Variation in quality report viewing by providers and correlation with NICU quality metrics.

BACKGROUND: To examine variation in quality report viewing and assess correlation between provider report viewing and neonatal intensive care unit (NICU) quality. METHODS: Variation in report viewing sessions for 129 California Perinatal Quality Care Collaborative NICUs was examined. NICUs were stratified into tertiles based on their antenatal steroid (ANS) use and hospital-acquired infection (HAI) rates to compare report viewing session counts. RESULTS: The number of report viewing sessions initiated by providers varied widely over a 2-year period (median=11; mean=25.5; s.d.=45.19 sessions). Report viewing was not associated with differences in ANS use. Facilities with low HAI rates had less frequent report viewing. Facilities with high report views had significant improvements in HAI rates over time. CONCLUSIONS: Available audit and feedback reports are utilized inconsistently across California NICUs despite evidence that report viewing is associated with improvements in quality of care delivery. Further studies are needed for reports to reach their theoretical potential.

Effects of delivery room quality improvement on premature infant outcomes.

OBJECTIVE: Delivery room management interventions have been successfully implemented via collaborative quality improvement (QI) projects. However, it is unknown whether these successes translate to reductions in neonatal morbidity and mortality. STUDY DESIGN: This was a prospective pre-post intervention study of three nonrandomized hospital groups within the California Perinatal Quality Care Collaborative. A collaborative QI model (Collaborative QI) was compared with a single-site QI model (NICU QI) and a non-participant population when implementing evidence-based delivery room practices. The intervention period was between June 2011 and May 2012. Infants born with gestational age between 22 weeks 0 days and 29 weeks 6 days and birth weight ⩽1500 g were included. Outcomes were mortality and select morbidities (bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP) and necrotizing enterocolitis (NEC)). Outcomes were compared between the baseline (January 2010 to May 2011) and post-intervention period (June 2012 to May 2013) within each comparison group. RESULTS: Ninety-five hospitals were included with 4222 infants in the baseline period and 4186 infants in the post-intervention period. The Collaborative QI group had significantly reduced odds of developing BPD post-intervention (odds ratio (OR) 0.8, 95% confidence interval (CI) 0.65 to 0.99) or composite BPD-death (OR 0.83, 95% CI 0.69 to 1.00). In both the Collaborative QI and non-participants there were also reductions in IVH, severe IVH, composite severe IVH-death, severe ROP and composite severe ROP-death. CONCLUSION: Hospitals dedicated to improving delivery room practices can impact neonatal outcomes.

Accounting for variation in length of NICU stay for extremely low birth weight infants.

OBJECTIVE: To develop a length of stay (LOS) model for extremely low birth weight (ELBW) infants. STUDY DESIGN: We included infants from the California Perinatal Quality Care Collaborative with birth weight 401 to 1000 g who were discharged to home. Exclusion criteria were congenital anomalies, surgery and death. LOS was defined as days from admission to discharge. As patients who died or were transferred to lower level of care were excluded, we assessed correlation of hospital mortality rates and transfers to risk-adjusted LOS. RESULTS: There were 2012 infants with median LOS 79 days (range 23 to 219). Lower birth weight, lack of antenatal steroids and lower Apgar score were associated with longer LOS. There was negligible correlation between risk-adjusted LOS and hospital mortality rates (r=0.0207) and transfer-out rates (r=0.121). CONCLUSION: Particularly because ELBW infants have extended hospital stays, identification of unbiased and informative risk-adjusted LOS for these infants is an important step in benchmarking best practice and improving efficiency in care.

Differences in NMDA receptor expression during human development determine the response of neurons to HIV-tat-mediated neurotoxicity.

HIV infection of the CNS can result in neurologic dysfunction in a significant number of infected individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV infection in neurons. Thus, neuronal damage and dropout are likely due to indirect effects of HIV infection of other CNS cells, through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivating protein tat. We and others demonstrated that tat induces apoptosis in differentiated mature human neurons. We now demonstrate that the high level of tat toxicity observed in human neurons involves specific developmental stages that correlate with N-methyl-D-aspartate receptor (NMDAR) expression, and that tat toxicity is also dependent upon the species being analyzed. Our results indicate that tat treatment of primary cultures of differentiated human neurons with significant amounts of NMDAR expression induces extensive apoptosis. In contrast, tat treatment induces only low levels of apoptosis in primary cultures of immature human neurons with low or minimal expression of NMDAR. In addition, tat treatment has minimal effect on rat hippocampal neurons in culture, despite their high expression of NMDAR. We propose that this difference may be due to low expression of the NR2A subunit. These findings are important for an understanding of the many differences among tissue culture systems and species used to study HIV-tat-mediated toxicity.

