arginine vasopressin ( avp ) has well - known roles in the regulation of varied biological systems , with effects mediated by one of three receptors : v1ar , v1br and v2r . v1ar is believed to predominate in the central nervous system ( cns )   and its involvement in affiliation and social communication in microtine rodents   and humans   is known to be male - specific and genetically determined . in humans , two studies using intranasal avp analogs observed no   or modest effects on acute pain . in rodents , avp produces clear but modest inhibition of acute , thermal pain after systemic , supraspinal or spinal injection in rodents , but most of the evidence suggests that this analgesia is mediated by the v1br   or the v2r . pain and analgesic responses in humans and rodents feature robust interindividual variability , and the responsible genes are just starting to be identified . an explicit rodent - to - human translational strategy was successfully employed on a few occasions in this domain . we describe herein the identification of the mouse avpr1a gene ( encoding v1ar ) as responsible for variable pain sensitivity to formalin and capsaicin . translation of this finding to humans revealed that v1ar 's role in pain interacts not only with genotype but with sex and acute stress , and this interaction was then confirmed and explained in new experiments in the mouse . standard inbred mouse strains display robust variability on virtually all nociceptive assays , including on the formalin test , a widely used assay of spontaneous , tonic chemical / inflammatory nociception . using an f2 intercross between the phenotypically divergent a / j ( resistant ) and c57bl/6j ( b6 ; sensitive ) strains , we previously identified a quantitative trait locus ( qtl ) , which we named nociq2 , on distal mouse chromosome 10 ( > 58 cm ) , associated with variability in the late / tonic phase ( 10 - 60 min post - injection ) of the formalin test . a recent qtl mapping study of the related acetic acid abdominal constriction test also identified significant linkage in this region . we used a variety of strategies to confirm and refine the position of the qtl , including the testing of recombinant congenic strains   and the breeding and testing of marker - assisted congenic and subcongenic strains . a comparison of the formalin test phenotype and chromosome 10 genotype of these strains confirmed that the responsible polymorphism(s ) could only lie between dna markers d10mit25 ( at 121.781 mb ) and d10mit103 ( at 125.176 mb ) ( figs . this 3.4-mb region of chromosome 10 contains only 11 unique transcripts , including five annotated genes ( table s1 ) . of these , the only obvious candidate was avpr1a , although no attempt was made to rule out the others functionally . instead , we conducted a haplotype mapping analysis comparing the late - phase formalin test phenotypes of 16 strains ( fig . we investigated snp haplotype patterns in the mouse phenome database ( mpd ) high - density ( 8 million ; cgd1 imputed 74-strain data set ) snp map ( http://phenome.jax.org/snp ) in the 3.4-mb linked region . we noted a large number of snps in the proximal part of this region featuring two haplotypes with perfect alignment with formalin sensitivity ( fig . of snps observed to be polymorphic among the 16 strains tested , a very high percentage showed perfect correspondence in the 121.7121.8 mb region ( table s2 ) , with smaller numbers of perfectly corresponding snps between 122.1122.3 mb ( fig . only two genes lie near these regions : avpr1a ( 121.886 mb ) and ppm1h ( 122.115 mb ) . strains with the b6-like ( high phenotypic ) haplotype at snps within the 121.7121.8 mb region ( e.g. , rs29348576 at 121.75 mb ) exhibited more than double the pain behavior of strains with the a / j - like ( low phenotypic ) haplotype ( 24.71.0 vs. 11.80.7% samples featuring licking ; t246 = 9.4 , p<0.001 ) . given that multiple polymorphic snps exist upstream of avpr1a , and that no snps are polymorphic between a / j and b6 in the coding region of the gene itself , it is likely that one or more of these snps are affecting the expression of avpr1a . quantitative real time rt - pcr experiments confirmed differential basal expression of avpr1a between the strains in multiple regions of the cns and the liver ( fig . since a / j , the pain - resistant strain , displays higher avpr1a expression than b6 , we tentatively concluded that higher basal expression is protective against pain , or analgesic . this would predict that a null mutant of avpr1a should be more sensitive to late - phase formalin nociception than wildtype ( b6 ) , and that is what we observed ( f2,38 = 3.4 , p<0.05 ) ( fig . there were no genotype differences in early - phase behavior or edema ( not shown ) . we tested the avpr1a mice on a battery of nociceptive assays ( data reported in ) , and no other significant genotype differences were observed , except on the capsaicin ( 2.5 g ) test , another chemical assay featuring spontaneous licking behavior , in which null mutants again displayed higher pain - related behavior ( f2,36 = 4.1 , p<0.05 ) ( fig . 