Spontaneous and evoked vesicle fusion: the emergence of a synapse

Neurons in the brain communicate with each other at specialized sites called synapses. During brain development a large number of neurons (about 10 billion) make an astonishingly large number of synaptic connections (about 100 trillion) to form a brain. Not surprisingly, an important question in neuroscience is to understand how synapses are formed and how these connections pass information from one neuron to the next. At the synapse, the transfer of information is carried out by the release of packets of neurotransmitter released from a presynaptic neuron onto specific receptors on a postsynaptic neuron. Neurotransmitter is stored inside membrane vesicles and is released by the fusion of vesicles with the plasma membrane, which expels the neurotransmitter towards the postsynaptic neuron. The membrane from the vesicle is subsequently retrieved from the plasma membrane to be refilled with neurotransmitter and recycled for use once again. The research proposed here will focus on understanding how a presynaptic terminal forms and matures to provide such an exquisitely controlled system for releasing neurotransmitter. For this purpose I will use especially designed fluorescent probes that report vesicle cycling in real time by showing flashes of fluorescence every time a vesicle fuses with the plasma membrane or by tagging the vesicle with coloured particles that make them visible under a fluorescent microscope. Exploiting these tools I will study how synapses gradually mature to release neurotransmitter, from the initial moments of the formation of a synaptic contact to the final stages of neuronal maturation. Understanding these events will help uncover how connections in the brain form during development and help us understand neurological diseases where this process has gone wrong.

Neurons transmit information within a network through synaptic connections. Neurotransmitter, stored in small clear vesicles, is released from a presynaptic terminal to activate specific postsynaptic receptors. Here, we set out to establish how these connections are formed, by focusing mainly on how the presynaptic terminal develops and gradually matures to relay information through a tightly controlled activity-dependent vesicle cycle. Our previous findings in mature terminals have found two independent pools of vesicles exist at a bouton: one that cycles in response to neuronal activity and another, distinct pool that cycles spontaneously. More importantly, we see a gradual switch in the modes of vesicle cycling, from a predominantly spontaneous form in early axons, to a mainly activity-dependent fusion mechanism in mature networks. Here, we aim to establish how vesicle pools originate in the axon and characterize how they develop into a mature presynaptic terminal. Our approach will be to assay how vesicle cycling matures throughout the different stages of the formation of a presynaptic terminal: from the original moments of growth cone motility and axon path-finding, to the stabilization of a growth cone and formation of a presynaptic terminal. For this purpose we will use genetically-encoded reporters of neuronal activity which will allow us to label distinct vesicle pools and assess their rates and modes of cycling. In early, extending axons we will image both synaptic vesicle precursors (SVPs) and active zone vesicle precursors (PTVs) to describe how and where along the axon they fuse with the plasma membrane. The interaction between these two macromolecular precursors is key to understanding how vesicles cycle in the absence of a postsynaptic partner and provides a unique way to measure synapse formation from a functional perspective.Aas synapses mature we will follow the progression of vesicle pool dynamics as they encounter a postsynaptic partner and establish how these pools are arranged within a mature bouton. Both the properties of pre- and post-synaptic compartments will be assessed to establish how this bicellular unit is functionally arranged. Finally, in mature synapses, we will characterize the properties of vesicle pool dynamics, their location within the synapse and how they are mobilized by calcium. This proposal encompasses the entire life of a synapses, from its immature beginnings within an extending axon to its final role as a site of information transfer between neurons.