Elissa J. Chesler

The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans

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.

Sex differences in thermal nociception and morphine antinociception in rodents depend on genotype

It has been appreciated for some time that the sexes can differ in their sensitivity to pain and its inhibition. Both the human and rodent literatures remain quite contentious, with many investigators failing to observe sex differences that others document clearly. Recent data from our laboratory have pointed to an interaction between sex and genotype in rodents, such that sex differences are observed in some strains but not others. However, these studies employed inbred mouse strains and are thus not directly relevant to existing data. We presently examined whether the observation of statistically significant sex differences in nociception and morphine antinociception might depend on the particular outbred rodent population chosen for study. Rats of both sexes and three common outbred strains were obtained from three suppliers (Long Evans, Simonsen; Sprague Dawley, Harlan; Wistar Kyoto, Taconic) and tested for nociceptive sensitivity on the 49 degrees C tail-withdrawal assay, and antinociception following morphine (1-10mg/kg, i.p.). In further studies, three outbred populations of mice (CD-1, Harlan; Swiss Webster, Harlan; Swiss Webster, Simonsen) were bred in our vivarium for several generations and tested for tail-withdrawal sensitivity and morphine antinociception (1-20male, and no significant difference. In a separate study in which the estrous cycle was tracked in female mice, we found evidence for an interaction between genotype and estrous phase relevant to morphine antinociception. However, estrous cyclicity did not explain the observed sex differences. These data are discussed with respect to the existing sex difference and pain literature, and also as they pertain to future investigations of these phenomena.

Identification and ranking of genetic and laboratory environment factors influencing a behavioral trait, thermal nociception, via computational analysis of a large data archive

Laboratory conditions in biobehavioral experiments are commonly assumed to be controlled, having little impact on the outcome. However, recent studies have illustrated that the laboratory environment has a robust effect on behavioral traits. Given that environmental factors can interact with trait-relevant genes, some have questioned the reliability and generalizability of behavior genetic research designed to identify those genes. This problem might be alleviated by the identification of the most relevant environmental factors, but the task is hindered by the large number of factors that typically vary between and within laboratories. We used a computational approach to retrospectively identify and rank sources of variability in nociceptive responses as they occurred in a typical research laboratory over several years. A machine-learning algorithm was applied to an archival data set of 8034 independent observations of baseline thermal nociceptive sensitivity. This analysis revealed that a factor even more important than mouse genotype was the experimenter performing the test, and that nociception can be affected by many additional laboratory factors including season/humidity, cage density, time of day, sex and within-cage order of testing. The results were confirmed by linear modeling in a subset of the data, and in confirmatory experiments, in which we were able to partition the variance of this complex trait among genetic (27%), environmental (42%) and genetic x environmental (18%) sources.

Acute administration of estrogen and progesterone impairs the acquisition of the spatial morris water maze in ovariectomized rats.

Although several markers of synaptic efficacy are enhanced during proestrus, spatial water maze performance is impaired. Because levels of both estrogen and progesterone are elevated in proestrus, the nature of their individual and combined effects on spatial learning was examined. Long-Evans hooded rats were ovariectomized postpubertally and pretrained on a water maze with a visible platform (nonspatial). Following pretraining, rats were administered estrogen (5 microg sc) or oil 48 and 24 h prior to testing and progesterone (500 microg sc) or oil 4 h prior to testing. Rats were tested on a water maze in a different room with a submerged platform (spatial) for 16 trials with random start location in a single testing day. Latency and path length to the target platform were significantly greater in estrogen plus progesterone-treated animals than in controls. Neither estrogen nor progesterone alone significantly impaired performance relative to controls on either measure. Swim speed was not significantly affected by any of the hormone treatments. Performance on a nonspatial cue task was not significantly altered by ovarian steroids. Thus, the combination of estrogen and progesterone produces deficits in the acquisition of the Morris water maze that may be specific to spatial reference memory. These deficits could be due to hormonal influences on extrahippocampal structures or to detrimental effects on behavior resulting from the increased synaptic activity intrinsic to the hippocampus proper.

