Transcription profiling of two mouse lines displaying different phenotypes on fear conditioning to identify identify differences in gene expression in two key brain regions: amygdala and hippocampus

Fear conditioning (FC) is a behavioral paradigm that measures an animal's ability to learn fear related information. FC is measured by pairing a mild foot-shock with the surroundings in which the shock was received. Upon being placed back in the context, mice exhibit freezing behavior, which is a species-specific response to fear. We have used selective breeding to produce lines of mice with high or low levels of freezing behavior. We are able to identify alleles that govern the genetic variability for FC by using chromosomal markers in these lines. Using microarrays, we will identify differences in gene expression in two key brain regions: amygdala and hippocampus. Gene expression differences and data regarding chromosomal regions involved in the behavior will be compared to identify particular genes that are both differentially expressed and whose expression is governed by alleles that fall into critical chromosomal regions.,We will compare gene expression in the amygdala and hippocampus (brain regions known to be relevant to fear behavior) from the two lines of mice and use Bayesian statistics in an effort to identify gene expression that affects fear behavior.,We hypothesize that selection has acted in part by changing the frequency of alleles that cause differential expression of key genes in the amygdala and hippocampus of our selected lines. Selective breeding changes the frequency of trait relevant (FC) alleles. A relevant allele is expected to increase in one selected line and decrease in the oppositely selected line. Some trait relevant alleles are expected cause changes in the level of expression at particular genes.,Amygdala and hippocampus will be rapidly dissected out of experimentally naive mice from each line. Naive mice will be used for expression studies since the behavior of the mice in the FC test can be reliably anticipated due to their lineage. We have practiced these procedures, and can accurately and reproducibly remove these regions in less than 5 minutes. Different mice will be used to collect each brain region, since the dissection of hippocampus disrupts the removal of amygdala. We will collect enough samples from each region to accommodate a total of 6 microarrays per brain region, per line, thus we will use a total of 24 microarrays. We anticipate that a single brain region will be sufficient to for a microarray. However, we propose to utilize three samples per microarray, because this will reduce variability due to environmental factors and due to slight variability in our dissection procedures. Once this tissue is removed, we will isolate RNA for shipment to the Microarray consortium. We will also collect spleens from each subject as a source of genomic DNA, in order to permit direct comparison of genotype and expression phenotypes. Once we have the results of the microarray analysis, we use WebQTL.org to identify the chromosomal locations of alleles that are know to influence the expression of genes for which we have found differential expression. We will then superimpose this information on trait relevant chromosomal regions identified from our selected lines. This will allow us to rapidly identify genes which may account for genetic variability in FC due to differential expression. Such genes will then be subjected to further study.naive恐惧条件反射（Fear conditioning, FC）是一类用于评估动物学习恐惧相关信息能力的经典行为范式。该实验通过将轻度足底电击与电击发生时所处的环境情境进行配对来完成测定。当小鼠被重新放回该电击情境时，会表现出僵立行为——这是啮齿类动物面对恐惧时的物种特异性反应。本研究通过选择性繁育技术，构建了僵立行为水平高低分化的两类小鼠品系。我们将借助这些品系中的染色体分子标记，筛选并鉴定出调控FC行为遗传变异的等位基因。 我们将利用基因芯片技术，鉴定杏仁核（amygdala）与海马体（hippocampus）这两个关键情绪相关脑区内的基因表达差异。随后将比对基因表达差异数据与该行为相关的染色体区域定位数据，以筛选出同时满足两项条件的候选基因：一是该基因在两类品系间存在显著表达差异；二是其表达水平受位于关键染色体区域内的等位基因调控。 我们将比对两类小鼠品系杏仁核与海马体（已知与恐惧行为调控密切相关的脑区）的全基因组表达谱，并运用贝叶斯统计方法，以期识别出与恐惧行为调控相关的基因表达特征。 我们提出如下研究假说：选择性繁育的部分作用机制，是通过改变调控所选品系杏仁核与海马体内关键功能基因差异表达的等位基因频率来实现的。选择性繁育会改变与FC性状相关的等位基因频率：这类功能相关等位基因在正向选育品系中频率会显著升高，而在反向选育的品系中频率则会降低。部分与FC性状相关的等位基因，可通过调控特定基因的表达水平来改变动物的恐惧相关行为。 我们将从每个品系的未经过实验处理的小鼠体内快速分离杏仁核与海马体组织。选用未接触过实验的小鼠进行表达谱研究，是因为该品系小鼠在FC行为测试中的表现可通过其遗传谱系进行可靠预测。我们已熟练掌握该解剖分离流程，可在5分钟内准确且可重复地获取这两个脑区的组织样本。由于海马体的解剖分离操作会破坏杏仁核的完整取出，因此我们将使用不同的小鼠分别采集这两个脑区的样本。我们将为每个脑区、每个品系收集足够的样本，以满足每个脑区开展6次基因芯片实验的需求，总计将使用24张基因芯片。我们预计单个脑区的样本量即可满足一次基因芯片实验的要求，但我们计划每张芯片使用三份生物学重复样本，以降低环境因素以及解剖操作细微差异所带来的实验变异。 组织分离完成后，我们将提取总RNA并送至基因芯片合作联盟进行检测。我们还将采集每只小鼠的脾脏组织作为基因组DNA的来源，以便后续直接比较基因型与基因表达表型的关联。获得基因芯片分析结果后，我们将通过WebQTL.org数据库，鉴定出已知可调控我们所发现的差异表达基因的等位基因的染色体定位信息。随后，我们会将该等位基因定位信息与从选育品系中鉴定出的FC性状相关染色体区域进行叠加比对，从而快速筛选出可能通过差异表达介导FC行为遗传变异的候选基因。这类候选基因后续将接受进一步的功能验证研究。