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inflammatory nociception in mice
Pain 96 (2002) 385–391 www.elsevier.com/locate/pain
Identification of quantitative trait loci for chemical/inflammatory nociception in mice Sonya G. Wilson a,1, Elissa J. Chesler a,1, Heather Hain b, Andrew J. Rankin a, Joel Z. Schwarz a, Stanford B. Call c, Michael R. Murray a, Erin E. West a, Cory Teuscher c, Sandra Rodriguez-Zas d, John K. Belknap b, Jeffrey S. Mogil a,* a
Department of Psychology and Neuroscience Program, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA Department of Behavioral Neuroscience and VA Medical Center, Oregon Health Sciences University, Portland, OR 97201, USA c Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
b
Received 18 September 2001; received in revised form 16 November 2001; accepted 13 December 2001
Abstract 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 ml 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. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Pain; Genetics; Gene mapping; Formalin test; Inflammation; Strain differences
1. Introduction The contribution of genetic factors to individual differences in pain and analgesic sensitivity is now widely accepted, and much progress is being made towards the identification of the responsible genes (see Mogil, 1999 for review). Our studies using a common set of inbred mouse strains have revealed a limited number of fundamental pain ‘types’ as defined by genetic correlations (Mogil et al., 1999a,b). The dimension appearing to underlie these types is stimulus modality: thermal, mechanical and chemical. That is, some strains are sensitive to any one of a number of assays of thermal nociception (e.g. hot-plate
* Corresponding author. Department of Psychology, McGill University, 1205 Dr. Penfield Avenue, Montreal, QC, Canada H3A 1B1. Tel.: 11-514398-6085; fax: 11-514-398-4896. E-mail address: [email protected] (J.S. Mogil). 1 These authors contributed equally to this manuscript.
test, tail-withdrawal test, Hargreaves’ test), whereas a different set of strains are sensitive to assays using a noxious chemical stimulus (e.g. formalin test, acetic acid abdominal constriction test, bee venom test). We have conducted quantitative trait locus (QTL) mapping studies of two thermal assays, the hot-plate test (Mogil et al., 1997) and the Hargreaves’ test of paw withdrawal (manuscript in preparation). In the former effort, we provided evidence for the existence of a trait-relevant gene on distal mouse chromosome 4; this gene may affect thermal nociception in males only, and may code for the d2-opioid receptor (Mogil et al., 1997). A QTL for autotomy, which shares a number of features with thermal nociception (see Mogil et al., 1999b), has also been recently reported on chromosome 15 (Seltzer et al., 2001). Thermal pain, of course, has very limited clinical significance, with the possible exception of burn pain. By contrast, a large majority of clinical pain complaints involve pain of inflammatory origin. Although inflammatory pain is
0304-3959/02/$20.00 q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00489-4
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comparatively sensitive to inhibition by opioids (see Kayser and Guilbaud, 1983; Stanfa and Dickenson, 1995) and thus can be well controlled clinically, the overwhelming incidence of this type of pain demands continued research attention especially in light of the well-known liabilities of opioid drugs. In a set of 11 inbred mouse strains, chemical/inflammatory assays displayed moderate-to-high heritability ðh2 ¼ 0:39–0:76Þ, and ranges of sensitivity among strains greatly exceed those of thermal assays (Mogil et al., 1999a). To investigate the genetic basis of chemical/ inflammatory pain, we chose to study the most commonly used assay in this class: the formalin test. The formalin test features a biphasic time course of behavioral and electrophysiological responses to the hindpaw injection of dilute formalin; subjects engage in vigorous recuperative behavior (e.g. licking) for approximately 5–10 min post-injection (the acute/early phase; Fearly), and then resume this behavior from approximately 15–90 min post-injection (the tonic/late phase; Flate). The purpose of the present study was to work towards identifying genes producing inherited variation in the formalin test, by using QTL mapping to detect genomic regions containing such genes. To this end, we constructed F2 hybrids from the most extreme-responding inbred strains for which microsatellite marker allele data were available (see Section 2), and performed a full-genome scan for linkage. Accordingly, we chose the A/J strain and the C57BL/6J strain as progenitors. These strains were the most resistant and second most sensitive strains of the 11 tested (Mogil et al., 1999a), and have been subsequently shown to display consistent differences in behavioral and neural responses to formalin, even though formalin-induced inflammation is equivalent in these strains (Mogil et al., 1998; Bon et al., 2002). 2. Methods 2.1. Subjects A/J (A) and C57BL/6J (B6) breeders were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). These mice were used as B6AF1 hybrid breeders (both reciprocals), which in turn were used as B6AF2 hybrid breeders (all reciprocals). The 203 B6AF2 hybrid mice tested represent the offspring of 28 litters from eight dams over a 14month period. All mice were housed in standard wiretopped, polypropylene shoebox cages with their same-sex littermates (after weaning at 18–21 days), two to five mice per cage (singlets were discarded), in a temperature- and humidity-controlled vivarium (12:12 h light cycle, lights on at 07:00 h), and with ad lib access to food (Harlan Teklad 8604) and tap water. Naı¨ve mice were tested in all cases between 6 and 9 weeks of age. 2.2. QTL mapping strategy A full-genome search for linkage was performed on this
B6AF2 intercross. The genome was covered with a set of 90 polymorphic microsatellite (simple sequence length/repeat polymorphism) markers spaced at approximately 22 cM on average. Genetic linkage maps of ordered markers were constructed using MAPMAKER (Lander et al., 1987). QTLs were then identified via interval mapping as implemented by QTL Cartographer (Zeng, 1993). The significance level of individual QTLs was assessed by genomewide permutation tests (Churchill and Doerge, 1994), and compared to Lander and Kruglyak’s (1995) suggested thresholds for significance. 2.3. Formalin test This assay (Dubuisson and Dennis, 1977) has been routinely adapted for use in mice (see Tjolsen et al., 1992 for review). Mice were habituated on a glass surface suspended 50 cm above a tabletop, within (30 cm high £ 30 cm diameter) transparent Plexiglas cylinders for 30 min, 24 h prior to testing, and for 30 min immediately prior to injection. All testing occurred near mid-photoperiod to reduce circadian variation in pain sensitivity (Kavaliers and Hirst, 1983). Using a 50 ml Hamilton microsyringe with a 30guage needle, 25 ml of 5% formalin was injected into the plantar surface of the right hindpaw. Following injection, mice were immediately returned to the observation cylinder and their behavior videorecorded from below for 90 min. After this period of time, mice were sacrificed by cervical dislocation under isoflurane anesthesia, both hindpaws were carefully severed at the ankle joint, and the spleen and liver removed and stored at 2208C. Videotapes were scored later by trained observers using The Observere Videotape Analysis Package software (Noldus, Inc.). We have previously demonstrated that the most appropriate nociceptive measure in the mouse is number of seconds spent licking/biting the injected hindpaw (Sufka et al., 1998); this dependent measure was quantified in 5-min time bins. Fearly and Flate were defined conservatively as 0–10 and 10–90 min, respectively, post-formalin injection. Hindpaws were weighed on a microbalance and measured for cross-sectional thickness using microcalipers within 2 min post-mortem. Two dependent measures of inflammation were analyzed: percent difference in weight between the two paws (%WD), and percent difference in thickness between the two paws (%TD). Data from seven mice were excluded from further analysis based on the absence of any evidence of inflammation (i.e. bad injection) from either of these measures. 2.4. DNA isolation and genotyping Genomic DNA was isolated from spleen and liver using a standard proteinase K digestion, phenol/chloroform extraction protocol. Primer pairs for microsatellite markers previously determined to be polymorphic between A and B6 mice (Wardell et al., 1995) were ordered from Research
