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.

GCaMP成像已广泛用于研究神经元Ca ²⁺动力学。在果蝇幼虫的神经肌肉接头（NMJ）由三种不同类型的电动机端子（Ib型，是和II）的。我们在推断内在Ca ^{2+的基础上}，研究了突触钮扣大小和GCaMP表达水平的可变性是否混淆了GCaMP读数的解释。处理这些功能上不同的突触之间的特性。多年来积累的大量数据集的分析建立了宽范围的钮扣大小和GCaMP基线荧光，在突触类别之间有很大的重叠。我们表明，在确定Ib型，Is型和II型突触之间的特征频率响应时，钮扣大小和GCaMP基线荧光不是混淆因素。更重要的是，高兴奋性突变优先出现在特定的突触类别中的剧烈表型，在物理尺寸和GCaMP表达水平上都不会被布顿异质性所掩盖。我们的数据使得能够通过遗传和药理学操作对GCaMP反应的远端-近端梯度进行广泛的分析。结果说明了破坏或增强远端至近端梯度的情况。例如，刚好在阈值水平以上的刺激频率在低Ca下产生了最陡峭的梯度²⁺（0.1 mM）盐水，而超阈值刺激使梯度变平。此外，膜超兴奋性突变（例如¹ sh ¹²⁰和para ^bss1）和二硝基苯酚（DNP）的线粒体抑制作用破坏了梯度。但是，长期DNP孵育后，出现了新的远端-近端衰减动力学梯度。我们进行了焦点记录，以评估在低Ca ²⁺水平下传播的失败率，这表明释放概率有轻微的远端至近端梯度。