Image acquisition of stained preparations and image quantification

Microscopy was performed at the School of Biomedical Sciences Imaging Facility and Queensland Brain Institute Advanced Microscopy Facility. Imaging of larval preparations was predominantly performed on an Olympus FV1000 upright scanning confocal microscope with NA 1.35 60× or NA 1.4 100× oil immersion objectives via photomultiplier tubes at room temperature. Fluoview software (Olympus America Inc.) was used to acquire images taken on the FV1000 system. Imaging was also performed on an Axio Observer Z1 (Zeiss) equipped with a CSU-W1 spinning-disk head (Yokogawa Corporation of America), ORCA-Flash4.0 v2 sCMOS camera (Hamamatsu Photonics), and NA 1.4 100× oil immersion objective. Huygens software (Scientific Volume Imaging) was used to acquire images taken on the spinning-disk system. Optical sectioning and laser settings were kept constant across all image data acquisition sessions for like experiments and depended on the type of experiment being performed. Images were taken from NMJs formed upon muscles 6 and 7 by MN6/7-Ib and MNSNb/d-Is in abdominal segments 3 and 4 (A3 and A4). No more than two NMJs were imaged per larva.

Analysis of all images was done using ImageJ (National Institutes of Health). To measure the number of boutons per NMJ, anti-Syn immunoreactivity was used to label individual boutons, which were manually counted. To measure the maximal cross-sectional area of muscles, a perimeter was traced around the perpendicular muscle cross section from maximal z-stack projections of phalloidin. Cross-sectional muscle area was then calculated from the traced perimeters. To measure bouton and subsynaptic reticulum (SSR) diameters, a line was drawn across the longest axis perpendicular to the main branch of HRP and anti-dlg immunoreactivity from maximal z-stack projections. All boutons on at least one Ib or Is branch per NMJ were measured and averaged to generate representative values. To measure GluRIIA fluorescence intensity, HRP immunostaining was used to generate “synaptic” regions of interest (ROIs) from which raw anti-GluRIIA pixel intensity was measured. Three random regions of similar cross-sectional area that did not intersect with synaptic ROIs were then determined as “nonsynaptic” ROIs and were averaged to determine the background fluorescence. The synaptic GluRIIA raw pixel values were then divided by nonsynaptic pixel values to generate normalized GluRIIA fluorescence values. These results were confirmed by manually measuring the size and average pixel values of at least 20 individual GluRIIA puncta per NMJ for every animal. A similar approach was taken for measuring relative Syt1 immunofluorescence intensity with the exception of having to define and measure puncta, as Syt1 immunoreactivity filled the entire bouton.

Measurement of PLCδPH-mCherry and 2xFYVE-GFP average pixel intensity per bouton was performed using ROIs that were made by drawing perimeters around HRP-positive boutons. For these genotypes, immunostaining was performed to amplify signals of the tags. The same methodology was used to quantify FM4-64FX immunofluorescence intensity, but ROIs were generated based on the FM4-64FX signal directly, instead of from HRP.