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Table 1 Responses to formalin injection in A and B6 mice, and their hybrids a Genotype
n
Fearly (s)
Flate (s)
%WD (mg/kg)
%TD (mm/kg)
A B6 B6AF1 B6AF2
28 25 17 203
80.9 (9.6)* 136.8 (10.7) 131.8 (13.8) 146.2 (4.8)
394.2 (64.1)* 782.7 (71.3) 695.9 (82.9) 751.7 (27.2)
41.5 (3.1) 38.9 (3.1) 27.4 (2.1)* 40.1 (0.9)
1.9 (0.1) 1.6 (0.2) 1.6 (0.2) 1.9 (0.04)
a Values are means; SEM values are in parentheses. Fearly, formalin test, acute/early phase; Flate, formalin test, tonic/late phase; %WD, percent weight difference between injected and non-injected hindpaw; %TD, percent dorsal–ventral thickness difference between injected and non-injected hindpaw. *Significantly lower than all other genotypes, P , 0:05.
Genetics (Huntsville, AL, USA). Polymerase chain reaction was performed in a volume of 25 ml in 96-well microtiter plates using a standard ‘hot-start’ protocol, and products were resolved on 7% non-denaturing polyacrylamide gels and visualized by laser detection of ethidium bromide (Nucleoscan 2000, Nucleotech Inc.). Each F2 mouse was scored for its genotype with reference to progenitor strain samples run concurrently. 2.5. Statistics To estimate heritability, a one-way analysis of variance (ANOVA) by non-segregating strain (B6, A and B6AF1 hybrids) was performed. The proportion of the total variance for each trait due to genotype (strain), or r 2, was calculated as SSstrain/SStotal, which provides an estimate of the narrow sense (additive) heritability (Belknap, 1998). For group comparisons, a criterion level of significance was chosen as a ¼ 0:05, corrected where appropriate for multiple comparisons. 3. Results 3.1. Phenotyping All populations displayed the expected temporally biphasic response to formalin. Descriptive statistics describing the behavioral and inflammatory responses of the B6AF2 population, progenitor and F1 populations tested simultaneously, are provided in Table 1. As can be seen, the two progenitor populations differed significantly on the two behavioral measures, but not on either of the measures of inflammation. Behavioral scores in B6AF1 mice resembled that of the B6 parent, indicative of dominant inheritance of these traits. With the exception of an abnormally low %WD mean in B6AF1 mice, there were no genotypic differences in the inflammation measures. Heritability estimates of Fearly and Flate were h2 ¼ 0:23 and 0.24, respectively. No significant sex differences in behavioral or inflammatory responses to formalin were observed in progenitor, B6AF1 or B6AF2 mice, although a trend towards higher licking values in females was seen in every case. As predicted by our previous genetic correlation findings
(Mogil et al., 1999b), Fearly was significantly correlated with Flate among B6AF2 mice (r ¼ 0:40, P , 0:001). Also as expected, the two inflammation measures were significantly correlated (r ¼ 0:48, P , 0:001). %WD was significantly correlated with Fearly (r ¼ 0:32, P , 0:001), but not with Flate (r ¼ 0:12, P ¼ 0:11). %TD was uncorrelated with either of the behavioral measures (rs ¼ 0:03 and 20.01). 3.2. QTL detection A linkage map of the genome was generated using MAPMAKER that showed fairly good agreement with published marker locations. No evidence for segregation distortion was noted in any region of the genome. To satisfy normality assumptions, a square root transformation of Fearly and Flate was performed prior to QTL analysis. Within this map, two significant QTLs were identified that exceeded significance thresholds both determined by permutation analysis and Lander and Kruglyak’s (1995) criteria. One of these, on distal chromosome 9, accounted for 27.3% of the overall phenotypic variance in Fearly, but did not significantly influence Flate (see Figs. 1 and 2; Table 2). The other, on distal chromosome 10, accounted for approximately 15% of the overall phenotypic variance in Fearly and Flate (see Figs. 1 and 2; Table 2). It should be noted that the percent variance in Fearly accounted for by these two QTLs exceeds the heritability of the trait. This seeming paradox may arise from the fact that estimates of variance accounted for are likely to be overestimates in studies of limited sample size (Beavis, 1998), or may be due to negative covariance between the QTLs. As shown in Fig. 2, B6AF2 mice inheriting two copies of the B6 allele at D10Mit35 spend approximately 4 more minutes (238.5 s) licking their hindpaws in response to formalin injection in Flate than do mice inheriting two copies of the A allele. Interval mapping was also performed separately on data from male and female mice, and yielded similar results (although with accordingly reduced power) for the QTLs described above. Some suggestive evidence for sex-specific QTLs was uncovered (P , 0:1; not shown), but none achieved statistical significance. Inflammation measures were log transformed to satisfy
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4. Discussion Although the heritability of pain sensitivity and susceptibility to the development of chronic pain syndromes appears to be highly dependent on the trait in question in humans, nociceptive sensitivity is clearly heritable in mice (see Mogil, 1999 for review). The present study and the recent effort of Seltzer et al. (2001) represent the first attempts in laboratory animals to identify genes responsible for individual differences in sensitivity to clinically relevant pain modalities. We have identified two significant QTLs, on chromosomes 9 and 10; we propose Nociq1 and Nociq2 as identifiers for these QTLs, respectively. 4.1. Genetic and environmental contributions to formalin test variability Although significantly different, the A versus B6 strain difference in formalin licking is less robust than reported
Fig. 1. Interval mapping results for Fearly and Flate, showing significantly linked regions. Curved lines represent logarithm of the odds (LOD) scores for linkage versus against linkage at successive chromosomal locations, as determined by QTL Cartographer and with reference to published marker locations. The two significant QTLs are dubbed Nociq1 and Nociq2. Nociq1, located on distal chromosome 9, is a significant QTL for Fearly (A). Nociq2, located on distal chromosome 10, is a significant QTL for both Fearly and Flate (B). The horizontal dashed line indicates significance threshold obtained from 1000 permutations of marker and trait data and corresponding to an experimentwise a ¼ 0:05. Each trait was permuted separately, producing slightly different significance thresholds (Fearly: LOD ¼ 3.2; Flate: LOD ¼ 3.4); for clarity the mean of these two thresholds is shown. Horizontal interval bars represent the 1-LOD drop-off confidence interval.
