Mark H. Ellisman - US grants

This project is focused on the node of Ranvier of myelinated nerve. The general goal is to extend our work on establishing a better understanding of how the node functions and to continue to identify the locations of macromolecules in the nodal complex that are important to conduction of the nerve impulse and development Specific aim I is to determine the sources, mechanisms and roles of[Ca2+]i transients and the sites of voltage transients in the nodal complex. The ultimate objectives of these studies are twofold: to gain a better understanding of (1) the roles of paranodal glia in the regulation of the nodal ionic milieu; and (2) the mechanisms underlying alterations in threshold and changes in conduction velocity that occur following impulse activity. Experiments are to be conducted using optical methods for recording of voltage and (Ca2+]i transients as well as time-lapse Nomarski imaging. In these experiments we will use optical methods to determine the structural and physiological effects of drugs known to alter specific ion conductances. Specific aim II involves studies of 5-HT receptors in Schwann cells. The objectives are to determine the type of 3-HT receptor responsible for Schwann cell [Ca2+]i transients in vitro and determine if these receptors are present in vivo. Specific aim IIl is to determine the sites of previously characterized and newly identified macromolecules in the node of Ranvier complex using immunolocalization techniques as well as to determine the sequence of appearance of macromolecules during development, regeneration and remyelination. The objectives of these studies are (1) to identify the precise locations of membrane proteins and other macromolecular constituents that we believe are important to nodal function and development; and (2) to study the developmental sequence for the appearance of the subset of these macromolecules that may be involved in defining the initial locations of nodes along premyelinated or remyelinating fibers. Our first experiments localizing macromolecules in the nodal complex win follow up on interesting preliminary results with newly characterized antibodies to K+ channels, ryanodine receptors, Na+/Ca2+ exchangers and H2O channels, as well as Na+/K+ ATPases, tyrosine kinases and proteins phosphorylated on tyrosine. Simultaneously we will conduct experiments aimed at gaining new insight into the initial stages of nodal development, remyelination and regeneration, using antibodies specific for the voltage dependent Na+ and K+ channels. In these experiments the aim is to determine the sequence of development of macromolecular components of the nodal complex. A special interest will be to determine if one of the constituents disclosed during the antibody localization studies appears prior to the arrival of myelinating cells thus participating in the early definition of the node of Ranvier. The results of these studies will provide a better understanding of the relationship between structure and function at the node of Ranvier and the underlying macromolecular basis for impaired conduction in demyelinating disease.

The long term goals of this project are to understand the molecular mechanisms underlying the formation and function of the triad junction in muscle cells. This is a junction between plasma and sarcoplasmic reticulum (SR) membranes that is thought to have a critical role in coupling excitation and muscle contraction. This research will establish the developmental history of a key element of the triad junction, the "foot" protein in embryonic and neonatal chick skeletal muscle, in order to gain insight into the role of this protein in junction formation, muscle morphogenesis and the biogenesis of SR membranes. The plant alkaloid, ryanodine, binds specifically to the "foot" protein; this compound is, thus, a useful experimental probe of this protein. The developmental time course of the appearance of the "foot" or ryanodine-binding protein (RBP) and of the manifestation of its ability to bind ryanodine and to alter the calcium permeability of SR membranes will be examined. The appearance of the "foot" protein will be measured by specific tritiated ryanodine binding, and detection of the 400-500 kDa RBP using SDS-PAGE and RBP specific antibodies. The appearance of "foot" protein function will be assayed by measurement of intracellular calcium transients and contractile activity. These events will be correlated with the development of a mature muscle morphology and SR function in pectoral muscle from embryonic and neonatal chicks and primary cultures of embryonic muscle as visualized using electron microscopy and immunocytochemical techniques. The results of this research should contribute substantially to our understanding of the mechanism by which excitation of the surface membrane of a striated muscle cell triggers calcium release from an intracellular compartment, the sarcoplasmic reticulum, as the key step in initiation of muscle contraction.

An advanced interactive serial reconstruction system will be developed, and programs will be implemented for the derivation and visualization of data from thick section electron microscopy tilt series. This award will provide an initial basis for these efforts. The facilities of the San Diego Supercomputer Center will be utilized to implement and explore computer graphic methods for the three dimensional reconstruction and visualization of data represented as a series of planes or cross sections through a three dimensional medium. The computer software tools will be available not only for biologists but also for meteorologists, oceanographers and geologists who wish to explore three dimensional distributional aspects of data collected in the form of layers or strata. The programs are being implemented on an Ardent Titan and a Silicon Graphics IRIS workstation. The electron microscopic images are being analyzed to obtain substantial three dimensional information about the structure of biological specimens. The methods employ tomographic reconstruction of two dimensional images and exploit prior developments for registration of digitized tilt series and three dimensional energy distribution. The computer advances will be applied to three dimensional reconstruction of cellular images from both electron and light microscopy, to images of molecular and macromolecular assemblies, and to neuroanatomical mapping systems.

A Regional Resource for computer-aided Intermediate Voltage Transmission Electron Microscopy (300-400 KV, IVEM) and 3-dimensional (3-D) image analysis will be established & serve the Southwest region of the country. These facilities would be made available immediately to investigators using established techniques and working on biological problems that could clearly benefit from the use of thicker specimens and 3-D imaging. We have directed our plan for core technological research toward the development of technologies that will contribute to research in structural neurobiology as well as cell biology and molecular biology. Our goals for collaboration, service, training and dissemination are all related to expanding the use ot these technologies so that they can become as valuable as possible for the biomedical community We efmphasize the use of computer aided methods for enhancement of image contrast, extraction of accurate 3-dimentional information from within single thick specimens and reconstruction of larger structural complexes with serial thich section analysis. We are planning to use a variety of contrast enhancing methods on model system/biological problems that will clearly benefit from the unique capabilities of a computer-coupled IVEM. These include core technological research activities to: provide new IVEM compatible macromolecular probes; improve penetration of probes into frozen sections; enhance selective staining of subcellular structure; and define the 3-D distributions of and relationships between submicron cell processes in nervous systems with quantitative accuracy. The capabilities to be developed and research accomplishments anticipated in all of the core project areas involving methods of specimen preparation will be augmented by features of the imaging system to be developed. These will include: user interactive modification of operating conditions of the IVEM in order to alter image contrast; microscope-coupled image processing and analysis; and semi-automated 3-D image acquisition and reconstruction facilities. Training of users in the application of the technology will occur at the Resource. In addition, yearly workshops and various other forms of scientific communication will be used to disseminate the to-be- developed Resource technologies.

This proposal details a research program designed to localize three specific macromolecules within intracellular membrane systems of both the cell body and the axon. Localization of these macromolecules will elucidate the involvement of specific somal membrane systems in the packaging of proteins destined for different target areas of the plasma membrane or extracellular space and the involvement of axonal membrane systems in axonal transport. The three target macromolecules to be studied are; 1) the Na+ +K+ ATPase; 2) the voltage dependent SODIUM CHANNEL; and 3) ACETYLCHOLINESTERASE (AChE). These macromolecules (their individuals constituent subunits or distinct molecular forms) will be localized with the aid of specific antibody probes visualized by light and electron microscopy using gold, ferritin, peroxidase, or fluorescent antibody conjugates. The membrane systems under study are both those of the cell body and of the axon. In the cell body, those involved in the biosynthesis and packaging of membrane proteins or secretion products; e.g., the rough endoplasmic reticulum (RER), the smooth endoplasmic reticulum (SER), and the Golgi apparatus. In the axon, there are at least three morphologically and functionally distinct membrane systems. One of these is clearly involved in anterograde rapid axonal transport, another in retrograde transport and the third which does not appear to be rapidly transported and whose function or relationship to the other two is unknown. These planned investigations into the biosynthesis and transport of all three of these macromolecules are now enabled by the recent production and characterization of specific antibodies to 10 the Na+ +K+ ATPase and its alpha and beta subunits from rat and eel (Electrophorus electricus) tissues, 2) the Na+ channel from eel, and 3) two of the major molecular forms of AChE from Torpedo. We have already proven antibodies to each of these macromolecules suitable for use in high resolution immunoelectron microscopic studies, immunofluorescent studies, or as biochemical probes with techniques including Western blotting, enzyme linked immunoadsorbent assays or radioimmunoassays. Species and tissue specificity of antibodies have been examined. All are capable of recognizing neuronal forms of each antigen and in most cases cytoplasmic labeling capacity has been already confirmed.

This proposal is to obtain a high resolution transmission electron microscope (JEOL JEM-1200EX-BIO) to replace a 20 year- old JEM-100B which is currently one of two standard transmission electron provided as "core facilities" to biological investigators at the University of California at San Diego (UCSD). These microscopes, along with facilities for specimen preparation, constitute the Laboratory for Neurocytology that is operated on a recharge basis in the School of Medicine at UCSD. There are over 20 research projects involving 12 established investigators currently dependent on the use of the electronmicroscopic facilities of the Laboratory for Neurocytology. The major user group consists of established scientists mainly from the School of Medicine with research interests in neurobiology. Other users from the general campus or the allied institutions also benefit from the special facilities for freeze-fracture, three-dimensional reconstruction, or cryosectioning, not available for use on a recharge basis elsewhere in this geographic area.

A Regional Resource for computer-aided Intermediate Voltage Transmission Electron Microscopy (300-400 KV, IVEM) and 3-dimensional (3-D) image analysis will be established & serve the Southwest region of the country. These facilities would be made available immediately to investigators using established techniques and working on biological problems that could clearly benefit from the use of thicker specimens and 3-D imaging. We have directed our plan for core technological research toward the development of technologies that will contribute to research in structural neurobiology as well as cell biology and molecular biology. Our goals for collaboration, service, training and dissemination are all related to expanding the use ot these technologies so that they can become as valuable as possible for the biomedical community We efmphasize the use of computer aided methods for enhancement of image contrast, extraction of accurate 3-dimentional information from within single thick specimens and reconstruction of larger structural complexes with serial thich section analysis. We are planning to use a variety of contrast enhancing methods on model system/biological problems that will clearly benefit from the unique capabilities of a computer-coupled IVEM. These include core technological research activities to: provide new IVEM compatible macromolecular probes; improve penetration of probes into frozen sections; enhance selective staining of subcellular structure; and define the 3-D distributions of and relationships between submicron cell processes in nervous systems with quantitative accuracy. The capabilities to be developed and research accomplishments anticipated in all of the core project areas involving methods of specimen preparation will be augmented by features of the imaging system to be developed. These will include: user interactive modification of operating conditions of the IVEM in order to alter image contrast; microscope-coupled image processing and analysis; and semi-automated 3-D image acquisition and reconstruction facilities. Training of users in the application of the technology will occur at the Resource. In addition, yearly workshops and various other forms of scientific communication will be used to disseminate the to-be- developed Resource technologies.

DESCRIPTION (provided by applicant): This project is focused on the structure and function of the node of Ranvier of myelinated nerve. The nodal complex contains axonal specializations with adjacent paranodal terminations of the myelin sheath, and glial processes that invest the nodal gap and the gap substance. Our goal is to extend our work on establishing a better understanding of how the node functions and to continue to identify the locations of macromolecules in the complex that are important to maintain the structural integrity and in conduction and coordination of the nerve impulse. In SPECIFIC AIM 1, we will extend the current knowledge of specific membrane, cytoskeletal and extracellular matrix structures of both CNS and PNS nodes by building high resolution 3D volumes using the improved methodology for higher voltage electron tomographic structure determination. We will explore the relationship between different nodal compartments and their interconnectivity from nerves and fiber tracts with differing conduction properties to test if the number and frequency of action potentials have an effect on the morphology of the nodal compartments. In SPECIFIC AIM 2, we will use novel new methods such as the photoconversion of novel fluorescent labels, double tilt tomography and high resolution electron microscopic imaging for obtaining accurate and high resolution information (50 A) in situ about the arrangements and locations of specific molecules using intermediate high voltage electron tomography. Specifically, we are interested in the relationships between ion channels found in the membranes, cytoskeletal anchoring proteins and cell-cell junctional elements and the relationships between. The data we obtain from these new structures will then be used to construct highly accurate models of the node of Ranvier complex. In SPECIFIC AIM 3, we will deposit this structural data into a recently developed cell-level microanatomical database system that provides a framework for the integration of systems, cellular, subcellular and molecular data from distributed data sources as well as use a computer simulator program of cellular physiology that will use our 3D structural models obtained from tomography to conduct in silico tests of physiological function. The results of these studies will provide a better understanding of the relationship between structure and function at the node of Ranvier and the underlying macromolecular basis for impaired conduction in demyelinating disease and nerve repair.

