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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7744-7751, July 1999 Colloquium Paper This paper was presented at the National Academy of Sciences colloquium "The Neurobiology of Pain," held December 11-13, 1998, at the Arnold and Mabel Beckman Center in Irvine, CA. The genetic mediation of individual differences in sensitivity to pain and its inhibition (nociception/gene mapping/strain differences) JEFFREY S. MOGIL* Department of Psychology and Neuroscience Program, University of Illinois at Urbana-Champaign, Champaign, IL 61820 ABSTRACT The underlying bases of the considerable interindividual variability in pain-related traits are starting to be revealed. Although the relative importance of genes versus experience in human pain perception remains unclear, rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. The identification and characterization of particularly divergent populations provides a powerful initial step in the genetic analysis of pain, because these models can be exploited to identify genes contributing to the behavior-level variability. Ultimately, DNA sequence differences representing the differential alleles at pain-relevant genes can be identified. Thus, by using a com- bination of "top-down" and "bottom-up" strategies, we are now able to genetically dissect even complex biological traits like pain. The present review summarizes the current progress toward these ends in both humans and rodents. Pain is considered both a sensation and an emotion (1) and shows considerable complexity and subjectivity, especially when compared with other sensory systems. Physical injury is neither necessary nor sufficient for the experience of pain, and in both clinical and laboratory settings, the perception of pain bears a notoriously poor relationship to the intensity of the noxious stimulus. Mildly noxious stimuli can be perceived as very painful whereas very noxious stimuli can produce no pain whatsoever (2~. Substantial advances in understanding why this might be have been made in the past few decades, including the discovery of pain-modulatory mechanisms (producing both analgesia and antianalgesia) and nervous system plasticity following exposure to noxious stimuli. Even as our understanding of pain physiology has been facilitated by such paradigmatic advancements, pain percep- tion in humans and animals nonetheless displays considerable, and unexplained, interindividual variability. The focus of this review is to examine the scope of individual differences in pain and analgesia in both humans and laboratory animals and to consider what is known and what remains to be determined regarding the genetic contributions to such variability. Null mutants (e.g., transgenic knockouts) will not be considered presently, because they have been recently reviewed (3~. The Scope of Individual Variability to Pain and Analgesia Human Studies. Variability in pain-related traits (or "phe- notypes"~- experimental pain sensitivity, propensity to de- velop painful pathologies, and sensitivity to analgesic manip- ulations has long been appreciated, although empirical val- idation of such variability is somewhat limited. In 1934, Libman (4) reported that pressure applied to the mastoid bone in the direction of the styloid process produces "marked" pain in PNAS is available online at www.pnas.org. 60-70% of his human patients, but no or little pain in 30-40%. Subsequent investigations over the next few decades with more quantitative methodology confirmed the presence of large individual differences in threshold sensitivity and tolerance to noxious pressure (5-8), heat (7, 9, 10), and electrical current (7, 11) applied to cutaneous tissues, and tolerance to visceral (6) and deep muscle (9, 10) pain. Impressive individual dif- ferences in sensitivity to opioid analgesics were also docu- mented during this period, typified by Lasagna and Beecher (121 observing a "success rate" of only 65% of the standard clinical dose of morphine, 10 mg (see also ref. 13~. Modern studies have confirmed that interindividual vari- ability in pain thresholds greatly exceeds intraindividual vari- ability for pressure pain threshold (e.g., refs. 14 and 15), cold pressor tolerance (e.g., ref. 16), and pain associated with manual palpation of tender points in fibromyalgia sufferers (17~. In the cold pressor test, Chen et al.~16, 18) consistently observe dichotomous responses, with a minority "pain- sensitive" group tolerating the test for a mean duration of 50 see and a majority "pain-tolerant" group able to tolerate the test for the full 300-sec duration. Similar findings have been obtained for opiate analgesics, which display large clinical (e.g., refs. 19 and 20) and experi- mental (e.g., ref. 21) variability in their efficacies, side effects, and tolerance liability. The analgesic actions of morphine on dental extraction pain also show evidence of bimodality, with morphine "responder" and "nonresponder" groups being eas- ily identified at a range of doses (22~. Individual differences in the actions of an opiate antagonist, naloxone, have also been demonstrated. Buchsbaum et al. (23), after dividing human subjects into pain-sensitive and pain-insensitive groups based on their sensitivity to electric shock to the forearm, noted that intravenous injection of 2.0 mg of naloxone produced hyper- algesia in the latter group and (paradoxically) analgesia in the former. Animal Studies. In the concluding comment of Libman's (4) seminal study of human pain variability, he posits that studies of animals may prove of value, because "in all the work hitherto performed it has been taken for granted that sensi- tiveness is the same in all animals of a given species" (p. 341~. Indeed, this is the working assumption of much biological research: that findings obtained from some presumed ran- domly bred sample of Rattus rattus will be representative of the "universal rat," and subsequently generalizable to all rats, and mice, and maybe to all humans. It remains an empirical question as to just how untenable this assumption is. Unfor- tunately for pain researchers (but fortunately for pain genet Abbreviations: RI, recombinant inbred; SIA, stress-induced analgesia; CIP, congenital insensitivity to pain; cM, centimorgan; QTL, quanti- tative trait locus; 5-MT, serotonin. *To whom reprint requests should be addressed. e-mail: jmogil@ s.psych.uiuc.edu. 7744