Genotype-dependence of gabapentin and pregabalin sensitivity: the pharmacogenetic mediation of analgesia is specific to the type of pain being inhibited

&NA; The antiepileptic drug, gabapentin, and another structurally related compound, pregabalin, are increasingly employed in the pharmacotherapy of chronic pain states, although their primary mechanism of action remains a topic of active study. A genomic approach to the study of these drugs may elucidate their potentially novel mechanisms. We examined the heritability of sensitivity to analgesia from gabapentin and pregabalin as a precursor to linkage mapping efforts. Accordingly, 11 inbred mouse strains were tested for inhibition of nociception by gabapentin or pregabalin (50–300 mg/kg, i.p.) in two different preclinical assays of inflammatory pain, the formalin test (5% formalin; 20 &mgr;l) and zymosan thermal hyperalgesia on the paw‐withdrawal test (3 mg/ml zymosan; 20 &mgr;l). Significant strain‐dependence of drug action was noted in each case, indicating that sensitivity to these analgesics is heritable. Furthermore, the pattern of strain sensitivities to gabapentin and pregabalin were mostly similar, supporting the notion that they act via similar genetic and physiological mechanisms. However, there was virtually no correlation between strain sensitivities to pregabalin inhibition of formalin nociception and zymosan thermal hyperalgesia. In light of previous data from our laboratory and others regarding morphine analgesia, we now establish and empirically demonstrate the general principle that pharmacogenetic mechanisms underlying analgesic sensitivity are specific to the type of pain being inhibited. This has considerable implications for ongoing pharmacogenetic investigations and, more generally, for the choices of preclinical models of pain used in drug development.

Identification of quantitative trait loci for chemical/inflammatory nociception in mice

&NA; Sensitivity to pain is widely variable, and much of this variability is genetic in origin. The specific genes responsible have begun to be identified, but only for thermal nociception. In order to facilitate the identification of polymorphic, pain‐related genes with more clinical relevance, we performed quantitative trait locus (QTL) mapping studies of the most common assay of inflammatory nociception, the formalin test. QTL mapping is a technique that exploits naturally occurring variability among inbred strains for the identification of genomic locations containing genes contributing to that variability. An F2 intercross was constructed using inbred A/J and C57BL/6J mice as progenitors, strains previously shown to display resistance and sensitivity, respectively, to formalin‐induced nociception. Following phenotypic testing (5% formalin, 25 &mgr;l intraplantar injection), mice were genotyped at 90 microsatellite markers spanning the genome. We provide evidence for two statistically significant formalin test QTLs – chromosomal regions whose inheritance is associated with trait variability – on distal mouse chromosomes 9 and 10. Identification of the genes underlying these QTLs may illuminate the basis of individual differences in inflammatory pain, and lead to novel analgesic treatment strategies.

Acute progesterone can recruit sex-specific neurochemical mechanisms mediating swim stress-induced and kappa-opioid analgesia in mice

There is a qualitative sex difference in the neurochemical mediation of stress-induced and kappa-opioid analgesia; these phenomena are dependent on N-methyl-d-aspartic acid (NMDA) receptors in males but not females. Progesterone modulation of this sex difference was examined in mice. Analgesia against thermal nociception was produced by forced cold water swim or by systemic administration of the kappa-opioid agonist, U50,488. As seen previously, the NMDA receptor antagonist MK-801 blocked both forms of analgesia in male but not female mice. Also as in previous studies, this sex difference was found to be dependent on ovarian hormones such that ovariectomy induced female mice to switch to the male-like, NMDAergic system. We now demonstrate that a single injection of progesterone (50 microg), systemically administered 30 min before analgesia assessment, is sufficient to restore female-specific mediation of analgesia (i.e., insensitivity to MK-801 blockade) in ovariectomized female mice. The rapidity of this neurochemical switching action of progesterone suggests mediation via cell surface receptors or the action of neuroactive steroid metabolites of progesterone.