Raphe-spinal neurons display an age-dependent differential capacity for neurite outgrowth compared to other brainstem-spinal populations

Functional regeneration of brainstem–spinal pathways occurs in the developing chick when the spinal cord is severed prior to embryonic day (E) 13. Functional spinal cord regeneration is not observed in animals injured after E13. This developmental transition from a permissive to a restrictive repair period may be due to the formation of an extrinsic inhibitory environment preventing axonal growth, and/or an intrinsic inability of mature neurons to regenerate. Here, we investigated the capacity of specific populations of brainstem–spinal projection neurons to regrow neurites in vitro from young (E8) versus mature (E17) brainstem explants. A crystal of carbocyanine dye (DiI) was implanted in ovo into the E5 cervical spinal cord to retrogradely label brainstem–spinal projection neurons. Three or 12 days later, discrete regions of the brainstem containing DiI-labeled neurons were dissected to produce explant cultures grown in serum-free media on laminin substrates. The subsequent redistribution of DiI into regenerating processes permitted the study of in vitro neurite outgrowth from identified brainstem–spinal neurons. When explanted on E8, i.e., an age when brainstem–spinal neurons are normally elongating through the spinal cord and are capable of in vivo functional regeneration, robust neurite outgrowth was observed from all brainstem populations, including rubro-, reticulo-, vestibulo-, and raphe–spinal neurons. In contrast, when explanted on E17, robust neurite outgrowth was seen only from raphe-spinal neurons. Neurite outgrowth from raphe-spinal neurons was 5-hydroxy-tryptamine immunoreactive. This study demonstrates that in growth factor-free environments with permissive growth substrates, neurite outgrowth from brainstem–spinal neurons is dependent on both neuronal age and phenotype.