficits associated with trauma and stroke. Finally, they may elucidate the developmental basis for normal aging effects on nervous system function and also for degenerative conditions such as Parkinson's and Alzheimer's diseases.

This project will utilize sophisticated optical imaging methods to explore activity-dependent regulation of both presynaptic and postsynaptic structures in rat hippocampus. The fluorescent marker FM 1-43 will be used to study the recycling vesicles of presynaptic boutons in cell culture preparations. Lipophillic carbocyanine stains will be used to study dendrites and spines in cell cultures and tissue slices. Fluorescence will be visualized in liver preparations using laser-scanning fluorescence microscopy (transmission, confocal and two-photon) while neural activity is manipulated by electric field stimulation and by drugs. Pharmacological analysis will be used to test for the involvement of specific receptor- medicated signaling pathways in structural dynamics. Retrospective immunohistochemistry and electron microscopy will be used for cell-type identification and to improve interpretation of fine structural events detected by fluorescence in live specimens. Specific questions about interpretation of fine structural events detected by fluorescence in live specimens. Specific questions about basic presynaptic and postsynaptic mechanisms and their use-dependent plasticities will be addressed as indicated below. 1. Presynaptic vesicle pool dynamics. A. Basic release and recycling mechanisms: (i) How long do synaptic vesicles remain open after transmitter release? Does open time depend on stimulation intensity? (ii) Do all synaptic vesicles take up FM 1-43 during transmitter release? (iii) How are vesicles compartmentalized during recycling? (iv) How do vesicle pool dynamics vary from synapse to synapse of the same and different cell types. B. Regulation and plasticity of synaptic vesicle pools. (i) How stable are synaptic vesicle pools over periods of hours to days? (ii) How does electrical activity influence the synaptic vesicle pool? (iii) Do presynaptic autoreceptors or adrenergic receptors modulate pool size or dynamic parameters? (iv) How do specific vesicle proteins influence the size, dynamics and regulation of vesicle pools? 2. Postsynaptic dendrite spine dynamics. (i) How stable are dendritic spine and branchlet structures over hours an days in mature, steady-state cultures? (ii) How is dendrite structure influenced by electrical activity? What signaling mechanisms link dendritic structure to activity? These studies of basic mechanisms of synaptic function and synapse structural plasticity should contribute to understanding and treating mental retardation, learning disorders and epilepsies. They may also help in devising means to promote recovery of function after nervous system trauma and stroke.

DESCRIPTION (provided by applicant): In vivo two-photon imaging methods will be used to explore the mechanisms of dendrite growth and synaptogenesis in the zebrafish retinotectal pathway. The project will focus especially on effects of visually stimulated activity on dendrite growth and synaptogenesis. The zebrafish tectum is very well suited to two-photon fluorescence imaging, and development of the retinotectal projection is known to depend on activity. Novel genetically encoded fluorescent protein (FP) markers and automated two-photon (2P) microscopy will be used to efficiently study dynamics of dendritic arbor growth and synaptogenesis at high time resolution over long time periods. The research involves four specific aims: (1) determine kinetic parameters of dendrite growth and synaptogenesis under baseline (dark, unstimulated) conditions, (2) measure effects of defined visual stimuli on kinetic parameters of dendrite growth and synaptogenesis, (3) test selective glutamate receptor antagonists for effects on growth and synaptogenesis parameters under baseline and visual stimulation conditions, and (4) measure and analyze dendritic Ca transients evoked by patterned visual stimulation. These studies will be among the first to integrate dynamic imaging of dendritic arbor growth with visualization of synaptogenesis processes. Experiments based on this new capability will test various current hypotheses relating the dynamics of dendrite growth and synaptogenesis to each other and to the activity-dependent development of functional neural circuitry. To understand normal or pathological brain development, it is obviously necessary to understand dendrite growth and synaptogenesis. It now appears that dendrite growth and synaptogenesis processes may persist in mature brains, as well, so our studies of these processes in developing zebrafish may advance understanding of the plasticity and pathologies of mature nervous systems. In addition, the developmental insights obtained may point to unexpected therapeutic opportunities for treating disorders of vision, and repairing effects of neurological trauma, neurodegeneration, and drug addiction.

0423076
Smith
The goal of this project is the design of a novel implantable brain imaging device called an IOS sensor. Based on a microscale integrated array of intermingled GaAs NIR emitters and detectors, this device will be optimized to image the intrinsic optical signal (IOS), a diffuse optical reflectance correlate of brain electrical activity rich in detail about sensory and motor information processing in mammalian cortex. The neurobiological utility of IOS imaging is already very well established, but all previous IOS imaging has used bulky, benchtop-scale instruments that require subjects to be immobilized and, almost always, anesthetized. Because of its small size, the IOS sensor will allow unprecedented imaging of cortical activity patterns in unanaesthetized and freely behaving subjects. Applications of the implantable IOS sensor will include neuroscience research, prosthetics for neurological injury patients, and drug discovery.

