Fluorescence lifetime imaging reveals regulation of presynaptic Ca2+ by glutamate uptake and mGluRs, but not somatic voltage in cortical neurons.

Brain function relies on vesicular release of neurotransmitters at chemical synapses. The release probability depends on action potential-evoked presynaptic Ca2+ entry, but also on the resting Ca2+ level. Whether these basic aspects of presynaptic calcium homeostasis show any consistent trend along the axonal path, and how they are controlled by local network activity, remains poorly understood. Here, we take advantage of the recently advanced FLIM-based method to monitor presynaptic Ca2+ with nanomolar sensitivity. We find that, in cortical pyramidal neurons, action potential-evoked calcium entry (range 10-300 nM), but not the resting Ca2+ level (range 10-100 nM), tends to increase with higher order of axonal branches. Blocking astroglial glutamate uptake reduces evoked Ca2+ entry but has little effect on resting Ca2+ whereas both appear boosted by the constitutive activation of group 1/2 metabotropic glutamate receptors. We find no consistent effect of transient somatic depolarization or hyperpolarization on presynaptic Ca2+ entry or its basal level. The results unveil some key aspects of presynaptic machinery in cortical circuits, shedding light on basic principles of synaptic connectivity in the brain.