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Summary
The biogenic amine serotonin (5-HT, 5-hydroxytryptamine) exerts powerful, modulatory control over multiple physiological functions in the brain and periphery, ranging from mood and appetite to vasoconstriction and gastrointestinal motility. In order to gain insight into shared and distinct molecular and phenotypic networks linked to variations in 5-HT homeostasis, we capitalized on the stable genetic variation present in recombinant inbred (RI) mouse strains. This family of strains, all derived from crosses between C57BL/6J and DBA/2J (BXD) parents, represent a unique, community resource with ∼40 years of assembled phenotype data that can be exploited to explore and test causal relationships in silico. We determined levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) from whole blood, midbrain, and thalamus/hypothalamus (diencephalon) of 38 BXD lines and both sexes. All 5-HT measures proved highly heritable in each region, although both gender and region significantly impacted between-strain correlations. Our studies identified both expected and novel biochemical, anatomical, and behavioral phenotypes linked to 5-HT traits, as well as distinct quantitative trait loci (QTL). Analyses of these loci nominate a group of genes likely to contribute to gender- and region-specific capacities for 5-HT signaling. Analysis of midbrain mRNA variations across strains revealed overlapping gene expression networks linked to 5-HT synthesis and metabolism. Altogether, our studies provide a rich profile of genomic, molecular and phenotypic networks that can be queried for novel relationships contributing risk for disorders linked to perturbed 5-HT signaling.
Experiment design
RNA Isolation and Microarray Analysis
Midbrain RNA was extracted using an RNeasy mini kit (Qiagen) according to manufacture's instructions. Samples were fragmented and hybridized using Agilent hybridization kit (#5188-5242). Arrays were hybridized for 17 hours at 65 C. Microarray analysis was performed on an Agilent-028005 SurePrint G3 Mouse GE 8×60K platform in the VANTAGE (Vanderbilt Technologies for Advanced Genomics) core laboratory. To make gene expression data comparable across samples, the processed signal values (gProcessedSignal) were subjected to a quantile normalization. Next, the data were log transformed and then gene-wise normalized using Z-scores. Data were rescaled to a mean of 8 units with a standard deviation of 2 units (the 2Z + 8 normalization used in GeneNetwork).
A subset of ∼50 probes with bimodal Mendelian patterns of expression were used as internal (sample-specific) genetic markers. These Mendelian probes are almost always associated with QTLs with LOD scores above 20 that map precisely to location of the cognate gene. (Rare bimodal probes with trans expression QTLs are usually associated with annotation errors or genome assembly errors and do not materially affect this QC step.) Our transcriptome studies were blinded to strain ID and then assigned based on the internal cis eQTL markers noted above. The final verified data has been deposited in GEO and in GN and reflects this systematic genotype QC process. Transcriptome-derived gene networks were generated using Cytoscape 2.8.3 (Cytoscape Consortium). Input data files (node and network) were generated from WebQTL correlation analysis module.
About cases
BXD RI mice were obtained from the Oak Ridge National Laboratories (ORNL) and the Jackson Laboratory (JAX). A total of 126 BXD recombinant inbred mice from 38 strains were used for neurochemical analyses. The average age of mice used was 74 days (range from 51 to 89 days), and comprised a total of 67 male and 59 female mice, respectively. Females were used irrespective of estrous cycle though they were never exposed to males or male bedding after weaning, typically required to initiate synchronized estrous cycling. A total of 129 male mice from 37 strains, selected independently of the 38 strains chosen for 5-HT analyses based on availability, were used for to obtain transcriptome profiles. All lines utilized are provided in Suppl File 1. Animals were grouped house and maintained on a 12:12 light/dark cycle with food and water available ad libitum.
