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GCaMP is an optogenetic Ca 2+ sensor widely used for monitoring neuronal activities but the precise physiological implications of GCaMP signals remain to be further delineated among functionally distinct synapses. The Drosophila neuromuscular junction (NMJ), a powerful genetic system for studying synaptic function and plasticity, consists of tonic and phasic glutamatergic and modulatory aminergic motor terminals of distinct properties. We report a first simultaneous imaging and electric recording study to directly contrast the frequency characteristics of GCaMP signals of the three synapses for physiological implications. Different GCaMP variants were applied in genetic and pharmacological perturbation experiments to examine the Ca 2+ influx and clearance processes underlying the GCaMP signal. Distinct mutational and drug effects on GCaMP signals indicate differential roles of Na + and K + channels, encoded by genes including paralytic ( para ), Shaker ( Sh ), Shab , and ether-a-go-go ( eag ), in excitability control of different motor terminals. Moreover, the Ca 2+ handling properties reflected by the characteristic frequency dependence of the synaptic GCaMP signals were determined to a large extent by differential capacity of mitochondria-powered Ca 2+ clearance mechanisms. Simultaneous focal recordings of synaptic activities further revealed that GCaMPs were ineffective in tracking the rapid dynamics of Ca 2+ influx that triggers transmitter release, especially during low-frequency activities, but more adequately reflected cytosolic residual Ca 2+ accumulation, a major factor governing activity-dependent synaptic plasticity. These results highlight the vast range of GCaMP response patterns in functionally distinct synaptic types and provide relevant information for establishing basic guidelines for the physiological interpretations of presynaptic GCaMP signals from in situ imaging studies.

eneuro

Unraveling Synaptic GCaMP Signals: Differential Excitability and Clearance Mechanisms Underlying Distinct Ca<sup>2+</sup>Dynamics in Tonic and Phasic Excitatory, and Aminergic Modulatory Motor Terminals in<i>Drosophila</i>

Unraveling Synaptic GCaMP Signals: Differential Excitability and Clearance Mechanisms Underlying Distinct Ca2+Dynamics in Tonic and Phasic Excitatory, and Aminergic Modulatory Motor Terminals inDrosophila

Unraveling Synaptic GCaMP Signals: Differential Excitability and Clearance Mechanisms Underlying Distinct Ca²⁺Dynamics in Tonic and Phasic Excitatory, and Aminergic Modulatory Motor Terminals inDrosophila