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Selective serotonin reuptake inhibitors (SSRIs) have an inhibitory effect on various ion channels including Ca2+ channels. We used fluorescent dye-based digital imaging, whole-cell patch clamping and cytotoxicity assays to examine whether dapoxetine, a novel rapid-acting SSRI, affect glutamate-induced calcium signaling, mitochondrial depolarization and neuronal cell death in cultured rat hippocampal neurons. Pretreatment with dapoxetine for 10 min inhibited glutamate-induced intracellular free Ca2+ concentration ([Ca2+]i) increases in a concentration-dependent manner (Half maximal inhibitory concentration=4.79 μM). Dapoxetine (5 μM) markedly inhibited glutamate-induced [Ca2+]i increases, whereas other SSRIs such as fluoxetine and citalopram only slightly inhibited them. Dapoxetine significantly inhibited the glutamate-induced [Ca2+]i responses following depletion of intracellular Ca2+ stores by treatment with thapsigargin. Dapoxetine markedly inhibited the metabotropic glutamate receptor agonist, (S)−3,5-dihydroxyphenylglycine-induced [Ca2+]i increases. Dapoxetine significantly inhibited the glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i responses in either the presence or absence of nimodipine. Dapoxetine also significantly inhibited AMPA-evoked currents. However, dapoxetine slightly inhibited N-methyl-D-aspartate (NMDA)-induced [Ca2+]i increases. Dapoxetine markedly inhibited 50 mM K+-induced [Ca2+]i increases. Dapoxetine significantly inhibited glutamate-induced mitochondrial depolarization. In addition, dapoxetine significantly inhibited glutamate-induced neuronal cell death and its neuroprotective effect was significantly greater than fluoxetine. These data suggest that dapoxetine reduces glutamate-induced [Ca2+]i increases by inhibiting multiple pathways mainly through AMPA receptors, voltage-gated L-type Ca2+ channels and metabotropic glutamate receptors, which are involved in neuroprotection against glutamate-induced cell death through mitochondrial depolarization.