Publications by authors named "Guo-Qiang Bi"

40 Publications

Structure and plasticity of silent synapses in developing hippocampal neurons visualized by super-resolution imaging.

Cell Discov 2020 Feb 25;6(1). Epub 2020 Feb 25.

Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui, 230027, China.

Excitatory synapses in the mammalian brain exhibit diverse functional properties in transmission and plasticity. Directly visualizing the structural correlates of such functional heterogeneity is often hindered by the diffraction-limited resolution of conventional optical imaging techniques. Here, we used super-resolution stochastic optical reconstruction microscopy (STORM) to resolve structurally distinct excitatory synapses formed on dendritic shafts and spines. The majority of these shaft synapses contained N-methyl-D-aspartate receptors (NMDARs) but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), suggesting that they were functionally silent. During development, as more spine synapses formed with increasing sizes and expression of AMPARs and NMDARs, shaft synapses exhibited moderate reduction in density with largely unchanged sizes and receptor expression. Furthermore, upon glycine stimulation to induce chemical long-term potentiation (cLTP), the previously silent shaft synapses became functional shaft synapses by recruiting more AMPARs than did spine synapses. Thus, silent shaft synapse may represent a synaptic state in developing neurons with enhanced capacity of activity-dependent potentiation.
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http://dx.doi.org/10.1038/s41421-019-0139-1DOI Listing
February 2020

Unique dynamics and exocytosis properties of GABAergic synaptic vesicles revealed by three-dimensional single vesicle tracking.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, 999077 Kowloon, Hong Kong;

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter to selectively label GABAergic (i.e., predominantly inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single GABAergic vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that GABAergic synaptic vesicles traveled a shorter distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed that GABAergic synaptic vesicles move more straightly to their release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and exocytosis properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated coordination between excitation and inhibition in the central nervous system.
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http://dx.doi.org/10.1073/pnas.2022133118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936280PMC
March 2021

Long-range GABAergic projections from the nucleus of the solitary tract.

Mol Brain 2021 02 19;14(1):38. Epub 2021 Feb 19.

CAS Key Laboratory of Brain Function and Disease, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.

The nucleus of the solitary tract (NTS) plays a crucial role in integrating peripheral information regarding visceral functions. Glutamate decarboxylase 2 (GAD2) inhibitory neurons are abundant in the NTS, and are known to form local and short-range projections within the NTS and nearby hindbrain areas. Here we performed whole-brain mapping of outputs from GAD2 neurons in the NTS using cell-type specific viral labeling together with ultrahigh-speed 3D imaging at 1-μm resolution. In addition to well-known targets of NTS GAD2 neurons including the principle sensory nucleus of the trigeminal (PSV), spinal nucleus of the trigeminal (SPV), and other short-range targets within the hindbrain, the high sensitivity of our system helps reveal previously unknown long-range projections that target forebrain regions, including the bed nuclei of the stria terminalis (BST) involved in stress and fear responses, and the paraventricular hypothalamic nucleus (PVH) involved in energy balance and stress-related neuroendocrine responses. The long-range projections were further verified by retrograde labeling of NTS GAD2 neurons with cholera toxin B (CTB) injections in the BST and PVH, and by Cre-dependent retrograde tracing with rAAV2-retro injections in the two regions of GAD2-Cre mice. Finally, we performed complete morphological reconstruction of several sparsely labeled neurons projecting to the forebrain and midbrain. These results provide new insights about how NTS might participate in physiological and emotional modulation.
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http://dx.doi.org/10.1186/s13041-021-00751-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893933PMC
February 2021

Biphasic exocytosis of herpesvirus from hippocampal neurons and mechanistic implication to membrane fusion.

Cell Discov 2020 Jan 14;6(1). Epub 2020 Jan 14.

California NanoSystems Institute, University of California, Los Angeles (UCLA), Los Angeles, CA, 90095-7227, USA.

