The melanocortin-1 receptor gene mediates female- specific mechanisms of analgesia in mice and humans. (2003) Proc. Natl. Acad. Sci USA. Mogil JS, Wilson SG, Chesler EJ, Rankin AL, Nemmani KV, Lariviere WR, Groce MK, Wallace MR, Kaplan L, Staud R, Ness TJ, Glover TL, Stankova M, Mayorov A, Hruby VJ, Grisel JE, Fillingim RB.

Dr. Jeffrey S. Mogil McGill University, Montreal

Background of Qualitative Sex Differences in Analgesia Swim stress-induced analgesia is reversed by NMDA Receptor antagonist (MK-801) in male mice only, suggesting that females have a descending pathway not mediated by NMDAR (4: 1993).

Background of Qualitative Sex Differences in Analgesia Swim stress-induced analgesia is reversed by NMDA Receptor antagonist (MK-801) in male mice only, suggesting that females have a descending pathway not mediated by NMDAR (4: 1993). The same sex-specific effect is observed in analgesia resulting from administration of  -opioid agonists (9: 1997).

Background of Qualitative Sex Differences in Analgesia Swim stress-induced analgesia is reversed by NMDA Receptor antagonist (MK-801) in male mice only, suggesting that females have a descending pathway not mediated by NMDAR (4: 1993). The same sex-specific effect is observed in analgesia resulting from administration of  -opioid agonists (9: 1997). A previous linkage study by the Mogil group implicates the distal portion of chromosome 8 as the QTL for swim stress- induced analgesia in mice (7: 1997).

Background of Qualitative Sex Differences in Analgesia Swim stress-induced analgesia is reversed by NMDA Receptor antagonist (MK-801) in male mice only, suggesting that females have a descending pathway not mediated by NMDAR (4: 1993). The same sex-specific effect is observed in analgesia resulting from administration of  -opioid agonists (9: 1997). A previous linkage study by the Mogil group implicates the distal portion of chromosome 8 as the QTL for swim stress- induced analgesia in mice (7: 1997). This paper looks at sex-specific  -opioid analgesia through QTL mapping (2003).

Stress-Induced Pain Inhibition Periaqueductal gray (PAG) of midbrain Brainstem Dorsal horn of spinal cord OW! Incoming pain information Nociceptor Modified pain information Actual or impending tissue damage in periphery

Abstract What was the research question of the Mogil group? Read the abstract above and click to reveal the answer. Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from κ-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mc1r) gene mediates κ-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered κ-opioid analgesia. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the κ-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human.

Abstract What was the research question of the Mogil group? Read the abstract above and click to reveal the answer. Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from κ-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mc1r) gene mediates κ-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered κ-opioid analgesia. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the κ-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human. What gene mediates female-specific κ-opioid analgesia in mice? Is this gene involved in sex differences in analgesia in humans, as well?

Hypothesis What was their hypothesis about the identity of the gene?

Hypothesis They believed it would be located somewhere in the distal portion of chromosome 8. Other than that, they had no idea what gene it would be! “I think nonhypothesis-driven research is probably more important than hypothesis-driven research.... You find genes that weren't high probability candidates beforehand.... We can find completely novel things, very surprising things." JSM

Stress-Induced Analgesia Differs Between Sexes NMDAR glutamate & aspartate Male Mouse: pain inhibition mediated by NMDAR opioid receptors Modified pain information

Stress-Induced Analgesia Differs Between Sexes ??? receptor NMDAR estrogen receptors glutamate & aspartate Male Mouse: pain inhibition mediated by NMDAR Female Mouse: an additional pathway mediated by an unknown receptor opioid receptors Modified pain information

Figure 1: Research Question Looking at Figure 1, what question was the Mogil group trying to answer when they designed this study?

Figure 1: Research Question What region of the genome contains genes that mediate female-specific stress-induced analgesia? Looking at Figure 1, what question was the Mogil group trying to answer when they designed this study? What method was used to answer this question?

