Thomas A. Blanpied, PhD - US grants

The release of neurotransmitters is a fundamental step in neuronal communication and is probably an important site of plasticity during development or learning. Neurotransmitters are released, from storage organelles called synaptic vesicles, via the process of calcium-regulated exocytosis. The availability of synaptic vesicles for exocytosis is tightly regulated and seems to be determined in part by "priming" processes that occur after a vesicle docks at the presynaptic membrane and before its Ca-dependent fusion with it. The general goal of this research is to define the molecular mechanisms underlying the exocytosis of synaptic vesicles focusing in particular on the process of vesicle priming. The experiments proposed here will test, in autaptically connected hippocampal neurons in culture, the hypothesis that an ATP- dependent priming process is involved in neurotransmitter release. The time course of transmitter release will be monitored following an instantaneous rise in the Ca concentration due to photolysis of a "caged" Ca compound. The hypothesis predicts that a multiphasic release time course will be evoked, arising from the sequential exocytosis of primed vesicles followed by unprimed ones, and that inhibition of ATP hydrolysis will inhibit only the release of unprimed vesicles. This kinetic assay will also be used to determine whether two cytosolic proteins critical in production of phosphoinositides play a role in priming. These experiments are of general interest because they will help identify the molecular basis for a process that is central to the ability of the brain to process information.

DESCRIPTION (provided by applicant): Activity-regulated modulation of synapse function lies at the heart of molecular theories of learning and neural development, and is disrupted by diseases and disorders including schizophrenia, autism spectrum disorders, and obsessive compulsive disorder. In glutamatergic synapses, multi-domain proteins establish the core of the postsynaptic density (PSD), the structure which links neurotransmitter receptors to the actin cytoskeleton and to intracellular signaling pathways. Though the structures of PSDs and synapses are known to change dramatically in diverse paradigms of synaptic plasticity, it is not known to what extent structure alone-rather than molecular content such as receptor number-can control synaptic strength. We have developed tools to address this. Thus, in cultured neurons from rat hippocampus, we will use single-molecule imaging approaches along with electrophysiology and new molecular tools to tackle this central question of synapse biology. First, we will follow up on intriguing work from our prior funding period indicating that scaffold molecules in the PSD are distributed with substantial peaks and valleys of density across the PSD parallel to the membrane. We hypothesize that these peaks establish a similar organization of other proteins at points that stretch from the presynaptic terminal too deep into the spine, and particularly that PSD structure guides protein organization within the active zone, thus assuring the optimal alignment of vesicle fusion apparatus with clustered postsynaptic receptors. Second, we ask whether PSD substructure-rather than its overall size-regulates receptor activation and thus synaptic strength. We test this using new techniques to monitor postsynaptic NMDAR or AMPAR activation during concurrent super-resolution imaging to measure postsynaptic structure. Third, using a newly developed method, we analyze for the first time the distribution of vesicle release sites within single active zones. For these aims, we test the role of the key scaffolding molecule Shank in maintaining transsynaptic structural organization, both to test fundamental mechanisms and because its failure to do so may contribute significantly to human disease. The answers to these questions provide a new and detailed view of how synaptic function arises from its notoriously detailed architecture. Perhaps most importantly, they will provide an important platform on which to test hypotheses regarding the molecular basis of disorders that disrupt synaptic transmission.

We propose to acquire a recently developed, dually configured confocal microscope (the LSM 5 Duo, Zeiss)[unreadable] to support the research of a group of 9 NIH-funded investigators at the University of Maryland School of[unreadable] Medicine. These investigators each have immediate need for high-speed, multi-color fluorescence confocal[unreadable] imaging during photoactivation, photobleaching, or long-distance Z motion. No equipment with the required[unreadable] capabilities exists on campus. The Zeiss Duo combines the well-established, high-resolution optical[unreadable] sectioning capabilities of the LSM 510 with the high-speed imaging capabilities of the more recent LSM[unreadable] 5Live. The two, independent beam-steering systems of the Duo create the synergistic capacity for[unreadable] simultaneous region-of-interest photomanipulation during rapid image acquisition. The Major Users each[unreadable] confirmed that the capabilities of the Duo directly and uniquely well meet their requirements. The new[unreadable] instrument will be incorporated into the long-standing Confocal Core Facility at the University, where it will[unreadable] replace an LSM410 that was purchased in 1994 and is neither suitable nor reliable for the live-cell[unreadable] experiments that now occupy the large majority of time on the confocal microscopes in the Facility. After[unreadable] evaluating possible system configurations from the major vendors, the Facility?s oversight committee also[unreadable] agreed that the Duo presented the only configuration that would meet the increasing demand for highspeed,[unreadable] live-cell imaging while providing consistent access to a high-resolution point-scanning confocal.[unreadable] Accordingly, institutional support is very strong at the Dean?s level and also spread widely among many[unreadable] Departments with extensive NIH-supported research. The presence of the Duo in the Confocal Core[unreadable] Facility would directly and strongly benefit the research of the Major Users; more broadly, it would add an[unreadable] important new capability for advancing the NIH-sponsored research of the many other investigators on[unreadable] campus studying biological processes in living cells and systems.

