Search

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.

Significance Trichoplax is a disk-shaped marine animal whose body plan and lifestyle could be similar to those of the Precambrian ancestor of many animals on earth today. It has only six cell types and no nervous system. It glides on substrates feeding on microalgae, propelled by asynchronously beating cilia. It can move in any direction, so we assume that its cilia do not have a uniform polarity and may be randomly oriented, unlike cilia in most epithelia. We show that Trichoplax exhibits chemotaxis and present a mathematical model to explain how directional movement could arise from collective actions of randomly oriented ciliated cells, each independently responding to a chemoattractant gradient but constrained by their attachments to other cells in the animal., Trichoplax adhaerens is a small, ciliated marine animal that glides on surfaces grazing upon algae, which it digests externally. It has no muscles or nervous system and only six cell types, all but two of which are embedded in its epithelium. The epithelial cells are joined by apical adherens junctions; neither tight junctions nor gap junctions are present. Monociliated epithelial cells on the lower surface propel gliding. The cilia beat regularly, but asynchronously, and transiently contact the substrate with each stroke. The animal moves in random directions in the absence of food. We show here that it exhibits chemotaxis, moving preferentially toward algae embedded in a disk of agar. We present a mathematical model to explain how coherent, directional movements could arise from the collective actions of a set of ciliated epithelial cells, each independently sensing and responding to a chemoattractant gradient. The model incorporates realistic values for viscoelastic properties of cells and produces coordinated movements and changes in body shape that resemble the actual movements of the animal. The model demonstrates that an animal can move coherently in search of food without any need for chemical signaling between cells and introduces a different approach to modeling behavior in primitive multicellular organisms.

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 ∼33 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.

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.

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.

Background: Peanut allergy has a rising prevalence in high-income countries, affecting 0.5–1.4% of children. This study aimed to better understand peanut anaphylaxis in comparison to anaphylaxis to other food triggers in European children and adolescents. Methods: Data was sourced from the European Anaphylaxis Registry via an online questionnaire, after in-depth review of food induced anaphylaxis cases in a tertiary paediatric allergy centre. Results: 3514 cases of food anaphylaxis were reported between July 2007 - March 2018, 56% in patients younger than 18 years. Peanut anaphylaxis was recorded in 459 children and adolescents (85% of all peanut anaphylaxis cases). Previous reactions (42% vs 38%; p=0.001), asthma comorbidity (47% vs 35%; p

The role of T-type calcium channels in animals without nervous systems is unknown. Smith et al. characterize TCav3 from Trichoplax adhaerens, finding expression in neurosecretory-like cells and preference for Ca2+ over Na+ via strong extracellular Ca2+ block, despite low selectivity for Ca2+ in the pore., Four-domain voltage-gated Ca2+ (Cav) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Cav channels, Cav1 (L-type), Cav2 (P/Q-, N- and R-type) and Cav3 (T-type), Cav3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Cav3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCav3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCav3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCav3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Cav3 family. We also characterize the channel’s cation permeation properties and find that its pore is less selective for Ca2+ over Na+ compared with the human homologue Cav3.1, yet it exhibits a similar potent block of inward Na+ current by low external Ca2+ concentrations (i.e., the Ca2+ block effect). A comparison of the permeability features of TCav3 with other cloned channels suggests that Ca2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca2+ block and higher Ca2+ selectivity. TCav3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Cav3 channel structure–function properties, ion selectivity, and cellular physiology.

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.

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.

Syntaxins are a family of transmembrane proteins that participate in SNARE complexes to mediate membrane fusion events including exocytosis. Different syntaxins are thought to participate in exocytosis in different compartments of the nervous system such as the axon, the soma/dendrites or astrocytes. It is well known that exocytosis of synaptic vesicles at axonal presynaptic terminals involves syntaxin 1 but distributions of syntaxins on neuronal somal and dendritic, postsynaptic or astroglial plasma membranes are less well characterized. Here, we use pre-embedding immunogold labeling to compare the distribution of two plasma membrane-enriched syntaxins (1 and 4) in dissociated rat hippocampal cultures as well as in perfusion-fixed mouse brains. Comparison of Western blots of neuronal cultures, consisting of a mixture of hippocampal neurons and glia, with glial cultures, consisting of mostly astrocytes, shows that syntaxin 1 is enriched in neuronal cultures, whereas syntaxin 4 is enriched in glial cultures. Electron microscopy (EM)-immunogold labeling shows that syntaxin 1 is most abundant at the plasma membranes of axons and terminals, while syntaxin 4 is most abundant at astroglial plasma membranes. This differential distribution was evident even at close appositions of membranes at synapses, where syntaxin 1 was localized to the plasma membrane of the presynaptic terminal, including that at the active zone, while syntaxin 4 was localized to nearby peri-synaptic astroglial processes. These results show that syntaxin 4 is available to support exocytosis in astroglia.