2c ) . we have not formally excluded a role for ppm1h in inflammatory pain , but this gene is not known to be expressed in any pain - relevant loci ( http://biogps.gnf.org/ ) . to investigate whether the human avpr1a gene plays a role in variable responses to pain in our species , we genotyped two snps within the small ( < 6.4 kb ) gene ( rs1042615 and rs10877969 ; contained in the single haplotype block covering the avpr1a region ) in available cohorts of human volunteers ( n=104 ) with recorded capsaicin pain ratings and banked genomic dna samples . no genetic association with either snp was seen in overall mean pain ratings ( fig . 3a and data not shown ) , nor in ratings of stress ( n=61 ) at the time of testing ( fig . 3b and data not shown ) . however , for the promoter snp , rs10877969 , we observed a significant three - way interaction between genotype , stress level and sex ( f1,53 = 5.2 , p<0.05 ) , such that in male subjects reporting stress , pain ratings were lower in those with the gg / ga genotype compared to the aa genotype ( fig . allele frequencies of rs10877969 differed in caucasian and asian versus african - american subjects , and the lower pain ratings of male gg / ga subjects was not observed among the latter racial group ( fig . the overall results remained significant regardless of whether data from african - american subjects were included . factor analysis of components explaining the temporally dynamic capsaicin pain response   revealed a factor representing the influence of stress on capsaicin pain ; we observed a significant sex  genotype interaction ( genotype effect in males only ) within this  stress - induced analgesia  factor ( fig . the demonstrated involvement of avpr1a in capsaicin pain suggested that vasopressin may have analgesic properties against this pain modality . we administered the synthetic vasopressin analog , desmopressin ( 1-desamino-8-d - arginine vasopressin ; 50 g ) , or saline ( 0.5 ml volume ) intranasally to human subjects 25 min prior to pain testing with topical capsaicin ( 8% , applied to a 3-cm area overlying the ventral forearm ) . subjects gave ratings on a 0 - 100 scale every 10 min post - capsaicin administration for 50 min , and these ratings were averaged . we also measured pressure and heat pain thresholds , and heat pain tolerance , before and after drug administration . critically , the experiment occurred on two testing days per subject ; the design was randomly counterbalanced such that half the subjects received desmopressin on the first day and half on the second day . overall , there was absolutely no effect of desmopressin on capsaicin pain ratings compared to saline ( fig . however , we noticed a striking and robust interaction between the apparent efficacy of desmopressin and the order in which subjects completed the experiment ( f1,36 = 15.4 , p=0.0004 ) : in those receiving saline on the first day and desmopressin on the second , desmopressin produced significant analgesia , whereas desmopressin was completely inefficacious in those receiving desmopressin on the first day and saline on the second ( fig . on the first testing day , subject stress levels might be higher  since subjects had no prior experience with capsaicin pain and might fear its intensity  leading to stress - induced analgesia . on the second testing day , having already experienced capsaicin and realizing that pain levels are modest ( < 30/100 at peak ) , stress levels would be low and thus no stress - induced analgesia would be present . indeed , regardless of drug , stress ratings ( immediately before capsaicin ) decreased significantly ( f1,37 = 5.7 , p<0.05 ) from the first to second testing day ( day 1 : 3.40.4/10 ; day 2 : 2.30.3/10 ) . we thus reanalyzed the data using a median split of stress ratings irrespective of testing order , and again we observed that desmopressin produced significant analgesia when stress levels were low , but not when stress levels were high ( f1,37 = 7.1 , p=0.01 ) ( fig . these effects were similar in both sexes , with the exception of a strongly trending ( p=0.06 ) interaction between drug and sex such that males given desmopressin on the first testing day displayed the highest stress levels ( 4.8/10 ) of all groups , in accordance with findings that intranasal vasopressin elevated anxiety to neutral stimuli in men but not women . we found the relationship between stress and desmopressin