Large-scale connectomics requires dense staining of neuronal tissue blocks for electron microscopy (EM). Here we report a large-volume dense en-bloc EM staining protocol that overcomes the staining gradients, which so far substantially limited the reconstructable volumes in three-dimensional (3D) EM. Our protocol provides densely reconstructable tissue blocks from mouse neocortex sized at least 1 mm in diameter. By relaxing the constraints on precise topographic sample targeting, it makes the correlated functional and structural analysis of neuronal circuits realistic.
3 3 a r t I C l e SA main rate-limiting step in synaptic transmission is the retrieval of synaptic vesicles from the presynaptic membrane for further rounds of use. Several modes of synaptic vesicle retrieval have been proposed, but the main pathway is considered to be clathrin-mediated endocytosis 1,2 . This process is relatively slow because after fusion the recycling machinery has to resort different vesicle membrane proteins in the right stoichiometry to generate fusion-competent synaptic vesicles 3 . As a result, this process occurs with a time constant of tens of seconds to minutes 4 . However, to sustain transmission during continuous activity, it was suggested that synaptic vesicles might 'kiss and run' with a time constant of 1-2 s, whereby the vesicles transiently fuse with the membrane without full collapse and hence retain their molecular identity [5][6][7][8][9] .The fast 'kiss and run' recycling mechanism not only would provide a kinetic advantage but would spatially and temporally couple exoand endocytosis. Using a green fluorescent protein (GFP) fused with the coat-forming clathrin light chain, we previously found evidence that during the first 10 s of prolonged stimulation, clathrin is not being recruited from the cytosol to form coated pits, although the rate of endocytosis measured with styryl (FM) dyes is high 3 , suggesting that vesicles during this first phase are either retrieved by a clathrin-independent mechanism (kiss and run) or by preassembled coat structures at the periphery of the active zone.Support for such a 'readily retrievable pool' (RRetP) of preassembled structures came from experiments using fusion constructs of the synaptic vesicle proteins synaptobrevin 2 (Syb2) and synaptotagmin 1 (Syt1) with a pH-sensitive GFP, pHluorin 10 . These studies showed that synaptic vesicles lose their protein complement after fusion, and the molecular identity of synaptic vesicles exocytosed and subsequently endocytosed is not conserved [11][12][13] . On the basis of these observations, we suggested that exocytosis and subsequent endocytosis are uncoupled and that there is a pool of preassembled vesicle proteins on the presynaptic surface that is preferentially retrieved on exocytosis 3,13 . Previous studies using activity-dependent markers in snake neuromuscular terminals have proposed that the accumulation of such probes at the bouton margins upon stimulation might represent endocytic active zones 14,15 . This is in agreement with other ultrastructural and high-resolution microscopy analyses that describe the presence of several synaptic vesicle proteins on the presynaptic membrane of resting synapses 16,17 . Likewise, the first reconstruction of the endocytic time course from electron micrographs of frog neuromuscular junctions quick-frozen at different times after stimulation revealed a first wave of clathrin-mediated endocytosis lasting ~10 s (ref. 18), in line with the notion of a preclustered pool being immediately available for this first wave of endocytosis upon stimulation 13 . In hi...
It is commonly thought that clathrin-mediated endocytosis is the rate-limiting step of synaptic transmission in small CNS boutons with limited capacity for synaptic vesicles, causing short-term depression during high rates of synaptic transmission. Here, we show by analyzing synaptopHluorin fluorescence that 200 action potentials evoke the same cumulative amount of vesicle fusion, irrespective of the frequency of stimulation (5-40 Hz), implying the absence of vesicle reuse, since the method used (alkaline-trapping) measures only first-round exocytosis. After blocking all slow or specifically clathrin-mediated endocytosis, however, the same stimulation patterns cause a rapid stimulation-frequency-dependent release depression. This form of depression does not reflect insufficient vesicle supply, but appears to be the result of slow clearance of vesicular components from the release site. Our findings uncover an important yet overlooked role of endocytic proteins for release site clearance in addition to their well-characterized role in endocytosis itself.
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