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.

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): 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.

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 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.

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.