Rat Astrocyte-Neuron Co-cultures versus purified co-cultured Astrocytes versus Astrocyte alone

Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that at least part of the involved astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-induced astrocyte genes in vitro, we tested the effectiveness of the ‘cold jet’, a new method for separation of neurons from co-cultured astrocytes. The cold jet method is performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis revealed changes in expression of a large number of mRNAs and biological processes, including novel findings. Thus, cold jet is an efficient method to separate astrocytes from neurons in co-culture, and in this study reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.过去十年来，星形胶质细胞（astrocyte）与神经元（neuron）之间的通信在神经元发育及突触可塑性（synaptic plasticity）中的重要性愈发明晰。由于神经元-星形胶质细胞互作属于高度动态且双向的过程，我们推测，至少部分相关的星形胶质细胞基因，可能会因与处于成熟过程中的神经元的相互作用而受到调控。为在体外（in vitro）鉴定此类由神经元诱导的星形胶质细胞基因，我们测试了“冷射流法”（cold jet method）的有效性——这是一种可从共培养星形胶质细胞中分离神经元的新方法。该冷射流法在冰寒条件下操作，无需采用蛋白酶介导的方式分离星形胶质细胞，也规避了耗时的离心步骤，可获得完整的星形胶质细胞信使RNA（mRNA），并去除约90%的神经元RNA。利用该方法，我们开展了全基因组表达谱分析（genome-wide profiling）：将仅含星形胶质细胞的培养物来源的RNA，与源自分化中神经元-星形胶质细胞共培养物的星形胶质细胞RNA进行对比。数据分析显示，大量信使RNA的表达及诸多生物学过程均发生了改变，其中涵盖多项全新发现。综上，冷射流法是一种可从共培养体系中高效分离星形胶质细胞与神经元的方法，本研究借助该方法证实，神经元可诱导共培养的星形胶质细胞发生显著的基因表达变化。