Different Modes of Synaptic Vesicle Recycling un Synaptophysin-pHluorin Transgenic Mouse Motor Nerve Terminals

Abstract

In the present work, a new tool for studying synaptic vesicle exo- and end ocytosis in real time has been developed (the SypHy mouse). By combining live imaging, electrophysiological recordings and immunostaining in SypHy mice we have investigated the functional and structural organization of a vertebrate model synapse, the neuromuscular junction (NMJ). The adult vertebrate NMJ is a highly reliable synapse that must exocytose a large number of vesicles with each stimulus to guarantee effective excitation of the postsynaptic muscle fiber. To maintain effective synaptic transmission, the motor nerve terminal must be able to reuse vesicles by subsequent endocytosis. Diverse modes of endocytosis seem to coexist in the same motor terminal although their relative contribution and regulation mechanisms are not completely clear yet. In the first part of this work, we used dynasore, a blocker of the GTPase activity of dynamin 1/2, to investigate the contribution of dynamin in endocytosis at the presynaptic terminal, during and following high frequency stimulation trains. If endocytosis during stimulation is dynamin 1/2-dependent, SypHy would remain in the plasma membrane and the fluorescence would be larger in the presence of the drug. On the other hand, if dynamin 1/2 is activated after the stimulus, slowness of the signal is expected. Our experiments suggest that dynamin 1/2 only modestly, and in a time-dependent manner, participates in endocytosis during repetitive stimulation. After stimulation, however, dynamin 1/2 is rapidly activated. In addition, we demonstrated by immunolabeling that dynamin 1 and dynamin 3 are co-expressed in the motor nerve terminals, suggesting that dynamin 1/2-independent mechanisms also exist in this synapse. In the second part of this work, the role of calcineurin on exo-/endocytosis was investigated. We found that calcineurin is essential for both exo- and endocytosis in a subset of motor nerve terminals. In the presence of FK506 or CsA, two calcineurin specific inhibitors, end-plate potentials and fluorescence signals were affected in a dose-dependent manner. Finally, in the third part of this work, we studied the structural organization of active zones (AZs) at the mouse motor nerve terminal in adult and during the postnatal maturation period. We used quantitative confocal microscopy of AZs immunostained with fluorescently tagged antibodies to bassoon and piccolo, two scaffolding proteins of the AZ. We found that the number of AZs increases exponentially from postnatal day two to adulthood but the density of AZs remains almost constant during the maturation process.