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The synaptic cleft is the extracellular part of the synapse, bridging the pre- and postsynaptic membranes. The geometry and molecular organization of the cleft is gaining increased attention as an important determinant of synaptic efficacy. The present study by electron microscopy focuses on short-term morphological changes at the synaptic cleft under excitatory conditions. Depolarization of cultured hippocampal neurons with high K+ results in an increased frequency of synaptic profiles with clefts widened at the periphery (open clefts), typically exhibiting patches of membranes lined by postsynaptic density, but lacking associated presynaptic membranes (18.0% open clefts in high K+ compared to 1.8% in controls). Similarly, higher frequencies of open clefts were observed in adult brain upon a delay of perfusion fixation to promote excitatory/ischemic conditions. Inhibition of basal activity in cultured neurons through the application of TTX results in the disappearance of open clefts whereas application of NMDA increases their frequency (19.0% in NMDA vs. 5.3% in control and 2.6% in APV). Depletion of extracellular Ca2+ with EGTA also promotes an increase in the frequency of open clefts (16.6% in EGTA vs. 4.0% in controls), comparable to that by depolarization or NMDA, implicating dissociation of Ca2+-dependent trans-synaptic bridges. Dissociation of transsynaptic bridges under excitatory conditions may allow perisynaptic mobile elements, such as AMPA receptors to enter the cleft. In addition, peripheral opening of the cleft would facilitate neurotransmitter clearance and thus may have a homeostatic and/or protective function.
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A-kinase anchoring protein 79-human/150-rodent (AKAP79/150) organizes signaling proteins to control synaptic plasticity. AKAP79/150 associates with the plasma membrane and endosomes through its N-terminal domain that contains three polybasic regions and two Cys residues that are reversibly palmitoylated. Mutations abolishing palmitoylation (AKAP79/150 CS) reduce its endosomal localization and association with the postsynaptic density (PSD). Here we combined advanced light and electron microscopy (EM) to characterize the effects of AKAP79/150 palmitoylation on its postsynaptic nanoscale organization, trafficking, and mobility in hippocampal neurons. Immunogold EM revealed prominent extrasynaptic membrane AKAP150 labeling with less labeling at the PSD. The label was at greater distances from the spine membrane for AKAP150 CS than WT in the PSD but not in extra-synaptic locations. Immunogold EM of GFP-tagged AKAP79 WT showed that AKAP79 adopts a vertical, extended conformation at the PSD with its N-terminus at the membrane, in contrast to extrasynaptic locations where it adopts a compact or open configurations of its N- and C-termini with parallel orientation to the membrane. In contrast, GFP-tagged AKAP79 CS was displaced from the PSD coincident with disruption of its vertical orientation, while proximity and orientation with respect to the extra-synaptic membrane was less impacted. Single-molecule localization microscopy (SMLM) revealed a heterogeneous distribution of AKAP150 with distinct high-density, nano-scale regions (HDRs) overlapping the PSD but more prominently located in the extrasynaptic membrane for WT and the CS mutant. Thick section scanning transmission electron microscopy (STEM) tomography revealed AKAP150 immunogold clusters similar in size to HDRs seen by SMLM and more AKAP150 labeled endosomes in spines for WT than for CS, consistent with the requirement for AKAP palmitoylation in endosomal trafficking. Hidden Markov modeling of single molecule tracking data revealed a bound/immobile fraction and two mobile fractions for AKAP79 in spines, with the CS mutant having shorter dwell times and faster transition rates between states than WT, suggesting that palmitoylation stabilizes individual AKAP molecules in various spine subpopulations. These data demonstrate that palmitoylation fine tunes the nanoscale localization, mobility, and trafficking of AKAP79/150 in dendritic spines, which might have profound effects on its regulation of synaptic plasticity.
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Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.
Assuntos
Evolução Biológica , Citofagocitose , Imunidade Inata , Placozoa/imunologia , Rodófitas/imunologia , Cicatrização , Animais , FilogeniaRESUMO
Trichoplax adhaerens is an enigmatic animal with an extraordinarily simple morphology and a cellular organization, which are the focus of current research. Protocols outlined here provide detailed descriptions of advanced techniques for light and electron microscopic studies of Trichoplax. Studies using these techniques have enhanced our understanding of cell type diversity and function in placozoans and have provided insight into the evolution, development, and physiology of this little understood group.
