Cortical ChAT + neurons co-transmit acetylcholine and GABA in a target- and brain-region-specific manner

The mouse cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the neurotransmitters released by cortical ChAT neurons and their synaptic connectivity are unknown. We show that the nearly all cortical ChAT neurons in mice are specialized VIP interneurons that release GABA strongly onto other inhibitory interneurons and acetylcholine sparsely onto layer 1 interneurons and other VIP /ChAT interneurons. This differential transmission of ACh and GABA based on the postsynaptic target neuron is reflected in VIP /ChAT interneuron pre-synaptic terminals, as quantitative molecular analysis shows that only a subset of these are specialized to release acetylcholine. In addition, we identify a separate, sparse population of non-VIP ChAT neurons in the medial prefrontal cortex with a distinct developmental origin that robustly release acetylcholine in layer 1. These results demonstrate both cortex-region heterogeneity in cortical ChAT interneurons and target-specific co-release of acetylcholine and GABA.