The triad junction is a complex structure involving plasma and sarcoplasmic reticulum membranes and at least seven proteins, and is the site where electrical excitation is transduced to release intracellular calcium, and thus has a critical role in coupling electrical excitation to muscle contraction. Additionally, it may interact with the cellular cytoskeleton to form muscle-specific morphology. Two key components of the triad junction, the dihydropyridine (DHPR) and ryanodine receptors (RR), are expressed early during embryonic chick skeletal muscle differentiation. The aim of this project is to determine how these proteins are incorporated into the triad junction and become active in the release of intracellular calcium in developing skeletal muscle, and whether the calcium release events mediated by the triad junction influence other aspects of muscle development. The specific aims are to: (1), define the experimental in detail by establishing the time course of the expression of the DHPR and RR in developing embryonic chick skeletal muscle; and (2), investigate whether these proteins influence skeletal muscle development by testing the hypothesis that DHPR and RR serve as organizers for the assembly of the triad junction and its integration into mature muscle structure. This will be done by defining the time course of incorporation of these and other junctional components into the junction, and assess the significance of interactions between junctional proteins and cytoskeletal elements. Dr. Ellisman's contribution to the project will be primarily with regard to the morphological aspects. Skeletal muscle provides the contractile force to move bones and other structures, effecting organ or whole organism movement. The signal which "tells" the muscle to contract and ensures synchronous contraction along the length of the muscle involves an electrical wave generated at the neuromuscular junction, which travels down the length of the muscle fiber along the plasma membrane. The conversion of the electrical signal to contraction occurs by transduction of the electrical signal to a "second messenger" signal, namely release of calcium from intracellular stores (sarcoplasmic reticulum) into the cytoplasm. At the "triad junction" of the muscle cell, the membrane of the sarcoplasmic reticulum is closely apposed to a specialized structure of the plasma membrane known as the transverse tubule. This highly specialized region contains a set of unique sarcoplasmic reticulum membrane proteins which interact with the closely apposed transverse tubule membrane to effect the coupling of the electrical signal to the opening of a calcium channel which allows the release of calcium from the sarcoplasmic reticulum to the cytoplasm. Neither the mechanism of this signal transduction event, nor the cellular development of this intricate subcellular structure, is well understood. The results of this research will increase our understanding of both of these very important processes in vertebrate muscle.

DESCRIPTION (provided by the applicant): New microscopy technologies will be developed and made available to biomedical researchers to bridge understanding of biological systems across gross anatomical and molecular scales. Project technology development efforts take on two important challenges: addressing the mesoscale range - from a few nanometers to 100 microns, required to see complexes in the context of cellular aggregates in tissues - and developing/advancing capabilities to enable multimodal imaging, that is, correlated imaging across a range of instruments (and their varying capabilities), to allow combining of data from the same specimens, providing new understanding of processes related to disease mechanisms. To achieve these two goals, activities will be in four related technology thrusts: probe development in Core 1, specimen development in Core 2, imaging instrument development in Core 3, and development of software tools to refine, integrate, analyze, and share microscopy data in Core 4. Core 1 will focus on labeling strategies for various new genetic or enzymatic probes. Core 2 will refine specimen-preparation protocols and materials to provide more suitable samples for examination. The combined outcome of these first two cores will be to streamline imaging using correlated microscopy by creating specimens better suited to a range of imaging modalities and enhancing the palette of in-situ markers to facilitate tracking areas of interest in related images and achieve their co-registration. Tracking methods, to be developed by activities of Cores 2 and 3, will be used to connect data in databases using new data analysis tools of Core 4, developing new tools for segmentation and annotation of images. Core 3 advances the imaging capabilities of multiple microscope types to deliver information not only with greater sensitivity and accuracy but more rapidly and seamlessly across scales. The work of all Cores, but especially the instrument development Core 3, will enable more sophisticated use of a next generation microscopes and their application to important biomedical research challenges. Core 4 develops software tools and infrastructure to refine, integrate, quantify, interpret, and add value to image data derived from the new instruments. The coordinated work among the four cores and collaborator projects will enable biomedical researchers to image wider, see deeper, increase contrast and differentiate complex structures, and observe complex phenomena at higher resolution and at faster time scales. Thus we will propel biomedical research requiring traversal of now difficult to navigate spatial scales, thereby facilitating new understanding of the molecular mechanisms underlying disease processes.

9318180 Ellisman The increased availability of High Performance Computing and Communications (HPCC) offers scientists the potential for effective remote interactive use of centralized, specialized, and expensive instrumentation and computers. The goal of this national challenge project is to design and implement a collaborative computational environment, or Collaboratory for Microscopic Digital Anatomy, providing a researcher at a remote site distributed interaction with unique instrumentation for the acquistion and manipulation of images of biological structure. The initial focus will be on the development of an integrated system for remote interactive acquistition and analysis of 2- and 3- dimensional light and electron microscopic data from digital image acquistion systems. To accomplish this task, a multidisciplinary team representing several institutions including the University of California , San Diego, Cornell University and the Center for Computer Graphics and Scientific Visualization, and the San Diego Supercomputer Center, has been assembled, including computer scientists specializing in HPCC and volume visualization, and biologists with expertise in computer-aided light and electron microscopy. This award will enable these activities. This project is being supported by several programs at NSF: New Technologies in Computer and Information Science, Cell Biology, Computational Neuroscience and Computational Biology.

We continued to expand the use of photo-oxidation as a method for selective staining of subcellular structures using both immunological and non-immunogenic probes. A summary of our progress with phalloidin-eosin as a marker for dendritic spines is provided under the highlights' section. We have also experienced a tremendous expansion of the use of tomography and selective stains to reconstruct organelles and other subcellular features. Guy Perkins, a post-doctoral researcher in the laboratory, produced spectacular reconstructions of mitochondrial cristae in collaboration with Dr. Terry Frey. Several of these projects deal with aspects of synaptic structure, because of the superiority of tomography for these types of three-dimensional analyses. A project with Drs. David Lenzi and William Roberts of the University of Oregon on the three-dimensional structure of the frog vestibular hair cell synapse has been completed, and a manuscript is in preparation. Drs. Thomas Schikorski and Charles Stevens of UCSD have produced several reconstructions of synapses onto dendritic spines in the rat hippocampus. Using software developed at NCMIR, they analyzed the distributions of synaptic vesicles and their relationships to the active zone. Finally, in collaboration wit h Drs. Bing Ren Hu and Justin Zivin of UCSD we have employed selective staining with phosphotungstic acid to stain post-synaptic densities in normal brain and in brains from animals subjected to transient ischemia followed by reperfusion. We observed a dramatic alteration in the size and appearance of synapses from post-ischemic hippocampus and cerebral cortex. published in the Journal of Neuroscience (Hu et al., J. Neurosci., 18: 625-633, 1998). Using thick sections and IVEM, we are able to visualize the entire 3-D structure of pre- and post-synaptic specializations. We performed tomographic reconstructions of synapses in the hippocampus of ischemic and control rats at different time points of reperfusion. These reconstructions clearly showed that synapses in area CA1 were more loosely configured in the ischemic brain than in control brains. The 3D images suggest that synapses in CA1 are undergin degenerative changes prior to obvious cell death. This work was presented at the Society for Neurosciences meeting in 1997 and a manuscript has been submitted to the Journal of Neuroscience.

The proposed projects concern the organization of calcium regulatory proteins within the neuronal endomembrane system. Transient rises in intracellular calcium serve as important signals regulating a host of cellular behaviors such as neurotransmitter release, activation of key enzymes, gene transcription and cell movement How neurons regulate intracellular calcium levels and produce calcium responses that are both temporally and spatially limited is a major research area in neurobiology. This information is critical not only for understanding normal neuronal function, but also for understanding various pathological processes, since abnormalities in intracellular calcium handling may be involved in several degenerative disorders such as Alzheimer's and Huntington's disease. A focus of this laboratory has been on defining the subcellular localization of proteins involved in the regulation of intracellular calcium pools. Neurons, like muscle cells, possess intracellular stores of calcium located within the endoplasmic reticulum that are capable of calcium release, storage and uptake. These stores, along with calcium fluxes across the plasma membrane, are believed to contribute to temporal and spatial regulation of calcium dynamics within neurons, although their precise function is unknown. These studies will employ single- and double- labeling immunolocalization techniques at both the light and electron microscopic levels to investigate the structure and distribution of these intracellular calcium pools. Structural aspects of the endoplasmic reticulum, such as the relative volume and surface area of the smooth endoplasmic reticulum within different portions of the Purkinje cell, will also be examined using three-dimensional reconstruction techniques and intermediate high voltage electron microscopy. Concurrently, cultured neurons will be used as model systems to investigate some functional aspects of intracellular calcium regulation.

DESCRIPTION (Taken from application abstract): New methods have led to rapid expansion of information on the molecular makeup of nerve cells and glia. Previously unknown proteins are being discovered at a rapid rate. Antibodies are now readily available to map the cellular and subcellular localization of these proteins, but tools for organizing and integrating these data with new and existing information are lacking. Computerized databases for accessing and visualizing the 3 dimensional (3-D) distributions of various macromolecules will provide important tools for the neurobiologist interested in modeling the brain's molecular complexity in health and disease. In this project we will develop techniques and software tools for the acquisition and representation of immunocytochemical data on the 3-D distribution of cellular constituents within realistic compartmental neuronal models. Macromolecules involved in inter- and intra-cellular signaling such as ion channels, neurotransmitter receptors and second messenger proteins will be localized using light microscopic (LM) and electron microscopic (EM) immunocytochemistry. Concurrently, realistic morphological representations of the neurons and neuronal compartments expressing these proteins will be obtained from intra cellularly injected neurons. The immunolocalization data will be combined with compartmental neuronal models based on these identified neurons and used to populate an atlas-based database for representation, storage and retrieval of information on neurons and the locations of their constituents. Initial studies will concentrate on the cerebellum with plans to study other brain regions later (e.g., neostriatum, hippocampus, olfactory bulb, cortex). The techniques and software developed will be made available to other neuroscientists to use in adding results from similar studies to the database. To accomplish these goals we will develop or implement: 1) a standard scheme for acquiring 3-D immunolocalization data by using double and triple label immunofluorescence and confocal LM; 2) methods for acquiring large 3-D datasets on neuronal form using intra cellularly filled neurons imaged with confocal LM and EM; 3) methods for combining immunocytochemical localization with intracellular filling to aid in determining the distribution of target molecules relative to cellular compartments; 4) software tools and methods for segmentation, editing, and data entry into a database for 3-D data on neuronal form acquired using LM and EM datasets; 5) software and methods for acquiring and merging multi resolution datasets; 6) a multi resolution database and visualization system integrating these data; and 7) software to access and query this database over the Internet.

technology /technique development; animal tissue; microscopy; nervous system; human tissue; drug adverse effect; biotechnology; biomedical resource;

The electron microscope is essential for resolving biological structures which are too complex for X-ray crystallographic methods and too small to be resolved with the light microscope. In contrast to the conventional instrument, the High-Voltage Electron Microscopes (HVEM) such as the instrument located at the National Center for Microscopy and Imaging Research (NCMIR) can image relatively thick specimens that contain substantial 3D structure. Tomographic methods can be applied to a set of images, acquired from different orientations by tilting the specimen, to derive a 3D representation of its biological structure. Tomography requires extensive computation and considerable processing time on conventional workstations in order to reconstruct typically large volumes from HVEM tilt series. Goals (1) Expedite tomographic processing by implementing electron microscope tomographic methods on parallel machines. (2) Provide transparent interactive access to these programs in a form easily used by structural biologists. 3) Investigate and implement alternative tomographic algorithms that may potentially provide improved reconstructions. Major achievements: We previously implemented the commonly used single axis tilt, R-weighted backprojection algorithm and two iterative reconstruction methods, algebraic reconstruction (ART) and simultaneous iterative reconstruction (SIRT) on the Intel Paragon. (1) During this last year we ported these programs to the Cray T3E and (2) completed parametric runs on the Paragon and the Cray T3E. With increasing numbers of nodes, Paragon performance is impaired by limited disk I/O bandwidth as more nodes attempt I/O at approximately at the same time. The I/O bottleneck is substantially less severe on the Cray. The Cray implementation is generally 2-3 times faster than the Paragon. Using only 16 nodes, the Cray is 10 times faster than a single processor SGI R10000 workstation. (3) We have examination of the parallel algorithms using specimens from NCMIR collaborative research projects involving changes in the structure of dendritic spines following loss of synaptic input, an analysis o f the complex three-dimensional structure of mitochondria, and changes in the 3D structure of cardiac muscle in a study of heart failure. These initial studies indicate that the iterative methods can produce superior reconstructions with fewer artifacts in comparison to the R-weighted algorithm. These evaluations are continuing. (4) To facilitate and encourage the use of these programs by biologists, we have constructed scripts to obviate the need to understand the NQS (Network Queuing System) facility, and to automatically handle data transfer to and from Cray local storage to the HPSS archival file storage at SDSC. The script and the high performance computing facilitate running multiple jobs on the same dataset to perform parameter manipulations for obtaining optimum reconstructions. We have also developed data format conversion programs to facilitate parallel processing of HDF file formatted data obtained using our telemicroscopy system. Collaboration and service: As described above, several biological projects associated with NCMIR are using the CrayT3E for tomographic reconstruction. We expect the number of users to increase as methods for access are further improved. The availability of these programs has been announced at an international meeting on electron microscope tomography and a paper based on this conference has been published (Perkins et al. 1997). Recently, we have provided our software and consultation to Drs. Carl Kessleman (Univ. of Southern California), Ian McNalty (Argonne National Laboratory), and Mark Rivers (U. Chicago) who plan to use our programs for tomographic reconstructions of data from advanced photon and other X-ray sources as part of their DOE Grand Challenge project "Supercomputer Solution of Massive Crystallographic and Microtomographic Structural Problems." Future plans: 1) Increase the number of parallel platforms by porting the software to the IBM SP2. 2) Implement and evaluate new iterative algorithms for improved reconstructions, which will benefit from the performance gains achievable with parallel computing. We plan to parallelize an algorithm developed by Gabor Herman and Jose Carazo using "blob" basis functions. In collaboration with these investigators, this algorithm has been implemented for single axis tilt tomography and is currently undergoing testing on conventional workstations. (3) Continue effort to interface these programs to a telemicroscopy system providing remote control of the HVEM for acquisition of tomographic data and transparent distribution of computationally intensive tasks to high performance computers on the network. (4) Develop web-based access.