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Colloquium Paper: Mogil icists!), the existing data regarding the nociceptive and anal- gesic sensitivity of laboratory rodent populations reveal great, and in some cases qualitative, variability. Rodent populations of use for genetic analysis can either be produced (e.g., inbred strains, recombinant inbred strains, artificially selected lines, transgenics) or identified (e.g., spon- taneous mutants). The characteristics of these genetic models have been reviewed elsewhere (e.g., ref. 24~. The most studied genetic rodent models of relevance to pain include the recom- binant inbred (RI) CXBK mouse strain (25, 26), the High Analgesia/Low Analgesia (HA/LA) mouse lines selectively bred for swim stress-induced analgesia (SIA) (27, 28), the High Analgesic Response/Low Analgesic Response (HAR/LAR) mouse lines selectively bred for levorphanol analgesia (29), the High Autotomy/Low Autotomy (HA/LA) rat lines selectively bred for autotomy (30), and the normotensive Wistar Kyoto versus Spontaneously Hypertensive Rat (WKY/SHR). The former models have been thoroughly reviewed (244. The literature regarding the relationship between nociception and genetic or experimentally induced hypertension has also been recently reviewed (31~. Strain differences. Although less well studied at present, the comparison of inbred strain responses is more relevant to the issue of the scope of individual differences than the afore- mentioned models. Inbred strains are derived by repeated Table 1. Rat strain differences of relevance to pain Proc. Natl. Acad. Sci. USA 96 (1999) 7745 (~20 generation) full-sibling (i.e., brother x sister) mating (32~. Mating of individuals with common ancestors increases the probability of offspring inheriting two copies of the same allele identical-by-descent. During inbreeding, therefore, ge- netic heterozygosity is progressively lost as alleles of initially segregating genes are fixed into a homozygous state. Tables 1 and 2 present some existing data regarding inbred (and outbred) strain differences of relevance to pain in rats and mice, respectively. The only obvious generalizations that can be made from Table 1 are the nociceptive sensitivity of the Lewis (LEW) inbred rat strain and the sensitivity to a wide variety of analgesic manipulations of the outbred Sprague- Dawley (SD) strain. Multistrain comparisons ("strain sur- veys") are far more common in the mouse because of the ready availability of over 30 major inbred strains. Obvious general- izations from mouse strain surveys are thus harder to make. One exception is the voluminous research demonstrating the relative sensitivity of the DBA/2 (D2) strain to opioid anal- gesia compared with the C57BL/6 (B6) strain (not shown in Table 2, but reviewed in refs. 33 and 34~. Although D2 mice display high, and B6 mice display low magnitudes of analgesia, the B6 strain is markedly more sensitive than the D2 strain to other opioid-mediated phenomena, including locomotor acti- vation, learning/memory, and muscular rigidity (Straub tail). Trait Parameters Administration Stimulus Strain Difference* Ref. Nociceptive sensitivity Thermal Electrical Mechanical Chemical Neuropathic Analgesia Morphine Codeine Clonidine TRH Serotonin Footshock Restraint Acupuncture Analgesic tolerance Morphine 0-15 mg/kg 0-10 mg/kg 0-10 mg/kg 0-20 mg/kg 0-10 mg/kg 0-10 mg/kg 50-400 ,umol/kg 0-60 ,ug/kg 10 ,ug 1 mg/kg 0-300 ,ug/kg 0.5 ,ug 1.5 mA 30 min in tubes 100 Hz, 1-3 mA 14 x 5-10 mglkg 8 x 10 mg/kg TW TF HP FJ CD VF CFA CFA NT NT NT 1.p. i.p. i.p. i.p. i.p. s.c. s.c. i.p. i.c.v. i.p. i.v. i.C.V. l.p. S.C. FJ TF TW FT TF HP TF FT TW TW TF TW TW TF TF TF HP WAG > F344 LE = LEW = WIS > F344 = SD LEW > F344 F344 > SD LEW > F344 F344 = LEW = WIS > SD LEW > SD LEW > AVN SD > WKY LE = SAB = SD = WKY > LEW BUF = SD > BN > WIS > LEW SD > F344! SD > WIS F344 > WAG F344 > LEW LE = SD 2 LEW = WIS 2 F344 SD > WKY SD > DA SD > WKY: WAG > F344 WAG > F344 SD > WKY F344 > WAG F344 > SD LE = SD > F344 = LEW = WIS P77PMC > WIS SD > WIS WKY > SD 117 118 119 120 121 42 122 123 124 125 126 120 127 117 41 118 128 116 129 130 131 132 130 133 118 134 127 128 Strain Abbreviations: BN, Brown Norway; BUF, Buffalo; DA, Dark Agouti; F344, Fischer 344; LE, Long-Evans (outbred); LEW, Lewis; SAB, Sabra (outbred); SD, Sprague-Dawley (outbred); WAG, Wistar Albino Glaxo (WAG/GSto); WIS, Wistar (outbred); WKY, Wistar Kyoto. Genealogical origins of all inbred rat strains can be found at http://www.informaticsjax.org/bin/strains/search. Other Abbreviations: CD, colorectal distention; CFA, complete Freund's adjuvant; FJ, flinchjump test; FT, formalin test; HP, hot-plate test; i.c.v., intracerebroventricular, i.p., intraperitoneal; NT, sciatic and saphenous nerve transection; TF, radiant heat tail-flick test; TRH, thyrotropin- releasing hormone; TW, hot water tail-immersion/withdrawal test. *Only studies with significant strain differences are reported. Excluded are studies involving selected lines [including the spontaneously hypertensive rat (SHR)l and mutants. iMorphine analgesia was significantly attenuated by pretreatment with p-chlorophenylalanine in SD, but not F344 rats. TClonidine analgesia was naloxone-reversible in SD rats but naloxone-insensitive in WKY rats. F344 rats also developed increased conditioned analgesia to footshock relative to SD rats.

