Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca2+ imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions.

GCaMP imaging is widely employed for investigating neuronal Ca2+ dynamics. The Drosophila larval neuromuscular junction (NMJ) consists of three distinct types of motor terminals (type Ib, Is and II). We investigated whether variability in synaptic bouton sizes and GCaMP expression levels confound interpretations of GCaMP readouts, in inferring the intrinsic Ca2+ handling properties among these functionally distinct synapses. Analysis of large data sets accumulated over years established the wide ranges of bouton sizes and GCaMP baseline fluorescence, with large overlaps among synaptic categories. We showed that bouton size and GCaMP baseline fluorescence were not confounding factors in determining the characteristic frequency responses among type Ib, Is and II synapses. More importantly, the drastic phenotypes that hyperexcitability mutations manifest preferentially in particular synaptic categories, were not obscured by bouton heterogeneity in physical size and GCaMP expression level. Our data enabled an extensive analysis of the distal-proximal gradient of GCaMP responses upon genetic and pharmacological manipulations. The results illustrate the conditions that disrupt or enhance the distal-proximal gradients. For example, stimulus frequencies just above the threshold level produced the steepest gradient in low Ca2+ (0.1 mM) saline, while supra-threshold stimulation flattened the gradient. Moreover, membrane hyperexcitability mutations (eag1 Sh120 and parabss1) and mitochondrial inhibition by dinitrophenol (DNP) disrupted the gradient. However, a novel distal-proximal gradient of decay kinetics appeared after long-term DNP incubation. We performed focal recording to assess the failure rates in transmission at low Ca2+ levels, which yielded indications of a mild distal-proximal gradient in release probability.