The present study used immunogold EM to examine distribution of endogenous synaptic vesicle (SV) proteins in young axons in dissociated hippocampal cultures at 3–6 DIV to document these proteins’ biosynthesis, axon transport, and eventual sorting into the SV. The main focus here is the contrast between the two types of SV proteins—SV integral membrane proteins vs. SV-associated proteins.
In the neuronal soma where proteins are synthesized, labels for SV integral membrane proteins (synaptophysin, SV2, VAM/synaptobrevin, and synaptotagmin) were localized at the Golgi complex and other membranous structures in the cytoplasm. In contrast, labels for SV-associated proteins (synapsin and synuclein) were not localized at the Golgi, consistent with earlier LM results . Here at the ultrastructural level, labels for synapsin I and -synuclein were dispersed in the cytoplasm, not associated with membranous vesicles/vacuoles.
SV integral membrane and SV-associated proteins were both polarized into axons early in development , but transported differently [1, 23]. SV integral membrane proteins are transported as a mixture of tubular-vesicular structures [6, 7] by fast axonal transport, predominantly carried by Kinesin 3 family . However, different SV membrane proteins may be sorted into different cargos  as synaptophysin and SV2 are transported separately in spinal nerve bundles  and in differentiated PC12 cells . On the other hand, axonal transport for SV-associated proteins (synapsin and synuclein) is even more complicated [1, 23] because these proteins are not constantly, but reversibly associated with SV membranes. The membrane-associated form may be part of the fast component of axonal transport, while the ones not associated with membranes may be in the slow component [1, 23]. The present study provided structural evidence that label for synapsin I was mostly cytosolic, and only became associated with vesicular membranes after clusters of SV-like vesicles were formed in axons. This finding is consistent with the notion that synapsin I plays a role in clustering the SV vesicles .
SVs with a full complement of their specific proteins are not formed in soma but only in axon, and can form in the absence of dendritic contact. These observations are consistent with earlier reports that SVs are formed only after undergoing exo- and endocytosis through specialized sorting at recycling endosomes in axons, and can form without dendritic contact [1, 6, 26, 27]. The common occurrence of clathrin-coated vesicles near the SV transport aggregates provides structural evidence of robust endocytosis at these locations. The fact that many clathrin-coated vesicles were of a similar size to the clusters of SV-like vesicles nearby are consistent with the possibility that these coated vesicles could shed the clathrin coating and become SV-like vesicles . Furthermore, the axolemmal labeling of SNAP–25, a part of the SNARE complex involved in exocytosis , is consistent with the idea that exocytosis can occur all along the axons, not just restricted to presynaptic active zone . Thus, the present study showed ultrastructural and immunogold illustrations that young axons could be capable of localized exocytosis and endocytosis, resulting in clusters of SV-like vesicles at non-synaptic sites.
In addition to SV proteins, the active zone (AZ) cytomatrix proteins, such as Bassoon and Piccolo, also have to be transported through axons to reach their final destination at the synapses [2, 3, 4, 5]. These AZ transport aggregates consist of 1–2 dense core vesicles (DCV) and 4–5 SV-like vesicles in single sections, and the average size of these AZ transport aggregates (~ 0.2 µm)  is much smaller than the SV membrane protein transport aggregates reported here, which often exceeds 1 µm in length (Additional File 3). Although LM immunolabeling showed partial colocalization of SV and AZ transport cargos [3, 28], the bulk of the SV membrane proteins has to be transported via the much larger sized SV membrane protein transport aggregates due to the sheer abundance of SV proteins contained in these aggregates.
Notably, DCVs are consistently present in AZ transport aggregates, and AZ proteins like Bassoon and Piccolo are associated with the outside of the DCV membrane . It has been proposed that a nascent presynaptic active zone can be formed by the exocytosis of a few DCV  or AZ transport aggregate . It is likely that the exocytosis of these DCVs would deposit the externally associated AZ material onto the cytosolic side of the plasma membrane, forming an AZ-like structure. Whether such AZ-like structures precede dendritic contact is still unresolved. If so, such “orphan” active zones would have Bassoon or Piccolo-labeled dark material localized to the cytoplasmic side of axonal plasma membrane without an apposed dendritic element. No such “orphan” AZ-like structures were seen in young axons 3–6 DIV by EM examination , but they could exist in cultures older than 10 DIV, where Bassoon-labeled “orphan” punctas are present by LM evidence . Finally, many more DCVs are present in developing than in mature axons [3, 5, 7, 21], and multiple DCVs are sometimes seen at nascent presynaptic terminals  but rarely in mature ones . The depletion of DCVs in mature axons suggests that DCVs are exocytosed during development, and could possibly play a role in synaptogenesis .
Interestingly, multivesicular body (MVB), a vacuole of the late endosome category , was frequently seen in close association with the SV protein transport aggregate. This observation is consistent with LM observations on axons from young hippocampal cultures that ~85% of anterogradely transported SV punctas colocalize with lysosome-related punctas . The lysosome-related marker used in that study is Lamp1, which labels MVBs even before their fusion with lysosomes . Thus, the MVB seen in the present study near SV membrane transport aggregates may represent the Lamp1-labeled “lysosome-related vesicles” . In that study, loss of the lysosomal kinesin adaptor led to accumulation of SV and AZ proteins in the soma and a decrease of these proteins in the presynaptic sites, suggesting that a lysosome-related organelle may be involved in presynaptic biogenesis . The present finding that these MVBs did not labeled for SV proteins suggests that SV proteins may not traffic through these MVBs.