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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 plasticity mechanisms are usually discussed in terms of changes in synaptic strength. The capacity of excitatory synapses to rapidly modify the membrane expression of glutamate receptors in an activity-dependent manner plays a critical role in learning and memory processes by re-distributing activity within neuronal networks. Recent work has however also shown that functional plasticity properties are associated with a rewiring of synaptic connections and a selective stabilization of activated synapses. These structural aspects of plasticity have the potential to continuously modify the organization of synaptic networks and thereby introduce specificity in the wiring diagram of cortical circuits. Recent work has started to unravel some of the molecular mechanisms that underlie these properties of structural plasticity, highlighting an important role of signaling pathways that are also major candidates for contributing to developmental psychiatric disorders. We review here some of these recent advances and discuss the hypothesis that alterations of structural plasticity could represent a common mechanism contributing to the cognitive and functional defects observed in diseases such as intellectual disability, autism spectrum disorders and schizophrenia. [ABSTRACT FROM AUTHOR]

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]

Experience-driven activity plays an essential role in the development of brain circuitry during critical periods of early postnatal life, a process that depends upon a dynamic balance between excitatory and inhibitory signals. Since general anesthetics are powerful pharmacological modulators of neuronal activity, an important question is whether and how these drugs can affect the development of synaptic networks. To address this issue, we examined here the impact of anesthetics on synapse growth and dynamics. We show that exposure of young rodents to anesthetics that either enhance GABAergic inhibition or block NMDA receptors rapidly induce a significant increase in dendritic spine density in the somatosensory cortex and hippocampus. This effect is developmentally regulated; it is transient but lasts for several days and is also reproduced by selective antagonists of excitatory receptors. Analyses of spine dynamics in hippocampal slice cultures reveals that this effect is mediated through an increased rate of protrusions formation, a better stabilization of newly formed spines, and leads to the formation of functional synapses. Altogether, these findings point to anesthesia as an important modulator of spine dynamics in the developing brain and suggest the existence of a homeostatic process regulating spine formation as a function of neural activity. Importantly, they also raise concern about the potential impact of these drugs on human practice, when applied during critical periods of development in infants. [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]

Relatively mild ischemic episode can initiate a chain of events resulting in delayed cell death and significant lesions in the affected brain regions. We studied early synaptic modifications after brief ischemia modeled in rats by transient vessels' occlusion in vivo or oxygen-glucose deprivation in vitro and resulting in delayed death of hippocampal CA1 pyramidal cells. Electron microscopic analysis of excitatory spine synapses in CA1 stratum radiatum revealed a rapid increase of the postsynaptic density (PSD) thickness and length, as well as formation of concave synapses with perforated PSD during the first 24 h after ischemic episode, followed at the long term by degeneration of 80% of synaptic contacts. In presynaptic terminals, ischemia induced a depletion of synaptic vesicles and changes in their spatial arrangement: they became more distant from active zones and had larger intervesicle spacing compared to controls. These rapid structural synaptic changes could be implicated in the mechanisms of cell death or adaptive plasticity. Comparison of the in vivo and in vitro model systems used in the study demonstrated a general similarity of these early morphological changes, confirming the validity of the in vitro model for studying synaptic structural plasticity. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described. Microsc. Res. Tech. 68:90-96, 2005. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Mutations of the gene coding for PAK3 (p21-activated kinase 3) are associated with X-linked, nonsyndromic forms of mental retardation (MRX) in which the only distinctive clinical feature is the cognitive deficit. The mechanisms through which PAK3 mutation produces the mental handicap remain unclear, although an involvement in the mechanisms that regulate the formation or plasticity of synaptic networks has been proposed. Here we show, using a transient transfection approach, that antisense and small interfering RNA-mediated suppression of PAK3 or expression of a dominant-negative PAK3 carrying the human MRX30 mutation in rat hippocampal organotypic slice cultures results in the formation of abnormally elongated dendritic spines and filopodia-like protrusions and a decrease in mature spine synapses. Ultrastructural analysis of the changes induced by expression of PAK3 carrying the MRX30 mutation reveals that many elongated spines fail to express postsynaptic densities or contact presynaptic terminals. These defects are associated with a reduced spontaneous activity, altered expression of AMPA-type glutamate receptors, and defective long-term potentiation. Together, these data identify PAK3 as a key regulator of synapse formation and plasticity in the hippocampus and support interpretations that these defects might contribute to the cognitive deficits underlying this form of mental retardation. [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]