Exocytosis is a crucial cellular process involved in the release of neural transmitters or signaling hormones, and disposal of waste or toxic materials. The relationship between structural transition and temporal progression of this process is poorly understood, partly due to lack of adequate tools to resolve such dynamic structures at sufficient resolution in 3D. Exocytosis can be hijacked by some viruses, exemplified by the widely used model α-herpesvirus pseudorabies virus (PRV), which relies on exocytosis for trans-synaptic spread across neurons. Here, we have used cryo electron tomography (cryoET) to capture 199 events of PRV exocytosis from cultured hippocampal neurons. We established cumulative frequency analysis to estimate the relative duration of an exocytosis stage based on the frequency of observed viral particles at that stage. This analysis revealed that PRV exocytosis is biphasic, including a fast, "release phase" driven by fusion proteins and fused membranes, and a slow, "recovery phase" driven by flattening of curved membranes. The biphasic property of exocytosis discovered here appears to be conserved for membrane fusion during viral entry, and our approach of cumulative frequency analysis should have general utility for characterizing other membrane fusion events.
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http://dx.doi.org/10.1038/s41421-019-0134-6DOI Listing
January 2020

Mesophasic organization of GABA receptors in hippocampal inhibitory synapses.

Nat Neurosci 2020 12 2;23(12):1589-1596. Epub 2020 Nov 2.

Center for Integrative Imaging, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.

Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are organized in situ remains largely unknown. In this study, template-free classification of oversampled sub-tomograms was used to analyze cryo-electron tomograms of hippocampal synapses. We identified type-A GABA receptors (GABARs) in inhibitory synapses and determined their in situ structure at 19-Å resolution. These receptors are organized hierarchically: from GABAR super-complexes with a preferred inter-receptor distance of 11 nm but variable relative angles, through semi-ordered, two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. These assemblies likely form via interactions among postsynaptic scaffolding proteins and receptors and align with putative presynaptic vesicle release sites. Such mesophasic self-organization might allow synapses to achieve a 'Goldilocks' state, striking a balance between stability and flexibility and enabling plasticity in information processing.
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http://dx.doi.org/10.1038/s41593-020-00729-wDOI Listing
December 2020

Structures of capsid and capsid-associated tegument complex inside the Epstein-Barr virus.

Nat Microbiol 2020 10 27;5(10):1285-1298. Epub 2020 Jul 27.

California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA.

As the first discovered human cancer virus, Epstein-Barr virus (EBV) causes Burkitt's lymphoma and nasopharyngeal carcinoma. Isolating virions for determining high-resolution structures has been hindered by latency-a hallmark of EBV infection-and atomic structures are thus available only for recombinantly expressed EBV proteins. In the present study, by symmetry relaxation and subparticle reconstruction, we have determined near-atomic-resolution structures of the EBV capsid with an asymmetrically attached DNA-translocating portal and capsid-associated tegument complexes from cryogenic electron microscopy images of just 2,048 EBV virions obtained by chemical induction. The resulting atomic models reveal structural plasticity among the 20 conformers of the major capsid protein, 2 conformers of the small capsid protein (SCP), 4 conformers of the triplex monomer proteins and 2 conformers of the triplex dimer proteins. Plasticity reaches the greatest level at the capsid-tegument interfaces involving SCP and capsid-associated tegument complexes (CATC): SCPs crown pentons/hexons and mediate tegument protein binding, and CATCs bind and rotate all five periportal triplexes, but notably only about one peri-penton triplex. These results offer insights into the EBV capsid assembly and a mechanism for recruiting cell-regulating factors into the tegument compartment as 'cargoes', and should inform future anti-EBV strategies.
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http://dx.doi.org/10.1038/s41564-020-0758-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546529PMC
October 2020

High frequency optogenetic activation of inputs to the lateral amygdala forms distant association with foot-shock.

Mol Brain 2020 03 20;13(1):44. Epub 2020 Mar 20.

CAS Key Laboratory of Brain Function and Disease, and School of Life Sciences, University of Science and Technology of China, Hefei, 230026, Anhui, China.

Aim: A hallmark of classical conditioning is that conditioned stimulus (CS) must be tightly coupled with unconditioned stimulus (US), often requiring temporal overlap between the two, or a short gap of several seconds. In this study, we investigate the temporal requirements for fear conditioning association between a strong artificial CS, high-frequency optogenetic activation of inputs into the lateral amygdala of rats, and a foot-shock to the animal with delays up to many minutes.

Methods: AAV-oChIEF-tdTomato viruses were injected into the auditory cortex and the medial geniculate nucleus of rats. An optical fiber was implanted just above the lateral amygdala of the animal. Optogenetic high-frequency stimuli (oHFS; containing five 1-s trains of 100 Hz laser pulses) were delivered to the lateral amygdala, before or after (with varying intervals) a foot-shock that elicits fear responses in the animal. Pre-trained lever-press behavior was used to assess the degree of fear recall by optogenetic test stimuli (OTS; 10 Hz for 2 min) 24 h after the association experiment.

