Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity

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1 Pain 97 (2002) Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity William R. Lariviere, Sonya G. Wilson, Tinna M. Laughlin, Anna Kokayeff, Erin E. West, Seetal M. Adhikari, You Wan, Jeffrey S. Mogil* Department of Psychology and Neuroscience Program, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA Received 28 August 2001; received in revised form 28 November 2001; accepted 15 December 2001 Abstract We and others have previously demonstrated that nociception in the mouse is heritable. A genetic correlation analysis of 12 common measures of nociception among a common set of inbred strains revealed three major clusters (or types ) of nociception in this species. In the present study, we re-evaluated the major types of nociception and their interrelatedness using ten additional assays of nociception and hypersensitivity, including: three thermal assays (tail withdrawal from 47.58C water or 2158C ethanol; tail flick from radiant heat), two chemical assays of spontaneous nociception (bee venom test; capsaicin test) and their subsequent thermal hypersensitivity states (including contralateral hypersensitivity in the bee venom test), a mechanical nociceptive assay (tail-clip test), and a mechanical hypersensitivity assay (intrathecal dynorphin). Confirming our earlier findings, the results demonstrate distinct thermal and chemical nociceptive types. It is now clear that mechanical hypersensitivity and thermal hypersensitivity are genetically dissociable phenomena. Furthermore, we now see at least two distinct types of thermal hypersensitivity: afferent-dependent, featuring a preceding significant period of spontaneous nociceptive behavior associated with afferent neural activity, and non-afferent-dependent. In conclusion, our latest analysis suggests that there are at least five fundamental types of nociception and hypersensitivity: (1) baseline thermal nociception; (2) spontaneous responses to noxious chemical stimuli; (3) thermal hypersensitivity; (4) mechanical hypersensitivity; and (5) afferent input-dependent hypersensitivity. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Genetic correlation; Strain difference; Stimulus modality; Hyperalgesia; Allodynia 1. Introduction An understanding of the relationships among laboratory animal assays of nociception and hypersensitivity informs us about the underlying neural mechanisms evoked by each model, and can suggest how the models relate to human clinical and experimental pain. One useful method for investigating such relationships is the determination of genetic correlations among heritable traits. The observation of similar patterns of sensitivity of a number of isogenic (i.e. inbred) strains across two or more traits is evidence of shared genetic determination of those traits (Hegmann and Possidente, 1981; Crabbe et al., 1990). In turn, if traits have overlapping genetic determination, the involvement of * Corresponding author. Department of Psychology, McGill University, 1205 Dr. Penfield Avenue, Montreal, Quebec, Canada H3A 1B1. Tel.: ; fax: address: jeff@hebb.psych.mcgill.ca (J.S. Mogil). common proteins and thus similar physiology can be evinced. Recently, by investigating genetic correlations of the sensitivity of 11 inbred strains of mice on 12 common assays of nociception and hypersensitivity (Mogil et al., 1999a), we provided evidence for three major types of nociception: (1) baseline thermal nociception; (2) spontaneous responses to noxious chemical stimuli; and (3) baseline mechanical sensitivity and cutaneous hypersensitivity (Mogil et al., 1999b). Inbred strains sensitive to one assay within a type (e.g. the formalin test of chemical/inflammatory nociception) were, generally speaking, also sensitive to other assays within the type (e.g. the acetic acid abdominal constriction test), but not necessarily to assays of a different type (e.g. the hot-plate test of thermal nociception). Although the 12 assays were selected to examine a wide range of dimensions on which nociceptive assays can vary (e.g. stimulus location, stimulus duration, behavioral response), the list was by no means exhaustive and the topic warrants further analysis. Therefore, the present study re-examines the relation /02/$20.00 q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S (01)

2 76 W.R. Lariviere et al. / Pain 97 (2002) ships among animal models of pain with the addition of ten new assays of nociception and hypersensitivity, lending greater power of resolution among assays, affording greater generalizability, and providing critical comparisons. We examined sensitivity to several additional thermal nociceptive stimuli, and to spontaneous nociceptive behaviors evoked by the chemical irritants, bee venom and capsaicin. Bee venom and capsaicin produce both spontaneous nociception and subsequent hypersensitivity, whereas the assays previously considered generally feature acute nociception only or hypersensitivity in the absence of measurable responses indicative of spontaneous pain (e.g. peripheral nerve injury). In the original study (Mogil et al., 1999b), the third major type of nociception was defined by a loose clustering of mechanical sensitivity to von Frey filaments with two assays of thermal hypersensitivity (following carrageenan inflammation and peripheral nerve injury) and one assay of mechanical hypersensitivity (following peripheral nerve injury). To test the strength of this finding, an additional assay of mechanical nociception, the tail-clip test, and an assay of mechanical hypersensitivity, intrathecal dynorphin-induced mechanical hypersensitivity, were examined. 2. Methods 2.1. Subjects Male mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in-house for a limited number of generations. The following strains were used (all J substrains): 129P3, A, AKR, BALB/c, C3H/He, C57BL/6, C57BL/10, C58, CBA, DBA/2, RIIIS, and SM. All 11 strains from our previous effort (Mogil et al., 1999a,b) were used (with exceptions for two traits; see below), and for new assays the C57BL/10 strain was added to increase statistical power. The 129P3/J strain is the same 129 substrain used previously, formerly designated 129/J (see Gene and Strain Nomenclature at ( jaxmice). Mice were housed with same-sex littermates in groups of four or less, and maintained on a 12-h light/dark cycle (lights on at 07:00 h), with food (Harlan Teklad Laboratory Diet) and tap water available ad libitum. All experiments with new assays were performed in the same laboratory during the light phase, and a single investigator performed all of the measurements for any given assay. Each mouse was used only once on a single assay and was returned to its home cage after testing Assays The reader is referred to Mogil et al. (1999a) for detailed protocols of the