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7746 Colloquium Paper: Mogil Table 2. Mouse strain differences of relevance to pain Proc. Natl. Acad. Sci. USA 96 (1999) Trait Parameters Administration Stimulus Strain Difference* Ref. Nociceptive sensitivity Thermal Chemical Neuropathic HP HP HP HP HT TW AC AC AC FT NT NT NT B6 = ICR = SW ~ BALB B6 > D2 >C3H B6 > 7 others RIIIS > 9 others > AKR B6 > 9 others > AKR B6 > 9 others > RIIIS CBA > C3H = D2 > B6 C3H = CBA > 6 others C3H > 9 others > 129 13 others > A 0F1 = BALB > NMRI > B6CBAF1 C3HeB > ICR B6 > 8 others > AKR = C58 135 136 62 59 59 59 137 62 59 138 139 140 59 Hypersensitivity Thermal CAR BALB > 9 others > SM 59 PNI AKR > 9 others > C58 59 Mechanical PNI 129 > 9 others > CBA 59 Analgesia Morphine 0-8 mg/kg s.c. HP CF-1 > CFW 141 0-320 mg/kg i.p. HP SW > B6 = BALB = ICR 135 0-2 mg/kg s.c. AC A = B6 = D2 > C3H = ICR = SW 142 75 mg pellet TF SW > ICR 143 75 mg pellet HP C3H = D2 > CD-1 144 5 mg/kg s.c. TF BALB > CD-1 > SW 145 5 mg/kg s.c. HP BALB > CD-1 > SW 145 0-100 mg/kg s.c. HP BALB = CBA = D2 > AKR = B6 = C3H 62 0-10 mg/kg s.c. AC B6 = D2 > C3H = CBA > AKR 62 0-12 ,ug i.c.v. TW C3H > 9 others > SWR! 58 U-50,488 5 mg/kg s.c. TF CD-1 > SW > BALB 145 NalBzoH 50 mg/kg s.c. TF CD-1 > SW > BALB 145 D-phenylalanine 0-400 mg/kg i.p. HP B6 = C3H > BALB = D2 146 Nitrous oxide 25-75% AC A > 8 others > D2 147 Alprazolam 0-100 mg/kg i.p. TF BALB > SW > B6 = CD-1 148 Nicotine 0-10 mg/kg i.p. TF CD-1 > CF-1 149 Epibatidine 0-0.1 mg/kg s.c. TW A > 7 others > C3H 150 Acetaminophen 0-250 mg/kg s.c. AC AKR > 6 others > BALB (unpublished data) Analgesic tolerance Morphine 9 x 50-100 mg/kg HP B6 = BALB = ICR > SW 135 75 mg pellet TF SW > ICR 143 75 mg pellet TF CD-1 > 129! 151 Strain Abbreviations (substrain identifiers are omitted; in most cases, inbred strains were obtained from The Jackson Laboratory: B6, C57BL/6; BALB, BALB/c; C3H, C3H/He; CD-1, Hsd:ICR (outbred); CF-1, Hsd:NSA (outbred); CFW, HsdWin:CFW1 (outbred); D2, DBA/2; ICR, Institute for Cancer Research stock (many suppliers; outbred); SW, Swiss Webster (outbred). Genealogical origins of all inbred mouse strains can be found in Festing (37) or at http://www.informatics.jax.org/bin/strains/search. Other Abbreviations: AC, abdominal constriction (writhing) test; CAR, carrageenan; FT, formalin test; i.c.v., intracerebroventricular; i.p., intraperitoneal; i.v., intravenous; HP, hot-plate test; NalBzoH, naloxone benzoylhydrazone (a ~3-opioid agonist); NT, sciatic and saphenous nerve transection; PNI, peripheral nerve injury (Chung model); TF, radiant heat tail-flick test; TW, hot water tail-immersion/withdrawal test; VF, von Frey fiber test. *Only studies with significant strain differences are reported. Excluded are studies involving selected lines, mutants, recombinant inbred (RI) strains, and non-extant populations. Also excluded are studies specifically comparing the B6 and D2 strains. TStrain differences observed varied with sex. TNo analgesic tolerance whatsoever developed in the 129/SvEv substrain used. Qualitat~ve strain differences. In addition to the quantitative strain differences compiled in Tables 1 and 2, some very intriguing qualitative strain differences of relevance to pain have been noted. For instance, a number of investigations have suggested that certain strains activate opioid analgesic systems after exposure to stress, whereas other strains produce ap- proximately equivalent amounts of SIA, but of a non-opioid (i.e., naloxone-insensitive) character (35-37~. Vaccarino et al. (38) reported that naloxone injection produced paradoxical analgesia on the formalin test in BALB/c mice, but not B6 or outbred CD-1 mice. Fujimoto and colleagues (39) have dem- onstrated convincingly that heroin analgesia is mediated by ,u-opioid receptor activation in outbred Institute for Cancer Research stock (ICR) mice, but by &-receptor activation in outbred Swiss Webster (SW) mice. The same workers have recently (40) identified ,u-, 3-, and K-type heroin responders among inbred mouse strains. Vaccarino and Couret, Jr. (41) observed that the presence of formalin-induced pain during tolerance induction wholly prevented tolerance development in the Fischer 344 (F344) strain but not in the LEW strain. Lee et al. (42) observed a complete blockade of neuropathic mechanical allodynia after treatment with the c~-adrenergic receptor antagonist, phentolamine, in LEW rats; this treat- ment was wholly ineffective in F344 rats. The authors con- cluded that this neuropathy was sympathetically maintained in the former strain only. Finally, Proudfit's laboratory has demonstrated (43) that electrical stimulation-produced anal- gesia is reversed by cx2-adrenergic antagonists in SD rats from

