CCVs are smaller in axons than in soma/dendrite
One of the striking features of synaptic vesicles (SV) is the uniformity of their size [4, 18], and this uniformity is across species. Glutamatergic SVs in mammalian brains are all ~ 40 nm in diameter in mouse, rat, cat and monkey . Examples of SVs from glutamatergic presynaptic terminals of different samples are shown in Fig. 1 (a, d, g). In perfusion-fixed 1-3 month-old mouse brains, the size of CCVs in presynaptic terminals (Fig. 1b1-4) is the same as that of SVs (Fig. 1a) at ~ 40 nm. The same is observed in perfusion-fixed adult rat brains (images not shown). This observation is as expected since SVs result from shedding of clathrin from CCVs [4, 18]. In dissociated rat cultures, the great majority of CCVs were also ~ 40 nm in diameter. However, there were occasional CCVs that were larger at ~70 nm (70.1 ± 1.0, n=11), and they were more prevalent in younger cultures at 3-6 DIV (Fig. 1h3) than at 3 wk in culture (Fig. 1e4). This observation suggests that larger axonal CCV may be associated with developing axons.
Although the “thickness” of the clathrin coat was the same at ~15 nm for axonal and dendritic CCVs, the average diameter of CCVs in neuronal soma and dendrites was conspicuously larger (~90 nm; Figs. 1c, f, and i) than CCVs in presynaptic terminals. The size distributions of somal/dendritic CCVs from the three different experimental materials were plotted into histograms (Fig. 1j). Although the mean diameter of CCVs from perfusion-fixed mouse brains was somewhat smaller than those from dissociated cultures of rat neurons, these differences did not reach statistical significance.
As expected , immunogold labeling of dissociated cultures demonstrated that CCVs in both axons and dendrites specifically labeled for clathrin and AP2, clathrin adaptor protein-2. Examples of dendritic CCVs labeled for clathrin and AP2 are illustrated in Fig. 1f1 and 1f2, respectively, consistent with LM observations that these two proteins co-localize as concentrated puncta representing CCVs . In contrast, label for transferrin receptor (TfR), a constitutively endocytosed membrane protein [1, 2], is present in dendritic CCVs (Fig. 1f3) but absent in axons , indicating differential sorting of cargos between axon and dendrite.
Presence of clustered CCPs on somal/dendritic plasma membrane
Although the majority of CCPs in thin sections of neuronal soma/dendrites were captured as individual pits, clustered CCPs consisting of 2-3 pits within~100 nm of each other were occasionally seen (Fig. 2). This finding is consistent with LM evidence suggesting that “hot spots” of GFP-tagged clathrin puncta support multiple endocytosis events , and that some optically stable endocytic sites can yield several CCVs within minutes .
The appearance of the clathrin coat was affected by specific EM fixation/staining reagents. Osmium tetroxide at a low concentration of 0.2% is enough to make the clathrin coat visible (Fig. 2a, b). Including tannic acid (1%) in the initial fixative along with glutaraldehyde enhanced the darkness of the clathrin coat (Fig. 2c), whereas adding 1% potassium ferrocyanide along with 1% osmium tetroxide in the postfixation reduced the visibility of the clathrin coat (Fig. 2d).
Somal/dendritic CCPs can be juxtaposed to different cellular elements
In perfusion-fixed brains, somal/dendritic CCPs were apposed to various cellular elements, including axons (Fig. 3a), another soma/dendrite (Fig. 3b), or astroglia (Fig. 3c). In dissociated cultures, somal/dendritic CCPs occurred on plasma membrane exposed to culture media without any juxtaposed cellular elements (Additional File 1b, Fig. 2a, b). Thus, formation of CCP in neuronal soma/dendrites appears to be an inherent property of the neuron, not dependent on the juxtaposed cellular elements.
