Motojiro Yoshihara

An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.

In this study, we tested a hypothesis that activation of calcineurin, Ca2+/calmodulin-dependent protein phosphatase 2B, is an initiating signal for synaptic vesicle endocytosis. We examined effects of calcineurin inhibitors, cyclosporin A or FK506 and calmodulin inhibitors on stimulus-induced FM1-43 uptake into nerve terminals of Drosophila larvae. Fluorescent FM1-43 labels recycling synaptic vesicles in nerve terminals. Pretreatment with cyclosporin A (5-40 microM) or with FK506 (5-10 microM) enhanced FM1-43 uptake induced by high (60 mM) K+ in a dose-dependent manner. The effect required some preincubation time of about 10 min. The nerve terminals loaded with FM1-43 were destained by electrical nerve stimulation in the cyclosporin A-pretreated preparations, confirming that FM1-43 was taken up into synaptic vesicles. Pretreatment with rapamycin (2 or 20 microM), a structural analog of FK506 which has no effect on calcineurin, or calyculin A (0.3-50 nM), an inhibitor of protein phosphatase 1 and 2A, had no detectable effect on FM1-43 uptake. On the other hand, pretreatment with trifluoperazine (1-50 microM) or with phenoxybenzamine (100 microM), inhibitors of calmodulin, enhanced FM1-43 uptake. Since endocytosis is coupled with exocytosis, it is possible that the enhancement of FM1-43 uptake results from facilitation of exocytosis. However, the frequency of spontaneous junctional potentials and the mean amplitude of evoked potentials did not change after the cyclosporin A treatment, suggesting that the exocytosis process was not significantly affected by the drug. Furthermore, we can temporally separate synaptic vesicle exocytosis and endocytosis in a Drosophila mutant, shibire (shi(ts1)). By taking advantage of this mutation, we showed that cyclosporin A and trifluoperazine enhanced synaptic vesicle recycling by directly acting on the endocytotic process. Present results are not compatible with the hypothesis, but suggest that calcineurin inhibits synaptic vesicle recycling.

Ultrastructural Correlates of Neuromuscular Junction Development

Publisher Summary The larval and late embryonic neuromuscular junctions found on the abdominal muscles of Drosophila are a very useful experimental model system for examining the normal developmental processes underlying synapse formation and for examining the effects of lethal or semi-lethal mutations producing defects in proteins important to synaptic function. However, because some of the defects may be subtle, involving only quantitative changes in the sizes or number of pre- and postsynaptic organelles, it has been important to characterize the normal ultrastructure of the different types of nerve terminals that innervate these muscle fibers and to characterize the morphological responses that the muscle fiber makes to the presence of each of them in turn. Each of the abdominal muscle fibers is innervated by a unique combination of the subtypes of motor and neuromodulatory neurons. The secondary motor nerve branch containing their axons typically contacts each muscle fiber at a particular spot, and the type I terminals then give rise to a fairly consistent number and placement of branches that spread out from this point on each muscle fiber. Other terminal types diverge in a less consistent pattern. This region, referred to as the nerve entry point, is thus interesting from the point of view that it is the target for the ingrowing axons during development, as well as the point from which their terminals must disassociate from each other and form a specific branch pattern on the muscle fiber. The group of terminals from the various motor and neuromodulatory neurons that innervate each fiber is referred to as the junctional aggregate.

Neuromuscular synaptic transmission in neuronal-synaptobrevin null mutants

Institute for Behavioral Sciences, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi 3 7 1-85 11 We examined effects of activation of metabotropic glutamate receptors (mGluRs) on synaptic transmission at the neuromuscular synapse of newly hatched Drosophila larvae, using the patch-clamp technique. In the absence of external Ca 2+, the frequency of miniature synaptic currents (mSCs) was low in the resting state, a few/min and transiently increased to about 30/min upon puff-application of 100 m of glutamate without affecting the mean amplitude. The similar effects were evoked by mGluR agonists, (S)-4C3HPG and DCG-IV, but not by ionotropic glutamate receptor agonists, NMDA, AMPA, or kainate. These effects of glutamate or agonists were blocked by an mGluR antagonist, MCCG-I. In the presence of external Ca2+, tetanic stimulation (10 Hz, 12 s) facilitated synaptic transmission during tetanus (facilitation ) and after tetanus (post-tetanic potentiation). MCCG-I reduced facilitation and blocked post-tetanic potentiation. Thus, it appears that during tetanic stimulation indigenous glutamate released activates presynap tic mGluRs and facilitates synaptic transmission.