Stephen J Smith

All Publications

• A scalable and modular automated pipeline for stitching of large electron microscopy datasets ELIFE Mahalingam, G., Torres, R., Kapner, D., Trautman, E. T., Fliss, T., Seshamani, S., Perlman, E., Young, R., Kinn, S., Buchanan, J., Takeno, M. M., Yin, W., Bumbarger, D. J., Gwinn, R. P., Nyhus, J., Lein, E., Smith, S. J., Reid, R., Khairy, K. A., Saalfeld, S., Collman, F., Macarico da Costa, N. 2022; 11

  Abstract

  Serial-section electron microscopy (ssEM) is the method of choice for studying macroscopic biological samples at extremely high resolution in three dimensions. In the nervous system, nanometer-scale images are necessary to reconstruct dense neural wiring diagrams in the brain, so -called connectomes. The data that can comprise of up to 108 individual EM images must be assembled into a volume, requiring seamless 2D registration from physical section followed by 3D alignment of the stitched sections. The high throughput of ssEM necessitates 2D stitching to be done at the pace of imaging, which currently produces tens of terabytes per day. To achieve this, we present a modular volume assembly software pipeline ASAP (Assembly Stitching and Alignment Pipeline) that is scalable to datasets containing petabytes of data and parallelized to work in a distributed computational environment. The pipeline is built on top of the Render Trautman and Saalfeld (2019) services used in the volume assembly of the brain of adult Drosophila melanogaster (Zheng et al. 2018). It achieves high throughput by operating only on image meta-data and transformations. ASAP is modular, allowing for easy incorporation of new algorithms without significant changes in the workflow. The entire software pipeline includes a complete set of tools for stitching, automated quality control, 3D section alignment, and final rendering of the assembled volume to disk. ASAP has been deployed for continuous stitching of several large-scale datasets of the mouse visual cortex and human brain samples including one cubic millimeter of mouse visual cortex (Yin et al. 2020); Microns Consortium et al. (2021) at speeds that exceed imaging. The pipeline also has multi-channel processing capabilities and can be applied to fluorescence and multi-modal datasets like array tomography.

  View details for DOI 10.7554/eLife.76534

  View details for Web of Science ID 000848544100001

  View details for PubMedID 35880860

  View details for PubMedCentralID PMC9427110

• Cell-type-specific neuromodulation guides synaptic credit assignment in a spiking neural network. Proceedings of the National Academy of Sciences of the United States of America Liu, Y. H., Smith, S., Mihalas, S., Shea-Brown, E., Sumbul, U. 1800; 118 (51)

  Abstract

  Brains learn tasks via experience-driven differential adjustment of their myriad individual synaptic connections, but the mechanisms that target appropriate adjustment to particular connections remain deeply enigmatic. While Hebbian synaptic plasticity, synaptic eligibility traces, and top-down feedback signals surely contribute to solving this synaptic credit-assignment problem, alone, they appear to be insufficient. Inspired by new genetic perspectives on neuronal signaling architectures, here, we present a normative theory for synaptic learning, where we predict that neurons communicate their contribution to the learning outcome to nearby neurons via cell-type-specific local neuromodulation. Computational tests suggest that neuron-type diversity and neuron-type-specific local neuromodulation may be critical pieces of the biological credit-assignment puzzle. They also suggest algorithms for improved artificial neural network learning efficiency.

  View details for DOI 10.1073/pnas.2111821118

  View details for PubMedID 34916291

• Transcriptomic evidence for dense peptidergic networks within forebrains of four widely divergent tetrapods CURRENT OPINION IN NEUROBIOLOGY Smith, S. J. 2021; 71: 100-109

  Abstract

  The primary function common to every neuron is communication with other neurons. Such cell-cell signaling can take numerous forms, including fast synaptic transmission and slower neuromodulation via secreted messengers, such as neuropeptides, dopamine, and many other diffusible small molecules. Individual neurons are quite diverse, however, in all particulars of both synaptic and neuromodulatory communication. Neuron classification schemes have therefore proven very useful in exploring the emergence of network function, behavior, and cognition from the communication functions of individual neurons. Recently published single-cell mRNA sequencing data and corresponding transcriptomic neuron classifications from turtle, songbird, mouse, and human provide evidence for a long evolutionary history and adaptive significance of localized peptidergic signaling. Across all four species, sets of at least twenty orthologous cognate pairs of neuropeptide precursor protein and receptor genes are expressed in individually sparse but heavily overlapping patterns suggesting that all forebrain neuron types are densely interconnected by local peptidergic signals.

  View details for DOI 10.1016/j.conb.2021.09.011

  View details for Web of Science ID 000740937800014

  View details for PubMedID 34775262

• A community-developed open-source computational ecosystem for big neuro data. Nature methods Vogelstein, J. T., Perlman, E., Falk, B., Baden, A., Gray Roncal, W., Chandrashekhar, V., Collman, F., Seshamani, S., Patsolic, J. L., Lillaney, K., Kazhdan, M., Hider, R. J., Pryor, D., Matelsky, J., Gion, T., Manavalan, P., Wester, B., Chevillet, M., Trautman, E. T., Khairy, K., Bridgeford, E., Kleissas, D. M., Tward, D. J., Crow, A. K., Hsueh, B., Wright, M. A., Miller, M. I., Smith, S. J., Vogelstein, R. J., Deisseroth, K., Burns, R. 2018

  View details for PubMedID 30377345

• Mapping synapses by conjugate light-electron array tomography. journal of neuroscience Collman, F., Buchanan, J., Phend, K. D., Micheva, K. D., Weinberg, R. J., Smith, S. J. 2015; 35 (14): 5792-5807

  Abstract

  Synapses of the mammalian CNS are diverse in size, structure, molecular composition, and function. Synapses in their myriad variations are fundamental to neural circuit development, homeostasis, plasticity, and memory storage. Unfortunately, quantitative analysis and mapping of the brain's heterogeneous synapse populations has been limited by the lack of adequate single-synapse measurement methods. Electron microscopy (EM) is the definitive means to recognize and measure individual synaptic contacts, but EM has only limited abilities to measure the molecular composition of synapses. This report describes conjugate array tomography (AT), a volumetric imaging method that integrates immunofluorescence and EM imaging modalities in voxel-conjugate fashion. We illustrate the use of conjugate AT to advance the proteometric measurement of EM-validated single-synapse analysis in a study of mouse cortex.

  View details for DOI 10.1523/JNEUROSCI.4274-14.2015

  View details for PubMedID 25855189

  View details for PubMedCentralID PMC4388933

• Fmr1 KO and Fenobam Treatment Differentially Impact Distinct Synapse Populations of Mouse Neocortex NEURON Wang, G. X., Smith, S. J., Mourrain, P. 2014; 84 (6): 1273-1286

  Abstract

  Cognitive deficits in fragile X syndrome (FXS) are attributed to molecular abnormalities of the brain's vast and heterogeneous synapse populations. Unfortunately, the density of synapses coupled with their molecular heterogeneity presents formidable challenges in understanding the specific contribution of synapse changes in FXS. We demonstrate powerful new methods for the large-scale molecular analysis of individual synapses that allow quantification of numerous specific changes in synapse populations present in the cortex of a mouse model of FXS. Analysis of nearly a million individual synapses reveals distinct, quantitative changes in synaptic proteins distributed across over 6,000 pairwise metrics. Some, but not all, of these synaptic alterations are reversed by treatment with the candidate therapeutic fenobam, an mGluR5 antagonist. These patterns of widespread, but diverse synaptic protein changes in response to global perturbation suggest that FXS and its treatment must be understood as a networked system at the synapse level.

  View details for DOI 10.1016/j.neuron.2014.11.016

  View details for Web of Science ID 000346574500018

  View details for PubMedID 25521380

• Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways NATURE Chung, W., Clarke, L. E., Wang, G. X., Stafford, B. K., Sher, A., Chakraborty, C., Joung, J., Foo, L. C., Thompson, A., Chen, C., Smith, S. J., Barres, B. A. 2013; 504 (7480): 394-?

  Abstract

  To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

  View details for DOI 10.1038/nature12776

  View details for Web of Science ID 000328575300043

  View details for PubMedID 24270812

• Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors NATURE Allen, N. J., Bennett, M. L., Foo, L. C., Wang, G. X., Chakraborty, C., Smith, S. J., Barres, B. A. 2012; 486 (7403): 410-?