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Colloquium Paper: Mogil the now defunct Sasco (Omaha, NE), but not in SD rats from Harlan-Sprague-Dawley. This difference may be explained by the differential projection routes and dorsal horn termination fields (laminae VII-X versus laminae I-IV, respectively) of pontospinal noradrenergic neurons in these substrains (44~. Heritability of Pain-Related Traits That individual differences in pain-related traits exist, of course, does not imply that these differences are necessarily attributable to genetic factors. Familial aggregation of pain pathologies and extremes of pain sensitivity has been repeat- edly noted in humans, but such findings have almost uniformly been attributed to shared environmental variance and/or familial modeling (e.g., refs. 45-49~. To separate genetic and environmental factors, twin studies have been conducted (some even featuring adoption), comparing the concordance rates of pain-related traits in monozygotic (identical) versus dizygotic (fraternal) twins (see ref. 50~. Heritability (i.e., the proportion of overall phenotypic variance accounted for by genetic factors) has been estimated as 39-58% for migraine (51-53), 55% for menstrual pain (54), 50% for back pain (55), and 21% for sciatica (56~. Only one report exists of a twin study of nonpathological, basal pain sensitivity. MacGregor et al. (57) assessed forehead pressure pain thresholds in 269 monozygotic pairs and 340 dizygotic pairs, and observed only a slight excess correlation in monozygotic versus dizygotic twins (r = 0.57 vs. 0.51, respec- tively). This excess corresponds to a heritability of this trait of only 10% and suggests that the twin correlations in pain thresholds are largely due to shared environmental factors. In contrast to this last study in humans, heritability estimates for nociceptive and analgesic sensitivity in mice are fairly high, ranging from 28% to 76% (29, 58-60) certainly well within the range considered for further genetic analysis. Because none of the murine nociceptive assays used are similar to the forehead pressure pain test used by MacGregor et al. (57), it is difficult at the present time to evaluate whether humans and mice truly differ in the contribution of genes to pain sensitivity. Even if genetic factors are ultimately demonstrated to play only a minor role in the determination of individual pain sensitivity in humans, genetic studies of pain may still prove highly valuable. Such studies, for example, may illuminate those components of pain processing circuitry in mice and humans that are especially amenable to alteration, knowledge likely to be useful for the development of novel analgesic strategies. Genetic Correlations Among Pain Phenotypes The tools of classical genetics can be used, even in advance of the identification of the relevant genes, to determine whether traits share genetic mediation (see ref. 61~. The fact that a given gene can influence more than one trait is known in genetic parlance as pleiotropy. Pleiotropic actions of genes result in the genetic correlation of traits, because allelic variation in a gene will influence all traits in which that gene participates. The determination of genetic correlation has proven to be very heuristic, leading to novel theories regarding the underlying physiological mediation of traits. A number of genetic correlations of pain-related phenotypes have been noted using selected lines and inbred strains (see ref. 24~. I would like to focus on two intriguing findings, as follows. Genetic Correlation of Nociception and Opiate Analgesia. It has been demonstrated by several groups that a negative genetic correlation exists between initial nociceptive sensitiv- ity and subsequent morphine analgesia (27, 62, 63~. That is, mice that are initially sensitive to noxious stimuli tend to exhibit modest analgesic responses to morphine, whereas mice that are relatively resistant to basal nociception exhibit robust morphine analgesia. In two separate studies using multiple Proc. Natl. Acad. Sci. USA 96 (1999) 7747 inbred strains, this correlation was estimated as r = -0.63 to -0.85 (62) and r = -0.61 (58~. Thus, mice are "doubly advantaged" or "doubly disadvantaged" with respect to noci- ception and its opiate inhibition. Interestingly, this negative correlation (albeit with n = 2 only) can also be observed when comparing the sexes. Genetic Correlations Among Nociceptive Assays. We re- cently tested 11 inbred strains on 12 separate measures of nociception in common use in the mouse (59, 609. The assays used can be placed on a number of dimensions, including etiology (nociceptive, inflammatory, neuropathic), modality (thermal, chemical, mechanical), duration (acute, tonic, chronic), and location (cutaneous, subcutaneous, visceral). We reasoned that inbred strain variation could be exploited to identify clusters of genetically correlated nociceptive assays. Similar genetic mediation implies similar physiological medi- ation of assays, suggesting that they measure the same "type of pain as defined mechanistically. Essentially, we were at tempting to produce a natural rather than artificial taxonomy of nociception in the mouse, similar to that called for by Woolf et al. (64~. The results of this effort were variously expected and surprising. By using multivariate analyses, we identified three obvious clusters of pain tests, in which within-cluster genetic correlations greatly exceeded between-cluster correlations: "thermal" (Hargreaves' test, hotplate test, tail-immersion/ withdrawal test, and, surprisingly, autotomy), "chemical" (ace- tic acid abdominal constriction, magnesium sulfate abdominal constriction, acute- and tonic-phase formalin test), and "me- chanical + hypersensitivity" (von Frey test, carrageenan ther- mal hypersensitivity, peripheral nerve injury thermal, and mechanical hypersensitivity) (59, 60~. Thus, the stimulus mo- dality dimension accounted for the obtained genetic correla- tions to a far greater degree than any other factor. The presence or absence of neuropathy or inflammation was found to be essentially irrelevant as was the site or duration of the stimulus. Identification of Pain-Related Genes The holy grail of pain genetics, of course, is the actual identification of pain-related genes and the polymorphisms within or near such genes that account for trait variability. Note that "pain-related gene" could be broadly defined as any gene encoding a protein of known pain relevance or of a gene whose null mutant exhibits a pain-related phenotype. Defined in this way, a large number of pain-related genes are known. However, if more properly defined as one in which allelic variation directly produces individual differences or pathology, only a handful of pain-related genes have been identified. Techniques. Essentially, there are two ways to identify genes associated with trait variability: (i) linkage analysis, including classical model-based linkage techniques and allele-sharing methods in humans and test-crosses in animals, and (ii) association studies (reviewed in ref. 65~. Linkage analyses follow familial inheritance patterns, whereas association stud- ies compare allele frequencies in defined populations. Given the increasingly large number of genes already cloned and mapped in Homo sapiens and Mus musculus, linkage studies may lead immediately to the identification of candidate genes, which can then be studied by using the latter approach once allelic variants are found. Candidate genes, of course, can also be evaluated by using nongenetic means, by investigating the physiology of the proteins they encode. Failing the identifica- tion of an already cloned candidate gene, positional cloning techniques (e.g., ref. 66) can be used to narrow the ~20- centimorgan (cM)-wide chromosomal region identified by linkage down to the OCR for page 7744
7748 Colloquium Paper: Mogil Human Studies. A handful of single gene pain pathologies have recently been, or are on the verge of being, explained on the DNA sequence level. Congenital insensitivity to pain. Over 40 cases of congenital insensitivity to pain (CIP) with preservation of all other sensory modalities have been reported since the original description of a carnival performer known as The Human Pincushion (see ref. 67~. Recently, the genetic basis of this neuropathy (CIP with anhidrosis; hereditary sensory and autonomic neuropathy type IV) was elucidated. Based in part on the striking similarities between CIP and the phenotype of null-mutant mice lacking the Ntrkl gene encoding the high affinity, nerve growth factor-specific tyrosine kinase receptor, Indo et al. (68) considered the homologous human gene, NTRK1 (previously known as TRIM), as a candidate for CIP. Direct sequencing of the coding region of TRKA in four unrelated CIP patients revealed three separate, exonic muta- tions: a single base (C) deletion, an A ~ C transversion, and a G ~ C transversion (68~. Other sensory neuropathies. Nicholson et al. (69) performed a microsatellite marker genome screen on 102 members of four Australian kindreds with multiple individuals with hereditary sensory neuropathy type I, a disease featuring loss of all sensory modalities but especially pain and temperature. Link- age was established to a series of markers encompassing a 5-cM region of chromosome 9. An obvious candidate gene, NTRK2, encoding the tyrosine kinase receptor type 2 that is bound by brain-derived neurotrophic factor and neurotrophin-4, was excluded by informative recombination events. Positional cloning efforts facilitated by the development of a yeast artificial chromosome-based transcript map are ongoing (704. Another hereditary sensory neuropathy (type II), featuring loss of pain sensation and autoamputation, was recently sub- jected to genetic analysis in a consanguineous family with two affected sisters (71~. These investigators used exclusion map- ping, a mixed linkage/association strategy, to exclude a variety of known neurotrophin-related genes as candidates for this disorder. Migraine. An important recent finding of relevance to a more prevalent painful condition, migraine, has been the attribution of familial hemiplegic migraine and migraine-like episodic ataxia type 2 to mutations in the P/Q-type, calcium channel or1 subunit gene, CACNL1A4 (72~. Previous linkage studies mapped the gene for these disorders to chromosome l9pl3 (e.g., ref. 73~. By using a technique called exon trapping, Ophoff et al. (72) cloned the 47-axon-long CACNL1A4 gene in this region, and identified four different missense point mu- tations