Introduction
Responses to drugs of abuse vary among individuals and are genetically influenced ( Mistry et al., 2014). Such drugs stimulate the brain reward pathway ( Gardner, 2011). Analysis of genetic variation in the behavioral and neurobiological processes can reveal molecular mechanisms that regulate or mediate the response to drugs, and neurobiological changes associated with chronic drug use. Many of the genes and signaling processes involved in the reward pathway in humans are conserved in mice ( Adinoff, 2004), and mouse genetic methods provide an efficient means of identifying these genes and loci.
Quantitative trait loci (QTL) analysis of recombinant inbred mouse strains (RIS) integrates phenotype and genotype data and is a widely used approach for studying the genetic basis of drug effects and addiction susceptibility ( Spence et al., 2005). The BXD RIS, derived from C57BL/6J (B) and DBA/2J (D), are a well-established genetic reference population used in behavioral neuroscience studies for mapping complex traits associated with drug use ( Crabbe et al., 1996; Dickson et al., 2016; Gora-Maslak et al., 1991; Peirce et al., 2004; Plomin et al., 1991).
In the present study we reanalyzed data from high throughput behavioral phenotyping of BXD RIS in the presence of morphine ( Philip et al., 2010). Our purpose was to identify additional morphine response related genetic loci beyond those initially reported. We employed composite interval mapping for two behavioral phenotypes, rearing and locomotion in response to morphine, and identified a QTL on mouse Chromosome 7 that was masked by a major QTL on Chromosome 10.
Methods
Experimental protocols have been described elsewhere ( Philip et al., 2010). BXD data were generated in the laboratory of Dr. Charles D. Blaha at the University of Memphis and obtained from GeneNetwork.org.
QTL mapping
QTL mapping was performed using GeneNetwork 1.0 with a composite interval mapping function using 2000 permutations. This is a forward regression approach in which a single locus with a major effect is included in a mapping model that scans for additional additive effects and interactions with the major locus. The technique will miss higher order interactions among loci that are not detectable as main effects, but is effective when a large, consistently observed major effect locus is present. Strain mean scores were Winsorized when statistical outliers were present. We first determined which marker had the highest LRS value, using a marker regression analysis and then performed composite interval mapping, controlling for the Chromosome 10 SNP rs3721803, one of the 3 markers with the highest LRS scores.
Bioinformatics
The MGI database ( http://www.informatics.jax.org/) was used to find information about SNPs and strain polymorphisms occurring within the Chromosome 7 interval (queried –June 20, 2016). This database now includes variants detected in the sequencing of 17 mouse genomes ( Keane et al., 2011). The 3 SNPs located at the Chromosome 7 QTL peak (rs13479451, rs3724540, rs6386601) are at ~114.5 Mb in GeneNetwork and ~107.6 Mb in the MGI and NCBI databases ( http://www.ncbi.nlm.nih.gov/gene). In GeneNetwork, the relevant QTL interval on Chromosome 7 was 110–125 Mb whereas this region corresponds to 103–118 Mb in the MGI database. As finer mapping methods are developed, the exact interval location as reported here, may change. DAVID version 6.8 ( https://david.ncifcrf.gov/) was used to obtain functional annotations and pathway information for the genes within the interval and GeneWeaver 1.0 ( http://www.geneweaver.org/) to identify other drug-related phenotypes associated with the candidate genes.
Results
Composite interval mapping
As reported by Philip et al. (2010), whole genome scans produce robust QTLs mapping to Chromosome 10 at 0–30 Mb ( Figure 1A-B). The positional and functional best gene candidate is the Oprm1 gene which encodes the opioid G-protein coupled receptor mu 1 ( Philip et al., 2010) which has been previously detected ( Bergeson et al., 2001; Doyle et al., 2014).
Figure 1.
