PMC

Quantification of Dlg width. a Anti-Dlg was used to immuno-label the SSR and images were analyzed with ImageJ software. An octagonal overlay was placed on the image and a line tool was used to quantify the average length of eight spokes, producing the average width of Dlg fluorescence for a single bouton type. b The quantification of Dlg width for each genotype, where each bar represents the mean ± SEM. n = 15 boutons for each genotype (One-way ANOVA Bonferroni post test, not significant n.s., **p < 0.001; ***p < 0.0001)

Electrophysiological analysis of spontaneous miniature potential (mEJP) amplitudes of all genotypes. mEJPs were recorded en masse from muscle abdominal segments A3–A5 of muscle 4 for all genotypes, except for genotypes Ral ^G0501 /y and Ral ^G0501 /+, where recordings were taken from muscle 6. Data is presented as the mean ± SEM amplitude of 50 mEJP for 10 larvae of each genotype (n = 10) (One-way ANOVA Bonferroni post test, there are no significant differences between groups)

Simultaneous extracellular and intracellular recordings. a Examples of extracellular (Ext) recordings in which transmitter release produces a corresponding postsynaptic response detected by the intracellular (Int) electrode. b Example traces with missing events. An extracellular trace is shown where there is a signal detected (Ext), but with the absence of an intracellular (Int) response (no mEJP) in the intracellular trace. Arrows indicate the presence of an extracellular event, but a missing intracellular event. c Muscle 4 of MHC::mCD8-GFP-Sh larvae under transmitted light microscope showing extracellular (single arrowhead) and intracellular microelectrode (double arrowhead) placement. d Same NMJ showing the fluorescence of CD8-Sh-GFP and how boutons were selected. e Cartoon representation of muscle, bouton and electrode placement

Influence of passive membrane properties on mEJPs. a The 20–80 % rise time of identified mEJPs was analyzed from each of the indicated genotypes and plotted against the measured SSR width. The linear regression line shows a positive slope of 2.4 ms/µm Dlg width with an r² of 0.64. The slope is statistically different from zero. b The amplitude of mEJPs collected from identified OreR Type 1b (973 mEJPs) and Type 1s (935 mEJPs) boutons were analyzed. The bars represent the mean ± SEM amplitude for each bouton type, with a significant difference between the two (Mann–Whitney test, p < 0.0001). c Cumulative frequency histogram showing the distribution of mEJP amplitudes for each bouton type. mEJPs from Type Ib boutons are more frequently smaller than from Type 1s

The relationship between missing events and SSR complexity. a The proportion of missing quanta from indicated genotype and bouton type. Each bar represents the mean ± SEM missing intracellular spontaneous miniature potential (mEJP) as a percentage. 50 extracellular events recorded from 20 Type 1b boutons of each genotype (n = 20), as well as 50 extracellular events recorded from 20 Type 1s boutons of of the OreR and MHC::mCD8-GFP-Sh genotype (n = 20), for a total of 1000 events analyzed per data point (One-way ANOVA, Bonferroni post tests for relevant genetic comparisons, not significant n.s., ***p < 0.0001). b The percent of missing events for individual bouton recordings of Type 1b and Type 1s OreR and MHC::mCD8-GFP-Sh larvae. Each point represents the percent of missing events for an individual bouton analyzed from 50 intracellular events of 20 boutons for each genotype (n = 20). Error bars represent the mean ± SEM (One-way ANOVA, Bonferroni post test, **p < 0.001). c The relationship between percent missing events and SSR size is shown in where each point represents the mean ± SEM of percent missing events and SSR complexity (using Dlg width (μm)). The linear regression line shows a positive slope of 10.4 % missing events per micron Dlg width and with an r² value of 0.8. The slope is statistically different from zero