Keller F, Levitt P
Institute of Pharmacology, University of Zurich, Switzerland.
Neuroscience. 1989;28(2):455-74. doi: 10.1016/0306-4522(89)90192-9.
In the present study we have examined the topographic and temporal patterns of expression of the limbic system associated membrane protein by light and electron microscopic immunocytochemistry in organotypic cultures of the rat brain. The regional, cellular and subcellular distribution of staining in young cultures was similar to that in the intact brain of corresponding age. Since the tissue in vitro is isolated both from afferents and targets, short-term protein expression appears to be regulated by factors intrinsic to the neuron. In culture, the protein was present on the surface of neurons which are physiologically interconnected, such as neurons belonging to the septohippocampal system (cholinergic neurons in the septum and pyramidal and granule cells in the hippocampus). It was also present on the surface of axons and growth cones during process outgrowth. Thus, the limbic system associated membrane protein is expressed in an appropriate spatial and temporal pattern for mediating interactions between growing axons and their targets. The expression of the protein in culture showed some important differences as compared to the intact brain. With increasing age, there was an increasing scattering and disappearance of immunoreactivity in cultures fixed with paraformaldehyde/glutaraldehyde. The decreased immunoreactivity in aged cultures does not appear to reflect decreased protein synthesis, because unfixed and acetone-fixed explants continued to show immunostaining. Furthermore, dot-blot assays showed similar amounts of immunoreactivity in culture as in the intact brain of corresponding age. Thus, the age-dependent decrease of immunoreactivity may reflect altered insertion of the protein into the membrane or a modification of the epitope recognized by the antibody. There was a rapid increase (within 1 hour) of immunostaining on the surface of sprouting processes following mechanical lesion of mature, unstained axons. The altered distribution after tissue injury could be a means of ensuring specificity of connectivity during nerve fiber regeneration. On the basis of the reported findings, we suggest that system-specific membrane proteins, including the limbic system associated membrane protein, may mediate the formation of specific connections in the brain. Furthermore, we suggest that the reinnervation processes taking place after central nervous system injury may exhibit a similar molecular basis to the development of neural pathways.
在本研究中，我们通过光镜和电镜免疫细胞化学方法，在大鼠脑器官型培养物中检测了边缘系统相关膜蛋白的表达的拓扑和时间模式。年轻培养物中染色的区域、细胞和亚细胞分布与相应年龄的完整大脑中的分布相似。由于体外组织与传入神经和靶组织均分离，短期蛋白质表达似乎受神经元内在因素调节。在培养物中，该蛋白存在于生理上相互连接的神经元表面，如属于隔海马系统的神经元（隔区的胆碱能神经元以及海马中的锥体细胞和颗粒细胞）。在轴突生长过程中，它也存在于轴突和生长锥表面。因此，边缘系统相关膜蛋白以适当的空间和时间模式表达，以介导生长中的轴突与其靶组织之间的相互作用。与完整大脑相比，该蛋白在培养物中的表达存在一些重要差异。随着年龄的增长，用多聚甲醛/戊二醛固定的培养物中免疫反应性的散射和消失增加。老年培养物中免疫反应性的降低似乎并不反映蛋白质合成的减少，因为未固定和丙酮固定的外植体仍显示免疫染色。此外，斑点印迹分析显示培养物中的免疫反应性与相应年龄的完整大脑中的相似。因此，免疫反应性的年龄依赖性降低可能反映了该蛋白插入膜中的改变或抗体识别的表位的修饰。成熟的未染色轴突受到机械损伤后，发芽过程表面的免疫染色在1小时内迅速增加。组织损伤后分布的改变可能是确保神经纤维再生过程中连接特异性的一种方式。基于所报道的发现，我们认为包括边缘系统相关膜蛋白在内的系统特异性膜蛋白可能介导大脑中特定连接的形成。此外，我们认为中枢神经系统损伤后发生的再支配过程可能与神经通路的发育具有相似的分子基础。
