Synaptic activity-regulated gene expression_embryonic day 14

Activity-dependent changes in synapses rely on functional changes in resident proteins and on gene expression. We addressed the relationship between synapse activity and the expression of synaptic genes by comparing RNA levels in the neocortex of normal mice versus synaptically silent munc18-1 null mutants, using microarray expression analysis, quantitative PCR and Northern blotting. We hypothesized that genes under control of synaptic activity are differentially expressed between mutants and controls and found that few synaptic signaling genes were differentially expressed. However, all neuropeptide genes with detectable expression on the microarray were differentially expressed, being 3-20 fold higher in control cortex. Genes encoding their receptors and most other synaptic components were not differentially expressed. Differential expression of the neuropeptide genes was confirmed by qPCR analysis. In situ hybridization indicated that the difference in neuropeptide expression was uniform and not due to the loss of specific cells in the mutant. In primary sensory neurons, which do not depend on synaptic activity for their input, the differential expression of neuropeptides was not observed. These data reveal no simple relation between activity of synapses and expression of their resident proteins, but instead identified a unique feature of neuropeptide gene expression, which appears to depend on synaptic activity. Keywords: munc18-1 null mutant mice are characterized by the complete loss of both evoked and spontaneous transmitter release 6 biological replicates of munc18-1 null mutants, hybridized with 6 pooled biological replicates. On three arrays the mutant sample was labeled with cy3 and on the other three with cy5.突触的活动依赖性改变依赖于突触驻留蛋白的功能变化与基因表达调控。本研究通过基因芯片表达分析（microarray expression analysis）、定量聚合酶链反应（quantitative PCR，简称qPCR）以及Northern印迹杂交（Northern blotting），对比正常小鼠与突触沉默型munc18-1纯合敲除突变小鼠（munc18-1 null mutant mice）的新皮层RNA水平，以此探究突触活动与突触基因表达之间的关联。我们曾假设受突触活动调控的基因在突变体与对照组间存在差异表达，结果发现仅有极少数突触信号传导基因呈现差异表达。然而，所有在基因芯片上可检测到表达的神经肽基因均存在差异表达，在对照组皮层中的表达量为突变体的3至20倍。编码其受体的基因以及绝大多数其他突触组分相关基因则未呈现差异表达。该神经肽基因的差异表达经qPCR分析得以证实。原位杂交（In situ hybridization）实验结果显示，神经肽表达的差异呈均一分布，并非由突变体中特定细胞丢失所致。在无需依赖突触活动获取输入信息的初级感觉神经元中，则未观察到神经肽的差异表达现象。上述研究结果表明，突触活动与其驻留蛋白表达之间并不存在简单的对应关系，反而揭示了神经肽基因表达的独特调控特征——该类基因的表达似乎依赖于突触活动。关键词：munc18-1纯合敲除突变小鼠以诱发性与自发性递质释放完全丧失为特征。本实验包含6份munc18-1纯合敲除突变体生物学重复样本，与6份混合生物学重复样本进行杂交。其中3张基因芯片的突变体样本采用Cy3标记，剩余3张则采用Cy5标记。