The objective of this core TR&D project is to improve EMmethods for in situ hybridization. We have achieved exciting progress in localizing nucleic acid sequences at the electron microscopic level. We are still encountering sensitivity problems at the level of detecting single messages. We are currently exploring amplification methods based on the tyramide system developed by Dupont. Tyramide is a substrate of peroxidase and when activated by this enzyme, it sticks to sites near the site of activation. Tyramide can be conjugated to biotin or to a fluorophore and thus can be used for signal amplification. In our initial studies with immuno detection, the tyramide system yielded a tremendous increase in signal detection. We have used this methodology routinely now in non-radioactive light microscopic detection of K+ channel transcripts in cultured neurons and tissue sections and are planning on extending this analysis to the electron microscopic level in the near future. We are also beginning to work with riboprobes instead of the cDNA and oligoprobes that we have used previously. Riboprobes should afford improved sensitivity although their use necessitates more stringent wash conditions which severely impact ultrastructure. We are considering the use of post-embedding methods as well as more robust fixation protocols to overcome expected morphological damage.

The objective of this core TR&D project is to improve methods for the selective staining of neurons for correlative light and electron microscopy and to develop methods that will help researchers to visualize and quantify neuronal shape and connectivity at EM resolution. Our efforts in this area have been driven by our collaborative efforts with Drs. Charles Wilson, Gordon Arbuthnott and Cali Ingham on obtaining quantitative information on structural changes in striatal dendrites and dendritic spines associated with partial deafferentation. Briefly, we have been using electron microscopic tomography to obtain volume reconstructions of spiny dendrites in both normal neostriatum and in neostriatum in which the dopaminergic input has been lesioned chemically. Spiny dendrites receive the bulk of dopaminergic input within the neostriatum and it was observed at the light microscopic level that loss of this input may lead to both a loss of spines and a change in their morphology. The pilot portion of this project which involved acquiring tomographic reconstructions and quantitative measurements from 20 dendrites has been completed. These 20 dendrites came from 4 animals from both the lesioned and unlesioned neostriata. This population of dendrites yielded length, surface area and volume measurements from over 200 dendritic spines. Two problems were encountered: 1) because of the large variability within each group, we have determined that it will be necessary to obtain additional reconstructions from several more animals per group; 2) it appeared that the spine densities that we were finding were lower than those reported by serial sectioning analysis. Because we were limited to a section thickness of about 2 5m at 400 keV, many spines were likely being cut off of the dendrite at the top and bottom of the section. We performed tomographic reconstructions of spiny dendrites contained within 3-4 5m thick sections from tilt series acquired with the 1meV high voltage microscope in Okasaki, Japan. We found that the spine density was on average 20-30% higher with these thicker specimens, suggesting that indeed, we are underestimating the spine density. To overcome the thickness limitation in calculating spine densities, we are determining the accuracy with which dendritic spines can be counted in Golgi-filled neurons at the light microscopic level by using correlated 3D light and electron microscopic examination. A through-focus series of images was obtained at the light microscopic level of Golgi-impregnated dendrites. Using image deconvolution, a 3D volume was produced and the individual spines segmented and counted. The dendrite was then embedded for electron microscopy, and portions of the dendrites were reconstructed using electron tomography. We have performed spine counts on two correlated volumes thus far and have found 10-20% higher spine densities in the electron microscopic volumes, mostly due to the difficulty in resolving overlapping spines in the light microscope. We are hoping to be able to derive some scaling factors that can be used to correct light microscopic estimates so that these types of analyses c an be performed at the light microscopic level. Another structure that we have been working on is the basket cell "pinceau" in the cerebellar cortex. The pinceau is formed by the axons of basket cells which descend from the molecular layer and completely encase the Purkinje cell axon initial segment and cell body. Despite the impressive size of this axonal ramification and its close association with the Purkinje cell, virtually no synapses are formed on the axonal initial segment. We are performing 3-dimensional reconstructions on this structure to gain some understanding of the relationship of individual axons in the pinceau to the Purkinje cell and to each other. These reconstructions will be used by modelers such as Jim Bower at Cal. Tech. to investigate the possible function of this mysterious and complex structure. We have reconstructed a large extent of the pinceau from 3 Purkinje neurons using relatively low magnification micrographs and have reconstructed several additional basket cell fiber terminations at high er magnification. This work was presented at the Society for Neurosciences meeting this past year. Simultaneous with the structural work, we are investigating the distribution of various macromolecules that are concentrated in the pinceau. We are interested in developing methods for representing the locations of various ion channels (in particular potassium channels), membrane proteins (e.g. PSD-95) and intracellular calcium regulatory proteins in reconstructions in a form useful to modelers and other investigators. We have recently received funding for the creation of a cell-centered database for this type of data.

technology /technique; infection; proteins; computers; biomedical resource; biological products;

The objective of this core TR&D project is to improve methods for the selective staining of neurons for correlative light and electron microscopy and to develop methods that will help researchers to visualize and quantify neuronal shape and connectivity at EM resolution. Our efforts in this area have been driven by our collaborative efforts with Drs. Charles Wilson, Gordon Arbuthnott and Cali Ingham on obtaining quantitative information on structural changes in striatal dendrites and dendritic spines associated with partial deafferentation. Briefly, we have been using electron microscopic tomography to obtain volume reconstructions of spiny dendrites in both normal neostriatum and in neostriatum in which the dopaminergic input has been lesioned chemically. Spiny dendrites receive the bulk of dopaminergic input within the neostriatum and it was observed at the light microscopic level that loss of this input may lead to both a loss of spines and a change in their morphology. The pilot portion of this project which involved acquiring tomographic reconstructions and quantitative measurements from 20 dendrites has been completed. These 20 dendrites came from 4 animals from both the lesioned and unlesioned neostriata. This population of dendrites yielded length, surface area and volume measurements from over 200 dendritic spines. Two problems were encountered: 1) because of the large variability within each group, we have determined that it will be necessary to obtain additional reconstructions from several more animals per group; 2) it appeared that the spine densities that we were finding were lower than those reported by serial sectioning analysis. Because we were limited to a section thickness of about 2 5m at 400 keV, many spines were likely being cut off of the dendrite at the top and bottom of the section. We performed tomographic reconstructions of spiny dendrites contained within 3-4 5m thick sections from tilt series acquired with the 1meV high voltage microscope in Okasaki, Japan. We found that the spine density was on average 20-30% higher with these thicker specimens, suggesting that indeed, we are underestimating the spine density. To overcome the thickness limitation in calculating spine densities, we are determining the accuracy with which dendritic spines can be counted in Golgi-filled neurons at the light microscopic level by using correlated 3D light and electron microscopic examination. A through-focus series of images was obtained at the light microscopic level of Golgi-impregnated dendrites. Using image deconvolution, a 3D volume was produced and the individual spines segmented and counted. The dendrite was then embedded for electron microscopy, and portions of the dendrites were reconstructed using electron tomography. We have performed spine counts on two correlated volumes thus far and have found 10-20% higher spine densities in the electron microscopic volumes, mostly due to the difficulty in resolving overlapping spines in the light microscope. We are hoping to be able to derive some scaling factors that can be used to correct light microscopic estimates so that these types of analyses c an be performed at the light microscopic level. Another structure that we have been working on is the basket cell "pinceau" in the cerebellar cortex. The pinceau is formed by the axons of basket cells which descend from the molecular layer and completely encase the Purkinje cell axon initial segment and cell body. Despite the impressive size of this axonal ramification and its close association with the Purkinje cell, virtually no synapses are formed on the axonal initial segment. We are performing 3-dimensional reconstructions on this structure to gain some understanding of the relationship of individual axons in the pinceau to the Purkinje cell and to each other. These reconstructions will be used by modelers such as Jim Bower at Cal. Tech. to investigate the possible function of this mysterious and complex structure. We have reconstructed a large extent of the pinceau from 3 Purkinje neurons using relatively low magnification micrographs and have reconstructed several additional basket cell fiber terminations at high er magnification. This work was presented at the Society for Neurosciences meeting this past year. Simultaneous with the structural work, we are investigating the distribution of various macromolecules that are concentrated in the pinceau. We are interested in developing methods for representing the locations of various ion channels (in particular potassium channels), membrane proteins (e.g. PSD-95) and intracellular calcium regulatory proteins in reconstructions in a form useful to modelers and other investigators. We have recently received funding for the creation of a cell-centered database for this type of data.

Microscopy is developed to the state where it can complement X-ray crystallography and multi-dimensional NMR by determining the three- dimensional localization of labeled proteins and nucleic acids in cells and subcellular proteins. Especially powerful is correlated light and electron microscopy in which labeled proteins can be found first at the subcellular or organellar level using laser scanning confocal and multi- photon microscopies and after additional specimen preparation, these labels can be visualized at high resolution with an intermediate high- voltage electron microscope (IVEM). The purpose of the Microscopy ore is to provide excellent facilities and the expertise to make it possible for all members of this program to apply state-of the-art microscopy techniques with no need for prior experience in microscopy methods. For suitable imaging studies the Microscopy Core will have access to the extensive facilities and highly knowledgeable staff of the National Center for Microscopy and Imaging Research at San Diego (NCMIR). Unique capabilities developed or improved at NCMIR include: (1) Fluorescence photo-oxidation, which has proven to be a valuable tool for studying the three-dimensional (3-D) organization of cellular constituents by confocal microscopy and intermediate high-voltage electron microscopy (2) Electron tomography, which can explore the 3 D distributions of immunolabeled components of kinase anchoring proteins and associated supramacromolecular and cellular components, (3) Multi-labeling (sometime called multi-color) immuno-confocal light microscopy to localize two or more molecular species using fluorophores with different excitation and/or emission wavelengths and (4) Several software packages which are critical for the visualization and interpretation of structural information derived from microscope images.

Ensembles of distributed communication, computation, and storage resources, also known as "Computational Grids", are emerging as a critical platform for high-performance computing. Grids are used effectively to support runs of distributed applications at a large enough scale to provide new disciplinary results to their developers. Researchers in almost every field of science and engineering are particularly interested in a class of applications particularly well suited to the Grid, scientific simulations where many parameterized instances of a give computation are performed. The development of accessible, efficient, fault-tolerant Grid-enabled versions of simulation software will enable disciplinary scientists to investigate wide-ranging scenarios and to obtain new results orders of magnitude faster than is currently possible.

Many scientists would like to view large-scale simulations as software instruments that support some level of user interaction. This would be effective only if simulations can be deployed easily and controlled dynamically, i.e. if the computation can be steered. A traditional scenario is for the user to steer the simulation based on partial results that evolve continuously during execution. The partial results provide an increasingly refined indicator of the final results of the simulation and can be used to identify mid-execution which parameter sets are most promising. Given the potential of wide-area, federated Grid environments to deliver the aggregate computational power, data storage and dissemination facilities for large-scale simulations, and the need for scientists to steer such computations, it is increasingly important to develop performance-efficient and steerable software instruments that target the Grid. This project will address the significant computer science problems that arise from the need to support steerable scientific simulations in large-scale Grid environments.

The project will design, develop, and prototype a virtual software instrument as a vehicle for designing and prototyping scalable, steerable scientific simulations for the Grid. It will use a Monte Carlo simulation program, MCell, as a prototype application for development and testing of the virtual instrument. The virtual instrument itself will consist of a set of software modules, libraries, interfaces, and steering-sensitive scheduling algorithms. The project will have impact on both the computer science and disciplinary science communities. It will foster new research in computer science through the development of event models, performance models, data management strategies, and adaptive scheduling and steering algorithms. It will also enable domain scientists to obtain new results in neuroscience.

0140644
Wysocki
This award provided support for a workshop entitled "Analytical Instrumentation for the New Millennium - Biological Sciences" to be held Dec 2-5, 2001 in Tucson, Arizona. The workshop will bring together scientists with broad interests in biological measurements, including both the biologists who need the measurements and the instrument developers who design and build instrumentation. The format of the workshop will include highlight presentations by cutting-edge researchers who will conclude with a brief description of goals for the future and the major obstacles to be faced in meeting those goals. These presentations will be followed by breakout sessions to critique/evaluate the proposed goals and obstacles. The expected outcomes of the workshop are a report to be written jointly by the breakout leaders and organizers and presentations to be made at national meetings. This workshop is jointly supported by the Directorate for Biological Sciences and the Directorate for Mathematical and Physical Sciences.

