Hippocampal Gene Expression in bred High Responder (bHR) vs. bred Low Responder (bLR) Rats: RNA-Seq Data from Generation F37

The strong pattern of comorbidity amongst psychiatric disorders is believed to be generated by a spectrum of latent liability, arising from a complex interplay of genetic risk and environmental factors, such as stress and childhood adversity. At one end of this spectrum are internalizing disorders, which are associated with neuroticism, anxiety, and depression. At the other end of the spectrum are externalizing disorders, which are associated with risk-taking and novelty-seeking, as seen in mania, substance abuse, and impulse-control disorders. We model the genetic contributions underlying both extremes of this spectrum by selectively breeding rats that react differently to a novel environment. “Bred high responder” (bHR) rats are highly exploratory with a disinhibited, novelty-seeking temperament, including hyperactivity, aggression, and drug-seeking. “Bred low responder” (bLR) rats are highly-inhibited, exhibiting reduced locomotor activity and anxious and depressive-like behavior. These behavioral propensities are robust and stable, beginning early in development similar to temperament in humans. This RNA-sequencing study examined gene expression in the hippocampus, a region critical for emotional regulation, in generation F37 adult male bHR rats and bLR rats (n=6/group), as well as in rats that showed an intermediate locomotor response to a novel field (“bred Intermediate Responder” or bIR rats, n=6), which were obtained by cross-breeding F37 bHR and bLR rats. Prior to sacrifice, the animals experienced behavioral testing. Locomotor response to a novel environment was assessed between age P50–75 as part of our selective breeding paradigm. We also measured anxiety-like behavior in adulthood (bHR/bLR: P160-P167; bIR: P65-75) using the percent time spent in the open arms of an Elevated Plus Maze (EPM; 5 min test). These behavioral testing results are provided here along with the gene expression data. Overall Design: This RNA-sequencing study examined gene expression in the hippocampus in generation F37 adult male bHR rats and bLR rats (n=6/group), as well as in adult male rats that showed an intermediate locomotor response to a novel environment (“bred Intermediate Responder” or bIR rats, n=6), which were obtained by cross-breeding F37 bHR and bLR rats. Behavioral Testing: Locomotor response to a novel environment was assessed between age P50–75 as part of our selective breeding paradigm (protocol: Stead et al., 2006, Behav Genet. 36: 697–712). We measured anxiety-like behavior in adulthood (bHR/bLR: P160-P167; bIR: P65-75) using the percent time spent in the open arms of an Elevated Plus Maze (EPM; 5 min test, protocol: Aurbach et al. 2015, Proc Natl Acad Sci USA. 112: 11953–11958). Sacrifice & RNA Extraction: The rats were sacrificed in adulthood (bHR/bLR=P160-P167, bIR=P126-134) by rapid decapitation and the whole hippocampus was extracted on ice, rapidly frozen, and stored at -80 degrees C. Nucleotides were extracted using Qiagen AllPrep DNA RNA miRNA Universal Kit 50. Extracted RNA was evaluated for total concentration and quality using a Nanodrop spectrophotometer (concentration range 285-432 ng/ul, 260/280 ratio range 1.61-1.80) and then sent to the University of Michigan DNA Sequencing Core (https://seqcore.brcf.med.umich.edu). RNA Sequencing: At the sequencing core, the RNA was re-assessed for quality using the TapeStation automated sample processing system (Agilent, Santa Clara, CA) and only samples with RNA integrity numbers (RINs) of >8 were included in the analysis. The cDNA library was constructed using 0.1-3ug of total RNA and the Illumina TruSeq Stranded mRNA Library Preparation kit (Catalog #s RS-122-2101, RS-122-2102) (Illumina, San Diego, CA). The final cDNA libraries were checked for quality once again by TapeStation (Agilent) as well as qPCR through the use of Kapa’s library quantification kit for Illumina Sequencing platforms (catalog # KK4835, Kapa Biosystems,Wilmington MA). The samples were clustered on a cBot automated cluster generation system (Illumina) for clonal amplification. The samples were then hybridized to the slide (“flow cell”) of a HiSeq 2000 (Illumina) with 6.66 samples per lane and underwent a 100 cycle paired end run in High Output mode using version 3 reagents. RNA-Seq Data Preprocessing: Following sequencing and demultiplexing, the RNA-Seq reads were aligned to the rat genome (Rnor_6.0) using the SubRead aligner (Liao et al. 2014, Bioinformatics. 30: 923–930) using default parameters with the exception of indel detection (maximum length of indel that could be detected=0). The featureCounts program (Liao et al. 2014, Bioinformatics. 30: 923–930) then generated the gene-level RNA-Seq count summaries for each sample based on ENSEMBL annotation (Ensembl v.81). This count summary dataset was then filtered to exclude rows of data from genes that did not meet a minimum threshold of 4 samples with greater than or equal to 10 counts. Rows that lacked official gene symbol annotation were also excluded. Our current analysis used the log2 fragments per million gene-level summary output for each sample provided by the voom() function (R package limma; Ritchie et al. 2015, Nucleic Acids Res. 43: e47). Quality control included 1) visualization of the overall log (base2) transformed transcript expression across all subjects via boxplot, 2) examination of the overall reads (mean and standard deviation) per subject, 3) visualization of a subject/subject correlation matrix to identify particularly atypical samples (R<.95). No outlier samples were identified.