( A) Trait data for locomotion (distance traveled) for 64 BXD strains post morphine injection (Trait id 11851). ( B) Whole genome scan showing a robust QTL on Chromosome 10 (Trait id 11851, SNP rs372180). The x-axis represents chromosome number and megabase position and the y-axis represents the likelihood ratio statistic (LRS) of linkage. Blue lines indicate LRS across the genome. The pink and gray horizontal lines are approximate threshold values which are used to assess whether a peak is significant (P<0.05) or suggestive (P<0.63), respectively. Red and green lines represent the additive genetic contribution: red lines indicate negative values (C57BL/6J alleles increasing trait values) and green lines indicate positive values (DBA/2J alleles increasing trait values). Gray lines are shown when the parental strain is unknown. The yellow bars represent the relative frequency of peak LRS at a given location from 2000 bootstrap resamples ( C) Composite interval mapping of locomotion (Trait id 11851) after correcting for the peak on Chromosome 10 reveals a significant QTL on Chromosome 7.
We remapped these data for each time point using composite interval mapping to identify QTLs potentially masked by this large QTL. This revealed a second significant QTL on Chromosome 7 for locomotion (LRS= 18.0, 114.7 Mb; Figure 1C) and, in an overlapping position, for rearing (LRS=16.5, 114.3 Mb). We also observed a time dependent decrease of the peak LRS values for both traits after the 60–75 minute time interval post-morphine injection. A suggestive QTL for the rearing trait data was already apparent on Chromosome 7 from the whole genome scan, but this was not the case for the locomotion trait data ( Philip et al., 2010).
The area under the QTL peak contains 280 protein coding genes (MGI Chromosome 7: 103–118 Mb, GeneNetwork Chromosome 7: 110–125 Mb; database query-June 20, 2016, Supplementary worksheet 2). Of these genes, 162 are associated with olfactory receptors. Of the other genes with functional annotations, 11 are connected with neural processes ( Adm: neural tube development, Appb1, Tub, Calca, Cckbr, Cnag4: sensory perception and cognition, Insc, Arntl: neurogenesis, Tpp1: neuromuscular control, Rras2: regulation of neuron death and Pde3b: morphine addiction (KEGG pathway 05032).
There are 3 SNPs mapped at the location of the LRS peak and two positional candidates: Synaptogamin 9 ( Syt9: rs13479451, rs3724540) and PPFIA binding protein 2 ( Ppfibp2, rs6386601). Both of these genes differ between the parental strains (MGI database query – June 20, 2016). Syt9 functions in vesicle traffic and Ca2+ triggered exocytosis ( Ncbi, 2016b) and Ppfibp2 is involved in the regulation and development of neuronal synapses ( Ncbi, 2016a). These two candidates are also differentially expressed in the striatum of the brains of mice from strains with distinct opioid sensitivity ( Korostynski et al., 2006; GeneWeaver: Syt 9, geneset #86830; Ppfibp2, geneset #86906).
Conclusions
Genetic analysis of the BXD RIS resulted in the detection of an additional locus for morphine induced locomotor activity and rearing on Chromosome 7 which may be associated with activation of the reward system pathway in response to morphine treatment. Whether this effect is opioid-specific or also occurs with other classes of drugs is not yet clear. Alternatively, this QTL may also be related to general locomotor activity or nociception, a central nervous system response to potentially harmful stimuli. The best gene candidates within the QTL interval, besides the positional candidates Syt9 and Ppfibp2, are Calca and Cckbr (sensory perception of pain) and Pde3b (morphine addiction).
Data availability
The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2016 Crusio WE et al.
All data are available in GeneNetwork ( www.genenetwork.org; see supplementary worksheet 1 for trait-ids and descriptions). Trait IDs: Locomotion: 11843, 11833, 11844, 11834, 11845, 11835, 11846, 11847, 11851, 11852, 11836, 11837, 11838, Rearing: 11884, 11885, 11886, 11887, 11888, 11878, 11854.
Data are also available in the Supplementary material.
Copyright: © 2016 Crusio WE et al. This work is licensed under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/3.0/) (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.