[unreadable] DESCRIPTION (provided by applicant): This proposal outlines specific enhancements to the process of very large-field 3D laser-scanning light microscopy and electron tomography to increase the throughput of generating structural data to be assembled into multi-resolution "visible" cells for computational neuroscience. For each processing stream, this proposal identifies data, computation time, and labor intensive tasks, and it outlines solutions enhanced by grid resources for increasing end-to-end performance, accelerating computation, enhancing visualization, and archiving and managing data. To foster modeling studies, this project also outlines the development of software solutions for improving the accuracy and continuity of rendered surfaces, aligning "serial" volumes, and isolating the areas containing specific densities or types of "effecter molecules" (post-synaptic densities, channels, proteins, etc.) and functional components derived from literature, light and/or EM studies. Each processing stream will be further integrated into a web-based application environment where users can securely and collaboratively perform each operation from data acquisition to database deposition, and provides an end-to-end solution for automatically scheduling instruments, performing the necessary data movements, coordinating and scheduling the data processing stages, acquiring specialized visualization resources, and managing and staging distributed data. [unreadable] [unreadable] The data produced by both imaging processes is of great value to researchers and students using simulation programs like MCell, Neuron, and GENESIS. The incorporation of realistic structure into the computational models increases the accuracy of the simulation output as well as the utility and validity of the results. Ultimately, the use of accurate derived "visible" neurons will create an unprecedented opportunity to probe the effects of structure in the behavior of the nervous system. Moreover, the technologies to richly integrate instruments, grid infrastructure and services, automated data management, and user-friendly web-based collaborative control will be broadly extensible to other scientific domains and will continue to enable and enrich opportunities for education and outreach. [unreadable] [unreadable]

This grant supports the purchase of a low temperature scanning tunneling microscope (STM) that will be used for investigations of defects and bonding sites in electronic and sensor materials. The low temperature STM provides a zero drift environment in which isolated molecules do not diffuse. In this quiet, stationary environment, the local electronic structure of single defects or bonding sites can be determined using current-voltage (dI/dV) measurements. The specific systems that will be studied with this instrument include: (a) defects at the semiconductor/gate oxide interface; (b) polysilole-based nanowire sensors; (c) halogen reactions with aluminum; (d) mixed, asymmetric metal phthalocyanine-based chemical sensors; and (e) fabrication of ordered arrays of single macromolecular assemblies. With the advent of commercial software (VASP) for calculating the electronic structures of molecules on surfaces, low temperature STM and STS studies can be directly compared to both simulated STM images and the partial density of states on single atoms. This comparison provides critical insights into the control of electronic structure on surfaces.

This instrument will have impact on a broad audience at the university. (a) The San Diego Fellowship program for entering graduate students: UCSD is donating 3 months of support for 4 entering graduate students. These fellowships will be used to support graduate students to increase the diversity of the chemistry and physics graduate programs. (b) Undergraduate research: Undergraduate students will be involved in the proposed research via the Howard Hughes Undergraduate Enrichment Program (HHUEP) and the UCSD Office of Academic Enrichment. (c) UCSD-TV and webcasts: Lectures that are appropriate for high school and college students will be recorded for both broadcast and webcast by the UCSD-TV education outreach program. (d) Teacher training: The funds support one high school science teacher per summer to work in a research laboratory at UCSD. The teacher will have the opportunity to learn how to use the telemetry system developed at the UCSD National Center for Microscopy and Imaging Research (NCMIR). The NCMIR facilities house an electron microscope that can be remotely controlled via the web, enabling teachers and students to use the SEM directly from their classrooms. (e) Industrial collaborations: The low temperature STM will be used in collaborative research projects that focus on device and sensor development and involve Motorola, Microsense, and IBM.

ABSTRACT NOT PROVIDED

Abstract not provided.

bioimaging /biomedical imaging; technology /technique development

bioimaging /biomedical imaging; technology /technique development

Abstract not provided.

DESCRIPTION (provided by applicant): The Biomedical Informatics Research Network (BIRN) is a recently-launched initiative to foster large-scale collaborations in biomedical science by utilizing the capabilities of the emerging national cyberinfrastructure (high speed networks, distributed high-performance computing and the necessary software integrative capabilities). Scientists at the San Diego Supercomputer Center and the School of Medicine at the University of California San Diego (UCSD) serve as the BIRN Coordinating Center (BIRN CC) for this large project. The BIRN CC serves the critical task of developing, deploying and maintaining key infrastructure components, including high bandwidth connectivity via Internet 2, Grid-based security, file management and computational services, techniques to federate databases and shared visualization and analysis environments. In its initial phase, the BIRN involves a consortium of 12 universities participating in test bed projects centered around brain imaging of human neurological disorders and associated animal models. Groups are working on large scale, cross-institutional imaging studies on Alzheimer's disease, depression and schizophrenia using structural and function MRI. Others are studying animal models relevant to the study of multiple sclerosis, attention deficit disorder, Parkinson's disease and brain cancer using MRI, whole brain histology and high-resolution light and electron microscopy. The BIRN CC provides the focal point for interactions among the BIRN partner sites, both coordinating and catalyzing the successful collaboration of a large group of highly talented scientists. This proposal describes continuation plans for the BIRN CC for the next five years. In the next phase, the BIRN CC will continue to support and enhance the existing infrastructure while fostering and facilitating collaboration among the BIRN participants. Development and implementation of new technologies will continue to be tightly coupled to the requirements of the various test bed projects and will be informed by lessons learned during the initial phase. At the same time, the BIRN infrastructure will continue to evolve in accordance with emerging Grid standards, anticipating the longevity and future expansion of the BIRN.

This core aims to provide the necessary support and services to enable resources (computation and data), users, and applications to interact seamlessly on the Grid. This activity will incorporate Grid services to provide seamless authentication and access to remote computation and data resources by taking advantage of a common software infrastructure being deployed by the Biomedical Informatics Research Network (BIRN;). Leveraging the BIRN infrastructure will also enable NAMIC to be able to seamlessly interoperate with other grid environments, such as the Distributed Terascale Facility (DTF). This activity will enable Grid computational resources to be utilized by the NAMIC project groups to handle applications and algorithms being developed as part of NAMIC (e.g., see the description of fast implementations of PDEs and deformable models in Core 1:5) and other activities in which the data constraints are beyond what is easily managed in workstation-level environments. Ensuring the compatibility between BIRN and NAMIC will allow for the seamless interoperation of biomedical codes within both these environments.

ABSTRACT NOT PROVIDED

Architecture; CRISP; Cells; Client; Computer Retrieval of Information on Scientific Projects Database; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Engineering / Architecture; Funding; Grant; Institution; Internet; Investigators; Java; Microscopy; NIH; National Institutes of Health; National Institutes of Health (U.S.); Process; Research; Research Personnel; Research Resources; Researchers; Resources; Services; Source; Standards; Standards of Weights and Measures; System; System, LOINC Axis 4; Systems Integration; Technology; United States National Institutes of Health; Update; WWW; clinical data repository; clinical data warehouse; data repository; detector; instrument; relational database; web; world wide web

DESCRIPTION (provided by applicant): The Imaging Core will provide resources and technologies to assess the effects of Superfund toxicants on cultured cells, tissues, and whole animals at the cellular and subcellular levels, in vivo and in vitro, by bioluminescence, confocal, and multiphoton laser light microscopies and also provide capabilities for subcellular to supramolecular imaging by advanced 3-dimensional electron microscopy. This core will leverage on the advanced instrumentation of the NCRR-supported National Center for Microscopy and Imaging Research (NCMTR), which Dr. Ellisman directs at UC San Diego. Working closely with Dr. Ellisman, Dr. Tsien will oversee the development and application of new fluorescence, biarsenical reporter, and tetracysteine-based methods for the analysis of gene and protein expression and detection both in cultured cells and tissues. The core is fully equipped with state-of-the-art instrumentation and is staffed with experienced scientists who shall assist in the development and execution of experiments. Personnel of the Imaging Core will provide training and assistance with novel labeling strategies, whole-animal imaging, immunofluorescence, in situ hybridization, EM immunolocalizations, EM tomography, and cryoelectron microscopy interfaced with 2D and 3D biological image analysis.

bioimaging /biomedical imaging; technology /technique development

This award will provide support for qualified undergraduates, graduate students, and postdoctoral researchers to attend the International Congress on Electron Tomography (ICET) in San Diego, CA, from November 5-8, 2006. This congress is the fourth in a series of workshops that was initiated in 1997 to begin to bring together practitioners developing and applying electron microscopic tomography. The meetings have provided a forum for intensive interactions and have catalyzed technical developments and an increased number of users. At this juncture, electron tomography is one of the most rapidly evolving approaches in multiscale 3D microscopy and is being employed for an increasingly broad range of applications, well beyond those envisioned at the time of the first conference.

Electron Tomography is a new research area that produces 3-dimensional quantitative images from electron microscopy. Electron tomography is moving from a specialized experimental technique practiced by a few laboratories to one that is delivering critical new information to cell biologists, structural biologists and neuroscientists. Because electron tomography has been identified as a growth field, it is anticipated that the graduate students and postdoctoral researchers being trained today will go on to become the next generation lead investigators.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 2005 Neuroscience meeting was held November 12-18, 2005 at Washington, D.C. NBCR, NCMIR, BIRN, and SCI participated in the booth activities. Telescience project was featured as a demonstration.

[unreadable] DESCRIPTION (provided by applicant): The International Congress on Electron Tomography will be held at Paradise Point Resort and Conference Center, San Diego, CA, from November 5-8, 2006. This congress is the fourth in a series of workshops that was initiated in 1997 to begin to bring together practitioners developing and applying electron microscopic tomography. The meetings have provided a forum for intensive interactions and have catalyzed technical developments and an increased the number of users. At this juncture, electron tomography is one of the most rapidly evolving approaches in multiscale 3D microscopy and is being employed for an increasingly broad range of applications, well beyond those envisioned at the time of the first conference. Hence we are inviting speakers from a broad range of approaches in order to stimulate a cross-fertilization of ideas. The sole purpose of this grant request is to obtain funds to help defray costs for invited speakers, program committee and session chairs. [unreadable] [unreadable] [unreadable]

This core aims to provide the necessary support and services to enable resources (computation and data), users, and applications to interact seamlessly on the Grid. This activity will incorporate Grid services to provide seamless authentication and access to remote computation and data resources by taking advantage of a common software infrastructure being deployed by the Biomedical Informatics Research Network (BIRN;). Leveraging the BIRN infrastructure will also enable NAMIC to be able to seamlessly interoperate with other grid environments, such as the Distributed Terascale Facility (DTF). This activity will enable Grid computational resources to be utilized by the NAMIC project groups to handle applications and algorithms being developed as part of NAMIC (e.g., see the description of fast implementations of PDEs and deformable models in Core 1:5) and other activities in which the data constraints are beyond what is easily managed in workstation-level environments. Ensuring the compatibility between BIRN and NAMIC will allow for the seamless interoperation of biomedical codes within both these environments.

1. Overall Objectives The goal of the Imaging Core is to assist PPG investigators in the use of novel molecular probes, labeling, microscopy, and image analysis crucial to achieving the aims of the efforts headed by Drs. Taylor, Newton, Scott, King, and Murphy through the use of advanced imaging approaches. These approaches require state-of-the-art instruments, are constantly evolving, and are practiced by highly specialized personnel with years of training. Because of this, it is cost-effective to have a centralized facility. An economy of effort is achieved by sharing the costs for development and knowledge gained across the 5 groups that will use the same or similar probes and/or technologies. 2. Summary of Services The Imaging Core will: provide training and assistance with confocal and multiphoton immunofluorescence experiments including the use of state-of-the-art reporter technologies such as the tetracysteine-based FIAsH and ReAsH labeling systems, kinase reporters for FRET, and quantum dots enable live-cell imaging and large-area mapping of cells and tissues using advanced light-microscopy instruments provide training and analysis for correlated light and electron microscopic immunolocalization studies including the use of quantum dots, tetracysteine-based photooxidation, and cryosectioning techniques for mitochondria, mitochondrial subfractions, cells, and tissues. provide training and enable supramolecular and molecular characterizations by electron microscopy including electron microscope tomography and high-pressure freezing/freeze substitution and cryoelectron microscopy. provide training and access to software programs for 2- and 3-dimensional biological image analysis.

[unreadable] DESCRIPTION (provided by applicant): This proposal requests funds to support the acquisition of a modern, high performance intermediate voltage transmission electron microscope (IVEM) to replace an aging Japanese Electron Optics (JEOL) JEM- 4000EX that has served as a production instrument for automated 3D imaging of thick sections (electron tomography, electron optical sectioning) for nearly 17 years. This instrument will physically supplant the JEM-4000EX at the National Center for Microscopy and Imaging Research (NCMIR) on the campus of the University of California, San Diego. The NCMIR is an NCRR/NIH Biomedical Technology Research Center established in 1989 to develop computer-aided advanced microscopy for acquisition of structural and functional data in the dimensional range of 1 nm3 to 100 um3, a dimensional range referred to as the "meso" or middle scales. This instrument will serve the primary function of enabling the continued support of an increasing base of driving application projects that require tailored performance and functionality in the area of meso-scale, 3D imaging of thick, 1 to 3 um sections of cells and biological tissues. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the previous funding period, we have invested considerable effort and time in merging tetracysteine tags with other reporter systems to generate hybrids with enhanced performance for both LM and EM detection. This report covers the progress we made in: (1) Enhancing the sensitivity and the signal/noise ratio of protein detection with biarsenicals. (2) Detecting nucleic acids and interacting proteins in correlated LM/EM studies. (3) Developing a photo-reversible protein aggregation tag for functional studies. (4) Labeling with biarsenicals after fixation. (5) Developing methods for the simultaneous preservation of fluorescent signals, ultrastructure and antigenicity. (6) Developing multiple labeling strategies using EEL spectroscopy.

