Molecular Mechanisms for Pathfinding of Spinal Primary Afferents

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In order to clarify the guidance mechanisms of projections of primary afferent axons from dorsal root ganglion (DRG) to target neurons in the spinal gray matter, we examined interactions between primary afferent axons and spinal cord using the cryoculture methods. DRG neurons were dissociaterd and cultured for 1-2 days on the cryosections of spinal cord at embryonic day 5 (E5) or at E9. Many E5 and E9 DRG neurons extended unipolar or bipolar neurites onto both spinal gray and white matters. When E5 DRG neurites reached cryosections of E5 spinal cord from outside, most of them ran along or within the white matter, whereas about half of E9 DRG neurites entered the gray matter through the white matter of E9 spinal cord. These outgrowth patterns of DRG neurites on spinal white and gray matters are similar to the pathway formation of primary afferent axons at comparable stages in vivo ; primary afferents run exclusively in the dorsal funiculus (white matter) at E5 and enter the gray matter at E9. DRG neurites in vitro, however, did not display any preference to dorsal funiculus among white matter. Furthermore, in the heterochronic culture in which E5 (E9) DRG neurons were grown on E9 (E5) spinal cord, the outgrowth patterns of DRG neurites were similar to those at the comparable stage of the spinal cord of the isochronic culture, suggesting that spinal cord rather than DRG may play a crucial role in the pattern formation of neurite outgrowth. Addition of anti-Ng-CAM or anti-Nr-CAM antibodies but not anti-NCAM antibody in the culture mediuim resulted in the increase of DRG neurites to enter the gray matter of E5 spinal cord, suggesting that both Ng-CAM and Nr-CAM is involved in the regulation of DRG neurites to run in the white matter and to invade target regions in the gray matter.