Abstract: To explore if prolonged – as opposed to acute – 5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (KATP) channels, we investigated in rat and mouse neocortical slices the effects of KATP channel blockers on electrically evoked [3H]-serotonin ([3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1μM), a KATP channel blocker, enhanced the electrically evoked [3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1μM), another KATP channel antagonist. After short-term blockade (45min) of 5-HT uptake, glibenclamide still increased the release of [3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [3H]-5-HT release after long-term uptake blockade (210min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1μM) prevented the facilitatory effect of glibenclamide. The Na+/K+-ATPase inhibitor ouabain (3.2μM) abolished the glibenclamide-induced increase in [3H]-5-HT release in both rat and mouse, suggesting that an operative Na+/K+-ATPase is a prerequisite for activation of KATP channels. The terminal 5-HT1B autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [3H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT1B autoreceptor ligands metitepin (1μM) and cyanopindolol (1μM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na+/K+-ATPase, which increases intracellular ATP and consequently closes KATP channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na+/K+-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close KATP channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers. [Copyright &y& Elsevier]