acetic acid and magnesium sulfate abdominal constriction (writhing) tests (AC AA,AC MS ), autotomy following sciatic and saphenous nerve transection (AUT), carrageenan-induced thermal hypersensitivity assessed with Hargreaves test (CAR HT ), the early/acute and late/tonic phases of the formalin test (F early,f late ), the hot-plate test (HP), Hargreaves et al. s (1988) thermal paw-withdrawal test (HT), the Kim and Chung (1992) model of peripheral nerve injury-induced thermal and mechanical hypersensitivity assessed with Hargreaves test and the von Frey test (PNI HT, PNI VF ), the 498C tail-withdrawal test (TW 49 ), and the von Frey monofilament test (VF) (Table 1). Table 1 Abbreviations used for nociception and hypersensitivity assays Abbreviation AC AA AC MS AUT BV BV HT BV CON CAP CAP TW CAR HT DYN VF F early F late HP HT PNI HT PNI VF TC TF TW 215 TW 47.5 TW 49 VF Assay Abdominal constriction (writhing) test acetic acid Abdominal constriction (writhing) test magnesium sulfate Autotomy following sciatic and saphenous nerve transection Bee venom-induced spontaneous pain behavior (licking) Bee venom-induced thermal hypersensitivity assessed with Hargreaves test (ipsilateral) Contralateral bee venom-induced thermal hypersensitivity assessed with Hargreaves test Capsaicin-induced spontaneous pain behavior (licking) Capsaicin-induced thermal hypersensitivity assessed with tail-withdrawal test Carrageenan-induced thermal hypersensitivity assessed with Hargreaves test Dynorphin-induced mechanical hypersensitivity assessed with von Frey monofilament test Early/acute phase of formalin test Late/tonic phase of formalin test Hot-plate test Hargreaves et al. s thermal paw-withdrawal test Peripheral nerve injury-induced thermal hypersensitivity assessed with Hargreaves test Peripheral nerve injury-induced mechanical hypersensitivity assessed with von Frey monofilament test Tail-clip test Tail-flick from radiant heat source Tail withdrawal from 2158C ethanol Tail withdrawal from 47.58C water Tail withdrawal from 498C water von Frey monofilament test

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4 78 W.R. Lariviere et al. / Pain 97 (2002) The new assays are described in Sections below Bee venom test (BV, BV HT,BV CON ) Subcutaneous injection of honey bee venom in the paw of the rat produces marked edema, spontaneous elevation, licking and shaking of the injected paw (Lariviere and Melzack, 1996). The pain response peaks at 5 10 min postinjection and continually declines thereafter, lasting for up to 1 h at doses #0.2 mg. Following this period of spontaneous pain behavior, significant ipsilateral mechanical hypersensitivity and bilateral thermal hypersensitivity can be observed, and can last for more than 8 h (Chen et al., 1999). In the present study, we evaluated bee venom-induced licking immediately following intraplantar injection (BV), and subsequent thermal hypersensitivity in both ipsilateral and contralateral hind paws (BV HT,BV CON ). Bee venom-induced mechanical hypersensitivity was found in pilot experiments to be too variable within strain for consideration in this study. Mice were habituated to transparent Plexiglas cylinders (15 cm diameter 46 cm high) on a glass floor for 30 min/ day for 3 days, including immediately before bee venom injection. They were then placed in a cloth/cardboard holder and injected in the left mid-plantar hind paw with 50 mg honey bee venom (lyophilized whole venom of Apis mellifera; #V3375, Sigma) dissolved in 25 ml of saline. The mice were returned to the cylinders and the total time spent licking/biting the injected paw was measured for 60 min (BV). This dependent measure was employed because in the mouse formalin test, which evokes the same set of individual behaviors, licking is the behavior best correlated with formalin concentration (Sufka et al., 1998). Mice were then placed in well-ventilated observation chambers ( cm 3 ) with transparent outer walls and ceiling, a 3/16th-inch-thick glass floor, and opaque inner walls that visually isolated each mouse from the others. The mice were habituated to the chamber for 2 h/ day for 3 days including immediately before baseline thermal sensitivity assessment, which occurred the day before bee venom injection. From 2 to 4 h after bee venom injection, thermal hypersensitivity in Hargreaves test was measured in both hind paws. In Hargreaves test, a highintensity beam from a projector lamp bulb (setting ¼ 3, < 45 W, IITC Model 336) set 6 cm below the glass floor is aimed at the plantar surface of the mid-hind paw of the inactive mouse, and the latency for the mouse to withdraw and/or lick the paw is measured to the nearest 0.1 s. Each mouse was tested six times on each hind paw, alternating sides with an interval of at least 5 min between determinations, and the mean of the six measures was used in the analysis. Percent hypersensitivity for each hind paw (BV HT, BV CON ) was calculated as: [(baseline latency 2 postinjection latency)/baseline latency] Capsaicin test (CAP, CAP TW ) Mice were habituated to Plexiglas cylinders (30 cm diameter 30 cm high) for 30 min prior to being placed in a cloth/cardboard holder and injected in the right mid-plantar hind paw with 2.5 mg capsaicin dissolved in 20 ml of vehicle (2% dimethyl sulfoxide (DMSO) in saline). The mice were returned to the cylinders and the amount of time spent licking the injected paw (CAP) was measured with a stopwatch to the nearest 0.1 s for 15 min, a time point determined by pilot experiments to represent the termination of the nociceptive episode. In a separate group of mice, capsaicin-induced thermal hypersensitivity was measured in the tail-withdrawal test following the procedure of Ko et al. (2000). The mice were taken from their home cages, placed in a cloth/cardboard holder, and the distal half of their tail was immersed into a circulating water bath maintained at 47 ^ 0:28C (Isotemp IC2100 Circulator, Fisher Scientific). The latency to vigorous withdrawal of the tail was measured with a stopwatch to the nearest 0.1 s. The mean of two determinations separated by at least 10 s was calculated and used in the analysis as the baseline. Immediately thereafter, 2.5 mg capsaicin dissolved in 5 ml of vehicle (2% DMSO in saline) was injected subcutaneously into the dorsal surface of the tail near the midpoint, and the mouse was returned to its home cage. Tail-withdrawal latency was then measured at 15, 30, 45, 60, 90, and 120 min postinjection and percent hypersensitivity calculated at each time point as: [(baseline latency 2 latency at time x)/baseline latency] 100. Total percent hypersensitivity (CAP TW ) was calculated as the area under the percent hypersensitivity time curve compared to the maximum possible hypersensitivity over the full time course Intrathecal dynorphin-induced mechanical hypersensitivity (DYN VF ) Only seven of the 12 strains