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.

Automated summary

In cortical pyramidal neurons, the amount of action potential-evoked Ca2+ entry tends to increase with the number of axonal branches, while the resting Ca2+ level remains stable. Inhibition of astroglial glutamate uptake reduces evoked Ca2+ entry but has minimal effect on resting Ca2+, whereas activation of group 1/2 metabotropic glutamate receptors enhances both. Transient somatic depolarization or hyperpolarization does not consistently affect presynaptic Ca2+ entry or its basal level. These findings provide insights into the basic principles of synaptic connectivity in the brain.