DESCRIPTION (provided by applicant): We propose to acquire an upright, multiphoton and confocal microscope to support the research of investigators at the University of Maryland in the School of Medicine, Dental School, School of Nursing, and Comprehensive Cancer Center. The investigators selected as Major Users, like many at the University, have immediate need for fluorescence imaging deep within intact or in vivo systems. However, they are currently limited in their ability to accomplish key aims of their research using only confocal microscopy, which penetrates only to shallow layers. Multiphoton microscopes represent the current standard configuration for in vivo and thick-tissue high-resolution imaging in universities around the world However, no equipment with the required capabilities is available to the users, and indeed no upright multiphoton microscope is present within the entire University of Maryland Baltimore campus. This has left a debilitating hole in our ability to conduct relevant research in many of our key areas, and the proposed microscope is designed to remedy this problem. During a series of on-campus demos involving microscopes from three major manufacturers, the Major Users collected substantial preliminary data, and each confirmed that the capabilities of the Zeiss 710NLO directly met their requirements. Zeiss supplies a number of multiphoton microscope configurations, but the 710NLO provides an excellent combination of capabilities: high-sensitivity detectors for time-lapse imaging in thick and scattering tissues; compact light path for preserving laser power and beam quality on the illumination side and conserving photons on the emission side; spectral detection for removal of autofluorescence; high ease of use due to familiarity with Zeiss software; an excellent new upright microscope design optimized for patch-clamp electrophysiology; dependable local service; as well as a top-quality pulsed laser tunable over a broad wavelength for diverse projects. The new instrument will be incorporated into the long-standing and successful Confocal Core Facility at the University. The presence of extensive on-campus expertise along with pledged support from experts at nearby institutions, promise swift and efficient utilization of the microscope. Funded by a major NCRR construction grant, the Core is currently undergoing extensive renovations to house our confocal microscopes. The system matches the long-term goals of the Deans of the School of Medicine, School of Nursing, and Dental School to build research resources, and accordingly, institutional support is very strong. The availability of the LSM710 NLO in the School's Confocal Core Facility would directly and strongly benefit the research of the Major Users. More broadly, it will add critically important new capabilities to advance NIH-sponsored research throughout a large and diverse medical research university. PUBLIC HEALTH RELEVANCE: The University of Maryland has established itself as an international leader in the area of biological microscopy. The instrument requested in this proposal will be used to explore new directions in our traditional areas of strength, including cardiology, neuroscience, hypertension, cancer biology, and diabetes. By permitting visualization of cellular- and organ-level structure and function within the most intact systems possible-including within living animals- this microscope will immediately and dramatically advance our efforts to bring life sciences research to bear on human health.

