Polyamines contribute to ethanol withdrawal-induced neurotoxicity in rat hippocampal slice cultures through interactions with the NMDA receptor

Background: Several reports demonstrate that withdrawal from long-term ethanol exposure is associated with significant central nervous system neurotoxicity, produced at least in part by increased activity of N-methyl-D-aspartate receptors (NMDARs). Recent evidence suggests that elevations in the synthesis and release of the polyamines spermidine and spermine, which are known modulators of NMDARs, contribute to the increased activity of the receptor during ethanol withdrawal. Therefore, the goal of this investigation was to examine what role, if any, spermidine and spermine have in the generation of ethanol withdrawal-induced neurotoxicity. Methods: Neurotoxicity (measured as fluorescence of the cell death indicator propidium iodide, PI), glutamate release (measured by high-performance liquid chromatography analysis), and polyamine concentrations (by high-performance liquid chromatography) were measured in rat hippocampal slice cultures undergoing withdrawal from chronic (10 day) ethanol exposure (100 mM). In addition, the effects of the polyamine synthesis inhibitor di-fluoro-methyl-ornithine (DFMO, 0.1-100 nM) and NMDAR polyamine-site antagonists ifenprodil, arcaine, and agmatine (1 nM-100 μM) on ethanol withdrawal- and NMDA-induced neurotoxicity were measured. Results: Ethanol withdrawal significantly increased glutamate release (peaking at 18 hr with a 53% increase), increased concentrations of putrescine and spermidine (136% and 139% increases, respectively, at 18 hr), and produced significant cytotoxicity in the CA1 hippocampal region (56% increase in PI staining relative to controls) of the cultures. The cell death produced by ethanol withdrawal was significantly inhibited by ifenprodil (IC 50 = 14.9 nM), arcaine (IC 50 = 37.9 nM), agmatine (IC 50 = 41.5 nM), and DFMO (IC 50 = 0.6 nM). NMDA (5 μM) significantly increased PI staining in the CA1 region of the hippocampal cultures (365% relative to controls), but ifenprodil, arcaine, agmatine, and DFMO all failed to significantly affect this type of toxicity. Conclusions: These data implicate a role for polyamines in ethanol withdrawal-induced neurotoxicity and suggest that inhibiting the actions of polyamines on NMDARs may be neuroprotective under these conditions.