Dopamine controls essential brain functions through volume transmission. Different from fast synaptic transmission, where neurotransmitter release and receptor activation are tightly coupled by an active zone, dopamine transmission is widespread and may not necessitate these organized release sites. Here, we determine whether striatal dopamine secretion employs specialized machinery for release. Using super resolution microscopy, we identified co-clustering of the active zone scaffolding proteins bassoon, RIM and ELKS in ∼30% of dopamine varicosities. Conditional RIM knockout disrupted this scaffold and, unexpectedly, abolished dopamine release, while ELKS knockout had no effect. Optogenetic experiments revealed that dopamine release was fast and had a high release probability, indicating the presence of protein scaffolds for coupling Ca influx to vesicle fusion. Hence, dopamine secretion is mediated by sparse, mechanistically specialized active zone-like release sites. This architecture supports spatially and temporally precise coding for dopamine and provides molecular machinery for regulation.

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane.

No abstract

In a presynaptic nerve terminal, synaptic vesicle exocytosis is restricted to specialized sites called active zones. At these sites, neurotransmitter release is determined by the number of releasable vesicles and their probability of release. Proteins at the active zone set these parameters by controlling the presynaptic Ca 2ϩ signal, and through docking and priming of synaptic vesicles. Vertebrate ELKS proteins are enriched at presynaptic active zones, but their functions are not well understood. ELKS proteins are produced by two genes in vertebrates, and each gene contributes ϳ50% to total brain ELKS. We generated knock-out mice for ELKS1 and found that its constitutive removal causes lethality. To bypass lethality, and to circumvent redundancy between ELKS1 and ELKS2 in synaptic transmission, we used a conditional genetic approach to remove both genes in cultured hippocampal neurons after synapses are established. Simultaneous removal of ELKS1 and ELKS2 resulted in a 50% decrease of neurotransmitter release at inhibitory synapses, paralleled by a reduction in release probability. Removal of ELKS did not affect synapse numbers or their electron microscopic appearance. Using Ca 2ϩ imaging, we found that loss of ELKS caused a 30% reduction in single action potential-triggered Ca 2ϩ influx in inhibitory nerve terminals, consistent with the deficits in synaptic transmission and release probability. Unlike deletion of the active zone proteins RIM, RIM-BP, or bruchpilot, ELKS removal did not lead to a measurable reduction in presynaptic Ca 2ϩ channel levels. Our results reveal that ELKS is required for normal Ca 2ϩ influx at nerve terminals of inhibitory hippocampal neurons.

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3-KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3-KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.