Molecular Mechanisms for Pathfinding of Spinal Primary Afferents

为了阐明初级传入轴突从背根神经节(DRG)投射到脊髓灰质靶神经元的导向机制，我们用冷冻培养的方法研究了初级传入轴突与脊髓之间的相互作用。在胚胎第5天(E5)和胚胎第9天(E9)脊髓冰冻切片上分离培养DRG神经元，培养1~2天。许多E5和E9背根节神经元将单极或双极突起延伸至脊髓灰质和白质。当E5背根节神经突起从外部到达E5脊髓冰冻段时，大部分沿白质或在白质内运行，而约有一半的E9背根节神经突起通过E9脊髓白质进入灰质。背根神经节突起在脊髓白质和灰质的这些突起的生长模式类似于体内类似时期初级传入轴突的路径形成；初级传入在E5时仅在背索(白质)运行，在E9时进入灰质。然而，体外培养的背根节轴突在白质中并没有表现出任何对背索的偏爱。此外，在E5(E9)背根节神经元生长于E9(E5)脊髓的异时培养中，背根神经节突起的生长模式与等时培养的可比脊髓相似，提示在突起生长模式的形成中，可能是脊髓而不是背根节起关键作用。在培养液中加入抗Ng-CAM或抗Nr-CAM抗体，但不加入抗NCAM抗体，可使DRG突起进入E5脊髓灰质的数量增加，提示Ng-CAM和Nr-CAM共同参与了DRG突起在白质内活动和侵袭灰质靶区的调节。

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.