Results: In contrast to the tight temporal requirement for classical conditioning with paired optogenetic moderate-frequency stimuli (oMFS; 10 Hz for 20 s) and foot-shock, oHFS followed by foot-shock with a 5-min or even 1-h (but not 3-h) interval could successfully establish an association to be recalled by OTS the next day. Meanwhile, foot-shock followed by oHFS with a 5-min (but not 1-h) interval could also establish the conditioning. Thus, distant association may be formed between temporally distant stimuli when the CS is strong.
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http://dx.doi.org/10.1186/s13041-020-00587-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082919PMC
March 2020

Structure and plasticity of silent synapses in developing hippocampal neurons visualized by super-resolution imaging.

Cell Discov 2020 25;6. Epub 2020 Feb 25.

1Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230027 China.

Excitatory synapses in the mammalian brain exhibit diverse functional properties in transmission and plasticity. Directly visualizing the structural correlates of such functional heterogeneity is often hindered by the diffraction-limited resolution of conventional optical imaging techniques. Here, we used super-resolution stochastic optical reconstruction microscopy (STORM) to resolve structurally distinct excitatory synapses formed on dendritic shafts and spines. The majority of these shaft synapses contained -methyl-d-aspartate receptors (NMDARs) but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), suggesting that they were functionally silent. During development, as more spine synapses formed with increasing sizes and expression of AMPARs and NMDARs, shaft synapses exhibited moderate reduction in density with largely unchanged sizes and receptor expression. Furthermore, upon glycine stimulation to induce chemical long-term potentiation (cLTP), the previously silent shaft synapses became functional shaft synapses by recruiting more AMPARs than did spine synapses. Thus, silent shaft synapse may represent a synaptic state in developing neurons with enhanced capacity of activity-dependent potentiation.
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http://dx.doi.org/10.1038/s41421-019-0139-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039918PMC
February 2020

Biphasic exocytosis of herpesvirus from hippocampal neurons and mechanistic implication to membrane fusion.

Cell Discov 2020 14;6. Epub 2020 Jan 14.

2California NanoSystems Institute, University of California, Los Angeles (UCLA), Los Angeles, CA 90095-7227 USA.

Exocytosis is a crucial cellular process involved in the release of neural transmitters or signaling hormones, and disposal of waste or toxic materials. The relationship between structural transition and temporal progression of this process is poorly understood, partly due to lack of adequate tools to resolve such dynamic structures at sufficient resolution in 3D. Exocytosis can be hijacked by some viruses, exemplified by the widely used model α-herpesvirus pseudorabies virus (PRV), which relies on exocytosis for trans-synaptic spread across neurons. Here, we have used cryo electron tomography (cryoET) to capture 199 events of PRV exocytosis from cultured hippocampal neurons. We established cumulative frequency analysis to estimate the relative duration of an exocytosis stage based on the frequency of observed viral particles at that stage. This analysis revealed that PRV exocytosis is biphasic, including a fast, "release phase" driven by fusion proteins and fused membranes, and a slow, "recovery phase" driven by flattening of curved membranes. The biphasic property of exocytosis discovered here appears to be conserved for membrane fusion during viral entry, and our approach of cumulative frequency analysis should have general utility for characterizing other membrane fusion events.
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http://dx.doi.org/10.1038/s41421-019-0134-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957672PMC
January 2020

Atomic structure of the human herpesvirus 6B capsid and capsid-associated tegument complexes.

Nat Commun 2019 11 25;10(1):5346. Epub 2019 Nov 25.

California NanoSystems Institute, University of California, Los Angeles (UCLA), Los Angeles, CA, 90095-7151, USA.

Human herpesvirus 6B (HHV-6B) belongs to the β-herpesvirus subfamily of the Herpesviridae. To understand capsid assembly and capsid-tegument interactions, here we report atomic structures of HHV-6B capsid and capsid-associated tegument complex (CATC) obtained by cryoEM and sub-particle reconstruction. Compared to other β-herpesviruses, HHV-6B exhibits high similarity in capsid structure but organizational differences in its CATC (pU11 tetramer). 180 "VΛ"-shaped CATCs are observed in HHV-6B, distinguishing from the 255 "Λ"-shaped dimeric CATCs observed in murine cytomegalovirus and the 310 "Δ"-shaped CATCs in human cytomegalovirus. This trend in CATC quantity correlates with the increasing genomes sizes of these β-herpesviruses. Incompatible distances revealed by the atomic structures rationalize the lack of CATC's binding to triplexes Ta, Tc, and Tf in HHV-6B. Our results offer insights into HHV-6B capsid assembly and the roles of its tegument proteins, including not only the β-herpesvirus-specific pU11 and pU14, but also those conserved across all subfamilies of Herpesviridae.
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http://dx.doi.org/10.1038/s41467-019-13064-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877594PMC
November 2019

DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV.