| Index | Sample ID | Sex | Strain Assignment L14 |
|---|---|---|---|
| 1 | 916-DCA-68 | M | BXD1 |
| 2 | 916-DCA-165 | M | BXD1 |
| 3 | 916-DCA-147 | M | BXD1 |
| 4 | 916-DCA-114 | M | BXD1 |
| 5 | 916-DCA-107 | M | BXD11 |
| 6 | 916-DCA-187 | M | BXD11 |
| 7 | 916-DCA-67 | M | BXD11 |
| 8 | 916-DCA-158 | M | BXD12 |
| 9 | 916-DCA-35 | M | BXD12 |
| 10 | 916-DCA-36 | M | BXD12 |
| 11 | 916-DCA-140 | M | BXD14 |
| 12 | 916-DCA-45 | M | BXD14 |
| 13 | 916-DCA-99 | M | BXD14 |
| 14 | 916-DCA-191 | M | BXD14 |
| 15 | 916-DCA-79 | M | BXD15 |
| 16 | 916-DCA-38 | M | BXD15 |
| 17 | 916-DCA-41 | M | BXD15 |
| 18 | 916-DCA-88 | M | BXD15 |
| 19 | 916-DCA-141 | M | BXD16 |
| 20 | 916-DCA-97 | M | BXD16 |
| 21 | 916-DCA-18 | M | BXD16 |
| 22 | 916-DCA-179 | M | BXD16 |
| 23 | 916-DCA-16 | M | BXD18 |
| 24 | 916-DCA-23 | M | BXD18 |
| 25 | 916-DCA-12 | M | BXD18 |
| 26 | 916-DCA-175 | M | BXD18 |
| 27 | 916-DCA-8 | M | BXD19 |
| 28 | 916-DCA-174 | M | BXD19 |
| 29 | 916-DCA-78 | M | BXD19 |
| 30 | 916-DCA-24 | M | BXD19 |
| 31 | 916-DCA-105 | M | BXD2 |
| 32 | 916-DCA-57 | M | BXD2 |
| 33 | 916-DCA-166 | M | BXD2 |
| 34 | 916-DCA-181 | M | BXD2 |
| 35 | 916-DCA-100 | M | BXD20 |
| 36 | 916-DCA-82 | M | BXD20 |
| 37 | 916-DCA-72 | M | BXD20 |
| 38 | 916-DCA-131 | M | BXD20 |
| 39 | 916-DCA-63 | M | BXD21 |
| 40 | 916-DCA-13 | M | BXD21 |
| 41 | 916-DCA-15 | M | BXD21 |
| 42 | 916-DCA-109 | M | BXD22 |
| 43 | 916-DCA-101 | M | BXD22 |
| 44 | 916-DCA-69 | M | BXD22 |
| 45 | 916-DCA-104 | M | BXD22 |
| 46 | 916-DCA-125 | M | BXD24 |
| 47 | 916-DCA-33 | M | BXD24 |
| 48 | 916-DCA-108 | M | BXD24 |
| 49 | 916-DCA-44 | M | BXD24 |
| 50 | 916-DCA-80 | M | BXD27 |
| 51 | 916-DCA-151 | M | BXD28 |
| 52 | 916-DCA-47 | M | BXD28 |
| 53 | 916-DCA-27 | M | BXD28 |
| 54 | 916-DCA-154 | M | BXD28 |
| 55 | 916-DCA-71 | M | BXD29 |
| 56 | 916-DCA-54 | M | BXD29 |
| 57 | 916-DCA-122 | M | BXD29 |
| 58 | 916-DCA-51.1 | M | BXD29 |
| 59 | 916-DCA-144 | M | BXD31 |
| 60 | 916-DCA-76 | M | BXD31 |
| 61 | 916-DCA-164 | M | BXD31 |
| 62 | 916-DCA-37 | M | BXD32 |
| 63 | 916-DCA-89 | M | BXD32 |
| 64 | 916-DCA-25 | M | BXD32 |
| 65 | 916-DCA-160 | M | BXD33 |
| 66 | 916-DCA-65 | M | BXD33 |
| 67 | 916-DCA-128 | M | BXD33 |
| 68 | 916-DCA-73 | M | BXD34 |
| 69 | 916-DCA-103 | M | BXD34 |
| 70 | 916-DCA-137 | M | BXD34 |
| 71 | 916-DCA-29 | M | BXD38 |
| 72 | 916-DCA-112 | M | BXD38 |
| 73 | 916-DCA-84 | M | BXD38 |
| 74 | 916-DCA-132 | M | BXD38 |
| 75 | 916-DCA-156 | M | BXD38 |
| 76 | 916-DCA-130 | M | BXD39 |
| 77 | 916-DCA-117 | M | BXD39 |
| 78 | 916-DCA-111 | M | BXD39 |
| 79 | 916-DCA-14 | M | BXD40 |
| 80 | 916-DCA-188 | M | BXD40 |
| 81 | 916-DCA-192 | M | BXD40 |
| 82 | 916-DCA-90 | M | BXD40 |
| 83 | 916-DCA-52 | M | BXD42 |
| 84 | 916-DCA-2 | M | BXD42 |
| 85 | 916-DCA-126 | M | BXD42 |
| 86 | 916-DCA-81 | M | BXD42 |
| 87 | 916-DCA-118 | M | BXD44 |
| 88 | 916-DCA-42 | M | BXD44 |
| 89 | 916-DCA-92 | M | BXD44 |
| 90 | 916-DCA-39 | M | BXD49 |
| 91 | 916-DCA-98 | M | BXD49 |
| 92 | 916-DCA-83 | M | BXD49 |
| 93 | 916-DCA-85 | M | BXD5 |
| 94 | 916-DCA-6 | M | BXD5 |
| 95 | 916-DCA-142 | M | BXD5 |
| 96 | 916-DCA-11 | M | BXD55 |
| 97 | 916-DCA-121 | M | BXD55 |
| 98 | 916-DCA-77 | M | BXD55 |
| 99 | 916-DCA-123 | M | BXD6 |
| 100 | 916-DCA-28 | M | BXD6 |
| 101 | 916-DCA-161 | M | BXD6 |
| 102 | 916-DCA-124 | M | BXD6 |