Autism; Autism, Early Infantile; Autism, Infantile; Autistic Disorder; Biomedical Informatics Research Network; CRISP; Computer Retrieval of Information on Scientific Projects Database; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Funding; Grant; Information Technology; Institution; Investigators; Kanner's Syndrome; NIH; National Institutes of Health; National Institutes of Health (U.S.); Research; Research Personnel; Research Resources; Researchers; Resources; Source; United States National Institutes of Health; clinical data repository; clinical data warehouse; data repository; relational database

The University of California-San Diego is awarded a grant to create a research coordination network to promote and integrate standards for genomic and metagenomic data and metadata within an international community. The network is based on the existing Genomic Standard Consortium and will be extended under this award to include ecological data standards such as Ecological Metadata Language, biodiversity standards such as Darwin Core, and environmental research programs such as the Global Lake Ecological Observatory Network and Long Term Ecological Research. Genome enabled research has transformed biological sciences for the entire breadth of scales from molecular to ecosystems. Expanding and fully incorporating early consensus building, technical standards and the institutional commitments inspired and implemented by the Genomic Standards Consortium, the creation of this RCN for the Genome Standards Consortium (RCN4GSC) will ensure a worldwide, community driven process for establishing standardized mechanisms for the electronic capture of genomic data and for obtaining willingness to participate; these are essential to ensure effective use of the sequence and associated data, to provide access for all biologists to all of the information, and to create interdisciplinary opportunities for discovery.
Intense collaborations around the maturation of GCDML, Genome Rosetta Stone and the Genome Catalogue will be an early focus. Productivity will be sustained year round by routine A.V. and web-enabled interactions, including small working group sessions and collaborations via the RCN's extant web hub at http://gensc.org. The activities will expand ongoing coordination required for implementation, including the Genome Catalog, GCDML, Genome Rosetta Stone software, Habitat-Lite and Environmental Ontology, and the eJournal "Standards in Genomic Sciences". The RCN will also sustain and build upon the very active, ongoing, two-way exchange between LTER and GSC, interactions arising from common goals. The RCN4GSC will be an open organization, welcoming inspired biologists with a commitment to community service, and will increase participation from Asia and the USA. Coordination with professional societies and communication to federal and foundation stakeholders will be among strategies for expanding participation; these will be managed by the Communications Committee, who will focus on enlisting further diversity in participation and in reaching out to young biologists.

DESCRIPTION (provided by applicant): The Cell Centered Database (CCDB) was released in 2002 as a web-accessible database for high resolution 2D, 3D and 4D data derived from light and electron microscopic imaging, including correlated light and electron microscopy. The CCDB encompasses and hosts many types of data in the dimensional range lying between gross morphology and macromolecular structure - the so-called "mesoscale". These data and the software infrastructure that hosts and serves them are unique in scope, presenting to the community data sets laden with information which is technically difficult to obtain but very rich in content, making them particularly valuable for developing computational models of structures and physiological processes that occur in cells and tissues. Envisioned as a grid and/or web-based federated database system from the beginning of the project in 1998, the CCDB pioneered the production of a distributed, connectable repository system for managing and sharing data for a growing research community of microscopists. This proposal aims to provide a stable base of support for a growing community of users of the Cell Centered Database, by refining core services and establishing the facilities needed for CCDB data and software to be openly shared, modified, extended, edited and documented by the expanding participant group. The proposed work is organized into three specific aims: Aim 1. Open Source CCDB: The current system infrastructure will be translated from a centrally supported research product with defined community requirements to an industrymodeled open source project tailored for community wide contribution, customization and refinement. Aim 2: CDB Core Services: The principal components (schema, core services) of the CCDB will be packaged for community distribution and refinement via open source distribution model. Documentaiton will be provided along with specifications for expanded "community contribution" to a repository/collection of core CCDB services. Aim 3: CCDB Client Tools: Existing CCDB client tools will be packaged and distributed for use by the end-user community.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ADVANCED TECHNOLOGIES FOR THE PROCESSING AND ANALYSIS OF MULTI-RESOLUTION, LIGHT AND ELECTRON MICROSCOPY DATA The aim of this technological research and development activity is to provide NCMIR researchers with an arsenal of reconstruction, visualization, and analysis tools specifically tailored to mesoscale investigations involving massive multi-modal 3D and 4D data. We will continue to follow a successful strategy of utilizing community tools where possible and partnering with leaders in image analysis and visualization. We direct our own development efforts to augmenting these tools where necessary and developing novel analysis approaches around the types of data generated by NCMIR and our collaborators. Specific work will involve enhancement of reconstruction algorithms for new imaging modes and specimen geometries;improvement of tools and approaches for semi-automated and manual segmentation of electron microscopic data;utilization of electron microscopic data to inform light microscopic analyses;and development of tools and platforms for visualization of large, multidimensional data sets. Where necessary, we will work with tool providers to adapt their tools to handle the massive data sets generated through new imaging modes and instrumentation described in core projects 4A2.1 and 4A2.2. The resulting products will be integrated within NCMIR's advanced cyberinfrastructure, and will be specifically designed to provide an integrated environment for diverse tools and imaging workflows that will improve our ability to manage large and complex multi-scale datasets. Each activity will also be brought into alignment with our established informatics infrastructure to be described in core project 4A3.2.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DEVELOPMENT AND APPLICATIONS OF NEW PROBES AND METHODS FOR CORRELATED LIGHT AND ELECTRON MICROSCOPY, LIVE IMAGING, AND FUNCTIONAL ANALYSIS In this subproject, our efforts are directed at further improving and refining probes and methodologies to enable correlated light and electron microscopy. These include the highly successful tetracysteine tag/biarsenical labeling technology acknowledged to be a powerful and versatile method for correlated time-lapse light and electron microscopy, and miniSOG, a new genetically encodable fluorescent protein designed specifically for correlated light and high- sensitivity electron microscopic localization of proteins and macromolecular complexes. We are also continuing our efforts to develop additional new probes for correlated microscopies.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DEVELOPMENT OF ADVANCED MULTIDIMENSIONAL MICROSCOPY TECHNOLOGIES FOR MESOSCALE IMAGING The correlation of data acquired across scales and technologies is undertaken to reveal relationships among subcellular components, the detailed organization of constituent protein building blocks, and the functional role of each in normal and abnormal biological systems. The continued development and deployment of enhancements to the resource imaging instrumentation is critical to the improvement and further acceleration of our capability to investigate biomedical research questions, especially those that offer insight into structural and functional relationships across scales. To this end, we are maximizing the impact of our recently deployed 300KV energy filtering TEM/STEM instrument, integrating its advanced production capabilities and commissioning new imaging modes enabled by NCMIR-specified customizations. This team is also refining our existing IVEM and ultra-wide-field light microscopy resources to solidify the diverse techniques performed at this unique facility. We continue to develop and integrate new instrumentation technologies to bridge resolution gaps and augment our ability to conduct seamless multi-scale, multi-resolution microscopy--in particular serial blockface scanning electron microscopy. For each instrument, we focus on TR&D toincrease imaging throughput and fidelity and extend the fields-of-view achievable at high resolution. These activities balance engineering development with collaborative use and connect efforts to exploit new labeling and specimen-development technologies as well as image analysis and database utilization.

DESCRIPTION (provided by applicant): This project will establish a modern environmental SEM platform and automated ultramicrotome system for serial block-face scanning electron microscopy (SBFSEM) at the National Center for Microscopy and Imaging Research. This unique resource will enable researchers to more effectively bridge the technology gap between correlated ultra-high resolution light microscopy and wide field electron microscopic imaging by providing a high throughput system for the automated reconstruction of 3D tissue structure over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles (Denk and Horstmann, 2004). This instrument will further propel pioneering activities to develop and apply emergent probe technologies for correlated light and electron microscopy of biological specimens. The shared use of this instrument will be driven by leading research projects that each require the specialized capabilities of the 3View system to advance multiscale studies ranging from cardiac mechanics, cancer (glioblastomas), mapping of neuronal circuitry, and the development of new probe chemistry for multiscale imaging.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DEVELOPMENT AND REFINEMENT OF HYBRID SPECIMEN PREPARTION METHODS FOR THE CREATION OF SPECIMEN GEOMETRIES MORE SUITABLE FOR 3-D IMAGING. The goal of this subproject is to extend resolution in electron tomography of resin embedded material by overcoming tilt-induced thickness increases inherent to single-axis, double-axis and conical tilt tomography of thick and thin sections by the creation of specimens with more suitable geometry. Our efforts are directed towards devising methods for creating specimens with new shapes to work with precise total rotation tilting specimen holders being developed at the NCMIR. These methods will increase the resolution and fidelity of the tomographic reconstructions from specimens developed using methods described in aims 4A1.1 and 4A1.2.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DEVELOPMENT OF HIGH-PERFORMANCE DIGITAL IMAGING SYSTEMS FOR MESOSCALE IVEM APPLICATIONS. This effort focuses on research, and development to improve technologies for digital imaging as a replacement for film. This is an enabling technology for high-throughput, wide-field mesoscale data acquisition and computer-integrated microscopy. Specifically, we continue to refine the hardware and software employed with an 8k x 8k ultra-wide-field lens-coupled CCD camera system for production use in microscopy. After significant work in the development of direct detection devices (DDD), we continue to explore and develop new circuitry and radiation-hardened devices for use in TEM. Similarly, we continue engineering and software development activities to integrate emerging DDD prototypes for routine data acquisition on our resource IVEMs. As a driver for the continued development and refinement of these systems, we cultivate mesoscale microscopy applications that exploit the performance characteristics of these devices and apply them to collaborative research. These goals are organized into the following specific aims: first, in order to accommodate the need for ultra-wide-field high-throughput image acquisition, we continued to refine the control software and automation of the NCMIR 8k x 8k lens-coupled CCD camera system commissioned on our JEM4000#2, and the 4k x 4k lens-coupled CCD camera system commissioned on our JEM4000#1. Second, to more accurately record information about biological specimens, we continued to refine technologies to directly record images using radiation-hardened CMOS cameras invented at NCMIR, and to field these DDDs on a variety of key resource platforms. Third, we utilized the development and integrations knowledge obtained in fielding the DDD and the lens-coupled systems to perform a major detector upgrade to the Titan FEI 80-300 300keV STEM/TEM.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. GENERALIZED INSTRUMENT CONTROL AND AUTOMATION SOFTWARE DEVELOPMENT This subproject's general objective is to enhance our ability to acquire data from light and electron microscopes (LM/EM) at the facility as well as at our allied resources, by developing, refining, and applying software technologies to integrate instrumentation, computing, and informatics technologies. Specifically, we aim to increase data acquisition and analysis throughput and increase the quality and extent of information logged about data collection and instrument configuration by leveraging on and expanding our scalable generalized telemicroscopy system (Molina et al., 2005). This system is designed to integrate, organize, and unify the control of imaging instruments and accessories (detectors, stages, etc.) and securely couple their use to advanced cyberinfrastructure, including resources for data management and storage, high-performance computing, and informatics. This software architecture is being extended to integrate instruments of unique value to the mesoscale imaging aims of the Center, in particular the incoming FEI Titan STEM/TEM. Data-driven and computationally enhanced automation schemes will be developed along with supporting services to organize and coordinate the increasingly large data volumes delivered from each instrument. Unifying interfaces are also be developed to simplify how manual, automated, basic, and advanced features are presented to the biomedical researcher. The steps necessary for achievement of these goals leverage, extend, and refine the GTS to integrate key multimodal imaging instruments (local and remote) to enable advanced control and automation for high-throughput mesoscale imaging;increasing our data handling performance and refining session state reporting through refinements to the capabilities of the GTS. Lastly, we shall develop a software prototyping environment to streamline, promote, and simplify the development of novel instrument-based applications, including automation and data-driven feedback schemes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. TECH R&D CORE SUPPORT FOR AIDS RESEARCH

DESCRIPTION (provided by applicant): Advanced instrumentation and cellular imaging techniques using high-throughput 3D electron microscopy are driving a new revolution in the exploration of complex biological systems by providing near seamless views across multiple scales of resolution. These datasets provide the necessary breadth and depth to analyze multicellular, cellular, and subcelluar structure across large swathes of neural tissue. While these new imaging procedures are generating extremely large datasets of enormous value, the quantities are such that no single user or even laboratory team can possibly analyze the full content of their own imaging activities through traditional means. To address this challenge, we propose to further develop and refine a prototype hybrid system for high-throughput segmentation of large neuropil datasets that: 1) advances automatic algorithms for segmentation of cellular and sub-cellular structures using machine learning techniques; 2) couples these techniques to a scalable and flexible process or tool suite allowing multiple users to simultaneously review, edit and curate the results of these automatic approaches; and, 3) builds a knowledge base of training data guiding and improving automated processing. This system will allow project scientists to select areas of interest, execute automatic segmentation algorithms, and distribute workload, curate data, and deposit final results into the Cell Centered Database (Martone et al. 2008) via accessible web-interfaces.

Contemporary information technology is ever more central to science and society in the midst of the deluge of complex data. The impact on bioscience is notable, where the pace of production and the data complexity means that a large amount of data is often not adequately analyzed by the data producers, yet researchers expect rapid dissemination of such types of data. To ensure effective impact, a solution promising to be transformational is to open "big data" analysis to the broader community. An avenue is provided by modern IT and the explosive growth and democratizing impact of the Internet, which, following the digitization of information and communication, has changed the pace of information exchange and opens up new channels for disseminating data and for engaging disparate disciplines in extended, productive collaborations. The result of this will be a platform with a customized pre-build interface that will significantly reduce the downside of the form-based data input approach. The input interface will be small, easy to use and readily accepted by users but still relevant to what a user might want to input. The interface will provide strong search ability to the controlled vocabulary and provide users with this information through "input hint", dropdown lists or auto-completion, according to what is most efficient for the specific extension and provides an effective, readily followed and precise process. Sustaining the free text input section will provide users with maximum freedom of data input. By enabling community collaboration via Web access and implementing a database resource linked with the knowledge collection interface together with free text entry format, this system will provide a venue for researchers among many communities, including those located at non-research intensive universities, community colleges and minority-serving institutions, in this Nation and worldwide, to contribute their insight to experimental research observations that currently requires expensive specialized equipment only available in a few centers around the world.