normality assumptions. No chromosomal regions showed linkage even approaching significance for either %WD or %TD. This is not surprising since the progenitor strains did not differ on either of these measures of inflammation, and so conditions were not optimal to detect such QTLs. There was no evidence whatsoever of linkage to either of these measures on chromosomes 9 or 10, suggesting that the formalin behavior QTLs there are not secondary to inherited variability in the degree of formalin-induced hindpaw inflammation.
Fig. 2. Effect of gene dosage (A/A, A/B6, B6/B6) at D9Mit182 (A) and D10Mit35 (B) in B6AF2 mice on acute tonic phase nociception, respectively, in the formalin test. F2 mice inherit the A/A, A/B6, and B6/B6 genotypes in the familiar 1:2:1 ratio; when a QTL exists near a marker such inheritance will affect the phenotype accordingly. Bars represent mean (^SEM) time spent licking in Flate. *Significant from A/A genotype at P , 0:05. **Significant from A/A genotype at P , 0:01. ***Significant from A/A genotype at P , 0:005. In neither case did heterozygotes (A/ B6) differ significantly from B6/B6 homozygote groups.
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Table 2 QTLs for formalin test nociception QTL
Fearly Flate a b c d e f
Linkage peak Chr. cM
LOD e
9 10 10
5.2 3.5 4.3
59.8 54.8 70.0
CI a (cM)
% Variance b
Model c
Significance d
44–68 34–64 58-end
27.3 14.2 15.0
Dominant Dominant Dominant/Additive f
,0.01 ,0.025 ,0.01
95% Confidence interval (CI) describing the likely location of the QTL, based on a 1-LOD drop-off. Percent of overall phenotypic variance explained by the QTL. Genetic model of highest likelihood among 1-df models (i.e. additive, dominant, recessive). Dominant refers to the B6 allele. Experimentwise significance level (P value) by genome-wide permutation analysis (Churchill and Doerge, 1994). LOD score based on interval mapping as implemented by QTL Cartographer. These models were equally likely.
previously (Mogil et al., 1998, 1999a). As a result, the heritabilities of Fearly and Flate as estimated herein (h2 ¼ 0:23 and 0.24, respectively) are lower than estimates previously obtained in a survey of 11 inbred strains (h2 ¼ 0:39 and 0.46, respectively) (Mogil et al., 1999a). Nevertheless, heritability of formalin test sensitivity remained high enough to successfully identify QTLs. It should be noted that different experimenters performed this and the previous study (Mogil et al., 1998). The individual performing formalin injections on all mice in the present study (S.G.W.) consistently observed increased licking behavior (and increased edema) than the previous injector (J.S.M.) in a large number of genotypes. In fact, the variable efficacy of the formalin injections themselves is a considerable (although poorly recognized) source of environmental variability in this assay. We have also found experimenter to be a major source of environmental variability in the tail-withdrawal assay (Chesler et al., submitted). Any number of factors are known to affect licking behavior in the formalin test, including, for example, locomotor activity of incompletely habituated animals (Aloisi et al., 1995). Thus, a possibility exists that we have mapped the location of genes mediating sensitivity to artefacts like locomotion rather than pain perception. Results from a recent study engender confidence in the latter, however (Bon et al., 2002). Testing a number of inbred and outbred strains, we found that in addition to differential formalin-induced licking behavior among strains, spinal cord expression of the immediate-early gene, c-fos, was also highly strain dependent. The correlation between Flate licking behavior and cfos expression in the neck of the dorsal horn (laminae V/VI) approached unity (r ¼ 0:94), suggesting that the latter neural phenomenon can be used as a proxy for the former behavioral phenomenon (Bon et al., 2002). If strain differences in formalin test licking were due to strain-dependent locomotor activity in a novel environment, one would not expect to see this relationship in a discrete nervous system locus associated with sensory processing. 4.2. Nociq1: a QTL for Fearly on chromosome 9 The QTL on mid-chromosome 9, Nociq1, accounts for
27% of the trait variance in Fearly. A search of the Mouse Genome Database (http://www.informatics.jax.org) in the region between 44 and 68 cM reveals any number of potential candidate genes that may underlie this QTL. Genes encoding proteins with recognized roles in the mediation or modulation of nociception include Gnai2 (59 cM; Gi protein, a2 subunit) and Htr1b (46 cM; serotonin-1B receptor). The gene coding for cholecystokinin (Cck; 71 cM) is also very nearby, and it should be noted that 1-logarithm of the odds (LOD) drop-off confidence intervals are often unduly optimistic in their precision. Given that inflammatory nociception in the formalin test has been shown to involve substances released from mast cells (Ribeiro et al., 2000; Parada et al., 2001), it is also intriguing that the Mcr gene, coding for a protein which controls mast cell clustering, is also found in this region (48 cM). Probably the most intriguing candidate genes, however, are the Scn10a and Scn11a genes (67 and 71 cM), encoding the SNS/PN3 and SNS2/NaN sodium channels, respectively. Both are voltage-gated, tetrodotoxin-resistant ion channels that are uniquely found in small-diameter sensory neurons (i.e. nociceptors) of the dorsal root ganglion (Akopian et al., 1996; Sangameswaran et al., 1996; DibHajj et al., 1998; Tate et al., 1998). Although evidence implicating especially SNS/PN3 in pain is increasing (see McCleskey and Gold, 1999), to our knowledge the role of these channels in the formalin test has never been directly examined. 4.3. Nociq2: a QTL for Fearly and Flate on chromosome 10 In contrast to Nociq1, which only showed linkage to Fearly, a region of chromosome 10 displayed linkage to Fearly and Flate. The peak of the chromosome 10 QTL for Fearly appeared 15 cM more proximally than the peak for Flate, suggesting that these may be independent QTLs. However, given the fact that the confidence intervals of these QTLs overlap, it seems at least equally likely that they represent the same QTL. More precise localization of the actual position of the QTL(s) should resolve this issue; it should be noted, however, that there are no informative microsatellite markers distal to D10Mit35 on chromosome 10.
