5-HT 3 R-sourced calcium enhances glutamate release from a distinct vesicle pool.

•5-HT3R activation increased sEPSCs.•5-HT3R activation did not alter voltage-activated calcium channel-mediated glutamate release.•5-HT3R activation delayed latencies of evoked synchronous events, often blocking transmission.•EGTA-AM attenuated 5-HT3R responses consistent with vesicles in both micro- and nanodomains.•5-HT3R-sourced calcium is isolated from CaV calcium domains.