Trichoplax adhaerens is a flat, millimeter-sized marine animal that adheres to surfaces and grazes on algae. Trichoplax displays a repertoire of different feeding behaviors despite the apparent absence of a true nervous system with electrical or chemical synapses. It glides along surfaces to find food, propelled by beating cilia on cells at its ventral surface, and pauses during feeding by arresting ciliary beating. We found that when endomorphin-like peptides are applied to an animal, ciliary beating is arrested, mimicking natural feeding pauses. Antibodies against these neuropeptides label cells that express the neurosecretory proteins and voltage-gated calcium channels implicated in regulated secretion. These cells are embedded in the ventral epithelium, where they comprise only 4% of the total, and are concentrated around the edge of the animal. Each bears a cilium likely to be chemosensory and used to detect algae. Trichoplax pausing during feeding or spontaneously in the absence of food often induce their neighbors to pause as well, even neighbors not in direct contact. Pausing behavior propagates from animal to animal across distances much greater than the signal that diffuses from just one animal, so we presume that the peptides secreted from one animal elicit secretion from nearby animals. Signal amplification by peptide-induced peptide secretion explains how a small number of sensory secretory cells lacking processes and synapses can evoke a wave of peptide secretion across the entire animal to globally arrest ciliary beating and allow pausing during feeding., Highlighted Article: Trichoplax, a primitive marine animal that lacks neurons and synapses, has neurosecretory cells that secrete a peptide into the ambient seawater to arrest ciliary beating during feeding.

Trichoplax adhaerens is the sole named member of Placozoa, an ancient metazoan phylum. This coin-shaped animal glides on ventral cilia to find and digest algae on the substrate. It has only six cell types, all but two of which are incorporated into the epithelium that encloses it. The upper epithelium is thin, composed of a pavement of relatively large polygonal disks, each bearing a cilium. The lower epithelium is thick and composed primarily of narrow ciliated cells that power locomotion. Interspersed among these cells are two different secretory cells: one containing large lipophilic granules that, when released, lyse algae under the animal; the other, less abundant, is replete with smaller secretory granules containing neuropeptides. All cells within both epithelia are joined by adherens junctions that are stabilized by apical actin networks. Cells are held in place during shape changes or under osmotic stress, but dissociate in low calcium. Neither tight, septate, nor gap junctions are evident, leaving only the adherens junction to control the permeability of the epithelium. Small (4 kDa) fluorescent dextrans introduced into artificial seawater readily penetrate into the animal between the cells. Larger dextrans enter slowly, except in animals treated with reduced calcium, indicating that the adherens junctions form a circumferential belt around each cell that impedes diffusion into the animal. During feeding, the limited permeability of the adherens junctions helps to confine material released from lysed algae within the narrow space under the animal, where it is absorbed by endocytosis.

Shank is a specialized scaffold protein present in high abundance at the postsynaptic density (PSD). Using pre-embedding immunogold electron microscopy on cultured hippocampal neurons, we had previously demonstrated further accumulation of Shank at the PSD under excitatory conditions. Here, using the same experimental protocol, we demonstrate that a cell permeable CaMKII inhibitor, tatCN21, blocks NMDA-induced accumulation of Shank at the PSD. Furthermore we show that NMDA application changes the distribution pattern of Shank at the PSD, promoting a 7-10 nm shift in the median distance of Shank labels away from the postsynaptic membrane. Inhibition of CaMKII with tatCN21 also blocks this shift in the distribution of Shank. Altogether these results imply that upon activation of NMDA receptors, CaMKII mediates accumulation of Shank, preferentially at the distal regions of the PSD complex extending toward the cytoplasm.

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. In order 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 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 ∼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.

Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains.

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.

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (∼106 e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

Densin is a scaffold protein known to associate with key elements of neuronal signaling. The present study examines the distribution of densin at the ultrastructural level in order to reveal potential sites that can support specific interactions of densin. Immunogold electron microscopy on hippocampal cultures shows intense labeling for densin at postsynaptic densities (PSDs), but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was ~27 nm, with the majority of label (90%) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (90 mM K+ for 2 min) promoted a slight shift of densin label within the PSD complex resulting in 77% of label remaining within 40 nm from the postsynaptic membrane. Densin molecules firmly embedded within the PSD may target a minor pool of CaMKII to substrates at the PSD core.

Hintergrund Im Anaphylaxie-Register werden ana-phylaktische Reaktionen in Deutschland, Osterreich und der Schweiz erfasst. Derzeit sind 75 allergologische Fachkliniken und Schwerpunktpraxen an das Register angebunden. Die anaphylaktischen Reaktionen werden uber einen passwortgeschutzten Onlinefragebogen gemeldet. Es werden ausschlieslich schwere Reaktionen mit pulmonalen und/oder kardiovaskularen Symptomen ausgewertet.

Biochemical studies have suggested that certain synaptic proteins associate with lipid rafts to perform key functions within the synapse. However, variability in biochemical preparations raises questions as to which synaptic proteins actually associate with lipid rafts. In the present study, we use both electron microscopy and biochemistry to investigate AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor localization in synaptic membrane subfractions prepared in two different ways, by Triton X-100 detergent treatment or without detergent by sonication at high pH. Immunogold electron microscopy shows that a detergent-resistant synaptosomal membrane subfraction consists of empty vesicles 0.1–1.0 μm in diameter. A subpopulation of these vesicles labelled for glycosphingolipid GM1 ganglioside, a marker of lipid rafts, and 46% of the labelled vesicles also labelled for the AMPA receptor subunit GluR2. This co-segregation into specific vesicles does not depend on effects of detergent because a similar distribution of label was found in vesicles isolated without the use of detergent. Our results suggest that AMPA receptors localize within specific regions of synaptic membranes rich in GM1 ganglioside.

Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.

Electron microscopic tomography is proving useful to examine the organization of protein molecules in large molecular machines at critical neural sites, such as synapses. Methods are still in a state of flux, and many steps exist between a neuron and a molecular model of one of its synapses. Here, we show our approach for doing tomography on synaptic structure, hoping thereby to shed light generally on the emerging field of EM tomography.

We recently showed by electron microscopy that the postsynaptic density (PSD) from hippocampal cultures undergoes rapid structural changes after ischemia-like conditions. Here we report that similar structural changes occur after delay in transcardial perfusion fixation of the mouse brain. Delay in perfusion fixation, a condition that mimics ischemic stress, resulted in 70%, 90%, and 23% increases in the thickness of PSDs from the hippocampus (CA1), cerebral cortex (layer III), and cerebellar cortex (Purkinje spines), respectively. In step with PSD thickening, the amount of PSD-associated alpha-calcium calmodulin-dependent protein kinase II (alpha- CaMKII) label increased more in cerebral cortical spines than in Purkinje spines. Although the Purkinje PSDs thickened only slightly after delayed fixation, they became highly curved, and many formed sub-PSD spheres approximately 80 nm in diameter that labeled for CaMKII. Delayed perfusion fixation also produced more cytoplamic CaMKII clusters ( approximately 110 nm in diameter) in the somas of pyramidal cells (from hippocampus and cerebral cortex) than in Purkinje cells. Thus a short delay in perfusion fixation produces cell-specific structural changes at PSDs and neuronal somas. Purkinje cells respond somewhat differently to delayed perfusion fixation, perhaps owing to their lower levels of CaMKII, and CaMKII binding proteins at PSDs. We present here a catalogue of structural changes that signal a perfusion fixation delay, thereby providing criteria by which to assess perfusion fixation quality in experimental structural studies of brain and to shed light on the subtle changes that occur in intact brain following metabolic stress.

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.