3-Dimensional; Age; Algorithms; Area; base; Binding; Biochemical; Biological; Biological Models; biological systems; calcium indicator; Cell physiology; Cells; cellular imaging; Chromatography; chromophore; Communities; Complex; Complex Mixtures; computerized tools; Data; Data Analyses; Databases; Dependence; Detection; Development; Disease; Dyes; Electron energy loss spectroscopy; Electron Microscopy; electron tomography; Electrons; Exposure to; expression vector; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; fluorophore; Freezing; Gel; Gene Expression Profiling; Image; Image Analysis; Imagery; In Situ; In Vitro; in vivo; Informatics; Injection of therapeutic agent; Instruction; instrumentation; Label; Lead; Life; Light; light microscopy; Maps; mass spectrometer; Mass Spectrum Analysis; Measurement; Metals; Microscope; Microscopic; Modeling; Molecular; Monitor; nanoparticle; Nucleic Acids; Oxygen; Peptides; Phosphorylation Site; Physiological; Physiology; phytochelatin; Plants; Play; Post-Translational Protein Processing; pressure; protein complex; protein expression; Proteins; Proteomics; Reagent; Relative (related person); remediation; Reporter; Research Personnel; Research Support; Resolution; Role; Scanning; Services; Singlet Oxygen; Solutions; Specific qualifier value; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Superfund; Technology; technology development; Time; Tissues; tomography; Toxic Environmental Substances; toxicant; Toxicant exposure; Training; Translational Research; transmission process;

DESCRIPTION (provided by applicant): We recently made the surprising discovery that mitochondria from retinal ganglion cell axons are passed onto neighboring astrocytes for degradation in the optic nerve head of normal mice. We present evidence that this pathway is conserved in species ranging from frogs to primates, and that it likely occurs widely throughout the mouse nervous system. Here, we propose to test the hypothesis that mitochondria transcellular degradation, or transmitophagy, is a common mechanism in the central nervous system through which normally-aged and stress-damaged neuronal mitochondria can be degraded. In addition, we will test the hypothesis that gamma- synuclein from axons plays a critical role in determining whether axonal mitochondria are degraded by the previously known cell-autonomous autophagy pathway, or rather by this newly discovered transcellular degradation pathway. The proposed studies will employ two vertebrate animal models, Mus musculus and Xenopus laevis, taking advantages of experimental approaches optimal in each, including live imaging in the latter. Both well established and highly novel methodologies, including new transgenic reagents of wide applicability, will be used to determine precisely where transmitophagy occurs, its cardinal structural features, and how long it takes. In addition, as a means to test the current working model, experiments will probe the cellular machinery underlying transmitophagy. Two accomplished laboratories with highly complementary expertise and technologies will come together to study transmitophagy at molecular and structural levels far beyond the capabilities of either laboratory, by using experimental designs that fully exploit the benefits of the collaborative approach. The proposed studies will comprehensively describe, and to some extent probe the mechanism of, a previously unknown degradation pathway in the central nervous system. Given the importance of both degradation pathways and mitochondria to nearly every neurodegenerative disorder, it is highly likely that transmitophagy will be found i the future to be a critically important pathway in a variety of neurodegenerative disorders. Thus, though the proposed experimental design aims to understand the basic cellular and molecular mechanisms involved in transmitophagy and is not directed at any disease, these studies are likely to a have a major impact on human health in the long-term.

DESCRIPTION (provided by applicant): This proposal outlines plans for continued development and maintenance of the OpenCCDB software suite, a set of tools for web-based management, visualization and annotation of large multidimensional light and electron microscopic imaging data sets. Modern analytical and molecularly-based biomedical investigations increasingly draw upon multiscale and multimodal cell-centered imaging to gain insight into spatially constrained interactions of molecular machinery of cells and tissues. The OpenCCDB was developed under the auspices of the Cell Centered Database (CCDB0 project, an on-line repository and platform for sharing microscopic imaging data of cells and tissues. The CCDB project was predicated on developing solutions for federating access to very large 3D imaging data sets, produced by diverse instrumentations and maintained across diverse technological platforms. The CCDB model is built on flexible and expandable data stores that can be fielded globally but accessed through CCDB's central metadata system and linked through data federations like the Neuroscience Information Framework (http://neuinfo.org). During the last funding period, the American Society for Cell Biology launched its own data repository, with support from ARRA funding. The Cell: An Image Library currently contains > 37,000 exemplary imaging data sets from light and electron microscopy, all available through an elegant cell-centered user interface. To provide a more unified and sustainable resource for research and education, the ASCB Council and Executive Committee voted in 2011 to partner their effort with the group at UCSD, combining their curatorial and interface design expertise and accomplishments with UCSD's database and software development expertise and accomplishments. In so doing, ASCB transitioned control of the development and future sustainability of their user portal environment, The Cell:An Image Library to the CCDB team at UCSD. Immediately, this new alliance applied CCDB's open source software framework, OpenCCDB, to the task of extending the functionality of The Cell: An Image Library system, adding new capability to upload and explore massive datasets, facilitate data sharing, and completely federate its underlying database with the CCDB. The resultant integrated environment offers the scientific community a more comprehensive solution to propel cell-centric biomedical research and education than that offered previously. In this proposal, we outline aims to develop and deploy additional personalized data management and annotation tools within the context of The Cell: An Image Library, by combining the power and tools of a social networking site with specialized tools for web-based management, sharing, annotation and federation of very large microscopic imaging data sets. PUBLIC HEALTH RELEVANCE: This renewal application outlines plans to harden and further develop the code base of the Open CCDB software platform in support of the new joint cell-centered microscopy resource established through the merging of the Cell Image Library and the Cell Centered Database.

Biodiversity comprises all variations of life at all levels of biological organization, most of which arise from genomic diversity. As genomic technologies become available across the biological sciences, a full characterization of biodiversity demands a full characterization of genomes. Similarly, data synthesis across the full range of biodiversity research domains demands development, implementation, integration and harmonization of data exchange standards. Such interoperable informatics would be transformational for our understanding of biology, with consequent impact on environmental and conservation policy. Adding to the transformational potential is the fact that the microbial world represents half of the world's biomass and nearly all of its biodiversity, yet is still effectively invisible and intractable to traditional biodiversity research. Metagenomic data are not amenable to the concepts, standards, semantics, and methods of traditional eukaryotic biodiversity, and therefore, require an alternate informatics framework.

The EAGER will transform the collaborations between two previously separate research communities: the informaticists of the traditional biodiversity community, who employ the Darwin Core (DwC) as a standard, and the informaticists of the Genomic Standards Consortium (GSC), who have developed the Minimal Information about any Sequence (MIxS) standard for genomics, metagenomics and marker genes. Together, these groups will engage in a unified informatics effort to develop three layers of interoperability. The EAGER will harmonize the observational (DwC) and genomic (MIxS) standards, building on a community dialogue and interdisciplinary networking hosted and established under an NSF Research Coordination Network. Standards interoperability is the basis for the next two layers. Syntactic interoperability (in the context of Internet APIs and a database Reference Model) will be supported. The EAGER will assemble experts from the two communities to (a) devise a database Reference Model that integrates the DwC and GSC MIxS standards; and (b) for effective data management, create specific implementations for different database platforms to foster adoption. The practical implementation of the reference model on/for different database systems will allow, for the first time, systematic comparative testing of technical performance and of use cases (e.g., which implementation best serves which complex data query). The EAGER will create task groups to establish the infrastructure for managing ontologies, and to construct a reference model on the purely semantic level in order to fuse the two worlds of data standards, both of which are advanced enough to engage in useful interoperability.

In developing an interdisciplinary information infrastructure to achieve data interoperability across domains, this EAGER would advance understanding of complex environmental phenomena and, thereby, inform future policy decisions. Indeed, by leading to an informatics standards platform to conceive a novel conceptual and theoretical framework for the world of microbial ?dark matter,? the EAGER would have a transformational impact beyond science.

This EAGER award to the University of California - San Diego, explores innovations to The Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis, (CAMERA, http://camera.calit2.net/). CAMERA is a widely used infrastructure that provides a single software system for depositing, locating, analyzing, visualization and sharing data about microbial biology. A heavily utilized and core component of CAMERA is the workflow driven computational system for data analysis. The award anticipates analysis needed for next generation sequence (NGS) data. BLASTx Work flows using CAMERA's Refseq Protein database) will require nearly 2.5 Million core hours to complete analysis of a typical NGS data set, making it impractical and costly to rely on a single computational resource for an entire workflow. The PI will prototype novel heuristic-based dynamic resource scheduling approaches to allowing distribution of workflow tasks among heterogeneous computing domains such as local systems, a variety of commercial clouds, and multiple national resources.

This research explores development of a multi-domain scheduling system where the individual component tasks of a workflow are not bound to a single computational resource or domain, but rather can be scheduled across multiple resources as well as across institutional domains. It benefits a rapidly growing segment of the scientific community in both large and modest laboratories whose instruments now generate massive amounts of genomic data at modest expense but who have insufficient access to resources needed for processing or analyzing these data. The cyberinfrastructure to be established will potentially be usable by other science communities facing similar computational challenges.

The Multiscale Imaging Core (Core B) will provide advanced structure and function capabilities. services and technical assistance to investigators who need to characterize how tissues. cells, organelles and molecules respond to kinase and phosphatase signaling. The leaders of Core B are leading experts in the development and use of labeling and indicator technology for physiological detection. quantitative imaging, and monitoring of biological processes. They are also recognized authorities on mitochondrial physiology and structure, key to the research goals of all four projects. A common theme among all projects is that perturbation of the precise balance between protein phosphorylation, catalyzed by protein kinases, and protein dephosphorylation, catalyzed by protein phosphatases, can lead to deregulation of insulin homeostasis. Deregulation of this balance can suppress signaling pathways, resulting in insulin resistance, a defining characteristic of type 2 diabetes mellitus and metabolic syndrome. The multi-faceted capabilities of Core B will assist researchers to address the underlying molecular mechanisms driving the assembly and dissociation of protein scaffolds during insulin resistance/insufficiency and are designed to provide novel insights into the organization of signaling networks that influence diabetes and metabolic syndrome. The Core Services Aims are: 1. Enable broad access to fluorescent probes, reporters, and indicators being developed for imaging biochemical and physiological functions of tissues and cells to investigate models of signaling. 2. Enable the probing of biological systems through long-duration, live-cell imaging, large-area imaging of cells and tissues, and correlated 3D light and electron microscopic mapping of probes. 3. Provide real-time, 4D imaging for the localization, monitoring, and characterizations of molecular and supramolecular complexes in vitro, in vivo and in situ. 4. Perform electron microscopy and tomography to characterize mitochondrial structural alterations upon scaffolding complex association/dissociation and component knockdown or knockout. 5. Assay the alteration of kinases, anchoring and scaffolding proteins on the bioenergetic function of mitochondria using state-of-the-art measurements on cells and isolated mitochondria. 6. Provide the supporting computational, visualization, and informatics technologies for functional and multidisciplinary analyses.

The Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA, http://camera.calit2.net/) is a semantically enabled database and distributed computational infrastructure that provides a single system for depositing, locating, analyzing, visualizing, and sharing microbial biology data. With the rapid advance of newer DNA sequencing methods, so called Next Generation Sequencing (NGS) technologies, such as Illumina HiSeq and MiSeq, it is becoming increasingly difficult for researchers using sequencing data to meet the computing requirements for large-scale NGS datasets with existing methods. In response to these aspects of the BIG DATA challenge, the CAMERA team is developing new bioinformatics algorithms, high performance computing solutions, visualization interfaces, and data resources to specifically address the NGS data analysis challenges. Here, the group proposes a crosscutting methodology for analyzing NGS data that marries innovative bioinformatics algorithms and workflows with leading edge computational methods for managing large scale distributed computing. The integration of XSEDE resources for BIG DATA analysis will provide the scale and specification necessary to drive the development of this system. This project will be conducted over two years. Year one will be focused on the refinement of core CAMERA CI (e.g. Panfish) and the continued development of core NGS workflows/algorithms. Specifically, CAMERA CI will be extended to take full advantage of two new NSF XSEDE resources to be commissioned in early 2015 (Wrangler at TACC & Comet at SDSC). Year 2 will be focused on the production integration of Wrangler and Comet and the subsequent deployment of the NGS workflows (via CAMERA CI) to the entire CAMERA community. These new software tools and pipelined processes facilitate the processing and analyze very large-scale metagenomic data on the scale of tens of GB per sample and provide comprehensive and unique functions such as 16S analysis[7], taxonomy binning[8], assembly, rRNA finding, clustering, filtering, function and pathway annotation, and visualization]. These next generation tools enable orders of magnitude faster computational process, more comprehensive analysis, integrated data output, and novel ways to investigate complex data, once made to operate in extensible HPC cloud environments. The Broader Impact is viewed as that currently, manual operations are necessary to complete analysis with these tools due to the complexity of the process and the large number of software tools involved. The goal of this project is to develop a series of fully integrated and easy-to-use analysis workflows encapsulating these tools. These new workflows of software tools will significantly improve NGS data analysis for researchers who use metagenomics as an investigative tool, researchers who are now impeded by challenges with regard to managing and analyzing BIG DATA.

