Global ischemia induces downregulation of Glur2 mRNA and increases AMPA receptor-mediated Ca2+ influx in hippocampal CA1 neurons of gerbil

Abstract

Transient, severe forebrain or global ischemia leads to delayed cell death of pyramidal neurons in the hippocampal CA1. The precise molecular mechanisms underlying neuronal cell death after global ischemia are as yet unknown. Glutamate receptor-mediated Ca2+ influx is thought to play a critical role in this cell death. In situ hybridization revealed that the expression of mRNA encoding GluR2 (the subunit that limits Ca2+ permeability of AMPA-type glutamate receptors) was markedly and specifically reduced in gerbil CA1 pyramidal neurons after global ischemia but before the onset of neurodegeneration. To determine whether the change in GluR2 expression is functionally significant, we examined the AMPA receptor-mediated rise in cytoplasmic free Ca2+ level ([Ca2+]i) in individual CA1 pyramidal neurons by optical imaging with the Ca2+ indicator dye fura-2 and by intracellular recording. Seventy-two hours after ischemia, CA1 neurons that retained the ability to fire action potentials exhibited a greatly enhanced AMPA-elicited rise in [Ca2+]i. Basal [Ca2+]i in these neurons was unchanged. These findings provide evidence for Ca2+ entry directly through AMPA receptors in pyramidal neurons destined to die. Downregulation of GluR2 gene expression and an increase in Ca2+ influx through AMPA receptors in response to endogenous glutamate are likely to contribute to the delayed neuronal death after global ischemia.

Fig. 1.

Global ischemia induces selective, delayed neuronal cell loss in the hippocampal CA1. Toluidine blue labeling of coronal brain sections at the level of the dorsal hippocampus from control (A, B) and experimental (C, D) gerbils revealed no detectable neuronal damage 72 hr after transient global ischemia. Global ischemia was induced by two-vessel occlusion as described in Materials and Methods. One week after ischemia, the pyramidal cell layer of the hippocampal CA1 exhibited virtually complete loss of neurons with only a few apparent survivors that may also have been deteriorating (E, F). A,C, E, lower magnification than inB, D, F. Scale bars:E, 100 μm; F, 50 μm.

Fig. 2.

Expression of GluR2 mRNA is reduced specifically in CA1 after ischemia. Photomicrographs of autoradiograms of GluR1, GluR2, and NR1 mRNA in situ hybridization in coronal sections of gerbil brain at the level of the dorsal hippocampus from control animals (A, C, E) and from experimental animals 72 hr after ischemia (B,D, F) are shown. GluR2 mRNA was dramatically reduced in the pyramidal cell layer of the vulnerable CA1 but not in the pyramidal cell layer of CA3 or in the granule cell layer of the dentate gyrus, areas that do not undergo neurodegeneration. GluR1 and NR1 mRNAs were somewhat reduced in CA1 72 hr after ischemia.

Fig. 3.

Progressive decrease of GluR2 mRNA expression in the hippocampal CA1 after global ischemia. A–D, Expression of GluR2 mRNA in the pyramidal cell layer of the hippocampal CA1 decreased with time after ischemia. Expression of GluR2 mRNA was little changed in CA3 and dentate gyrus. Autoradiograms are described in Figure 2.

Fig. 4.

Time course of reduction in GluR2 mRNA expression in the CA1 pyramidal cell layer after ischemia. Glutamate receptor mRNA expression at the level of the hippocampus was measured as mean optical density within regions of interest in film autoradiograms from experimental and control animals. A, Values for GluR1, GluR2, and NR1 mRNA expression in CA1 of animals 24, 48, and 72 hr after ischemia are plotted as percent ± SEMs of the corresponding values for control animals. Twenty-four hours after ischemia, GluR2 mRNA expression was decreased relative to the corresponding control value; the reduction in GluR2 was significantly greater than the reduction in GluR1 (p < 0.01) and NR1 (p < 0.01) mRNA expression. At 48 hr GluR2 had decreased to 48 ± 6% of the corresponding control value; GluR1 and NR1 were only slightly decreased. Seventy-two hours after ischemia, GluR1 and NR1 were decreased to 45 ± 4 and 54 ± 3%, respectively, of control values; GluR2 was decreased to 17 ± 10% of control values (*p < 0.01 for each mRNA). Numbers of animals in each group are indicated inparentheses. B, Ratios of GluR2 to GluR1 mRNA expression in the hippocampal CA1, CA3, and dentate gyrus (DG) 24, 48, and 72 hr after ischemia are normalized to the control values. In CA1, the normalized ratio decreased progressively to 0.38 at 72 hr (asterisks indicate significance). In CA3 and dentate gyrus, the ratio remained constant.