  Abstract

  In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

  View details for DOI 10.1038/nature11059

  View details for Web of Science ID 000305466800045

  View details for PubMedID 22722203

  View details for PubMedCentralID PMC3383085

• Deep molecular diversity of mammalian synapses: why it matters and how to measure it NATURE REVIEWS NEUROSCIENCE O'Rourke, N. A., Weiler, N. C., Micheva, K. D., Smith, S. J. 2012; 13 (6): 365-379

  Abstract

  Pioneering studies in the middle of the twentieth century revealed substantial diversity among mammalian chemical synapses and led to a widely accepted classification of synapse type on the basis of neurotransmitter molecule identity. Subsequently, powerful new physiological, genetic and structural methods have enabled the discovery of much deeper functional and molecular diversity within each traditional neurotransmitter type. Today, this deep diversity continues to pose both daunting challenges and exciting new opportunities for neuroscience. Our growing understanding of deep synapse diversity may transform how we think about and study neural circuit development, structure and function.

  View details for DOI 10.1038/nrn3170

  View details for Web of Science ID 000304197000007

  View details for PubMedID 22573027

  View details for PubMedCentralID PMC3670986

• Single-Synapse Analysis of a Diverse Synapse Population: Proteomic Imaging Methods and Markers NEURON Micheva, K. D., Busse, B., Weiler, N. C., O'Rourke, N., Smith, S. J. 2010; 68 (4): 639-653

  Abstract

  A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

  View details for DOI 10.1016/j.neuron.2010.09.024

  View details for Web of Science ID 000285079500005

  View details for PubMedID 21092855

  View details for PubMedCentralID PMC2995697

• Array tomography: A new tool for Imaging the molecular architecture and ultrastructure of neural circuits NEURON Micheva, K. D., Smith, S. J. 2007; 55 (1): 25-36

  Abstract

  Many biological functions depend critically upon fine details of tissue molecular architecture that have resisted exploration by existing imaging techniques. This is particularly true for nervous system tissues, where information processing function depends on intricate circuit and synaptic architectures. Here, we describe a new imaging method, called array tomography, which combines and extends superlative features of modern optical fluorescence and electron microscopy methods. Based on methods for constructing and repeatedly staining and imaging ordered arrays of ultrathin (50-200 nm), resin-embedded serial sections on glass microscope slides, array tomography allows for quantitative, high-resolution, large-field volumetric imaging of large numbers of antigens, fluorescent proteins, and ultrastructure in individual tissue specimens. Compared to confocal microscopy, array tomography offers the advantage of better spatial resolution, in particular along the z axis, as well as depth-independent immunofluorescent staining. The application of array tomography can reveal important but previously unseen features of brain molecular architecture.

  View details for DOI 10.1016/j.neuron.2007.06.014

  View details for Web of Science ID 000248010700006

  View details for PubMedID 17610815

  View details for PubMedCentralID PMC2080672

• Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., Frommer, W. B. 2005; 102 (24): 8740-8745

  Abstract

  Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.

  View details for Web of Science ID 000229807200061

  View details for PubMedID 15939876

  View details for PubMedCentralID PMC1143584

• Functional imaging reveals rapid development of visual response properties in the zebrafish tectum NEURON Niell, C. M., Smith, S. J. 2005; 45 (6): 941-951

  Abstract

  The visual pathway from the retina to the optic tectum in fish and frogs has long been studied as a model for neural circuit formation. Although morphological aspects, such as axonal and dendritic arborization, have been well characterized, less is known about how this translates into functional properties of tectal neurons during development. We developed a system to provide controlled visual stimuli to larval zebrafish, while performing two-photon imaging of tectal neurons loaded with a fluorescent calcium indicator, allowing us to determine visual response properties in intact fish. In relatively mature larvae, we describe receptive field sizes, visual topography, and direction and size selectivity. We also characterize the onset and development of visual responses, beginning when retinal axons first arborize in the tectum. Surprisingly, most of these properties are established soon after dendrite growth and synaptogenesis begin and do not require patterned visual experience or a protracted period of refinement.

  View details for DOI 10.1016/j.neuron.2005.01.047

  View details for PubMedID 15797554

• In vivo imaging evidence for strong effects of synaptogenesis on the growth of both dendritic and axonal arbors in zebrafish optic tectum Smith, S. J., Niell, C. M., Meyer, M. P. FEDERATION AMER SOC EXP BIOL. 2005: A786–A787

  View details for Web of Science ID 000227610705464

• Stability and plasticity of developing synapses in hippocampal neuronal cultures JOURNAL OF NEUROSCIENCE Hopf, F. W., Waters, J., Mehta, S., Smith, S. J. 2002; 22 (3): 775-781

  Abstract

  To explore mechanisms governing the formation, stability, and elimination of synapses during neuronal development, we used FM 1-43 fluorescence imaging to track vesicle turnover at >7000 individually identified developing synapses between embryonic rat hippocampal neurons in culture. The majority of presynaptic boutons were stable in efficacy and position over a period of 1.5 hr. Activity, evoked by burst-patterned field stimulation, decreased presynaptic function across the population of boutons, an effect that required NMDA receptor activation. Decreased FM 1-43 staining correlated with low synapsin-I and synaptophysin immunoreactivities, suggesting that decreased presynaptic function was commensurate with synaptic disassembly. These observations provide new information on the stability of developing presynaptic function and suggest that NMDA receptor activation may regulate the stability of developing synapses.

  View details for Web of Science ID 000173660800027

  View details for PubMedID 11826107

• Optical detection of a quantal presynaptic membrane turnover NATURE Ryan, T. A., Reuter, H., Smith, S. J. 1997; 388 (6641): 478-482

  Abstract

  Exploration of the mechanisms and plasticity of synaptic transmission has been hindered by the lack of a method to measure single vesicle turnover directly in individual presynaptic boutons at isolated nerve terminals. Although postsynaptic electrical recordings have provided a wealth of invaluable basic information about quantal presynaptic processes, this approach has often proved difficult to apply at most central nervous system synapses. Here we describe the direct optical detection of single quantal events in individual presynaptic boutons of cultured hippocampal neurons. Using the fluorescent dye FM 1-43 as a tracer for presynaptic endocytosis, we have characterized both evoked and spontaneous components of presynaptic function at the level of individual quanta. Our results are consistent with quantal interpretations of previous electrophysiological analyses and provide new information about the unitary membrane recycling event and its coupling to individual action potential stimuli, about spontaneous vesicle turnover at individual boutons, and about the numbers of vesicles recycling at individual boutons.

  View details for PubMedID 9242407

• Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion JOURNAL OF CELL BIOLOGY Adams, C. L., NELSON, W. J., Smith, S. J. 1996; 135 (6): 1899-1911

  Abstract

  Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.

  View details for PubMedID 8991100

• Evidence for a role of dendritic filopodia in synaptogenesis and spine formation NEURON Ziv, N. E., Smith, S. J. 1996; 17 (1): 91-102

  Abstract

  Axo-dendritic synaptogenesis was examined in live hippocampal cell cultures using the fluorescent dyes DiO to label dendrites and FM 4-64 to label functional presynaptic boutons. As the first functional synaptic boutons appeared in these cultures, numerous filopodia (up to 10 micron long) were observed to extend transiently (mean lifetime 9.5 min) from dendritic shafts. With progressively increasing numbers of boutons, there were coincident decreases in numbers of transient filopodia and increases in numbers of stable dendritic spines. Dendritic filopodia were observed to initiate physical contacts with nearby axons. This sometimes resulted in filopodial stabilization and formation of functional presynaptic boutons. These findings suggest that dendritic filopodia may actively initiate synaptogenic contacts with nearby (5-10 micron) axons and thereafter evolve into dendritic spines.

  View details for Web of Science ID A1996UY98100010

  View details for PubMedID 8755481

• The dynamics of dendritic structure in developing hippocampal slices JOURNAL OF NEUROSCIENCE Dailey, M. E., Smith, S. J. 1996; 16 (9): 2983-2994

  Abstract

  Time-lapse fluorescence confocal microscopy was used to directly visualize the formation and dynamics of postsynaptic target structures (i.e., dendritic branches and spines) on pyramidal neurons within developing tissue slices. Within a 2 week period of time, pyramidal neurons in cultured slices derived from early postnatal rat (postnatal days 2-7) developed complex dendritic arbors bearing numerous postsynaptic spines. At early stages (1-2 d in vitro), many fine filopodial protrusions on dendrite shafts rapidly extended (maximum rate approximately 2.5 microM/minute) and retracted (median filopodial lifetime, 10 min), but some filopodia transformed into growth cones and nascent dendrite branches. As dendritic arbors matured, the population of fleeting lateral filopodia was replaced by spine-like structures having a low rate of turnover. This developmental progression involved a transitional stage in which dendrites were dominated by persistent (up to 22 hr) but dynamic spiny protrusions (i.e., protospines) that showed substantial changes in length and shape on a timescale of minutes. These observations reveal a highly dynamic state of postsynaptic target structures that may actively contribute to the formation and plasticity of synaptic connections during CNS development.

