Activation of the metabotropic glutamate receptor is neuroprotective during nitric oxide

0304-3940/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95)11’ 753-G
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K. Maiese et al. /Neuroscience
Letters 194 (1995) 173-l 76
medium and incubated in dissociation medium containing trypsin inhibitor (10-20 U/ml) three times, for 5 min each. The cells were washed in growth medium (Leibovitz’ s L- 15 Medium (Gibco BRL, Gaithersburg, MD) with 6% sterile rat serum (Bioproducts for Science, Indianapolis, IN), 150 mM NaHCOs, 2.25 mg/ml transferrin, 2.5 ,ug/ml insulin, 10 nM progesterone, 90pM putrescine, 15 nM selenium, 35 mM glucose, 1 mM Lglutamine, penicillin (50 U/ml), streptomycin (5Opg/ml), and supplemented with vitamins. The dissociated cells were plated at a density of approximately 15 x lo5 cells/ mm* in 35 mm* polylysine-laminin coated plates (Falcon Labware, Lincoln Park, NJ). Cells were maintained in growth medium at 37°C in a humidified atmosphere of 5% CO2 and 95% room air. All experiments were performed with cultured neurons that had been in culture for 2-3 weeks. Non-neuronal cells accounted for less than 20% of the total cell population. NO administration was performed by replacing the culture media with media containing 3OOpM sodium
Neuroscience Letters 194 (1995) 173-176
Activation of the metabotropic glutamate receptor is neuroprotective during nitric oxide toxicity in primary hippocampal neurons of rats
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Log [L-AP4J @Ml Fig. 1. Reduction in NO toxicity by L-AP4. Hippocampal cultures were pretreated with increasing concentrations of L-API1 1 h prior to a 5-min exposure of sodium nitroprusside (SNP) (3OOpM) (closed symbols) or in the absence of SNP (open symbols). Each data point represents the mean and SEM of n = 6 determinations (culture plates) from three separate experimental preparations. Neuronal survival was based on the percentage of the total number of neurons (viable + non-viable) and determined by trypan blue exclusion 24 h following exposure to SNP or 25 h following exposure to LAP4 without SNP administration. In cultures receiving only SNP, 25 * 7% (mean and SEM of n = 6 determinations (culture plates) from three separate experimental preparations) of the neurons were viable.
duced in hippocampal astrocytes [3] and mutant mice deficient in neuronal NOS activity have been shown to experience reduced infarct volumes [7]. Glutamate receptors have recently been reported to stimulate NO production in neurons [6,16]. Modulation of metabotropic glutamate receptor activity in the hippocampus [5] may represent one of the pathways that mediates NO toxicity during cerebral ischemia. Therefore, we examined whether the neuroprotective effect of mGluR activation was dependent upon the NO pathway. We focused on the mGluR4 receptor since this subtype has been associated with the inhibition of calcium currents [23] which could subsequently alter the generation and toxicity of NO [ 151. The hippocampi were obtained from l-day-old Sprague-Dawley rat pups and incubated in dissociation medium (90 mM Na$O+ 30 mM KzS04, 5.8 mM MgCl*, acid, and 1 mM 0.25 mM CaC12, 10 mM kynurenic HEPES with pH adjusted to 7.4) containing papain (10 U/ml) and cysteine (3 mmol/l) for two 20-min periods. The hippocampi were then rinsed in dissociation
Received 11 April 1995; revised version received 12 June 1995; accepted 12 June 1995
Abstract Metabotropic glutamate receptors (mGluRs) can influence neuronal survival and have been shown to be neuroprotective during glutamate toxicity in retinal cells and in cortical neurons. The mechanisms that mediate protection by this group of receptors are not clear. Since nitric oxide (NO) production can lead to neuronal cell death during excessive glutamate release, we examined whether neuronal survival was directly linked to mGluR activity and the NO pathway. Treatment with the mGluR4 receptor subtype agonist, L(+)-2-amino-4-phosphonobutyric acid, in hippocampal cell cultures protected neurons during NO exposure. Treatment with L-(+)-2amino-3-phosphonopropionic acid, an antagonist of the mGluRI receptr of inositol trisphosphate formation, did not significantly alter neuronal survival during NO administration. We conclude that activation of the mGluR4 receptor protects hippocampal neurons from NO toxicity and that the mechanism of NO induced neurodegeneration does not appear to involve inhibition of the mGluR1 receptor subtype activity or the phosphoinositide system.