Simultaneous GCaMP imaging and focal recording of tonic and phasic synapses: Probing short-term plasticity within a defined microenvironment
GCaMP fluorescence has been widely used to monitor intracellular Ca2+. However, the physiological significance of the GCaMP signal in presynaptic terminals remains to be further elucidated. We investigated how the dynamics of GCaMP signals correlates with the activity dependence of short-term plasticity in synaptic transmission. We devised a local manipulation protocol that minimizes interference from muscular contraction during simultaneous Ca2+ imaging and focal recording at the Drosophila larval neuromuscular junction (NMJ), where the tonic and phasic excitatory synapses can be compared side-by-side. By confining the local ionic microenvironment, this protocol enabled stable measurements across extended concentration ranges of Ca2+ or Sr2+ in saline. Compared to tonic synapses, phasic synapses displayed stronger GCaMP signals, along with faster facilitation and more severe depression. Upon repetitive stimulation (40 Hz), facilitation of transmission occurred during or immediately prior to the early rising phase (0.25 s) of the GCaMP signal, which could subsequently convert into a depression phase of transmission decline, most evident during a steeper and longer rise of GCaMP signals in higher Ca2+ saline. Typically, deepest depression occurred when GCaMP signals rose to a plateau. Phasic synapses with stronger GCaMP signal and deeper depression, more often exhibited lingering post-stimulation releases. In both tonic and phasic synapses, replacing Ca2+ with Sr2+ induced extreme asynchronous transmission coupled with post-stimulation lingering releases during the decay of GCaMP signals. Further applications of this focal recording-local manipulation protocol may help to probe additional mechanisms underlying synaptic transmission and plasticity.