Induction of long-term potentiation and application of short periods of anoxia/hypoglycemia result in the growth of dendritic filopodia and formation of new spines. Here we investigated whether these conditions also affected the morphology of presynaptic structures. Using confocal imaging of DiI-labeled axons, electron microscopy, and stereological analyses, we show that short anoxia/hypoglycemia and theta burst stimulation induced rapid, calcium-dependent growth of presynaptic filopodia-like protrusions and remodeling of presynaptic varicosities. Three-dimensional reconstruction of axonal outgrowths revealed that, within 30 min, they made contacts and triggered the formation of a postsynaptic density on the target cell. Interestingly, these axonal filopodia first established synapses with the dendritic shaft and later mostly with spines. They also contributed to the formation of multi-innervated spines. Because these presynaptic growth mechanisms depended on NMDA receptor activation, we investigated whether a diffusing messenger could be involved. We found that blockade of nitric oxide synthase prevented these changes, and conversely, a nitric oxide donor could reproduce them. A model is presented that proposes that activation of NMDA receptors and subsequent release of nitric oxide could trigger the growth of presynaptic filopodia, which, in turn, play an active role in synaptogenesis and spine formation. [ABSTRACT FROM AUTHOR]

Integrins are a major family of heterodimeric surface glycoproteins that act as adhesion molecules, have a spectrum of extracellular matrix (ECM) molecules as their ligands, and regulate a variety of cellular functions. Integrins are known to be critical to embryonic brain development, and recent studies have indicated their essential role in adult brain function, although their role in postnatal brain development and function has not been examined. Here, we used the organotypic slice culture system to investigate the role of integrins in postnatal hippocampal development by exposing the tissue to either an integrin competitive antagonist, the peptide GRGDSP containing Arg–Gly–Asp (RGD) attachment site, or to function-blocking β1-integrin antibodies to disrupt integrin interactions. These experiments revealed that β1-integrin antibodies interfered with spreading of the culture, resulting in a rapid and marked diminution of slice area. β1-integrin antibodies and RGD peptide disrupted cell adhesion, causing cell detachment and migration of glial cells from the explant. The majority of the detached cells were of macroglial origin and switched to expression of the intermediate filament proteins vimentin and nestin, suggesting a developmental regression. The organotypic organization of slice cultures was not affected, although exposure to either integrin antagonist or antibody resulted in a statistically significant reduction in the number of synapses measured in the apical dendrites of CA1 pyramidal neurons. The results demonstrate that integrins markedly affect postnatal CNS development, in both ultrastructural construction and organizational processes. [Copyright &y& Elsevier]

Summary Background Excitatory synapses in the CNS are highly dynamic structures that can show activity-dependent remodeling and stabilization in response to learning and memory. Synapses are enveloped with intricate processes of astrocytes known as perisynaptic astrocytic processes (PAPs). PAPs are motile structures displaying rapid actin-dependent movements and are characterized by Ca 2+ elevations in response to neuronal activity. Despite a debated implication in synaptic plasticity, the role of both Ca 2+ events in astrocytes and PAP morphological dynamics remain unclear. Results In the hippocampus, we found that PAPs show extensive structural plasticity that is regulated by synaptic activity through astrocytic metabotropic glutamate receptors and intracellular calcium signaling. Synaptic activation that induces long-term potentiation caused a transient PAP motility increase leading to an enhanced astrocytic coverage of the synapse. Selective activation of calcium signals in individual PAPs using exogenous metabotropic receptor expression and two-photon uncaging reproduced these effects and enhanced spine stability. In vivo imaging in the somatosensory cortex of adult mice revealed that increased neuronal activity through whisker stimulation similarly elevates PAP movement. This in vivo PAP motility correlated with spine coverage and was predictive of spine stability. Conclusions This study identifies a novel bidirectional interaction between synapses and astrocytes, in which synaptic activity and synaptic potentiation regulate PAP structural plasticity, which in turn determines the fate of the synapse. This mechanism may represent an important contribution of astrocytes to learning and memory processes. [ABSTRACT FROM AUTHOR]

Abstract: Synaptic activity, such as long-term potentiation (LTP), has been shown to induce morphological plasticity of excitatory synapses on dendritic spines through the spine head and postsynaptic density (PSD) enlargement and reorganization. Much less, however, is known about activity-induced morphological modifications of inhibitory synapses. Using an in vitro model of rat organotypic hippocampal slice cultures and electron microscopy, we studied activity-related morphological changes of somatic inhibitory inputs triggered by a brief oxygen–glucose deprivation (OGD) episode, a condition associated with a synaptic enhancement referred to as anoxic LTP and a structural remodeling of excitatory synapses. Three-dimensional reconstruction of inhibitory axo-somatic synapses at different times before and after brief OGD revealed important morphological changes. The PSD area significantly and markedly increased at synapses with large and complex PSDs, but not at synapses with simple, macular PSDs. Activity-related changes of PSD size and presynaptic bouton volume developed in a strongly correlated manner. Analyses of single and serial sections further showed that the density of inhibitory synaptic contacts on the cell soma did not change within 1 h after OGD. In contrast, the proportion of the cell surface covered with inhibitory PSDs, as well as the complexity of these PSDs significantly increased, with less macular PSDs and more complex, segmented shapes. Together, these data reveal a rapid activity-related restructuring of somatic inhibitory synapses characterized by an enlargement and increased complexity of inhibitory PSDs, providing a new mechanism for a quick adjustment of the excitatory–inhibitory balance. This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’. [Copyright &y& Elsevier]

