Modulation of Glutamate-Induced Intracellular Energy Failure in Neonatal Cerebral Cortical Slices by Kynurenic Acid, Dizocilpine, and NBQX

Abstract

The severity and rapidity of acute, glutamate-induced energy failure were compared in live cerebral cortical slices. In each experiment 80 live cerebral cortical slices (350 μm thick) were obtained from neonatal Sprague–Dawley rats, suspended and perfused in a nuclear magnetic resonance (NMR) tube, and studied at 4.7 T with interleaved 31P/1H NMR spectroscopy. NMR spectra, obtained continually, were determined as 5-min averages. Slices were perfused for 60 min with artificial cerebrospinal fluid (ACSF) containing either glutamate alone or glutamate mixed with one of three glutamate-receptor antagonists: kynurenate, dizocilpine (MK-801), and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( F)quinoxaline (NBQX). Dose-dependent decreases in high-energy phosphates were studied during glutamate exposure (0.5 to 10 m M), with and without antagonist protection. Energy recovery after glutamate exposures was measured during a 60-min washout with glutamate-free, antagonist-free ACSF. Reversible and irreversible energy failures were characterized by changes in intracellular pH, and by changes in relative concentrations of ATP, phosphocreatine (PCr), and inorganic phosphate. No changes were observed in intracellular levels of N-acetylaspartate and lactate. Some special studies were also done using R-(–)-2-amino-5-phosphonovaleric acid (100 μ M) and tetrodotoxin (1 m M) to examine glutamate receptor specificity in this tissue model. Dizocilpine (150 μ M) best ameliorated the energy failure caused by 2.0 m M glutamate. With dizocilpine the maximum ATP decrease was only 6 ± 5%, instead of 35 ± 7%. Additionally, the dizocilpine-induced recovery of ATP levels, complete after 30 min of glutamate exposure, lasted througout 30 additional min of glutamate exposure and 60 additional min of washout with glutamate-free ACSF. Although dizocilpine did not alter the maximum decrease that occurred in PCr (to 36 ± 4% of control), dizocilpine did cause PCr levels to return to within 7 ± 5% of the control after 30 min of glutamate exposure. PCr levels stayed at this value throughout 30 additional min of glutamate exposure. During the washout period PCr immediately rose to a value 5 ± 2% above the control and then remained constant during the rest of the 60-min washout. During the first 20 min of glutamate administration, kynurenic acid (1.0 m M) best improved the high-energy phosphate levels. NBQX (6.0 μ M), reported to protect the brain from ischemic injury, decreased PCr depletion during glutamate exposure without affecting the loss of ATP. After 60 min of glutamate washout, PCr levels with kynurenate (84 ± 6% of control) and NBQX (84 ± 2% of control) were significantly higher (p < 0.001) than with glutamate alone (42 ± 6% of control), although ATP levels were not significantly improved by either drug. Acute energy failure in our brain slice model, intended to simulate oxygenated penumbral tissue, probably occurs primarily in neurons. The reason that dizocilpine best preserves high-energy phosphate levels might relate to its mechanism of N-methyl-d-aspartate receptor blockade. Additional energy protection from dizocilpine might also arise from a partial blockade of voltage-dependent Na+ channels, which is possible at the concentration used.