2008 — 2010 |
Ostroff, Linnaea E |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Synaptic Tagging in the Lateral Amygdala Fear Conditioning Circuit
[unreadable] DESCRIPTION (provided by applicant): Auditory fear conditioning, a learning paradigm in which a rat can form an association between a previously neutral tone and a painful stimulus, is believed to occur via the persistent strengthening of synaptic connections, termed long-term potentiation (LTP), in the lateral amygdala (LA). Protein synthesis is necessary for both consolidation and reconsolidation of fear conditioning memory, and this may suggest a role for synapse remodeling. Auditory information enters the LA via plastic synapses from both the thalamus and cortex, but the functional and anatomical relationship between these synapses is unclear. The proposed project will begin to investigate the relative roles of these synapses in fear conditioning memory and their structural underpinnings. First, the protein synthesis-dependence of LTP consolidation and reconsolidation will be compared between the cortical and thalamic input pathways using a behaviorally relevant induction protocol. Second, serial section electron microscopy will be used to examine ultrastructural changes in LA synapses that occur during consolidation of fear conditioning and LTP, including changes in synapse size, synapse number, and local protein synthesis. Tracer injections will allow comparison of cortical and thalamic synapses under all conditions. A better understanding of the synaptic basis of the formation and maintenance of fear memories is important for the development of interventions for psychiatric disorders involving fear associations, such as phobias. [unreadable] [unreadable] [unreadable]
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1 |
2011 — 2012 |
Ostroff, Linnaea E |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Genetically Encoded Neural Tracers For Electron Microscopy
DESCRIPTION (provided by applicant): The connections between neurons compose the basic structure of the brain, through which all of its functions are expressed. Mapping and characterizing these connections is fundamental to neuroscience, as no brain system can be understood if its architecture is unknown. Neuroanatomical tracers have been used for decades to reveal connectivity in much of the brain, but the information they provide is limited. Tracers are compounds which are transported along axons and allow their origin, destination, or both to be visualized. It is difficult to control the specificity of existing tracers, which leads to inconsistent and unreliable results. To accurately map connectivity, the existence of synaptic connections between identified neurons must be verified at the electron microscopy (EM) level. Furthermore, EM studies of synapse morphology should ideally be carried out on labeled connections. The few existing tracers that are compatible with EM are not compatible with morphological studies due to severely compromised ultrastructure. We have conducted extensive morphological and neuroanatomical tracer studies at the EM level on our system of interest, the adult rat lateral amygdala. We propose to develop novel tracers which will label specific cells for light microscopy and EM while preserving high quality ultrastructure for morphological studies. Using a viral vector, we will express a membrane-targeted form of the EM label horseradish peroxidase (HRP) in adult rat neurons. Unlike current tracers, HRP can be visualized without subjecting tissue to detergents which damage EM ultrastructure. This allows good preservation of tissue morphology, while restricting the HRP to the membrane prevents the label from obscuring any cellular organelles. We will place the membrane-bound HRP gene under the control of one of two different promoters. The first, the calcium/calmodulin-dependent protein kinase II promoter, will restrict expression of the label to excitatory neurons. Combined with the fact that viral transfection is restricted to cell bodies (which conventional tracer uptake is not), this will be the most spatially and functionally specific tracer available. The second promoter will be from the activity-regulated cytoskeleton-associated protein Arc, which is expressed in response to strong synaptic activation and behavioral experience. This will allow identification of the axons and dendrites of cells activated during learning and plasticity experiments such that their synapses can be specifically examined. The tools we propose to create will have a broad range of applications in neuroanatomy, neurobiology, and plasticity studies throughout the brain. PUBLIC HEALTH RELEVANCE: Mapping and characterizing the connectivity between brain cells is fundamental to understanding how the brain works. Many neurological diseases involve dysfunction of particular brain circuits or connections, and it is essential to elucidate and examine these connections in both normal and diseased brains. The goal of this project is to develop novel tools to map neural connections and visualize them clearly at the microscopic level.