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Our confidence in the existence of a gene or genes in this region of relevance to chemical/inflammatory nociception is increased by the fact that two of us (H.H. and J.K.B.), in a completely independent QTL mapping study performed simultaneously, have collected evidence that this same region of chromosome 10 is linked to variability in the chemical/inflammatory acetic acid abdominal constriction (writhing) test (unpublished data). This study used recombinant inbred and F2 hybrid mice derived from a C57BL/ 6 £ DBA/2 intercross, and also found dominant inheritance of the C57BL/6 allele. This is not a straight replication of our findings, but the fact that the same QTL may have been detected using both the formalin test and the writhing test represents strong support of our previous contention that chemical nociception represents a fundamental nociceptive ‘type’ (Mogil et al., 1999b). Thus, Nociq2 may be broadly relevant to chemical/inflammatory nociception, and may represent a major reason for the high correlation of the performance of inbred strains on different chemical assays of nociception. As can be seen from Table 2 and Fig. 1, the confidence interval generally describing Nociq2 is on the distal half of chromosome 10. Genes in this region encoding proteins with recognized roles in the mediation or modulation of inflammation and/or nociception include: Ifng (67 cM; interferon-g) and Igf1 (48 cM; insulin-like growth factor). The former is a particularly intriguing candidate gene given its role in the induction of inflammation, and the increasing appreciation of the role of neuroimmune molecules in persistent pain states (see Watkins and Maier, 2000; DeLeo and Yezierski, 2001). We were, however, unable to detect any difference between mutant mice lacking Ifng and their wildtype controls on either the formalin or writhing test (data not shown). This negative finding does not exclude this gene as a candidate, of course, since in the mutant mice compensatory mechanisms may have obscured the true effect of the gene deletion. It is also of interest that QTLs affecting arthritis severity have been mapped to distal mouse chromosome 10 (Dracheva et al., 1999; Yang et al., 1999). It is tempting to suggest that the same gene(s) influencing inflammatory nociceptive sensitivity in the formalin test may also influence disease severity in arthritis. If true, the successful identification of Nociq2 may have implications in the understanding of this clinically important pain syndrome. 4.4. Future directions A number of options are available to work towards the identification of the genes and DNA variants underlying these QTLs. We are currently testing candidate gene hypotheses and attempting to further resolve the confidence intervals containing these QTLs using a congenic strategy (i.e. positional cloning). Practical linkage disequilibrium strategies using single nucleotide polymorphisms (SNPs) are almost at hand as well.
Through application of gene expression, transgenic, and antisense oligonucleotide experiments, no less than 20 different genes and their corresponding protein products have been implicated in the mediation of nociception on the formalin test (see Mogil and McCarson, 2000 for review). Virtually none of these genes are located in chromosomal regions defined by Nociq1 and Nociq2. Our failure to find evidence for linkage does not in any way exclude an important role of these genes in pain processing. For one thing, our study lacked sufficient statistical power to definitively rule out the existence of linkage anywhere on the genome. We also lacked statistical power to detect QTLs explaining a small percentage of trait variance. Third, QTL mapping is only able to detect the influence of genes that are polymorphic between the progenitor strains employed. Finally, genes can be trait-relevant without being responsible for individual variability on that trait. Nociq1 and Nociq2 belong to an important subset of the larger number of genes relevant to chemical/inflammatory nociception, in that they are both trait-relevant and polymorphic, affecting the magnitude of pain behavior (and perhaps, then, the perceived intensity of that pain) on these assays. The eventual identification of these particular genes, therefore, may have important implications for the prediction of analgesic requirements in humans, and may lead to novel targets for analgesic development. Acknowledgements Thanks to Rabia Akram, Camron Bryant, Natalie Coleman, Mark Gaudio, Jae Haas, Anna Kokayeff, Karen O’Mara, Neha Mehta, Kim Melton, Bryan Mitton, Jeane Palmer, and Ian Robberechts for assistance with data scoring. This work was supported by PHS grants DA11394 and DE12735 (J.S.M.). S.W.G. was supported by an NRSA award (DA6000). References Akopian AN, Silvotti L, Wood JN. A tetrodotoxin-resistant sodium channel expressed by C-fibre-associated sensory neurons. Nature 1996;379: 257–262. Aloisi AM, Albonetti ME, Carli G. Behavioral effects of different intensities of formalin pain in rats. Physiol Behav 1995;58:603–610. Beavis WD. QTL analyses: power, precision and accuracy. In: Patterson C, editor. Molecular Dissection of Complex Traits, Boca Raton, FL: CRC Press, 1998. pp. 145–162. Belknap JK. Effect of within-strain sample size on QTL detection and mapping using recombinant inbred mouse strains. Behav Genet 1998;28:29–38. Bon K, Wilson SG, Mogil JS, Roberts WJ. Genetic evidence for the correlation of deep dorsal horn Fos protein immunoreactivity with tonic formalin pain behavior. J Pain 2002 (in press). Churchill GA, Doerge RW. Empirical threshold values for quantitative trait mapping. Genetics 1994;138:963–971. DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune activation in persistent pain. Pain 2001;90:1–6. Dib-Hajj SD, Tyrrell L, Black JA, Waxman SG. NaN, a novel voltage-gated
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Identification of quantitative trait loci for chemical/inflammatory nociception in mice Sonya G. Wilson a,1, Elissa J. Chesler a,1, Heather Hain b, Andrew J. Rankin a, Joel Z. Schwarz a, Stanford B. Call c, Michael R. Murray a, Erin E. West a, Cory Teuscher c, Sandra Rodriguez-Zas d, John K. Belknap b, Jeffrey S. Mogil a,* a
Department of Psychology and Neuroscience Program, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA Department of Behavioral Neuroscience and VA Medical Center, Oregon Health Sciences University, Portland, OR 97201, USA c Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
b
Received 18 September 2001; received in revised form 16 November 2001; accepted 13 December 2001
Abstract 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 ml 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. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Pain; Genetics; Gene mapping; Formalin test; Inflammation; Strain differences
1. Introduction The contribution of genetic factors to individual differences in pain and analgesic sensitivity is now widely accepted, and much progress is being made towards the identification of the responsible genes (see Mogil, 1999 for review). Our studies using a common set of inbred mouse strains have revealed a limited number of fundamental pain ‘types’ as defined by genetic correlations (Mogil et al., 1999a,b). The dimension appearing to underlie these types is stimulus modality: thermal, mechanical and chemical. That is, some strains are sensitive to any one of a number of assays of thermal nociception (e.g. hot-plate
* Corresponding author. Department of Psychology, McGill University, 1205 Dr. Penfield Avenue, Montreal, QC, Canada H3A 1B1. Tel.: 11-514398-6085; fax: 11-514-398-4896. E-mail address: [email protected] (J.S. Mogil). 1 These authors contributed equally to this manuscript.
