Maximal Fusion Capacity and Efficient Replenishment of the Dense Core Vesicle Pool in Hippocampal Neurons

Abstract

Neuropeptides and neurotrophins, stored in dense core vesicles (DCVs), are together the largest currently known group of chemical signals in the brain. Exocytosis of DCVs requires high-frequency or patterned stimulation, but the determinants to reach maximal fusion capacity and for efficient replenishment of released DCVs are unknown. Here, we systematically studied fusion of DCV with single vesicle resolution on different stimulation patterns in mammalian CNS neurons. We show that tetanic stimulation trains of 50-Hz action potential (AP) bursts maximized DCV fusion, with significantly fewer fusion event during later bursts of the train. This difference was omitted by introduction of interburst intervals but did not increase total DCV fusion. Interburst intervals as short as 5 s were sufficient to restore the fusion capacity. Theta burst stimulation (TBS) triggered less DCV fusion than tetanic stimulation, but a similar fusion efficiency per AP. Prepulse stimulation did not alter this. However, low-frequency stimulation (4 Hz) intermitted with fast ripple stimulation (200 APs at 200 Hz) produced substantial DCV fusion, albeit not as much as tetanic stimulation. Finally, individual fusion events had longer durations with more intense stimulation. These data indicate that trains of 50-Hz AP stimulation patterns triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.SIGNIFICANCE STATEMENTNeuropeptides and neurotrophins modulate multiple regulatory functions of human body like reproduction, food intake or mood. They are packed into dense core vesicles (DCVs) that undergo calcium and action potential (AP) fusion with the plasma membrane. In order to study the fusion of DCVsin vitro, techniques like perfusion with buffer containing high concentration of potassium or electric field stimulation are needed to trigger the exocytosis of DCVs. Here, we studied the relationship between DCVs fusion properties and different electric field stimulation patterns. We used six different stimulation patterns and showed that trains of 50-Hz action potential bursts triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.