Gap junction hemichannels in astrocytes of the CNS.

Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemichannels or just hemichannels. Because some gap junction channels are permeable to negatively and/or positively charged molecules up to approximately 1kDa in size, it was thought that hemichannels should not open to the extracellular space. A growing amount of evidence indicates that opening of hemichannels does occur under both physiological and pathological conditions in astrocytes and other cell types. Electrophysiological studies indicate that hemichannels have a low open probability under physiological conditions but may have a much higher open probability under certain pathological conditions. Some of the physiological behaviours of astrocytes that have been attributed to gap junctions may, in fact, be mediated by hemichannels. Hemichannels constituted of Cx43, the main connexin expressed by astrocytes, are permeable to small physiologically significant molecules, such as ATP, NAD+ and glutamate, and may mediate paracrine as well as autocrine signalling. Hemichannels tend to be closed by negative membrane potentials, high concentrations of extracellular Ca2+ and intracellular H+ ions, gap junction blockers and protein phosphorylation. Hemichannels tend to be opened by positive membrane potentials and low extracellular Ca2+, and possibly by as yet unidentified cytoplasmic signalling molecules. Exacerbated hemichannel opening occurs in metabolically inhibited cells, including cortical astrocytes, which contributes to the loss of chemical gradients across the plasma membrane and speeds cell death.

Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca(2+), and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca(2+) concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice.

Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

Activation of metabotropic glutamate receptor 1 accelerates NMDA receptor trafficking.

Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluRs) is known to play a critical role in synaptic transmission. The molecular mechanisms underlying mGluR1-mediated potentiation of NMDARs are as yet unclear. The present study shows that in Xenopus oocytes expressing recombinant receptors, activation of mGluR1 potentiates NMDA channel activity by recruitment of new channels to the plasma membrane via regulated exocytosis. Activation of mGluR1alpha induced (1) an increase in channel number times channel open probability, with no change in mean open time, unitary conductance, or reversal potential; (2) an increase in charge transfer in the presence of NMDA and the open channel blocker MK-801, indicating an increased number of functional NMDARs in the cell membrane; and (3) increased NR1 surface expression, as indicated by cell surface Western blots and immunofluorescence. Botulinum neurotoxin A or expression of a dominant negative mutant of synaptosomal associated protein of 25 kDa molelcular mass (SNAP-25) greatly reduced mGluR1alpha-mediated potentiation, indicating that receptor trafficking occurs via a SNAP-25-mediated form of soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor-dependent exocytosis. Because group I mGluRs are localized to the perisynaptic region in juxtaposition to synaptic NMDARs at glutamatergic synapses in the hippocampus, mGluR-mediated insertion of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Expanded Prussian blue analogues incorporating [Re6Se8(CN)6](3-/4-) clusters: adjusting porosity via charge balance.