Fig. 5.

Changes in GluR2 mRNA expression are cell-specific. Emulsion-dipped coronal sections of the hippocampus from control and experimental animals 72 hr after ischemia show silver grains densely clustered over CA1 pyramidal neurons. A, Sections of control brain revealed dense clusters of silver grains overlying individual pyramidal neurons in the CA1; virtually all neurons in the field exhibited intense labeling. B, Seventy-two hours after ischemia, GluR2 labeling was dramatically reduced for all CA1 neurons. Sections were counterstained with hematoxylin and eosin. Arrows indicate representative pyramidal neurons. At this time, histological analysis showed no cell loss (see Fig. 1).

Fig. 6.

AMPA-elicited inward current and rise in [Ca²⁺]_i in a CA1 pyramidal neuron after ischemia. A, Inward current elicited by AMPA [30 μm with 10 μm cyclothiazide (CTZ)] in a CA1 pyramidal neuron in a hippocampal slice from an animal 72 hr after ischemia. AMPA and CTZ were bath-applied for 30 sec (red bar). Then the AMPA was washed out with saline containing the NMDA receptor and the Ca²⁺ and Na⁺ channel blockers. After ∼5 min, CNQX (20 μm) was added to the other blockers to cause more rapid recovery. In the control neuron illustrated in B, the AMPA-elicited inward current in the presence of 30 μm CTZ was of somewhat lower amplitude but was similar in time course.B, Optical imaging (350 nm excitation images) of individual CA1 pyramidal neurons injected with fura-2 in hippocampal slices from a control animal (upper row) and an experimental animal 72 hr after ischemia (lower row, same cell as in A). a–b, Time of imaging indicated in current trace above. a, Image taken before bath application of agonist. b, Image taken at peak inward current after application of AMPA (100 μm with 10 μm CTZ). c, Image taken after recovery to near baseline current. Colorrepresents [Ca²⁺]_i determined from the ratio of fluorescence obtained at two excitation wavelengths (350 and 380 nm); calibration is at right. AMPA elicited little change in [Ca²⁺]_i in the control neuron. In contrast, AMPA elicited a rise in [Ca²⁺]_i in the soma of the postischemic neuron and a smaller increase in its proximal dendrites.Red circles in the 350 nm excitation images indicate representative sites of Ca²⁺ measurement in cell somata and dendrites for data presented in Figures 7 and 8.

Fig. 7.

AMPA-elicited rises in [Ca²⁺]_i in CA1 neurons are increased 72 hr after global ischemia. A, Basal and AMPA-elicited rise in [Ca²⁺]_i in the somatic region of individual CA1 pyramidal neurons in hippocampal slices of control animals and ischemic animals 48 and 72 hr after reperfusion. Basal [Ca²⁺]_i did not significantly differ in control and postischemic neurons (open bars). AMPA (30 μm with 10 μm CTZ to reduce desensitization) induced a slight rise in [Ca²⁺]_i in control neurons and neurons from animals 48 hr after ischemia (filled bars). AMPA elicited a large rise in [Ca²⁺]_iin neurons 72 hr after ischemia relative to that elicited in control neurons (p < 0.005) or to that in neurons 48 hr after ischemia (p < 0.025).B, The peak AMPA-induced current did not differ significantly in control versus postischemic neurons at 48 hr or at 72 hr.

Fig. 8.

AMPA-elicited rises in [Ca²⁺]_i in individual CA1 pyramidal neurons are increased 72 hr after ischemia. Peak inward currents and rises in [Ca²⁺]_i elicited by AMPA (30 μm with 10 μm CTZ) are indicated for individual pyramidal neurons in slices from control animals and experimental animals 48 and 72 hr after ischemia. [In the two control neurons exhibiting the largest AMPA-elicited currents (open circles), the CTZ concentration was increased to 30 μm to elicit a current comparable with the large currents observed in three neurons 72 hr after ischemia.] AMPA-elicited rises in [Ca²⁺]_i were greater in neurons from animals at 72 hr than in neurons from animals at 48 hr after ischemia or from control animals. A, Data from somata.B, Data from proximal dendrites.