Long-term intake of ethanol produces adaptive alterations in multiple transmitter systems in the hippocampal formation that likely contribute to ethanol withdrawal-induced seizure and excitotoxicity. The present studies were designed to examine the role of N-methyl-d-aspartate receptor activation and cytosolic Ca(2+) accumulation in the neurotoxic effects of ethanol withdrawal. Further, these studies investigated the role of hippocampal network excitation in promoting both Ca(2+) accumulation and neurotoxicity during ethanol withdrawal. Chronic, continuous (11 day) exposure to ethanol (91 mM starting concentration) did not produce neurotoxicity in any region of organotypic hippocampal explants, as measured by uptake of the non-vital fluorescent marker propidium iodide. Withdrawal from chronic (10 day) ethanol exposure was associated with rapid (30 min) and significant increases in intracellular Ca(2+), assessed by visualization of Calcium-Orange fluorescence, in each region of hippocampal explants. However, neurotoxicity was observed 24 h after initiation of withdrawal and was only seen in the cornu ammonis 1 (CA1) region. Exposure to MK-801 (20 microM) at the start of ethanol withdrawal markedly attenuated Ca(2+) entry in all regions, as well as, CA1 region neurodegeneration. Further, treatment of explants with tetrodotoxin (500 nM) as well as surgical transection of mossy fiber or Schaffer collateral projections immediately prior to ethanol withdrawal blocked both regional increases in Ca(2+) accumulation and CA1 neurotoxicity. These data suggest that neurodegeneration observed during ethanol withdrawal is dependent upon polysynaptic propagation of action potentials ("network excitation") and whole-hippocampal excitation of glutamatergic systems.