Figure 1: Research Question What region of the genome contains genes that mediate female-specific stress-induced analgesia? Looking at Figure 1, what question was the Mogil group trying to answer when they designed this study? What method was used to answer this question? Quantitative trait locus (QTL) mapping

Figure 1: Quantitative Trait Locus (QTL) Mapping Microsatellites used as markers for linkage mapping of a certain trait –Microsatellite: dinucleotide repeats of variable lengths –At the same microsatellite locus on the genome, the number of repeats varies between individuals (or, in this case, between strains) “The technique is blind. The disadvantage is it takes a bloody long time.” JSM

Figure 1: Quantitative Trait Locus (QTL) Mapping Microsatellites used as markers for linkage mapping of a certain trait –Microsatellite: dinucleotide repeats of variable lengths –At the same microsatellite locus on the genome, the number of repeats varies between individuals (or, in this case, between strains) For each microsatellite locus –PCR using primers for DNA on either side of microsatellite marker –PCR produces will vary in size depending on the number of repeats “The technique is blind. The disadvantage is it takes a bloody long time.” JSM

Figure 1: Creation of B6D2F2 mice B6D2 DBA/J2: MK-801-insensitive strain C57BL/6J: MK-801 sensitive X

Figure 1: Creation of B6D2F2 mice B6D2 DBA/J2: MK-801-insensitive strain C57BL/6J: MK-801 sensitive X B6D2F1 X F1B6D2 All heterozygous

Figure 1: Creation of B6D2F2 mice B6D2 F2 DBA/J2: MK-801-insensitive strain C57BL/6J: MK-801 sensitive X B6D2F1 B6D2F2 B6D2F1 X B6 B6B6 B6D2 D2D2 F1B6D2 Due to independent assortment and crossing over, each F2 mouse could be B6/B6, B6/D2, or D2/D2 at each of the microsatellite markers genotyped. 1:2:1 All heterozygous D2

Figure 1: Results and Conclusion Mice were injected with U50,488 (  - opiod agonist) to activate the descending analgesia pathways and nociceptive sensitivity was measured using a hot-water tail-withdrawal assay. A statistical linkage analysis was performed for the microsatellite genotypes and the analgesia phenotypes. likelihood of QTL What can be concluded from these data? at D8Mit56 Microsatellite Markers Genotype at D8Mit56

Figure 1: Results and Conclusion Mice were injected with U50,488 (  - opiod agonist) to activate the descending analgesia pathways and nociceptive sensitivity was measured using a hot-water tail-withdrawal assay. A statistical linkage analysis was performed for the microsatellite genotypes and the analgesia phenotypes. κ-opiod analgesia is linked to the distal portion of chromosome 8 (near 67cM) in females. There is no linkage of κ-opioid analgesia in this region for males. likelihood of QTL What can be concluded from these data? at D8Mit56 Microsatellite Markers Genotype at D8Mit56

Candidate Gene: Melanocortin-1 Receptor (Mc1r) The Mogil group decided to test Mc1r from among the candidate genes near the QTL because one of the lab members happened upon a paper that demonstrated MC1R expression in the areas of the brain associated with analgesia.

Candidate Gene: Melanocortin-1 Receptor (Mc1r) eumelanin (black) phaeomelanin (red/yellow) α-MSH MC1R cAMP The Mogil group decided to test Mc1r from among the candidate genes near the QTL because one of the lab members happened upon a paper that demonstrated MC1R expression in the areas of the brain associated with analgesia. MC1R is commonly known for its role in the control of melanin synthesis. (α-melanocyte-stimulating hormone)

Testing the Candidate Gene After picking MC1R as their candidate gene, the Mogil group set out to determine whether its gene product is actually required for activity of the female-specific analgesia pathway.

