Labels for SV integral membrane proteins are localized at the Golgi complex in the soma and sorted into vesicular structures in axon
Dissociated hippocampal neuronal cultures at 3–6 DIV were fixed and labeled with antibodies against four different SV integral membrane proteins: synaptophysin, SV2, VAMP & synaptotagmin. Synaptophysin and SV2 antibodies produced the most consistent and high efficiency labeling under many different fixation conditions, and thus, were illustrated to a greater extent in the present study.
As expected of integral membrane proteins, labels for synaptophysin (Fig. 1a) and SV2 (Fig. 2a) were localized at the Golgi complex . In neuronal somas, labels for both antibodies were also specifically localized at membranous structures of various size and shape, scattered in the cytoplasm as individual entities (arrows in Figs. 1a & 2a). Many of these labeled vesicles/vacuoles became aggregated in the axons (arrows in Figs. 1b & 2b), but not in soma and dendrites. These labeled aggregates are termed as “SV membrane protein transport aggregate” in this paper. The overall size of the labeled aggregates ranged widely from several vesicles/vacuoles (~0.2 µm, arrows in Additional File 2a & b) to often exceeding 1 µm in length (arrows in Figs. 1b & 2b), and sometimes greater than 2 µm (Additional File 3a). Higher mag images of these labeled aggregates showed a mixture of tubular (arrows in Figs. 1c & 2c) and vesicular structures of variable size and shape.
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Notably, clusters of vesicles of uniform size at ~40 nm were also labeled in axons (Fig. 1d; circled area in Fig. 2c). These vesicle clusters resemble synaptic vesicles (SV) clusters in the presynaptic terminals , and are termed as “clusters of SV-like vesicles” here. The number of vesicles in these clusters ranged from 4–30 in single sections, and a few examples from small to larger clusters are illustrated in Fig. 3. Interestingly, clathrin-coated vesicles were often present near these SV-like vesicle clusters (arrows in Fig. 3c), indicating occurrence of endocytoses .
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Serial thin sections demonstrated that some labeled aggregates were clearly not part of synapses, and many axons containing these labeled aggregates did not even come in contact with dendrites (Fig. 4; Additional File 4). Thus, the aggregation of these labeled vesicles/vacuoles was intrinsic to axon and not induced by external contact with dendritic elements. Some of these aggregates consisted mostly of tubular vesicular structures (aggregate “a” in Fig. 4), and others mostly of SV-like vesicles (aggregate “c” in Fig. 4). Interestingly, clathrin-coated vesicles were often seen among both types of labeled aggregates (thick arrows in Fig. 4), indicating active endocytosis near both types. Due to crowding of the vesicles/vacuoles, it is difficult to discern whether clathrin-coated vesicles were specifically labeled. However, on occasion, some coated vesicles appeared to be labeled (arrow in Additional File 4, section #4).
Multivesicular bodies (MVB, open arrows in Fig. 2b; Additional File 2c; Additional File 4, section # 4 & 5), which are classified as late endosome that may be en route to fuse with lysosome , were often present among the labeled aggregates of vesicles/vacuoles. However, the great majority of these MVBs were not labeled for SV integral membrane proteins. Notably, no late stage lysosomes, such as lipofuscin bodies or vacuoles containing multilamellar structures or electron dense material , were observed in the vicinity of labeled SV protein transport aggregates.
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Labels for two other SV integral membrane proteins, VAMP/synaptobrevin and synaptotagmin, were also localized at the Golgi complex (images not shown), and at individual and aggregated vesicles/vacuoles in the axons (Additional File 2c, d). Thus, the four SV integral membrane proteins studied here had similar distribution patterns in soma and in axon. However, the present preembedding immunogold labeling method does not allow double labeling, and thus, cannot determine whether these four proteins are colocalized in the same vesicle/vacuoles. Notably, not all vesicles/vacuoles were labeled even when they were in the vicinity of the labeled aggregates (boxed area in Figs.1b, 2c and Additional File 2).
Labels for SV-associated proteins are cytosolic in soma and become associated with SV clusters in axon
In contrast to labels for SV integral membrane proteins, labels for two SV-associated proteins, synapsin I and -synuclein, were not localized at the Golgi complex (Figs. 5a & 6a) in the soma, but dispersed in cytoplasm, not associated with any membranous structures. The synapsin I antibody used here produced better labeling efficiency than the -synuclein antibody, and thus, generated more detailed observations here.
In mature synapses, labels for synapsin I and -synuclein are localized to clusters of SVs in the presynaptic terminals . Here in young neuronal cultures before robust synaptogenesis occurs, most labels for synapsin I and -synuclein were cytosolic in somas and axons, but became concentrated on clusters of SV-like vesicles in axons (arrow in Figs. 5b & 6b). However, no other membranous structures, such as tubular or pleomorphic vacuoles were conspicuously labeled for synapsin I in the axons. Thus, other than being colocalized in SV-like vesicles, the distribution of labels for SV-associated proteins were very different from those of the SV integral membrane proteins.
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Label for SNAP 25 is sorted to axolemma early in development
SNAP–25, synaptosomal associated protein of 25 kDa, is part of the SNARE complex involved in exocytosis of synaptic vesicles . In mature neurons, label for SNAP–25 is polarized to axon and localized to plasma membrane along the entire axon [19, 20]. Here, in young neuronal cultures, labeling pattern of SANP–25 was compared to those of the SV proteins illustrated above. Label for SNAP–25 was localized at the Golgi complex in soma (Fig. 7a), and became sorted to axolemma (Fig. 7b, c) as early as 4 DIV. In contrast to SV proteins, clusters of SV-like vesicles were clearly not labeled (Fig. 7c).
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Presynaptic terminals of immature synapses contain a full complement of SV and AZ proteins
Nascent synapses were seen as early as 3 DIV (Fig. 8a) and synaptic profiles appeared more frequently in subsequent days. The immature synapses contained fewer number of SV than mature synapses [5, 21], but already showed the characteristic synaptic cleft with a uniform gap at ~20 nm, and the postsynaptic density of variable prominence (Fig. 8). As in the case of mature synapses , the SVs were labeled for both the SV integral membrane proteins (Figs. 8a & b) and SV-associated proteins (Fig. 8c). Thus, even though SV integral membrane proteins and SV-associated proteins were transported via different routes from soma through axons, they ended up in the same final compartment, the SVs, at the presynaptic terminal upon synapse formation. Furthermore, labels for AZ cytomatrix proteins were also localized at active zone in these nascent synapses  (Fig. 8d).
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Dense core vesicles are frequent in developing axons, and contain some but not all SV proteins
Dense core vesicles (DCV) are more frequently seen in young axons than in mature samples, both in animals  and in cell cultures [3, 5, 7]. These DCVs sometimes existed in groups, intermingled with some SV-like vesicles (Fig. 9). Because occurrence of these vesicle mixtures of multiple DCVs and SVs were relatively infrequent compared to the occurrence frequencies of SV or AZ protein transport aggregates, it is difficult to capture them for serial section analysis. Thus, it cannot be determined whether these vesicle mixtures are part of a developing presynaptic specialization or existed in isolation in the absence of dendritic contact. While the SV-like vesicles labeled for all SV proteins, the vesicular membranes of DCV only labeled for some, such as SV2 (Fig. 9a) and synaptotagmin (Fig. 9b), but not for all SV membrane proteins. For example, DCV were mostly negative for synaptophysin (Fig. 9c) or VAMP (image not shown), and labels for AZ cytomatrix proteins were localized to dark material outside of DCV (Figs. 9d) .
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