analgesia to also interact with sex and genotype , with a significant correlation between stress and analgesia ( r = 0.62 , p<0.01 ) only observed in men with the aa genotype of rs10877969 ( fig . all these effects were specific to capsaicin pain ; we observed no analgesic effects of desmopressin nor interactions with sex , stress or genotype , for heat or pressure pain ( fig . we then designed experiments to investigate whether vasopressin analgesia interacted with stress and genotype in mice as well as in humans . it is well known in the rat that the novelty of the testing situation can produce stress - induced analgesia that can be ameliorated by prior habituation to the environment , and this struck us as highly analogous to the apparent situation in the human desmopressin study . we thus explicitly compared the ability of avp ( 0.1 mg / kg , s.c . ) to inhibit capsaicin - induced licking behavior in male mice that were either habituated to the testing room and observation cylinders six times ( each for 60 min ) or were nave to the testing room ( i.e. , not habituated ) . in a striking parallel to the human study , avp was highly efficacious in habituated mice but produced no significant analgesia in non - habituated mice ( drug  habituation : f1,23 = 4.7 , p<0.05 ) ( fig . the avp analgesia was mediated via the v1ar , as no analgesia could be evinced in avpr1a mice ( fig . s7 ) . decreased licking behavior of saline - treated mice in the non - habituated condition compared to the habituated condition revealed the existence of stress - induced analgesia in the former ( fig . the influence of avpr1a gene deletion on capsaicin pain behavior ( and formalin pain behavior ; data not shown ) depended on habituation status ; we replicated the increased sensitivity of knockout mice in a new cohort of non - habituated male mice , but no genotype differences were seen in habituated mice ( genotype  habituation : f1,13 = 5.3 , p<0.05 ) ( fig . 5b ) . that habituation reduces stress was demonstrated by changes in fecal boli , plasma corticosterone levels and plasma vasopressin levels ( fig . the finding that only non - habituated avpr1a mutants would differ from wildtypes would be predicted if mutant mice were unable to produce stress - induced analgesia that would otherwise decrease pain behavior . accordingly , we demonstrated that male avpr1a mice were deficient in the opioid - mediated ( i.e. , naloxone - reversible ) stress - induced analgesia on the 56 c hot - plate test   produced by forced swimming in 32 c water ( genotype  drug : f1,21 = 8.2 , p<0.01 ) ( fig . finally , we retested male a / j and b6 mice on the formalin test with and without habituation ; the expected strain difference was observed in the non - habituated condition , but no strain difference was seen if mice were habituated ( genotype  habituation : f1,17 = 5.3 , p<0.05 ) ( fig . 5d ) . as predicted by results in the null mutants , the high avpr1a - expressing a / j strain displayed evidence of stress - induced analgesia in the non - habituated condition , whereas the lower - expressing b6 strain did not . we have previously demonstrated a relative deficit in opioid - mediated stress - induced analgesia in male b6 mice . thus , ironically , the elucidation of avpr1a as the gene underlying nociq2 was wholly reliant on testing - related stress ; if habituation had been used originally no qtl would have been apparent . nociq2 is not actually a pain qtl , but rather a stress - induced analgesia qtl . notably , the interaction between avp / v1ar and stress appeared in every case described above to be specific to male mice : in female mice , avp analgesia was seen regardless of habituation status ; capsaicin pain and stress - induced analgesia were unaltered in avpr1a mice ; and the a / j versus b6 strain difference was observed in both the habituated and non - habituated conditions ( fig . standard inbred mouse strains display robust variability on virtually all nociceptive assays , including on the formalin test , a widely used assay of spontaneous , tonic chemical / inflammatory nociception . using an f2 intercross between the phenotypically divergent a / j ( resistant ) and c57bl/6j ( b6 ; sensitive ) strains , we previously identified a quantitative trait locus ( qtl ) , which we named nociq2 , on distal mouse chromosome 10 ( > 58 cm ) , associated with variability in the late / tonic phase ( 10 - 60 min post - injection ) of the formalin test . a recent qtl mapping study of the related acetic acid abdominal constriction test also identified significant linkage in this region . we used a variety of strategies to confirm and refine the position of the qtl , including the testing of recombinant congenic strains   and the breeding and testing of marker - assisted congenic and subcongenic strains . a comparison of the formalin test phenotype and chromosome 10 genotype of these strains confirmed that the responsible polymorphism(s ) could only lie between dna markers d10mit25 ( at 121.781 mb ) and d10mit103 ( at 125.176 mb ) ( figs . this 3.4-mb region of chromosome 10 contains only 11 unique transcripts , including five annotated genes ( table s1 ) . of these , the only obvious candidate was avpr1a , although no attempt was made to rule out the others functionally . instead , we conducted a haplotype mapping analysis comparing the late - phase formalin test phenotypes of 16 strains ( fig . we investigated snp haplotype patterns in the mouse phenome database ( mpd ) high - density ( 8 million ; cgd1 imputed 74-strain data set ) snp map ( http://phenome.jax.org/snp ) in the 3.4-mb linked region . we noted a large number of snps in the proximal part of this region featuring two haplotypes with perfect alignment with formalin sensitivity ( fig . of snps observed to be polymorphic among the 16 strains tested , a very high percentage showed perfect correspondence in the 121.7121.8 mb region ( table s2 ) , with smaller numbers of perfectly corresponding snps between 122.1122.3 mb ( fig . only two genes lie near these regions : avpr1a ( 121.886 mb ) and ppm1h ( 122.115 mb ) . strains with the b6-like ( high phenotypic ) haplotype at snps within the 121.7121.8 mb region ( e.g. , rs29348576 at 121.75 mb ) exhibited more than double the pain behavior of strains with the a / j - like ( low phenotypic ) haplotype ( 24.71.0 vs. 11.80.7% samples featuring licking ; t246 = 9.4 , p<0.001 ) . given that multiple polymorphic snps exist upstream of avpr1a , and that no snps are polymorphic between a / j and b6 in the coding region of the gene itself , it is likely that one or more of these snps are affecting the expression of avpr1a . quantitative real time rt - pcr experiments confirmed differential basal expression of avpr1a between the strains in multiple regions of the cns and the liver ( fig . 2a ) . since a / j , the pain - resistant strain , displays higher avpr1a expression than b6 , we tentatively concluded that higher basal expression is protective against pain , or analgesic . this would predict that a null mutant of avpr1a should be more sensitive to late - phase formalin nociception than wildtype ( b6 ) , and that is what we observed ( f2,38 = 3.4 , p<0.05 ) ( fig . there were no genotype differences in early - phase behavior or edema ( not shown ) . we tested the avpr1a mice on a battery of nociceptive assays ( data reported in ) , and no other significant genotype differences were observed , except on the capsaicin ( 2.5 g ) test , another chemical assay featuring spontaneous licking behavior , in which null mutants again displayed higher pain - related behavior ( f2,36 = 4.1 , p<0.05 ) ( fig . 2c ) . we have not formally excluded a role for ppm1h in inflammatory pain , but this gene is not known to be expressed in any pain - relevant loci ( http://biogps.gnf.org/ ) . to investigate whether the human avpr1a gene plays a role in variable responses to pain in our species , we genotyped two snps within the small ( < 6.4 kb ) gene ( rs1042615 and rs10877969 ; contained in the single haplotype block covering the avpr1a region ) in available cohorts of human volunteers ( n=104 ) with recorded capsaicin pain ratings and banked genomic dna samples . no genetic association with either snp was seen in overall mean pain ratings ( fig . 3a and data not shown ) , nor in ratings of stress ( n=61 ) at the time of testing ( fig . however , for the promoter snp , rs10877969 , we observed a significant three - way interaction between genotype , stress level and sex ( f1,53 = 5.2 , p<0.05 ) , such that in male subjects reporting stress , pain ratings were lower in those with the gg / ga genotype compared to the aa genotype ( fig . 3c ) . we saw no effects of stress or genotype in women ( fig . allele frequencies of rs10877969 differed in caucasian and asian versus african - american subjects , and the lower pain ratings of male gg / ga subjects was not observed among the latter racial group ( fig . the overall results remained significant regardless of whether data from african - american subjects were included . factor analysis of components explaining the temporally dynamic capsaicin pain response   revealed a factor representing the influence of stress on capsaicin pain ; we observed a significant sex  genotype interaction ( genotype effect in males only ) within this  stress - induced analgesia  factor ( fig . the demonstrated involvement of avpr1a in capsaicin pain suggested that vasopressin may have analgesic properties against this pain modality . we administered the synthetic