Co‐expression of VGLUT1 and VGAT sustains glutamate and GABA co‐release and is regulated by activity in cortical neurons

In adult neocortex, VGLUT1, the main glutamate vesicular transporter, and VGAT, the GABA vesicular transporter, are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, where they are sorted to the same vesicles. However, the functional consequence of this co-localization in cortical neurons has not been clarified. Here, we tested the hypothesis that cortical axon terminals co-expressing VGLUT1 and VGAT can evoke simultaneously monosynaptic glutamate and GABA responses and investigated whether the amount of terminals co-expressing VGLUT1 and VGAT is affected by perturbations of excitation-inhibition balance. In rat primary cortical neurons, we found that a proportion of synaptic and autaptic responses were indeed sensitive to consecutive application of selective glutamate and GABAA receptor blockers. These "mixed" synapses exhibited paired-pulse depression. Notably, reducing the activity of the neuronal network by glutamate receptor antagonists decreased the amount of "mixed" synapses, whereas reducing spontaneous inhibition by bicuculline increased them. These synapses may contribute to homeostatic regulation of excitation/inhibition balance.