were available to be tested in this assay (see Fig. 1b). Intrathecal injection of dynorphin evokes chronic mechanical hypersensitivity via a nonopioid, N-methyl-d-aspartate (NMDA) receptor-mediated mechanism (Laughlin et al., 1997, 2000). Prior to dynorphin injection, the baseline mechanical threshold was measured with the von Frey test using the up down method of Dixon following 1 h of habituation to the testing chambers as previously described (Chaplan et al., 1994; Mogil et al., Fig. 1. Mean responses of 12 inbred mouse strains on ten behavioral measures of nociception and hypersensitivity (BV, BV HT,BV CON, CAP, CAP TW, DYN VF, TC, TF, TW 47.5,TW 215, see Section 2). Bars indicate mean ^ SEM (n ¼ 4 51 mice/strain/assay). A significant effect of genotype is seen in all assays ðp, 0:01Þ. Heritability estimates (h 2 ) are shown on each graph. Tail diameter strain means are shown inset in (g), with strains in the same order from left to right. Tail size is not correlated with nociceptive sensitivity in the tail-clip test ðr s ¼ 20:15Þ. n.t. ¼ not tested. The data shown in (h,i,j) have been published previously (Mogil and Adhikari, 1999; Wan et al., 2001).

5 W.R. Lariviere et al. / Pain 97 (2002) a). Awake mice were injected with 3 nmol dynorphin (Tocris Inc.) dissolved in 5 ml of saline into the intrathecal space of the lumbosacral spine through the skin with a 30- gauge needle (Hylden and Wilcox, 1980). Three days later, the von Frey test was re-administered and percent hypersensitivity was calculated as: [(baseline threshold postinjection threshold)/baseline threshold] Tail-clip test (TC) A modification of Haffner s tail-pinch test was employed (Takagi et al., 1966). Each mouse was placed in a cloth/ cardboard holder, the diameter of the tail 1 cm from the base was measured with precision calipers, and the mouse returned to its home cage. Thirty minutes later, mice were again placed in the holder and an alligator clip with rubber cuffs around the teeth of each jaw (exerting 500 g of force) was applied to the tail 1 cm from the base. The mouse was immediately removed from the holder and the latency to lick, bite, grab, or bring the nose to within 1 cm of the clip was measured with a stopwatch to the nearest 0.1 s, after which the clip was immediately removed. A maximum cut-off latency of 60 s was imposed, at which time the clip was removed to prevent the possibility of injury Tail-flick test (TF) All strains except CBA and DBA/2 were tested in this assay, a slight modification of the classic tail-flick test of D Amour and Smith (1941). These data were collected as baseline values in a published inbred mouse strain survey of electroacupuncture analgesia (Wan et al., 2001). Briefly, mice were restrained in well-ventilated, cylindrical Plexiglas restrainers, with the tail extended from one end. After insertion of acupuncture needles in the hind legs and 30 min of habituation (with no current applied), focused light from a 12.5-W projection bulb was applied directly to the middle of the tail, and a digital timer connected in series measured the tail-flick latency to the nearest 0.1 s as soon as the mouse flicked its tail. To avoid the possibility of tissue injury, the bulb was turned off at 10 s if the mouse did not respond. Three baseline threshold measurements were taken at 5-min intervals and the mean value calculated (TF) Tail-withdrawal tests (TW 47.5,TW 215 ) Mean baseline latency to withdraw the tail from circulating water held at 47:5 ^ 0:58C (TW 47.5 ) or ethanol at 215 ^ 18C (TW 215 ) was determined in a previously published genetic correlation study of hot and cold thermal nociception (Mogil and Adhikari, 1999). The method was exactly as described in Section for capsaicin-induced thermal hypersensitivity baseline values, but with an interval of 5 min between the two determinations Data analysis Effect of genotype and heritability estimates Significance of the effect of genotype on each trait was determined with analysis of variance (ANOVA). A criterion a level of 0.05 was adopted for purposes of establishing statistical significance. Estimates of narrow-sense trait heritability were calculated by comparing the ANOVA-derived between-strain variance to the total variance: h 2 ¼ V A =ðv A 1 V E Þ; where V A is the allelic (between-strain) variance and V E is the error or environmental (within-strain) variance (Falconer and Mackay, 1996; Mogil et al., 1999a) Correlations between assays Correlation coefficients were calculated using Spearman s statistic (r s ) for strain mean rank data as previously described (Mogil et al., 1999b). All data were corrected for sign, such that higher ranks (i.e. closer to 1) represented greater sensitivity in each assay. Pearson correlations were not used in this study since instances of non-linearity of the relationship between variables were observed (even after transformations), rendering this statistic inappropriate. With 11 or 12 strains and a ¼ 0:05, uncorrected critical values of r s are 0.62 and 0.59, respectively. However, no attempt was made to evaluate the significance of the correlations, since a Bonferroni correction for the 231 correlations examined would present an almost insurmountable obstacle. It should be noted that such correction is probably unduly conservative, and that the number of instances of uncorrected significant correlations herein is far larger than what would be expected by chance based on a Poisson distribution. Ultimately, the major purpose of the present study should be considered exploratory. This issue is discussed at length in our previous manuscript (Mogil et al., 1999b) Multivariate analyses Two multivariate analysis methods, multidimensional scaling (MDS) and principal components analysis (PCA), were then used to produce two-dimensional graphical representations of the cross-correlation among assays as previously described (Mogil et al., 1999b). Briefly, in MDS, coordinates in two-dimensional space for a set of objects representing the assays are computed such that the distances between pairs of objects fit as closely as possible to measured similarities, in our case, Spearman correlations. High positive correlations are represented as small inter-object distances, and high negative correlations are represented as large inter-object distances. Uncorrelated assays have intermediate inter-object distances. In PCA, two linear combinations of the assays are constructed, and the weights of the linear combinations are plotted in a two-dimensional space to produce a vector for each assay. High positive correlations are represented as angles between the vectors close to 08, and high negative correlations as angles close to Uncorrelated assays have angles between their vectors close to 908. In addition, the length of the vector indicates the extent to which the variance in the assay is explained by the two dimensions.

