Depolarization-induced Ca2+ entry preferentially evokes release of large quanta in the developing Xenopus neuromuscular junction.

The amplitude histogram of spontaneously occurring miniature synaptic currents (mSCs) is skewed positively at developing Xenopus neuromuscular synapses formed in culture. To test whether the quantal size of nerve-evoked quanta (eSCs) distributes similarly, we compared the amplitude histogram of single quantum eSCs in low external Ca(2+) with that of mSCs and found that nerve stimulation preferentially released large quanta. Depolarization of presynaptic terminals by elevating [K(+)] in the external solution or by direct injection of current through a patch pipette increased the mSC frequency and preferentially, but not exclusively, evoked the release of large quanta, resulting in a second broad peak in the amplitude histogram. Formation of the second peak under these conditions was blocked by the N-type Ca(2+) channel blocker, ω-conotoxin GVIA. In contrast, when the mSC frequency was elevated by thapsigargin- or caffeine-induced mobilization of internal Ca(2+), formation of the second peak did not occur. We conclude that the second peak in the amplitude histogram is generated by Ca(2+) influx through N-type Ca(2+) channels, causing a local elevation of internal Ca(2+). The mSC amplitude in the positively skewed portion of the histogram varied over a wide range. A competitive blocker of acetylcholine (ACh) receptors, d-tubocurarine, reduced the amplitude of smaller mSCs in this range relatively more than that of larger mSCs, suggesting that this variation in the mSC amplitude is due to variable amounts of ACh released from synaptic vesicles. We suggest that Ca(2+) influx through N-type Ca(2+) channels preferentially induces release of vesicles with large ACh content.

Roles of SNARE proteins and synaptotagmin I in synaptic transmission: studies at the Drosophila neuromuscular synapse.

The roles of SNARE proteins, i.e. neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx 1A), and Synaptotagmin I (Syt I) in synaptic transmission have been studied in situ using mutant embryos or larvae that lack these molecules or have alterations in them. Because of the ease of genetic manipulation, the Drosophila neuromuscular synapse is widely used for these studies. The functional properties of synaptic transmission have been studied in mutant embryos using the patch-clamp technique, and in larvae by recording with microelectrodes. A major vesicular membrane protein, n-Syb, is indispensable for nerve-evoked synaptic transmission. Spontaneous synaptic currents (minis), however, are present even in embryos totally lacking n-Syb (N-SYB). Furthermore, Ca(2+)-independent enhancement of mini frequency induced by hypertonic sucrose solutions (hypertonicity response) is totally absent in N-SYB. Embryos that have defects in SNAP-25 (SNAP-25) have similar but milder phenotypes than N-SYB. The phenotype in synaptic transmission was most severe in the synapse lacking Syx 1A. Neither nerve-evoked synaptic currents nor minis occur in embryos lacking Syx 1A (SYX 1A). No hypertonicity response was observed in them. Syt I binds Ca(2+) in vitro and probably serves as a Ca(2+) sensor for nerve-evoked synaptic transmission, since nerve-evoked synaptic currents were greatly reduced in embryos lacking Syt I (SYT I). Also, Syt I has a role in vesicle recycling. Interestingly, the Ca(2+)-independent hypertonicity response is also greatly reduced in SYT I. Minis persist in mutant embryos lacking any of these proteins (n-Syb, SNAP-25 and Syt I), except Syx, suggesting that minis have a distinct fusion mechanism from that for fast and synchronized release. It appears that these SNARE proteins and Syt I are coordinated for fast vesicle fusion. Minis, on the other hand, do not require SNARE complex nor Syt I, but Syx is absolutely required for vesicle fusion. The SNARE complex and Syt I are indispensable for the hypertonicity response. None of these molecules seem to serve for selective docking of synaptic vesicles to the release site. For further studies on synaptic transmission, the Drosophila neuromuscular synapse will continue to be a useful model.

Two synaptic vesicle pools, vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction.

Drosophila neuromuscular junctions ( D NMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K(+). The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca(2+). Mobilization of internal Ca(2+) stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and D NMJs are suitable for further studies.