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States that the membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. Step taken to probe the physiological importance of these interactions; Reference to both reagents inhibiting synaptic transmission.

The study of hepatitis C virus (HCV) replication in cell culture is mainly based on cloned viral isolates requiring adaptation for efficient replication in Huh7 hepatoma cells. The analysis of wild-type (WT) isolates was enabled by the expression of SEC14L2 and by inhibitors targeting deleterious host factors. Here, we aimed to optimize cell culture models to allow infection with HCV from patient sera. We used Huh7-Lunet cells ectopically expressing SEC14L2, CD81, and a GFP reporter with nuclear translocation upon cleavage by the HCV protease to study HCV replication, combined with a drug-based regimen for stimulation of non-modified wild-type isolates. RT-qPCR-based quantification of HCV infections using patient sera suffered from a high background in the daclatasvir-treated controls. We therefore established an automated image analysis pipeline based on imaging of whole wells and iterative training of a machine learning tool, using nuclear GFP localization as a readout for HCV infection. Upon visual validation of hits assigned by the automated image analysis, the method revealed no background in daclatasvir-treated samples. Thereby, infection events were found for 15 of 34 high titer HCV genotype (gt) 1b sera, revealing a significant correlation between serum titer and successful infection. We further show that transfection of viral RNA extracted from sera can be used in this model as well, albeit with so far limited efficiency. Overall, we generated a robust serum infection assay for gt1b isolates using semi-automated image analysis, which was superior to conventional RT-qPCR-based quantification of viral genomes.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines., Competing Interests: The authors have declared that no competing interests exist.

1. A fast ATP-mediated synaptic current was identified in CA3 pyramidal cells in organotypic hippocampal slice cultures. In the presence of inhibitors for ionotropic glutamate and GABA receptors, extracellular stimulation in the pyramidal cell layer evoked fast synaptic currents that reversed near 0 mV, reflecting an increase in a non-selective cationic conductance. This response was mimicked by focal application of ATP. Antagonists of ionotropic P2X receptors reduced both synaptic and ATP-induced currents. 2. Using a pharmacological approach, the source of synaptically released ATP was determined. Synaptic ATP responses were insensitive to presynaptic blockade of GABAergic transmission between interneurons and CA3 pyramidal cells with the mu-opioid receptor agonist D-Ala(2),MePhe(4),Met(O)(5)-ol-enkephalin (FK33-824), but were blocked by adenosine, which inhibits glutamate release from synaptic terminals in the hippocampus. However, selective inhibition of mossy fibre glutamatergic transmission with the metabotropic glutamate receptor group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not affect the response. This result points to the associational fibres as the source of the ATP-mediated synaptic response. 3. These results suggest that ATP, coreleased with glutamate, induces a synaptic response in CA3 pyramidal cells that is observed mainly under conditions of synchronous discharge from multiple presynaptic inputs.

Two classes of receptors transduce neurotransmitter signals: ionotropic receptors and heptahelical metabotropic receptors. Whereas the ionotropic receptors are structurally associated with a membrane channel, a mediating mechanism is necessary to functionally link metabotropic receptors with their respective effectors. According to the accepted paradigm, the first step in the metabotropic transduction process requires the activation of heterotrimeric G-proteins. An increasing number of observations, however, point to a novel mechanism through which neurotransmitters can initiate biochemical signals and modulate neuronal excitability. According to this mechanism metabotropic receptors induce responses by activating transduction systems that do not involve G-proteins.

Synaptically released glutamate activates ionotropic and metabotropic receptors at central synapses. Metabotropic glutamate receptors (mGluRs) are thought to modulate membrane conductances through transduction cascades involving G proteins. Here we show, in CA3 pyramidal cells from rat hippocampus, that synaptic activation of type 1 mGluRs by mossy fiber stimulation evokes an excitatory postsynaptic response independent of G-protein function, while inhibiting an afterhyperpolarization current through a G-protein-coupled mechanism. Experiments using peptide activators and specific inhibitors identified a Src-family protein tyrosine kinase as a component of the G-protein-independent transduction pathway. These results represent the first functional evidence for a dual signaling mechanism associated with a heptahelical receptor such as mGluR1, in which intracellular transduction involves activation of either G proteins or tyrosine kinases.

