Genome-Wide Significant Loci for Morphine-Induced Locomotion or Naloxone-Induced Withdrawal

Time series of QTLs for morphine-induced locomotor response.

Locomotion data were quantile normalized and mapped against WGS-based genotype using GEMMA in GeneNetwork.org. QTL for male (a) and female (b) are stacked up by increasing 15 min time intervals. The dotted lines represent the genome-wide significance level of ∼3.77. The color shaded areas indicate consistent associations across time bins for the Chr 10 locus (red) and the Chr 16 locus (blue). Strong correlation in morphine-induced locomotor response between male and female BXD strains shown in Pearson correlation scatterplots during the 45–60 min (c) and 165–180 min (d) time frames.

QTLs of morphine-induced locomotion between 45–60 min on Chr 10.

(a) QTL for males with a peak –log10P of 10.06 (n = 63 strains). (b) QTL for females with a peak –log10P of 9.60 (n = 64 strains). (c) Zoomed-in view of QTL in males. (d) Zoomed-in view of QTL in females. (e) Cis-eQTL in the NAc of the BXDs, with a peak –log10P of 10.01 (n = 34 strains). (f) Cis-eQTL in the hippocampus of the BXDs, with a peak –log10P 9.7 (n = 67 strains) on Chr 10 at 5.6Mb. (g) Oprm1 neighborhood in BXD family with SNP densities. (h) Map of Haplotypes showing gene expression. The “B” of BXD is the mother, and “D” is the father. GEMMA with LOCO was used for all association mapping.

QTLs of morphine-induced locomotion between 165–180 min on Chr 16.

(a) QTL in males with a peak –log10P of 4.28 (n = 63 strains). (b) QTL for females with a peak –log10P of 10.56 (n = 64 strains). (c) Zoomed-in view of the QTL in males. (d) Zoomed-in view of the QTL in females. (e) Cis-eQTL in the striatum of the BXDs, with a peak –log10P of 4.43. (f) Cis-eQTL in the VTA of the BXDs, with a peak –log10P of 4.06. (g) Histogram of the normalized expression of Fgf12 in striatum and zoomed in view of the QTL region. (h) Histogram of the normalized expression of Fgf12 in VTA and zoomed in view of the QTL region. GEMMA with LOCO was used for all association mapping.

List of Candidate Genes

Chr 16 locus at 165-180 min after morphine injection.

(a) Zoomed in view of the Chr 16 locus, from 26-29 Mb, showing individual SNPs (blue dots) and genes (purple horizontal lines) in the region. (b) A screenshot of GeneNetwork showing SNPs that inhabit this region. Almost all B vs D SNPs (orange hash along x-axis) are restricted to two regions.

Epistatic Interaction Among Genotype Combinations.

(a) Males and (b) females with different combinations of the “B” (i.e., B/B) or “D” (i.e. D/D) genotypes yield different distances traveled after morphine injection over 120 mins. Distance traveled (m) between the two genotypes for each loci is shown for each timepoint in (c-f) males and (g-j) females.

Oprm1 and Fgf12 are positively correlated in rat NAc suggested by snRNA-seq.

(a) UMAP visualization of cell clusters from 4495 nuclei from rat nucleus accumbens core. (b) Dot plot showing the expression level of cell type marker genes in cell clusters. The shade of dots denotes normalized and scaled average expression and the size of dots denotes the percentage of cells expressing the gene in each cell cluster. (c-d) Violin plots indicating the normalized and scaled expression level of Oprm1 (c) and Fgf12 (d) across cell clusters. (e-g) Scatter plots showing the correlation relationship between Oprm1 and Fgf12 in all cells (e), D1-MSN-3 (f), and D1-MSN-2 (g). Nuclei counts (n), Pearson’s correlation coefficients (r), and p-values (p) are labeled on each panel. UMAP, Uniform Manifold Approximation and Projection; D1-MSN, Drd1-expressing medium spiny neuron; D2-MSN, Drd2-expressing medium spiny neuron; GABA, GABAergic inhibitory neuron; Glut, glutamatergic excitatory neuron; Ol, oligodendrocyte; OPC, oligodendrocyte precursor cell; Ast, astrocyte, Mg, microglial cells

Modeling the mechanistic interactions between genetic variants and phenotypes using Bayesian network.

This network illustrates the relationship between genetic variants in the Chr 10 and Chr 16 QTL regions and the differential expression of candidate genes Oprm1 and Fgf12 in the BXD family. Additional gene expression data of MAP kinases were retrieved from GeneNetwork.org. Morphine induced locomotion responses at different time bins are also included in the network. This causal hypothesis was developed using the Bayesian network framework available in Genenetwork, where the arrow is indicative of the direction of the causal relationship. Additional genes included in the input of the network construction but had no connection to the final network were excluded in the illustration.

