Paradoxical network excitation by glutamate release from VGluT3⁺ GABAergic interneurons

In violation of Dale’s principle several neuronal subtypes utilize more than one classical neurotransmitter. Molecular identification of vesicular glutamate transporter three and cholecystokinin expressing cortical interneurons (CCK⁺VGluT3⁺INTs) has prompted speculation of GABA/glutamate corelease from these cells for almost two decades despite a lack of direct evidence. We unequivocally demonstrate CCK⁺VGluT3⁺INT-mediated GABA/glutamate cotransmission onto principal cells in adult mice using paired recording and optogenetic approaches. Although under normal conditions, GABAergic inhibition dominates CCK⁺VGluT3⁺INT signaling, glutamatergic signaling becomes predominant when glutamate decarboxylase (GAD) function is compromised. CCK⁺VGluT3⁺INTs exhibit surprising anatomical diversity comprising subsets of all known dendrite targeting CCK⁺ interneurons in addition to the expected basket cells, and their extensive circuit innervation profoundly dampens circuit excitability under normal conditions. However, in contexts where the glutamatergic phenotype of CCK⁺VGluT3⁺INTs is amplified, they promote paradoxical network hyperexcitability which may be relevant to disorders involving GAD dysfunction such as schizophrenia or vitamin B6 deficiency.