in affected individuals that segregated with the disease in five families. Although familial hemiplegic migraine and episodic ataxia type 2 are rare, genetic factors are known to play a role in "normal" migraine as well, and common allelic variants of this or other ion channel genes may (or may not, ref. 74) contribute to its etiology (75~. Of interest as well is a report of a familial migraine susceptibility locus on the X chromo- some, which may explain the preponderance of this condition in females (76~. Pathologies linked to human Iymphocyte antigens. Other preliminary genetic investigations of painful pathologies with familial aggregation including reflex sympathetic dystrophy/ complex regional pain syndrome (77), rheumatoid arthritis (e.g., ref. 78), and fibromyalgia (e.g., ref. 79) have demon- strated at least provisional linkage to or association with various human lymphocyte antigen regions or antigens. This system does not seem to be linked, however, to familial predisposition to discogenic low-back pain (80~. It is expected that full-scale genetic investigations of these and other com- plex pain traits are ongoing or imminent. What are less likely to occur are investigations of nonpathological pain traits, owing to the added complexity introduced by a continuous phenotype. For genetic investigation of "normal" pain sensi Proc. Natl. Acad. Sci. USA 96 (1999' tivity and sensitivity to analgesia, animal studies provide much-needed statistical power. Animal Studies. At the present time, three published studies exist (although many more are ongoing in my laboratory and others) demonstrating linkage of a pain-related trait to chro- mosomal locations in the mouse. A two-step quantitative trait locus (QTL) mapping approach has been chosen in each case (see refs. 81 and 82 for a detailed description). First, RI strains of the 26-strain BxD set, developed by Taylor (83) from an F2 intercross between B6 and D2 mice, were phenotyped to identify provisional linkages. These linkages were then inde- pendently confirmed or disconfirmed ~v usin~ new (BS x n21 F2 hybrids. Morphine analges~a. Belknap and Crabbe (84) tested BxD RI strains for a number of systemic morphine responses, including analgesia on the hot-plate test. Of eight broad chromosomal regions provisionally found to be linked with morphine anal- gesic magnitude, two have subsequently been confirmed be- yond the level of "suggestive linkage" (P < .0016) as proposed by Lander and Kruglyak (85~: the MpmvS region (0-20 cM) of mouse chromosome 10 (86) and the MyoSa region (30-50 cM) of mouse chromosome 9 (87~. The results to date of this ongoing QTL mapping study nicely illustrate the utility of the approach. In the MpmvS region lies the Oprm gene (8 cM) encoding the mouse ,u-opioid receptor type. Oprm is an obvious candidate gene for mor- phine analgesic magnitude, implicated via pharmacological (see ref. 88) and transgenic (89, 90) studies. Allelic variation at this QTL accounts for 28-33% of the observed genetic variability, and F2 mice inheriting two copies of the D2 allele at this QTL exhibit 4-fold more analgesia from a 16 mg/kg dose of morphine than do F2 mice inheriting two copies of the B6 allele (86~. This QTL has been statistically associated with other opioid traits as well, including morphine consumption (91) and whole-brain t3Hinaloxone binding (86~. One may have expected a pr~ori that the gene encoding the ,u-opioid receptor would be associated with morphine analge- sic magnitude. The candidate gene on mouse chromosome 9 is perhaps more heuristic. Within this region lies the Htrlb gene (46 cM), encoding the serotonin-lB (5-HT~B) receptor subtype (the mouse analog of the human 5-HT~D`3 receptor). Based on the hypothesis that Htrlb might represent the QTL for mor- phine analgesia on chromosome 9, we conducted a series of pharmacological experiments that provided substantial sup- port for the involvement of spinal 5-HT~B receptors (87~. Although data exist indicating a relationship between 5-HT~B receptors and opioid analgesia (e.g., ref. 92), there is still much confusion regarding the specific role of the 5-HT~A versus 5-HT~B subtypes, and until very recently subtype-specific ligands were decidedly lacking (93~. Thus, it is unlikely that the studies we conducted would have been conceived of in the absence of the QTL mapping data. Basal nociceptive sens~tivity. By using similar methodology to that described above, we recently mapped basal thermal nociceptive sensitivity by using the hot-plate test (94~. The most promising of six putative linkages in BxD RI strains the D4Mit71 region (50-70 cM) of mouse chromosome 4 was largely confirmed by using F2 mice. This QTL displayed evidence of sex specificity, with a combined BxD/F2 P value of 0.005 for males but only 0.085 for females. The identification of a candidate gene in this region, the Oprdl gene (65 cM) encoding the mouse &-opioid receptor type, inspired a simple pharmacological experiment in which B6 and D2 mice of both sexes were administered ,~-, ~-, and 8-specific antagonists prior to assessment of hot-plate sensitivity. As predicted by the hypothesis of Oprdl as a male-specific QTL for this trait, we observed a sex- and strain-dependent pattern of responses, with the lo-specific antagonist, naltrindole, lowering nocicep- tive latencies in the following order of efficacy: D2 male > B6 male ~ D2 female ~ B6 female (94~. , c, . . ~ _