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1 |
2019 — 2020 |
Ostroff, Linnaea E |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Methods For Serially Multiplexed Labeling in Em Reconstructions of Brain Tissue @ University of Connecticut Storrs
Project Summary/Abstract Tremendous technological progress in the use of serial electron microscopy (EM) for brain circuit mapping has been made over the last decade, and it is possible to directly visualize synaptic connectivity in larger volumes of brain tissue than ever before. Meanwhile, advances in molecular biology have shed new light on the diversity among neurons, particularly with respect to their patterns of gene expression. Currently, there are no methods available to efficiently integrate molecular labels into serial EM reconstructions. The ability to distinguish many molecular cell type markers in serial EM volumes would greatly enhance our ability to study circuit function, neuronal diversity, and neuroplasticity, and to determine how these are affected in disease states. The major barrier to visualizing molecules in serial EM is that the methods used to preserve morphology and generate contrast for serial EM are incompatible with most labels. Several methods have been developed to accommodate this limitation, mainly by using genetic tools to introduce labels before tissue samples are prepared for EM. These approaches are restrictive in that only a few labels can be used in a single sample, genetic manipulation is required, and endogenous molecules cannot be localized. This project will develop approaches that allow many different molecular labels to be differentiated within a single serial EM tissue volume. An innovative strategy will be used: instead of working around the standard serial EM protocol by designing labels that are compatible with it, the focus will be on replacing the incompatible elements of the standard protocol. Circuit reconstruction by serial EM requires a high degree of morphological preservation, which is typically accomplished with harsh chemicals that damage and denature molecules. However, all that is fundamentally required to preserve morphology is to retain as many molecules in the tissue as possible, which is also necessary for molecular labeling. Therefore, tissue preservation protocols will be developed to minimize extraction of molecules without damage or irreversible denaturation. A combination of strategies will be employed, including novel combinations of chemical crosslinkers and embedding resins. These new approaches will offer a means of revealing valuable information about circuit organization and neuronal diversity that is presently inaccessible.
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0.954 |
2020 |
Ostroff, Linnaea E |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Quantum Dot Probes For Electron Microscopy @ University of Connecticut Storrs
Project Summary/Abstract The lack of comprehensive maps of brain architecture from molecules to circuits is a critical barrier to progress in neuroscience, and better, more routine methods for accurately localizing molecules at the subcellular level are needed. Brain tissue presents a twofold challenge for molecular mapping: in addition to the obvious need for high-resolution imaging, accurate localization of molecules also requires a means of visualizing the surrounding cellular and tissue structure to identify not only which subcellular compartment contains a given molecule, but which cell. Super-resolution fluorescence microscopy has achieved single- molecule resolution, but reveals only probes, not tissue structure. Electron microscopy (EM) readily reveals comprehensive tissue structure at sub-nanometer resolution. Methods for molecular imaging at the EM level, however, remain inefficient and are often unreliable. Newly developed transgenic approaches can facilitate localization of specific targets by EM, but these require genetic manipulation, offer very limited multiplexing, and do not reveal endogenous molecules. Postembedding immuno-EM, in which antibody labeling is performed directly on EM sections, is a much more efficient and versatile approach, but is technically challenging to the point that it is largely avoided in neurobiology. A crucial unique feature of postembedding EM labeling, in contrast to the routine, widely used methods for immunolabeling of fixed tissue, is the use of gold particles for antibody detection. The premise of this proposal is that gold probes are an underappreciated cause of failure in postembedding labeling, based on the observation that EM sections are amenable to labeling with fluorescent antibody probes using simple, routine procedures. In contrast to popular fluorescent antibody probes, gold probes suffer from unfavorable stoichiometry, stearic hinderance, and instability of the gold-antibody complexes. The central aim of this project is to develop reagents for antibody detection on EM sections that circumvent these problems. Quantum dots, which are semiconductor nanocrystals that are visible by EM, are an excellent alternative to gold as they are simple to synthesize in a variety of sizes, shapes, and elemental compositions, which facilitates both probe optimization and multiplexed labeling. To avoid reliance on bulky, unstable protein-metal complexes that limit both sensitivity and signal amplification, a catalyzed reporter deposition (CARD) approach will be used. CARD employs antibody-linked peroxidase enzymes to catalyze covalent attachment of probe molecules to proteins at the antibody binding site. Functionalizing quantum dots for use as CARD substrates uncouples the antibody binding step from detection, so that the relatively bulky EM probe does not interfere with sensitivity, and enzyme-based probe deposition allows amplification to proceed across time without the limitation of binding-site saturation. This approach is innovative in that it does not simply replace one label for another, but instead addresses multiple known causes of poor performance in the existing probes.
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0.954 |