Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin-associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1-day-old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1-to 22-day-old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1- and 15-day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15-day cord overwhelmed permissive or growth promoting molecules in membranes from 1-day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth.