Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility

In 2008 and 2009, acute mortalities occurred in France among Pacific cupped oyster, Crassostrea gigas, spat. Different hypothesis including the implication of environmental factors, toxic algae and/or pathogens have been explored. Diagnostic tests indicated that OsHV-1 including a particular genotype, termed OsHV-1 μVar, was detected in most of samples and especially in moribund oysters with the highlighting of virus particles looking like herpes viruses by TEM examination. In this study, an experimental protocol to reproduce OsHV-1 infection in laboratory conditions was developed. This protocol was based on the intramuscular injection of filtered (0.22 μm) tissue homogenates prepared from naturally OsHV-1 infected spat collected on French coasts during mortality outbreaks in 2008. Results of the experimental trials showed that mortalities were induced after injection. Moreover, filtered tissue homogenates induced mortalities whereas the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that oyster spat mortalities require the presence of a UV sensitive agent. Furthermore, analysis of injected oyster spat revealed the detection of high amounts of OsHV-1 DNA by real-time quantitative PCR. Finally, TEM analysis demonstrated the presence of herpes virus particles. The developed protocol allowed to maintain sources of infective virus which can be useful for the development of further studies concerning the transmission and the development of OsHV-1 infection

A comparative analysis of the mobility of 45 proteins in the synaptic bouton

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology

Ultrastructural and functional fate of recycled vesicles in hippocampal synapses

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution

Plasticity of Astrocytic Coverage and Glutamate Transporter Expression in Adult Mouse Cortex

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton–spine interface and an increase in glutamate transporter expression in astrocytic processes

Dendrodendritic synapses in the mouse olfactory bulb external plexiform layer

Odor information relayed by olfactory bulb projection neurons, mitral and tufted cells (M/T), is modulated by pairs of reciprocal dendrodendritic synaptic circuits in the external plexiform layer (EPL). Interneurons, which are accounted for largely by granule cells, receive depolarizing input from M/T dendrites and in turn inhibit current spread in M/T dendrites via hyperpolarizing reciprocal dendrodendritic synapses. Because the location of dendrodendritic synapses may significantly affect the cascade of odor information, we assessed synaptic properties and density within sublaminae of the EPL and along the length of M/T secondary dendrites. In electron micrographs the M/T to granule cell synapse appeared to predominate and was equivalent in both the outer and inner EPL. However, the dendrodendritic synapses from granule cell spines onto M/T dendrites were more prevalent in the outer EPL. In contrast, individual gephyrin-immunoreactive (IR) puncta, a postsynaptic scaffolding protein at inhibitory synapses used here as a proxy for the granule to M/T dendritic synapse was equally distributed throughout the EPL. Of significance to the organization of intrabulbar circuits, gephyrin-IR synapses are not uniformly distributed along M/T secondary dendrites. Synaptic density, expressed as a function of surface area, increases distal to the cell body. Furthermore, the distributions of gephyrin-IR puncta are heterogeneous and appear as clusters along the length of the M/T dendrites. Consistent with computational models, our data suggest that temporal coding in M/T cells is achieved by precisely located inhibitory input and that distance from the soma is compensated for by an increase in synaptic density.Fil: Bartel, Dianna L.. University Of Yale. School Of Medicine; Estados UnidosFil: Rela, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Hsieh, Lawrence. University Of Yale. School Of Medicine; Estados UnidosFil: Greer, Charles A. . University Of Yale. School Of Medicine; Estados Unido

A preferentially segregated recycling vesicle pool of limited size supports neurotransmission in native central synapses

At small central synapses, efficient turnover of vesicles is crucial for stimulus-driven transmission, but how the structure of this recycling pool relates to its functional role remains unclear. Here we characterize the organizational principles of functional vesicles at native hippocampal synapses with nanoscale resolution using fluorescent dye labeling and electron microscopy. We show that the recycling pool broadly scales with the magnitude of the total vesicle pool, but its average size is small (∼45 vesicles), highly variable, and regulated by CDK5/calcineurin activity. Spatial analysis demonstrates that recycling vesicles are preferentially arranged near the active zone and this segregation is abolished by actin stabilization, slowing the rate of activity-driven exocytosis. Our approach reveals a similarly biased recycling pool distribution at synapses in visual cortex activated by sensory stimulation in vivo. We suggest that in small native central synapses, efficient release of a limited pool of vesicles relies on their favored spatial positioning within the termina

Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca2+ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca2+ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca2+ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized

Dendritic position is a major determinant of presynaptic strength

Different regulatory principles influence synaptic coupling between neurons, including positional principles. In dendrites of pyramidal neurons, postsynaptic sensitivity depends on synapse location, with distal synapses having the highest gain. In this paper, we investigate whether similar rules exist for presynaptic terminals in mixed networks of pyramidal and dentate gyrus (DG) neurons. Unexpectedly, distal synapses had the lowest staining intensities for vesicular proteins vGlut, vGAT, Synaptotagmin, and VAMP and for many nonvesicular proteins, including Bassoon, Munc18, and Syntaxin. Concomitantly, distal synapses displayed less vesicle release upon stimulation. This dependence of presynaptic strength on dendritic position persisted after chronically blocking action potential firing and postsynaptic receptors but was markedly reduced on DG dendrites compared with pyramidal dendrites. These data reveal a novel rule, independent of neuronal activity, which regulates presynaptic strength according to dendritic position, with the strongest terminals closest to the soma. This gradient is opposite to postsynaptic gradients observed in pyramidal dendrites, and different cell types apply this rule to a different extent