Monoamine-induced diacylglycerol signaling rapidly accumulates Unc13 in nanoclusters for fast presynaptic potentiation.

Abstract

Neuromodulators control mood, arousal, and behavior by inducing synaptic plasticity via G-protein-coupled receptors. While long-term presynaptic potentiation requires structural changes, mechanisms enabling potentiation within minutes remain unclear. Using the <i>Drosophila</i> neuromuscular junction, we show that octopamine, the invertebrate analog of norepinephrine, potentiates evoked neurotransmitter release on the timescale of one minute via a G-protein-coupled pathway involving presynaptic OAMB receptors and phospholipase C. This fast potentiation correlates with elevated signals of the release factor Unc13A and the scaffolding protein Bruchpilot. Live, single-molecule imaging of endogenously tagged Unc13 revealed its instantly reduced motility and increased concentration in synaptic nanoclusters with potentiation. Presynaptic knockdown of Unc13A fully blocked fast potentiation. Moreover, deleting its N-terminal localization sequence mislocalized the protein fragment to the cytosol, but still allowed for rapid plasma membrane recruitment by diacylglycerol (DAG) analog phorbol esters and octopamine, implicating a role of more C-terminal domains. A point mutation of endogenous Unc13 in its DAG-binding C1 domain blocked plasticity-induced nanoscopic enrichment and synaptic potentiation. The mutation increased basal neurotransmission but reduced Unc13 levels, revealing a gain of function and potential homeostatic compensation. The mutation also blocked phorbol ester-induced potentiation, decreased the calcium sensitivity of neurotransmission, and caused short-term synaptic depression. Homeostatic potentiation induced by postsynaptic receptor block mirrored octopamine-induced Unc13 recruitment and required presynaptic OAMB receptors, indicating overlapping machinery. Thus, rapid Unc13 immobilization and nanoscale compaction are salient features of fast presynaptic potentiation.