6 80 W.R. Lariviere et al. / Pain 97 (2002) Table 2 Spearman coefficients of rank correlation between the ten newly added assays (top row) and all 22 nociceptive measures studied thus far (left column) a Assay BV CAP TF TW 215 TW 47.5 TC DYN VF BV HT BV CON CAP TW AC AA AC MS BV CAP F early F late HP HT TF TW TW TW TC VF DYN VF PNI VF CAR HT PNI HT AUT BV HT BV CON CAP TW a Assays are grouped based on clusters identified in Fig. 3, except that VF is grouped with TC. 3. Results 3.1. Strain differences and heritability estimates Strain means, standard errors (reflecting environmental variability), and heritability estimates for the ten newly added assays are illustrated in Fig. 1 (see Mogil et al., 1999a,b for strain means of the original 12 assays). As can be seen, sensitivity varies greatly across mouse strain, and a significant effect of genotype was obtained in all assays ðp, 0:01Þ. As in our previous report, no strain is consistently most sensitive or least sensitive in all assays. However, as discussed elsewhere as it pertains to transgenic studies of pain and analgesia, 129P3 mice and C57BL/6 mice are usually significantly different from each other and from many of the other strains examined, whereas the DBA/2 mice are the most moderately responding strain overall (Lariviere et al., 2001). Also consistent with our previous effort, heritability estimates of the new assays range from h 2 ¼ 0:31 0:69, with a median of h 2 ¼ 0:46 for all 22 assays studied so far Correlations between assays Spearman correlations (r s ) between the ten new assays and all 22 assays examined to date are shown in Table 2 (see Mogil et al., 1999b for correlations between the 12 previously examined assays). Tail diameter was found to be uncorrelated with sensitivity in the tail-clip test ðr s ¼ 20:15Þ, suggesting that simple physical features of the tail do not explain the sensitivity. Scatterplots of ranked strain means shown in Fig. 2a, b, respectively, illustrate the seemingly paradoxical negative correlations between sensitivity in the two mechanical assays, the VF and TC tests ðr s ¼ 20:36Þ, and between BV HT and CAR HT ðr s ¼ 20:44Þ Multivariate analyses The total variance explained by the MDS and PCA plots was 83 and 58%, respectively. Both plots yielded obvious clustering of assays, and overall, a very high degree of convergence between the clustering patterns produced by the MDS and PCA algorithms was observed (see Fig. 3). We also examined the three-dimensional (three-factor) representations of the MDS and PCA analyses, both with and without rotation (not shown; accounting for 89 and 69% of the total variance, respectively). The two-dimensional plots in Fig. 3 represent a view of the three-dimensional plots from above very well, since the assays cluster similarly in the third dimension. Thus, the two-dimensional MDS and PCA plots provide an accurate representation of the clustering of assays. In both plots in Fig. 3, a large grouping of assays can be observed along the right side that includes the thermal nociception assays (HP, HT, TF, TW 47.5,TW 49, and TW 215 )in the upper quadrant of the plot and the chemical nociception

7 W.R. Lariviere et al. / Pain 97 (2002) nociception in mice, and we believe further validates the utility of genetic correlation analysis in the investigation of the interrelationship among laboratory animal models of pain. The inclusion of additional assays has replicated our previous findings of obvious clustering (and hence, genetic codetermination) of nociceptive assays into types (see Section 4.3 for further discussion). In addition, this study has provided more detailed information regarding the clustering of the assays, and has shown that seemingly similar assays may nonetheless differ genetically Strain differences and heritability estimates Confirming earlier reports of the heritability of baseline nociception and hypersensitivity, we observed statistically significant and usually marked variability in responses between strains in every assay. Inbred strain differences the effects of differential allelic fixation at trait-relevant genes accounted for up to 76% (for AC AA ) of the phenotypic response variability in the assays we examined, with a median of about 46% of the variability for all assays investigated so far. Similar heritability estimates have also been derived in our laboratory for multiple analgesia traits (unpublished data) Correlations and clusters Fig. 2. Scatterplot of the paradoxical negative genetic correlations between (a) VF and TC ðr s ¼ 20:36Þ and (b) BV HT and CAR HT ðr s ¼ 20:44Þ. Each point represents the ranked mean of an inbred strain on each assay (see Fig. 1 for strain names), with rank 1 indicating highest sensitivity. Note in (a) the presence of a clear outlier strain (CBA; with rank of 11 for VF); CBA mice were observed to be particularly agitated in the VF observation chambers. If this strain is removed, the correlation between VF and TC is r s ¼ 20:68. assays (AC AA,AC MS, BV, CAP, F early, and F late ) in the lower quadrant of the plot. On the left side of both plots, DYN VF and PNI VF comprise a cluster in the upper quadrant, as do CAR HT and PNI HT in the lower quadrant. Notably, TC does not cluster with VF at the extreme left, but instead with the baseline nociception assays on the right side of both plots. BV HT,BV CON, CAP TW (and AUT) group close to the large cluster on the upper right in the MDS plot and not with CAR HT and PNI HT on the bottom left, as might be expected. In the PCA plot, one can also see that BV HT,BV CON, CAP TW (and AUT) are positively correlated with the thermal assays, and negatively correlated with CAR HT and PNI HT. 4. Discussion This study provides further evidence of the heritability of Our latest