DESCRIPTION (provided by applicant): Our ability to store and retain new information rests upon the brain's immense plastic capabilities. Experimental evidence suggests that morpho-chemical modifications at the level of single dendritic spines may contribute to learning and memory but we lack both a quantitative and a mechanistic understanding of how spines function. To address this deficit, the proposed project aims to explore how the ultra-structural three-dimensional architecture of dendritic spines shapes their electro-chemical signal transduction and how structural changes alter the transduction and thus affect synaptic efficacy. State-of-the-art 3D electron microscope (EM) reconstructions endowed with precise ionotropic receptor kinetics and accurate biophysical models will be used to construct a nanometer-resolution model for dendritic spines. To simulate the electro-chemical dynamics within such a complex multi-scale environment, advanced numeric schemes such as finite-element discretization and fast multi-level solvers will be employed. By performing detailed experiments in silico, the primary factors that influence ionic current conduction in spines will be identified and then used to systematically derive a low-dimensional spine model amenable to exact mathematical analysis. Both, the full and the reduced model will allow the consortium to study the sub-cellular information-processing capabilities of single spines, and to compare the results with in vivo and in vitro data. The models will enable researchers to develop and test spine-related experimental hypotheses and to interpret data recorded in healthy and disease-modified tissue within a unified framework. Objective 1: Reconstruct Dendritic Spines in 3D at Nanometer Resolution; Objective 2: Establish a Biophysically Realistic Nanometer-Resolution Spine Model; Objective 3: Develop High-Performance Numerical Methods to Simulate the Spine Model; Objective 4: Use Simulations and Theory to Study the Computations of Dendritic Spines. The project aims to relate the multi-scale biological organization of dendritic spines to possible functional consequences at the macroscopic and systems level. In the era of ever-increasing super-computing capabilities, any mechanistic model of synaptic transmission and postsynaptic integration should start with a clear insight into precisely how biophysics orchestrates the signal transduction at the smallest scales. Deciphering the impact of micro-structural features on spine dynamics will be a stepping-stone towards understanding neural signal propagation and synaptic plasticity, and likely reveal novel sub-cellular computational principles. The findings wil deepen our understanding of neural information processing in healthy and disease-modified brains and may lead to new designs for neuromorphic devices. Alterations in spine morphology are seen in various brain diseases [1] including Down?s syndrome and fragile X syndrome [2]. Similarly, changes of the intra-spine calcium dynamics and homeostasis have been documented for Alzheimer?s disease [3]. Developing new cures and therapies for these diseases will profit from a better understanding of the relation between the dynamical structure and the function of dendritic spines. To reach this goal, in continuation of past NSF-supported projects, we will recruit and train young scientists to meet the interdisciplinary challenges of modern multi-scale and multi-modal data-driven biology, where progess is driven not only by neuroscience, but also engineering, mathematics and physical sciences, computational science and neuroinformatics The planned collaboration between the three laboratories in the US and Germany will generate international training opportunities for graduate students and postdoctoral researchers, and the participation of researchers in programs that encourage underrepresented minorities to pursue career paths in STEM disciplines.

? DESCRIPTION (provided by applicant): The biological functions and activity of our genomes is not determined by linear DNA sequence information alone. To fit within the nucleus, DNA assembles with nucleosomes to form chromatin that coils into spatially defined territories that determine if genes are active or silent through poorly understood mechanisms. The local and global organization of chromatin are integrated in the nucleus to determine gene activity and genome function. To visualize the different structural scales of genome function in intact cells, ChromEM has been developed that exploits a cell permeable fluorescent small molecule that binds specifically to DNA and upon excitation paints the surface of chromatin with an osmiophillic polymer that can be visualized using electron microscopy (EM). By combining ChromEM with new advances in multi-tilt EM tomography (EMT), chromatin ultrastructure and 3D organization can be visualized at nucleosome resolutions as a continuum through unprecedented nuclear volumes. In Aim 1, ChromEM will be used to visualize chromatin structure-function in human embryonic stem cells and differentiated lung epithelial cells at nucleosome resolutions and genomic scales. We will qualitatively and quantitatively analyze the large-scale changes in chromatin structure and organization in response to Adenovirus infection and HDAC/DNA methyl transferase inhibitors. We will expand the scale and 3D nuclear volumes using serial section EMT and serial block face EM. A major goal will be to reconstruct entire sister chromatid pairs in metaphase cells to determine if they have identical chromosome architectures and chromatin organization. In Aim 2, the EM equivalent of `multi-color' fluorescence will be developed. We will implement `multi-color EM' by using sequential excitation cycles of miniSOG and ChromEM to photo-oxidize different metal chelates of DAB that can be discriminated by elemental mapping using electron energy loss spectroscopy (EELS). Multi-color EM will be used to visualize the structural interactions of viral oncoprotein and PML bodies with chromatin that regulate he silencing and activation of p53 and anti-viral genes. Also, workflow will be extended to incorporate live imaging to visualize the spatiotemporal dynamics of PML and chromatin associated interactions that are disrupted by wild type and mutant adenovirus infections in primary cells. In Aim 3, novel probes will be developed that enable a single copy gene to be identified in a sea of chromatin within the nucleus while preserving native ultrastructure and sequence context. Synthetic self- assembling nanoparticles will be engineered with different architectures, sizes, metal and fluorescent properties so that multiple genes can be labeled and visualized using live imaging, X-Ray microscopy and high resolution 3D EM. These labels will enable GFP-tagged loci to be visualized by EMT together with endogenous gene loci using dCAS9 fusions. The chromatin ultrastructure of telomeres and critical growth regulatory genes will be visualized using live imaging and 3D EM in the dynamics of the cell cycle and viral infection. These studies will change the understanding of the nucleus and chromatin structure-function.

DESCRIPTION (provided by applicant): We propose to refine and exploit powerful new genetically encoded labeling systems to visualize proteins by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally applicable genetically encoded labels until our recent development of miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM. We aim to quantify the sensitivity and spatial resolution of miniSOG and to extend its applicability to low-temperature methods for sample preparation and imaging. Alternative genetically encoded labels will be developed and characterized to allow for two or more proteins of interest to be distinguished in a single EM image by electron energy loss spectroscopy of reaction deposits from oxidation of diaminobenzidine conjugated to different lanthanides. We have also developed reporters, based on the drug- controllable cis-acting protease from hepatitis C viral protease, to distinguish between old and newly synthesized copies of a genetically specified protein of interest. These fusion tags are visible by correlated LM and EM and will be applied to plasticity and disease-related synaptic proteins to reveal their localized appearance and turnover, initially in culture bt eventually in intact mammalian brain. Viral and Cre/lox modular targeting vectors will be created to make cell-type-selective expression of the above EM/LM tags robust and widely applicable to systems as complex as mouse models of disease and learning. We have chosen cell types and proteins important in liver fibrosis and synaptic plasticity as test cases because these biological processes are diverse, engage outstanding local collaborators, and have great biomedical importance. With collaborators we will investigate how hepatocytes, hepatic stellate cells, and endothelial cells change ultrastructural morphology and location of key proteins during fibrogenic injury. Another collaboration will focus on activity-induced changes in morphology and adhesion molecules at synapses in the amygdala during fear conditioning and memory consolidation. Such extension of these new tools into transgenic animals will make it possible to relate ultrastructural location and metabolic turnover of genetically specified proteins to whole- animal behavior and disease.

DESCRIPTION (provided by applicant): This proposal first aims to improve, develop, and test powerful new molecular techniques to monitor and manipulate dynamic signal transduction in neurons. Goals for voltage indicators based on photoinduced electron transfer include greater sensitivity, genetic targeting, and longer wavelengths. New far-red fluorescent proteins engineered from phycobiliproteins are promising building blocks for in vivo indicators of cell cycle status, Ca2+, and protease activity. A novel alternative approach to measuring and manipulating neuronal activity is to engineer an artificial transcription factor activated by simultaneous high [Ca2+] and illumination, greatly improving on endogenous activity reporters such as c-fos. The genetically encoded snapshot reporter will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, then drive expression of effector genes to mark those cells and allow selective excitation, inhibition, or ablation to test their functional importance. A chimeric channelrhodopsi activatable by red light permits optogenetic excitation of deep neurons through the intact skull, but its peak wavelength should be further increased and its residual sensitivity to blue light suppressed. Optogenetic inhibition of synaptic release will be improved by a more efficient singlet-oxygen generating protein, and introducing a singlet- oxygen-sensing GFP to map the spatial extent of inhibition. Both optogenetic tools will be applied to dissect amygdalar circuits n fear conditioning. The singlet-oxygen generating protein can now be split into two complementary fragments that only become photoactive after being brought together by chimeric partners. This complementation system may allow protein-protein interactions and kinase and protease activity to be captured for subsequent visualization by electron microscopy. A genetically encoded tag that marks proteins made during a pharmacologically defined period may become applicable to image synthesis and degradation of proteins in intact brain, thanks to development of nanoparticles that deliver small molecule drugs across the blood-brain barrier. Such nanoparticles may also aid clinical drug delivery to the brain. Such techniques will be used to test a new hypothesis that very long-term memories such as fear conditioning are stored as the pattern of holes in the perineuronal net (PNN), a specialized extracellular matrix that envelops mature neurons and restricts synapse formation. The 3-D intertwining of PNN and synapses will be imaged by serial-section electron microscopy. Lifetimes of PNN vs. intrasynaptic components will be compared by pulse-chase 15N labeling in mice and 14C content in human cadaver brains. Genetically encoded indicators and anti-neoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. New techniques including genetic knockouts, better pharmacological inhibitors, and the snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Biosynthesis of PNN components and proteases will be imaged.

? DESCRIPTION (provided by applicant): Recent advances in light and electron microscopy, along with new probes and labeling methods for cellular and molecular imaging, are driving a revolutionary expansion in biomedical research activities that involve exploration of complex biological systems. These new capabilities in instruments and methods are providing nearly seamless views across multiple scales of resolution of biological specimens. At the same time, researchers are increasingly generating extremely large and diverse datasets of enormous value, which are driving renewed demand for software and tools to facilitate aggregation, curation, refinement, and spatial and semantic assembly of these data. A key consumer of these data is the computational modeling and simulations community, which requires more accurate in-silico representations of supramolecular complexes and organelles in the context of complete cells and cells deployed in tissues to better model the impact of molecular changes found in diseases on the functioning of a cell. Building on prior success, we propose to continue to support, maintain, and extend the capabilities of the Cell Centered Database, which we have recently renamed the Cell Image Library (CIL). The CIL is a cell- centered community repository for storing, managing, and sharing multi-scale microscopy data encompassing cellular networks, cellular and subcellular microdomains, and their macromolecular components. The CIL is comprised of software for researchers to upload, organize, process, and share project data prior to publication. It also provides a searchable, public-facing website for users to disseminate their results and openly distribute their data for reuse by others. Our extended development plans include integration of computational workflows to facilitate generation of fully segmented, 3D structural models ready for meshing, discretization, and use in assembling more complete, realistic, and accurate models for simulations. We will also harden and extend our unique capabilities for supporting large data, in particular massive individual 3D datasets, to enable biomedical researchers to not only store, but to process and visualize their data at scale with tools that are easy to use and easily accessible.

PROJECT SUMMARY/ABSTRACT This proposal requests funding for the National Center for Microscopy and Imaging Research (NCMIR) at UCSD to acquire a 300-keV FEI state-of-the-art intermediate-high voltage electron microscope (IVEM) and integrate it with massive data storage and computational resources. The result, to benefit the research and technology development aims of NIH biomedical investigators, will be an integrated big data appliance for volume visualization (to nanometer resolution) through extensive three-dimensional (3D) volumes. This coupled microscope / computational system will be equipped to perform automated large-volume 3D imaging required for a range of biomedical research projects, including imaging of whole cells at resolutions sufficient to pinpoint the molecular building blocks comprising macromolecular complexes, intra-nuclear and cytoplasmic structures, and characterization of nanostructures of biological materials in the contexts of their functional environments. This proposal includes eight major and three minor research projects that exemplify the transformative science that can be accomplished with this new one-of-a-kind instrument. Because this instrument will generate massive 3D datasets requiring substantial resources for computation-based refinement and quantitative analysis, it will be coupled tightly to a large, scalable data- storage and high-performance computing environment, leveraging the significant resources at the San Diego Supercomputer Center and the California Institute for Telecommunications and Information Technology (both at UCSD). Prominent national, international, and local users will broaden the application of this platform to achieve maximum impact in advancing scientific discovery and human health. They will work together and with our management team to commission, use, and refine the capabilities of this microscope. They will also help others benefit fully from its capabilities. Our experiences in designing and commissioning high-end instruments and those of our staff and scientists using them have been and will be communicated to the manufacturers with the goal of guiding the advance of the capabilities of future instruments in directions of most benefit to the biomedical research community.

