AUTHOR=Paul Mila M. , Pauli Martin , Ehmann Nadine , Hallermann Stefan , Sauer Markus , Kittel Robert J. , Heckmann Manfred 

TITLE=Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=9

YEAR=2015

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2015.00029

DOI=10.3389/fncel.2015.00029

ISSN=1662-5102

ABSTRACT=<p>The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at <italic>Drosophila melanogaster</italic> neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in <italic>brp</italic> null mutants (<italic>brp<sup>69</sup></italic>). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at <italic>brp<sup>69</sup></italic> AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (<italic>syt<sup>KD</sup></italic>) in wildtype (wt), <italic>brp<sup>69</sup></italic> and <italic>rab3</italic> null mutants (<italic>rab3<sup>rup</sup></italic>), where Brp is concentrated at a small number of AZs. At wt and <italic>rab3<sup>rup</sup></italic> synapses, <italic>syt<sup>KD</sup></italic> lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, <italic>syt<sup>KD</sup></italic> did not alter EPSC amplitude at <italic>brp<sup>69</sup></italic> synapses, but shortened delay and rise time. In fact, following <italic>syt<sup>KD</sup></italic>, these kinetic properties were strikingly similar in wt and <italic>brp<sup>69</sup></italic>, which supports the notion that Syt protracts release at <italic>brp<sup>69</sup></italic>synapses. To gain insight into this surprising role of Syt at <italic>brp<sup>69</sup></italic> AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after <italic>syt<sup>KD</sup></italic>. Notably, <italic>syt<sup>KD</sup></italic> boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via <italic>d</italic>STORM revealed that <italic>syt<sup>KD</sup></italic> increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of <italic>Drosophila</italic> AZs requires concerted action of Brp and Syt.</p>