Synaptic activity-regulated gene expression_embryonic day 16

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 4 biological replicates of munc18-1 null mutants, hybridized with 4 pooled biological replicates. On one array the mutant sample was labeled with cy3 and on the other three with cy5.突触的活动依赖性变化依赖于驻留蛋白的功能改变以及基因表达。本研究通过基因芯片表达分析、定量PCR（qPCR）和Northern印迹法，对比正常小鼠与突触沉默型munc18-1敲除突变体（munc18-1 null mutants）新皮层的RNA水平，以此探究突触活动与突触基因表达之间的关联。我们提出假说：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；研究发现，仅有少数突触信号基因呈现差异表达。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，其在对照组皮层中的表达量为突变体的3~20倍。编码其受体的基因以及绝大多数其他突触组分基因均未出现差异表达。神经肽基因的差异表达经qPCR分析得以验证。原位杂交结果显示，神经肽表达的差异呈均匀分布，并非由突变体中特定细胞丢失所致。在输入不依赖突触活动的初级感觉神经元中，未观察到神经肽的差异表达。上述数据表明，突触活动与其驻留蛋白的表达之间并无简单的线性关联，反而揭示了神经肽基因表达的独特特征——其表达似乎依赖于突触活动。关键词：munc18-1敲除突变体小鼠（munc18-1 null mutant mice）以完全丧失诱发型与自发型递质释放为特征。本研究包含4份munc18-1敲除突变体的生物学重复样本，与4份混合生物学重复样本进行杂交：其中一张芯片中，突变体样本以cy3标记，其余三张芯片则以cy5标记。