Embryonic cortical layer 5 pyramidal neurons form an active, transient circuit motif perturbed by autism-associated mutations

Summary Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that embryonic layer 5 pyramidal neurons, identified through single cell transcriptomics, display two phases of circuit assembly in vivo. At E14.5, a multi-layered circuit motif, composed of a single layer 5 cell type, forms. This motif is transient, switching to a second circuit motif, involving all three types, by E17.5. In vivo targeted single cell recordings and two-photon calcium imaging of embryonic layer 5 neurons reveal that, in both phases, neurons have active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses. Embryonic layer 5 neurons strongly express autism-associated genes, and perturbing these genes disrupts the switch between the two motifs. Hence, layer 5 pyramidal neurons form transient active pyramidal-to-pyramidal circuits, at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.