Synaptic activity-regulated gene expression_embryonic day 18

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 11 biological replicates of munc18-1 null mutants, hybridized with 11 pooled biological replicates. On six arrays the mutant sample was labeled with cy3 and on the other five with cy5.突触的活动依赖性变化依赖于突触驻留蛋白的功能改变以及基因表达过程。本研究采用微阵列表达分析、定量PCR（quantitative PCR, qPCR）与Northern印迹杂交（Northern blotting）技术，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）大脑新皮层的RNA水平，旨在探究突触活动与突触基因表达之间的关联。我们提出假说：受突触活动调控的基因在突变体与野生型对照间存在差异表达；实验结果显示，仅有少量突触信号通路相关基因呈现差异表达。然而，所有在微阵列上可检测到表达的神经肽基因均出现显著差异表达，在对照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表现出差异表达。上述神经肽基因的差异表达经qPCR分析得到验证。原位杂交实验结果表明，神经肽表达的差异呈均匀分布，并非由突变体中特定细胞的丢失所导致。在不依赖突触活动获取输入信号的初级感觉神经元中，未观察到神经肽的差异表达现象。本研究数据显示，突触活动与其驻留蛋白的表达之间并不存在简单的线性关联；相反，本研究揭示了神经肽基因表达的一项独特特征——其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11份munc18-1纯合敲除突变小鼠的生物学重复样本，与11份混合生物学重复样本进行芯片杂交；其中6张芯片的突变样本采用cy3荧光标记，剩余5张采用cy5荧光标记。