vasopressin analog , desmopressin ( 1-desamino-8-d - arginine vasopressin ; 50 g ) , or saline ( 0.5 ml volume ) intranasally to human subjects 25 min prior to pain testing with topical capsaicin ( 8% , applied to a 3-cm area overlying the ventral forearm ) . subjects gave ratings on a 0 - 100 scale every 10 min post - capsaicin administration for 50 min , and these ratings were averaged . we also measured pressure and heat pain thresholds , and heat pain tolerance , before and after drug administration . critically , the experiment occurred on two testing days per subject ; the design was randomly counterbalanced such that half the subjects received desmopressin on the first day and half on the second day . overall , there was absolutely no effect of desmopressin on capsaicin pain ratings compared to saline ( fig . however , we noticed a striking and robust interaction between the apparent efficacy of desmopressin and the order in which subjects completed the experiment ( f1,36 = 15.4 , p=0.0004 ) : in those receiving saline on the first day and desmopressin on the second , desmopressin produced significant analgesia , whereas desmopressin was completely inefficacious in those receiving desmopressin on the first day and saline on the second ( fig . the interaction between pain and stress noted previously suggested a hypothesis . on the first testing day , subject stress levels might be higher  since subjects had no prior experience with capsaicin pain and might fear its intensity  leading to stress - induced analgesia . on the second testing day , having already experienced capsaicin and realizing that pain levels are modest ( < 30/100 at peak ) , stress levels would be low and thus no stress - induced analgesia would be present . indeed , regardless of drug , stress ratings ( immediately before capsaicin ) decreased significantly ( f1,37 = 5.7 , p<0.05 ) from the first to second testing day ( day 1 : 3.40.4/10 ; day 2 : 2.30.3/10 ) . we thus reanalyzed the data using a median split of stress ratings irrespective of testing order , and again we observed that desmopressin produced significant analgesia when stress levels were low , but not when stress levels were high ( f1,37 = 7.1 , p=0.01 ) ( fig . these effects were similar in both sexes , with the exception of a strongly trending ( p=0.06 ) interaction between drug and sex such that males given desmopressin on the first testing day displayed the highest stress levels ( 4.8/10 ) of all groups , in accordance with findings that intranasal vasopressin elevated anxiety to neutral stimuli in men but not women . we found the relationship between stress and desmopressin analgesia to also interact with sex and genotype , with a significant correlation between stress and analgesia ( r = 0.62 , p<0.01 ) only observed in men with the aa genotype of rs10877969 ( fig . all these effects were specific to capsaicin pain ; we observed no analgesic effects of desmopressin nor interactions with sex , stress or genotype , for heat or pressure pain ( fig . we then designed experiments to investigate whether vasopressin analgesia interacted with stress and genotype in mice as well as in humans . it is well known in the rat that the novelty of the testing situation can produce stress - induced analgesia that can be ameliorated by prior habituation to the environment , and this struck us as highly analogous to the apparent situation in the human desmopressin study . we thus explicitly compared the ability of avp ( 0.1 mg / kg , s.c . ) to inhibit capsaicin - induced licking behavior in male mice that were either habituated to the testing room and observation cylinders six times ( each for 60 min ) or were nave to the testing room ( i.e. , not habituated ) . in a striking parallel to the human study , avp was highly efficacious in habituated mice but produced no significant analgesia in non - habituated mice ( drug  habituation : f1,23 = 4.7 , p<0.05 ) ( fig . the avp analgesia was mediated via the v1ar , as no analgesia could be evinced in avpr1a mice ( fig . s7 ) . decreased licking behavior of saline - treated mice in the non - habituated condition compared to the habituated condition revealed the existence of stress - induced analgesia in the former ( fig . the influence of avpr1a gene deletion on capsaicin pain behavior ( and formalin pain behavior ; data not shown ) depended on habituation status ; we replicated the increased sensitivity of knockout mice in a new cohort of non - habituated male mice , but no genotype differences were seen in habituated mice ( genotype  habituation : f1,13 = 5.3 , p<0.05 ) ( fig . that habituation reduces stress was demonstrated by changes in fecal boli , plasma corticosterone levels and plasma vasopressin levels ( fig . the finding that only non - habituated avpr1a mutants would differ from wildtypes would be predicted if mutant mice were unable to produce stress - induced analgesia that would otherwise decrease pain behavior . accordingly , we demonstrated that male avpr1a mice were deficient in the opioid - mediated ( i.e. , naloxone - reversible ) stress - induced analgesia on the 56 c hot - plate test   produced by forced swimming in 32 c water ( genotype  drug : f1,21 = 8.2 , p<0.01 ) ( fig . finally , we retested male a / j and b6 mice on the formalin test with and without habituation ; the expected strain difference was observed in the non - habituated condition , but no strain difference was seen if mice were habituated ( genotype  habituation : f1,17 = 5.3 , p<0.05 ) ( fig . the high avpr1a - expressing a / j strain displayed evidence of stress - induced analgesia in the non - habituated condition , whereas the lower - expressing b6 strain did not . we have previously demonstrated a relative deficit in opioid - mediated stress - induced analgesia in male b6 mice . thus , ironically , the elucidation of avpr1a as the gene underlying nociq2 was wholly reliant on testing - related stress ; if habituation had been used originally no qtl would have been apparent . nociq2 is not actually a pain qtl , but rather a stress - induced analgesia qtl . notably , the interaction between avp / v1ar and stress appeared in every case described above to be specific to male mice : in female mice , avp analgesia was seen regardless of habituation status ; capsaicin pain and stress - induced analgesia were unaltered in avpr1a mice ; and the a / j versus b6 strain difference was observed in both the habituated and non - habituated conditions ( fig . taken together , these results suggest that  in both mice and humans  vasopressin , acting on the v1ar , produces inhibition of capsaicin - induced pain by activating endogenous ( stress - induced ) analgesic systems unless those same systems have already been activated by stress itself . the likelihood of stress - induced analgesia being present , affecting both pain and desmopressin inhibition of that pain , is in turn strongly influenced by sex and avpr1a genotype . s10 . it appears , therefore , that desmopressin analgesia in males results from a three - way interaction between genotype , drug , and acute stress . similarly , capsaicin ( and in mice , formalin ) pain sensitivity is also influenced , in males , by an interaction between genotype and stress . we believe this is the first explicit evidence of the dependence of analgesic efficacy with the acute emotional state of the subject at the time of administration . if the pharmacological efficacy of drugs interacts with both genotype and stress ( especially chronic stress ) , this could have widespread implications for both pharmacotherapy and the design of pharmacological studies . moreover , these findings strikingly demonstrate the utility of the bidirectional ( bench to bedside and back to bench ) translational approach , since all three research components ( murine genetics , human genetics , human pharmacology ) provided a crucial piece of information clarifying the analysis of the others , and successfully predicted the results of new , more complex experiments . although we observe the role of v1ars in pain to be specific to the chemical / inflammatory modality , most chronic pain states feature inflammation , and the capsaicin test is thought to be an excellent model of human clinical pain . finally , these data represent yet another dramatic example of qualitative differences in the operation of pain mechanisms between the sexes .
quantitative trait locus mapping of chemical / inflammatory pain in the mouse identified the avpr1a gene , encoding the vasopressin-1a receptor ( v1ar ) , as responsible for strain - dependent pain sensitivity to formalin and capsaicin . 
 a genetic association study in humans revealed the influence of a single nucleotide polymorphism ( rs10877969 ) within avpr1a on capsaicin pain levels , but only in male subjects reporting stress at the time of testing . 
 the analgesic efficacy of the vasopressin analog , desmopressin , revealed a similar interaction between the drug and acute stress , as desmopressin inhibition of capsaicin pain was seen only in non - stressed subjects . 
 additional experiments in mice confirmed the male - specific interaction of v1ar and stress , leading to the conclusion that vasopressin activates endogenous analgesia mechanisms unless they have already been activated by stress . 
 these findings represent the first explicit demonstration of analgesic efficacy depending on the emotional state of the recipient , and illustrate the heuristic power of a bench - to - bedside - to - bench translational strategy .