  View details for Web of Science ID A1996UF71100012

  View details for PubMedID 8622128

• THE KINETICS OF SYNAPTIC VESICLE RECYCLING MEASURED AT SINGLE PRESYNAPTIC BOUTONS NEURON Ryan, T. A., Reuter, H., Wendland, B., Schweizer, F. E., Tsien, R. W., Smith, S. J. 1993; 11 (4): 713-724

  Abstract

  We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a t1/2 approximately 60 s (approximately 24 degrees C); once internalized, vesicles become reavailable for exocytosis in approximately 30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Ca2+.

  View details for Web of Science ID A1993MD06800014

  View details for PubMedID 8398156

• Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses. eNeuro Micheva, K. D., Gong, B., Collman, F., Weinberg, R. J., Smith, S. J., Trimmer, J. S., Murray, K. D. 2023

  Abstract

  Antibody-based imaging techniques rely on reagents whose performance may be application-specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique.Significance StatementArray tomography (AT) is a powerful volume microscopy technique for high-dimensional analysis of complex protein populations in cells and organelles, including synapses. AT involves the use of ultrathin serial sections embedded in resin and subjected to multiple rounds of immunofluorescence antibody (Ab) labeling and imaging. AT relies on antibody-based detection of proteins but because commercial antibodies are typically validated for other applications they often fail for AT. To identify antibodies with high probability of success in AT we developed a novel screening strategy and used this to create a comprehensive database of AT-validated antibodies for neuroscience.

  View details for DOI 10.1523/ENEURO.0290-23.2023

  View details for PubMedID 37945352

• A synapse census for the ages. Science (New York, N.Y.) Micheva, K. D., Weinberg, R. J., Smith, S. J. 2020; 369 (6501): 253–54

  View details for DOI 10.1126/science.abc9555

  View details for PubMedID 32675362

• Progress and Future Goals for Neuroscience NEURON Moser, M., Gallant, J. L., Hu, H., Buckner, R. L., Stevens, M. M., Smith, S. J. 2018; 100 (1): 14–15

  Abstract

  Scientists reflect on how neuroscience has quickly progressed and continues to open new avenues for great discovery with improvements in imaging, interrogating, and interfacing with the nervous system of experimental model systems and humans.

  View details for DOI 10.1016/j.neuron.2018.09.037

  View details for Web of Science ID 000446862000006

  View details for PubMedID 30308167

• Distinctive Structural and Molecular Features of Myelinated Inhibitory Axons in Human Neocortex. eNeuro Micheva, K. D., Chang, E. F., Nana, A. L., Seeley, W. W., Ting, J. T., Cobbs, C., Lein, E., Smith, S. J., Weinberg, R. J., Madison, D. V. 2018; 5 (5)

  Abstract

  Numerous types of inhibitory neurons sculpt the performance of human neocortical circuits, with each type exhibiting a constellation of subcellular phenotypic features in support of its specialized functions. Axonal myelination has been absent among the characteristics used to distinguish inhibitory neuron types; in fact, very little is known about myelinated inhibitory axons in human neocortex. Here, using array tomography to analyze samples of neurosurgically excised human neocortex, we show that inhibitory myelinated axons originate predominantly from parvalbumin-containing interneurons. Compared to myelinated excitatory axons, they have higher neurofilament and lower microtubule content, shorter nodes of Ranvier, and more myelin basic protein (MBP) in their myelin sheath. Furthermore, these inhibitory axons have more mitochondria, likely to sustain the high energy demands of parvalbumin interneurons, as well as more 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein enriched in the myelin cytoplasmic channels that are thought to facilitate the delivery of nutrients from ensheathing oligodendrocytes. Our results demonstrate that myelinated axons of parvalbumin inhibitory interneurons exhibit distinctive features that may support the specialized functions of this neuron type in human neocortical circuits.

  View details for PubMedID 30406183

• A Computational Synaptic Antibody Characterization Tool for Array Tomography FRONTIERS IN NEUROANATOMY Simhal, A. K., Gong, B., Trimmer, J. S., Weinberg, R. J., Smith, S. J., Sapiro, G., Micheva, K. D. 2018; 12

  View details for DOI 10.3389/fnana.2018.00051

  View details for Web of Science ID 000438942600001

• A Computational Synaptic Antibody Characterization Tool for Array Tomography. Frontiers in neuroanatomy Simhal, A. K., Gong, B., Trimmer, J. S., Weinberg, R. J., Smith, S. J., Sapiro, G., Micheva, K. D. 2018; 12: 51

  Abstract

  Application-specific validation of antibodies is a critical prerequisite for their successful use. Here we introduce an automated framework for characterization and screening of antibodies against synaptic molecules for high-resolution immunofluorescence array tomography (AT). The proposed Synaptic Antibody Characterization Tool (SACT) is designed to provide an automatic, robust, flexible, and efficient tool for antibody characterization at scale. SACT automatically detects puncta of immunofluorescence labeling from candidate antibodies and determines whether a punctum belongs to a synapse. The molecular composition and size of the target synapses expected to contain the antigen is determined by the user, based on biological knowledge. Operationally, the presence of a synapse is defined by the colocalization or adjacency of the candidate antibody punctum to one or more reference antibody puncta. The outputs of SACT are automatically computed measurements such as target synapse density and target specificity ratio that reflect the sensitivity and specificity of immunolabeling with a given candidate antibody. These measurements provide an objective way to characterize and compare the performance of different antibodies against the same target, and can be used to objectively select the antibodies best suited for AT and potentially for other immunolabeling applications.

  View details for DOI 10.3389/fnana.2018.00051

  View details for PubMedID 30065633

  View details for PubMedCentralID PMC6057115

• Probabilistic fluorescence-based synapse detection. PLoS computational biology Simhal, A. K., Aguerrebere, C., Collman, F., Vogelstein, J. T., Micheva, K. D., Weinberg, R. J., Smith, S. J., Sapiro, G. 2017; 13 (4)

  Abstract

  Deeper exploration of the brain's vast synaptic networks will require new tools for high-throughput structural and molecular profiling of the diverse populations of synapses that compose those networks. Fluorescence microscopy (FM) and electron microscopy (EM) offer complementary advantages and disadvantages for single-synapse analysis. FM combines exquisite molecular discrimination capacities with high speed and low cost, but rigorous discrimination between synaptic and non-synaptic fluorescence signals is challenging. In contrast, EM remains the gold standard for reliable identification of a synapse, but offers only limited molecular discrimination and is slow and costly. To develop and test single-synapse image analysis methods, we have used datasets from conjugate array tomography (cAT), which provides voxel-conjugate FM and EM (annotated) images of the same individual synapses. We report a novel unsupervised probabilistic method for detection of synapses from multiplex FM (muxFM) image data, and evaluate this method both by comparison to EM gold standard annotated data and by examining its capacity to reproduce known important features of cortical synapse distributions. The proposed probabilistic model-based synapse detector accepts molecular-morphological synapse models as user queries, and delivers a volumetric map of the probability that each voxel represents part of a synapse. Taking human annotation of cAT EM data as ground truth, we show that our algorithm detects synapses from muxFM data alone as successfully as human annotators seeing only the muxFM data, and accurately reproduces known architectural features of cortical synapse distributions. This approach opens the door to data-driven discovery of new synapse types and their density. We suggest that our probabilistic synapse detector will also be useful for analysis of standard confocal and super-resolution FM images, where EM cross-validation is not practical.

  View details for DOI 10.1371/journal.pcbi.1005493

  View details for PubMedID 28414801

• Sub-synaptic, multiplexed analysis of proteins reveals Fragile X related protein 2 is mislocalized in Fmr1 KO synapses. eLife Wang, G. X., Smith, S. J., Mourrain, P. 2016; 5

  Abstract

  The distribution of proteins within sub-synaptic compartments is an essential aspect of their neurological function. Current methodologies, such as electron microscopy (EM) and super-resolution imaging techniques, can provide the precise localization of proteins, but are often limited to a small number of one-time observations with narrow spatial and molecular coverage. The diversity of synaptic proteins and synapse types demands synapse analysis on a scale that is prohibitive with current methods. Here, we demonstrate SubSynMAP, a fast, multiplexed sub-synaptic protein analysis method using wide-field data from deconvolution array tomography (ATD). SubSynMAP generates probability distributions for that reveal the functional range of proteins within the averaged synapse of a particular class. This enables the differentiation of closely juxtaposed proteins. Using this method, we analyzed 15 synaptic proteins in normal and Fragile X mental retardation syndrome (FXS) model mouse cortex, and revealed disease-specific modifications of sub-synaptic protein distributions across synapse classes and cortical layers.