Assuntos
Microscopia Eletrônica/métodos , Microscopia/métodos , Placozoa/ultraestrutura , Animais , Criopreservação/métodos , Imuno-Histoquímica/métodos , Microtomia/métodos , Placozoa/citologia , Fixação de Tecidos/métodosRESUMO
Decision making is an inherent, complex and vital component of the work of managers. Its importance and role in operationalizing the activities of an organisation are well-evidenced in management literature. Yet, there is a dearth of literature about the processes used by nurse managers to make decisions. The principal aim of this paper is to identify the different types of decisions made by nurse managers and explore the related decision-making processes. A 'dependency model' is proposed, which illustrates the factors affecting the art of decision making. Structured interviews were conducted to identify types of decisions made by nurse managers in different health-care settings and the factors underpinning these decisions. The research focused on an intensive study of a small group of nurse managers working in rural and regional health institutions in South Australia. The sample included nurse unit managers, after-hours coordinators and directors of nursing. Hermeneutic principles and interpretive research were used to conduct interviews with nurse managers who make numerous and varied types of decisions, though often without following a step-by-step approach. The study identified dependency factors that influence how decisions are made, and developed a model based on eight key variables: (1) the situation to be addressed; (2) the time period in which the decision has to be made; (3) required inputs from colleagues; (4) complexity of the task and the environment, (5) the duration and time it takes to make a decision, (6) availability of resources, (7) the decision-making environment, and (8) personal characteristics. These eight variables are interrelated and have both direct and indirect impacts on how decisions are made. Nurse managers make pragmatic decisions reflecting the complexity of their roles and responsibilities. Awareness of the factors on which decisions depend helps understanding of how they navigate through decision-making processes. The findings are presented as a model that can be used to support decision making by nurse managers in various health settings.
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The disk-shaped millimeter-sized marine animal, Trichoplax adhaerens, is notable because of its small number of cell types and primitive mode of feeding. It glides on substrates propelled by beating cilia on its lower surface and periodically pauses to feed on underlying microorganisms, which it digests externally. Here, a combination of advanced electron and light microscopic techniques are used to take a closer look at its secretory cell types and their roles in locomotion and feeding. We identify digestive enzymes in lipophils, a cell type implicated in external digestion and distributed uniformly throughout the ventral epithelium except for a narrow zone near its edge. We find three morphologically distinct types of gland cell. The most prevalent contains and secretes mucus, which is shown to be involved in adhesion and gliding. Half of the mucocytes are arrayed in a tight row around the edge of the ventral epithelium while the rest are scattered further inside, in the region containing lipophils. The secretory granules in mucocytes at the edge label with an antibody against a neuropeptide that was reported to arrest ciliary beating during feeding. A second type of gland cell is arrayed in a narrow row just inside the row of mucocytes while a third is located more centrally. Our maps of the positions of the structurally distinct secretory cell types provide a foundation for further characterization of the multiple peptidergic cell types in Trichoplax and the microscopic techniques we introduce provide tools for carrying out these studies.
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Analysis of affinity-purified PSD-95 complexes had previously identified a 'hypothetical protein', product of the gene FAM81A [1]. The present study examined the tissue and subcellular distribution of FAM81A protein and its expression levels during development. Comparison of different organs indicates selective expression of FAM81A protein in brain. FAM81A is expressed late in development, with a post-natal gradual increase in brain levels that parallels the expression of PSD-95. Comparison of subcellular fractions from adult brain shows that the distribution of FAM81A protein is similar to that of PSD-95, with a drastic enrichment in the postsynaptic density fraction. Immuno-electron microscopy of adult brain tissue reveals specific immunogold labeling for FAM81A protein at postsynaptic densities in the forebrain. The label for FAM81A protein is concentrated at the cytoplasmic edge of the electron-dense core of the postsynaptic density, with a mean distance of â¼33 nm from the postsynaptic membrane. These observations firmly establish FAM81A protein as a component of the postsynaptic density in the adult brain, suggesting a role in synaptic function.