analyses now suggest that there are at least five fundamental types of nociception and hypersensitivity: (1) baseline thermal nociception; (2) spontaneous responses to noxious chemical stimuli; (3) thermal hypersensitivity; (4) mechanical hypersensitivity; and (5) afferent input-dependent hypersensitivity. The independent status of baseline mechanical nociception as a fundamental type is now in question, as will be discussed in Section 4.5 below Thermal and chemical nociceptive types The inclusion of three additional thermal assays (TF, TW 47.5, and TW 215 ) and two additional chemical assays (BV and CAP) demonstrates the robustness of the previously identified thermal and chemical types of baseline nociception. This replication of previous findings can be considered a validation of the method of genetic correlation analysis for pain-related traits, and a partial refutation of claims that behavioral genetics studies are prone to unreliability by environmental differences (see Crabbe et al., 1999). Genetic correlations among assays within the identified clusters were preserved despite the fact that data were collected in different laboratories, by different experimenters, and over a several-year period (also see Crabbe et al., 1999). Although, it could be argued (from Fig. 3) that the thermal and chemical assays comprise a single large cluster of assays, it should be noted that there is no instance of a thermal assay within the group of chemical assays, nor vice versa, suggesting that despite some shared genetic

8 82 W.R. Lariviere et al. / Pain 97 (2002) Fig. 3. Multivariate analyses illustrating cross-correlations among inbred strain means for 22 assays of nociception and hypersensitivity. In the MDS plot (a), Euclidean distances between objects are representative of their correlation; objects with higher positive correlations are closer (see text). A Kruskal loss function with monotonic regression was employed, resulting in a final stress of The proportion of total variance accounted for is In the PCA plot of the first two unrotated principal components (b), the angles between rays projecting to objects are representative of their correlation (see text). The proportion of total variance accounted for is As can be seen, the clusters emerging from each analysis show a high degree of convergence. determination the types are also genetically distinct. However, TW 215 appears to bridge the thermal and chemical clusters (with thermal cluster: r s ¼ 0:20 0:73; with chemical cluster: r s ¼ 0:28 0:67). This may be due to a chemical reaction of ethanol with the tail; pure ethanol can defat skin and produce dermatitis with drying and fissuring (Sittig, 1985). Similarly, CAP correlates moderately with the thermal assays ðr s ¼ 0:18 0:71Þ and even more highly with the chemical nociception assays ðr s ¼ 0:45 0:76Þ. This might be predicted by the ability of both heat and acidic solutions to activate the endogenous receptor for capsaicin, the vanilloid receptor-1 (VR-1) (Caterina et al., 1997), and by the dependence of several chemical assays on capsaicin-sensitive afferent fibers or VR-1 activation (Santos and Calixto, 1997; Caterina et al., 2000; Chen and Chen, 2001) Independent genetic mediation of thermal hypersensitivity and mechanical hypersensitivity With the inclusion of an additional assay (DYN VF )itis now apparent that mechanical hypersensitivity truly is distinct from thermal hypersensitivity with respect to their underlying genetic mediation in these strains (see Fig. 3). This is best demonstrated still by the complete lack of correlation between mechanical and thermal hypersensitivity evoked by the same procedure (PNI VF and PNI HT, respectively; see Mogil et al., 1999b and Fig. 3). Although it may be surprising that sensitivity to two different sequellae of the same injury would be genetically distinct, in fact the neural substrates underlying mechanical hypersensitivity and thermal hypersensitivity have been dissociated in a number of ways (see Meller, 1994; also see Mogil et al., 1999b). Within the thermal hypersensitivity cluster, there is a good correlation between assays ðr s ¼ 0:53Þ despite the fact that in one case the hypersensitivity was induced by an inflammatory chemical and in the other by nerve injury. One possibility for this positive correlation is that nerve injury models generally feature inflammation around the site of injury, which may ultimately be responsible for the development of hypersensitivity (see DeLeo and Yezierski, 2001). Thermal hypersensitivity following the Bennett and Xie (1988) model of chronic constriction injury can be ameliorated with systemic steroid treatment (Clatworthy et al., 1995) or anti-inflammatory cytokine (interleukin-10) treatment (Wagner et al., 1998). Thus, it is conceivable that the genetic polymorphism producing similar strain sensitivities on both of these assays is to be found in a gene or genes affecting inflammatory pain. It is also possible that inflammation may hold the key to the mechanical hypersensitivity cluster. Cui et al. (2000) performed an intriguing experiment in rats subjected to Bennett model and Seltzer et al. (1990) model neuropathies. These injuries produced significant mechanical hypersensitivity in only 38% of Bennett model rats and 29% of Seltzer model rats. Subsequent to the development (or lack thereof) of hypersensitivity, sciatic nerve immunohistochemistry revealed that the number of cells featuring inflammatory