Face-capped octahedral [Re(6)Se(8)(CN)(6)](3-/4-) clusters are used in place of octahedral [M(CN)(6)](3-/4-) complexes for the synthesis of microporous Prussian blue type solids with adjustable porosity. The reaction between [Fe(H(2)O)(6)](3+) and [Re(6)Se(8)(CN)(6)](4-) in aqueous solution yields, upon heating, Fe(4)[Re(6)Se(8)(CN)(6)](3).36H(2)O (4). A single-crystal X-ray analysis confirms the structure of 4 to be a direct expansion of Prussian blue (Fe(4)[Fe(CN)(6)](3).14H(2)O), with [Re(6)Se(8)(CN)(6)](4-) clusters connected through octahedral Fe(3+) ions in a cubic three-dimensional framework. As in Prussian blue, one out of every four hexacyanide units is missing from the structure, creating sizable, water-filled cavities within the neutral framework. Oxidation of (Bu(4)N)(4)[Re(6)Se(8)(CN)(6)] (1) with iodine in methanol produces (Bu(4)N)(3)[Re(6)Se(8)(CN)(6)] (2), which is then metathesized to give the water-soluble salt Na(3)[Re(6)Se(8)(CN)(6)] (3). Reaction of [Co(H(2)O)(6)](2+) or [Ni(H(2)O)(6)](2+) with 3 in aqueous solution affords Co(3)[Re(6)Se(8)(CN)(6)](2).25H(2)O (5) or Ni(3)[Re(6)Se(8)(CN)(6)](2).33H(2)O (6). Powder X-ray diffraction data show these compounds to adopt structures based on the same cubic framework present in 4, but with one out of every three cluster hexacyanide units missing as a consequence of charge balance. In contrast, reaction of [Ga(H(2)O)(6)](3+) with 3 gives Ga[Re(6)Se(8)(CN)(6)].6H(2)O (7), wherein charge balance dictates a fully occupied cubic framework enclosing much smaller cavities. The expanded Prussian blue analogues 4-7 can be fully dehydrated, and retain their crystallinity with extended heating at 250 degrees C. Consistent with the trend in the frequency of framework vacancies, dinitrogen sorption isotherms show porosity to increase along the series of representative compounds 7, Ga(4)[Re(6)Se(8)(CN)(6)](3).38H(2)O, and 6. Furthermore, all of these phases display a significantly higher sorption capacity and surface area than observed in dehydrated Prussian blue. Despite incorporating paramagnetic [Re(6)Se(8)(CN)(6)](3-) clusters, no evidence for magnetic ordering in compound 6 is apparent at temperatures down to 5 K. Reactions related to those employed in preparing compounds 4-6, but carried out at lower pH, produce the isostructural phases H[cis-M(H(2)O)(2)][Re(6)Se(8)(CN)(6)].2H(2)O (M = Fe (8), Co (9), Ni (10)). The crystal structure of 8 reveals a densely packed three-dimensional framework in which [Re(6)Se(8)(CN)(6)](4-) clusters are interlinked through a combination of protons and Fe(3+) ions.

Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein.

We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< approximately 2 ms) transitions and a slow gate that fully closes channels via slow transitions (> approximately 10 ms). Here we demonstrate that transjunctional voltage (V(j)) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V(j) gating to a residual state but show slow V(j) gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO(2) and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V(j) gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V(j) gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V(j) gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.

mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C.

Potentiation of ionotropic glutamate receptor activity by metabotropic glutamate receptors (mGluRs) is thought to modulate activity at glutamatergic synapses in the hippocampus. However, the precise pathway by which this modulation occurs is not well understood. The present study tests the hypothesis that mGluR1-mediated potentiation of N-methyl-D-aspartate receptors (NMDARs) occurs via a phospholipase C (PLC)-initiated cascade. NMDAR functional activity was examined by whole-cell recording from Xenopus oocytes expressing recombinant NMDARs and mGluR1alpha. The mGluR1 agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) significantly potentiated NMDA-elicited currents. mGluR1alpha-mediated potentiation of NMDA responses was eliminated by the PLC inhibitor U-73122. Buffering of intracellular Ca2+ by BAPTA-AM or depletion of intracellular Ca2+ by the Ca2+/ATPase inhibitor thapsigargin greatly reduced ACPD potentiation. ACPD potentiation was reduced by the specific protein kinase C (PKC) inhibitor Ro-32-0432 and eliminated by the broad spectrum kinase inhibitor staurosporine. ACPD produced no further potentiation after potentiation of NMDARs by the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Thus, Group I mGluRs potentiate NMDA responses via activation of PLC; at least part of the potentiation is due to rise in intracellular Ca2+ and stimulation of PKC. Cytochalasin D, which disrupts the actin cytoskeleton, blocked ACPD-elicited chloride currents and ACPD-induced potentiation of NMDAR currents, consistent with a role for cytoskeletal protein(s) in the signaling pathway. As Group I mGluRs are localized to the perisynaptic region in juxtaposition to NMDARs at glutamatergic synapses, mGluR-mediated potentiation of NMDAR activity may play a role in synaptic transmission and plasticity including LTP.

Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease.

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-gamma and tumor necrosis factor-alpha.

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma) or tumor necrosis factor-alpha (TNF-alpha) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-gamma plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-alpha antibody, suggesting the release and autocrine action of TNF-alpha. Treatment with INF-gamma plus TNF-alpha also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18 alpha-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-gamma plus LPS or INF-gamma plus TNF-alpha. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Insulin promotes rapid delivery of N-methyl-D- aspartate receptors to the cell surface by exocytosis.

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nP(o)) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or P(o); (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in P(o); and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Protein kinase C modulates NMDA receptor trafficking and gating.

