Although the clathrin-coated pits accumulating in the DKO had a size similar to that of synaptic vesicles, they were less densely packed than synaptic vesicles (Figure 6B versus Figure 6C). Furthermore, the overall loss of synaptic vesicles was not fully accounted for by the increase in clathrin-coated pits (Figure 6F), suggesting that synaptic vesicle membranes might be partially trapped within the axonal plasma membrane outside of pits. Such a possibility agrees with the increased steady-state plasma membrane abundance of the vGlut1-pHlourin reporter in DKO neurons (Figure 4A). As a result, immunofluorescence for intrinsic membrane proteins

of synaptic vesicles (synaptophysin, synaptobrevin, synaptotagmin, and SV2), which are expected to be enriched in these pits, was less punctate in DKO Selleckchem MEK inhibitor nerve terminals than in control

nerve terminals where the puncta correspond to abundant and highly clustered synaptic vesicles (Figure 7A and data not shown). However, not surprisingly, given the loss of synaptic vesicles, the most striking change was observed for synapsin 1 and Rab3a, two peripheral proteins of synaptic vesicles (De Camilli et al., 1990 and Fischer von Mollard et al., VX-809 concentration 1990) that dissociate from the vesicle prior to, or in parallel with, exocytosis and then reassociate with newly reformed synaptic vesicles once the endocytic/recycling journey is completed (Chi et al., 2001, Giovedi et al., 2004 and Star et al., 2005). Immunoreactivities for these proteins lost their normally highly punctate enrichment within presynaptic terminals and became more diffusely spread out along the axonal length (Figure 7A). Biochemical analysis of the subcellular localization

of synaptic vesicle proteins (synaptotagmin 1 and synaptophysin) by a cell surface biotinylation-based strategy Edoxaban confirmed an increase in their plasma membrane levels (Figure 7B). The ultrastructural changes of many DKO nerve terminals described above (Figure 6) revealed a near-complete switch from a “secretion-ready mode” to an “endocytic mode,” where the large clusters of synaptic vesicles, which represent the defining morphological feature of synapses, are replaced by a massive accumulation of clathrin-coated pits. We asked whether these dramatic changes are reversible upon silencing of electrical activity (Ferguson et al., 2007). Neurons were first allowed to differentiate over 14 days in culture so that they would exhibit a strong accumulation of endocytic intermediates, and then exposed to tetrodotoxin (TTX; 1μM, overnight) to silence neuronal network activity. After TTX treatment, immunofluorescence for α-adaptin (and other clathrin coat components) became diffuse (Figures 8A–8C) and electron microscopy showed a reduction of clathrin-coated pit number (Figures 8D–8F).