Most Early-Born Subplate Neurons Persist as Layer 6b Neurons in the Adult Mouse Neocortex

Subplate neurons (SpNs) are early-born neurons in the mammalian neocortex and play a pivotal role in cortical development. It is widely believed that SpNs are predominantly lost due to postnatal cell death, with only a few remnants contributing to layer 6b (L6b) neurons in the adult neocortex, but the extent to which SpNs persist as L6b neurons remains largely unknown. To address this, we conducted a comprehensive birthdate analysis using multiple ethynyl deoxyuridine (EdU) injections over 24 h, thereby precisely tracking the survival and distribution of SpNs in the adult mouse neocortex. We further estimated the total number of SpNs and L6b neurons based on the total volumes of the subplate and L6b, quantified by employing 3D tissue clearing. Our findings reveal that a significant proportion of L6b neurons are remnants of SpNs and comprise diverse subtypes reminiscent of those in the postnatal period (SpNs: 319,588 ± 49,263, L6b neurons: 316,720 ± 11,268). This study provides direct evidence showing that most L6b neurons in the adult mouse neocortex originate as SpNs during the earliest stages of neurogenesis and persist throughout life.