Testing the Candidate Gene After picking MC1R as their candidate gene, the Mogil group set out to determine whether its gene product is actually required for activity of the female-specific analgesia pathway. Formal proof that they picked the correct gene would require the group to engineer their own knock-out mouse and then rescue melanocortin-1 receptor function. “A dead boring task.... A waste of time and taxpayer money.” JSM

Testing the Candidate Gene After picking MC1R as their candidate gene, the Mogil group set out to determine whether its gene product is actually required for activity of the female-specific analgesia pathway. Formal proof that they picked the correct gene would require the group to engineer their own knock-out mouse and then rescue melanocortin-1 receptor function. “A dead boring task.... A waste of time and taxpayer money.” JSM The Mogil group decided to depart from tradition and designed a group of independent experiments that all set out to answer the same question: Does MC1R mediate the female-specific stress-induced analgesia pathway?

Testing the Candidate Gene After picking MC1R as their candidate gene, the Mogil group set out to determine whether its gene product is actually required for activity of the female-specific analgesia pathway. Formal proof that they picked the correct gene would require the group to engineer their own knock-out mouse and then rescue melanocortin-1 receptor function. “A dead boring task.... A waste of time and taxpayer money.” JSM The Mogil group decided to depart from tradition and designed a group of independent experiments that all set out to answer the same question: Does MC1R mediate the female-specific stress-induced analgesia pathway? Their data can be grouped into three main lines of evidence: Mutant Data (Table 1 and Figure 2) Pharmacological Data (Figure 3) Human Data (Table 2 and Figure 4)

Figure 2: Research Question What method did they use to answer their question?

Figure 2: Research Question What method did they use to answer their question? Comparison of B6 and Mc1r mutant mouse strains. In which sex do you hypothesize that the mutation will have no effect on analgesia?

Figure 2: Research Question What method did they use to answer their question? Comparison of B6 and Mc1r mutant mouse strains. In which sex do you hypothesize that the mutation will have no effect on analgesia? Male e/e and B6 mice should have an identical analgesia phenotypes because their descending analgesia pathways should not be mediated by MC1R.

Figure 2: Mc1r Null Mutant e/e “recessive yellow” mouse B6 The Mogil group used the pre-existing e/e “recessive yellow” mouse, which is a Mc1r mutant in B6 background. The Mc1r allele is the only known mutation in the e/e mouse.

Figure 2: Mutant Data ? NMDAR estrogen receptors opioid receptors

Figure 2: Mutant Data NMDAR antagonist (MK-801) ? NMDAR estrogen receptors opioid receptors

Figure 2: Mutant Data NMDAR antagonist (MK-801) Stimulate stress response (U50,488) What can be concluded from these data? ? NMDAR estrogen receptors opioid receptors

Figure 2: Mutant Data NMDAR antagonist (MK-801) Stimulate stress response (U50,488) e/e females display typically male MK-801 sensitivity. Since the only known difference between e/e and B6 strains is the MC1R genotype, it seems that MC1R is mediating female-specific analgesia. What can be concluded from these data? ? NMDAR estrogen receptors opioid receptors

Figure 3: Pharmacological Data Outbred Crl:CD-1 “mutt” mice ANT peptide = MC1R antagonist

Figure 3: Pharmacological Data Outbred Crl:CD-1 “mutt” mice ANT peptide = MC1R antagonist Males: ANT has no effect

Figure 3: Pharmacological Data Outbred Crl:CD-1 “mutt” mice ANT peptide = MC1R antagonist Males: ANT has no effect MK-801 sensitive

Figure 3: Pharmacological Data Outbred Crl:CD-1 “mutt” mice ANT peptide = MC1R antagonist Males: ANT has no effect MK-801 sensitive Females: antagonizing MC1R results in MK-801 sensitivity

Figure 3: Pharmacological Data Outbred Crl:CD-1 “mutt” mice ANT peptide = MC1R antagonist Males: ANT has no effect MK-801 sensitive Females: antagonizing MC1R results in MK-801 sensitivity Why: ANT blocked female- specific pathway, causing mouse to “switch systems” and have male-type NMDAergic analgesia profile

Melanocortin-1 Receptor in Humans "The fun thing about this finding was it could have been any gene. It wasn't any gene; it was the redhead gene." JSM

Melanocortin-1 Receptor in Humans eumelanin (black) phaeomelanin (red/yellow) α-MSH MC1R cAMP "The fun thing about this finding was it could have been any gene. It wasn't any gene; it was the redhead gene." JSM People with very fair skin and red hair tend to have certain null allele variations of MC1R.

