Ultrastructural characterization of hippocampal inhibitory synapses under resting and stimulated conditions.

The present study uses electron microscopy to document ultrastructural characteristics of hippocampal GABAergic inhibitory synapses under resting and stimulated conditions in three experimental systems. Synaptic profiles were sampled from stratum pyramidale and radiatum of the CA1 region from (1) perfusion fixed mouse brains, (2) immersion fixed rat organotypic slice cultures, and from (3) rat dissociated hippocampal cultures of mixed cell types. Synapses were stimulated in the brain by a 5 min delay in perfusion fixation to trigger an ischemia-like excitatory condition, and by treating the two culture systems with 90 mM high K for 2-3 min to depolarize the neurons.

Modification of the synaptic cleft under excitatory conditions.

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.

Palmitoylation of A-kinase anchoring protein 79/150 modulates its nanoscale organization, trafficking, and mobility in postsynaptic spines.

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).

Placozoan fiber cells: mediators of innate immunity and participants in wound healing.

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.

Microscopy Studies of Placozoans.

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.

The ventral epithelium of deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides.

The disk-shaped millimeter-sized marine animal, , 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.

FAM81A protein, a novel component of the postsynaptic density in adult brain.

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.

Identification of PSD-95 in the Postsynaptic Density Using MiniSOG and EM Tomography.

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.

Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.

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.

Zinc Stabilizes Shank3 at the Postsynaptic Density of Hippocampal Synapses.

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.

Depolarization of Hippocampal Neurons Induces Formation of Nonsynaptic NMDA Receptor Islands Resembling Nascent Postsynaptic Densities.

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.

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density.

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.

Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens.

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.

Electron tomography on γ-aminobutyric acid-ergic synapses reveals a discontinuous postsynaptic network of filaments.

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.

PSD-95 is required to sustain the molecular organization of the postsynaptic density.

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.

Sequestration of CaMKII in dendritic spines in silico.

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.

Trafficking of AMPA receptors at plasma membranes of hippocampal neurons.

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.

Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons.

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.

Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity.

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.

Organization of the core structure of the postsynaptic density.

Much is known about the composition and function of the postsynaptic density (PSD), but less is known about its molecular organization. We use EM tomography to delineate the organization of PSDs at glutamatergic synapses in rat hippocampal cultures. The core of the PSD is dominated by vertically oriented filaments, and ImmunoGold labeling shows that PSD-95 is a component of these filaments.

Changes in the distribution of calcium calmodulin-dependent protein kinase II at the presynaptic bouton after depolarization.

Phosphorylation of synapsin I by CaMKII has been reported to mobilize synaptic vesicles from the reserve pool. In the present study, the distributions of alpha-CaMKII and of synapsin I were compared in synaptic boutons of unstimulated and stimulated hippocampal neurons in culture by immunogold electron microscopy. CaMKII and synapsin I are located in separate domains in presynaptic terminals of unstimulated neurons.

Strong calcium entry activates mitochondrial superoxide generation, upregulating kinase signaling in hippocampal neurons.

Large increases in cytosolic free Ca2+ ([Ca2+]i) activate several kinases that are important for neuronal plasticity, including Ca2+/calmodulin-dependent kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC). Because it is also known, mainly in non-neuronal systems, that superoxide radicals (O2-) activate these (and other) kinases and because O2- generation by mitochondria is in part [Ca2+]i dependent, we examined in hippocampal neurons the relationship between Ca2+ entry, O2- production, and kinase activity. We found that, after large stimulus-induced [Ca2+]i increases, O2- selectively produced by mitochondria near plasmalemmal sites of Ca2+ entry acts as a modulator to upregulate the two kinases, namely, CaMKII and PKA, whose activities are directly or indirectly phosphorylation dependent.

Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons.

In neurons, excitotoxic stimulation induces mitochondrial calcium overload and the release of pro-apoptotic proteins, which triggers delayed cell death. The precise mechanisms of apoptogen release, however, remain controversial. To characterize the linkage between mitochondrial calcium load and cell vulnerability, and to test the hypothesis that only a subpopulation of mitochondria damaged by calcium overload releases apoptogens, we have measured directly the concentrations of total Ca (free plus bound) in individual mitochondria and monitored in parallel structural changes and the subcellular localization of pro-apoptotic cytochrome c after NMDA overstimulation in cultured hippocampal neurons.