The mechanisms underlying neurotoxicity mediated by cytotoxic T lymphocytes (CTL) and their secretory granule proteins perforin and granzymes remain unclear. We evaluated the possible role of the neurotransmitter glutamate in cell death observed in differentiated neurons exposed to CTL-derived granules. Excitotoxicity induced by excessive concentrations of extracellular glutamate is associated with a rise in intracellular calcium and can lead to generation of NO through the activation of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Consistent with an involvement of glutamate, we found that cell death in mature cerebral granule cells was inhibited by 65-80% by two NMDA receptor blockers (MK-801 and D-2-amino-5-phosphonovaleric acid) or a NO synthase blocker (N(G)-nitro-L-arginine methylester). Furthermore, neurons treated with secretory granules responded with a biphasic rise in the intracellular calcium concentration ([Ca2+]i). Whereas MK-801 did not interfere with the immediate rise of [Ca2+]i, the second wave of calcium accumulation starting at 40 min was delayed by 20 min and reduced in amplitude in the presence of MK-801. In immature, NMDA receptor-negative neurons, MK-801 prevented neither the cytotoxicity nor the calcium influx observed 5 min after addition of cytotoxic granules. The demonstration that NMDA receptors and NO are involved in granule-mediated killing of mature neurons opens new avenues in the treatment of neuronal cell death in CTL-mediated diseases such as viral encephalitis.

Gene transfer into nervous tissue is a powerful tool for the analysis of gene function. By using a rat hippocampal slice culture preparation, we show here that Semliki Forest virus (SFV) and Sindbis virus (SIN) vectors are useful for the effective infection of neurons. The stratum pyramidale and/or the granular cell layer were injected with recombinant virus encoding beta-galactosidase (LacZ) or green fluorescent protein (GFP). By using low concentrations of injected SFV-LacZ or SIN-LacZ, we detected LacZ staining of pyramidal cells, interneurons, and granule cells. About 60% of the infected cells showed clear neuronal morphology; thus, relatively few glial cells expressed the transgene. Expression of GFP from SFV and SIN vectors gave similar results, with an even higher percentage (>90%) of the GFP-positive cells identified as neurons. Infected pyramidal cells were readily recognized in living slices, displaying GFP fluorescence in dendrites of up to fourth order and in dendritic spines. They appeared morphologically normal and viable at 1-5 days postinfection. We conclude that both SFV and SIN vectors efficiently transfer genes into neurons in hippocampal slice cultures. In combination with the GFP reporter, SFV and SIN vectors will allow the physiological examination of identified neurons that have been modified by overexpression or suppression of a specific gene product.

Biochemical evidence indicates that the exocytotic release of neurotransmitters involves both evolutionary conserved membrane proteins, the SNAREs, as well as ubiquitous cytosolic fusion proteins, NSF and SNAPs. We have analyzed the biochemical properties and the physiological effects of these proteins. Our data suggest models how NSF, SNAPs and SNAREs may function in neurotransmitter exocytosis.

The distribution of fiber size in the posterior articular nerve (PAN) and medial articular nerve (MAN) of the cat's knee joint was studied by light and electron microscopy. The myelinated fibers of the PAN ranged from approximately 1 to 18 micron, with maxima at 3-4 micron and 8-9 micron. According to the classification of Boyd and Davey (1968), the PAN contained about 34% Group III fibers, 56% Group II fibers, and 10% Group I fibers. In contrast, the MAN showed a unimodal, skewed distribution, with a range from approximately 1 to 14 micron and a maximum at 3-4 micron. According to the Boyd and Davey classification, the PAN contained about 69% Group III fibers, 30% Group II fibers, and 1% Group I fibers. Unmyelinated fibers examined in the MAN showed a unimodal distribution, with a range of from 0.1 to 1.5 micron and a maximum at 0.4-0.5 micron. To differentiate between afferent and sympathetic fibers, a sympathectomy or ganglionectomy was performed on one side. The fiber size distribution indicated a considerable overlap in the diameter of the afferent and sympathetic unmyelinated fibers. Most sympathetic fibers had a diameter of between 0.8 and 0.9 micron, whereas afferent fibers showed a maximum at 0.3-0.4 micron. When data were combined for myelinated and unmyelinated fibers in the PAN, about 74% of the afferent fibers were found to belong to Groups III and IV; they are thought to terminate in noncorpuscular endings. The other 26% were found to belong to Groups I and II; they terminate in corpuscular receptors and muscle spindle primary and secondary endings.(ABSTRACT TRUNCATED AT 250 WORDS)