Extrasynaptic glutamate release through cystine/glutamate antiporter contributes to ischemic damage
Federico N. Soria, … , Carlos Matute, María Domercq
Federico N. Soria, … , Carlos Matute, María Domercq
Published July 18, 2014
Citation Information: J Clin Invest. 2014;124(8):3645-3655. https://doi.org/10.1172/JCI71886.
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Research Article

Extrasynaptic glutamate release through cystine/glutamate antiporter contributes to ischemic damage

Abstract

During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.

Authors

Federico N. Soria, Alberto Pérez-Samartín, Abraham Martin, Kiran Babu Gona, Jordi Llop, Boguslaw Szczupak, Juan Carlos Chara, Carlos Matute, María Domercq

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Figure 1

Blockade of EAATs during OGD contributes to extracellular glutamate accumulation in acute slices and organotypic cultures.

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Blockade of EAATs during OGD contributes to extracellular glutamate accu...
(A) Voltage-clamp recording of cortical neurons at 30 mV in acute slices demonstrates activation of a large current after OGD (n = 32). Ionotropic glutamate receptor antagonists AP5 (100 μM) plus CNQX (30 μM) significantly inhibited the OGD-activated current (n = 9). Nonsubstrate broad-spectrum EAAT inhibitor DL-TBOA (100 μM) did not significantly change (but tended to increase) the amplitude of the OGD-induced current and shortened the onset of AD in cortical neurons (n = 12), an effect abolished when applied concomitantly with AP5 plus CNQX (n = 7). (B) Histograms showing the average amplitude (pA ± SEM) and latency (minutes ± SEM) of the OGD-induced current for each condition. Latency was significantly reduced in the presence of TBOA. *P < 0.05 versus OGD. (C) TBOA increased the OGD-activated current when applied after the onset of AD (n = 11). (D) Inhibition of EAATs exacerbated OGD-induced cell death in organotypic slices, as demonstrated by increased LDH release in cultures subjected to 45 minutes of OGD and 24 hours of reoxygenation in the presence or absence of TBOA (100 μM). Data are expressed as the mean ± SEM (n = 3–5). **P < 0.01 and *P < 0.05 versus OGD. (E) Representative fields demonstrate propidium iodide labeling in cortical layers of slices treated as in D. Scale bar: 1 mm.