QTLs for naloxone-induced morphine withdrawal responses in males and female BXD mice.

Behavior data were quantile normalized and mapped against WGS-based genotypes using GEMMA in GeneNetwork.org. (a) Number of jumps 15 minutes after naloxone injection in males. (b) Number of jumps 15 minutes after naloxone injection in females. (c) Change in locomotion measured by the last 15 min of morphine locomotion response minus the first 15 min after naloxone injection in males, with a peak on Chr 10 and a peak on Chr 16 (d) Change in locomotion measured by the last 15 min of morphine locomotion response minus the first 15 min after naloxone injection in females, with a peak on Chr 16. (e) Horizontal activity (distance traveled) 0–15 min after naloxone injection for males. (f) Horizontal activity (distance traveled) 0–15 min after naloxone injection for females, with a peak on Chr 6, and a peak on Chr 12. (g) Number of beam breaks in an open field 0–15 min after naloxone injection in females, with a peak on Chr 6 and a peak on Chr 12. (h) Salivation level in females. Dashed lines: genome wide significance threshold. Dotted lines: threshold for suggestive significance.

Distribution of raw locomotion data after morphine injection.

Distribution of raw data for locomotion after morphine injection at different time intervals in females (orange A-K) and males (blue L-V). Data for 135 to 150 minutes were lost for both sexes.

Distribution of quantile-normalized locomotion data after morphine injection.

Data are plotted for each time bin for both females (orange A-K) and males (blue L-V). Data for 135 to 150 minutes were lost for both sexes.

Pairwise linkage statistics across the genome.

(a) Genome-wide two-dimensional heat map showing all pairwise marker combinations, illustrating both additive (main) effects and epistatic (interaction) effects. The upper-left triangle displays LOD scores for interaction terms, while the rightmost column shows the main effect (single-locus) genome scan. (b) Zoomed view of linkage statistics between chromosomes 10 and 16. The red band along the bottom represents a strong additive effect on chromosome 16.

Quality control (QC) of snRNA-seq and number of cells per cluster.

(a-e) Violin plots showing QC parameters used for selecting high-quality nuclei in each sample: (a) Unique gene numbers per nuclei (nFeature_RNA). (b) Log-transformed total read numbers per nuclei (log-nCount_RNA). (c) Percentage of mitochondrial gene reads. (d) Percentage of small 40s ribosomal (Rps) gene reads. (e) Percentage of large 60s ribosomal (Rpl) gene reads. (f) Number of nuclei per cell cluster. D1-MSN, Drd1-expressing medium spiny neuron; D2-MSN, Drd2-expressing medium spiny neuron; GABA, GABAergic inhibitory neuron; Glut, glutamatergic excitatory neuron; Ol, oligodendrocyte; OPC, oligodendrocyte precursor cell; Ast, astrocyte, Mg, microglial cells.

Levels of Oprm1, Fgf12, and MAP kinases proteins in the brains of BXD parental strains and F1s.

Effect of genotype on associated MAP kinases and genes for both parental strains and F1 generations. The genotype effect shows the B allele is associated with high expression of Oprm1 and the MAP kinases Mapk8ip2, Map3k11, and Map3k12, but low expression of Fgf12, when compared to the D allele.

Gene Expression of D1 and D2 Type Medium Spiny Neuron Subtypes.

Expression of Oprm1 and Fgf12 in acute and repeated nucleus accumbens and in primary striatal culture from the Ratlas database. (a) Expression of Oprm1 in acute NAc. (b) Expression of Fgf12 in acute NAc. (c) Expression of Oprm1 in both acute and repeated NAc. (d) Expression of Fgf12 in both acute and repeated NAc. (e) Expression of Oprm1 in primary striatal culture. (f) Expression of Fgf12 in primary striatal culture.

Genome-wide significant loci associated with morphine and naloxone responses (quantile-normalized).

List of Locomotion Trait IDs.

Naloxone Phenotypes vs. Genotypes.

The turquoise points show morphine-induced locomotion (distance traveled in cm) between 165–180 min after injection. Orange points show the change in locomotion measured by the last 15 min of morphine locomotion response minus the first 15 min after naloxone injection. (a) Effect of genotype on two naloxone phenotypes in males. (b) Effect of genotype on number of beam breaks in an open field 0–15 min after naloxone injection in males. (c) Effect of genotype on two naloxone phenotypes in females. (d) Effect of genotype on the number of beam breaks in an open field 0–15 min after naloxone injection in females.