LAUSR

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.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.