Spontaneous and evoked vesicle cycling at a presynaptic terminal

The presynaptic vesicle cycle is crucial for chemical neurotransmission. In response to an action potential, neurotransmitter-filled vesicles are exocytosed from the pre-synaptic terminal and activate specific receptors at the post-synaptic membrane. To sustain neuronal activity, vesicles are then retrieved by endocytosis and refilled with neurotransmitters for further rounds of release. Neurotransmitter release can also occur spontaneously in the absence of action potentials. The current assumption is that both types of release use the same set of vesicles. To check this hypothesis, I developed a genetically encoded probe, biosyn, to specifically label pre-synaptic vesicles in living neurons. It consists of a fusion protein between VAMP2 and a biotin acceptor peptide (BAP). Co-transfection of biosyn with BirA, an enzyme that specifically recognizes the BAP sequence, results in the biotinylation of VAMP2. The subsequent addition of fluorescently-labeled streptavidin enables the visualization of newly exocytosed vesicles. I show in cultured hippocampal neurons that this approach successfully tagged synaptic vesicles that had undergone a cycle of exocytosis and re-endocytosis without affecting synaptic function. Using different stimulation protocols, I then revealed the existence of two distinct pools of vesicles that recycle independently and with different kinetics at the presynaptic terminal. One pool is mobilized in response to neuronal activity whereas the other fuses spontaneously with the plasma membrane. I also report that the resting pool of vesicles, which cannot be mobilized by neuronal activity, is the source of spontaneous vesicle release. Finally, I describe a switch in the modes of synaptic vesicle fusion during development. Young neurons (before synapse formation) showed high levels of spontaneous, but not activity-dependent, cycling, whereas in mature neurons activity-dependent release was predominant over spontaneous fusion.<br/>These findings suggest that the spontaneous and activity-dependent modes of release may play different roles in synaptic function and during neuronal development.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Ben Fredj, Naila

Advisors dc:contributor.advisor

• Burrone, Juan
• Eickholt, Britta

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/73d947b1-31e0-4d5e-bd48-1be223d78334

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/73d947b1-31e0-4d5e-bd48-1be223d78334