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...

Synaptic vesicle recycling involves AP‐2/clathrin‐mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue‐specific AP‐1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP‐1–σ1A complex mediates protein sorting between the trans‐Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B‐deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B‐deficient mice have reduced motor coordination and severely impaired long‐term spatial memory. These data reveal a molecular mechanism for a severe human X‐chromosome‐linked mental retardation.

A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling.

BackgroundDifferent non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria.ResultsWe have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response.ConclusionLabeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.