AIDA-1 is highly enriched in postsynaptic density (PSD) fractions and is considered a major component of the PSD complex. In the present study, immunogold electron microscopy was applied to determine localization as well as the activity-induced redistribution of AIDA-1 at the PSD using two antibodies that recognize two different epitopes. In cultured rat hippocampal neurons under basal conditions, immunogold label for AIDA-1 is mostly located within the dense core of the PSD, with a median distance of ~30 nm from the postsynaptic membrane. Under excitatory conditions, such as depolarization with high K+ (90 mM, 2 min) or application of NMDA (50 μM, 2 min), AIDA-1 label density at the PSD core is reduced to 40% of controls and the median distance of label from the postsynaptic membrane increases to ~55 nm. The effect of excitatory conditions on the postsynaptic distribution of AIDA-1 is reversed within 30 minutes after returning to control conditions. The reversible removal of AIDA-1 from the PSD core under excitatory conditions is similar to the redistribution of another abundant PSD protein, SynGAP. Both SynGAP-alpha1 and AIDA-1 are known to bind PSD-95. Activity-induced transient translocation of these abundant proteins from the PSD core could promote structural flexibility, vacate sites on PSD-95 for the insertion of other components and thus may create a window for synaptic modification.

Background Anaphylaxis in children and adolescents is a potentially life-threatening condition. Its heterogeneous clinical presentation and sudden occurrence in virtually any setting without warning have impeded a comprehensive description. Objective We sought to characterize severe allergic reactions in terms of elicitors, symptoms, emergency treatment, and long-term management in European children and adolescents. Methods The European Anaphylaxis Registry recorded details of anaphylaxis after referral for in-depth diagnosis and counseling to 1 of 90 tertiary allergy centers in 10 European countries, aiming to oversample the most severe reactions. Data were retrieved from medical records by using a multilanguage online form. Results Between July 2007 and March 2015, anaphylaxis was identified in 1970 patients younger than 18 years. Most incidents occurred in private homes (46%) and outdoors (19%). One third of the patients had experienced anaphylaxis previously. Food items were the most frequent trigger (66%), followed by insect venom (19%). Cow's milk and hen's egg were prevalent elicitors in the first 2 years, hazelnut and cashew in preschool-aged children, and peanut at all ages. There was a continuous shift from food- to insect venom– and drug-induced anaphylaxis up to age 10 years, and there were few changes thereafter. Vomiting and cough were prevalent symptoms in the first decade of life, and subjective symptoms (nausea, throat tightness, and dizziness) were prevalent later in life. Thirty percent of cases were lay treated, of which 10% were treated with an epinephrine autoinjector. The fraction of intramuscular epinephrine in professional emergency treatment increased from 12% in 2011 to 25% in 2014. Twenty-six (1.3%) patients were either admitted to the intensive care unit or had grade IV/fatal reactions. Conclusions The European Anaphylaxis Registry confirmed food as the major elicitor of anaphylaxis in children, specifically hen's egg, cow's milk, and nuts. Reactions to insect venom were seen more in young adulthood. Intensive care unit admissions and grade IV/fatal reactions were rare. The registry will serve as a systematic foundation for a continuous description of this multiform condition.

Electron microscopy has revealed an abundance of material in the clefts of synapses in the mammalian brain, and the biochemical and functional characteristics of proteins occupying synaptic clefts are well documented. However, the detailed spatial organization of the proteins in the synaptic clefts remains unclear. Electron microscope tomography provides a way to delineate and map the proteins spanning the synaptic cleft because freeze substitution preserves molecular details with sufficient contrast to visualize individual cleft proteins. Segmentation and rendering of electron dense material connected across the cleft reveals discrete structural elements that are readily classified into five types at excitatory synapses and four types at inhibitory synapses. Some transcleft elements resemble shapes and sizes of known proteins and could represent single dimers traversing the cleft. Some of the types of cleft elements at inhibitory synapses roughly matched the structure and proportional frequency of cleft elements at excitatory synapses, but the patterns of deployments in the cleft are quite different. Transcleft elements at excitatory synapses were often evenly dispersed in clefts of uniform (18 nm) width but some types show preference for the center or edges of the cleft. Transcleft elements at inhibitory synapses typically were confined to a peripheral region of the cleft where it narrowed to only 6 nm wide. Transcleft elements in both excitatory and inhibitory synapses typically avoid places where synaptic vesicles attach to the presynaptic membrane. These results illustrate that elements spanning synaptic clefts at excitatory and inhibitory synapses consist of distinct structures arranged by type in a specific but different manner at excitatory and inhibitory synapses.