9 W.R. Lariviere et al. / Pain 97 (2002) markers increased much more in the allodynic subset than the non-allodynic subset of rats. That is, there was a clear correlation between inflammation and hypersensitivity development that related directly to individual differences in these models (Cui et al., 2000). Unfortunately, the Chung model was not included in these studies, so the direct relevance to our data is unknown. However, we note that dynorphin-induced mechanical hypersensitivity can be reduced by the administration of interleukin-10 or antagonists of pro-inflammatory cytokines (interleukin-1) (Laughlin et al., 2000). It should be noted that if inflammation is indeed the factor that these assays hold in common, it still remains unclear why thermal hypersensitivity and mechanical hypersensitivity should be uncorrelated genetically Negative genetic correlation between VF and TC test There appears to be a lack of consensus in the literature and among current pain researchers as to whether the application of a von Frey monofilament at paw withdrawal threshold truly evokes nociception in the uninjured animal. Whereas the protracted withdrawal (often accompanied by shaking and licking) of an inflamed or neuropathic paw provides convincing evidence of pain in the animal, the withdrawal of the normal paw may simply represent a desire of the rodent to avoid being touched. In fact, in our laboratory we can reliably evoke withdrawal responses by tickling the mouse with clearly non-noxious von Frey filaments lightly rubbed across the hindpaw digits. In humans, of course, there are two thresholds to stimulation with von Frey filaments, a sensation threshold and a pain threshold (e.g. Bouhassira et al., 1999). The negative correlation of VF with all baseline nociceptive assays (r s ¼ 20:31 to 20.83) may be interpreted as supporting the independent status of the VF test relative to the others. By contrast, the TC test displayed uniformly positive genetic correlation with the thermal assays ðr s ¼ 0:10 0:61Þ. Therefore, our original contention that thermal and mechanical assays are negatively correlated genetically (Mogil et al., 1999b) appears to be wrong. Nonetheless, it seems premature to rule out a distinct mechanical nociception type until more assays are tested. If the apparent negative correlation between VF and TC is found to be reliable (see Fig. 2a), it would indicate that the same genes affect variability in these assays, but that allelic differences at these genes exert opposite effects on each (see Mogil et al., 1999b). Sensitivity to painful stimulation in the von Frey test in humans has been shown to be determined by the relative activity of afferent fibers of different sizes and myelination status (Andrew and Greenspan, 1999). Thus, some obvious candidate genetic mechanisms that might explain the negative genetic correlation between VF and TC include developmental genes contributing to the relative number of Ab,Ad, and C fibers in the adult mouse, and/or to the myelination of such afferents. For instance, it is possible that genotypic differences in the number or functioning of Ab fibers could increase sensitivity of a strain to nonnoxious von Frey filament stimulation while simultaneously decreasing sensitivity to painful mechanical stimulation in the TC test Dissociation of thermal hypersensitivity models A surprising finding of the present study is that bee venom- and capsaicin-induced thermal hypersensitivity (BV HT, BV CON and CAP TW ) do not correlate with the CAR HT and PNI HT assays, despite the fact that they are all assays of hypersensitivity to evoked thermal stimuli. These new assays cluster on the right side of the plots, close to, but not within, the thermal nociception assays (Fig. 3a). We believe that the spontaneous nociceptive behavior preceding the hypersensitivity in the new assays is responsible for the dissociation with CAR HT and PNI HT. It has recently been shown that activity in capsaicinsensitive peripheral afferents is necessary for the development of primary, secondary, and contralateral bee venominduced thermal hypersensitivity (Chen and Chen, 2001). In fact, a certain amount of afferent input must be transmitted to the spinal cord prior to sciatic nerve transection for the development of contralateral thermal hypersensitivity (Chen et al., 2001). Once initiated, however, the hypersensitivity is no longer dependent on afferent input. CAP TW also requires a significant contribution of afferent input (Baumann et al., 1991; Simone et al., 1991); as with bee venom, if capsaicin-induced afferent activity is blocked, the development of the centrally maintained hypersensitivity is also blocked (LaMotte et al., 1991). In contrast, CAR HT and PNI HT may not feature enough afferent activity to evoke the same set of central changes, although these injuries certainly produce plasticity of their own. In our original report, we observed little evidence of spontaneous licking, biting, or scratching of the injected paw in any strain of mouse following subcutaneous injection of 0.4 mg carrageenan into the mouse plantar hind paw (Mogil et al., 1999a). Similarly, mice with Chung model neuropathies were rarely observed performing behaviors indicative of spontaneous pain. In the Sprague Dawley rat, 0.5 mg carrageenan administered into the dorsal hind paw evokes approximately ten paw flinches and 140 s of licking over 2 h (Wheeler-Aceto et al., 1990), far below the threshold amount of afferent input (170 flinches and 300 s of lifting/licking in 10 min) required for the induction of contralateral bee venom-evoked thermal hypersensitivity in the same strain (Chen et al., 1999, 2001). Nonetheless, it is possible that if carrageenan-induced hypersensitivity were assessed later than 4 h postinjection as in our previous study (Mogil et al., 1999a), effects on spinal cord mechanisms that are observed at 20 h (but not at 2 6 h) post-injection (Rygh et al., 2001) might then contribute to the sensitivity across mouse strains and affect the genetic correlations accordingly.

