Molecular Mechanisms for Pathfinding of Spinal Primary Afferents

为了阐明背根神经节（dorsal root ganglion，DRG）初级传入轴突向脊髓灰质靶神经元投射的导向机制，我们采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）或第9天（E9）分离DRG神经元，在脊髓冰冻切片上培养1-2天。许多E5和E9背根神经节神经元在脊髓灰质和白色物质上延伸出单极或双极神经突。当E5 DRG神经突起从外部到达E5脊髓冷冻切片时，大多数神经突起沿着或在白色物质内行进，而约一半的E9 DRG神经突起通过E9脊髓的白色物质进入灰质。脊髓白色和灰质上DRG神经突的这些生长模式类似于体内可比较阶段初级传入轴突的通路形成;初级传入在E5时仅在背索（白色物质）中运行，并在E9时进入灰质。然而，在体外培养的DRG神经突起中，没有显示出对白色物质中的背索的任何偏好。此外，在E5（E9）DRG神经元生长在E9（E5）脊髓上的异时培养中，DRG神经突的生长模式与等时培养的脊髓的可比阶段相似，这表明脊髓而不是DRG可能在神经突生长模式的形成中起关键作用。在培养液中加入抗Ng-CAM或抗Nr-CAM抗体而不加入抗NCAM抗体，可使DRG神经突进入E5脊髓灰质的数量增加，提示Ng-CAM和Nr-CAM均参与调节DRG神经突进入白色物质和侵入灰质靶区。

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.