test, tail-withdrawal test, Hargreaves’ test), whereas a different set of strains are sensitive to assays using a noxious chemical stimulus (e.g. formalin test, acetic acid abdominal constriction test, bee venom test). We have conducted quantitative trait locus (QTL) mapping studies of two thermal assays, the hot-plate test (Mogil et al., 1997) and the Hargreaves’ test of paw withdrawal (manuscript in preparation). In the former effort, we provided evidence for the existence of a trait-relevant gene on distal mouse chromosome 4; this gene may affect thermal nociception in males only, and may code for the d2-opioid receptor (Mogil et al., 1997). A QTL for autotomy, which shares a number of features with thermal nociception (see Mogil et al., 1999b), has also been recently reported on chromosome 15 (Seltzer et al., 2001). Thermal pain, of course, has very limited clinical significance, with the possible exception of burn pain. By contrast, a large majority of clinical pain complaints involve pain of inflammatory origin. Although inflammatory pain is
0304-3959/02/$20.00 q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00489-4
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comparatively sensitive to inhibition by opioids (see Kayser and Guilbaud, 1983; Stanfa and Dickenson, 1995) and thus can be well controlled clinically, the overwhelming incidence of this type of pain demands continued research attention especially in light of the well-known liabilities of opioid drugs. In a set of 11 inbred mouse strains, chemical/inflammatory assays displayed moderate-to-high heritability ðh2 ¼ 0:39–0:76Þ, and ranges of sensitivity among strains greatly exceed those of thermal assays (Mogil et al., 1999a). To investigate the genetic basis of chemical/ inflammatory pain, we chose to study the most commonly used assay in this class: the formalin test. The formalin test features a biphasic time course of behavioral and electrophysiological responses to the hindpaw injection of dilute formalin; subjects engage in vigorous recuperative behavior (e.g. licking) for approximately 5–10 min post-injection (the acute/early phase; Fearly), and then resume this behavior from approximately 15–90 min post-injection (the tonic/late phase; Flate). The purpose of the present study was to work towards identifying genes producing inherited variation in the formalin test, by using QTL mapping to detect genomic regions containing such genes. To this end, we constructed F2 hybrids from the most extreme-responding inbred strains for which microsatellite marker allele data were available (see Section 2), and performed a full-genome scan for linkage. Accordingly, we chose the A/J strain and the C57BL/6J strain as progenitors. These strains were the most resistant and second most sensitive strains of the 11 tested (Mogil et al., 1999a), and have been subsequently shown to display consistent differences in behavioral and neural responses to formalin, even though formalin-induced inflammation is equivalent in these strains (Mogil et al., 1998; Bon et al., 2002). 2. Methods 2.1. Subjects A/J (A) and C57BL/6J (B6) breeders were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). These mice were used as B6AF1 hybrid breeders (both reciprocals), which in turn were used as B6AF2 hybrid breeders (all reciprocals). The 203 B6AF2 hybrid mice tested represent the offspring of 28 litters from eight dams over a 14month period. All mice were housed in standard wiretopped, polypropylene shoebox cages with their same-sex littermates (after weaning at 18–21 days), two to five mice per cage (singlets were discarded), in a temperature- and humidity-controlled vivarium (12:12 h light cycle, lights on at 07:00 h), and with ad lib access to food (Harlan Teklad 8604) and tap water. Naı¨ve mice were tested in all cases between 6 and 9 weeks of age. 2.2. QTL mapping strategy A full-genome search for linkage was performed on this
B6AF2 intercross. The genome was covered with a set of 90 polymorphic microsatellite (simple sequence length/repeat polymorphism) markers spaced at approximately 22 cM on average. Genetic linkage maps of ordered markers were constructed using MAPMAKER (Lander et al., 1987). QTLs were then identified via interval mapping as implemented by QTL Cartographer (Zeng, 1993). The significance level of individual QTLs was assessed by genomewide permutation tests (Churchill and Doerge, 1994), and compared to Lander and Kruglyak’s (1995) suggested thresholds for significance. 2.3. Formalin test This assay (Dubuisson and Dennis, 1977) has been routinely adapted for use in mice (see Tjolsen et al., 1992 for review). Mice were habituated on a glass surface suspended 50 cm above a tabletop, within (30 cm high £ 30 cm diameter) transparent Plexiglas cylinders for 30 min, 24 h prior to testing, and for 30 min immediately prior to injection. All testing occurred near mid-photoperiod to reduce circadian variation in pain sensitivity (Kavaliers and Hirst, 1983). Using a 50 ml Hamilton microsyringe with a 30guage needle, 25 ml of 5% formalin was injected into the plantar surface of the right hindpaw. Following injection, mice were immediately returned to the observation cylinder and their behavior videorecorded from below for 90 min. After this period of time, mice were sacrificed by cervical dislocation under isoflurane anesthesia, both hindpaws were carefully severed at the ankle joint, and the spleen and liver removed and stored at 2208C. Videotapes were scored later by trained observers using The Observere Videotape Analysis Package software (Noldus, Inc.). We have previously demonstrated that the most appropriate nociceptive measure in the mouse is number of seconds spent licking/biting the injected hindpaw (Sufka et al., 1998); this dependent measure was quantified in 5-min time bins. Fearly and Flate were defined conservatively as 0–10 and 10–90 min, respectively, post-formalin injection. Hindpaws were weighed on a microbalance and measured for cross-sectional thickness using microcalipers within 2 min post-mortem. Two dependent measures of inflammation were analyzed: percent difference in weight between the two paws (%WD), and percent difference in thickness between the two paws (%TD). Data from seven mice were excluded from further analysis based on the absence of any evidence of inflammation (i.e. bad injection) from either of these measures. 2.4. DNA isolation and genotyping Genomic DNA was isolated from spleen and liver using a standard proteinase K digestion, phenol/chloroform extraction protocol. Primer pairs for microsatellite markers previously determined to be polymorphic between A and B6 mice (Wardell et al., 1995) were ordered from Research