  View details for DOI 10.7554/eLife.20560

  View details for PubMedID 27770568

  View details for PubMedCentralID PMC5098911

• Sub-synaptic, multiplexed analysis of proteins reveals Fragile X related protein 2 is mislocalized in Fmr1 KO synapses ELIFE Wang, G. X., Smith, S. J., Mourrain, P. 2016; 5

  Abstract

  The distribution of proteins within sub-synaptic compartments is an essential aspect of their neurological function. Current methodologies, such as electron microscopy (EM) and super-resolution imaging techniques, can provide the precise localization of proteins, but are often limited to a small number of one-time observations with narrow spatial and molecular coverage. The diversity of synaptic proteins and synapse types demands synapse analysis on a scale that is prohibitive with current methods. Here, we demonstrate SubSynMAP, a fast, multiplexed sub-synaptic protein analysis method using wide-field data from deconvolution array tomography (ATD). SubSynMAP generates probability distributions for that reveal the functional range of proteins within the averaged synapse of a particular class. This enables the differentiation of closely juxtaposed proteins. Using this method, we analyzed 15 synaptic proteins in normal and Fragile X mental retardation syndrome (FXS) model mouse cortex, and revealed disease-specific modifications of sub-synaptic protein distributions across synapse classes and cortical layers.

  View details for DOI 10.7554/eLife.20560

  View details for Web of Science ID 000388096400001

  View details for PubMedCentralID PMC5098911

• Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target BRAIN Hiu, T., Farzampour, Z., Paz, J. T., Wang, E. H., Badgely, C., Olson, A., Micheva, K. D., Wang, G., Lemmens, R., Tran, K. V., Nishiyama, Y., Liang, X., Hamilton, S. A., O'Rourke, N., Smith, S. J., Huguenard, J. R., Bliss, T. M., Steinberg, G. K. 2016; 139: 468-480

  Abstract

  Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem's potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery.

  View details for DOI 10.1093/brain/awv360

  View details for Web of Science ID 000370205100025

  View details for PubMedID 26685158

  View details for PubMedCentralID PMC4805083

• A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons. eLife Micheva, K. D., Wolman, D., Mensh, B. D., Pax, E., Buchanan, J., Smith, S. J., Bock, D. D. 2016; 5

  Abstract

  Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits.

  View details for DOI 10.7554/eLife.15784

  View details for PubMedID 27383052

  View details for PubMedCentralID PMC4972537

• A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons. eLife Micheva, K. D., Wolman, D., Mensh, B. D., Pax, E., Buchanan, J., Smith, S. J., Bock, D. D. 2016; 5

  Abstract

  Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits.

  View details for DOI 10.7554/eLife.15784

  View details for PubMedID 27383052

  View details for PubMedCentralID PMC4972537

• Generation of a Functional Human Cortex from Pluripotent Stem Cells Sloan, S., Pasca, A., Clarke, L., Tian, Y., Kim, C., Park, J., O'Rourke, N., Smith, S., Geschwind, D., Barres, B., Pasca, S. NATURE PUBLISHING GROUP. 2015: S524

  View details for Web of Science ID 000366597700854

• Knowing a synapse when you see one FRONTIERS IN NEUROANATOMY Burette, A., Collman, F., Micheva, K. D., Smith, S. J., Weinberg, R. J. 2015; 9

  Abstract

  Recent years have seen a rapidly growing recognition of the complexity and diversity of the myriad individual synaptic connections that define brain synaptic networks. It has also become increasingly apparent that the synapses themselves are a major key to understanding the development, function and adaptability of those synaptic networks. In spite of this growing appreciation, the molecular, structural and functional characteristics of individual synapses and the patterning of their diverse characteristics across functional networks have largely eluded quantitative study with available imaging technologies. Here we offer an overview of new computational imaging methods that promise to bring single-synapse analysis of synaptic networks to the fore. We focus especially on the challenges and opportunities associated with quantitative detection of individual synapses and with measuring individual synapses across network scale populations in mammalian brain.

  View details for DOI 10.3389/fnana.2015.00100

  View details for Web of Science ID 000360431900001

  View details for PubMedCentralID PMC4517447

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods Pasca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J., O'Rourke, N. A., Nguyen, K. D., Smith, S. J., Huguenard, J. R., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015; 12 (7): 671-678

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedID 26005811

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods Pasca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J., O'Rourke, N. A., Nguyen, K. D., Smith, S. J., Huguenard, J. R., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015; 12 (7): 671-678

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedID 26005811

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D cultures. Nature Methods Pasca, A. M., Sloan, S., Clarke, L. E., Tian, Y., Makinson, C., Huber, N., Kim, C., Park, J., O’Rourke, N., Nguyen, K., Smith, S. J., Huguenard, J., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015: 671–78

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedCentralID PMC4489980

• Knowing a synapse when you see one. Frontiers in neuroanatomy Burette, A., Collman, F., Micheva, K. D., Smith, S. J., Weinberg, R. J. 2015; 9: 100-?

  Abstract

  Recent years have seen a rapidly growing recognition of the complexity and diversity of the myriad individual synaptic connections that define brain synaptic networks. It has also become increasingly apparent that the synapses themselves are a major key to understanding the development, function and adaptability of those synaptic networks. In spite of this growing appreciation, the molecular, structural and functional characteristics of individual synapses and the patterning of their diverse characteristics across functional networks have largely eluded quantitative study with available imaging technologies. Here we offer an overview of new computational imaging methods that promise to bring single-synapse analysis of synaptic networks to the fore. We focus especially on the challenges and opportunities associated with quantitative detection of individual synapses and with measuring individual synapses across network scale populations in mammalian brain.

  View details for DOI 10.3389/fnana.2015.00100

  View details for PubMedID 26283929

• Synaptic molecular imaging in spared and deprived columns of mouse barrel cortex with array tomography. Scientific data Weiler, N. C., Collman, F., Vogelstein, J. T., Burns, R., Smith, S. J. 2014; 1: 140046-?

  Abstract

  A major question in neuroscience is how diverse subsets of synaptic connections in neural circuits are affected by experience dependent plasticity to form the basis for behavioral learning and memory. Differences in protein expression patterns at individual synapses could constitute a key to understanding both synaptic diversity and the effects of plasticity at different synapse populations. Our approach to this question leverages the immunohistochemical multiplexing capability of array tomography (ATomo) and the columnar organization of mouse barrel cortex to create a dataset comprising high resolution volumetric images of spared and deprived cortical whisker barrels stained for over a dozen synaptic molecules each. These dataset has been made available through the Open Connectome Project for interactive online viewing, and may also be downloaded for offline analysis using web, Matlab, and other interfaces.

  View details for DOI 10.1038/sdata.2014.46

  View details for PubMedID 25977797

  View details for PubMedCentralID PMC4411012

• Synaptic molecular imaging in spared and deprived columns of mouse barrel cortex with array tomography SCIENTIFIC DATA Weiler, N. C., ColIman, F., Vogelstein, J. T., Burns, R., Smith, S. J. 2014; 1

  Abstract

  A major question in neuroscience is how diverse subsets of synaptic connections in neural circuits are affected by experience dependent plasticity to form the basis for behavioral learning and memory. Differences in protein expression patterns at individual synapses could constitute a key to understanding both synaptic diversity and the effects of plasticity at different synapse populations. Our approach to this question leverages the immunohistochemical multiplexing capability of array tomography (ATomo) and the columnar organization of mouse barrel cortex to create a dataset comprising high resolution volumetric images of spared and deprived cortical whisker barrels stained for over a dozen synaptic molecules each. These dataset has been made available through the Open Connectome Project for interactive online viewing, and may also be downloaded for offline analysis using web, Matlab, and other interfaces.

  View details for DOI 10.1038/sdata.2014.46

  View details for Web of Science ID 000209843500042

  View details for PubMedCentralID PMC4411012

• Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature Chung, W., Clarke, L. E., Wang, G. X., Stafford, B. K., Sher, A., Chakraborty, C., Joung, J., Foo, L. C., Thompson, A., Chen, C., Smith, S. J., Barres, B. A. 2013; 504 (7480): 394-400

  Abstract

  To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

  View details for DOI 10.1038/nature12776

  View details for PubMedID 24270812

• Accelerated Experience-Dependent Pruning of Cortical Synapses in Ephrin-A2 Knockout Mice NEURON Yu, X., Wang, G., Gilmore, A., Yee, A. X., Li, X., Xu, T., Smith, S. J., Chen, L., Zuo, Y. 2013; 80 (1): 64-71

  Abstract

  Refinement of mammalian neural circuits involves substantial experience-dependent synapse elimination. Using in vivo two-photon imaging, we found that experience-dependent elimination of postsynaptic dendritic spines in the cortex was accelerated in ephrin-A2 knockout (KO) mice, resulting in fewer adolescent spines integrated into adult circuits. Such increased spine removal in ephrin-A2 KOs depended on activation of glutamate receptors, as blockade of the N-methyl-D-aspartate (NMDA) receptors eliminated the difference in spine loss between wild-type and KO mice. We also showed that ephrin-A2 in the cortex colocalized with glial glutamate transporters, which were significantly downregulated in ephrin-A2 KOs. Consistently, glial glutamate transport was reduced in ephrin-A2 KOs, resulting in an accumulation of synaptic glutamate. Finally, inhibition of glial glutamate uptake promoted spine elimination in wild-type mice, resembling the phenotype of ephrin-A2 KOs. Together, our results suggest that ephrin-A2 regulates experience-dependent, NMDA receptor-mediated synaptic pruning through glial glutamate transport during maturation of the mouse cortex.

