Pyramidal Neurons Form Active, Transient, Multilayered Circuits Perturbed by Autism-Associated Mutations at the Inception of Neocortex

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi- layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a secondmotif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calciumimaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.