[unreadable] DESCRIPTION (provided by investigator): The proposed research explores the usefulness of a new high-resolution tissue molecular imaging method called Array Tomography (AT) to the understanding, diagnosis, prevention, and cure of Alzheimers Disease (AD). As part of this effort, a new set of array tomographic molecular labeling and image analysis methods, called Array Tomographic Single-Synapse Analysis (AT/SSA), will be developed for the purpose of quantifying and classifying individual synapses in tissues of mouse and human cerebral and hippocampal cortex. Loss of cortical synapses is widely believed to be the proximal cause of cognitive dysfunction in AD and many other neurodegenerative disorders, but quantitative information about such cortical synapse deficiencies has been limited, due to the technical limitations of available methodology. Moreover, cortical synapses are now recognized as being highly diverse, and again, relevant quantitative information is scarce. These shortcomings are consequential to human mental health because of strong evidence that different neurochemically defined synapse types exhibit differential pathologies in AD and therefore represent distinct therapeutic target discovery opportunities. AT/SAA is expected to provide new quantitative information about cortical synapse populations and their diversity that could fill critical gaps in our understanding of AD and lead to the development of novel diagnostic and therapeutic strategies. AT/SSA is based on the ability of array tomographic immunofluorescence imaging to resolve and quantify individual synaptic puncta in three-dimensional volumes of cortical neuropil, and the use of high-order antibody multiplexing to discriminate amongst various molecularly defined synapse types. This project will continuing efforts to make array tomography imaging methods faster, easier, more reliable and more informative and to develop high-throughput array tomographic classification tools for discrimination and quantification of synapse types. To establish a baseline quantifying normal cortical synapse populations, AT/SSA methods will be applied first to tissue specimens from wild-type mice and from autopsies and biopsies of humans unaffected by AD. The same methods will then be applied to specimens from transgenic, disease-model mice and from neurologically characterized AD patients. The information to be obtained is expected to provide new insights into the cellular and molecular basis of AD disease progression in human patients and to identify new ways that disease model mice may be used to help develop and test new drugs, vaccines, or other treatments aimed at the prevention and cure of AD. PUBLIC HEALTH RELEVANCE: The proposed research explores the potential usefulness of a new high-resolution molecular imaging technique called Array Tomography (AT) to the understanding, diagnosis, prevention, and cure of Alzheimers Disease (AD). A new set of AT-based methods will be developed and used to compare cortical synapses from the brains normal and AD-affected humans and from normal mice and mice genetically modified to mimic the human AD condition. The information to be obtained is expected to provide new insights into the cellular and molecular basis of AD disease progression in human patients and to identify new ways that disease model mice may be used to help develop and test new drugs, vaccines, or other treatments aimed at the prevention and cure of AD. [unreadable] [unreadable]

Recent developments in molecular neuroscience allow scientists to both optically record neuronal activity and to cause neurons to fire by stimulating them with light. In genetic model organisms like zebrafish and mice, these technologies can be used to observe and manipulate the activity patterns of thousands of neurons at once using non-invasive all-optical methods. While such techniques promise to revolutionize how we look at brain circuits, neural circuits are fundamentally three-dimensional structures. As a result, optical tools able to both image and selectively stimulate 3D volumes of brain tissue must be developed.

This project will develop a device that can record a volume of neurons at each camera exposure, extract information from thousands of these neurons over time, and then use this information to choose which groups of individual neurons in the volume to stimulate with light. This feedback loop will allow scientists to test causal hypotheses about brain network function and its relationship to behavior in a fast and powerful way, leveraging feedback-control technologies currently used in robotics and aeronautics to build and refine dynamical models of the brain online. At the core of this device are new developments in computational microscopy: the light field microscope (LFM), which can computationally reconstruct an entire volume from a single snapshot, and the light field illuminator (LFI), which can create (nearly) arbitrary patterns of light in three dimensions. The project will couple these two devices and accelerate their performance using commercial graphics cards (GPUs) to allow real-time control of biological neural networks in behaving animals.

Project outcomes, including scientific findings resulting from the application of the device to biological specimens, detailed directions on how to construct the physical device, and free, open-source software to run the device, will be provided online at http://graphics.stanford.edu/projects/lfmicroscope/.

