Multiplex imaging of quantal glutamate release and presynaptic Ca2+ at multiple synapses in situ
Information processing by brain circuits depends on Ca2+-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 Ca2+. 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. Imaging of multiple synapses in an identified neural circuit reveals that fluctuations both in spike-evoked Ca2+ transients and in resting presynaptic Ca2+ can affect release efficacy. At the sub-microscopic level within individual presynaptic boutons, we detected no consistent co-localisation of presynaptic Ca2+ 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.
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