| 103 | 916-DCA-189 | M | BXD62 |
| 104 | 916-DCA-143 | M | BXD62 |
| 105 | 916-DCA-116 | M | BXD62 |
| 106 | 916-DCA-159 | M | BXD73 |
| 107 | 916-DCA-40 | M | BXD73 |
| 108 | 916-DCA-169 | M | BXD73 |
| 109 | 916-DCA-120 | M | BXD8 |
| 110 | 916-DCA-64 | M | BXD8 |
| 111 | 916-DCA-93 | M | BXD8 |
| 112 | 916-DCA-148 | M | BXD86 |
| 113 | 916-DCA-55 | M | BXD86 |
| 114 | 916-DCA-170 | M | BXD86 |
| 115 | 916-DCA-50 | M | BXD89 |
| 116 | 916-DCA-127 | M | BXD89 |
| 117 | 916-DCA-32 | M | BXD89 |
| 118 | 916-DCA-62 | M | BXD89 |
| 119 | 916-DCA-53 | M | BXD9 |
| 120 | 916-DCA-138 | M | BXD9 |
| 121 | 916-DCA-180 | M | BXD9 |
| 122 | 916-DCA-46 | M | BXD9 |
| 123 | 916-DCA-74 | M | BXD96 |
| 124 | 916-DCA-110 | M | BXD96 |
| 125 | 916-DCA-94 | M | BXD96 |
| 126 | 916-DCA-49 | M | BXD96 |
| 127 | 916-DCA-119 | M | BXD98 |
| 128 | 916-DCA-135 | M | BXD98 |
| 129 | 916-DCA-58 | M | BXD98 |
About tissue
All tissue procurements were performed under approved IACUC protocols of the Oak Ridge National Laboratory or Vanderbilt University. Brains were procured following rapid decapitation in the absence of anesthesia to limit artifactual alterations in 5-HT, 5-HIAA and mRNA levels. Trunk blood was obtained from the same animals used for brain harvests. Regions dissected from brain were determined based on landmarks provided by the Paxinos and Watson Atlas of the Mouse Brain (Watson and Paxinos 2010). Midbrain was collected to provide measures of 5-HT and metabolite levels in the region of the dorsal and median raphe nuclei and their local projects and was defined as brain tissue lying between the anterior and posterior margins of the inferior and superior colliculi, respectively (AP approximately -3.25 to -5.02 mm relative to Bregma). The diencephalon, constituted primarily by the thalamus and hypothalamus, was isolated to provide a measure of 5-HT and metabolite levels in a significant projection area of raphe neurons, and was defined as tissue lying between the anterior margin of the third ventricle and the anterior margin of the midbrain segment noted above, minus the basal ganglia (AP approximately -0.80 to -3.25 mm relative to Bregma). For assessment of 5-HT and 5-HIAA levels, sections were homogenized, using an Omni Tissue Homogenizer, in 100-750 μl of 0.1M trichloroacetic acid (TCA), 10 mM sodium acetate, 0.1 mM EDTA, 5 ng/ml isoproterenol (as internal standard) and 10.5 % methanol (pH 3.8). Whole blood from BXD mice was collected in 1.5 mL venoject tubes containing dry EDTA. Equal amounts of sample were mixed with extraction solvent (0.8 M perchloric acid, 0.1 M ascorbic acid and 10 mM EDTA), vortexed for 15 seconds and spun in a microfuge at 10,000 ×g for 10 minutes with supernatants collected. 5-HT and 5-HIAA were determined by HPLC through the Neurochemistry Core of the Vanderbilt Brain Institute, utilizing an Antec Decade II (oxidation: 0.5) electrochemical detector operated at 33° C. Twenty μl samples of the supernatant were injected using a Water 717+ autosampler onto a Phenomenex Nucleosil (5u, 100A) C18 HPLC column (150 × 4.60 mm). Samples were eluted with a mobile phase consisting of 89.5% 0.1 M TCA, 10 mM sodium acetate, 0.1 mM EDTA and 10.5 % methanol (pH 3.8). Solvent was delivered at 0.6 ml/min using a Waters 515 HPLC pump. HPLC control and data acquisition were managed by Millennium 32 software.