Clathrin patches on multivesicular bodies have a different structural organization than those of CCVs
In addition to CCVs and CCPs, multivesicular body (MVB), an organelle of late endosomal origin , also labeled for clathrin on its limiting membrane. Label for clathrin was concentrated on a patch of dark material attached to the cytoplasmic side of the MVB (Fig. 4a, Additional File 4c). Here in dissociated hippocampal neuronal cultures, MVBs were seen throughout the neuron, like in brains . It is also confirmed here that the dark patches on MVB consisted of two layers (Fig. 4c) at a thickness of ~ 30 nm [22, 23]. These patches labeled for clathrin (Fig. 4a), but not for AP2 (Fig. 4b), findings consistent with those reported in non-neuronal cells . MVBs in astroglia displayed similar features as in neurons (Additional File 4). Notably, the labeling pattern of clathrin on MVB is different from those of CCVs in axons (Fig. 4d, f) or in dendrites (Fig. 4e, g) where assembled clathrin molecules are evenly distributed in a single layer around the entire vesicles, and these CCVs label for both clathrin (Fig. 4d, e) and AP2 (Fig. 4f, g).
Double-layered, clathrin-labeled patches were never seen on any other organelles except on MVB. The length of these clathrin-labeled patches on MVB in a single section was typically ~200 nm, and in some sections could be as long as 300-400 nm (Additional File 4C). The area of such a clathrin-labeled patch is sufficient to support the formation of a CCV. However, no budding of vesicles, either into the lumen of MVB or into the cytoplasm were observed from these patches . Thus, these patches are large enough to be resolved by fluorescence LM as puncta of concentrated clathrin signals, but the present evidence suggests that these patches are not involved in budding of coated vesicles.
Increase of CCP and CCV in presynaptic axon terminals under excitatory conditions
CCPs and CCVs are preferentially located at the periphery of presynaptic active zones of frog neuromuscular junctions , lamprey giant axon terminals , and mouse dissociated cortical cultures . In all three experimental systems, CCPs and CCVs are rarely seen in resting synapses but become more frequent upon stimulation. Here, archived images of perfusion-fixed adult rat and mouse brains [15, 16] were examined to see if the number of CCV and CCP in presynaptic terminals is affected by the activity state of the synapses caused by the particular perfusion fixation conditions.
A 5-8 min delay in perfusion fixation has been shown to trigger activity in neurons [15, 16]. Thus, synapses are under a resting state after “fast” perfusion fixation, and under a stimulated state after “delayed” perfusion fixation, which induces ischemic stress . CCVs and CCPs were consistently more abundant in delayed (Fig. 5b) than in fast perfusion-fixed brains (Fig. 5a), and these CCVs were typically located at the periphery of SV clusters (Fig. 5b). Notably, CCVs outnumber CCPs, perhaps reflecting their respective residence time. When CCVs and CCPs were scored from seven pairs of samples from different regions of the brain, their average number per 100 presynaptic profiles increased by 7.5 fold upon delayed perfusion fixation (Fig. 5e upper panel, Additional File 5A).
In 3 week-old dissociated hippocampal cultures, depolarization with high K+ at 90 mM for 2-3 min causes dispersion and depletion of SVs . These findings are consistent with the idea that high K+-treated synaptic terminals are highly stimulated, resulting in massive exocytosis of SVs. However, conspicuous increase of CCVs and CCPs was only detected in some of the high K+-treated samples. For example, more CCVs and CCPs were observed in high K+-treated samples (Fig. 5d) than in controls (Fig. 5c) in some experiments (exp 1 and 2 in Additional File 5B) but not in others (exp 3 and 4 of Additional File 5B). Bar graphs in lower panel of Fig. 5e represent means of 4 experiments. Since exocytosis takes place in milliseconds and endocytosis requires minutes , It is possible that the formation of CCVs needs more time than the acute 2-3 min of treatment carried out in the present study. Indeed, a previous EM study of dissociated mouse neuronal cultures reported an increase of CCVs in presynaptic terminals upon 10 min of high K+ treatment .