  View details for DOI 10.1016/j.neuron.2013.07.014

  View details for Web of Science ID 000326305300008

  View details for PubMedID 24094103

• Accelerated Experience-Dependent Pruning of Cortical Synapses in Ephrin-A2 Knockout Mice NEURON Yu, X., Wang, G., Gilmore, A., Yee, A. X., Li, X., Xu, T., Smith, S. J., Chen, L., Zuo, Y. 2013; 80 (1): 64-71

  Abstract

  Refinement of mammalian neural circuits involves substantial experience-dependent synapse elimination. Using in vivo two-photon imaging, we found that experience-dependent elimination of postsynaptic dendritic spines in the cortex was accelerated in ephrin-A2 knockout (KO) mice, resulting in fewer adolescent spines integrated into adult circuits. Such increased spine removal in ephrin-A2 KOs depended on activation of glutamate receptors, as blockade of the N-methyl-D-aspartate (NMDA) receptors eliminated the difference in spine loss between wild-type and KO mice. We also showed that ephrin-A2 in the cortex colocalized with glial glutamate transporters, which were significantly downregulated in ephrin-A2 KOs. Consistently, glial glutamate transport was reduced in ephrin-A2 KOs, resulting in an accumulation of synaptic glutamate. Finally, inhibition of glial glutamate uptake promoted spine elimination in wild-type mice, resembling the phenotype of ephrin-A2 KOs. Together, our results suggest that ephrin-A2 regulates experience-dependent, NMDA receptor-mediated synaptic pruning through glial glutamate transport during maturation of the mouse cortex.

  View details for DOI 10.1016/j.neuron.2013.07.014

  View details for Web of Science ID 000326305300008

  View details for PubMedID 24094103

  View details for PubMedCentralID PMC3792401

• Automated Analysis of a Diverse Synapse Population PLOS COMPUTATIONAL BIOLOGY Busse, B., Smith, S. 2013; 9 (3)

  Abstract

  Synapses of the mammalian central nervous system are highly diverse in function and molecular composition. Synapse diversity per se may be critical to brain function, since memory and homeostatic mechanisms are thought to be rooted primarily in activity-dependent plastic changes in specific subsets of individual synapses. Unfortunately, the measurement of synapse diversity has been restricted by the limitations of methods capable of measuring synapse properties at the level of individual synapses. Array tomography is a new high-resolution, high-throughput proteomic imaging method that has the potential to advance the measurement of unit-level synapse diversity across large and diverse synapse populations. Here we present an automated feature extraction and classification algorithm designed to quantify synapses from high-dimensional array tomographic data too voluminous for manual analysis. We demonstrate the use of this method to quantify laminar distributions of synapses in mouse somatosensory cortex and validate the classification process by detecting the presence of known but uncommon proteomic profiles. Such classification and quantification will be highly useful in identifying specific subpopulations of synapses exhibiting plasticity in response to perturbations from the environment or the sensory periphery.

  View details for DOI 10.1371/journal.pcbi.1002976

  View details for Web of Science ID 000316864200047

  View details for PubMedID 23555213

  View details for PubMedCentralID PMC3610606

• Increaed GABA(A) Mediated Synaptic Activity and Structural Remodeling in Peri-infarct Cortex Layer 5 in the Post-stroke Rodent Brain. Hiu, T., Bliss, T. M., Farzampour, Z., Paz, J. T., Olson, A., Micheva, K. D., Wang, E. H., Wang, G., Manley, N., Nishiyama, Y., Arac, A., O'Rourke, N., Huguenard, J. R., Smith, S. J., Steinberg, G. K., Tran, K. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000330540200438

• Human Neural Stem Cells Enhance Synaptic Structural Remodeling in the Ischemic Brain. Hiu, T., Bliss, T. M., Manley, N., Wang, E. H., Wang, G., Micheva, K. D., Olson, A., Smith, S. J., Steinberg, G. K. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000330540201322

• Multiple Antibody Colocalization Imaging of Skeletal Muscle Ghochani, M., Humphrey, G., Farrington, J., Miyake, K., Micheva, K., Busse, B., Blank, P., Smith, S., Zimmerberg, J. CELL PRESS. 2013: 485A

  View details for DOI 10.1016/j.bpj.2012.11.2675

  View details for Web of Science ID 000316074304460

• The Open Connectome Project Data Cluster: Scalable Analysis and Vision for High-Throughput Neuroscience. Scientific and statistical database management : International Conference, SSDBM ... : proceedings. International Conference on Scientific and Statistical Database Management Burns, R., Roncal, W. G., Kleissas, D., Lillaney, K., Manavalan, P., Perlman, E., Berger, D. R., Bock, D. D., Chung, K., Grosenick, L., Kasthuri, N., Weiler, N. C., Deisseroth, K., Kazhdan, M., Lichtman, J., Reid, R. C., Smith, S. J., Szalay, A. S., Vogelstein, J. T., Vogelstein, R. J. 2013

  Abstract

  We describe a scalable database cluster for the spatial analysis and annotation of high-throughput brain imaging data, initially for 3-d electron microscopy image stacks, but for time-series and multi-channel data as well. The system was designed primarily for workloads that build connectomes- neural connectivity maps of the brain-using the parallel execution of computer vision algorithms on high-performance compute clusters. These services and open-science data sets are publicly available at openconnecto.me. The system design inherits much from NoSQL scale-out and data-intensive computing architectures. We distribute data to cluster nodes by partitioning a spatial index. We direct I/O to different systems-reads to parallel disk arrays and writes to solid-state storage-to avoid I/O interference and maximize throughput. All programming interfaces are RESTful Web services, which are simple and stateless, improving scalability and usability. We include a performance evaluation of the production system, highlighting the effec-tiveness of spatial data organization.

  View details for PubMedID 24401992

• Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens PLOS COMPUTATIONAL BIOLOGY Wang, G., Smith, S. J. 2012; 8 (8)

  Abstract

  Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA(2) (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is -220 nm and -600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50-100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.

  View details for DOI 10.1371/journal.pcbi.1002671

  View details for Web of Science ID 000308553500046

  View details for PubMedID 22956902

  View details for PubMedCentralID PMC3431294

• Three-Dimensional Microstructural Changes in Murine Abdominal Aortic Aneurysms Quantified Using Immunofluorescent Array Tomography JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY Saatchi, S., Azuma, J., Wanchoo, N., Smith, S. J., Yock, P. G., Taylor, C. A., Tsao, P. S. 2012; 60 (2): 97-109

  Abstract

  This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.

  View details for DOI 10.1369/0022155411433066

  View details for Web of Science ID 000299481300001

  View details for PubMedID 22140132

  View details for PubMedCentralID PMC3351119

• High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy NATURE PROTOCOLS Tapia, J. C., Kasthuri, N., Hayworth, K. J., Schalek, R., Lichtman, J. W., Smith, S. J., Buchanan, J. 2012; 7 (2): 193-206

  Abstract

  Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

  View details for DOI 10.1038/nprot.2011.439

  View details for Web of Science ID 000300402300001

  View details for PubMedID 22240582

• Bidirectional Regulation of Dendritic Voltage-Gated Potassium Channels by the Fragile X Mental Retardation Protein NEURON Lee, H. Y., Ge, W., Huang, W., He, Y., Wang, G. X., Rowson-Baldwin, A., Smith, S. J., Jan, Y. N., Jan, L. Y. 2011; 72 (4): 630-642

  Abstract

  How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.

  View details for DOI 10.1016/j.neuron.2011.09.033

  View details for Web of Science ID 000297180100013

  View details for PubMedID 22099464

  View details for PubMedCentralID PMC3433402

• Large-Scale Automated Histology in the Pursuit of Connectomes JOURNAL OF NEUROSCIENCE Kleinfeld, D., Bharioke, A., Blinder, P., Bock, D. D., Briggman, K. L., Chklovskii, D. B., Denk, W., Helmstaedter, M., Kaufhold, J. P., Lee, W. A., Meyer, H. S., Micheva, K. D., Oberlaender, M., Prohaska, S., Reid, R. C., Smith, S. J., Takemura, S., Tsai, P. S., Sakmann, B. 2011; 31 (45): 16125-16138

  Abstract

  How does the brain compute? Answering this question necessitates neuronal connectomes, annotated graphs of all synaptic connections within defined brain areas. Further, understanding the energetics of the brain's computations requires vascular graphs. The assembly of a connectome requires sensitive hardware tools to measure neuronal and neurovascular features in all three dimensions, as well as software and machine learning for data analysis and visualization. We present the state of the art on the reconstruction of circuits and vasculature that link brain anatomy and function. Analysis at the scale of tens of nanometers yields connections between identified neurons, while analysis at the micrometer scale yields probabilistic rules of connection between neurons and exact vascular connectivity.

