Multiplex imaging relates quantal glutamate release to presynaptic Ca(2+) homeostasis at multiple synapses in situ

Information processing by brain circuits depends on Ca(2+)-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(2+) in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca(2+) indicator Cal-590 is Ca(2+)-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca(2+) 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(2+) or spike-evoked Ca(2+) entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca(2+) 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.