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

Abstract

Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn(2+) in CA1 neurons just before the onset of histologically detectable cell death. Here we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca(2+)/Zn(2+)-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn(2+) and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn(2+) in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn(2+) and death of CA1 neurons, although a direct action at the time of the rise in Zn(2+) is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans.

Fig. 1.

Global ischemia induces REST and suppresses GluR2 mRNA expression in hippocampal CA1. RT-PCR products amplified from DNase-treated RNA from the hippocampal CA1 of control animals at 48 h after sham operation and experimental animals at 6, 12, 24, and 48 h after global ischemia. (a) Representative agarose gel electrophoresis of RT-PCR products obtained with primers specific to REST, GluR2, and actin mRNAs. The REST mRNA signal was low in control CA1 and increased by 6 h after ischemia; it reached a plateau at 12 h that was maintained at least until 48 h. The GluR2 mRNA signal was prominent in control CA1 and was decreased by 6 h after ischemia; it was further decreased at 12 and 24 h and remained decreased until at least 48 h. No changes were detected in actin mRNA expression. (b and c) Quantitation of REST (b) and GluR2 (c) mRNA abundance after global ischemia (n = 5 per time point). Band densities for REST and GluR2 were normalized to the corresponding band density for actin and expressed as the ratio of the band density for the experimental sample to the band density of the corresponding control sample. Bars represent means ± SEMs. Statistical significance was assessed by Student's unpaired t test of each band density vs. its control (^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001). cntrl, control.

Fig. 2.

Synaptic currents in postischemic CA1 neurons exhibit properties of GluR2-lacking AMPARs. (a and b) Representative AMPA EPSCs and I-V relations of the peak responses at Schaffer collateral synapses on CA1 pyramidal cells recorded in acute hippocampal slices. The external solution contained 25 μM d-2-amino-5-phosphonovaleric acid and 100 μM picrotoxin to block NMDA and GABA_A receptors, respectively. (a) AMPA EPSCs recorded in a neuron from a control animal exhibited a linear I-V relation. (b) AMPA EPSCs recorded from postischemic neurons 42 h after reperfusion exhibited reduced current at positive potentials (Left) and an inwardly rectifying I-V relation (1 mM permine was included in pipette to maintain rectification) (Right). (c) The rectification index [(EPSC amplitude at +40 mV/EPSC amplitude at -60 mV) × 1.5] was significantly reduced in neurons from postischemic animals (P < 0.001, n = 6 per group). (d) AMPA EPSCs in postischemic neurons were reduced but not blocked by Naspm (250 μM) (Left), an antagonist selective for GluR2-lacking AMPARs, and nearly completely blocked by the general AMPAR antagonist GYKI-53655 (50 μM) (Right). The neurons were voltage-clamped at -60 mV. (e) Percentage inhibition of AMPA EPSCs by Naspm (n = 6) and GYKI-53655 (n = 4) in hippocampal slices from postischemic animals. The effect of Naspm on AMPA EPSCs in postischemic neurons was significantly different from that of GYKI-53655 (P < 0.001).

Fig. 3.

Block of GluR2-lacking AMPARs protects CA1 neurons from postischemic neurodegeneration. (a-j) Toluidine blue-stained coronal brain sections at the level of the dorsal hippocampus from control (a and b) and experimental animals subjected to Naspm injection (c and d), global ischemia (e and f), or to global ischemia, followed by Naspm at 30 or 40 h (g-j) and killed 5 d after reperfusion. The sections are from maximally protected animals. (k) Summary data for control (n = 8) and Naspm injections given at 9 h (n = 8), 14 h (n = 13), 20 h (n = 6), 30 h (n = 8), or 40 h (n = 7) after ischemia. Injection of Naspm at all times except 20 h after ischemia significantly protected CA1 neurons; saline was ineffective (n = 10). Statistical significance was assessed by a nonparametric Kruskal-Wallis test (^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001). (Scale bars: a, c, e, and g, 500 μm; b, d, f, and h, 50 μm.)

Fig. 4.

Block of GluR2-lacking AMPARs can prevent the late rise in TSQ-reactive Zn²⁺ in postischemic CA1 neurons. Coronal sections stained with TSQ showing Zn²⁺ fluorescence (Left and Center), and adjacent sections were stained with acid fuchsin (Right). Sections from animals subjected to sham operation (a-c), to sham operation followed by Naspm injection (d-f), to global ischemia (g-i), or to global ischemia followed by Naspm (j-l). (a-c) In control hippocampus, TSQ labeling revealed intense fluorescence in axon terminals of dentate granule (DG) neurons in the hilus and stratum lucidum of CA3 and faint fluorescence in the stratum radiatum (sr) and stratum oriens (so) of CA1 and CA3 (a and b); acid fuchsin staining was minimal (c). (d-f) Injection of Naspm at 40 h after sham operation did not detectably alter the Zn²⁺ fluorescence (d and e) or acidophilia (f) assessed at 72 h after surgery. (g-i) Global ischemia induced a pronounced increase in Zn²⁺ fluorescence in the cell bodies of CA1 stratum pyramidale (sp) at 72 h (g, arrows, and h) and pronounced acid fuchsin staining (i). (j-l) Injection of Naspm at 40 h after ischemia markedly reduced the ischemia-induced rise in Zn²⁺ fluorescence (j and k) and acid fuchsin staining (l) in CA1. (n = 5 per group). (Scale bars: Left, 500 μm; Right and Center, 50 μm.)