Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss

Abstract

Kainic acid (KA) induces status epilepticus and delayed neurodegeneration of CA3 hippocampal neurons. Downregulation of glutamate receptor 2 (GluR2) subunit mRNA [the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) subunit that limits Ca2+ permeability] is thought to a play role in this neurodegeneration, possibly by increased formation of Ca2+ permeable AMPA receptors. The present study examined early hippocampal decreases in GluR2 mRNA and protein following kainate‐induced status epilepticus and correlated expression changes with the appearance of dead or dying cells by several histological procedures. At 12 h, in situ hybridization followed by emulsion dipping showed nonuniform decreases in GluR2 mRNA hybridization grains overlying morphologically healthy‐appearing CA3 neurons. GluR1 and N‐methyl‐D‐aspartate receptor mRNAs were unchanged. At 12–16 h, when little argyrophilia or cells with some features of apoptosis were detected by silver impregnation or electron microscopy, single immunohistochemistry with GluR2 and GluR2/3 subunit‐specific antibodies demonstrated a pattern of decreased GluR2 receptor protein within CA3 neurons that appeared to predict a pattern of damage, similar to the mRNA observations. Double immunolabeling showed that GluR2 immunofluorescence was depleted and that GluR1 immunofluorescence was sustained in clusters of the same CA3 neurons. Quantitation of Western blots showed increased GluR1:GluR2 ratios in CA3 but not in CA1 or dentate gyrus subfields. Findings indicate that the GluR1:GluR2 protein ratio is increased in a population of CA3 neurons prior to significant cell loss. Data are consistent with the “GluR2 hypothesis” that reduced expression of GluR2 subunits will increase formation of AMPA receptors permeable to Ca2+ and predict vulnerability to a particular subset of pyramidal neurons following status epilepticus. Hippocampus 1998;8:511–525. © 1998 Wiley‐Liss, Inc.

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

“…This hypothesis was supported in vivo using the kainic acid (KA) model of delayed neurodegeneration since in adult rats, KA induces status epilepticus and pronounced decreases in GluR2 mRNA (Friedman et al, 1994) and protein in the vulnerable CA3 subregion but prior to cell loss (Friedman, 1998). Our study of KA-induced seizures in development also supported the GluR2 hypothesis, since at young ages, rat pups that are relatively resistant to CA3 damage do not show decreases in GluR2 expression (Friedman et al, 1997).…”

“…Dissociated cultures plated onto coverslips were processed for GluR1 and GluR2 immunocytochemistry within flat well dishes as described (Friedman, 1998). All antisera are commercially available (Chemicon Int.).…”

“…To solubilize membranes, cultures were scraped and homogenized in 200 l of homogenization buffer supplemented with 1% Triton-X 100 and incubated 30 min at 4 • C similar to that described previously (Friedman, 1998). After centrifugation (16,000 rpm for 20 min) the supernatant was frozen at −70 • C. SDS-PAGE was performed using 5% polyacrylamide mini gels (Laemmli, 1970).…”

Forebrain-Specific Glutamate Receptor B Deletion Impairs Spatial Memory But Not Hippocampal Field Long-Term Potentiation

“…The remaining GluR-A and GluR-C homomers or GluR-A/-C heteromers (Sans et al, 2003) are sensitive to voltage-dependent polyamine block, exhibit increased single-channel conductance, and are Ca 2ϩ permeable (Jia et al, 1996). This AMPA receptormediated Ca 2ϩ entry can lead to neuronal cell death when induced by either kainic acid-mediated status epilepticus or transient forebrain ischemia (Pollard et al, 1993;Friedman, 1998;Friedman and Koudinov, 1999;Grooms et al, 2000).…”

Blockade of calcium-permeable AMPA receptors protects hippocampal neurons against global ischemia-induced death

“…Principal neurons of the hippocampus express primarily GluR2-containing AMPARs (21); thus, an acute change in the level of GluR2 expression could have significant consequences for neuronal survival. The relative expression of GluR2 in neurons is not static but is regulated in a cell-specific manner during development (22) and is altered after seizures (23,24), ischemic insult (25,26), or treatment with antipsychotics, drugs of abuse, or corticosteroids (for review, see ref. 27).…”