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

Abstract

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 para^bss1) 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.

Keywords: Ca2+ clearance; GCaMP baseline fluorescence; Proximal-distal gradient; bouton size; facilitation; frequency response; ion channels; mitochondria; octopamine; phasic; tonic; type II synapses; type Ib; type Is.