Glutamate co-release at GABA/glycinergic synapses is crucial for the refinement of an inhibitory map

Many non-glutamatergic synaptic terminals in the mammalian brain contain the vesicular glutamate transporter 3 (VGLUT3), indicating that they co-release the excitatory neurotransmitter glutamate. However, the functional role of glutamate co-transmission at these synapses is poorly understood. In the auditory system, VGLUT3 expression and glutamate co-transmission are prominent in a developing GABA/glycinergic sound localization pathway. Here we show that mice with a genetic deletion of VGLUT3 exhibit disrupted glutamate-co-transmission and severe impairment in the refinement of this inhibitory pathway. Specifically, loss of glutamate co-transmission disrupts synaptic silencing and the strengthening of GABA/glycinergic connections that normally occur with maturation. Functional mapping studies further revealed that these deficits markedly degrade the precision of tonotopy in this inhibitory auditory pathway. These results demonstrate the crucial role of glutamate co-transmission in the synaptic reorganization and topographic specification of a developing inhibitory circuit.