The mechanisms of Ca2+ responses evoked in hippocampal glial cells in situ, by local application of glutamate and by synaptic activation, were studied in slices from juvenile rats using the membrane permeant fluorescent Ca2+ indicator fluo-3AM and confocal microscopy. Ca2+ responses induced by local application of glutamate were unaffected by the sodium channel blocker tetrodotoxin and were therefore due to direct actions on glial cells. Glutamate-evoked responses were significantly reduced by the L-type Ca2+ channel blocker nimodipine, the group I/II metabotropic glutamate receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), and the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)2-amino-5-phosphonopentanoic acid (APV). However, glutamate-induced Ca2+ responses were not significantly reduced by the non-NMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). These results indicate that local application of glutamate increases intracellular Ca2+ levels in glial cells via the activation of L-type Ca2+ channels, NMDA receptors, and metabotropic glutamate receptors. Brief (1 s) tetanization of Schaffer collaterals produced increases in intracellular Ca2+ levels in glial cells that were dependent on the frequency of stimulation (> or =50 Hz) and on synaptic transmission (abolished by tetrodotoxin). These Ca2+ responses were also antagonized by the L-type Ca2+ channel blocker nimodipine and the metabotropic glutamate receptor antagonist MCPG. However, the non-NMDA receptor antagonist CNQX significantly reduced the Schaffer collateral-evoked Ca2+ responses, while the NMDA antagonist APV did not. Thus, these synaptically mediated Ca2+ responses in glial cells involve the activation of L-type Ca2+ channels, group I/II metabotropic glutamate receptors, and non-NMDA receptors. These findings indicate that increases in intracellular Ca2+ levels induced in glial cells by local glutamate application and by synaptic activity share similar mechanisms (activation of L-type Ca2+ channels and group I/II metabotropic glutamate receptors) but also have distinct components (NMDA vs. non-NMDA receptor activation, respectively). Therefore, neuron-glia interactions in rat hippocampus in situ involve multiple, complex Ca2+-mediated processes that may not be mimicked by local glutamate application.