  View details for DOI 10.1523/JNEUROSCI.4077-11.2011

  View details for Web of Science ID 000296799700012

  View details for PubMedID 22072665

• The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels CELLULAR AND MOLECULAR BIOENGINEERING Saatchi, S., Wanchoo, N., Azuma, J., Smith, S. J., Tsao, P. S., Yock, P. G., Taylor, C. A. 2011; 4 (2): 311-323

  View details for DOI 10.1007/s12195-011-0165-z

  View details for Web of Science ID 000290959200016

• Characterization of genetically targeted neuron types in the zebrafish optic tectum FRONTIERS IN NEURAL CIRCUITS Robles, E., Smith, S. J., Baier, H. 2011; 5

  Abstract

  The optically transparent larval zebrafish is ideally suited for in vivo analyses of neural circuitry controlling visually guided behaviors. However, there is a lack of information regarding specific cell types in the major retinorecipient brain region of the fish, the optic tectum. Here we report the characterization of three previously unidentified tectal cell types that are specifically labeled by dlx5/6 enhancer elements. In vivo laser-scanning microscopy in conjunction with ex vivo array tomography revealed that these neurons differ in their morphologies, synaptic connectivity, and neurotransmitter phenotypes. The first type is an excitatory bistratified periventricular interneuron that forms a dendritic arbor in the retinorecipient stratum fibrosum et griseum superficiale (SFGS) and an axonal arbor in the stratum griseum centrale (SGC). The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS. The third type is a GABAergic periventricular projection neuron that extends a dendritic arbor into the SGC and a long axon to the torus semicircularis, medulla oblongata, and anterior hindbrain. Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale. This approach revealed several novel aspects of tectal circuitry, including: (1) a glutamatergic mode of transmission from the superficial, retinorecipient neuropil layers to the deeper, output layers, (2) the presence of interneurons with mixed dendrite/axon arbors likely involved in local processing, and (3) a heretofore unknown GABAergic tectofugal projection to midbrain and hindbrain. These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system.

  View details for DOI 10.3389/fncir.2011.00001

  View details for Web of Science ID 000289442400001

  View details for PubMedID 21390291

  View details for PubMedCentralID PMC3046383

• Novel 3-Dimensional Microscopy Methodology Development to Study Regional Microstructural Changes over the Time Course of Abdominal Aortic Aneurysm Development Scientific Sessions on Arteriosclerosis, Thrombosis and Vascular Biology Saatchi, S., Azuma, J., Wanchoo, N., Tsao, P. S., Smith, S. J., Yock, P. G., Taylor, C. A. LIPPINCOTT WILLIAMS & WILKINS. 2010: E277–E277

  View details for Web of Science ID 000283234800423

• Array tomography: imaging stained arrays. Cold Spring Harbor protocols Micheva, K. D., O'Rourke, N., Busse, B., Smith, S. J. 2010; 2010 (11): pdb prot5526-?

  Abstract

  Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are imaged using conventional wide-field fluorescence microscopy. Images can be captured manually or, with the appropriate software and hardware, the process can be automated.

  View details for DOI 10.1101/pdb.prot5526

  View details for PubMedID 21041399

• Array tomography: production of arrays. Cold Spring Harbor protocols Micheva, K. D., O'Rourke, N., Busse, B., Smith, S. J. 2010; 2010 (11): pdb prot5524-?

  Abstract

  Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time consuming and require some practice to perfect. This protocol describes the sectioning of embedded tissues and the mounting of the serial arrays. The procedures require some familiarity with the techniques used for ultramicrotome sectioning for electron microscopy.

  View details for DOI 10.1101/pdb.prot5524

  View details for PubMedID 21041397

• Array tomography: immunostaining and antibody elution. Cold Spring Harbor protocols Micheva, K. D., O'Rourke, N., Busse, B., Smith, S. J. 2010; 2010 (11): pdb prot5525-?

  Abstract

  Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are prepared for imaging by tagging with primary antibodies against specific cellular targets, followed by labeling with fluorescent secondary antibodies. Alternatively, fluorescent proteins that have been introduced into the tissue before dissection can be used.

  View details for DOI 10.1101/pdb.prot5525

  View details for PubMedID 21041398

• Array tomography: rodent brain fixation and embedding. Cold Spring Harbor protocols Micheva, K. D., O'Rourke, N., Busse, B., Smith, S. J. 2010; 2010 (11): pdb prot5523-?

  Abstract

  Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. This protocol describes the fixation and processing required to prepare tissues for immunofluorescence array tomography.

  View details for DOI 10.1101/pdb.prot5523

  View details for PubMedID 21041396

• Array tomography: semiautomated image alignment. Cold Spring Harbor protocols Micheva, K. D., O'Rourke, N., Busse, B., Smith, S. J. 2010; 2010 (11): pdb prot5527-?

  Abstract

  Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. Successful array tomography requires that the captured images be properly stacked and aligned, and the software to achieve these ends is freely available. This protocol describes the construction of volumetric image stacks from images of fluorescently labeled arrays for three-dimensional image visualization, analysis, and archiving.

  View details for DOI 10.1101/pdb.prot5527

  View details for PubMedID 21041400

• Array tomography: high-resolution three-dimensional immunofluorescence. Cold Spring Harbor protocols Micheva, K. D., O'Rourke, N., Busse, B., Smith, S. J. 2010; 2010 (11): pdb top89-?

  Abstract

  Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Although the fabrication procedures can be relatively difficult, the high resolution, depth invariance, and molecular discrimination offered by array tomography justify the effort involved. Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture.

  View details for DOI 10.1101/pdb.top89

  View details for PubMedID 21041404

• Circadian and Homeostatic Regulation of Structural Synaptic Plasticity in Hypocretin Neurons NEURON Appelbaum, L., Wang, G., Yokogawa, T., Skariah, G. M., Smith, S. J., Mourrain, P., Mignot, E. 2010; 68 (1): 87-98

  Abstract

  Neurons exhibit rhythmic activity that ultimately affects behavior such as sleep. In living zebrafish larvae, we used time-lapse two-photon imaging of the presynaptic marker synaptophysin in hypocretin/orexin (HCRT) neurons to determine the dynamics of synaptic modifications during the day and night. We observed circadian rhythmicity in synapse number in HCRT axons. This rhythm is regulated primarily by the circadian clock but is also affected by sleep deprivation. Furthermore, NPTX2, a protein implicated in AMPA receptor clustering, modulates circadian synaptic changes. In zebrafish, nptx2b is a rhythmic gene that is mostly expressed in hypothalamic and pineal gland cells. Arrhythmic transgenic nptx2b overexpression (hcrt:NPTX2b) increases synapse number and abolishes rhythmicity in HCRT axons. Finally, hcrt:NPTX2b fish are resistant to the sleep-promoting effects of melatonin. This behavioral effect is consistent with NPTX2b-mediated increased activity of HCRT circuitry. These data provide real-time in vivo evidence of circadian and homeostatic regulation of structural synaptic plasticity.

  View details for DOI 10.1016/j.neuron.2010.09.006

  View details for Web of Science ID 000283704200010

  View details for PubMedID 20920793

  View details for PubMedCentralID PMC2969179

• Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain PLOS ONE Li, L., Tasic, B., Micheva, K. D., Ivanov, V. M., Spletter, M. L., Smith, S. J., Luo, L. 2010; 5 (7)

  Abstract

  Proper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons.In this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs.The ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits.

  View details for DOI 10.1371/journal.pone.0011503

  View details for Web of Science ID 000279715300014

  View details for PubMedID 20634890

  View details for PubMedCentralID PMC2901335

• Sleep-wake regulation and hypocretin-melatonin interaction in zebrafish PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Appelbaum, L., Wang, G. X., Maro, G. S., Mori, R., Tovin, A., Marin, W., Yokogawa, T., Kawakami, K., Smith, S. J., Gothilf, Y., Mignot, E., Mourrain, P. 2009; 106 (51): 21942-21947

  Abstract

  In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep-wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr-/-). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr-/- pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr-/- fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr-/- fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

  View details for DOI 10.1073/pnas.906637106

  View details for Web of Science ID 000272994200086

  View details for PubMedID 19966231

  View details for PubMedCentralID PMC2799794

• Classical MHCI Molecules Regulate Retinogeniculate Refinement and Limit Ocular Dominance Plasticity NEURON Datwani, A., McConnell, M. J., Kanold, P. O., Micheva, K. D., Busse, B., Shamloo, M., Smith, S. J., Shatz, C. J. 2009; 64 (4): 463-470

  Abstract

  Major histocompatibility complex class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-K(b) and H2-D(b), ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-K(b) and H2-D(b) are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN), where protein localization correlates strongly with synaptic markers and complement protein C1q. In K(b)D(b-/-) mice, developmental refinement of retinogeniculate projections is impaired, similar to C1q(-/-) mice. These phenotypes in K(b)D(b-/-) mice are strikingly similar to those in beta2 m(-/-)TAP1(-/-) mice, which lack cell surface expression of all MHCIs, implying that H2-K(b) and H2-D(b) can account for observed changes in synapse plasticity. H2-K(b) and H2-D(b) ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.