Depolarization induces redistribution of clathrin in presynaptic axon terminals
Distribution of clathrin molecules was studied by pre-embedding immunogold labeling of 3 week-old dissociated hippocampal cultures. Under control conditions, label for clathrin was absent from the active zone and typically concentrated outside of SV clusters (Fig 6a, Additional File 2). Upon depolarization with high K+, label for clathrin became dispersed among the de-clustered SVs (Fig. 6b).
Measurement of density of label for clathrin within 200 nm of the presynaptic membrane showed low labeling densities, ~2-5 particles per µm of active zone (Additional File 6). Upon high K+ treatment, the density increased by ~3.7 fold, on average (Additional File 6). The median distance of clathrin label, averaged from 3 experiments, decreased from 150 nm under control conditions to 88 nm upon high K+ treatment (Additional File 6). Fig. 6c shows histograms of distance measurement from one representative experiment. These results indicate that upon depolarization, more clathrin molecules moved into the measurement area within 200 nm of the presynaptic membrane.
Depolarization induces a decrease of CCPs and CCVs in soma/dendrites
In neuronal soma and dendrites under control conditions, the most striking feature of label for clathrin was the abundant clusters of aggregated labels (arrows and circles in Fig. 7a), which was lacking in axons. Serial section analysis revealed that many such clusters of tightly aggregated clathrin labels are indeed CCVs sectioned at the edge of vesicles (Additional File 3). Thus, each tightly aggregated clathrin labels can be reasonably assumed to represent a CCV. Notably, many clathrin labels also appeared as individual particles representing unassembled clathrin molecules dispersed in the cytoplasm [5, 7, 10].
Upon depolarization with high K+, the tightly aggregated label for clathrin disappeared, and the great majority of clathrin labels appeared as individual particles (Fig. 7b). Likewise, the number of clathrin-labeled CCPs and CCVs near plasma membrane of neuronal soma/dendrites decreased to ~28% of control values (Additional File 7). The disappearing of tightly aggregated clathrin labels in the cytoplasm suggests that clathrin molecules disassembled from CCVs upon depolarization.
Whether depolarization also induced a decrease in clathrin-mediated endocytosis (CME) was tested in another set of experiments where 3 week-old dissociated cultures were fixed with glutaraldehyde for better structural preservation. Plasma membrane of neuronal somas were traced to score the number of CCPs, which were identified by their characteristic coat on the omega figure (Additional File 1b), and which represent bona fide CME. The number of CCPs decreased to ~ 42% of control values upon depolarization (Additional File 8). These results indicate that in addition to increased disassembly of clathrin from CCVs, depolarization also induced a decrease in CME in neuronal soma.
Number of peri-PSD CCP is not significantly affected by depolarization
It has been proposed that there are specialized “endocytic zones” near synapses in spines that may facilitate the internalization of glutamate receptors [5, 6]. However, there are also reports that suggest CCPs near postsynaptic densities (PSD) may not be particularly involved in endocytosis of glutamate receptors . In the present EM study, I defined only CCPs located immediately adjacent to (within 30 nm of) the PSD as being peri-PSD (Fig. 8a), a definition different from the “endocytic zone” reported by previous LM studies which included clathrin puncta within 300 nm of the PSD . Notably, peri-PSD CCP existed in both excitatory (Fig. 8a) and inhibitory (Fig. 8b) synapses. It should also be noted that no CCP was ever detected at the PSD itself [7, 10] or the inhibitory postsynaptic specialization, indicating that clathrin cannot assemble at these specialized postsynaptic junctional membranes, and that the closest site where CME can take place is at these peri-synaptic locations.
The number of peri-PSD pits of glutamatergic excitatory synapses was scored from archived images  of dissociated cultures to see if depolarization induces any change in the occurrence frequency of these peri-PSD pits. The number pooled from 10 experiments did not change (Additional file 9) between control and high K+-treated samples (15.4 vs. 14.6 peri-PSD pits/1000 synaptic profiles, respectively).