Both synthesis and reuptake are critical for replenishing the releasable serotonin pool in Drosophila.

The two main sources of serotonin available for release are expected to be newly synthesized serotonin and serotonin recycled after reuptake by the serotonin transporter. However, their relative importance for maintaining release and the time course of regulation are unknown. We studied serotonin signaling in the ventral nerve cord of the larval Drosophila CNS. Fast-scan cyclic voltammetry at implanted microelectrodes was used to detect serotonin elicited by channelrhodopsin2-mediated depolarization. The effects of reuptake were probed by incubating in cocaine, which is selective for the serotonin transporter in Drosophila. p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase2, was used to investigate the effects of synthesis. Stimulations were repeated at various intervals to assess the time course of recovery of the releasable pool. Reuptake is important for the rapid replenishment of the releasable pool, on the 1 min time scale. Synthesis is critical to the longer-term replenishment (10 min) of the releasable pool, especially when reuptake is also inhibited. Concurrent synthesis and reuptake inhibition decreased both serotonin tissue content measured by immunohistochemistry (by 50%) and the initial amount of evoked serotonin (by 65%). Decreases in evoked serotonin are rescued by inhibiting action potential propagation with tetrodotoxin, implicating endogenous activity in the depletion. These results show synthesis is necessary to replenish part of the releasable serotonin pool that is depleted after reuptake inhibition, suggesting that regulation of synthesis may modulate the effects of serotonin reuptake inhibitors.