Assuntos
Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Densidade Pós-Sináptica/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Proteína 4 Homóloga a Disks-Large/biossíntese , Feminino , Masculino , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Ratos , Distribuição TecidualRESUMO
Combining tomography with electron microscopy (EM) produces images at definition sufficient to visualize individual protein molecules or molecular complexes in intact neurons. When freeze-substituted hippocampal cultures in plastic sections are imaged by EM tomography, detailed structures emerging from 3D reconstructions reveal putative glutamate receptors and membrane-associated filaments containing scaffolding proteins such as postsynaptic density (PSD)-95 family proteins based on their size, shape, and known distributions. In limited instances, structures can be identified with enhanced immuno-Nanogold labeling after light fixation and subsequent freeze-substitution. Molecular identification of structure can be corroborated in their absence after acute protein knockdown or gene knockout. However, additional labeling methods linking EM level structure to molecules in tomograms are needed. A recent development for labeling structures for TEM employs expression of endogenous proteins carrying a green fluorescent tag, miniSOG, to photoconvert diaminobenzidine (DAB) into osmiophilic polymers. This approach requires initial mild chemical fixation but many of structural features in neurons can still be discerned in EM tomograms. The photoreaction product, which appears as electron-dense, fine precipitates decorating protein structures in neurons, may diffuse to fill cytoplasm of spines, thus obscuring specific localization of proteins tagged with miniSOG. Here we develop an approach to minimize molecular diffusion of the DAB photoreaction product in neurons, which allows miniSOG tagged molecule/complexes to be identified in tomograms. The examples reveal electron-dense clusters of reaction product labeling membrane-associated vertical filaments, corresponding to the site of miniSOG fused at the C-terminal end of PSD-95-miniSOG, allowing identification of PSD-95 vertical filaments at the PSD. This approach, which results in considerable improvement in the precision of labeling PSD-95 in tomograms without complications due to the presence of antibody complexes in immunogold labeling, may be applicable for identifying other synaptic proteins in intact neurons.
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Trichoplax adhaerens has only six cell types. The function as well as the structure of crystal cells, the least numerous cell type, presented an enigma. Crystal cells are arrayed around the perimeter of the animal and each contains a birefringent crystal. Crystal cells resemble lithocytes in other animals so we looked for evidence they are gravity sensors. Confocal microscopy showed that their cup-shaped nuclei are oriented toward the edge of the animal, and that the crystal shifts downward under the influence of gravity. Some animals spontaneously lack crystal cells and these animals behaved differently upon being tilted vertically than animals with a typical number of crystal cells. EM revealed crystal cell contacts with fiber cells and epithelial cells but these contacts lacked features of synapses. EM spectroscopic analyses showed that crystals consist of the aragonite form of calcium carbonate. We thus provide behavioral evidence that Trichoplax are able to sense gravity, and that crystal cells are likely to be their gravity receptors. Moreover, because placozoans are thought to have evolved during Ediacaran or Cryogenian eras associated with aragonite seas, and their crystals are made of aragonite, they may have acquired gravity sensors during this early era.
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Carbonato de Cálcio/metabolismo , Gravitação , Placozoa/metabolismo , Animais , Carbonato de Cálcio/química , Cristalização , Corantes Fluorescentes , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Neurônios , Placozoa/citologia , Análise Espectral/métodos , SinapsesRESUMO
Shank3 is a postsynaptic density (PSD) scaffold protein of the Shank family. Here we use pre-embedding immunogold electron microscopy to investigate factors influencing the distribution of Shank3 at the PSD. In dissociated rat hippocampal cultures under basal conditions, label for Shank3 was concentrated in a broad layer of the PSD, ~20-80 nm from the postsynaptic membrane. Upon depolarization with high K+ (90 mM, 2 min), or application of NMDA (50 µM, 2 min), both the labeling intensity at the PSD and the median distance of label from the postsynaptic membrane increased significantly, indicating that Shank3 molecules are preferentially recruited to the distal layer of the PSD. Incubation in medium supplemented with zinc (50 µM ZnCl2, 1 hr) also significantly increased labeling intensity for Shank3 at the PSD, but this addition of Shank3 was not preferential to the distal layer. When cells were incubated with zinc and then treated with NMDA, labeling intensity of Shank3 became higher than with either treatment alone and manifested a preference for the distal layer of the PSD. Without zinc supplementation, NMDA-induced accumulation of Shank3 at the PSD was transient, reversing within 30 min after return to control medium. However, when zinc was included in culture media throughout the experiment, the NMDA-induced accumulation of Shank3 was largely retained, including Shank3 molecules recruited to the distal layer of the PSD. These results demonstrate that activity induces accumulation of Shank3 at the PSD and that zinc stabilizes PSD-associated Shank3, possibly through strengthening of Shank-Shank association.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Densidade Pós-Sináptica/metabolismo , Sinapses/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Microscopia Eletrônica/métodos , Neurônios/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Depolarization of neurons in 3-week-old rat hippocampal cultures promotes a rapid increase in the density of surface NMDA receptors (NRs), accompanied by transient formation of nonsynaptic NMDA receptor clusters or NR islands. Islands exhibit cytoplasmic dense material resembling that at postsynaptic densities (PSDs), and contain typical PSD components, including MAGUKS (membrane-associated guanylate kinases), GKAP, Shank, Homer, and CaMKII detected by pre-embedding immunogold electron microscopy. In contrast to mature PSDs, islands contain more NMDA than AMPA receptors, and more SAP102 than PSD-95, features that are shared with nascent PSDs in developing synapses. Islands do not appear to be exocytosed or endocytosed directly as preformed packages because neurons lacked intracellular vacuoles containing island-like structures. Islands form and disassemble upon depolarization of neurons on a time scale of 2-3 min, perhaps representing an initial stage in synaptogenesis.