10 84 W.R. Lariviere et al. / Pain 97 (2002) Although autotomy is not usually considered a model of hypersensitivity, it also likely features aberrant central summation of afferent input (Rappaport and Devor, 1990). For instance, the application of local anesthetic to the contralateral limb reduces the degree of pre-established autotomy behaviors (Bileviciute-Ljungar, 2000). Furthermore, like bee venom- and capsaicin-induced hypersensitivity, the development of autotomy following sciatic nerve transection is in part dependent on afferent input, as local anesthetic pretreatment of the transected nerve delays the onset of autotomy behavior (Kauppila and Pertovaara, 1991). In contrast, hypersensitivity in the Chung model is only transiently affected by nerve block (Abdi et al., 2000), suggesting that the development of this hypersensitivity state is largely independent of afferent input. Thus, we have shown that models of thermal hypersensitivity can differ dramatically. At present, the most obvious distinction between the groups seems to be the dependence on peripheral afferent input. It remains to be determined if the same relationship exists for mechanical hypersensitivity models. Unfortunately, such experiments present practical difficulties since neither bee venom nor capsaicin produced robust and reliable enough mechanical hypersensitivity to be included in this analysis Generalizability across species Although the direct transfer of results from the rodent to the human is always problematic, these results can elucidate relationships that may also exist in humans. First, the demonstration of significant strain differences and at least moderate heritability of all pain-related traits examined to date suggests that the same is likely true for quantifiable, continuous pain-related traits in humans (see, however, MacGregor et al., 1997). That is, individual differences in humans are partially due to inherited factors. The demonstration of such heritability in humans is complicated by the relative genetic and environmental heterogeneity of the human population compared to that of inbred strains of mice in the laboratory. Although a number of human pain syndromes (especially sensory neuropathies) have recently been attributed to mutations in specific genes (Indo et al., 1996; Ophoff et al., 1998; Wu et al., 2000; Anderson et al., 2001; Bejaoui et al., 2001; Dawkins et al., 2001; Slaugenhaupt et al., 2001), these are all rare single-gene disorders. The vastly more common multigenic pain disorders are proving harder to study using current human genetic techniques. The recent demonstration of linkage of particular genomic loci to hot-plate test, formalin test, and autotomy trait variability in the mouse (Mogil et al., 1997; Seltzer et al., 2001; Wilson et al., 2002), suggests that laboratory animal genetics may be a more efficacious route to the identification of pain-related genes in humans. Secondly, the present study provides information on how different pain-related phenomena might be related to each other. Of particular relevance is the possibility that certain experimental assays of baseline nociceptive sensitivity may predict susceptibility to painful pathology in humans. Indeed, intriguing new data from a prospective study suggests that there is a correlation between experimental sensitivity to basal nociception and subsequent susceptibility to temporomandibular disorders in women (Maixner et al., poster presentation at International Association for Dental Research annual meeting, Chiba, Japan, 2001). The goal, of course, is the early detection of predisposed individuals and the administration of preventive treatments when indicated. Such testing, combined with knowledge of relevant genetic alleles, would go a long way towards truly individualized pain therapy. It should be noted, however, that recent attempts to conduct genetic correlation analyses in the rat have suggested that important species differences may exist. Using eight rat strains (two inbred, two outbred, and four selected lines), Shir et al. (2001) were unable to replicate our finding of a negative correlation between touch (using von Frey fibers) and thermal nociception (using a CO 2 laser), finding a positive correlation instead. They also observed no correlation between thermal nociception and autotomy. However, their data did support our contention that thermal hypersensitivity and mechanical hypersensitivity are genetically independent, seeing no correlation between these phenomena in rats given identical Seltzer et al. (1990) model neuropathic injuries (Shir et al., 2001). In another recent study of five rat populations, Xu et al. (2001) noted a weak but positive correlation between hot-plate test and von Frey test sensitivity. In agreement with the present study, however, they noted genetic correlations between ipsilateral and contralateral hypersensitivity and genetic dissociations between thermal hypersensitivity and mechanical hypersensitivity induced by an ischemic sciatic nerve injury (Xu et al., 2001). It should be noted that a fundamental limitation of the genetic correlation approach is that it considers only genes that exist in polymorphic form within the set of strains chosen. Genes that are not polymorphic whether or not they would be of value in helping to delineate physiological relationships among these nociceptive assays are effectively invisible to this analysis. Nonetheless, if the relationships among the animal models observed here are shown to be generalizable to other strains of mice and to humans, they may be useful to determine which assays are most appropriate for the study of specific clinically relevant sensory phenomena and pain syndromes. For instance, our data suggest that the standard von Frey test should not be used as a model of mechanical pain sensitivity, but rather for the assessment of innocuous mechanical sensitivity and mechanical hypersensitivity. A method akin to the TC test might be more appropriate as a model of mechanical pain. Similarly, the appropriate match of available hypersensitivity assays should be made to the particular clinical hypersensitivity syndrome one wishes to model. Our findings suggest that injury-related hypersensitivity with an initial

