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Showing 1-1 of 1 resultsInformation processing by brain circuits depends on Ca-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca indicator Cal-590 is Ca-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca or spike-evoked Ca entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.