Abstract This study aims to understand how the ensemble activity and network architecture of a neuronal population guides natural and learned behavior. The model system is the midbrain localization pathway of the owl. Ensemble recordings, microcircuit analysis, behavioral measurements and computational modeling will be used to analyze the neural representation of auditory space and the head-orienting movement driven by it. The compact volume of tissue commanding this behavior makes a complete understanding of information processing tractable with high-throughput electrophysiological and microanatomical methods. How information about sound location is readout to guide orienting behaviors has not been demonstrated in any species. This project has the potential to fill this gap. Aim 1 will investigate the relationship between orienting behavior and activity in the neuronal population representing auditory space, in which frontal space is overrepresented. The hypothesis is based on recent work showing that sound localization can be explained by statistical inference, computed by integrating activity across the entire population. Microelectrode arrays (MEAs) will be used to map the activity of the population upon presentation of sounds. Population decoders will be constructed to determine how the population activity is readout to drive behavior. In Aim 2, the network architecture supporting the activity pattern will be studied with light and electron microscopy. Network models will combine the data to explain how connectivity and cellular computations result in the population activity and correlated firing that drives behavior. When auditory-visual cues are modified, the midbrain representation of auditory space adapts over time, and consequently drives a learned behavior. Aim 3 will directly examine this link. MEA recordings, microcircuit analysis and behavioral measurements will be made in owls adapted to prismatic spectacles. Population decoders will be used to test the hypothesis that population activity in the learned condition maintains a non-uniform population code with an overrepresentation of frontal space. Network models will be used to examine how local re-wiring may explain changes in the distribution of activity across the population. This would be the first time that neural activity and network architecture underlying sound localization are approached from the complete-population down to single-cell level, before and after learning. This integrative approach holds potential for understanding principles of population coding, plasticity and learning that operate across species and brain circuits.

Project Summary / Abstract Traditionally, neurological diseases have been classified into mechanistically distinct categories, such as neurodegenerative, inflammatory, and vascular. However, recent insights have led to a reassessment of the complex relationship between diseases and their mechanisms. Emerging evidence supports a role for vascular dysfunction as an early feature in AD that is an equal and independent predictor for cognitive decline compared to amyloid and correlates with worse prognosis in AD. However, the cellular and molecular mechanisms at the neurovascular interface that promote cognitive decline are poorly characterized. Furthermore, whether and how vascular alterations contribute to neuronal network and synaptic dysfunction, one of the earliest manifestations of AD, is unknown. A fundamental change at the neurovascular interface in AD is the deposition of the blood coagulation factor fibrinogen, which is deposited as insoluble fibrin in the AD brain. Our ultimate goal is to determine the dynamic interactions between innate immunity, vascular, and blood-derived signals and their causal relationships in regulating impaired synaptic activity as a prerequisite for devising novel therapies to improve synaptic and cognitive functions after vascular impairment. Studies from our laboratory and others have shown that genetic or pharmacologic depletion of the blood coagulation factor fibrinogen protects from neuroinflammation in several models of neurological disease. Our preliminary data demonstrate that dendritic spine elimination occurs around fibrinogen deposits in AD mice and fibrinogen-CD11b signaling promotes dendritic spine loss and cognitive impairment in AD mice. The four specific aims will are designed to determine the role of fibrinogen/CD11b signaling in microglial-synapse interactions and neuronal network abnormality, determine the mechanisms underlying fibrin-induced innate-immune driven neuronal dysfunction, and the therapeutic implications of targeting fibrin-microglia interactions in protecting from spine elimination and neuronal dysfunction. Our experimental design is based on a cutting-edge multi-pronged experimental approach consisting of in vivo two-photon imaging of neuronal activity and microglial dynamics, EM co-registration, iDISCO, unbiased transcriptomics and proteomics, and combined two-photon imaging with in vivo EEG recordings. The proposed studies will set the foundation how neurovascular dysfunction regulates synapse elimination and neuronal activity and the outcomes of this research would be applicable for the understanding of the etiology and the development of new treatments for vascular cognitive impairment including in AD and related conditions.

The cochlear nucleus is the gateway for central nervous system processing of auditory information in mammals. It has been proposed that parallel processing channels are set up in the CN, and these form the basis for further computation at higher stations of the auditory system. Despite decades of study, enumeration of CN cell types is incomplete and CN circuitry is described only superficially. In neuroscience generally, classification and naming of neurons has relied primarily upon qualitative approaches based upon human observational capabilities. We have implemented and in some cases developed novel high-throughput and unbiased techniques for labeling, segmenting and classifying neurons in 3D, generated from large-scale electron microscopy image volumes. We propose to deliver a nanoscale map, or connectome, of the mouse CN with enumerated and localized cell types and their synaptic connections. This effort is unbiased because all neurons will be sampled. To achieve this goal, we bring together four parallel modes of tissue analysis for neuron classification: morphology, connectivity, molecular identity and function. We propose that connectivity analysis will define long-proposed parallel processing circuits that will be tested functionally using realistic biophysical models of identified cell types. Notably, the cochlear nucleus contains both amorphous and layered organizations of cells, which serve as templates for all other brain regions. By investigating the fundamental structure of this sensory center, we will establish principles of neural computation and methods for structural and functional phenotyping that will apply to other brain regions regardless of their particular neural architecture.

PROJECT SUMMARY/ABSTRACT This project will expand the acquisition, reconstruction, analysis, and dissemination of 3D electron microscopic (3D EM) reference data, disclosing key ultrastructural details preserved within a remarkable collection of legacy biopsy brain samples from patients suffering from Alzheimer?s Disease (AD). These samples were originally collected, characterized and archived by neuropathologists R.D. Terry and N. Gonatas (at A. Einstein in the 1960?s), with later samples taken as part of a cerebrospinal fluid (CSF) drug infusion study involving S. Mirra (at Emory in the 1980?s). They were re-examined by Ellisman, Masliah, Terry, and Mirra in the 1980s, using early 3D EM methods, and were found to manifest excellent preservation of ultrastructure, showing paired helical filaments (PHF) and amyloid accumulations as well as modifications to subcellular organelles and cytoskeletons of the cell bodies, axonal and dendritic processes. Here, we will exploit recent advances in high throughput, automated 3D EM to massively scale the examination of these precious samples, reconstructing 100s of brain cells with and without PHF, tracking axons (and mapping glia and synapses) through much greater brain volumes than was feasible previously. Our goal is to target areas associated with both plaques and tangles, attending to locations where existing findings suggest cell and network vulnerability and contain molecular interactions suspected by some to underlie the initiation and progression of AD. Supporting investigations into the progression of soma/dendritic degeneration, we will target cells operationally defined to represent a progression of neuronal decline as seen in AD; determining the volume fraction of PHF in the cytoplasm as a practical staging measure and linking this to the characterization of quantitative changes in the microstructure of major subcellular constituents. Likewise, we will analyze the progression of axonal degeneration in and near plaques as data obtained suggests that axons may become dystrophic before their parent cell bodies and their dendrites degenerate.

PROJECT SUMMARY This focused technology development project will advance into first practice a transformative new imaging technology for multicolor electron microscopy (ColorEM), enabling new capabilities for high resolution and 3 dimensional (3D) localization and differentiation of different molecular complexes in cells and tissues. With this technology, we will substantively improve upon presently practiced, multiple labeling strategies involving antibody labelling approaches detected using colloidal gold, quantum dots, etc.. We will further provide a more accessible, higher throughput, and dose efficient alternative to spectroscopic methods, like energy dispersive x-ray (EDX) and electron energy loss spectroscopy (EELS) / energy filtered transmission electron microscopy (EFTEM). At the heart of this effort, we will exploit the performance characteristics of a next- generation, ultra-high speed direct detection device (DDD), now being optimized and extended (by us with an industry partner) for use with scanning transmission electron microscopy (STEM), a broadly deployed capability on modern EM columns. The optimized application of this new pixelated STEM detector technology, in combination with an extended palette of elemental probes and advanced software analysis, will allow for unprecedented new capabilities for performing atomic number (Z) contrast imaging ? a process we call ColorSTEM. To bring this new integrated methodology into first practice, we will build on recent feasibility studies and surmount remaining technical hurdles to 1) make multi-labelled EM specimens with optimized elemental probe combinations; 2) troubleshoot the first use of a potentially transformative new sensor technology and work out the methodology for employing it for Z contrast imaging; and 3) devise the associated analytical/computational methodology for processing the very large resultant data sets to enable unambiguous differentiation of molecular complexes selectively marked with elemental probes.

PROJECT SUMMARY AND ABSTRACT The National Center for Microscopy and Imaging Research (NCMIR) at UC San Diego is a vital and unique NIGMS supported biomedical technology development research resource (BTRR). NCMIR is completing its 31st and final funded year of continuous operation and is thereby ineligible for renewal under new guidelines governing the P41 program. With this proposal, we request stabilizing support to help ease the transition of the Center to independent sustainability and to allow for the ordered completion of the resource's existing pool of supported biomedical research projects. NCMIR's mission is to advance the coordinated use of specialized molecular probes, novel specimen preparation methods, leading-edge microscopy and imaging technologies, and scalable computational tools to help investigators traverse difficult to navigate spatial and temporal scales, deliver new insight into multiscale structure/function relationships, and provide a fundamental understanding of the macromolecular mechanisms underlying many topics in health, disease, and aging. In transitioning the resource to a self-sustaining community-wide scientific resource, we provide details of our `Operation and Maintenance Plan' to govern how the resource supports ongoing research users, prepares developed technologies for broad dissemination, and modifies its approach to community engagement to communicate offered technologies, services, and resources. We further establish a `Transition Plan' to scale existing cost- recovery processes and put into place new mechanisms to stabilize support for the operation of the resource, buttressed by commitments from stakeholders within our home institution at the University of California, San Diego and the greater La Jolla mesa. Last, we establish an `Evaluation Plan' to quantify and assess resource performance and impact on the community, putting forth both Short-term Internal Metrics, to optimize resource utilization and make ongoing improvements, and Long-term Objective Metrics, to gauge overall impact on the scientific community.

SUMARY Circadian rhythms depend on the molecular transcription/translation negative feedback loop (TTL) operating in clock neurons, and on the network properties of these neurons. Among the properties that could be recruited by the circadian clock are changes in the identity of pre/post synaptic partners and/or strength of the connectivity between clock neurons, a property collectively termed as circadian structural plasticity. Our central hypothesis is that circadian structural plasticity within the central circadian clocks of mammals and Drosophila are part of the time-encoding mechanisms. We will employ mouse and fly genetics combined with state-of-the- art quantitative 3D light and electron microscopy techniques to address the extent of structural plasticity within specific neurons of the mouse suprachiasmatic nucleus (SCN) and the Drosophila circadian network. Specific aim 1 will assess how widespread structural plasticity is in the Drosophila circadian network as well as which are the functional consequences of those structural changes. We will explore the extent of circadian neuronal remodeling of subsets of PDF and non-PDF pacemaker neurons using CM and SBEM (sub-aims 1A i and ii). We will examine time-of-day dependent functional connectivity changes among clock neurons through chemogenetic GCamP6-reporting (sub-aim 1B). Sub-aim 1C will examine the behavioral consequences of impairing structural remodeling; sub-aim 1D will further investigate the molecular mechanisms underlying circadian structural plasticity. Specific aim 2 will examine the degree of circadian structural remodeling in SCN VIPergic neurons, which are an essential component of the timekeeping mechanism, through virally mediated sparse-labeling (CM) (sub- aim 2A), or serial block-face scanning electron microscopy (SBEM) with a marker that enables the analysis of dendritic ultrastructure (sub-aim 2C). Finally, we will assess if circadian oscillations in VIP neuronal processes rely on the TTL by repeating experiments in 1A in VIP-specific Bmal1-/- mice (sub-aim 2B). Specific aim 3 will explore if connectivity of VIPergic neurons changes throughout the 24-h cycle. Using GFP reconstitution across synaptic partners (GRASP), we will investigate if these connections change with circadian time through immunocytochemistry and CM analysis in fixed tissue (sub-aim 3A) as well as ex vivo in SCN slices (sub- aim 3B). We will also determine whether GRASP-detected rhythms depend on the canonical TTL by repeating experiments in 2A and 2B in VIP- or SCN astrocyte-specific Bmal1-/- mice (sub-aim 3C). Our experiments test predictions of the hypothesis that circadian structural plasticity represents a defining feature of central neuronal circadian pacemakers. Support for this hypothesis would provide a critical new perspective to understand how these pacemakers encode time at the network level. Furthermore, the experiments we propose represent a unique opportunity for research capacity building in Argentina, where the foreign principal investigator is located, and where students and postdocs will be trained in techniques that are still not fully developed in that country.

PROJECT SUMMARY/ABSTRACT This project aims to disseminate validated technologies and resources of the National Center for Microscopy and Imaging Research (NCMIR) at UC San Diego to advance the completion of strategic goals of the BRAIN Initiative. The proposed technology integration and dissemination resource will provide the neuroscience community with technical help to obtain and manage large scale data, broadening the access to and incorporation of high throughput multiscale imaging tools and leading-edge analysis strategies in their studies. Richly integrated resources will be offered, including molecular probes, imaging platforms and data analysis tools certain to enable and hasten brain research. The types of research projects we will support, include: 1) investigations requiring the traversal of spatiotemporal scales to reveal new insight and understanding of specific neural populations; 2) investigations which aim to mark and track neuronal processes and/or visualize targeted connectomes of local circuits within large volumes of nervous tissue in multiple animal models; 3) projects which seek to perform nano-histological assessment of cellular and subcellular level alterations associated with learning, physiological state, or disease; and 4) projects performing higher resolution 3D morphometric analysis of subcellular underpinnings of function, with particular emphasis on synaptic function as influenced by subcellular constituents. This new center will leverage an administrative framework and information technology cyberinfrastructure which is already in place and has been refined over many years. In addition to providing a management structure, this framework includes a community outreach, project review, selection, and onboarding process refined and long practiced by the PI (and the assembled team). Additional mechanisms for tool/technology dissemination and training, data management and sharing, and mechanisms for cost-recovery and long-term sustainability will also be leveraged and expanded so as to maximize the reach and impact of our technologies and resources.