Optical measurements of exo- and endocytosis in neurons provide useful information of presynaptic function. In particular, a proton-quenchable variant of green fluorescent protein (pHluorin), tagged to the luminal domain of synaptic vesicle proteins, is a valuable tool to monitor the synaptic vesicle cycle.

Aim: 

To generate transgenic mice expressing synaptophysin-pHluorin (syphy) to monitor with high resolution exo-endocytosis in real time.

Methods, Results and Conclusion: 

We have generated two transgenic Syn-Synaptophysin-pHluorin (sypHy) founder lines (L1&L2).Both, showed specific positive GFP immunohistochemical staining distributed specifically in different areas of the CNS. L1 showed expression in substantia nigra, superficial gray layer of superior colliculus, retrosplenial granular cortex, hippocampus, striatum, globus pallidus and some thalamic nuclei. L2 showed expression in the inferior and superior colliculus, dentate gyrus of hippocampus, subiculum, lateral entorhinal cortex, and thalamic nuclei. Immunostaining of hippocampal neurons in culture from L1 & L2 mice showed spotty GFP expression in the soma, without co-localization with SV2 which marked synaptic vesicles. Functional analysis of hippocampal neurons in culture revealed that, upon minimal electrical stimulation, the fluorescence of somatic spots increased transiently. This response is consistent with rapid and local exo-endocytosis of a single DCV, i.e. kiss-and-run. Upon sustained stimulation, DVCs fused permanently with the plasma membrane as no endocytosis was observed. These results clearly two modes of endocytosis of DCVs in CNS neurons in culture.