van den Pol A N, Spencer D D
Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520, USA.
Neuroscience. 2000;95(2):603-16. doi: 10.1016/s0306-4522(99)00430-3.
In the present study, we used co-culture of astrocytes from one species with neurons from a different species to examine neuritic outgrowth. We include a focus on human cells. Three types of neuron were used, including rat hippocampal dentate granule cells, rat hypothalamic neurons and human cortical neurons. To visualize neuronal processes, neurons were either immunostained with GABA antiserum or transfected with the jellyfish green fluorescent protein gene. The entire axonal and dendritic fields of single neurons could be quantitatively analysed based on their strong green fluorescent protein label. Astrocytes were obtained from rat hippocampus or hypothalamus, chicken cortex, normal human cortex, human cortex lesion, and from the sclerotic human hippocampus after surgery for intractable temporal lobe epilepsy. In the absence of astrocytes, isolated neurons died within three to four days. In contrast, neurons from both rat and human brains survived and extended dendrites and axons on rat, chicken and human astrocytes or in their conditioned medium. Astrocytes from interspecies cultures were not only capable of enhancing the survival of neuron co-cultures, but neuronal neurite extension in some cases was even greater on heterospecific astrocytes than on homospecific astrocytes. To support the hypothesis that synaptogenesis of rat hippocampal neurons was accelerated by a substrate of human astrocytes, we used a functional assay based on time-lapse confocal laser or digital imaging of calcium responses to transmitter release; synaptic responses were found earlier when rat neurons were grown on rat or human astrocytes than in the absence of these astrocytes. These data indicate that rodent glial cells enhance human neurite extension, and that rat neurite outgrowth can be used as a type of bioassay for the neurite promoting capacity of different derivations of human glia.
在本研究中，我们将来自一个物种的星形胶质细胞与来自不同物种的神经元进行共培养，以检测神经突生长情况。我们重点关注人类细胞。使用了三种类型的神经元，包括大鼠海马齿状颗粒细胞、大鼠下丘脑神经元和人类皮层神经元。为了观察神经元突起，神经元要么用γ-氨基丁酸抗血清进行免疫染色，要么用海蜇绿色荧光蛋白基因进行转染。基于单个神经元强烈的绿色荧光蛋白标记，可以对其整个轴突和树突区域进行定量分析。星形胶质细胞取自大鼠海马或下丘脑、鸡皮层、正常人脑皮层、人脑皮层损伤部位以及难治性颞叶癫痫手术后硬化的人海马。在没有星形胶质细胞的情况下，分离的神经元在三到四天内死亡。相比之下，大鼠和人类大脑的神经元在大鼠、鸡和人类星形胶质细胞上或在它们的条件培养基中存活并延伸树突和轴突。种间共培养的星形胶质细胞不仅能够提高神经元共培养物的存活率，而且在某些情况下，神经元在异种星形胶质细胞上的神经突延伸甚至比在同种星形胶质细胞上更大。为了支持人类星形胶质细胞底物加速大鼠海马神经元突触形成这一假设，我们使用了一种基于延时共聚焦激光或对递质释放的钙反应进行数字成像的功能测定方法；当大鼠神经元在大鼠或人类星形胶质细胞上生长时，比在没有这些星形胶质细胞的情况下更早发现突触反应。这些数据表明，啮齿动物的神经胶质细胞可增强人类神经突的延伸，并且大鼠神经突生长可作为一种生物测定方法，用于检测人类神经胶质细胞不同衍生物的神经突促进能力。