Cell 2019 09 22;178(6):1329-1343.e12. Epub 2019 Aug 22.

Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an ∼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.
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http://dx.doi.org/10.1016/j.cell.2019.07.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753055PMC
September 2019

Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome.

Nature 2019 06 29;570(7760):257-261. Epub 2019 May 29.

California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA, USA.

Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid-with triangulation number (T) equal to 16-encapsidating a tightly packed double-stranded DNA (dsDNA) genome. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion. Although this process has been studied in dsDNA phages-with which herpesviruses bear some similarities-a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal-a longstanding mystery in icosahedral viruses-and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging.
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http://dx.doi.org/10.1038/s41586-019-1248-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732574PMC
June 2019

Postsynaptic protein organization revealed by electron microscopy.

Curr Opin Struct Biol 2019 02 21;54:152-160. Epub 2019 Mar 21.

Center for Integrative Imaging, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui, China; School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China; CAS Center for Excellence in Brain Science and Intelligence Technology, University of Science and Technology of China, Hefei, Anhui, China. Electronic address:

Neuronal synapses are key devices for transmitting and processing information in the nervous system. Synaptic plasticity, generally regarded as the cellular basis of learning and memory, involves changes of subcellular structures that take place at the nanoscale. High-resolution imaging methods, especially electron microscopy (EM), have allowed for quantitative analysis of such nanoscale structures in different types of synapses. In particular, the semi-ordered organization of neurotransmitter receptors and their interacting scaffolds in the postsynaptic density have been characterized for both excitatory and inhibitory synapses by studies using various EM techniques such as immuno-EM, electron tomography of high-pressure freezing and freeze-substituted samples, and cryo electron tomography. These techniques, in combination with new correlative approaches, will further facilitate our understanding of the molecular organization underlying diverse functions of neuronal synapses.
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http://dx.doi.org/10.1016/j.sbi.2019.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753054PMC
February 2019

Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.

PLoS Pathog 2018 12 3;14(12):e1007452. Epub 2018 Dec 3.

Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, United States of America.

Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain.
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http://dx.doi.org/10.1371/journal.ppat.1007452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307773PMC
December 2018

Electrically Controlled Neurochemical Release from Dual-Layer Conducting Polymer Films for Precise Modulation of Neural Network Activity in Rat Barrel Cortex.

Adv Funct Mater 2018 Mar 11;28(12). Epub 2017 Dec 11.

Department of Bioengineering, University of Pittsburgh, 5057 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.

Implantable microelectrode arrays (MEAs) are important tools for investigating functional neural circuits and treating neurological diseases. Precise modulation of neural activity may be achieved by controlled delivery of neurochemicals directly from coatings on MEA electrode sites. In this study, a novel dual-layer conductive polymer/acid functionalized carbon nanotube (fCNT) microelectrode coating is developed to better facilitate the loading and controlled delivery of the neurochemical 6,7-dinitroquinoxaline-2,3-dione (DNQX). The base layer coating is consisted of poly(3,4-ethylenedioxythiophene/fCNT and the top layer is consisted of polypyrrole/fCNT/DNQX. The dual-layer coating is capable of both loading and electrically releasing DNQX and the release dynamic is characterized with fluorescence microscopy and mathematical modeling. In vivo DNQX release is demonstrated in rat somatosensory cortex. Sensory-evoked neural activity is immediately (<1s) and locally (<446 µm) suppressed by electrically triggered DNQX release. Furthermore, a single DNQX-loaded, dual-layer coating is capable of inducing effective neural inhibition for at least 26 times without observable degradation in efficacy. Incorporation of the novel drug releasing coating onto individual MEA electrodes offers many advantages over alternative methods by increasing spatial-temporal precision and improving drug selection flexibility without increasing the device's size.
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http://dx.doi.org/10.1002/adfm.201703988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242295PMC
March 2018

Correlative light and electron microscopy for complex cellular structures on PDMS substrates with coded micro-patterns.

Lab Chip 2018 12;18(24):3840-3848

Center for Integrative Imaging, National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.