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Hipocampo/metabolismo , Neurônios/metabolismo , Densidade Pós-Sináptica/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Guanilato Quinases/análise , Guanilato Quinases/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/análise , Sinapses/metabolismoRESUMO
The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Densidade Pós-Sináptica/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , RatosRESUMO
BACKGROUND: Trichoplax adhaerens is the best-known member of the phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types. RESULTS: We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior, where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide. CONCLUSIONS: Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior.
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Grânulos Citoplasmáticos/metabolismo , Células Epiteliais/metabolismo , Neurônios/metabolismo , Neurossecreção/fisiologia , Placozoa/anatomia & histologia , Placozoa/citologia , Animais , Células Epiteliais/classificação , Epitélio/metabolismo , Neurônios/classificaçãoRESUMO
The regulation of synaptic strength at γ-aminobutyric acid (GABA)-ergic synapses is dependent on the dynamic capture, retention, and modulation of GABA A-type receptors by cytoplasmic proteins at GABAergic postsynaptic sites. How these proteins are oriented and organized in the postsynaptic cytoplasm is not yet established. To better understand these structures and gain further insight into the mechanisms by which they regulate receptor populations at postsynaptic sites, we utilized electron tomography to examine GABAergic synapses in dissociated rat hippocampal cultures. GABAergic synapses were identified and selected for tomography by using a set of criteria derived from the structure of immunogold-labeled GABAergic synapses. Tomography revealed a complex postsynaptic network composed of filaments that extend ⼠100 nm into the cytoplasm from the postsynaptic membrane. The distribution of these postsynaptic filaments was strikingly similar to that of the immunogold label for gephyrin. Filaments were interconnected through uniform patterns of contact, forming complexes composed of 2-12 filaments each. Complexes did not link to form an integrated, continuous scaffold, suggesting that GABAergic postsynaptic specializations are less rigidly organized than glutamatergic postsynaptic densities.
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Encéfalo/citologia , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/ultraestrutura , Rede Nervosa/ultraestrutura , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Tomografia com Microscopia Eletrônica , Técnica de Fratura por Congelamento , Glutamato Descarboxilase/metabolismo , Glutamato Descarboxilase/ultraestrutura , Proteínas de Membrana/metabolismo , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-Dawley , Membranas Sinápticas/ultraestrutura , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
Calcium calmodulin dependent kinase II (CaMKII) is sequestered in dendritic spines within seconds upon synaptic stimulation. The program Smoldyn was used to develop scenarios of single molecule CaMKII diffusion and binding in virtual dendritic spines. We first validated simulation of diffusion as a function of spine morphology. Additional cellular structures were then incorporated to simulate binding of CaMKII to the post-synaptic density (PSD); binding to cytoskeleton; or their self-aggregation. The distributions of GFP tagged native and mutant constructs in dissociated hippocampal neurons were measured to guide quantitative analysis. Intra-spine viscosity was estimated from fluorescence recovery after photo-bleach (FRAP) of red fluorescent protein. Intra-spine mobility of the GFP-CaMKIIα constructs was measured, with hundred-millisecond or better time resolution, from FRAP of distal spine tips in conjunction with fluorescence loss (FLIP) from proximal regions. Different FRAP \ FLIP profiles were predicted from our Scenarios and provided a means to differentiate binding to the PSDs from self-aggregation. The predictions were validated by experiments. Simulated fits of the Scenarios provided estimates of binding and rate constants. We utilized these values to assess the role of self-aggregation during the initial response of native CaMKII holoenzymes to stimulation. The computations revealed that self-aggregation could provide a concentration-dependent switch to amplify CaMKII sequestration and regulate its activity depending on its occupancy of the actin cytoskeleton.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citoesqueleto/enzimologia , Espinhas Dendríticas/enzimologia , Modelos Neurológicos , Transmissão Sináptica/fisiologia , Actinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Cultura Primária de Células , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/citologia , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Sinapses , Animais , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Feminino , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lentivirus/fisiologia , Masculino , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão/métodos , Modelos Biológicos , Interferência de RNA/fisiologia , Ratos , Receptores de AMPA/metabolismo , Receptores de AMPA/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Transfecção/métodosRESUMO
The number of AMPA receptors at synapses depends on receptor cycling. Because receptors diffuse rapidly in plasma membranes, their exocytosis and endocytosis need not occur near synapses. Here, pre-embedding immunogold electron microscopy is applied to dissociated rat hippocampal cultures to provide sensitive, high-resolution snapshots of the distribution of surface AMPA receptors in spines, dendrites, and cell bodies that will be informative about trafficking of AMPA receptors. The density of the label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSDs), where it is typically higher. Glutamate receptor 2 (GluR2) labels at PSDs significantly increase after synaptic activation by glycine treatment and increase further upon depolarization by high K(+). Islands of densely packed labels have consistent size and density but vary in frequency under different experimental conditions. These patches of label, which occur on plasma membranes of cell bodies and dendrites but not near PSDs, are taken to be the aftermath of exocytosis of AMPA receptors. A subpopulation of clathrin-coated pits in cell bodies and dendrites label for GluR2, and the number and amount of label in individual pits increase after NMDA treatment. Coated pits near synapses typically lack GluR2 label under basal conditions, but â¼40% of peri-PSD pits label for GluR2 after NMDA treatment. Thus, exocytosis and endocytosis of AMPA receptors occur mainly at extrasynaptic locations on cell bodies and dendrites. Receptors are not preferentially exocytosed near PSDs, but may be removed via endocytosis at peri-PSD locations after activation of NMDA receptors.
