The synaptic vesicle cycle is mediated by a molecular machinery that allows nerve terminals to operate continuously. Cysteine String Protein-alpha (CSP-alpha) is a synaptic vesicle protein that prevents neurodegeneration of presynaptic terminals as a mechanism to overcome the use-dependent damage of proteins. Presumably, CSP-alpha is a co-chaperone that rescue proteins aged by the effect of maintained synaptic activity. CSP-KO mice show a strong neurodegenerative phenotype and early lethality. We are interested in understanding which steps of the synaptic vesicle cycle are specially sensitive to the synaptic use-dependent stress. We have generated Thy-1 transgenic mice specifically express in central and peripheral neurons the protein SynaptopHluorin, a pH-sensitive GFP fused to the luminal part of synaptobrevin/VAMP2. Then, we have crossed both lines to obtain CSP-KO mice that express Synaptophluorin. Those mice show the same phenotype as CSP mutants. We have imaged changes in synaptopHluorin fluorescence due to exo- and endocytosis at the neuromuscular junction in CSP-KO mice. The approach is sensitive enough to detect that evoked fluorescence responses are remarkably diminished in KO mice compared to WT. The phenotype appears after the second week of life and it is nearly indistinguishable in younger mice. We are currently investigating alterations of synaptic vesicle cycle at age windows where genuine functional changes are not masked by strong structural neurodegeneration of nerve terminals. Supported by: Human Frontiers Science Program and Spanish Ministry of Education and Science (BFU2005-08130, Juan de la Cierva and FPU Programs).