DESCRIPTION (provided by applicant): Communication between cells in the nervous system underlies all complex behaviors, and occurs at specialized regions of the nerve cell called synapses. Synapses work by releasing chemical transmitter from a region called the active zone, which activates a neighboring cell. We propose to characterize the relationship between active zone function and structural organization within frog and mouse neuromuscular synapses. We hypothesize that neuromuscular active zones are assembled from a basic transmitter release building block: the unreliable single-vesicle release site consisting of a docked synaptic vesicle and its associated Ca2+ channels. We further hypothesize that major aspects of synaptic function and presynaptic homeostatic plasticity can be explained by changes in the number and organization of these single-vesicle release sites within active zones. Our approach is characterized by a seamless collaboration between three labs with expertise in computer simulations of cellular physiology (Dittrich lab), synaptic anatomy, physiology, and Ca2+ imaging (Meriney lab), and super-resolution imaging of the number and spatial distribution of synaptic proteins (Blanpied lab). Importantly, as part of this proposal, trainees from all three laboratories will receive crosstraining in each lab. We will use this collaborative approach to develop a comprehensive MCell computer model of the presynaptic transmitter release site that will significantly increase our understanding of the relationship between active zone organization and synaptic function. This insight will not only lead to a better understanding of presynaptic mechanisms of homeostatic plasticity but also aid in our understanding of synaptic diseases, which are known to underlie a large number of neurological disorders. Intellectual Merit: A significant number of neurological diseases are known to affect the synapse by targeting synaptic organization and function. While most research on this important topic has to date focused on postsynaptic adaptations, it has become increasingly clear that presynaptic homeostatic changes are likely to be just as important. Thus, a better understanding of the role of presynaptic structure and organization in synaptic function under both control and disease conditions is needed. Broader Impacts: The MCell model that we will develop will enhance our teaching mission in many ways. It will provide an example of unprecedented scale and realism for the illustration of nerve terminal structure and function. This material will be used in courses and programs at the University of Pittsburgh, the University of Maryland, and Carnegie Mellon University. These include undergraduate and graduate Neuroscience courses, a Computational Biology PhD program that spans PITT and Carnegie Mellon University, summer workshops, and web-based tutorials (www.mcell.org). These simulations will expand previous models that already have been converted into instructive 3D movies, which are routinely shown to a broad range of audiences during open houses, student visits or classroom teaching. This work will also provide source material for teaching examples tailored to high school outreach programs at the Pittsburgh Supercomputing Center, particularly the CMIST program (Computational Modules in Science Teaching, www.cmist.org) of the National Resource for Biomedical Supercomputing (NRBSC) directed by Dr. Dittrich. Our proposed work will have a broad impact on K-12 education, undergraduate teaching and training, graduate and post-graduate training, community outreach, STEM teaching, training at underrepresented minority institutions, and knowledge of synaptic function in the field. Dr. Meriney is a member of the Neuroscience outreach committee at the University of Pittsburgh (PITT), which organizes a variety of community events. Dr. Meriney's laboratory is in the Arts and Sciences College, so the proposed research would contribute to undergraduate teaching via undergraduate research participation in the proposed work, and changes to content for undergraduate courses based on new research insights. Dr. Dittrich will also train undergraduate students in his laboratory as participants in the proposed work. He is training faculty in the NSF funded TECBio REU program at the PITT and typically mentors 1-2 students in computational projects as part of the program. In addition, Dr. Dittrich is a training faculty in the PA Governors School for the Sciences, an intense summer program for talented high school students in Pennsylvania. Drs. Dittrich, Meriney, and Blanpied will bring graduate researchers and postdoctoral fellows into their labs who will directly participate in the proposed experiments, receive cross training in all three laboratories, and receive career training. Lastly, Dr. Ulises Ricoy (an under-represented minority faculty member) from Northern New Mexico College will visit during each summer to learn new research, teaching, and training tools to bring back to underrepresented minority undergraduates at Northern New Mexico College. This will expose these underrepresented minority students to an intense academic research environment and aid in their training and career planning.

DESCRIPTION (provided by applicant): Activity-regulated modulation of synapse function lies at the heart of molecular theories of learning and neural development, and is disrupted by diseases and disorders including schizophrenia, autism spectrum disorders, and obsessive compulsive disorder. In glutamatergic synapses, multi-domain proteins establish the core of the postsynaptic density (PSD), the structure which links neurotransmitter receptors to the actin cytoskeleton and to intracellular signaling pathways. Though the structures of PSDs and synapses are known to change dramatically in diverse paradigms of synaptic plasticity, it is not known to what extent structure alone-rather than molecular content such as receptor number-can control synaptic strength. We have developed tools to address this. Thus, in cultured neurons from rat hippocampus, we will use single-molecule imaging approaches along with electrophysiology and new molecular tools to tackle this central question of synapse biology. First, we will follow up on intriguing work from our prior funding period indicating that scaffold molecules in the PSD are distributed with substantial peaks and valleys of density across the PSD parallel to the membrane. We hypothesize that these peaks establish a similar organization of other proteins at points that stretch from the presynaptic terminal too deep into the spine, and particularly that PSD structure guides protein organization within the active zone, thus assuring the optimal alignment of vesicle fusion apparatus with clustered postsynaptic receptors. Second, we ask whether PSD substructure-rather than its overall size-regulates receptor activation and thus synaptic strength. We test this using new techniques to monitor postsynaptic NMDAR or AMPAR activation during concurrent super-resolution imaging to measure postsynaptic structure. Third, using a newly developed method, we analyze for the first time the distribution of vesicle release sites within single active zones. For these aims, we test the role of the key scaffolding molecule Shank in maintaining transsynaptic structural organization, both to test fundamental mechanisms and because its failure to do so may contribute significantly to human disease. The answers to these questions provide a new and detailed view of how synaptic function arises from its notoriously detailed architecture. Perhaps most importantly, they will provide an important platform on which to test hypotheses regarding the molecular basis of disorders that disrupt synaptic transmission.

