Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels triggers marked intracellular Ca2+ rises and consequent oxygen radical production

Abstract

The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca2+ ions. Microfluorometric techniques were performed by using the oxidation-sensitive dye hydroethidine (HEt) to determine whether the relatively rapid Ca2+ flux through AMPA/kainate channels expressed on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA. Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most cultured cortical neurons, whereas high K+ (50 mM) exposures (causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels) caused little fluorescence change. In contrast, kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic population. The effect of NMDA, kainate, and high K+ exposures on oxygen radical production paralleled the effect of these exposures on intracellular Ca2+ levels when they were monitored with the low-affinity Ca2+-sensitive dye fura-2FF, but not with the high-affinity dye fura-2. Inhibition of mitochondrial electron transport with CN- or rotenone almost completely blocked kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid Ca2+ influx through Ca2+-permeable AMPA/kainate channels also may result in mitochondrial Ca2+ overload and consequent injurious oxygen radical production.

Fig. 1.

Glutamic acid decarboxylase (GAD)-immunoreactive cortical neurons exhibit kainate-stimulated Co²⁺uptake. Cultures were subjected to kainate-stimulated Co²⁺ loading (see Materials and Methods), followed by processing for GAD immunocytochemistry. Then selected immunostained fields were photographed (400× magnification) before (A) and again after (B) development of the Co²⁺ stain. Co²⁺(+) neurons can be identified by adarkening in the cell body and processes. Scale bar, 50 μm.

Fig. 2.

NMDA, kainate, and high K⁺exposures produce distinct patterns of oxygen radical generation. Cortical cultures were exposed to NMDA (200 μm + 10 μm NBQX) (A), kainate (200 μm + 10 μm MK-801) (B), or high K⁺ (50 mm + 10 μm MK-801/NBQX) (C). In each experiment the images were obtained under visible light before exposure (column 1), under fluorescence for oxidized hydroethidine (HEt; see Materials and Methods) 10 min before (column 2) and 20 min after (column 3) drug application, and again after immunostaining for GAD (column 4). Note the widespread fluorescence increases after NMDA exposure and the relatively selective increases in GAD(+) neurons after kainate exposure. As indicated by the pseudocolor scale bar, HEt images represent fluorescent intensity on an eight-bit/0–256 scale. Scale bar, 50 μm.

Fig. 3.

Time course of oxygen radical generation after high K⁺, NMDA, and kainate exposures. HEt-loaded cultures were imaged for 10 min before and 20 min after drug application. For each cell the HEt fluorescence at each time point (F_x) was normalized to the mean fluorescence for that cell during the 10 min baseline period (F₀). Cultures were exposed to either normal HSS alone or high K⁺ modified HSS (+ 10 μm MK-801/NBQX) (A), to NMDA (200 μm + 10 μm NBQX) (B), or to kainate (200 μm + 10 μm MK-801) (C, D). Immediately after imaging the cultures were processed for GAD immunocytochemistry (A–C) or for kainate-stimulated Co²⁺ labeling (D). All traces represent the means ± SEM of 15–30 GAD(+)/Co²⁺(+) and 100–200 GAD(−)/Co²⁺(−) neurons, derived from at least four experiments.

Fig. 4.

Kainate-triggered oxygen radical production is Ca²⁺-dependent. After a 20 min baseline recording the HEt-loaded cultures were exposed to kainate (200 μm + 10 μm MK-801) in the presence of the indicated extracellular Ca²⁺ concentration. The traces represent the means ± SEM of >30 GAD(+) and 100 GAD(−) neurons from four experiments.

Fig. 5.

Measured [Ca²⁺]_irises induced by exposure to NMDA, kainate, or high K⁺ vary markedly, depending on the affinity of the fluorescent Ca²⁺ indicator that was used. Cultures were loaded with either fura-2 (A, C) or fura-2FF (B, D), as described (see Materials and Methods). Fura-2 [Ca²⁺]_ilevels were expressed as fluorescence ratios, whereas fura-2FF [Ca²⁺]_i levels were expressed as calibrated values. After baseline imaging the cultures were exposed for 15 min (starting at time 0) to NMDA (200 μm + 10 μm NBQX), kainate (200 μm + 10 μm MK-801), or high K⁺ (50 mm + 10 μmMK-801/NBQX), as indicated, and were processed for GAD immunocytochemistry. Traces show the means ± SEM of [Ca²⁺]_i responses in GAD(+) (A, B) and in GAD(−) neurons (C, D) [15–25 GAD(+) neurons and >150 GAD(−) neurons in three experiments].

