Application of this method to different embryonic stages can visualize neurons in most layers, and allows us to examine initiation of primary axons that differentiate into divergent projections.Here, we analyzed the axonal outgrowth of lateral cortex neurons labeled at different times during the main period of cortical neurogenesis.For example, in rats, layer V subcerebral projection neurons establish area-specific projections by initially forming a primary descending axon toward the spinal cord, developing a common set of collateral branches along the primary axon, and eliminating specific branches in an area-dependent manner (O'Leary and Terashima 1988).Similarly, a population of neurons in layers II/III and V of the rat sensorimotor cortex establishes association projections by first extending a primary axon to the contralateral cortex, forming projections to the ipsilateral cortical area, and finally losing some of the initial callosal projections (Mitchell and Macklis 2005).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.
Although this diversity may develop from primary axons, how many types of axons initially occur remains unknown.
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).
This labeling method permits the examination of morphological changes in developing excitatory cortical neurons at the single-cell level.
Using this method, we showed that neurons labeled at embryonic day (E)12.5 initiated directed axonal outgrowth in the intermediate zone (IZ) before migrating into the cortical plate (CP; Hatanaka and Yamauchi 2013).