Deciphering neural coding will require deconstructing the complex and intertwined signaling mechanisms that drive cellular excitability, synaptic plasticity, and circuit dynamics in the brain. This fundamental objective has been extremely challenging because unraveling the temporal and spatial interactions of multiple signaling pathways requires coordinated observation of multiple networks within individual cells and multiple neurons within intact circuits. Large gaps in knowledge remain because our current tools for tracking the dynamics of molecular activity are poorly suited for investigating more than one reporter at a time. Here, we propose to tackle this constraint through development of a novel methodology for simultaneous optical imaging of multiple quantitative FRET biosensors within single neurons, using FLuorescence Anisotropy Reporters (FLAREs). Numerous FLAREs targeting canonical signaling pathways, including calcium, cAMP, and the MAPK cascade, have been constructed in several colors allowing simultaneous imaging of up to three sensors in a single preparation, either in the same or complimentary pathways. We propose three aims to validate and further develop this technology to tailor it for studying cells and circuitry in acute and cultured slices from the mouse brain during neural coding. We will first adapt an optical sectioning microscopy method that is highly advantageous for fluorescence polarization imaging, known as dual-inverted Selective Plane Illumination Microscopy (diSPIM), for FLARE imaging. We will also expand the FLARE palette to include key regulators of synaptic function (Rac, CaMKII) and membrane excitability (voltage). Construction of the FLARE-SPIM instrument will enable proof of principle studies on two high-value neuronal circuits. First, pushing the limits of subcellular spatial resolution, FLARE-SPIM imaging will be performed on key signaling molecules in single dendritic spines in acute hippocampal brain slices during induction of long-term potentiation. Second, pushing the limits of cellular temporal resolution, we will track the rhythmic fluctuations of voltage, calcium, PKA and ERK activities during circadian oscillations of neuronal activity exhibited in organotypically-cultured suprachiasmatic nucleus brain slices. Together, these studies will lay the foundation for systematic exploration of neuromodulation within cells and neuronal circuitry, providing critical and unprecedented new insights for the spatial and temporal interactions between signaling pathways. Through collaboration with other Brain Initiative groups working on similar problems, this foundational work will be scalable to add suites of sensors that visualize nodes of coordinated cellular activity and reveal and measure the complexity of neural coding within intact brain circuits.

