Multiplex imaging relates quantal glutamate release to presynaptic Ca²⁺ homeostasis at multiple synapses in situ

Information processing by brain circuits depends on Ca²⁺-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 Ca²⁺ in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca²⁺ indicator Cal-590 is Ca²⁺-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca²⁺ 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 Ca²⁺ or spike-evoked Ca²⁺ entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca²⁺ 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.