The function and efficacy of synaptic transmission are determined not only by the composition and activity of pre- and postsynaptic components but also by the environment in which a synapse is embedded. Glial cells constitute an important part of this environment and participate in several aspects of synaptic functions. Among the glial cell family, the roles played by astrocytes at the synaptic level are particularly important, ranging from the trophic support to the fine-tuning of transmission. Astrocytic structures are frequently observed in close association with glutamatergic synapses, providing a morphological entity for bidirectional interactions with synapses. Experimental evidence indicates that astrocytes sense neuronal activity by elevating their intracellular calcium in response to neurotransmitters and may communicate with neurons. The precise role of astrocytes in regulating synaptic properties, function, and plasticity remains however a subject of intense debate and many aspects of their interactions with neurons remain to be investigated. A particularly intriguing aspect is their ability to rapidly restructure their processes and modify their coverage of the synaptic elements. The present review summarizes some of these findings with a particular focus on the mechanisms driving this form of structural plasticity and its possible impact on synaptic structure and function.

Background: Recent observations demonstrate that anesthetics rapidly impair synaptogenesis during neuronal circuitry development. Whether these effects are lasting and depend on the developmental stage at which these drugs are administered remains, however, to be explored., Methods: Wistar rats received propofol anesthesia at defined developmental stages during early postnatal life. The acute and long-term effects of these treatments on neuronal cytoarchitecture were evaluated by Neurolucida and confocal microscopy analysis after iontophoretic injections of Lucifer Yellow into layer 5 pyramidal neurons in the medial prefrontal cortex. Quantitative electron microscopy was applied to investigate synapse density., Results: Layer 5 pyramidal neurons of the medial prefrontal cortex displayed intense dendritic growth and spinogenesis during the first postnatal month. Exposure of rat pups to propofol at postnatal days 5 and 10 significantly decreased dendritic spine density, whereas this drug induced a significant increase in spine density when administered at postnatal days 15, 20, or 30. Quantitative electron microscopy revealed that the propofol-induced increase in spine density was accompanied by a significant increase in the number of synapses. Importantly, the propofol-induced modifications in dendritic spine densities persisted up to postnatal day 90., Conclusion: These new results demonstrate that propofol anesthesia can rapidly induce significant changes in dendritic spine density and that these effects are developmental stage-dependent, persist into adulthood, and are accompanied by alterations in synapse number. These data suggest that anesthesia in the early postnatal period might permanently impair circuit assembly in the developing brain.

Our current understanding of the mechanisms of information processing and storage in the brain, based on the concept proposed more than fifty years ago by D. Hebb, is that a key role is played by changes in synaptic efficacy induced by coincident pre- and postsynaptic activity. Decades of studies of the properties of long-term potentiation (LTP) have shown that this form of plasticity adequately fulfills these requirements and is likely to contribute to several models of learning and memory. Recent analyses of the molecular events implicated in LTP are consistent with the view that modifications of receptor properties or insertion of new receptors account for the potentiation of synaptic transmission. These experiments, however, have also uncovered an unexpected structural plasticity of synapses. Dendritic spines appear to be dynamic structures that can be formed, modified in their shape or eliminated under the influence of activity. Furthermore, recent studies suggest that LTP, in addition to changes in synaptic function, is also associated with mechanisms of synaptogenesis. We review here the evidence pointing to this activity-dependent remodeling and discuss the possible role of this structural plasticity for synaptic potentiation, learning and memory.

Cerebral ischemia is a major cause of brain dysfunction. Using a model of delayed death induced by a brief, transient oxygen and glucose deprivation, we studied here how this affected the structural organization of hippocampal synaptic networks. We report that brief anoxic-hypoglycemic episodes rapidly modified the structure of synapses. This was characterized, at the electron microscopic level, by a transient increase in the proportion of perforated synapses, followed after 2 hr by an increase in images of multiple synapse boutons. These changes were considerable because 10-20% of all synapses were affected. This structural remodeling was correlated by three kinds of modifications observed using two-photon confocal microscopy: the growth of filopodia, occurring shortly (5-20 min) after anoxia-hypoglycemia, enlargements of existing spines, and formation of new spines, both seen mainly 20-60 min after the insult. All of these structural changes were calcium and NMDA receptor dependent and thus reproduced, to a larger scale, those associated with synaptic plasticity. Concomitantly and related to the severity of anoxia-hypoglycemia, we could also observe spine loss and images of spine, dendrite, or presynaptic terminal swellings that evolved up to membrane disruption. These changes were also calcium dependent and reduced by NMDA receptor antagonists. Thus, short anoxic-hypoglycemic episodes, through NMDA receptor activation and calcium influx, resulted in a profound structural remodeling of synaptic networks, through growth, formation, and elimination of spines and synapses.