DESCRIPTION (provided by applicant): Synaptic plasticity in many different forms is widely recognized as essential to normal brain development, to homeostasis of the mature brain, and to our abilities to adapt to changing environments and injury and to learn. There is relatively little information, however, to link particular known forms of synaptic plasticity to particular forms or sites of behaviorally relevant brain circuit plasticity. This gap in our knowledge prevents us from efficiently pinpointing synapses of known functional relevance as we explore the molecular, synaptic, and circuit bases of memory and its disorders. The experiments proposed here will exploit unique advantages of the mouse whisker sensory system to explore basic mechanisms of neocortical synaptic plasticity. The work will apply a powerful new high-resolution proteomic imaging method called "array tomography" (AT) to measure molecular and structural characteristics of cortical synapse populations at the level of individual synapses. AT has unique abilities to resolve individual synapses in native circuit tissue context, to measure dozens of distinctive molecular markers (e.g., diverse receptor, transporter, signaling, scaffolding and adhesion proteins) at each synapse, and to do so with very high experimental throughput. Thus, AT can determine a high dimensional molecular signature for each individual synapse in very large populations and differentiate specific synapse subpopulations on the basis of such molecular signatures. The proposed research will develop and apply a novel AT-based screening strategy to search in an unbiased fashion for patterns of structural and molecular change occurring in specific mouse neocortical synapses in reaction to specific sensory adaptation and associative conditioning procedures. PUBLIC HEALTH RELEVANCE: This research will apply a powerful new high-resolution proteomic imaging method called array tomography to pinpoint specific sites of synaptic plasticity associated with particular sensory adaptation and associative learning paradigms. The resulting new information on brain plasticity mechanisms will contribute to the development of improved drug treatments for neurodevelopmental and neurodegenerative disorders.

DESCRIPTION (provided by applicant): This is a collaborative project between Northwestern University (Nelson Spruston and Bill Kath), Stanford University (Stephen Smith), and the University of Bonn (Stefan Remy). The project will lead to an improved understanding of neurons in the hippocampus, which were selected because of their roles in learning and memory as well as a number of cognitive disorders. Studies of these neurons will also offer insight into neurons in other areas of the brain, many of which have shared structural and functional properties. The goals of the project are as follows: We will collect functional data from hippocampal CA1 pyramidal neurons using patch-clamp recording in brain slices combined with two-photon uncaging of glutamate and two-photon calcium imaging. We will also collect structural and molecular data from the same dendritic branches using array tomography, which provides the highest possible resolution using light microscopy. We will examine the distribution of excitatory synaptic weights, as well as the distribution of inhibitory synapses from different interneuron subtypes. All experiments will be performed for dendrites in different dendritic compartments (e.g., basal versus apical dendrites). By performing both functional and structural experiments in the same neurons, we will be able to correlate and integrate the data sets. We will construct compartmental models of CA1 pyramidal neurons, using the data from the experiments to inform improvements on our existing models of these neurons. The models will be used to generate experimentally testable predictions concerning the integration of synaptic inputs. These predictions will extend beyond the range of experiments performed to constrain the model, so they will constitute predictions designed to inform future work on these neurons. Spruston and Kath have a record of using such predictions to design and perform experiments that lead to new discoveries. We will use the models developed in Aim 2 to examine whether stochastic activation of thousands of excitatory and inhibitory synaptic inputs, combined with the excitable properties of the dendrites and synaptic plasticity rules based on the resulting dendritic voltage changes, can lead to non-uniform gradients of excitatory synaptic weights in CA1 pyramidal neurons. Our working hypothesis is that the natural gradients of voltage that exist in CA1 dendrites can contribute to the development of non-uniform synaptic weights. We will compare the results of these simulations to the results from array tomography studies as a means of determining which activity patterns and synaptic plasticity rules best explain the observed distribution of synaptic weights. Collaboration: All team members will exchange data and interact on a regular basis. The Spruston and Remy labs will perform experiments using patch-clamp recording and two-photon uncaging and imaging. Filled cells from these experiments will be sent to Stanford for array tomography in the Smith lab. Spruston, Kath, Smith and Remy will supervise the integration of array tomography data with functional data, working together with the postdoc and student supported by this project. All members of the group will meet regularly to discuss progress and future plans. Intellectual Merit: The project will provide critical data concerning the structure and function of pyramidal neurons in the hippocampus, which will be used to generate computational models of unprecedented detail. The models will be used to advance our understanding of synaptic integration in dendrites and the contribution of excitable dendrites to synaptic plasticity and the distribution of excitatory synaptic weights in the dendritic tree. The underlying philosophy is that the function of neural circuits, as well as diseases that affect them, cannot be understood without an accurate understanding of the structure and function of the component parts in the circuit. Broader Impacts: The broader impacts of this work include international collaboration and international and multi-disciplinary training of students and postdocs. In addition, our experimental data and computational models will be shared with the larger research community. We will also work with Michael Kennedy, Director of Northwestern's Science in Society program, to use our data to generate interactive, web-based educational tools targeting high-school students as well as post-secondary students. Our goal will be to develop visually exciting tools that appeal to a teenage audience. The tools will be promoted through the Science in Society website and through Kennedy's personal interactions with Chicago Public Schools and the Boys & Girls club of Chicago, both of which have large populations of under-served minorities. Stefan Remy will promote these educational tools in Germany. Long term, we believe that these tools could reach national and international audiences.