Assuntos
Membrana Celular/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Animais , Membrana Celular/ultraestrutura , Células Cultivadas , Endocitose/fisiologia , Exocitose/fisiologia , Feminino , Hipocampo/ultraestrutura , Masculino , Neurônios/ultraestrutura , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Hippocampal CA1 pyramidal neurons are selectively vulnerable to ischemia, while adjacent CA3 neurons are relatively resistant. Although glutamate receptor-mediated mitochondrial Ca(2+) overload and dysfunction is a major component of ischemia-induced neuronal death, no direct relationship between selective neuronal vulnerability and mitochondrial dysfunction has been demonstrated in intact brain preparations. Here, we show that in organotypic slice cultures NMDA induces much larger Ca(2+) elevations in vulnerable CA1 neurons than in resistant CA3. Consequently, CA1 mitochondria exhibit stronger calcium accumulation, more extensive swelling and damage, stronger depolarization of their membrane potential, and a significant increase in ROS generation. NMDA-induced Ca(2+) and ROS elevations were abolished in Ca(2+)-free medium or by NMDAR antagonists, but not by zinc chelation. We conclude that Ca(2)(+) overload-dependent mitochondrial dysfunction is a determining factor in the selective vulnerability of CA1 neurons.
Assuntos
Isquemia Encefálica/metabolismo , Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/metabolismo , Cálcio/metabolismo , Degeneração Neural/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Animais Recém-Nascidos , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Região CA3 Hipocampal/patologia , Região CA3 Hipocampal/fisiopatologia , Cálcio/toxicidade , Sinalização do Cálcio/fisiologia , Causalidade , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
Overactivation of NMDA receptors (NMDARs) is a critical early step in glutamate-evoked excitotoxic injury of CNS neurons. Distinct NMDAR-coupled pathways specified by, for example, receptor location or subunit composition seem to govern glutamate-induced excitotoxic death, but there is much uncertainty concerning the underlying mechanisms of pathway selection. Here we ask whether, and if so how, route-specific vulnerability is coupled to Ca(2+) overload and mitochondrial dysfunction, which is also a known, central component of exitotoxic injury. In cultured hippocampal neurons, overactivation of only extrasynaptic NMDARs resulted in Ca(2+) entry strong enough to promote Ca(2+) overload, which subsequently leads to mitochondrial dysfunction and cell death. Receptor composition per se appears not to be a primary factor for specifying signal coupling, as NR2B inhibition abolished Ca(2+) loading and was protective only in predominantly NR2B-expressing young neurons. In older neurons expressing comparable levels of NR2A- and NR2B-containing NMDARs, amelioration of Ca(2+) overload required the inhibition of extrasynaptic receptors containing both NR2 subunits. Prosurvival synaptic stimuli also evoked Ca(2+) entry through both N2A- and NR2B-containing NMDARs, but, in contrast to excitotoxic activation of extrasynaptic NMDARs, produced only low-amplitude cytoplasmic Ca(2+) spikes and modest, nondamaging mitochondrial Ca(2+) accumulation. The results--showing that the various routes of excitotoxic Ca(2+) entry converge on a common pathway involving Ca(2+) overload-induced mitochondrial dysfunction--reconcile and unify many aspects of the "route-specific" and "calcium load-dependent" views of exitotoxic injury.