1. The effects of culturing hypothalamic neurons in glutamate receptor antagonists were studied with fura-2 Ca2+ digital imaging of groups of synaptically coupled neurons. Removal of D-2-amino-5-phosphonovalerate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from cultures chronically blocked for periods of 14-188 days caused a dramatic increase in neuronal Ca2+ to abnormally high levels 5- to- 10 fold greater than the normal intracellular levels of 50-100 nM. In most cases AP5/CNQX removal initiated spontaneous synchronized Ca2+ oscillations. 2. Ca2+ rises and oscillations were blocked by the reintroduction of AP5/CNQX or by the addition of tetrodotoxin to block action potentials. These data indicate that hypothalamic neurons were the source of the excitatory transmitter that activated glutamate receptors and consequently led to the Ca2+ hyperexcitability. 3. The Ca2+ spike amplitude and frequency increased in response to the removal of Mg2+ from the perfusion solution to facilitate N-methyl-D-aspartate (NMDA) receptor responses. Picrotoxin, a GABAA-receptor blocker, also increased Ca2+ activity. 4. Blocking either NMDA (with AP5) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type (with CNQX) glutamate receptors reduced the level of Ca2+, but blocking both types was necessary for chronically blocked neurons to return to their basal Ca2+ level. 5. The survival of a large percentage of chronically blocked neurons was dependent on the presence of glutamate receptor blockade. Removal of AP5/CNQX from the tissue culture medium induced an immediate increase in Ca2+ levels in the majority of chronically blocked neurons and, with prolonged withdrawal of AP5/CNQX (3 h), 50% of the neurons lost the immediate ability to regulate internal Ca2+ levels. Excitotoxic cell death was induced in 40% of the neurons within 40 h of the removal of AP5/CNQX from neurons chronically blocked for 30 days. The number of neurons that survived for 70 days doubled when cultures were maintained in AP5/CNQX. 6. Relative to control cultures of the same period in vitro, chronically blocked neurons showed an enhanced Ca2+ influx when stimulated with the glutamate receptor agonists kainate (+70%), NMDA (+62%), or glutamate (+34%) in the presence of tetrodoxin. When the data from control and chronically blocked cultures stimulated with glutamate receptor agonists were pooled, without exception all the smallest responses were found in the control neurons. Compared with controls, chronically blocked neurons showed an exaggerated response to glutamate in the presence of nimodipine, indicating that Ca2+ hyperexcitability was not due to changes in voltage activated L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)