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Colloquium Paper: Mogil Nonopioid SIA. It has long been known that many forms of analgesia are resistant to antagonism by naloxone, represent- ing the recruitment of non-opioid mechanisms (95~. To shed light on the mediation of these powerful but little understood systems, we conducted a QTL mapping experiment of non- opioid SIA resulting from 3-min forced swims in 15C water (96~. Six putative QTLs were identified in the BxD RI phase, of which four were subsequently disconfirmed by using F2 hybrids. Of the two remaining QTLs, one on chromosome 8 (50-80 cM) was confirmed beyond Lander and Kruglyak's (85) threshold for significant linkage. This QTL (dubbed Siafql) exhibited compelling evidence of sex specificity, reach- ing a combined BxD/F2 P value of 0.00000012 for females but only 0.038 for males. Female F2 mice inheriting two copies of the D2 allele at this locus displayed 3-fold more SIA than those inheriting two copies of the B6 allele (96~. This finding of a female-specific QTL for SIA is of special interest because we (36, 97) and others (e.g., ref. 98) had previously demonstrated the existence of qualitative sex differences in the neurochem- ical mediation of this trait. Sex-Specific Genetic Mediation of Pain and Analgesia The two findings of sex-specific QTLs described above a male-specific QTL for baseline thermal nociceptive sensitivity and a female-specific QTL for non-opioid SIA exemplify a phenomenon we and others have found repeatedly, i.e., sex/ genotype interactions of relevance to nociception and its modulation. The discovery of sex-specific QTLs on autosomes, first reported by Melo et al. (99) for alcohol preference, was surprising to many, but now more and more examples are being uncovered. It should be emphasized that the existence of autosomal, sex-specific QTLs does not imply that the sexes possess or express different genes, but rather that different genes are associated with trait variability in each sex. The existence of sex-specific QTLs does, however, imply that males and females possess at least partially independent physiolog- ical mechanisms underlying the traits in question. Sex differences in nociception and analgesia are controver- sial, but when differences are found, males of a number of species consistently display higher thresholds, tolerance, and analgesic sensitivity (see refs. 100 and 101 for reviews). The inability of some to observe these sex differences has been attributed to estrus cycle variability, test specificity, and ex- perimental parameters (e.g., ref. 1024. Recent data from my laboratory suggest that an important factor contributing to variable results in this literature has been overlooked, i.e., genotype of the test subjects. For example, a recent survey of supraspinal morphine analgesia in 11 inbred strains revealed no significant sex differences in morphine analgesic potency in seven of these strains (58~. In three strains (AKR, B6, and SWR), males exhibited 3.5- to 7-fold higher sensitivity to intracerebroventricularly administered morphine than their female counterparts. Finally, one strain (CBA) was identified in which females were 5-fold more sensitive to morphine than males. In another, just completed study specifically comparing outbred mouse strains, we found that a large male-vs.-female difference in baseline tail-flick latencies can be seen in SW mice obtained from Simonsen Laboratories (Gilroy, CA), but the analogous sex difference in SW and CD-1 mice from Harlan-Sprague-Dawley is either absent or too small to detect statistically with n = 16-32 (unpublished data). This "vendor effect" between SW mice from two different suppliers is likely due to genetic factors, because both populations have been bred in my vivarium for several generations. Ultimately, then, the failure of some to detect sex differences may simply be due to the fact that in the subject population chosen, there is no sex difference to detect. Another intriguing sex/genotype interaction is that demon- strated by Rady and Fujimoto (1034. As described above, these Proc. Natl. Acad. Sci. USA 96 (1999J 7749 investigators have determined that heroin analgesia is medi- ated by ,u-opioid receptors in ICR mice of both sexes but by 3-opioid receptors in SW mice of both sexes (39~. An analysis of reciprocal (ICR x SW) F~ hybrid mice revealed that male offspring displayed an ICR-like phenotype and female off- spring displayed a SW-like phenotype. That is, in F, males, heroin analgesia was blocked by ,u-opioid- but not b-opioid- specific antagonists, whereas the reverse was true for F~ females (103~. This is likely an example of sex-influenced autosomal dominant inheritance (see ref. 104~. Further anal- ysis of this phenomenon, if it can be replicated in inbred