The Confocal Core Facility at the University of Maryland School of Medicine proposes to acquire a set of microscopy components to support the research of investigators in the School of Medicine, as well as the Schools of Dentistry and Nursing. The investigators proposing projects as Major and Minor Users, like many others at the University, have immediate need for live confocal imaging in cell cultures and ex vivo systems. However, they have been drastically and increasingly limited in their ability to accomplish key aims of their research due to the aging of our microscope best suited to live-cell imaging. Indeed, no microscope optimized for high-speed imaging has come into the Core since 2008, which has left a widening hole in our ability to conduct relevant research in many of the areas of historic strength at the University. With this in mind, the departments of the Major and Minor Users with additional support from the Dean?s Office, Comprehensive Cancer Center and others, have purchased a new Nikon CSU-W1 spinning disk confocal microscope. This base system was installed in the core just within the past month, and provides a platform for modern, live-cell and high-speed imaging particularly in cell cultures and dissociated cells. This proposal now seeks funds to acquire specific, additional components that will allow the fully configured system to address the users? research demands, including ratiometric imaging and TIRF, as well as optical manipulation during imaging. All the proposed components will be permanently integrated into the W1 system. The new instrument will be incorporated into the long-standing and successful Confocal Core Facility at the University. The presence of extensive on-campus expertise along with pledged support from experts at nearby institutions promises swift and efficient utilization of these new components and full use of the W1 microscope with these new components. The W1 is being integrated into a core with stable and well tested policies for training, and the expert staff Manager at the core has several years of experience training users on a variety of microscopes most especially for live imaging. Institutional support for this request is extremely strong, as the school just purchased the base W1 microscope outright and has long-standing and continuing commitment to support the needs of the Core for space, personnel, and administrative services. Funded by a major NCRR construction grant, the Core has recently undergone extensive renovations to house our confocal microscopes including the W1, so the instrument is in excellent physical facility located central to the Major Users. The system matches the long-term goals of the Deans of the School of Medicine and Dental School to build research resources, including commitments to encourage use of the instrument and the tracking of its productivity. Overall, the availability of this new technology in the School?s Confocal Core Facility would directly and strongly benefit the research of the Major Users. More broadly, it will add critically important new capabilities to advance NIH- sponsored research throughout a large and diverse medical research university.

The synaptic cleft is a conserved and integral component of central synapses. It is comprised of protein complexes that span across it, and their adhesive interactions and signaling roles guide synapse development. Their relevance is underscored by the mutations in synapse-organizing proteins that are linked to complex brain disorders including autism-spectrum disorders and schizophrenia. Yet, the molecular patterning and dynamics of synaptic cleft components remain largely unknown. This contrasts with our understanding of the nanoscale organization of pre- and post-synaptic compartments, which is elucidating our understanding of synaptic structure and function. Moreover, acute synapse-organizing roles of cleft proteins at mature synapses and the functional interplay of trans-synaptic interaction systems remain to be defined. Our central hypothesis is that the properties and plasticity of mature synapses are dynamically instructed by select cleft components. This proposal builds on preliminary and previously published results by the collaborating groups that synaptic adhesion complexes are differentially localized within the cleft, shape this compartment, can undergo rapid changes in synaptic abundance upon plasticity induction, and alter long-term synaptic plasticity. Three specific aims will be pursued to test our hypothesis. First, we will test roles of trans-synaptic interactions in acutely instructing pre- and postsynaptic function. Second, it is our aim to determine the molecular and organizational dynamics of the cleft during long-term plasticity. Third, we will identify cleft proteins that guide changes during plasticity. Our approaches include tools to acutely perturb trans-synaptic interactions, proximity labeling to identify cleft-resident molecules and monitor them during plasticity, superresolution imaging to map their cleft locations, and cell biological and physiological functional assays. We anticipate to determine the molecular patterning and dynamics of the synaptic cleft and to identify how trans-synaptic interactions actively shape synaptic function. This expected progress is significant because it will define the cleft as a molecularly organized and acutely controlled cellular compartment that instructs synaptic properties. Moreover, this research can determine how a dynamic remodeling of the cleft architecture underlies the activity-dependent plastic changes at synapses. Synaptic organization and function are disrupted in neurodevelopmental and neurological disorders, and this program will provide information for defining how these diseases impact the cleft and may even originate in it to alter synaptic properties.