Fig. 6.

NMDA- or kainate-triggered [Ca²⁺]_i elevations that were assessed by fura-2FF predict oxygen radical production. Part A,Pseudocolor images, Cultures were loaded with both fura-2FF and HEt, and visible light images were obtained (A, A′). Fluorescent fura-2FF (B, C and B′, C′), and HEt (E, F and E′, F′) images were obtained 10 min before (B, B′ and E, E′) and 15 min after (C, C′ and F, F′) exposure to NMDA (200 μm + 10 μm NBQX) (top) or kainate (200 μm + 10 μm MK-801) (bottom). Then the cultures were immunostained for GAD (D, D′). Note that, although NMDA triggers strong HEt and fura-2FF fluorescence increases in nearly all neurons, with kainate exposures the fluorescence of both dyes was increased selectively in the GAD(+) neurons. As indicated by the scale bars, fura-2FF images represent fluorescence ratios, and HEt images represent fluorescent intensity on an eight-bit/0–256 scale. Scale bar, 50 μm. Part B,Time course, Cultures were loaded with both fura-2FF and HEt as above. After baseline imaging the cultures were exposed to kainate (200 μm + 10 μm MK-801), and changes in [Ca²⁺]_i levels (left axis, red lines) and oxygen radical production (right axis, blue lines) were monitored for an additional 15 min (see Materials and Methods). Traces show the means ± SEM of 7 GAD(+) and 25 GAD(−) neurons from one experiment, which is representative of three.

Fig. 7.

Electron transport inhibitors attenuate NMDA- or kainate-triggered oxygen radical production. After baseline recordings the cultures were exposed to 3 mm CN⁻for 15 min before NMDA (200 μm + 10 μmNBQX) (A) or kainate (200 μm + 10 μm MK-801) (B) was added in the continuing presence of CN⁻ (solid traces). For a comparison of agonist effects between CN⁻-treated and normal conditions, fluorescence intensities for each neuron were normalized to its average fluorescence during the 10 min immediately before agonist addition. Other HEt-loaded cultures were preexposed to rotenone (10 μm) for 40 min before kainate (200 μm + 10 μm MK-801) was added in the continued presence of rotenone (solid traces, C). For the purposes of comparison, in each experiment the sister cultures were exposed identically in the absence of the electron transport inhibitor (broken traces). Note that, because kainate caused little oxygen radical production in GAD(−) neurons, B andC illustrate the effect of electron transport inhibitors on kainate-triggered oxygen radical production only on GAD(+) neurons.

Fig. 8.

Kainate causes mitochondrial depolarization in GAD(+) neurons, but not in GAD(−) neurons. Cultures were loaded with 0.1 μm TMRE for 30 min (see Materials and Methods). After baseline recordings (F₀) the cultures were exposed to NMDA (200 μm + 10 μm NBQX;A), to kainate (200 μm + 10 μm MK-801; B), or to kainate (200 μm + 10 μm MK-801) in the presence of rotenone (10 μm; C) (see Materials and Methods); the fluorescence was monitored for 20 min more. Traces show the means ± SEM of TMRE fluorescence in all neurons (A) or in GAD(+) neurons (B, C, solid traces) and in GAD(−) neurons (B, C, broken traces) [30–70 GAD(+) neurons and >150 GAD(−) neurons, more than or equal to three experiments].

Fig. 9.

Antioxidants are neuroprotective against NMDA- or kainate-induced neuronal injury. Cultures were exposed to kainate (100 μm for 20 min) or NMDA (100 μm for 10 min) in the presence or absence of an antioxidant (3 mm trolox or 20 μm U74500; see Materials and Methods), and injury to the overall neuronal population (open bars) and to the GAD(+) neuronal population (filled bars) was assessed the next day. Values represent the means ± SEM compiled from at least four experiments; n = 12–20 cultures per condition. An ampersand indicates neuronal loss significantly different from that caused by agonist alone; anasterisk indicates GAD(+) neuronal loss significantly different from total neuronal loss (p < 0.01 by two-tailed t test).