Fluorescence light microscopy (FLM) is commonly used for localizing specific cellular and subcellular targets. Electron microscopy (EM), on the other hand, can reveal ultrastructural details of cellular architectures beyond the limit of optical resolution. Correlative light and electron microscopy (CLEM) that combines the two techniques has proven valuable in various cell biological applications that require both specificity and resolution. Here, we report an efficient and easy-to-use CLEM system, and its applications in studying neuronal synapses. The system utilizes patterned symbols to encode coordinates on micro-fabricated polydimethylsiloxane (PDMS) substrates, on which dissociated primary hippocampal neurons grow and form synaptic connections. After imaging and localizing specifically labeled synapses with FLM, samples are embedded in resin blocks and sectioned for EM analysis. The patterned symbols on PDMS substrates provide coordinate information, allowing efficient co-registration between FLM and EM images with high precision. A custom-developed software package achieves automated EM image collection, FLM/EM alignment, and EM navigation. With this CLEM system, we have obtained high quality electron tomograms of fluorescently labeled synapses along dendrites of hippocampal neurons and analyzed docking statistics of synaptic vesicles (SVs) in different subtypes of excitatory synapses. This technique provides an efficient approach to combine functional studies with ultrastructural analysis of heterogeneous neuronal synapses, as well as other subcellular structures in general.
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http://dx.doi.org/10.1039/c8lc00703aDOI Listing
December 2018

Cryo-EM structure of the human α5β3 GABA receptor.

Cell Res 2018 09 23;28(9):958-961. Epub 2018 Aug 23.

Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang, 310058, P. R. China.

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http://dx.doi.org/10.1038/s41422-018-0077-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123483PMC
September 2018

Accumulation of Dense Core Vesicles in Hippocampal Synapses Following Chronic Inactivity.

Front Neuroanat 2018 11;12:48. Epub 2018 Jun 11.

Center for Integrative Imaging, National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.

The morphology and function of neuronal synapses are regulated by neural activity, as manifested in activity-dependent synapse maturation and various forms of synaptic plasticity. Here we employed cryo-electron tomography (cryo-ET) to visualize synaptic ultrastructure in cultured hippocampal neurons and investigated changes in subcellular features in response to chronic inactivity, a paradigm often used for the induction of homeostatic synaptic plasticity. We observed a more than 2-fold increase in the mean number of dense core vesicles (DCVs) in the presynaptic compartment of excitatory synapses and an almost 20-fold increase in the number of DCVs in the presynaptic compartment of inhibitory synapses after 2 days treatment with the voltage-gated sodium channel blocker tetrodotoxin (TTX). Short-term treatment with TTX and the N-methyl-D-aspartate receptor (NMDAR) antagonist amino-5-phosphonovaleric acid (AP5) caused a 3-fold increase in the number of DCVs within 100 nm of the active zone area in excitatory synapses but had no significant effects on the overall number of DCVs. In contrast, there were very few DCVs in the postsynaptic compartments of both synapse types under all conditions. These results are consistent with a role for presynaptic DCVs in activity-dependent synapse maturation. We speculate that these accumulated DCVs can be released upon reactivation and may contribute to homeostatic metaplasticity.
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http://dx.doi.org/10.3389/fnana.2018.00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004418PMC
June 2018

Excitation wavelength optimization improves photostability of ASAP-family GEVIs.

Mol Brain 2018 06 4;11(1):32. Epub 2018 Jun 4.

Hefei National Laboratory for Physical Sciences at the Microscale, CAS Center for Excellence in Brain Science and Intelligence Technology, and School of Life Sciences, University of Science and Technology of China, Hefei, China.

Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.
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http://dx.doi.org/10.1186/s13041-018-0374-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987426PMC
June 2018

Corticosterone Signaling and a Lateral Habenula-Ventral Tegmental Area Circuit Modulate Compulsive Self-Injurious Behavior in a Rat Model.

J Neurosci 2018 06 14;38(23):5251-5266. Epub 2018 May 14.

Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China,

Self-injurious behavior (SIB) is commonly observed in patients with neuropsychiatric disorders, as well as in nonclinical populations with stress-related mental-health problems. However, the exact circuitry mechanisms underlying SIB have remained poorly understood. Here, with bilateral injection of muscimol into the entopeduncular nucleus (EP), we established a rat model of SIB. Following the muscimol injection, the male rats exhibited in a dose-dependent manner stereotypic self-biting behavior that lasted for hours and often resulted in wounds of various severities. The SIB was associated with an elevated level of serum corticosterone and could be exacerbated by enhancing the corticosterone signaling and, conversely, alleviated by inhibiting the corticosterone signaling. Activity mapping using c-fos immunostaining, combined with connectivity mapping using herpes simplex virus-based anterograde tracing from the EP and pseudorabies virus-based retrograde tracing from the masseter muscle, revealed the potential involvement of many brain areas in SIB. In particular, the lateral habenula (LHb) and the ventral tegmental area (VTA), the two connected brain areas involved in stress response and reward processing, showed a significant increase in neuronal activation during SIB. Furthermore, suppressing the LHb activity or modulating the GABAergic transmission in the VTA could significantly reduce the occurrence of SIB. These results demonstrate the importance of stress hormone signaling and the LHb-VTA circuit in modulating SIB resulting from EP malfunction, and suggest potential targets for therapeutic intervention of SIB and related disorders. Self-injurious behavior (SIB) occurs in ∼4% of the general population, with substantially higher occurrence among adolescents and patients of neuropsychiatric disorders. Stress has been linked to the occurrence of SIB, yet the underlying mechanisms have remained unclear. Using a rat model of SIB induced by disruption of activity in the entopeduncular nucleus (EP), we found that the behavior is regulated by stress and linked to corticosterone signaling. Viral tracing and c-fos immunostaining revealed the involvement of various subcortical areas, especially the EP-lateral habenula (LHb)-ventral tegmental area (VTA) circuit, in SIB. Furthermore, regulating activity in the LHb or the VTA alleviates SIB. These results may have implications in the development of new strategies for treating SIB.
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http://dx.doi.org/10.1523/JNEUROSCI.2540-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596003PMC
June 2018

Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy.

J Neurosci 2018 02 8;38(6):1493-1510. Epub 2018 Jan 8.

National Laboratory for Physical Sciences at the Microscale,

As key functional units in neural circuits, different types of neuronal synapses play distinct roles in brain information processing, learning, and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Here, by combining cryo-electron tomography and cryo-correlative light and electron microscopy, we distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, and visualized the 3D organization of synaptic organelles and macromolecules in their native state. Quantitative analyses of >100 synaptic tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density (PSD) with thickness ranging from 20 to 50 nm. In contrast, the PSD in inhibitory synapses assumes a thin sheet-like structure ∼12 nm from the postsynaptic membrane. On the presynaptic side, spherical synaptic vesicles (SVs) of 25-60 nm diameter and discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta phase plate and electron filtering and counting reveal glutamate receptor-like and GABA receptor-like structures that interact with putative scaffolding and adhesion molecules, reflecting details of receptor anchoring and PSD organization. These results provide an updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate the potential of our approach to gain insight into the organizational principles of cellular architecture underlying distinct synaptic functions. To understand functional properties of neuronal synapses, it is desirable to analyze their structure at molecular resolution. We have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Our approach shows that inhibitory synapses contain uniform thin sheet-like postsynaptic densities (PSDs), while excitatory synapses contain previously known mesh-like PSDs. We discovered "discus-shaped" ellipsoidal synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic types are characterized. High-resolution tomograms further allowed identification of putative neurotransmitter receptors and their heterogeneous interaction with synaptic scaffolding proteins. The specificity and resolution of our approach enables precise analysis of ultrastructural organization underlying distinct synaptic functions.
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http://dx.doi.org/10.1523/JNEUROSCI.1548-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815350PMC
February 2018

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

J Gen Virol 2017 11 16;98(11):2837-2849. Epub 2017 Oct 16.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA.

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a three-dimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the α-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9 Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsid-associated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsid-tegument interface.
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http://dx.doi.org/10.1099/jgv.0.000903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718256PMC
November 2017

Dendritic mitoflash as a putative signal for stabilizing long-term synaptic plasticity.

Nat Commun 2017 06 26;8(1):31. Epub 2017 Jun 26.

Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, 230027, China.

Mitochondrial flashes (mitoflashes) are recently discovered excitable mitochondrial events in many cell types. Here we investigate their occurrence in the context of structural long-term potentiation (sLTP) at hippocampal synapses. At dendritic spines stimulated by electric pulses, glycine, or targeted glutamate uncaging, induction of sLTP is associated with a phasic occurrence of local, quantized mitochondrial activity in the form of one or a few mitoflashes, over a 30-min window. Low-dose nigericin or photoactivation that elicits mitoflashes stabilizes otherwise short-term spine enlargement into sLTP. Meanwhile, scavengers of reactive oxygen species suppress mitoflashes while blocking sLTP. With targeted photoactivation of mitoflashes, we further show that the stabilization of sLTP is effective within the critical 30-min time-window and a spatial extent of ~2 μm, similar to that of local diffusive reactive oxygen species. These findings indicate a potential signaling role of dendritic mitochondria in synaptic plasticity, and provide new insights into the cellular function of mitoflashes.Mitoflashes are dynamic events in mitochondria, associated with depolarization and release of reactive oxygen species, and have been associated with several cellular functions. The authors now show that in neurons, dendritic mitoflashes are involved in structural postsynaptic changes during LTP.
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http://dx.doi.org/10.1038/s41467-017-00043-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484698PMC
June 2017

Homeostatic regulation of spontaneous and evoked synaptic transmission in two steps.

