Vandenberghe W, Van Den Bosch L, Robberecht W
Laboratory of Neurobiology, University of Leuven, Campus Gasthuisberg, B-3000, Leuven, Belgium.
Brain Res. 1998 Oct 5;807(1-2):1-10. doi: 10.1016/s0006-8993(98)00569-1.
AMPA/kainate receptor-mediated excitotoxicity is believed to play a pathogenic role in amyotrophic lateral sclerosis. To further characterize the mechanisms involved in AMPA/kainate receptor-mediated motoneuron injury, we investigated the influence of spinal glial cells on kainate-induced motoneuron death in vitro. A motoneuron-enriched neuronal population was obtained from embryonic mouse spinal cord by metrizamide density centrifugation. This population was cultured either on a pre-established glial feeder layer of ventral spinal origin (coculture) or in glia-free conditions (monoculture). Glial feeder layers significantly enhanced basal survival of neurons, and supported neuronal differentiation as judged by neuronal morphology and expression of the motoneuron markers peripherin and SMI-32. Neuronal vulnerability to kainate was two- to three-fold higher in coculture than in monoculture, and increased significantly with time in coculture. The effects of glial feeder layers on neuronal basal survival, differentiation and kainate vulnerability were not mimicked by conditioned medium from glial cells. The increase in neuronal kainate vulnerability with time in coculture was associated with a marked rise in the proportion of cocultured neurons possessing Ca2+-permeable AMPA/kainate receptors, as determined by kainate-activated Co2+-uptake. Neurons in monoculture were unstained by kainate-activated Co2+-uptake. Neurons were immunoreactive to specific antibodies against the AMPA receptor subunits GluR1 and GluR2 both in monoculture and coculture. This study indicates that motoneuron differentiation in coculture is associated with increased vulnerability to kainate and increased expression of Ca2+-permeable AMPA/kainate receptors. In this paradigm glial cells support basal survival and differentiation of neurons, but potentiate kainate-induced neuronal death.
AMPA/海人酸受体介导的兴奋性毒性被认为在肌萎缩侧索硬化症中起致病作用。为了进一步阐明AMPA/海人酸受体介导的运动神经元损伤的机制，我们在体外研究了脊髓胶质细胞对海人酸诱导的运动神经元死亡的影响。通过甲泛葡胺密度离心法从胚胎小鼠脊髓中获得富含运动神经元的神经元群体。该群体在预先建立的源自脊髓腹侧的胶质饲养层上培养（共培养）或在无胶质条件下培养（单培养）。胶质饲养层显著提高了神经元的基础存活率，并通过神经元形态以及运动神经元标志物外周蛋白和SMI-32的表达判断其支持神经元分化。共培养中神经元对海人酸的易损性比单培养高两到三倍，并且在共培养中随时间显著增加。胶质饲养层对神经元基础存活、分化和对海人酸易损性的影响不能被胶质细胞的条件培养基模拟。共培养中神经元对海人酸易损性随时间的增加与具有Ca2+通透性AMPA/海人酸受体的共培养神经元比例的显著增加相关，这是通过海人酸激活的Co2+摄取确定的。单培养中的神经元未被海人酸激活的Co2+摄取染色。在单培养和共培养中，神经元对针对AMPA受体亚基GluR1和GluR2的特异性抗体均具有免疫反应性。本研究表明，共培养中的运动神经元分化与对海人酸的易损性增加以及Ca2+通透性AMPA/海人酸受体的表达增加有关。在这种模式下，胶质细胞支持神经元的基础存活和分化，但增强海人酸诱导的神经元死亡。