  View details for DOI 10.1016/j.neuron.2009.10.015

  View details for PubMedID 19945389

• Gabapentin Receptor alpha 2 delta-1 Is a Neuronal Thrombospondin Receptor Responsible for Excitatory CNS Synaptogenesis CELL Eroglu, C., Allen, N. J., Susman, M. W., O'Rourke, N. A., Park, C. Y., Oezkan, E., Chakraborty, C., Mulinyawe, S. B., Annis, D. S., Huberman, A. D., Green, E. M., Lawler, J., Dolmetsch, R., Garcia, K. C., Smith, S. J., Luo, Z. D., Rosenthal, A., Mosher, D. F., Barres, B. A. 2009; 139 (2): 380-392

  Abstract

  Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

  View details for DOI 10.1016/j.cell.2009.09.025

  View details for Web of Science ID 000270857500020

  View details for PubMedID 19818485

  View details for PubMedCentralID PMC2791798

• New technologies Editorial overview CURRENT OPINION IN NEUROBIOLOGY Isacoff, E., Smith, S. J. 2009; 19 (5): 511–12

  View details for DOI 10.1016/j.conb.2009.10.012

  View details for Web of Science ID 000272922900009

  View details for PubMedID 19896828

• Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Koffie, R. M., Meyer-Luehmann, M., Hashimoto, T., Adams, K. W., Mielke, M. L., Garcia-Alloza, M., Micheva, K. D., Smith, S. J., Kim, M. L., Lee, V. M., Hyman, B. T., Spires-Jones, T. L. 2009; 106 (10): 4012-4017

  Abstract

  Synapse loss correlates with a cognitive decline in Alzheimer's disease (AD), but whether this is caused by fibrillar deposits known as senile plaques or soluble oligomeric forms of amyloid beta (Abeta) is controversial. By using array tomography, a technique that combines ultrathin sectioning of tissue with immunofluorescence, allowing precise quantification of small structures, such as synapses, we have tested the hypothesis that oligomeric Abeta surrounding plaques contributes to synapse loss in a mouse model of AD. We find that senile plaques are surrounded by a halo of oligomeric Abeta. Analysis of >14,000 synapses (represented by PSD95-stained excitatory synapses) shows that there is a 60% loss of excitatory synapses in the halo of oligomeric Abeta surrounding plaques and that the density increases to reach almost control levels in volumes further than 50 microm from a plaque in an approximately linear fashion (linear regression, r(2) = 0.9; P < 0.0001). Further, in transgenic cortex, microdeposits of oligomeric Abeta associate with a subset of excitatory synapses, which are significantly smaller than those not in contact with oligomeric Abeta. The proportion of excitatory synapses associated with Abeta correlates with decreasing density (correlation, -0.588; P < 0.0001). These data show that senile plaques are a potential reservoir of oligomeric Abeta, which colocalizes with the postsynaptic density and is associated with spine collapse, reconciling the apparently competing schools of thought of "plaque" vs. "oligomeric Abeta" as the synaptotoxic species in the brain of AD patients.

  View details for DOI 10.1073/pnas.0811698106

  View details for Web of Science ID 000264036900066

  View details for PubMedID 19228947

  View details for PubMedCentralID PMC2656196

• ELASTIC SOURCE SELECTION FOR IN VIVO IMAGING OF NEURONAL ENSEMBLES IEEE International Symposium on Biomedical Imaging - From Nano to Macro Grosenick, L., Anderson, T., Smith, S. J. IEEE. 2009: 1263–1266

  View details for Web of Science ID 000270678400323

• Seeing Circuits Assemble NEURON Lichtman, J. W., Smith, S. J. 2008; 60 (3): 441-448

  Abstract

  Developmental neurobiology has been greatly invigorated by a recent string of breakthroughs in molecular biology and optical physics that permit direct in vivo observation of neural circuit assembly. The imaging done thus far suggests that as brains are built, a significant amount of unbuilding is also occurring. We offer the view that this tumult is the result of the intersecting behaviors of the many single-celled creatures (i.e., neurons, glia, and progenitors) that inhabit brains. New tools will certainly be needed if we wish to monitor the myriad cooperative and competitive interactions at play in the cellular society that builds brains.

  View details for DOI 10.1016/j.neuron.2008.10.040

  View details for Web of Science ID 000260983800015

  View details for PubMedID 18995818

  View details for PubMedCentralID PMC2661113

• Low-frequency noise characterization of near-IR VCSELs for functional brain imaging Conference on Photonic Therapeutics and Diagnostics IV Lee, T. T., Lim, P. G., Harris, J. S., Shenoy, K. V., Smith, S. J. SPIE-INT SOC OPTICAL ENGINEERING. 2008

  View details for DOI 10.1117/12.764143

  View details for Web of Science ID 000255314100050

• The classical complement cascade mediates CNS synapse elimination CELL Stevens, B., Allen, N. J., Vazquez, L. E., Howell, G. R., Christopherson, K. S., Nouri, N., Micheva, K. D., Mehalow, A. K., Huberman, A. D., Stafford, B., Sher, A., Litke, A. M., Lambris, J. D., Smith, S. J., John, S. W., Barres, B. A. 2007; 131 (6): 1164-1178

  Abstract

  During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.

  View details for DOI 10.1016/j.cell.2007.10.036

  View details for PubMedID 18083105

• Circuit reconstruction tools today CURRENT OPINION IN NEUROBIOLOGY Smith, S. J. 2007; 17 (5): 601-608

  Abstract

  To understand how a brain processes information, we must understand the structure of its neural circuits-especially circuit interconnection topologies and the cell and synapse molecular architectures that determine circuit-signaling dynamics. Our information on these key aspects of neural circuit structure has remained incomplete and fragmentary, however, because of limitations of the best available imaging methods. Now, new transgenic tool mice and new image acquisition tools appear poised to permit very significant advances in our abilities to reconstruct circuit connection topologies and molecular architectures.

  View details for DOI 10.1016/j.conb.2007.11.004

  View details for Web of Science ID 000252835100015

  View details for PubMedID 18082394

  View details for PubMedCentralID PMC2693015

• Integrated semiconductor optical sensors for cellular and neural imaging Biomedical Optics Topical Meeting of the Optical-Society-of-America Levi, O., Lee, T. T., Lee, M. M., Smith, S. J., Harris, J. S. OPTICAL SOC AMER. 2007: 1881–89

  Abstract

  We review integrated optical sensors for functional brain imaging, localized index-of-refraction sensing as part of a lab-on-a-chip, and in vivo continuous monitoring of tumor and cancer stem cells. We present semiconductor-based sensors and imaging systems for these applications. Measured intrinsic optical signals and tissue optics simulations indicate the need for high dynamic range and low dark-current neural sensors. Simulated and measured reflectance spectra from our guided resonance filter demonstrate the capability for index-of-refraction sensing on cellular scales, compatible with integrated biosensors. Finally, we characterized a thermally evaporated emission filter that can be used to improve sensitivity for in vivo fluorescence sensing.

  View details for PubMedID 17356634

• Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons MOLECULAR PHARMACOLOGY Micheva, K. D., Taylor, C. P., Smith, S. J. 2006; 70 (2): 467-476

  Abstract

  Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

  View details for DOI 10.1124/mol.106.023309

  View details for Web of Science ID 000239117900007

  View details for PubMedID 16641316

• Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms JOURNAL OF NEUROSCIENCE Meyer, M. P., Smith, S. J. 2006; 26 (13): 3604-3614

  Abstract

  To explore the relationship between axon arbor growth and synaptogenesis, developing retinal ganglion cell (RGC) axon arbors in zebrafish optic tectum were imaged in vivo at high temporal and spatial resolution using two-photon microscopy. Individual RGC axons were dually labeled by expression of a cytosolic red fluorescent protein (DsRed Express) to mark arbor structure and a fusion of the synaptic vesicle protein synaptophysin with green fluorescent protein (Syp:GFP) to mark presynaptic vesicles. Analysis of time-lapse sequences acquired at 10 min intervals revealed unexpectedly rapid kinetics of both axon branch and vesicle cluster turnover. Nascent axonal branches exhibited short average lifetimes of 19 min, and only 17% of newly extended axonal processes persisted for periods exceeding 3 h. The majority (70%) of Syp:GFP puncta formed on newly extended axonal processes. Syp:GFP puncta also exhibited short average lifetimes of 30 min, and only 34% of puncta were stabilized for periods exceeding 3 h. Moreover, strongly correlated dynamics of Syp:GFP puncta and branch structure suggest that synaptogenesis exerts strong influences on both the extension and the selective stabilization of nascent branches. First, new branches form almost exclusively at newly formed Syp:GFP puncta. Second, stabilized nascent branches invariably bear Syp:GFP puncta, and the detailed dynamics of branch retraction suggest strongly that nascent synapses can act at branch tips to arrest retraction. These observations thus provide evidence that synaptogenesis guides axon arbor growth by first promoting initial branch extension and second by selective branch stabilization.

