Baird D H, Hatten M E, Mason C A
Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
J Neurosci. 1992 Feb;12(2):619-34. doi: 10.1523/JNEUROSCI.12-02-00619.1992.
The contributions of cell-cell interactions to the establishment of specific patterns of innervation within target brain regions are not known. To provide an experimental analysis of the regulation of afferent axonal growth, we have developed an in vitro assay system, based on the developing mouse cerebellum, in which afferent axons from a brainstem source of mossy fiber afferents, the basilar pontine nuclei, were cocultured with astroglia or granule neurons purified from the cerebellum. In the absence of cells from the cerebellum, pontine explants produced axons that fasciculated and extended rapidly on a culture surface treated with poly-lysine or laminin. When pontine neurites grew onto cerebellar astroglial cells, outgrowth was more abundant than on substrates alone, suggesting that glial cells provide a positive signal for axon extension. Time-lapse video microscopy indicated that the rate of neurite extension increased from less than 50 microns/hr to more than 100 microns/hr when axonal growth cones moved from the culture substratum onto an astroglial-cell surface. Acceleration of neurite extension was also observed as pontine neurites grew onto other pontine neurites. By contrast, when pontine neurites grew on granule neurons, the appropriate targets of mossy fibers, the length of pontine neurites was greatly reduced. As growing axons terminated on granule neurons, the target cells appeared to provide a "stop-growing signal" for axon extension. The length of pontine neurites decreased with increasing granule neuron density. Two lines of evidence suggested that the stop signal was contact mediated. First, video microscopy showed that pontine growth cones stopped extending after contacting a granule neuron. Second, the length of afferent axons was not reduced when pontine neurites grew at a distance from granule neurons. Competition experiments where both astroglia and granule neurons were plated together suggested that the growth arrest signal provided by granule neurons could override the growth-promoting signal provided by astroglial cells. These results suggest that specific cell-cell interactions regulate the growth of pontine afferent axons within their cerebellar target, with axoaxonal and axoglial interactions promoting axon extension and axon-target cell interactions interrupting axon extension.
细胞间相互作用对靶脑区内特定神经支配模式建立的贡献尚不清楚。为了对传入轴突生长的调控进行实验分析，我们基于发育中的小鼠小脑开发了一种体外检测系统，其中来自苔藓纤维传入神经的脑干来源（基底桥核）的传入轴突与从小脑纯化的星形胶质细胞或颗粒神经元共培养。在没有小脑细胞的情况下，脑桥外植体产生的轴突在经聚赖氨酸或层粘连蛋白处理的培养表面上成束并快速延伸。当脑桥神经突生长到小脑星形胶质细胞上时，其生长比仅在底物上更为旺盛，这表明胶质细胞为轴突延伸提供了正向信号。延时视频显微镜显示，当轴突生长锥从培养底物移到星形胶质细胞表面时，神经突延伸速率从每小时不到50微米增加到每小时超过100微米。当脑桥神经突生长到其他脑桥神经突上时，也观察到神经突延伸加速。相比之下，当脑桥神经突生长在颗粒神经元（苔藓纤维的合适靶细胞）上时，脑桥神经突的长度大大缩短。随着生长的轴突终止于颗粒神经元，靶细胞似乎为轴突延伸提供了“停止生长信号”。脑桥神经突的长度随着颗粒神经元密度的增加而缩短。两条证据表明该停止信号是接触介导的。第一，视频显微镜显示脑桥生长锥在接触颗粒神经元后停止延伸。第二，当脑桥神经突在与颗粒神经元有一定距离处生长时，传入轴突的长度并未缩短。将星形胶质细胞和颗粒神经元一起接种的竞争实验表明，颗粒神经元提供的生长抑制信号可以覆盖星形胶质细胞提供的生长促进信号。这些结果表明，特定的细胞间相互作用调节脑桥传入轴突在其小脑靶区内的生长，轴突-轴突和轴突-胶质细胞相互作用促进轴突延伸，而轴突-靶细胞相互作用则中断轴突延伸。
