BioProject

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: 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.