Novel in vitro platform to study molecular mechanisms of neurotransmitter release and synaptic plasticity

Rapid release of neurotransmitters stored in small vesicles at the nerve terminals forms the basis of information transfer in the brain. This process is essential for learning and memory and is disrupted in many neurological disorders. At nerve terminals, Ca2+-sensing proteins (synaptotagmins) couple vesicular release machinery (SNAREs) to Ca2+ signals, thus synchronising neurotransmission to neuronal firing. How the vesicular release machinery decodes Ca2+ signals and translates them into the complex patterns of neurotransmitter release remains enigmatic.
At present, the mechanisms of Ca2+-evoked neurotransmitter release are predominantly studied in live synapses, using a combination of electrophysiology, fluorescence imaging and genetic manipulations. However, due to intrinsic variability, experiments in live synapses require large numbers of animals. Moreover, genetic deletion of presynaptic proteins often results in severe or even lethal phenotypes. The interpretation of experiments in live synapses is further complicated by the expression of multiple protein isoforms and compensatory homeostatic mechanisms. This calls for developing new technologies that can effectively replace experiments in live synapses and reduce the number of animals used in the field of synaptic physiology.
One way to reduce the number of animals is to replace experiments in live neurons with biochemically defined reconstituted vesicle fusion assays. This reductionist approach, where the variables are limited, and the components can be precisely defined, has the potential to gain direct mechanistic insights into Ca2+-regulated synaptic vesicle fusion. However, the low temporal resolution and lack of precise Ca2+ control limit the application of the classical in vitro fusion setups.
In this project, we propose to develop a novel experimental platform that combines a single vesicle fusion assay with the fast and precise control of the Ca2+ signal. This setup will faithfully recapitulate synaptic architecture and physiology under cell-free conditions and will allow studying Ca2+-evoked vesicle fusion with millisecond precision. The developed technology will be of significant interest to the academics involved in the research of synaptic physiology, which is a large and dynamic research field. The implementation of this new innovative platform has a large potential to replace many experiments that are now only possible in animal preparations and therefore to allow for the replacement of experiments that require the production of over several tens of thousands of transgenic animals per year.

Ca2+-controlled release of neurotransmitters stored in small vesicles at the synaptic nerve terminals forms the basis of information transfer in the brain. Modulation of this process (synaptic plasticity) underpins learning and memory and is disrupted in many neurological disorders. How Ca2+ signals are translated into the complex vesicular release patterns remains poorly understood, in large part due to the limitations of available genetic/physiological tools and the lack of cell-free reconstitution systems required to achieve a mechanistic understanding. We aim to address this need by developing an innovative experimental platform based on a biochemically-defined single-vesicle fusion system. By combining this fusion setup with Ca2+ uncaging from photolysable Ca2+ chelator DM-nitrophen, we seek to reconstitute the fast Ca2+-evoked vesicular release of neurotransmitters under in vitro conditions. We will implement protocols for mimicking complex presynaptic Ca2+ dynamics observed during physiological bursts of neuronal activity to study synaptic plasticity in vitro. We anticipate that implementing this novel methodology would allow us and the other research groups to replace many experiments in live neurons, which will lead to a significant reduction in animal use in the field of synaptic physiology.