DESCRIPTION (provided by applicant): Synapses of the mammalian central nervous system (CNS) are very deeply diverse in both molecular and functional properties. At present, unfortunately, our understanding of this diversity is rudimentary, and quantitative data on the subject are very few. Left unfathomed, CNS synapse diversity poses formidable obstacles to better understanding of the development, function and disorders of the brain's synaptic circuitry. The major reason for the persistence of this distressing state of ignorance lies in the fact that tools for exploring synapse populations at the level of individual synapses are few and limited in their capabilities. To address the challenges synapse diversity poses to both basic and clinical neuroscience, this project aims to develop a superlative new proteometric imaging platform capable of analyzing very large synapse populations in situ with single- synapse resolution. Deployment and dissemination of this platform will facilitate study and treatment of the many neurodevelopmental, mental and neurodegenerative disorders linked to specific synapse subpopulations, as well as opening new perspectives on molecular mechanisms, circuit architectures and disorders of CNS memory encoding, storage and retrieval. The platform will be based on immunofluorescence array tomography (IAT) and involve development of novel antibody standardization protocols and novel image acquisition hardware and software. These innovations will improve the reproducibility, quantitative reliability, and speed of IAT by large margins and overcome limitations that have so far prevented proteometric analysis of large synapse populations at the single-synapse level. The platform would be capable of proteometric census of a million of more synapses per hour, at 50 or more markers per synapse, while maintaining precise neuroanatomical and molecular coordinates for each synapse. The new platform will be demonstrated by a 7-marker proteometric survey of an adult mouse cortex barrel column that would enumerate each of the tens of millions of synapses in the column and allow classification of each synapse based on neurotransmitter type and a set of neurons type markers. The results will be disseminated via methods publications, open-source PUBLIC HEALTH RELEVANCE: Abnormalities of individual synapses and of the brain's large and diverse synapse populations are widely believed to account for many or most neurodevelopmental, neurodegenerative and substance-abuse-related brain disorders. This work will address major gaps in present knowledge of such abnormalities by developing superlative new tools for the very rapid and highly detailed quantitative survey of large synapse populations in both research animal and human brain specimens.

DESCRIPTION (provided by applicant): The synapse is the principle active signaling component of the brain's neuronal circuitry. Synapses are highly complex, plastic, strongly modulated and deeply diverse entities, and their molecular complexity and diversity are fundamental to all synaptic circuit development and function. Moreover, many or most neurodevelopmental, psychiatric, and neurodegenerative disorders are rooted in abnormalities of the brain's vast and highly heterogeneous synapse populations. Unfortunately, such disorders are poorly understood and difficult to diagnose, prevent, and treat because we lack adequate tools to measure the brain's vast and highly diverse synapse populations, and because most of the limited tools in use today can be applied only to experimental animals such as mice. An interdisciplinary consortium comprising neurobiologists, biophysicists, clinicians, mathematicians and computer scientists here proposes development of a very ambitious synaptomic analysis pipeline that will transform the science of synaptic network function and disorders in both experimental animal and human brains. This novel high-throughput pipeline, based on powerful new array tomography methods, will enable measurement, analysis, and modeling of heterogeneous synapse and neuromodulatory fiber populations with unprecedented precision. The synaptomic pipeline will be demonstrated initially by developing synaptomes to model the heterogeneous synapse populations of mouse and human frontal and temporal lobes. Pipeline resources and data will then be shared via an Open Synaptome Project that will facilitate the development of synaptomes describing synapse populations of additional brain regions and species. These efforts are expected to provide a new foundation for understanding the basic mechanisms of mammalian brain function, and to offer new quantitative perspectives on both similarities and differences between mouse and human brain that will be critical to leveraging animal research opportunities for the improvement of human mental health. Because abnormalities of synapses and their neuromodulation are prime suspects in numerous human mental health disorders, the development and sharing of synaptomic pipeline resources and data proposed here are likely to catalyze rapid progress in clinical neuroscience.