  View details for DOI 10.1523/JNEUROSCI.0223-06.2006

  View details for PubMedID 16571769

• Integrated semiconductor optical sensors for chronic, minimally-invasive imaging of brain function. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference Lee, T. T., Levi, O., Cang, J., Kaneko, M., Stryker, M. P., Smith, S. J., Shenoy, K. V., Harris, J. S. 2006; 1: 1025-1028

  Abstract

  Intrinsic optical signal (IOS) imaging is a widely accepted technique for imaging brain activity. We propose an integrated device consisting of interleaved arrays of gallium arsenide (GaAs) based semiconductor light sources and detectors operating at telecommunications wavelengths in the near-infrared. Such a device will allow for long-term, minimally invasive monitoring of neural activity in freely behaving subjects, and will enable the use of structured illumination patterns to improve system performance. In this work we describe the proposed system and show that near-infrared IOS imaging at wavelengths compatible with semiconductor devices can produce physiologically significant images in mice, even through skull.

  View details for PubMedID 17946016

• Integrated semiconductor optical sensors for chronic, minimally-invasive imaging of brain function 28th Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Lee, T. T., Levi, O., Cang, J., Kaneko, M., Stryker, M. P., Smith, S. J., Shenoy, K. V., Harris, J. S. IEEE. 2006: 2443–2446

  View details for Web of Science ID 000247284702192

• Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals JOURNAL OF NEUROSCIENCE Micheva, K. D., Smith, S. J. 2005; 25 (33): 7481-7488

  Abstract

  Most cellular processes are known to be strongly temperature dependent. Nevertheless, a large fraction of studies of mammalian synaptic function have been and are performed near room temperature (i.e., at least 10 degrees C below physiological temperature). Here, we examined the effects of temperature on presynaptic function in primary cultures of rat hippocampal neurons. FM dyes, VAMP (vesicle-associated membrane protein)-GFP (green fluorescent protein) transfection, and HRP uptake were used to quantify various aspects of synaptic vesicle recycling. Our results show that there are very substantial differences in synaptic vesicle recycling at physiological temperature as opposed to the common, lower experimental temperatures. At 37 degrees C, compared with 23 degrees C, the speed of both exocytosis and endocytosis was higher. The size of the recycling vesicle pool (in both number of vesicles and spatial extent) was twofold larger at 37 degrees C. In addition, although repeated 10 Hz electrical stimulation caused an NMDA receptor-dependent enlargement (averaging 170%) of the measurable recycling vesicle pool at 23 degrees C, the same stimulus repetition had no effect at 37 degrees C. These results show that it is potentially misleading to extend conclusions drawn about vesicle function or presynaptic plasticity at lowered experimental temperature to physiological conditions and that much new experimental work at the higher physiological temperature range will be needed to understand the true parameters of presynaptic functions.

  View details for DOI 10.1523/JNEUROSCI.1801-05.2005

  View details for PubMedID 16107635

• Regulation of axon growth in vivo by activity-based competition NATURE Hua, J. Y., Smear, M. C., Baier, H., Smith, S. J. 2005; 434 (7036): 1022-1026

  Abstract

  The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby 'competing' RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.

  View details for DOI 10.1038/nature03409

  View details for PubMedID 15846347

• Greater than 10(6) optical isolation in integrated optoelectronic fluorescence sensor. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference Thrush, E., Levi, O., Cook, L., Smith, S., Harris, J. 2004; 3: 2080-2081

  Abstract

  Integrated optoelectronic sensors hold much potential for bio-medical applications. Our work focuses on the use of semiconductor lasers, photodetectors and filters to create a monolithically integrated near-infrared fluorescence sensor. Previous research has found that the close integration of these components results in large laser background levels from spontaneous emission emitted from the side of the laser and limits sensor sensitivity. This work presents an improved optical blocking structure between the laser and photodetector which results in greater than 10(6) optical isolation. This level of isolation will allow for sensitive fluorescence detection and shows that optoelectronic components can be successfully integrated for such purposes.

  View details for PubMedID 17272131

• Laser background characterization in a monolithically integrated biofluorescence sensor Conference on Advanced Biomedical and Clinical Diagnostic Systems II Thrush, E., Levi, O., Cook, L. J., Deich, J., Smith, S. J., Moerner, W. E., Harris, J. S. SPIE-INT SOC OPTICAL ENGINEERING. 2004: 59–65

  View details for DOI 10.1117/12.525133

  View details for Web of Science ID 000223125300007

• High throughput integration of optoelectronics devices for biochip fluorescent detection Conference on Microfluidics, BioMEMS, and Medical Microsystems Thrush, E., Levi, O., Wang, K., Harris, J. S., Smith, S. J. SPIE-INT SOC OPTICAL ENGINEERING. 2003: 162–169

  View details for Web of Science ID 000181864600022

• Changes in synapsin localization during synaptogenesis 42nd Annual Meeting of the American-Society-for-Cell-Biology Buchanan, J., Micheva, K., Smith, S. J. AMER SOC CELL BIOLOGY. 2002: 396A–396A

  View details for Web of Science ID 000179569102230

• Spatio-temporal dynamics and functions of endogenous Rac1 complexes during transition between migratory and cell-cell adherent states (part I) Ehrlich, J. S., Hansen, M. D., Smith, S. J., Nelson, W. J. AMER SOC CELL BIOLOGY. 2001: 263A

  View details for Web of Science ID 000172372501436

• Characterization of transport packets in hippocampal neurons undergoing synaptogenesis using HVEM tomography. Buchanan, J., Deerinck, T., Ahmari, S., Ellisman, M. H., Smith, S. J. AMER SOC CELL BIOLOGY. 2000: 471A

  View details for Web of Science ID 000165525902448

• Spatio-temporal dynamics of Rac small GTPase at MDCK cell-cell contacts Ehrlich, J. S., Smith, S. J., Nelson, W. J. AMER SOC CELL BIOLOGY. 2000: 172A

  View details for Web of Science ID 000165525900896

• Immunolocalization of GFP fusion proteins in hippocampal neurons using anti-GFP antibodies and nanogold labeling. Buchanan, J., Micheva, K., Smith, S. J. AMER SOC CELL BIOLOGY. 2000: 277A

  View details for Web of Science ID 000165525901444

• Regulation of synaptic phosphatidylinositol 4,5-biphosphate by neuronal activity Micheva, K. D., Holz, R. W., Smith, S. J. AMER SOC CELL BIOLOGY. 2000: 277A

  View details for Web of Science ID 000165525901445

• Two-photon fluorescence imaging of actin dynamics in live MDCK cells expressing mutant small GTPases Ehrlich, J. S., Smith, S. J., Nelson, W. J. AMER SOC CELL BIOLOGY. 1999: 29A

  View details for Web of Science ID 000083673500169

• Subcellular localization and dynamics of APC and beta-catenin in epithelial and neuronal cells. Barth, A. I., Ahmari, S. E., de Hostos, E. L., Smith, S. J., Nelson, W. J. AMER SOC CELL BIOLOGY. 1998: 35A

  View details for Web of Science ID 000076906700203

• Potentiation of evoked vesicle turnover at individually resolved synaptic boutons NEURON Ryan, T. A., Ziv, N. E., Smith, S. J. 1996; 17 (1): 125-134

  Abstract

  We have studied synaptic plasticity in hippocampal cell cultures using a new imaging approach that allows unambiguous discrimination of presynaptic function at the level of single synaptic boutons. Employing a protocol designed to test for use-dependent plasticity resembling N-methyl-D-aspartate receptor-dependent long-term potentiation (NMDA-type LTP), we find that brief tetanic stimuli induce a potentiation of evoked synaptic vesicle turnover that lasts for at least 1 hr. Induction of this clearly presynaptic potentiation is blocked by putative postsynaptic glutamate receptor antagonists, suggesting that a retrograde induction signal might be involved. Potentiation appears to occur approximately equally at boutons of low and high initial release probabilities, and evidently does not involve an increase in the size of the total recycling synaptic vesicle pool.

  View details for Web of Science ID A1996UY98100013

  View details for PubMedID 8755484