Pregnant mice were injected intraperitoneally with 5-ethynyl-2′-deoxyuridine (Ed U, 25 mg/kg; Invitrogen, Carlsbad, CA, USA; Salic and Mitchison 2008) 3 times (4-h intervals) from the time at 0, 24, or 48 h after electroporation at E12.5.
The diverse projections of these neurons develop in 3 phases: primary axon extension, delayed collateral branch formation, and selective axon elimination (O'Leary and Koester 1993).
However, it is unknown how many types of axonal projections initially develop from excitatory neurons because the retrograde-labeling method used in previous studies did not allow examinations of initial axonal outgrowth from these neurons.
Recently, we successfully sparsely labeled mouse excitatory cortical neurons by in utero electroporation (Hatanaka and Yamauchi 2013).
We demonstrated that these neurons commonly initiated axonal outgrowth when located in the IZ but that the outgrowth direction was lateral for early-born neurons and medial for later-born neurons.
Therefore, cortical projection divergence appears to originate from two populations.
The day of birth was designated as postnatal day (P)0.
All the experiments adhered to the guidelines for the use of laboratory animals of the Nara Institute of Science and Technology and the National Institute for Physiological Sciences.For dense labeling with green fluorescent protein (GFP; flat-mount preparations, population studies of axonal outgrowth and immunohistochemistry for postnatal brains), , 4–5 ng/μL; Mizuno et al.2014), which label cells sparsely but more strongly, were used.Notably, elimination of supernumerary axons from a subset of initial callosal projections has also been observed in the developing rhesus monkey (Lamantia and Rakic 1990).Therefore, it is likely that these divergent projections originate from a restricted number of primary axons with identical structural properties.2004; Hatanaka and Yamauchi 2013) except that the number of electric pulses was reduced from 5 to 4.