Multiplex imaging relates quantal glutamate release to presynaptic Ca2+ 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. How neurotransmitter release relates to presynaptic calcium dynamics is not fully understood. Here the authors develop an approach based on FLIM and optical quantal analysis for simultaneous imaging of calcium dynamics and glutamate release at presynaptic boutons.