SummaryThe mechanisms controlling synapse growth and maintenance are of critical importance for learning and memory. The MAGUK family of synaptic scaffolding proteins is abundantly expressed at glutamatergic central synapses, but their importance in controlling the synaptic content of glutamate receptors is poorly understood. Here, we use a chained RNAi-mediated knockdown approach to simultaneously remove PSD-93, PSD-95, and SAP102, the MAGUKs previously shown to be responsible for synaptic localization of glutamate receptors. We find that MAGUKs are specifically responsible for creating functional synapses after initial spine formation by filling functionally silent spines with glutamate receptors. Removal of the MAGUKs causes a transient reduction in AMPA receptor quantal size followed by synaptic consolidation resulting in a normalization of quantal size at the few remaining functional synapses. Consolidation requires signaling through L-type calcium channels, CaM kinase kinase, and the GluA2 AMPA receptor subunit, akin to a homeostatic process.

Shank and GKAP are scaffold proteins and binding partners at the postsynaptic density (PSD). The distribution and dynamics of Shank and GKAP were studied in dissociated hippocampal cultures by pre-embedding immunogold electron microscopy. Antibodies against epitopes containing their respective mutual binding sites were used to verify the expected juxtapositioning of Shank and GKAP. If all Shank and GKAP molecules at the PSD were bound to each other, the distribution of label for the two proteins should coincide. However, labels for the mutual binding sites showed significant differences in distribution, with a narrow distribution for GKAP located close to the postsynaptic membrane, and a wider distribution for Shank extending deeper into the cytoplasm. Upon depolarization with high K+, neither the intensity nor distribution of label for GKAP changed, but labeling intensity for Shank at the PSD increased to ~150% of controls while the median distance of label from postsynaptic membrane increased by 7.5 nm. These results indicate a preferential recruitment of Shank to more distal parts of the PSD complex. Conversely, upon incubation in Ca2+-free medium containing EGTA, the labeling intensity of Shank at the PSD decreased to ~70% of controls and the median distance of label from postsynaptic membrane decreased by 9 nm, indicating a preferential loss of Shank molecules in more distal parts of the PSD complex. These observations identify two pools of Shank at the PSD complex, one relatively stable pool, closer to the postsynaptic membrane that can bind to GKAP, and another more dynamic pool at a location too far away to bind to GKAP.

We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD.

The majority of hippocampal neurons in dissociated cultures and in intact brain exhibit clustering of calcium/calmodulin-dependent protein kinase II (CaMKII) into spherical structures with an average diameter of 110 nm when subjected to conditions that mimic ischemia and excitotoxicity [Neuroscience 106 (2001) 69]. Because clustering of CaMKII would reduce its effective concentration within the neuron, it may represent a cellular strategy to prevent excessive CaMKII-mediated phosphorylation during episodes of Ca2+ overload. Here we employ a relatively mild excitatory stimulus to promote sub-maximal clustering for the purpose of studying the conditions for the formation and disappearance of CaMKII clusters. Treatment with 30 microM N-methyl-D-aspartic acid (NMDA) for 2 min produced CaMKII clustering in approximately 15% of dissociated hippocampal neurons in culture, as observed by pre-embedding immunogold electron microscopy. These CaMKII clusters could be labeled with antibodies specific to the phospho form (Thr286) of CaMKII, suggesting that at least some of the CaMKII molecules in clusters are autophosphorylated. To test whether phosphorylation is involved in the formation and maintenance of CaMKII clusters, the phosphatase inhibitors calyculin A (5 nM) or okadaic acid (1 microM) were included in the incubation medium. With inhibitors more neurons exhibited CaMKII clusters in response to 2 min NMDA treatment. Furthermore, 5 min after the removal of NMDA and Ca2+, CaMKII clusters remained and could still be labeled with the phospho-specific antibody. In contrast, in the absence of phosphatase inhibitors, no clusters were detected 5 min after the removal of NMDA and Ca2+ from the medium. These results suggest that phosphatases type 1 and/or 2A regulate the formation and disappearance of CaMKII clusters.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIalpha translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIalpha in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha accumulation at synapses is a memory trace of past synaptic activity.