Simultaneous GCaMP imaging and focal recording of tonic and phasic synapses: Probing short-term plasticity within a defined microenvironment

Key point summary

• We developed a protocol for stable simultaneous focal recording and GCaMP imaging on identified motor synaptic boutons in the Drosophila neuromuscular preparation with minimized muscular movements in extended concentration ranges of extracellular Ca ²⁺ and Sr ²⁺ .

• This approach directly demonstrated temporal correlation between the dynamics of cytosolic residual Ca ²⁺ and activity-dependent synaptic plasticity in tonic and phasic synapses.

• Our data demonstrated presynaptic GCaMP signals markedly lagged behind and poorly correlated with the concurrent transmitter release, but reliably indicated the immediate states of short-term plasticity. GCaMP’s physical-chemical properties allow information extraction on cytosolic Ca ²⁺ levels during facilitation and depression phases.

• The decay phase of GCaMP signal often coincided with lingering vesicular releases after stimulation, more pronounced in phasic than tonic synapses and exaggerated in Sr ²⁺ -containing saline. Lingering releases were coupled with the tendency of asynchronous transmission.

GCaMP fluorescence has been widely used to monitor intracellular Ca ²⁺ . 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 Ca ²⁺ 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 Ca ²⁺ or Sr ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ with Sr ²⁺ 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.