Figure 4: From Mice to Humans Pentazocine analgesia by sex and MC1R genotype in humans Pentazocine: analgesic. κ-opioid receptor agonist. Humans were genotyped for their MC1R alleles and were tested for thermal and ischemic pain before and after administration of pentazocine or saline. What can be concluded from these data?more analgesia Ischemic pain less analgesia Thermal pain

Figure 4: From Mice to Humans Pentazocine analgesia by sex and MC1R genotype in humans Pentazocine: analgesic. κ-opioid receptor agonist. “redhead” Humans were genotyped for their MC1R alleles and were tested for thermal and ischemic pain before and after administration of pentazocine or saline. What can be concluded from these data?more analgesia Ischemic pain less analgesia Thermal pain

Figure 4: From Mice to Humans Pentazocine analgesia by sex and MC1R genotype in humans Pentazocine: analgesic. κ-opioid receptor agonist. “redhead” Humans were genotyped for their MC1R alleles and were tested for thermal and ischemic pain before and after administration of pentazocine or saline. What can be concluded from these data? Women with two variant alleles of MC1R were more sensitive to pentazocine analgesia than women of other genotypes. There was no significant effect of MC1R genotype in males. more analgesia Ischemic pain less analgesia Thermal pain

Methodology: To clone or not to clone… "We didn't do what a lot of people in the mouse genetics field think is the final step: [positional cloning]. What I did instead is regarded by some as cheating. I was able to get my philosophy about [the methodology] down on paper into a fairly prominent journal. Had this gotten into Science or Nature, I wouldn't have had the space, and there's no way they would have accepted such heresy" JSM

Methodology: To clone or not to clone… “I don’t really care why C57/B6 mice are different than DBA2 mice. That’s not why we’re doing this. We’re doing this to find out why some people are different from other people.... Once you get good enough evidence that it’s likely to be a particular gene in humans, simply start the human study.... And as far as I’m concerned once you have the human finding, it’s completely moot now to find the actual C that changed to a T that makes the difference between one mouse strain and another mouse strain. So I’m permanently leaving that left untied and that drives most mouse geneticists absolutely insane.” JSM

Supplementary Figure 5 Fig. 5. Switching of analgesia mechanisms in female B6 and e/e mice depending on estrogenic status. C57BL/6 (B6) and mutant (e/e) mice of both sexes (n = 4–14 per genotype per sex per status per drug) were either left intact or ovariectomized (OVX), via surgical removal (dorsal incision) of both ovaries under isoflurane/oxygen anesthesia. No testing occurred for at least 2 weeks after surgery. Some OVX mice were given chronic estrogen replacement (OVX+E2; 5.0 μg/day, i.p., in sesame oil vehicle, for 6–7 days), following our original protocol (1). After baseline testing on the 49ΊC tail-withdrawal test, all mice received a s.c. injection of MK-801 (0.075 mg/kg) or saline (10 ml/kg), followed immediately by an i.p. injection of U50,488 (50 mg/kg). Percent analgesia scores were calculated as described. *, Significant blockade of analgesia by MK-801, P < †, Significantly different from intact male and OVX female group, P < Note that both B6 and e/e females display evidence of estrogenic control of analgesic magnitude. Only B6 females display evidence of neurochemical switching of analgesia (i.e., MK-801 sensitivity), because e/e mice appear to be using the male-like, MK-801 sensitive system regardless of hormonal status. 1. Mogil, J. S., Sternberg, W. F., Kest, B., Marek, P. & Liebeskind, J. C. (1993) Pain 53, 17–25. Supplementary materials are from:

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