11 W.R. Lariviere et al. / Pain 97 (2002) painful period and associated afferent input, such as a sprained ankle (Melzack and Wall, 1996) may be best modeled by the BV HT or CAP TW tests, whereas clinical hypersensitivity states without an identifiable preceding phase of afferent input, for example diabetic neuropathy (Benbow et al., 1999), may be best modeled with animal models that share this property, such as CAR HT and PNI HT. Acknowledgements This research was supported by PHS grants DA11394 and DE12735 (J.S.M.), and a Fonds pour la Formation de Chercheurs et l Aide a la Recherche of Quebec postdoctoral fellowship (W.R.L.). Thanks to Dr Marshall Devor for helpful discussions regarding this manuscript, and to the anonymous reviewers for their excellent suggestions. References Abdi S, Lee DH, Park SK, Chung JM. Lack of pre-emptive analgesic effects of local anaesthetics on neuropathic pain. Br J Anaesth 2000;85: Anderson SL, Coli R, Daly IW, Kichula EA, Rork MJ, Volpi SA, Ekstein J, Rubin BY. Familial dysautonomia is caused by mutations of the IKAP gene. Am J Hum Genet 2001;68: Andrew D, Greenspan JD. Peripheral coding of tonic mechanical cutaneous pain: comparison of nociceptor activity in rat and human psychophysics. J Neurophysiol 1999;82: Baumann TK, Simone DA, Shain CN, LaMotte RH. Neurogenic hyperalgesia: the search for the primary cutaneous afferent fibers that contribute to capsiacin-induced pain and hyperalgesia. J Neurophysiol 1991;66: Bejaoui K, Wu C, Scheffler MD, Haan G, Ashby P, Wu L, de Jong P, Brown Jr RH. SPTLC1 is mutated in hereditary sensory neuropathy, type 1. Nat Genet 2001;27: Benbow SJ, Cossins L, MacFarlane IA. Painful diabetic neuropathy. Diabet Med 1999;16: Bennett GJ, Xie Y-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33: Bileviciute-Ljungar IL. Contralateral but not systemic administration of bupivacaine reduces acute inflammation in the rat hindpaw. Somatosens Mot Res 2000;17: Bouhassira D, Attal N, Willer J-C, Brasseur L. Painful and painless peripheral sensory neuropathies due to HIV infection: a comparison using quantitative sensory evaluation. Pain 1999;80: Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389: Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen- Zeitz KR, Koltzenburg M, Basbaum AI, Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288: Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia evoked by unilateral ligation of the fifth and sixth lumbar nerves in the rat. J Neurosci Methods 1994;53: Chen J, Chen HS. Pivotal role of capsaicin-sensitive primary afferents in development of both heat and mechanical hyperalgesia induced by intraplantar bee venom injection. Pain 2001;91: Chen J, Luo C, Li H, Chen H. Primary hyperalgesia to mechanical and heat stimuli following subcutaneous bee venom injection into the plantar surface of hindpaw in the conscious rat: a comparative study with the formalin test. Pain 1999;83: Chen HS, Chen J, Chen J, Guo WG, Zheng MH. Establishment of bee venom-induced contralateral heat hyperalgesia in the rat is dependent upon central temporal summation of afferent input from the site of injury. Neurosci Lett 2001;298: Clatworthy AL, Illich PA, Castro GA, Walters ET. Role of peri-axonal inflammation in the development of thermal hyperalgesia and guarding behavior in a rat model of neuropathic pain. Neurosci Lett 1995;184:5 8. Crabbe JC, Phillips TJ, Kosobud A, Belknap JK. Estimation of genetic correlation: interpretation of experiments using selectively bred and inbred animals. Alcohol Clin Exp Res 1990;14: Crabbe JC, Wahlsten D, Dudek BC. Genetics of mouse behavior: interactions with laboratory environment. Science 1999;284: Cui J-G, Holmin S, Mathiesen T, Meyerson BA, Linderoth B. Possible role of inflammatory mediators in tactile hypersensitivity in rat models of mononeuropathy. Pain 2000;88: D Amour FE, Smith DL. A method for determining loss of pain sensation. J Pharmacol Exp Ther 1941;72: Dawkins JL, Hulme DJ, Brahmbhatt SB, Auer-Grumbach MN. GA Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I. Nat Genet 2001;27: DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune activation in persistent pain. Pain 2001;90:1 6. Falconer DS, Mackay TFC. Introduction to quantitative genetics. Essex: Longman, Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32: Hegmann JP, Possidente B. Estimating genetic correlations from inbred strains. Behav Genet 1981;11: Hylden JLK, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67: Indo Y, Tsurata Y, Karim MA, Ohta K, Kawano T, Mitsubuchi H, Tonoki H, Awaya Y, Matsuda I. Mutations in the TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis. Nat Genet 1996;13: Kauppila T, Pertovaara A. Effects of different sensory and behavioral manipulations on autotomy caused by a sciatic lesion in rats. Exp Neurol 1991;111: Kim SH, Chung JM. An experimental model for peripheral neuropathic produced by segmental spinal nerve ligation in the rat. Pain 1992;50: Ko MCH, Tuchman JE, Johnson MD, Wiesenauer K, Woods JH. Local administration of mu or kappa opioid agonists attenuates capsaicininduced thermal hyperalgesia via peripheral opioid receptors in rats. Psychopharmacology 2000;148: LaMotte RH, Shain CN, Simone DA, Tsai E-FP. Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. J Neurophysiol 1991;66: Lariviere WR, Melzack R. The bee venom test: a new tonic-pain test. Pain 1996;66: Lariviere WR, Chesler EJ, Mogil JS. Transgenic studies of pain and analgesia: mutation or background phenotype? J Pharmacol Exp Ther 2001;297: Laughlin TM, Vanderah TW, Lashbrook J, Nichols ML, Ossipov MH, Porreca F, Wilcox GL. Spinally administered dynorphin A produces long-lasting allodynia: involvement of NMDA but not opioid receptors. Pain 1997;72: Laughlin TM, Bethea JR, Yezierski RP, Wilcox GL. Cytokine involvement in dynorphin-induced allodynia. Pain 2000;84: MacGregor AJ, Griffiths GO, Baker J, Spector TD. Determinants of pressure pain threshold in adult twins: evidence that shared environmental influences predominate. Pain 1997;73: Meller ST. Thermal and mechanical hyperalgesia: a distinct role for differ-