qPCR: Bmp4 expression in the hippocampus of selectively-bred High Responder (bHR) and Low Responder (bLR) rats at age P14 and adulthood

This data release accompanies the manuscript <b>"Genetic liability for internalizing versus externalizing behavior manifests in the developing and adult hippocampus: Insight from a meta-analysis of transcriptional profiling studies in a selectively-bred rat model"</b> by Birt, Hagenauer, et al. <br>https://doi.org/10.1016/j.biopsych.2020.05.024<br><b>Overview:</b>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 quantitative polymerase chain reaction (qPCR) study examined the expression of Bone morphogenetic protein 4 (Bmp4) in the hippocampus, a brain region critical for emotional regulation, in bHR and bLR rats. Whole hippocampus tissue was dissected from male bHR and bLR rats aged P14 and P90 (n=6/group per age, generations F51 and F55). Results indicate that Bmp4 was more highly expressed in the hippocampus of bLRs than bHRs at both ages.<br><br><b>Detailed Methods:</b><br><b>Animal husbandry and tissue collection: </b>Male bHR and bLR rats were sacrificed at ages P14 and P90 (n=6/group per age). For the P14 collection, F55 generation rats were sacrificed within 3-5 min of separation from the dam. The F51 generation rats designated for the P90 collection were weaned, housed, and tested for locomotor activity in a novel field as part of our standard selective breeding paradigm (protocol: Stead et al. 2006, Behav Genet. 36: 697–712) prior to sacrifice. Sacrifice was performed via rapid decapitation without anesthesia. Brains were immediately hemisected and flash frozen by submersion in -30C 2-methylbutane.<br><b>RNA extraction and cDNA synthesis: </b>Brains were stored at -80C for fewer than 6 months before processing. Hippocampus was dissected from one hemisphere and homogenized using a QIAshredder kit (Qiagen #79654), and RNA was extracted using an RNeasy Mini Kit (Qiagen #74104). cDNA was synthesized using a 20 μL reaction containing 400 ng of RNA template (iScript cDNA Synthesis Kit, Biorad#1708891).<br><b>qPCR:</b> The primers were custom-designed to target Bmp4 (ACC# NM_012827.2; forward primer: 5’-CCCTGGTCAACTCCGTTAAT-3’, start = 1214; reverse primer: 5’-AACACCACCTTGTCGTACTC-3’, start = 1319) and the reference gene Gapdh (ACC# NM_017008.4; forward primer: 5’- GTTTGTGATGGGTGTGAACC-3’, start = 459; reverse primer: 5’-TCTTCTGAGTGGCAGTGATG-3’, start = 628). Calibration curves for the Bmp4 and Gapdh primers were constructed using a standard dilution analysis of stock cDNA that had been previously synthesized from a mixture of adult and P14 bHR and bLR hippocampi (H20, 0.1 uL, 0.5 uL, 1 uL, 2 uL) in triplicate using iQTM SYBR® Green Supermix. The bLR/bHR samples were then analyzed using a similar procedure in triplicate, with the samples from each time point processed within a separate batch.<br><b>Data analysis: </b>The calibration curves revealed efficiencies close to 1 (Bmp4: R2=0.98, Gapdh: R2=0.99), therefore the sample data for each time point was analyzed using the traditional Livak method (Livak and Schmittgen 2001, Methods. 25: 402–408; Yuan et al. 2006, BMC Bioinformatics. 7: 85). After averaging the triplicate quantification cycle (Cq) values for each sample for each probe, the data were normalized by subtracting the Cq for the reference gene (Gapdh) from the target (Bmp4) for each sample (ΔCq). Group differences in ΔCq were assessed using Welch’s two sample t-test (Yuan et al. 2006, BMC Bioinformatics. 7: 85). Group differences in the Cq values for the reference gene (Gapdh) were also examined as a control (Yuan et al. 2006, BMC Bioinformatics. 7: 85).<br><b>Results:</b>bLRs showed greater hippocampal Bmp4 expression than bHRs at both P14 and adulthood (Welch’s t-test: P14: Log(2)FC=-3.74, T(5.60)=-6.10, p=0.00115; P90: T(8.74)=-6.87, p=8.44E-05).<br><b>Content of the Data Release:</b>The bHR and bLR qPCR data for the target gene (Bmp4) and reference gene (Gapdh) are released in an Excel file (“HRLR_Bmp4_DataRelease_v2.xlsx”) that includes a spreadsheet for the dataset from each age group (P14, P90). The sample from each rat (denoted with an ID number in the sample column: #1, #2, etc) was analyzed in triplicate for each probe. The number of qPCR reaction cycles that were necessary to reach threshold fluorescence (quantitation cycle or Cq) is provided for both the Bmp4 and Gapdh probes for each triplicate. When interpreting the results, lower Cq values mean higher initial copy numbers of the target.<br>