The Netrin-1 receptor DCC is a regulator of maladaptive responses to chronic morphine administration

Abstract

Background: Opioids are the cornerstone of treatment for moderate to severe pain, but chronic use leads to maladaptations that include: tolerance, dependence and opioid-induced hyperalgesia (OIH). These responses limit the utility of opioids, as well as our ability to control chronic pain. Despite decades of research, we have no therapies or proven strategies to overcome this problem. However, murine haplotype based computational genetic mapping and a SNP data base generated from analysis of whole-genome sequence data (whole-genome HBCGM), provides a hypothesis-free method for discovering novel genes affecting opioid maladaptive responses.

Results: Whole genome-HBCGM was used to analyze phenotypic data on morphine-induced tolerance, dependence and hyperalgesia obtained from 23 inbred strains. The robustness of the genetic mapping results was analyzed using strain subsets. In addition, the results of analyzing all of the opioid-related traits together were examined. To characterize the functional role of the leading candidate gene, we analyzed transgenic animals, mRNA and protein expression in behaviorally divergent mouse strains, and immunohistochemistry in spinal cord tissue. Our mapping procedure identified the allelic pattern within the netrin-1 receptor gene (Dcc) as most robustly associated with OIH, and it was also strongly associated with the combination of the other maladaptive opioid traits analyzed. Adult mice heterozygous for the Dcc gene had significantly less tendency to develop OIH, become tolerant or show evidence of dependence after chronic exposure to morphine. The difference in opiate responses was shown not to be due to basal or morphine-stimulated differences in the level of Dcc expression in spinal cord tissue, and was not associated with nociceptive neurochemical or anatomical alterations in the spinal cord or dorsal root ganglia in adult animals.

Conclusions: Whole-genome HBCGM is a powerful tool for identifying genes affecting biomedical traits such as opioid maladaptations. We demonstrate that Dcc affects tolerance, dependence and OIH after chronic opioid exposure, though not through simple differences in expression in the adult spinal cord.

Figure 1

Opioid-induced hyperalgesia in 23 strains of inbred mice. For each strain, mice were tested for mechanical nociceptive thresholds at baseline and after 4 days of morphine treatment. Nociceptive thresholds after morphine treatment were divided by baseline values to obtain the fraction of baseline values. For each strain n = 8 mice. Mean values are displayed +/- S.E.M.

Figure 2

Whole-genome computational genetic mapping. The strain-specific mechanical OIH data was used for whole genome halplotypic mapping. The top panel provides the input data for the 23 strains used. The bottom panel provides the name of the genes to which the mapped haplotype blocks belong, the p-value for association of the block with the trait data, the maximum genetic effect attributable to the mapped block, and the color-coded haplotypes for each of the 23 strains. The three highlighted genes contain SNPs predicted to change amino acid sequence. In addition, chromosomal location of the mapped blocks is provided.

Figure 3

Spinal cord protein levels of DCC in wild type and heterozygous mice. Lumbar spinal cord tissue from wild type and heterozygous Dcc mice were analyzed using Western analysis. The quantification of the DCC specific band in the immunoblots is shown. For these experiments n = 5 mice per genotype. Data are displayed as mean values +/- S.E.M., ***p < 0.001.

Figure 4

The effects of chronic morphine treatment on Dcc wild-type and heterozygous mice. In these experiments littermate Dcc wild-type (C57BL/6 J) and Dcc heterozygous mice were tested using the same chronic morphine paradigm used for the inbred strain mapping experiments. Panel A: Mechanical OIH measurements; Panel B: Morphine dose–response relationships before and after chronic morphine treatment; Panel C: Physical dependence measured using naloxone-precipitated jumping behavior. For these experiments n = 11 mice per genotype. Data are displayed as mean values +/- S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Intact distribution and central projections of peptidergic and non-peptidergic nociceptors in Dcc heterozygous mice. Panel A: Triple immunolabeling experiment showing similar distribution of CGRP-immunoreactivity, substance P-immunoreactivity, and binding of isolectin B4 (IB4) in dorsal root ganglion sections from Dcc wildtype and heterozygous littermates. Panel B: Triple immunolabeling experiment indicating that in the spinal cord dorsal horn the typical laminar organization of central terminals of peptidergic (CGRP- and substance P-expressing) and non-peptidergic (IB4-binding) nociceptors is intact in Dcc heterozygous mice, compared to wildtype littermates.

Figure 6

Expression of Dcc in spinal cord tissue after chronic morphine treatment in C57/6 J and 129S1 mice. Panel A: The mRNA levels of Dcc were unchanged after chronic morphine treatment in both C57/6 J and 129S1 mice. Values are displayed as the mean ± SEM, n = 6. ns, no significant difference. Panel B: Western blot assay demonstrated that there was no significant difference in baseline levels of DCC in spinal cord tissue from C57/6 J and 129S1 mice. Panel C: The spinal cord protein levels of DCC were unchanged after chronic morphine treatment in both C57/6 J and 129S1 mice. Values are displayed as the mean ± S.E.M., n = 4. ns, no significant difference. Veh = vehicle, Mor = morphine treatment.