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.

Molecular determinants of membrane potential dependence in vertebrate gap junction channels.

The conductance, g(j), of many gap junctions depends on voltage between the coupled cells (transjunctional voltage, V(j)) with little effect of the absolute membrane potential (V(m)) in the two cells; others show combined V(j) and V(m) dependence. We examined the molecular determinants of V(m) dependence by using rat connexin 43 expressed in paired Xenopus oocytes. These junctions have, in addition to V(j) dependence, V(m) dependence such that equal depolarization of both cells decreases g(j). The dependence of g(j) on V(m) was abolished by truncation of the C-terminal domain (CT) at residue 242 but not at 257. There are two charged residues between 242 and 257. In full-length Cx43, mutations neutralizing either one of these charges, Arg243Gln and Asp245Gln, decreased and increased V(m) dependence, respectively, suggesting that these residues are part of the V(m) sensor. Mutating both residues together abolished V(m) dependence, although there is no net change in charge. The neutralizing mutations, together or separately, had no effect on V(j) dependence. Thus, the voltage sensors must differ. However, V(j) gating was somewhat modulated by V(m), and V(m) gating was reduced when the V(j) gate was closed. These data suggest that the two forms of voltage dependence are mediated by separate but interacting domains.

The AMPAR subunit GluR2: still front and center-stage.

Abnormal influx of Ca(2+) through AMPA-type glutamate receptors (AMPARs) is thought to contribute to the neuronal death associated with a number of brain disorders. AMPARs exist as both Ca(2+)-impermeable and Ca(2+)-permeable channels. AMPARs are encoded by four genes designated GluR1 (GluR-A) through GluR4 (GluR-D). The presence of the GluR2 subunit renders heteromeric AMPA receptor assemblies Ca(2+)-impermeable. Molecular diversity of AMPARs under physiological and pathological conditions is generated by differential spatio-temporal patterns of GluR expression, by alternative RNA splicing and editing and by targeting and trafficking of receptor subunits at dendritic spines. The GluR2 gene is under transcriptional control by the RE1 element specific transcription factor, a gene silencing factor which renders it neuron-specific. GluR2 transcripts are edited by ADAR2 (double-stranded RNA-specific editase 1). AMPAR targeting and trafficking to spines are regulated by synaptic activity and are critical to synaptic plasticity. Recent studies involving animal models of transient forebrain ischemia and epilepsy show that GluR2 mRNA and GluR2 subunit expression are downregulated in vulnerable neurons prior to cell death. Ca(2+) imaging and electrical recording from individual pyramidal neurons in hippocampal slices reveal changes in AMPAR functional properties after ischemia. In slices from post-ischemia animals, CA1 neurons with robust action potentials exhibit greatly enhanced AMPA-elicited rises in intracellular Ca(2+). Excitatory postsynaptic currents in post-ischemic CA1 exhibit an enhanced Ca(2+)-dependent component that appears to be mediated by Ca(2+)-permeable AMPARs. These studies provide evidence for Ca(2+) influx through AMPARs in neurons destined to die. To examine whether acute GluR2 downregulation, even in the absence of a neurological insult, can induce neuronal death, we performed knockdown experiments in rats and gerbils with antisense oligonucleotides targeted to GluR2 mRNA. GluR2 antisense oligonucleotide induced neuronal cell death of pyramidal neurons and enhanced pathogenicity of brief ischemic episodes. These observations provide evidence for Ca(2+) influx through AMPARs in neurons destined to die and implicate Ca(2+)-permeable AMPARs in the pathogenesis of ischemia-induced neuronal death.

Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia.

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca(2+) influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca(2+)-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Electrical synapses, a personal perspective (or history).

Gap junctions are the morphological substrate of one class of electrical synapse. This memoir records the author's involvement in the development of our knowledge of the physiology and ultrastructure of electrical synapses. The answer to whether neurotransmission is electrical or chemical is either. One lesson is that Occam's razor sometimes cut too deep; the nervous system does its operations in a number of different ways and a unitarian approach can lead one astray [M.V.L. Bennett, Nicked by Occam's razor: unitarianism in the investigation of synaptic transmission, Biol. Bull. 168 (1985) 159-167]. Electrical synapses can do many things that chemical synapses can do, and do them just as slowly. The new molecular, cellular and physiological techniques will clarify where gap junctions and electrical coupling do and do not occur and permit experimental manipulation with high specificity.