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Table 1 Responses to formalin injection in A and B6 mice, and their hybrids a Genotype
n
Fearly (s)
Flate (s)
%WD (mg/kg)
%TD (mm/kg)
A B6 B6AF1 B6AF2
28 25 17 203
80.9 (9.6)* 136.8 (10.7) 131.8 (13.8) 146.2 (4.8)
394.2 (64.1)* 782.7 (71.3) 695.9 (82.9) 751.7 (27.2)
41.5 (3.1) 38.9 (3.1) 27.4 (2.1)* 40.1 (0.9)
1.9 (0.1) 1.6 (0.2) 1.6 (0.2) 1.9 (0.04)
a Values are means; SEM values are in parentheses. Fearly, formalin test, acute/early phase; Flate, formalin test, tonic/late phase; %WD, percent weight difference between injected and non-injected hindpaw; %TD, percent dorsal–ventral thickness difference between injected and non-injected hindpaw. *Significantly lower than all other genotypes, P , 0:05.
Genetics (Huntsville, AL, USA). Polymerase chain reaction was performed in a volume of 25 ml in 96-well microtiter plates using a standard ‘hot-start’ protocol, and products were resolved on 7% non-denaturing polyacrylamide gels and visualized by laser detection of ethidium bromide (Nucleoscan 2000, Nucleotech Inc.). Each F2 mouse was scored for its genotype with reference to progenitor strain samples run concurrently. 2.5. Statistics To estimate heritability, a one-way analysis of variance (ANOVA) by non-segregating strain (B6, A and B6AF1 hybrids) was performed. The proportion of the total variance for each trait due to genotype (strain), or r 2, was calculated as SSstrain/SStotal, which provides an estimate of the narrow sense (additive) heritability (Belknap, 1998). For group comparisons, a criterion level of significance was chosen as a ¼ 0:05, corrected where appropriate for multiple comparisons. 3. Results 3.1. Phenotyping All populations displayed the expected temporally biphasic response to formalin. Descriptive statistics describing the behavioral and inflammatory responses of the B6AF2 population, progenitor and F1 populations tested simultaneously, are provided in Table 1. As can be seen, the two progenitor populations differed significantly on the two behavioral measures, but not on either of the measures of inflammation. Behavioral scores in B6AF1 mice resembled that of the B6 parent, indicative of dominant inheritance of these traits. With the exception of an abnormally low %WD mean in B6AF1 mice, there were no genotypic differences in the inflammation measures. Heritability estimates of Fearly and Flate were h2 ¼ 0:23 and 0.24, respectively. No significant sex differences in behavioral or inflammatory responses to formalin were observed in progenitor, B6AF1 or B6AF2 mice, although a trend towards higher licking values in females was seen in every case. As predicted by our previous genetic correlation findings
(Mogil et al., 1999b), Fearly was significantly correlated with Flate among B6AF2 mice (r ¼ 0:40, P , 0:001). Also as expected, the two inflammation measures were significantly correlated (r ¼ 0:48, P , 0:001). %WD was significantly correlated with Fearly (r ¼ 0:32, P , 0:001), but not with Flate (r ¼ 0:12, P ¼ 0:11). %TD was uncorrelated with either of the behavioral measures (rs ¼ 0:03 and 20.01). 3.2. QTL detection A linkage map of the genome was generated using MAPMAKER that showed fairly good agreement with published marker locations. No evidence for segregation distortion was noted in any region of the genome. To satisfy normality assumptions, a square root transformation of Fearly and Flate was performed prior to QTL analysis. Within this map, two significant QTLs were identified that exceeded significance thresholds both determined by permutation analysis and Lander and Kruglyak’s (1995) criteria. One of these, on distal chromosome 9, accounted for 27.3% of the overall phenotypic variance in Fearly, but did not significantly influence Flate (see Figs. 1 and 2; Table 2). The other, on distal chromosome 10, accounted for approximately 15% of the overall phenotypic variance in Fearly and Flate (see Figs. 1 and 2; Table 2). It should be noted that the percent variance in Fearly accounted for by these two QTLs exceeds the heritability of the trait. This seeming paradox may arise from the fact that estimates of variance accounted for are likely to be overestimates in studies of limited sample size (Beavis, 1998), or may be due to negative covariance between the QTLs. As shown in Fig. 2, B6AF2 mice inheriting two copies of the B6 allele at D10Mit35 spend approximately 4 more minutes (238.5 s) licking their hindpaws in response to formalin injection in Flate than do mice inheriting two copies of the A allele. Interval mapping was also performed separately on data from male and female mice, and yielded similar results (although with accordingly reduced power) for the QTLs described above. Some suggestive evidence for sex-specific QTLs was uncovered (P , 0:1; not shown), but none achieved statistical significance. Inflammation measures were log transformed to satisfy
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4. Discussion Although the heritability of pain sensitivity and susceptibility to the development of chronic pain syndromes appears to be highly dependent on the trait in question in humans, nociceptive sensitivity is clearly heritable in mice (see Mogil, 1999 for review). The present study and the recent effort of Seltzer et al. (2001) represent the first attempts in laboratory animals to identify genes responsible for individual differences in sensitivity to clinically relevant pain modalities. We have identified two significant QTLs, on chromosomes 9 and 10; we propose Nociq1 and Nociq2 as identifiers for these QTLs, respectively. 4.1. Genetic and environmental contributions to formalin test variability Although significantly different, the A versus B6 strain difference in formalin licking is less robust than reported
Fig. 1. Interval mapping results for Fearly and Flate, showing significantly linked regions. Curved lines represent logarithm of the odds (LOD) scores for linkage versus against linkage at successive chromosomal locations, as determined by QTL Cartographer and with reference to published marker locations. The two significant QTLs are dubbed Nociq1 and Nociq2. Nociq1, located on distal chromosome 9, is a significant QTL for Fearly (A). Nociq2, located on distal chromosome 10, is a significant QTL for both Fearly and Flate (B). The horizontal dashed line indicates significance threshold obtained from 1000 permutations of marker and trait data and corresponding to an experimentwise a ¼ 0:05. Each trait was permuted separately, producing slightly different significance thresholds (Fearly: LOD ¼ 3.2; Flate: LOD ¼ 3.4); for clarity the mean of these two thresholds is shown. Horizontal interval bars represent the 1-LOD drop-off confidence interval.
