To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses.
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We thank all the members of the lab for discussions, M. Jones for providing us electrophysiological data, J.-I. Nakai (RIKEN Brain Science Institute) for providing us the GCaMP2 plasmid and Martin Meyer (King's College London) for providing us the а-tubulin:Gal4/VP16 and 5UAS:EGFP-N1 plasmids. The Virtual Cell simulation environment is supported by a US National Institutes of Health grant P41RR013186 from the National Center for Research Resources. This work was supported by the Medical Research Council and the Wellcome Trust.
Supplementary Figures 1–2, Supplementary Tables 1–2, Supplementary Note, Supplementary Methods (PDF 1716 kb)
Synaptic activity in cultured hippocampal neurons. Real-time movie recorded from rat hippocampal cultured neurons transfected with SyGCaMP2 (corresponding to Fig. 2a,b). The cells were stimulated by field stimulation with a train of 10 AP delivered at 20 Hz (white square in upper left corner). Intrinsic network activity was inhibited using blockers of excitatory synaptic transmission (CNQX and APV). All regions of interest (ROIs) identified, corresponding to active synapses, are displayed. Each ROI shows its averaged relative change in fluorescence, ΔF/F0 (color-coded). Differences in response to the same stimulus between individual synapses can be observed. Scale bar, 20 μm. (MOV 529 kb)
Synaptic activity in a tectal neuron responding to electric field stimulation: imaging in vivo. Real-time movie of SyGCaMP2 signals in tectal neurons in a zebrafish (9 d after fertilization). The whole fish was stimulated by applying an electric field in the pattern shown in Figure 5e (stimulus indicated by white square in upper left corner). ROIs corresponding to 100 synaptic boutons within a single optical plane were monitored (Fig. 5c). Calcium transients of each synapse, plotted in Figure 5e, are expressed as relative change in fluorescence, ΔF/F0 on a pseudo-color scale. Scale bar, 20 μm. (MOV 879 kb)
Synaptic activity in a tectal neuron responding to electric field stimulation: imaging in vivo. Real-time movie of SyGCaMP2 signals in tectal neurons in a zebrafish (9 d after fertilization). The whole fish was stimulated by applying an electric field in the pattern shown in Figure 5e (stimulus indicated by white square in upper left corner). ROIs corresponding to 100 synaptic boutons within a single optical plane were monitored (Fig. 5c). Calcium transients of each synapse, plotted in Figure 5e, are expressed as relative change in fluorescence, ΔF/F0 on a pseudo-color scale. Scale bar, 20 μm. (MOV 369 kb)
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Dreosti, E., Odermatt, B., Dorostkar, M. et al. A genetically encoded reporter of synaptic activity in vivo. Nat Methods 6, 883–889 (2009). https://doi.org/10.1038/nmeth.1399
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