A diverse group of 9 NIH-funded teams and investigators at the University of Maryland School of Medicine and School of Nursing request funds to purchase a Carl Zeiss Lightsheet 7 fluorescence microscope. This new state- of-the-art system will be situated in the UMB Core Confocal Facility, a well-established core facility that will oversee use, training, and service for the microscope. Lightsheet fluorescence microscopy occupies a unique but important niche for imaging intact organoids, organs, tissues, and organisms, both in fixed, cleared samples or in live specimens and embryos. This imaging modality fills an important gap in the technical arsenal of the university, which has excellent facilities for imaging at scales from electron microscopy to PET/CT and MRI but lacks a microscope optimized for high-resolution, large-volume imaging. This has left a widening hole in our ability to conduct relevant research in many of the areas of historic strength. With this in mind, the Core Facility organized a series of instrument demos, and user labs have dedicated substantial effort to adopt and adapt several tissue clearing methods suited to lightsheet imaging. After evaluation of 5 systems, the Major and Minor Users were unanimous in their selection of the Lightsheet 7 (LS7) as the instrument which would best meet their collective needs. Institutional support for this request is broad and deep, with the Dean?s Office, the Core and its host Department of Physiology, other user departments, the UM Greenebaum Comprehensive Cancer Center and including a substantial group of faculty committing funds and numerous other forms of support to purchase and maintain this new LS7. The new instrument will be incorporated into the long-standing and successful Confocal Core Facility at the University and will thus benefit from stable and well-tested policies for training. The presence of extensive on-campus expertise along with pledged support from experts at nearby institutions promises swift and efficient utilization of this new technology. The expert Manager at the Core not only has several years of experience training users on a variety of microscopes for live and fixed imaging but has even built an alternative lightsheet microscope (diSPIM). The school has long-standing and continuing commitment to support the needs of the Core for space, personnel, and administrative services. Funded by a major NCRR construction grant, the Core has undergone extensive renovations to house our microscopes including the LS7, so the LS7 will be in an excellent facility located central to the Major Users. The system meets the long-term goals of the School to build research resources, including commitments to encourage use of the instrument and the tracking of its productivity. In sum, the availability of this new technology in the School?s Confocal Core Facility would directly and strongly benefit the research of the Major Users. More broadly, by incorporation to annual graduate course offerings and supporting numerous training grants run by and contributed to by our users, the LS7 will add critically important new capabilities to advance all aspects of NIH-sponsored research throughout a large and diverse medical research university.

Activation of NMDA-type glutamate receptors (NMDARs) drives signaling and neuronal plasticity that mediates brain circuit wiring and learning. However, NMDAR overactivation can trigger neuronal cell death and is linked to neurodegeneration, and hypofunction of NMDARs is a leading model of the etiology of schizophrenia and other cognitive disorders. Further, the subsynaptic organization of NMDARs influences the probability of their activation during synaptic transmission, and extrasynaptic receptors are thought to play critical roles in cell death and gene expression. Thus, neurons utilize a variety of mechanisms to control the abundance of NMDARs on the cell surface and particularly in synapses. These mechanisms are complex in part because NMDARs are tetramers, formed from two obligatory GluN1 subunits and two subunits typically from the GluN2 family. Notably, NMDARs with different GluN2 compositions display different biophysical characteristics, and the GluN2 subunits also guide different protein interactions and signaling. Accordingly, different subtypes of NMDARs drive vastly different forms of physiological plasticity and are linked to different disorders. Thus, identifying how neurons control abundance of specific NMDAR subtypes in synapses has been a longstanding goal in neuroscience. Unfortunately, our understanding of these mechanisms has been crucially restricted by inability to visualize one of the key classes of NMDARs in neurons, the triheteromeric receptors, which contain GluN1 and two different GluN2 subunits (most commonly GluN2A+GluN2B). Triheteromeric receptors are thought to be the majority of NMDARs in adult brain, but there are as yet no tools to distinguish them from other NMDAR subtypes in neurons. Thus, their distribution with neurons remains mysterious, and the mechanisms controlling their subcellular trafficking remain almost totally unknown. To overcome this, we here introduce a new tool to visualize triheteromeric NMDARs in neurons. Our strategy is based on bimolecular complementation, and we tagged GluN2A and GluN2B with two parts of a modified fluorescent protein that complement to produce fluorescence only when an NMDAR is assembled containing both the GluN2A and GluN2B subunits (split-tagged NMDARs). Preliminary data demonstrate that split-tagged receptors traffic normally within neurons and accumulate strongly within synapses, and whole-cell recordings demonstrate that their activation by glutamate is unaltered by the presence of the tags. Proceeding from these results, we propose to develop and validate several versions of split-tagged triheteromeric NMDARs useful for different experiments. We will use confocal and super-resolution imaging to provide the first maps of triheteromeric NMDAR distribution in neurons. Finally, because the rate of NMDAR turnover in synapses is critical for determining synaptic strength and plasticity, we will use FRAP and single-molecule tracking to provide the first measures of synaptic exchange of triheteromeric NMDARs. This work will fill longstanding gaps in our knowledge of NMDARs and lay necessary groundwork for investigation of other aspects of triheteromeric NMDAR trafficking in healthy neurons and disease models.