normality assumptions. No chromosomal regions showed linkage even approaching significance for either %WD or %TD. This is not surprising since the progenitor strains did not differ on either of these measures of inflammation, and so conditions were not optimal to detect such QTLs. There was no evidence whatsoever of linkage to either of these measures on chromosomes 9 or 10, suggesting that the formalin behavior QTLs there are not secondary to inherited variability in the degree of formalin-induced hindpaw inflammation.
Fig. 2. Effect of gene dosage (A/A, A/B6, B6/B6) at D9Mit182 (A) and D10Mit35 (B) in B6AF2 mice on acute tonic phase nociception, respectively, in the formalin test. F2 mice inherit the A/A, A/B6, and B6/B6 genotypes in the familiar 1:2:1 ratio; when a QTL exists near a marker such inheritance will affect the phenotype accordingly. Bars represent mean (^SEM) time spent licking in Flate. *Significant from A/A genotype at P , 0:05. **Significant from A/A genotype at P , 0:01. ***Significant from A/A genotype at P , 0:005. In neither case did heterozygotes (A/ B6) differ significantly from B6/B6 homozygote groups.
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389
Table 2 QTLs for formalin test nociception QTL
Fearly Flate a b c d e f
Linkage peak Chr. cM
LOD e
9 10 10
5.2 3.5 4.3
59.8 54.8 70.0
CI a (cM)
% Variance b
Model c
Significance d
44–68 34–64 58-end
27.3 14.2 15.0
Dominant Dominant Dominant/Additive f
,0.01 ,0.025 ,0.01
95% Confidence interval (CI) describing the likely location of the QTL, based on a 1-LOD drop-off. Percent of overall phenotypic variance explained by the QTL. Genetic model of highest likelihood among 1-df models (i.e. additive, dominant, recessive). Dominant refers to the B6 allele. Experimentwise significance level (P value) by genome-wide permutation analysis (Churchill and Doerge, 1994). LOD score based on interval mapping as implemented by QTL Cartographer. These models were equally likely.
previously (Mogil et al., 1998, 1999a). As a result, the heritabilities of Fearly and Flate as estimated herein (h2 ¼ 0:23 and 0.24, respectively) are lower than estimates previously obtained in a survey of 11 inbred strains (h2 ¼ 0:39 and 0.46, respectively) (Mogil et al., 1999a). Nevertheless, heritability of formalin test sensitivity remained high enough to successfully identify QTLs. It should be noted that different experimenters performed this and the previous study (Mogil et al., 1998). The individual performing formalin injections on all mice in the present study (S.G.W.) consistently observed increased licking behavior (and increased edema) than the previous injector (J.S.M.) in a large number of genotypes. In fact, the variable efficacy of the formalin injections themselves is a considerable (although poorly recognized) source of environmental variability in this assay. We have also found experimenter to be a major source of environmental variability in the tail-withdrawal assay (Chesler et al., submitted). Any number of factors are known to affect licking behavior in the formalin test, including, for example, locomotor activity of incompletely habituated animals (Aloisi et al., 1995). Thus, a possibility exists that we have mapped the location of genes mediating sensitivity to artefacts like locomotion rather than pain perception. Results from a recent study engender confidence in the latter, however (Bon et al., 2002). Testing a number of inbred and outbred strains, we found that in addition to differential formalin-induced licking behavior among strains, spinal cord expression of the immediate-early gene, c-fos, was also highly strain dependent. The correlation between Flate licking behavior and cfos expression in the neck of the dorsal horn (laminae V/VI) approached unity (r ¼ 0:94), suggesting that the latter neural phenomenon can be used as a proxy for the former behavioral phenomenon (Bon et al., 2002). If strain differences in formalin test licking were due to strain-dependent locomotor activity in a novel environment, one would not expect to see this relationship in a discrete nervous system locus associated with sensory processing. 4.2. Nociq1: a QTL for Fearly on chromosome 9 The QTL on mid-chromosome 9, Nociq1, accounts for
27% of the trait variance in Fearly. A search of the Mouse Genome Database (http://www.informatics.jax.org) in the region between 44 and 68 cM reveals any number of potential candidate genes that may underlie this QTL. Genes encoding proteins with recognized roles in the mediation or modulation of nociception include Gnai2 (59 cM; Gi protein, a2 subunit) and Htr1b (46 cM; serotonin-1B receptor). The gene coding for cholecystokinin (Cck; 71 cM) is also very nearby, and it should be noted that 1-logarithm of the odds (LOD) drop-off confidence intervals are often unduly optimistic in their precision. Given that inflammatory nociception in the formalin test has been shown to involve substances released from mast cells (Ribeiro et al., 2000; Parada et al., 2001), it is also intriguing that the Mcr gene, coding for a protein which controls mast cell clustering, is also found in this region (48 cM). Probably the most intriguing candidate genes, however, are the Scn10a and Scn11a genes (67 and 71 cM), encoding the SNS/PN3 and SNS2/NaN sodium channels, respectively. Both are voltage-gated, tetrodotoxin-resistant ion channels that are uniquely found in small-diameter sensory neurons (i.e. nociceptors) of the dorsal root ganglion (Akopian et al., 1996; Sangameswaran et al., 1996; DibHajj et al., 1998; Tate et al., 1998). Although evidence implicating especially SNS/PN3 in pain is increasing (see McCleskey and Gold, 1999), to our knowledge the role of these channels in the formalin test has never been directly examined. 4.3. Nociq2: a QTL for Fearly and Flate on chromosome 10 In contrast to Nociq1, which only showed linkage to Fearly, a region of chromosome 10 displayed linkage to Fearly and Flate. The peak of the chromosome 10 QTL for Fearly appeared 15 cM more proximally than the peak for Flate, suggesting that these may be independent QTLs. However, given the fact that the confidence intervals of these QTLs overlap, it seems at least equally likely that they represent the same QTL. More precise localization of the actual position of the QTL(s) should resolve this issue; it should be noted, however, that there are no informative microsatellite markers distal to D10Mit35 on chromosome 10.
