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 second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging 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.

皮层回路主要由锥体到锥体的神经元连接组成，但它们在胚胎发育过程中的组装尚不清楚。我们表明小鼠胚胎 Rbp4-Cre 皮层神经元在转录组学上最接近第 5 层锥体神经元，在体内显示电路组装的两个阶段。在 E14.5，它们形成一个多层电路基序，仅由胚胎近投射型神经元组成。到 E17.5，这过渡到涉及所有三种胚胎类型的第二个基序，类似于三种成人第 5 层类型。体内从 E14.5 开始，胚胎 Rbp4-Cre 神经元的膜片钳记录和双光子钙成像揭示了活跃的胞体和轴突、河豚毒素敏感的电压门控电导和功能性谷氨酸能突触。胚胎 Rbp4-Cre 神经元强烈表达自闭症相关基因，扰乱这些基因会干扰两个基序之间的转换。因此，锥体神经元在新皮层开始形成活跃的、短暂的、多层的锥体到锥体回路，研究这些回路可以深入了解自闭症的病因。