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The ventral tegmental area (VTA) and nucleus accumbens (NAc) are essential for learning about environmental stimuli associated with motivationally relevant outcomes. The task of signalling such events, both rewarding and [...]

Cryo-electron microscopy of vitreous section makes it possible to observe cells and tissues at high resolution in a close-to-native state. The specimen remains hydrated; chemical fixation and staining are fully avoided. There is minimal molecular aggregation and the density observed in the image corresponds to the density in the object. Accordingly, organotypic hippocampal rat slices were vitrified under high pressure and controlled cryoprotection conditions, cryosectioned at a final thickness of [approximately equal to] 70 nm and observed below -170[degrees]C in a transmission electron microscope. The general aspect of the tissue compares with previous electron microscopy observations. The detailed analysis of the synapse reveals that the density of material in the synaptic cleft is high, even higher than in the cytoplasm, and that it is organized in 8.2-nm periodic transcleft complexes. Previously undescribed structures of presynaptic and postsynaptic elements are also described. Cryo-electron microscopy of vitreous section | high-pressure freezing | hippocampus

Recent developments in confocal imaging techniques have opened new avenues for investigating how synaptic networks evolve over time. These studies have revealed not only that synaptic structures are motile but also, most importantly, that a fraction of synapses undergo a continuous elimination and formation process, and that these mechanisms are markedly enhanced by activity. Turnover of dendritic spines was recently demonstrated in the somatosensory cortex upon sensory stimulation. De Paola et al. have now provided evidence for correlated remodelling of presynaptic structures. These results, together with others, indicate that activation of neuronal networks enhances dynamic mechanisms at both the presynaptic and the postsynaptic level, resulting in an increased turnover of synapses and, accordingly, a reorganization of synaptic circuitry.

Synaptic scaffolding proteins from membrane-associated guanylate kinases (MAGUK) family are implicated in synapse formation and functioning. To better understand the role of one of the proteins of this family, SAP97, we studied with electron microscopy the effects of its overexpression on spine and synapse morphology in CA1 pyramidal neurons of rat organotypic hippocampal slice cultures. Dramatic spine enlargement induced by SAP97 overexpression was accompanied by marked morphological changes, with spines enwrapping and engulfing presynaptic terminals. The size and complexity of the PSD was also significantly increased. Similar to PSD-95, SAP97 promoted formation of multi-innervated spines (MIS). In addition, both MAGUK proteins induced multiple excitatory contacts on dendritic shafts suggesting a mechanism for shaft synapse formation. Formation of MIS and shaft synapses was blocked by the nitric oxide synthase (NOS) inhibitor L-NAME. Immunochemistry revealed that overexpression of SAP97 was associated with overexpression of PSD-95 and recruitment of nNOS to the synapse. These data provide evidence for both common and distinct structural alterations produced by overexpression of SAP97 and PSD-95 and demonstrate strong interactions between these two proteins to regulate contact formation through nitric oxide signaling. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Patterns of activity that induce synaptic plasticity at excitatory synapses, such as long-term potentiation, result in structural remodeling of the postsynaptic spine, comprising an enlargement of the spine head and reorganization of the postsynaptic density (PSD). Furthermore, spine synapses represent complex functional units in which interaction between the presynaptic varicosity and the postsynaptic spine is also modulated by surrounding astroglial processes. To investigate how activity patterns could affect the morphological interplay between these three partners, we used an electron microscopic (EM) approach and 3D reconstructions of excitatory synapses to study the activity-related morphological changes underlying induction of synaptic potentiation by theta burst stimulation or brief oxygen/glucose deprivation episodes in hippocampal organotypic slice cultures. EM analyses demonstrated that the typical glia-synapse organization described in in vivo rat hippocampus is perfectly preserved and comparable in organotypic slice cultures. Three-dimensional reconstructions of synapses, classified as simple or complex depending upon PSD organization, showed significant changes following induction of synaptic potentiation using both protocols. The spine head volume and the area of the PSD significantly enlarged 30 min and 1 h after stimulation, particularly in large synapses with complex PSD, an effect that was associated with a concomitant enlargement of presynaptic terminals. Furthermore, synaptic activity induced a pronounced increase of the glial coverage of both pre- and postsynaptic structures, these changes being prevented by application of the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid. These data reveal dynamic, activity-dependent interactions between glial processes and pre- and postsynaptic partners and suggest that glia can participate in activity-induced structural synapse remodeling. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Expression of the neural cell adhesion molecule (NCAM) has been shown to promote long-term potentiation (LTP) and stabilization of synapses during early synaptogenesis. Here, we searched for the mechanisms of synaptogenic activity of NCAM, focusing on the role of polysialic acid (PSA), an unusual carbohydrate preferentially associated with NCAM. We show that enzymatic removal of PSA with endoneuraminidase-N (endo-N) abolished preferential formation of synapses on NCAM-expressing cells in heterogenotypic cocultures of wild-type and NCAM-deficient hippocampal neurons. Transfection of NCAM-deficient neurons with either of three major NCAM isoforms (different in intracellular domains but identical in extracellular domains and carrying PSA) stimulated preferential synapse formation on NCAM isoform-expressing neurons. Enzymatic removal of heparan sulfates from cultured neurons and a mutation in the heparin-binding domain of NCAM diminished synaptogenic activity of neuronally expressed PSA-NCAM, suggesting that interaction of NCAM with heparan sulfate proteoglycans mediates this activity. PSA-NCAM-driven synaptogenesis was also blocked by antagonists to fibroblast growth factor receptor and NMDA subtype of glutamate receptors but not by blockers of non-NMDA glutamate receptors and voltage-dependent Na+ channels. Enzymatic removal of PSA and heparan sulfates also blocked the increase in the number of perforated spine synapses associated with NMDA receptor-dependent LTP in the CA1 region of organotypic hippocampal cultures. Thus, neuronal PSA-NCAM in complex with heparan sulfate proteoglycans promotes synaptogenesis and activity-dependent remodeling of synapses. [ABSTRACT FROM AUTHOR]

Zinc is concentrated in certain CNS excitatory tracts, especially in hippocampal mossy fibres where it has been suggested to modulate synaptic transmission and plasticity. Using rat mossy fibre synaptosomes depolarized by 4-aminopyridine, we show here that low zinc concentrations restore the membrane potential and reduce glutamate release. Both effects arose from activation of ATP-sensitive potassium channels (KATP), since they were mimicked by the KATP opener diazoxide and antagonized by the KATP blocker tolbutamide. Using recombinant channels expressed in COS-7 cells, we confirmed that micromolar zinc did activate KATP of the type found in hippocampus. We tested the hypothesis that this action of zinc could be beneficial during an ischaemic challenge by using organotypic hippocampal slice cultures. When zinc was applied at micromolar concentrations during a brief anoxic-hypoglycaemic episode, it significantly attenuated the ensuing neuronal death, whereas chelation of endogenous zinc markedly aggravated cell damage. Protective effect of zinc was mediated through KATP, as was shown by using the opener diazoxide and the blocker tolbutamide. Thus, by activating pre-synaptic KATP channels, zinc protects neurones from hyper-excitation, excessive transmitter release and exitotoxicity, and may thus act as an endogenous neuroprotector in conditions such as epilepsy or stroke. [ABSTRACT FROM AUTHOR]