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Our confidence in the existence of a gene or genes in this region of relevance to chemical/inflammatory nociception is increased by the fact that two of us (H.H. and J.K.B.), in a completely independent QTL mapping study performed simultaneously, have collected evidence that this same region of chromosome 10 is linked to variability in the chemical/inflammatory acetic acid abdominal constriction (writhing) test (unpublished data). This study used recombinant inbred and F2 hybrid mice derived from a C57BL/ 6 £ DBA/2 intercross, and also found dominant inheritance of the C57BL/6 allele. This is not a straight replication of our findings, but the fact that the same QTL may have been detected using both the formalin test and the writhing test represents strong support of our previous contention that chemical nociception represents a fundamental nociceptive ‘type’ (Mogil et al., 1999b). Thus, Nociq2 may be broadly relevant to chemical/inflammatory nociception, and may represent a major reason for the high correlation of the performance of inbred strains on different chemical assays of nociception. As can be seen from Table 2 and Fig. 1, the confidence interval generally describing Nociq2 is on the distal half of chromosome 10. Genes in this region encoding proteins with recognized roles in the mediation or modulation of inflammation and/or nociception include: Ifng (67 cM; interferon-g) and Igf1 (48 cM; insulin-like growth factor). The former is a particularly intriguing candidate gene given its role in the induction of inflammation, and the increasing appreciation of the role of neuroimmune molecules in persistent pain states (see Watkins and Maier, 2000; DeLeo and Yezierski, 2001). We were, however, unable to detect any difference between mutant mice lacking Ifng and their wildtype controls on either the formalin or writhing test (data not shown). This negative finding does not exclude this gene as a candidate, of course, since in the mutant mice compensatory mechanisms may have obscured the true effect of the gene deletion. It is also of interest that QTLs affecting arthritis severity have been mapped to distal mouse chromosome 10 (Dracheva et al., 1999; Yang et al., 1999). It is tempting to suggest that the same gene(s) influencing inflammatory nociceptive sensitivity in the formalin test may also influence disease severity in arthritis. If true, the successful identification of Nociq2 may have implications in the understanding of this clinically important pain syndrome. 4.4. Future directions A number of options are available to work towards the identification of the genes and DNA variants underlying these QTLs. We are currently testing candidate gene hypotheses and attempting to further resolve the confidence intervals containing these QTLs using a congenic strategy (i.e. positional cloning). Practical linkage disequilibrium strategies using single nucleotide polymorphisms (SNPs) are almost at hand as well.
Through application of gene expression, transgenic, and antisense oligonucleotide experiments, no less than 20 different genes and their corresponding protein products have been implicated in the mediation of nociception on the formalin test (see Mogil and McCarson, 2000 for review). Virtually none of these genes are located in chromosomal regions defined by Nociq1 and Nociq2. Our failure to find evidence for linkage does not in any way exclude an important role of these genes in pain processing. For one thing, our study lacked sufficient statistical power to definitively rule out the existence of linkage anywhere on the genome. We also lacked statistical power to detect QTLs explaining a small percentage of trait variance. Third, QTL mapping is only able to detect the influence of genes that are polymorphic between the progenitor strains employed. Finally, genes can be trait-relevant without being responsible for individual variability on that trait. Nociq1 and Nociq2 belong to an important subset of the larger number of genes relevant to chemical/inflammatory nociception, in that they are both trait-relevant and polymorphic, affecting the magnitude of pain behavior (and perhaps, then, the perceived intensity of that pain) on these assays. The eventual identification of these particular genes, therefore, may have important implications for the prediction of analgesic requirements in humans, and may lead to novel targets for analgesic development. Acknowledgements Thanks to Rabia Akram, Camron Bryant, Natalie Coleman, Mark Gaudio, Jae Haas, Anna Kokayeff, Karen O’Mara, Neha Mehta, Kim Melton, Bryan Mitton, Jeane Palmer, and Ian Robberechts for assistance with data scoring. This work was supported by PHS grants DA11394 and DE12735 (J.S.M.). S.W.G. was supported by an NRSA award (DA6000). References Akopian AN, Silvotti L, Wood JN. A tetrodotoxin-resistant sodium channel expressed by C-fibre-associated sensory neurons. Nature 1996;379: 257–262. Aloisi AM, Albonetti ME, Carli G. Behavioral effects of different intensities of formalin pain in rats. Physiol Behav 1995;58:603–610. Beavis WD. QTL analyses: power, precision and accuracy. In: Patterson C, editor. Molecular Dissection of Complex Traits, Boca Raton, FL: CRC Press, 1998. pp. 145–162. Belknap JK. Effect of within-strain sample size on QTL detection and mapping using recombinant inbred mouse strains. Behav Genet 1998;28:29–38. Bon K, Wilson SG, Mogil JS, Roberts WJ. Genetic evidence for the correlation of deep dorsal horn Fos protein immunoreactivity with tonic formalin pain behavior. J Pain 2002 (in press). Churchill GA, Doerge RW. Empirical threshold values for quantitative trait mapping. Genetics 1994;138:963–971. DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune activation in persistent pain. Pain 2001;90:1–6. Dib-Hajj SD, Tyrrell L, Black JA, Waxman SG. NaN, a novel voltage-gated
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