19F NMR calcium changes, edema and histology in neonatal rat brain slices during glutamate toxicity

Respiring neonatal cerebrocortical slices (350 microns thick), loaded with the free calcium indicator 5F-BAPTA, were perfused in a 20-mm-diameter glass NMR tube with oxygenated artificial CSF, exposed to extracellular glutamate and studied at 4.7 Tesla with 19F NMR spectroscopy. 31P/1H NMR spectra, obtained concurrently, were used to assess slice integrity from determinations of intracellular pH, ATP, PCr, lactate and N-acetylaspartate. 60-min periods were induced of recoverable and nonrecoverable glutamate toxicity-defined from changes in NMR metabolites. In other NMR studies, where 5F-BAPTA was not used, metabolic toxicity was modulated by three glutamate receptor antagonists: dizocilpine, NBQX and kynurenic acid. Outcome measurements were made of edema, determined invasively in isolated slices from % swelling and water content and from histological changes in Nissl stains of slice sections. Edema was (1) detectable in all slices within minutes after onset of glutamate exposure, though never in untreated control slices, and (2) modulated differently by dizocilpine, NBQX and kynurenate. Correlations were observed between edema and NMR decreases in PCr and ATP. Nissl stains of sections from slices treated with the most protective agent, dizocilpine, showed preservation of neuronal processes. As was expected in 7-day-old rats with immature NMDA receptors, 19F NMR spectroscopy revealed only small increases in free intracellular calcium ([Ca2+]i). These occurred late during glutamate exposure and reversed early during glutamate washout. The studies demonstrate that it is possible to study correlations between repeated noninvasive NMR spectra in ensembles of brain slices and invasive measures of early cellular responses.