The Importance of Mobile Vesicles

In the presented work I demonstrated that synaptic vesicles exhibit high- and low-mobility states in the vesicle cycle. Low-mobility values of the vesicle cluster have already been found in the past; however, vesicles with a high-mobility state have only been observed after non-physiological treatments (e.g. okadaic acid treatment). The importance of mobile vesicles can only be surmised. The mobile recycling pool vesicles can without any difficulties reach the AZ for repeated neurotransmission. This in turn also keeps them in the recycling pool and thus mobile (see above). It was shown here that synaptic activity does not explicitly guide the vesicles towards the release site, indicating that (random?) vesicle movements guarantee reaching the AZ and the subsequent release, i.e. synaptic neurotransmission.

Furthermore, it has been hypothesized that synapses work as individual units with the synaptic vesicles being restricted to their host synapse. This view changed as substantial exchanges of synaptic vesicles or complete vesicle clusters between neighboring synapses have been observed (Krueger et al., 2003; Darcy et al., 2006; Chen et al., 2008; Staras et al., 2010). These studies all took advantage of FM dye labeling, and therefore investigated only the movements of recycling vesicles. However, it has also been shown that resting vesicles are shared between synapses (pHluorin experiments from Fernandez-Alfonso and Ryan, 2008). Moreover, with an ultrastructural analysis of FM dye labeled recycling vesicles (via

FM photo-oxidation and electron microscopy) Darcy and co-workers showed that unlabeled vesicles colocalize with labeled recycling vesicles, possibly belonging to moving vesicles of the resting pool. These results are in full agreement with the presented work, showing the movement of both the recently endocytosed and the immobile resting vesicles along the axon, and their integration into pre-existing vesicle clusters at neighboring release sites. The substantial lateral exchange of both the recycling pool vesicles and the resting pool vesicles across neighboring release sites can in principle represent an additional multi-synapse vesicle pool. This extra-synaptic pool was previously named “superpool” (Westphal et al., 2008;

Staras et al., 2010). The superpool hypothesis is based on observations from recycling pool vesicles which are dynamically exchanged with a turnover rate of >4% of the total synaptic vesicle pool of a synapse (Staras et al., 2010). As presented in this work, between 1-3 vesicles entered the imaged area per second. Taking this observation at its lowest value, if only one vesicle moves through a synapse, then in 3 minutes around 180 vesicles would have passed through which equals the amount of vesicles that are housed in the synaptic bouton of these type of neuron (Schikorski and Stevens, 1997). Furthermore, the vesicles participate with normal kinetics in neurotransmission at their new release site (Krueger et al., 2003;

Darcy et al., 2006; Staras et al., 2010), indicating that the synapses of the nerve cell are intensely inter-linked.

Consequently, one can assume that the mobile superpool vesicles may be simply an additional supporting reserve pool that helps to sustain neurotransmission at individual release sites - the vesicles travel across multiple boutons and (where necessary) they fuse or integrate into the existing vesicle cluster. However, the resting vesicles are also shared between synapses (Fernandez-Alfonso and Ryan, 2008). Thus, it is questionable whether the exchange of recycling vesicles and resting vesicles is used for distinct purposes. In general, as vesicles disappear or get incorporated in the vesicle clusters the exchange may balance the vesicle populations of the inter-linked synapses over a longer period (Darcy et al., 2006).

What would cause the release of the resting vesicles from the cluster and make them move along the axon? A simple explanation for this is missing, but some observations made clear that additional molecules other than synapsin might be present to manage the migration from and the entry into the cluster (as shown by additional cross-linking filaments (Siksou et al., 2007)).

Surprisingly, the vesicle exchange was exclusively observed in cultured hippocampal neurons. It may thus thought to be an effect of synapse maturation or neuron development rather than of importance for native tissues (Matteoli et al., 1992). However, one recent investigation strengthened the superpool hypothesis as also vesicles are exchanged in hippocampal tissue slices (Staras et al., 2010). Moreover, Shepherd and Harris performed serial sectioning of the connection between the CA3-CA1 hippocampus area of adult rat CNS slices (Shepherd and Harris, 1998). Their three-dimensional reconstructions revealed loose clusters of vesicles in the axons, indicating the existence of vesicle packages traveling between release sites in mature neurons. Nevertheless, it is not known if vesicle exchange is limited to the hippocampus or whether it also takes place in other parts of the CNS.

Although synaptic vesicles are shared inter-synaptically across multiple boutons, synaptic vesicle membrane proteins are as well exchanged after strong stimulation by lateral movements across the axonal plasma membrane (Li and Murthy, 2001). Vesicle or protein exchange may therefore be an additional way to redistribute synaptic vesicle components across several release sites.

Finally, not only synaptic vesicles or their components are constitutively exchanged between neighboring nerve terminals. Also components of the cytomatrix at the AZ are transported from one synaptic release site to the next, e.g. bassoon (Tsuriel et al., 2009), but also synapsin 1 (Tsuriel et al., 2006). Furthermore, entire orphan release sites (fully functional release sites formed in the absence of axon-dendritic contacts) arise from or coalesce with stable vesicle clusters (Krueger et al., 2003). This illustrates the importance of mobile presynaptic components for the organization and the maintenance of the functional and structural properties of the synapse.

Taken together, individual synapses cannot be seen anymore as autonomous units, as previously believed. Synapses are even more inter-linked with a substantial fraction of their vesicles (recycling and resting pool vesicles) being affiliated to a common mobile

“superpool” to serve multiple release sites.