strains, may help to illuminate the basis of sex/genotype interactions in analgesia. Also potentially enlightening is our ongoing mapping study of supraspinal morphine analgesia in (AKR x CBA) F2 mice, focusing specifically on the identification of sex-specific QTLs. Male mice of these two strains exhibit equipotent analgesic sensitivity to morphine, whereas the ADsos of female mice differ by a factor of 35 (58~. Allelic Variants of Pain-Related Genes Once the genes mediating a trait have been positively identi- fied, a crucial task still remains the identification of alternate alleles of that gene giving rise to the original phenotypic difference between individuals and/or populations. This effort is again rendered more difficult when considering complex genetic traits, because the allelic variants are more likely to be single base pair changes (single-nucleotide polymorphisms) than chromosomal rearrangements or large deletions. Also, whereas the mutations giving rise to disease phenotypes are likely to occur in the coding region of a gene, allelic variation causing subtle changes in gene expression can occur outside (even far outside) the coding region. Nonetheless, some suc- cess has been reported. Opioid Receptor Genes. The coding and much of the regulatory and intronic regions of human opioid receptor genes have been sequenced (e.g., ref. 105~. By using direct sequencing of hundreds of individuals, three separate investi- gations identified two common variants of the OPRM gene coding for the ,u-opioid receptor (106-108), with allele fre- quencies estimated to be 6.6-11%. An A118G variant (i.e., an A ~ G substitution in nucleotide 118 of exon 1, resulting in a Asn > Asp change in amino acid residue 40) was found to be present in a lower proportion of opioid-dependent subjects than controls, whereas the C17T variant was more common in opioid-dependent subjects (106, 1074. The A118G variant was found not to be associated with susceptibility to alcohol dependence (108~. An A118G ,u-opioid receptor constructed by using site-directed mutagenesis and stably transfected into cell lines displayed higher binding affinity for [3-endorphin than the more common wild-type receptor (106~. An allelic variant (T307C) of the OPRD gene encoding the b-opioid receptor has also been found (109~. Although the amino acid sequence remains unchanged by this substitution, the investigators found that heroin addicts were significantly more likely than controls to possess a CC genotype and less likely to possess a TT genotype. It was concluded that, although by unknown mechanisms, the C allele predisposes to heroin abuse. The direct relevance of any such opioid receptor variants to pain or analgesic sensitivity is as yet unpublished, although this work is no doubt underway in several laborato- r~es. Cytochrome P450. One genetic polymorphism of well doc- umented relevance to pain is of the gene coding for the neuronal cytochrome P450IID6 (CYP2D6; sparteine/ debrisoquine oxygenase) enzyme (see ref. 110 for review). This enzyme is in fact absent in ~7-10% of Caucasians, who are thus unable to convert codeine to morphine by O- demethylation (111~. Because much evidence indicates that codeine produces analgesic effects by being biotransformed to

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7750 Colloquium Paper: Mogil morphine, these "poor metabolizers" will receive minimal therapeutic benefit from administration of codeine but are generally subject nonetheless to its side effects (112, 113~. It has been shown as well that poor metabolizers report increased pain compared with "extensive metabolizers" in the cold presser test (114~. An animal model of this phenomenon exists, with the female Dark Agouti (DA) rat showing a poor metabolizes phenotype (115, 116~. This well known example should serve to remind that much individual variability in drug response may be due to polymorphisms related to pharmaco- kinetics rather than pharmacodynamics. Future Directions The construction of high-density genomic maps and the initial priority of the Mouse and Human Genome Projects will greatly facilitate (and already has) the identification of the 50,000-100,000 mammalian genes. Given the high degree of redundancy and pleiotropy known to exist in biological sys- tems, the determination of which genes participate in which physiological mechanisms will remain a daunting task, occu- pying scientists for decades to come. New, high-throughput genomic technologies (e.g., "gene chips") may further accel- erate the rate of discovery. It is likely that the focus in Homo sapiens will be on pathology, whereas animal models like the mouse will continue to be used to investigate more subtle questions involving the normal range of behavior. Although the use of genetic techniques naturally garners much excite- ment, it must be borne in mind that even variability in pain pathologies is largely determined by environmental factors. Thus, investigations into the psychosocial determinants of pain tolerance and pain behaviors must continue unabated. 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