Mol Brain 2013 Aug 22;6:38. Epub 2013 Aug 22.

Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Background: During development both Hebbian and homeostatic mechanisms regulate synaptic efficacy, usually working in opposite directions in response to neuronal activity. Homeostatic plasticity has often been investigated by assaying changes in spontaneous synaptic transmission resulting from chronic circuit inactivation. However, effects of inactivation on evoked transmission have been less frequently reported. Importantly, contributions from the effects of circuit inactivation and reactivation on synaptic efficacy have not been individuated.

Results: Here we show for developing hippocampal neurons in primary culture that chronic inactivation with TTX results in increased mean amplitude of miniature synaptic currents (mEPSCs), but not evoked synaptic currents (eEPSCs). However, changes in quantal properties of transmission, partially reflected in mEPSCs, accurately predicted higher-order statistical properties of eEPSCs. The classical prediction of homeostasis--increased strength of evoked transmission--was realized after explicit circuit reactivation, in the form of cells' pairwise connection probability. In contrast, distributions of eEPSC amplitudes for control and inactivated-then-reactivated groups matched throughout.

Conclusions: Homeostatic up-regulation of evoked synaptic transmission in developing hippocampal neurons in primary culture requires both the inactivation and reactivation stages, leading to a net increase in functional circuit connectivity.
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http://dx.doi.org/10.1186/1756-6606-6-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765453PMC
August 2013

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.

Proc Natl Acad Sci U S A 2012 Aug 13;109(35):13978-83. Epub 2012 Aug 13.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.
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http://dx.doi.org/10.1073/pnas.1201882109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435176PMC
August 2012

Measuring action potential-evoked transmission at individual synaptic contacts.

J Neural Eng 2012 Jun;9(3):036014

Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

In the neuronal culture experimental system, the total synaptic connection between two neurons can consist of large numbers of synaptic sites, each behaving probabilistically. Studies of synaptic function with paired recordings typically consider the summed response across all of these sites and from this infer the average response. Understanding of synaptic transmission and plasticity could be improved by examination of activity at as few synaptic sites as possible. To this end, we develop a system for recording responses from individual contacts. It relies on a precisely regulated pneumatic/hydrostatic pressure system to create a microenvironment within which individual synapses are active, and an acoustic signature method to monitor the stability of this microenvironment noninvasively. With this method we are able to record action potential-evoked postsynaptic currents consistent with individual quanta. The approach does not distort synaptic current waveforms and permits stable recording for several hours. The method is applied to address mechanisms of short-term plasticity, the variability of latency at individual synaptic sites and, in a preliminary experiment, the independence of nearby synapses on the same axon.
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http://dx.doi.org/10.1088/1741-2560/9/3/036014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049561PMC
June 2012

Ring-shaped neuronal networks: a platform to study persistent activity.

Lab Chip 2011 Mar 4;11(6):1081-8. Epub 2011 Feb 4.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Persistent activity in the brain is involved in working memory and motor planning. The ability of the brain to hold information 'online' long after an initiating stimulus is a hallmark of brain areas such as the prefrontal cortex. Recurrent network loops such as the thalamocortical loop and reciprocal loops in the cortex are potential substrates that can support such activity. However, native brain circuitry makes it difficult to study mechanisms underlying such persistent activity. Here we propose a platform to study synaptic mechanisms of such persistent activity by constraining neuronal networks to a recurrent loop like geometry. Using a polymer stamping technique, adhesive proteins are transferred onto glass substrates in a precise ring shape. Primary rat hippocampal cultures were capable of forming ring-shaped networks containing 40-60 neurons. Calcium imaging of these networks show evoked persistent activity in an all-or-none manner. Blocking inhibition with bicuculline methaiodide (BMI) leads to an increase in the duration of persistent activity. These persistent phases were abolished by blockade of asynchronous neurotransmitter release by ethylene glycol tetraacetic acid (EGTA-AM).
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http://dx.doi.org/10.1039/c0lc00450bDOI Listing
March 2011

Temporal modulation of spike-timing-dependent plasticity.

