Christine A. Winters

Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A

Potentiation by cyclothiazide of recombinant glutamate receptor responses in Xenopus oocytes showed absolute selectivity for AMPA versus kainate receptors. In contrast, concanavalin A strongly potentiated responses at kainate but not AMPA receptors. Rapid desensitization in HEK 293 cells transfected with AMPA receptors was blocked by cyclothiazide, but only weakly attenuated by concanavalin A. Desensitization at kainate receptors was blocked by concanavalin A but unaffected by cyclothiazide. Selective effects of these modulators following coexpression of subunits from different families suggest independent assembly of functional AMPA and kainate receptors. Northern blot analysis of mRNA for dorsal root ganglia revealed a predominant expression of GluR5, indicating that modulation of desensitization by concanavalin A but not cyclothiazide in sensory neurons accurately predicts subunit expression for native glutamate receptors.

Glial cells of the oligodendrocyte lineage express both kainate- and AMPA-preferring subtypes of glutamate receptor

mRNAs for AMPA- and kainate-preferring glutamate receptor subunits are expressed abundantly in the CNS, yet functional studies of neurons and glia from brain suggest selective expression of AMPA receptors. We now show that glial cells of the O-2A lineage express rapidly desensitizing responses to kainate, mRNAs for GluR6, GluR7, KA-1, and KA-2, rapidly desensitizing responses to AMPA, and mRNAs for GluR-B, -C, and -D. Analysis of glutamate receptor currents in single cells reveals two receptor populations with high and low affinity for kainate and different sensitivity for potentiation by concanavalin A and for block of desensitization by cyclothiazide. Our experiments describe the characterization of native kainate-preferring receptors in glia and reveal coexpression in single cells of functional AMPA- and kainate-preferring receptors.

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. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 10–20 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function.

Glutamate-induced transient modification of the postsynaptic density

Depolarization of rat hippocampal neurons with a high concentration of external potassium induces a thickening of postsynaptic densities (PSDs) within 1.5–3 min. After high-potassium treatment, PSDs thicken 2.1-fold in cultured neurons and 1.4-fold in hippocampal slices compared with their respective controls. Thin-section immunoelectron microscopy of hippocampal cultures indicates that at least part of the observed thickening of PSDs can be accounted for by an accumulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) on their cytoplasmic faces. Indeed, PSD-associated gold label for CaMKII increases 5-fold after depolarization with potassium. The effects of high-potassium treatment on the composition and structure of the PSDs are mimicked by direct application of glutamate. In cultures, glutamate-induced thickening of PSDs and the accumulation of CaMKII on PSDs are reversed within 5 min of removal of glutamate and Ca2+ from the extracellular medium. These results suggest that PSDs are dynamic structures whose thickness and composition are subject to rapid and transient changes during synaptic activity.

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 Ca2+ 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 Ca2+ entry strong enough to promote Ca2+ 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 Ca2+ 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 Ca2+ overload required the inhibition of extrasynaptic receptors containing both NR2 subunits. Prosurvival synaptic stimuli also evoked Ca2+ entry through both N2A- and NR2B-containing NMDARs, but, in contrast to excitotoxic activation of extrasynaptic NMDARs, produced only low-amplitude cytoplasmic Ca2+ spikes and modest, nondamaging mitochondrial Ca2+ accumulation. The results—showing that the various routes of excitotoxic Ca2+ entry converge on a common pathway involving Ca2+ overload-induced mitochondrial dysfunction—reconcile and unify many aspects of the “route-specific” and “calcium load-dependent” views of exitotoxic injury.

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. Beyond transient elevation of cytosolic calcium and perturbation of Na+/K+ homeostasis, NMDA stimulation induced dramatic, but mainly reversible, changes in mitochondria, including strong calcium elevation, membrane potential depolarization, and variable swelling. Elevation of matrix Ca in the approximately one-third of mitochondria that were strongly swollen, as well as the absence of swelling when Ca2+ entry was abolished, indicate an essential role for Ca overload. Shortly after NMDA exposure, cytochrome c, normally localized to mitochondria, became diffusely distributed in the cytoplasm, coincident with the appearance of severely swollen mitochondria with ruptured outer membranes; under these conditions, cytochrome c was retained in intact mitochondria, implying that it was released mainly from damaged mitochondria. Consistent with the role of mitochondrial Ca overload, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone decreased Ca accumulation, prevented cytochrome c release, and was neuroprotective. These results support a mechanism in which delayed excitotoxic death involves apoptogen release from a subpopulation of calcium-overloaded mitochondria, whereas other, undamaged mitochondria maintain normal function.

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. 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.

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. The common mechanism involves O2- inhibition of inactivating protein phosphatases. Conversely, because small [Ca2+]i increases do not promote mitochondrial respiration and O2- generation, weak stimuli favor enhanced phosphatase activity, which therefore leads to suppressed kinase activity. Enhanced O2- production also promoted PKC activity but by a phosphatase-independent pathway. These results suggest that Ca2+-dependent upregulation of mitochondrial O2- production may be a general mechanism for linking Ca2+ entry to enhanced kinase activity and therefore to synaptic plasticity. This mechanism also represents yet another way that mitochondria, acting as calcium sensors, can play a role in neuronal signal transduction.

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. 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.

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. 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.