About data processing
Statistical Analyses
Biochemical data were analyzed using standard analysis of variance (ANOVA) and correlation analysis (Stata 11, College Station, TX) with P < 0.05 taken as significant. As RI strains lack heterozygous samples and thus overestimate additive genetic variance, adjusted heritability (h2) of the traits were estimated using Hegmann and Possidente's method (Hegmann and Possidente 1981). Significance of heritability was calculated by the F-test comparing within-strain vs. between-strain variances. Quantitative trait locus mapping was performed using the WebQTL module of GeneNetwork (http://www.genenetwork.org). Quantitative trait loci (QTL) were calculated from the likelihood ratio statistic (LRS) and logarithm of odds (LOD) scores. Statistical significance was determined on data sets subjected to 2,000 permutations. Significant QTLs were defined as the LRS value that represents a genome-wide P value of 0.05. Suggestive QTLs were defined as having an LRS value a genome-wide P value of 0.63 since this value yields, on average, one false positive per genome scan (Lander and Kruglyak 1995). Thus, roughly one-third of scans at this threshold will yield no false positive, one-third will yield one false positive, and one-third will yield two or more false positives. Although a permissive threshold, such estimates call attention to loci that may derive from underpowered analyses and can be pursued with additional lines/approaches. Confidence intervals were determined as the location at which the LOD value is reduced by 1.5. A 1.5 LOD cutoff has been estimated to reflect a 95% confidence that genes driving the QTL lay within this range (Visscher et al. 1996; Dupuis and Siegmund 1999). Phenotypic correlation analyses were performed by comparing measured 5-HT traits with archived BXD phenotypes in the GeneNetwork database, with correlation coefficients and P values calculated using Spearman's rank tests (uncorrected P < 0.05 was considered significant).
To assess whether midbrain gene products identified as correlates of 5-HT homeostasis are likely to reflect cell-autonomous associations with neurochemical measures, we asked whether these genes are 1) exhibit colocalization in the adult mouse brain with Tph2 or Slc6a4 gene expression (http://mouse.brain-map.org) using the Anatomic Gene Expression Atlas and the NeuroBlast tool (Hawrylycz et al. 2011), or 2) expressed selectively in day 12.5 embryonic 5-HT neurons (flow-sorted, ePET-1:YFP positive), relative to non-ePET-1:YFP expressing cells in the midbrain (Wylie et al. 2010)
Online Data Access
All neurochemical measurements and midbrain gene expression data for the BXD strains reported in this study have been deposited into the GeneNetwork database at http://www.genenetwork.org. Neurochemical traits are stored under the category of “Central nervous system: Neurochemistry”. Our midbrain gene expression data set “VU BXD Midbrain Agilent SurePrint G3 Mouse GE (May12) Quantile” is archived under “Midbrain mRNA” with GN accession number GN381.
Acknowledgment
We acknowledge the expert support of Chris Svitek, Jane Wright, Qiao Han, Sarah Whitaker, Angela Steele, Tracy Moore-Jarrett, and Kathryn Lindler in general laboratory oversight. This work was supported by NIH Award MH096972 (RDB).