Front Synaptic Neurosci 2010 17;2:19. Epub 2010 Jun 17.

Molecular Neurobiology Program, Departments of Otolaryngology and Physiology/Neuroscience, The Helen and Martin Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine New York, NY, USA.

Spike-timing-dependent plasticity (STDP) has attracted considerable experimental and theoretical attention over the last decade. In the most basic formulation, STDP provides a fundamental unit - a spike pair - for quantifying the induction of long-term changes in synaptic strength. However, many factors, both pre- and postsynaptic, can affect synaptic transmission and integration, especially when multiple spikes are considered. Here we review the experimental evidence for multiple types of nonlinear temporal interactions in STDP, focusing on the contributions of individual spike pairs, overall spike rate, and precise spike timing for modification of cortical and hippocampal excitatory synapses. We discuss the underlying processes that determine the specific learning rules at different synapses, such as postsynaptic excitability and short-term depression. Finally, we describe the success of efforts toward building predictive, quantitative models of how complex and natural spike trains induce long-term synaptic modifications.
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http://dx.doi.org/10.3389/fnsyn.2010.00019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059714PMC
July 2011

Gain in sensitivity and loss in temporal contrast of STDP by dopaminergic modulation at hippocampal synapses.

Proc Natl Acad Sci U S A 2009 Aug 20;106(31):13028-33. Epub 2009 Jul 20.

Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

Spike-timing-dependent plasticity (STDP) is considered a physiologically relevant form of Hebbian learning. However, behavioral learning often involves action of reinforcement or reward signals such as dopamine. Here, we examined how dopamine influences the quantitative rule of STDP at glutamatergic synapses of hippocampal neurons. The presence of 20 muM dopamine during paired pre- and postsynaptic spiking activity expanded the effective time window for timing-dependent long-term potentiation (t-LTP) to at least -45 ms, and allowed normally ineffective weak stimuli with fewer spike pairs to induce significant t-LTP. Meanwhile, dopamine did not affect the degree of t-LTP induced by normal strong stimuli with spike timing (ST) of +10 ms. Such dopamine-dependent enhancement in the sensitivity of t-LTP was completely blocked by the D1-like dopamine receptor antagonist SCH23390, but not by the D2-like dopamine receptor antagonist sulpiride. Surprisingly, timing-dependent long-term depression (t-LTD) at negative ST was converted into t-LTP by dopamine treatment; this conversion was also blocked by SCH23390. In addition, t-LTP in the presence of dopamine was completely blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid, indicating that D1-like receptor-mediated modulation appears to act through the classical NMDA receptor-mediated signaling pathway that underlies STDP. These results provide a quantitative and mechanistic basis for a previously undescribed learning rule that depends on pre- and postsynaptic ST, as well as the global reward signal.
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http://dx.doi.org/10.1073/pnas.0900546106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713390PMC
August 2009

Calcium and synaptic dynamics underlying reverberatory activity in neuronal networks.

Phys Biol 2007 Jun 11;4(2):91-103. Epub 2007 Jun 11.

School of Physics and Astronomy, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv, Israel.

Persistent activity is postulated to drive neural network plasticity and learning. To investigate its underlying cellular mechanisms, we developed a biophysically tractable model that explains the emergence, sustenance and eventual termination of short-term persistent activity. Using the model, we reproduced the features of reverberating activity that were observed in small (50-100 cells) networks of cultured hippocampal neurons, such as the appearance of polysynaptic current clusters, the typical inter-cluster intervals, the typical duration of reverberation, and the response to changes in extra-cellular ionic composition. The model relies on action potential-triggered residual pre-synaptic calcium, which we suggest plays an important role in sustaining reverberations. We show that reverberatory activity is maintained by enhanced asynchronous transmitter release from pre-synaptic terminals, which in itself depends on the dynamics of residual pre-synaptic calcium. Hence, asynchronous release, rather than being a 'synaptic noise', can play an important role in network dynamics. Additionally, we found that a fast timescale synaptic depression is responsible for oscillatory network activation during reverberations, whereas the onset of a slow timescale depression leads to the termination of reverberation. The simplicity of our model enabled a number of predictions that were confirmed by additional analyses of experimental manipulations.
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http://dx.doi.org/10.1088/1478-3975/4/2/003DOI Listing
June 2007