Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons

Abstract

Synaptic transmission from a neuron to its target cells occurs via neurotransmitter release from dozens to thousands of presynaptic release sites whose strength and plasticity can vary considerably. We report an in vivo imaging method that monitors real-time synaptic transmission simultaneously at many release sites with quantal resolution. We applied this method to the model glutamatergic system of the Drosophila melanogaster larval neuromuscular junction. We find that, under basal conditions, about half of release sites have a very low release probability, but these are interspersed with sites with as much as a 50-fold higher probability. Paired-pulse stimulation depresses high-probability sites, facilitates low-probability sites, and recruits previously silent sites. Mutation of the small GTPase Rab3 substantially increases release probability but still leaves about half of the sites silent. Our findings suggest that basal synaptic strength and short-term plasticity are regulated at the level of release probability at individual sites.

Oral Sessions

“…In brp KO mutants, this gradient dissolves confirming the strong coherence between the presence of BRP and synaptic strength (0.52 ± 0.05 nA, 1.44 ± 0.06 ms vs. 0.50 ± 0.07 nA, 1.56 ± 0.08 ms). These data are in accordance with previous findings that synaptic strength is regulated at every single release site (Peled & Isacoff, 2011). To clarify the impact of AZ assembly on release we performed recordings in brp KO mutants with reduced levels of Synaptotagmin (Syt), a vesicle protein essential for sensing Ca 2+ (Südhof 2004).…”

Transmission, Development, and Plasticity of Synapses

“…These observations indicate single vesicle fusion at individual AZs is the primary mechanism for evoked release for isolated action potentials. Similar to the nonuniform nature of release probability per AZ for spontaneous fusion, evoked release probabilities were highly variable across the population as well (Figure 3) (Peled and Isacoff 2011;Melom et al 2013). Evoked release probabilities varied from 0.01 to 0.55 across individual AZs, with an average probability of 0.04.…”

“…Each AZ is spatially separated and opposed by a distinct cluster of ionotropic GluRs. The tight coupling of release sites and clustered receptors allows imaging of single SV fusion events at individual release sites by measuring Ca 2+ influx through postsynaptic GluRs following neurotransmitter release (Desai and Lnenicka 2011;Peled and Isacoff 2011;Melom et al 2013;Peled et al 2014). This allows the generation of release probability maps for individual release sites (Figure 3), a technique that has not been achieved at synaptic connections in other model systems.…”

“…Two genetically encoded Ca 2+ sensors have proven useful in this area, including a myristoylated (membrane-tethered) GCaMP (Melom et al 2013) and a transmembrane tethered GCaMP containing the Shaker K + channel PDZ interaction domain (Peled and Isacoff 2011). By concentrating these sensors at the postsynaptic plasma membrane, one can observe robust and spatially segregated signals for Ca 2+ influx through GluRs that occurs following SV fusion.…”

“…AZs are readily identifiable by electron microscopy (EM) as electron-dense T-bar structures (Feeney et al 1998) (Figure 1A). The precise matching of AZ to GluR cluster has allowed optical imaging of synaptic vesicle (SV) release at individual release sites at the fly NMJ (Peled and Isacoff 2011;Melom et al 2013), yielding an experimental system that offers unprecedented tools for analyzing how synaptic transmission is organized and regulated at individual AZs.…”