Multiplex imaging of quantal glutamate release and presynaptic Ca2+ at multiple synapses in situ

Information processing by brain circuits depends on Ca 2+ -dependent, stochastic release of the excitatory neurotransmitter glutamate. Optical glutamate sensors have enabled detection of evoked and spontaneous synaptic discharges. However, monitoring presynaptic function and its underpinning machinery in situ requires simultaneous readout of quantal glutamate release and nanomolar presynaptic Ca 2+ . 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. Imaging of multiple synapses in an identified neural circuit reveals that fluctuations both in spike-evoked Ca 2+ transients and in resting presynaptic Ca 2+ can affect release efficacy. At the sub-microscopic level within individual presynaptic boutons, we detected no consistent co-localisation of presynaptic Ca 2+ entry and glutamate release sites, suggesting loose coupling between the two. The present approach broadens qualitatively our horizon in understanding release machinery of central synapses.