Ultrastructure-function properties of recycling synaptic vesicles in acute hippocampal slices
Synaptic vesicles are the substrate of neurotransmission in most nerve terminals in the central nervous system. These small membrane spheres fuse with the synaptic membrane in an activity-dependent manner and release neurotransmitter into the synaptic cleft. Subsequently, vesicles are reclaimed through endocytosis prior to reuse. This recycling process is key to supporting ongoing signalling in the brain. \ud While substantial effort has gone into defining basic characteristics of vesicle recycling, for example elucidating the timing of vesicle turnover, key questions remain unanswered. An important area with significant knowledge deficits relates to the relationship between vesicle function and ultrastructural organisation in the terminal. The aim of this thesis is to address this issue, exploiting new methodologies which provide novel insights into function-structure relationships of vesicle populations in acute brain slices. Specifically, this study considers organisational principles of three defined vesicle pools as well as examining the impact of an established plasticity protocol on pool properties. The first results chapter, Chapter 3, outlines and validates the novel protocol used for fluorescently labelling functional recycling vesicle populations in acute rat brain slices using the vesicle-labelling dye FM1-43 and new antibody based probes (syt1-Oyster, CypHer5E). Reporter-labelling and release properties are compared to similar approaches using cultured neurons. We conclude that this approach provides a more physiologically relevant method to study the functional properties of cells than used previously in cultured neurons.\ud Chapter 4 outlines experiments utilising the capability of FM 1-43 to be photoconverted\ud to an electron-dense form to allow a defined vesicle population, the readily releasable pool (RRP), to be characterised ultrastructurally. The RRP is arguably the most significant pool class, released first in response to an activity train. Functional assays and time-stamped electron microscopy are used to define basic properties of this pool, including its size, functional release kinetics, and temporal organisation. Specifically, the results demonstrate that retrieved vesicles are close to the active zone after stimulation, but mixed randomly in the terminal volume over 20 min. These findings address fundamental questions about vesicle reuse, the composition of future vesicle pools, and thus the mechanism of ongoing signalling in the brain. \ud The same approach was used in Chapter 5 to examine the influence of Long Term Depression (LTD) on pool function and ultrastructure. LTD was induced in presynaptic terminals in CA1 via Schaffer collateral activation, and the following effects were observed: 1) a change in release kinetics; 2) a reduction in the total recycling pool size; and 3) no change in the composition of the docked pool. These findings demonstrate that there is a presynaptic component to LTD and that vesicle recruitment into the recycling pool appears to be an important possible substrate. However, the results suggest that such changes appear to be selective for specific pool subsets. Overall, work in this chapter offers new insights into fundamental principles supporting synaptic plasticity.\ud Chapter 6 expands on previous studies which have demonstrated that recycling vesicles are constitutively shared between neighbours. This sharing of a ‘superpool’ of vesicles has implications for the ability of synapses to adapt to changes in input weighting. In this chapter, the methods outlined above, as well as a new 3D EM technology, are used to define the size, positional organisation, and clustering properties of this pool in native hippocampal slice system. The findings in this chapter reveal that extrasynaptic vesicles appear to show a greater degree of motility than vesicles which remain in the intrasynaptic cluster, perhaps implying differential interactions with structural proteins in the synapse. Characterising the superpool is increasingly relevant, as it is now implicated in models of plasticity and disease.\ud Taken together, these results show that the ultrastructural arrangement of recycling vesicles is highly activity-dependent, and that the cytoarchitecture plays a large role in determining the functionality of individual vesicles and synapses.