精神疾病间强烈的共病（comorbidity）模式被认为由一系列潜在易感性（latent liability）所驱动，该易感性源于遗传风险与环境因素（如压力与童年逆境）的复杂相互作用。该谱系的一端为内化障碍（internalizing disorders），与神经质、焦虑及抑郁相关；另一端则为外化障碍（externalizing disorders），与冒险行为、寻求新奇特质相关，此类特质可见于躁狂、物质滥用及冲动控制障碍中。
本研究通过选育对新奇环境反应不同的大鼠，对该谱系两端的遗传贡献进行建模。“高选育反应型（bred high responder, bHR）”大鼠具有高度探索性，表现出脱抑制、寻求新奇的气质特征，包括多动、攻击行为与药物觅求行为。“低选育反应型（bred low responder, bLR）”大鼠则高度抑制，运动活动减少，并表现出焦虑及类抑郁行为。这些行为倾向稳定且持久，在发育早期即显现，与人类的气质特征类似。
本项RNA测序（RNA-sequencing）研究检测了第37代（F37）成年雄性bHR大鼠与bLR大鼠（每组n=6），以及通过杂交F37代bHR与bLR大鼠获得的、对新奇环境表现出中等运动反应的“中等选育反应型（bred Intermediate Responder, bIR）”大鼠（n=6）的海马体（hippocampus）基因表达情况——海马体是情绪调控的关键脑区。处死动物前，所有大鼠均接受行为学测试。作为选育范式的一部分，我们在大鼠P50–75日龄时评估其对新奇环境的运动反应（Stead等人，2006，Behav Genet. 36: 697–712）。我们还使用高架十字迷宫（Elevated Plus Maze, EPM；5分钟测试）检测了成年大鼠的类焦虑行为：bHR/bLR组为P160-P167日龄，bIR组为P65-75日龄，检测指标为大鼠在开放臂中停留的时间占比（Aurbach等人，2015，Proc Natl Acad Sci USA. 112: 11953–11958）。本文同步提供了上述行为学测试结果与基因表达数据。
总体实验设计：本项RNA测序研究检测了第37代成年雄性bHR大鼠与bLR大鼠（每组n=6），以及通过杂交F37代bHR与bLR大鼠获得的、对新奇环境表现出中等运动反应的成年雄性“中等选育反应型（bIR）”大鼠（n=6）的海马体基因表达情况。
行为学测试：作为选育范式的一部分，我们在大鼠P50–75日龄时评估其对新奇环境的运动反应（实验方案：Stead等人，2006，Behav Genet. 36: 697–712）。我们使用高架十字迷宫（EPM；5分钟测试）检测了成年大鼠的类焦虑行为：bHR/bLR组为P160-P167日龄，bIR组为P65-75日龄，检测指标为大鼠在开放臂中停留的时间占比（实验方案：Aurbach等人，2015，Proc Natl Acad Sci USA. 112: 11953–11958）。
处死与RNA提取：所有大鼠均在成年后处死（bHR/bLR组为P160-P167日龄，bIR组为P126-134日龄），采用快速断头法处死。随后在冰上剥离全海马体，快速冷冻后保存于-80℃。使用Qiagen AllPrep DNA RNA miRNA Universal Kit 50提取核苷酸。使用Nanodrop分光光度计检测提取RNA的总浓度与质量（浓度范围285-432 ng/ul，260/280比值范围1.61-1.80），随后将样本送至密歇根大学DNA测序核心实验室（https://seqcore.brcf.med.umich.edu）。
RNA测序：在测序核心实验室，使用TapeStation自动化样本处理系统（Agilent, Santa Clara, CA）再次评估RNA质量，仅保留RNA完整性数（RNA integrity numbers, RINs）>8的样本用于后续分析。使用0.1-3μg的总RNA与Illumina TruSeq Stranded mRNA Library Preparation kit（货号RS-122-2101、RS-122-2102，Illumina, San Diego, CA）构建cDNA文库。
最终构建的cDNA文库再次通过TapeStation（Agilent）以及使用Kapa’s library quantification kit for Illumina Sequencing platforms（货号KK4835, Kapa Biosystems,Wilmington MA）进行qPCR检测质量。随后使用cBot自动化簇生成系统（Illumina）对样本进行聚类以实现克隆扩增。将样本与HiSeq 2000（Illumina）的测序玻片（"flow cell"）进行杂交，每泳道上样6.66个样本，使用v3试剂以高输出模式进行100个循环的双端测序。
RNA测序数据预处理：测序与双索引拆分（demultiplexing）完成后，使用SubRead aligner（Liao等人，2014，Bioinformatics. 30: 923–930）将RNA测序读数比对至大鼠基因组（Rnor_6.0），除插入缺失检测参数外均采用默认设置（可检测的插入缺失最大长度=0）。随后使用featureCounts程序（Liao等人，2014，Bioinformatics. 30: 923–930）基于ENSEMBL注释（Ensembl v.81）生成每个样本的基因水平RNA测序计数汇总表。对该计数汇总数据集进行过滤，剔除未满足“至少4个样本计数≥10”阈值的基因行，同时剔除无官方基因符号注释的行。
本研究当前分析使用了由voom()函数（R包limma；Ritchie等人，2015，Nucleic Acids Res. 43: e47）生成的每个样本的log₂（每百万片段数）基因水平汇总输出结果。质量控制步骤包括：1）通过箱线图可视化所有受试者的log₂（以2为底）转换后的转录本表达整体分布；2）检查每个受试者的总读数均值与标准差；3）绘制受试者间相关矩阵以识别异常样本（R<0.95）。本研究未发现异常样本。