Genotype and dose-frequency determine acute and chronic effects of oxytocin on amygdala fear responses in humans: therapeutic implications

Genotype and dose-frequency may critically determine the therapeutic efficacy of chronic oxytocin treatment in humans | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Genotype and dose-frequency may critically determine the therapeutic efficacy of chronic oxytocin treatment in humans Juan Kou , Yingying Zhang , Feng Zhou , Cornelia Sindermann , Christian Montag , View ORCID Profile Benjamin Becker , Keith M Kendrick doi: https://doi.org/10.1101/493387 Juan Kou 1 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Laboratory for Neuroinformation, University of Electronic Science and Technology of China , Chengdu 611731, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yingying Zhang 1 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Laboratory for Neuroinformation, University of Electronic Science and Technology of China , Chengdu 611731, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Feng Zhou 1 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Laboratory for Neuroinformation, University of Electronic Science and Technology of China , Chengdu 611731, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cornelia Sindermann 2 Department of Molecular Psychology, Institute of Psychology and Education, Ulm University , Ulm, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christian Montag 2 Department of Molecular Psychology, Institute of Psychology and Education, Ulm University , Ulm, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Benjamin Becker 1 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Laboratory for Neuroinformation, University of Electronic Science and Technology of China , Chengdu 611731, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Benjamin Becker Keith M Kendrick 1 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Laboratory for Neuroinformation, University of Electronic Science and Technology of China , Chengdu 611731, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: kkendrick{at}uestc.edu.cn Abstract Full Text Info/History Metrics Preview PDF Abstract Chronic intranasal oxytocin administration daily is increasingly proposed as a therapy for social dysfunction but some clinical trials have reported small or no beneficial outcomes. No empirical evidence proves that this is optimal therapeutically or whether oxytocin receptor genotype influences treatment sensitivity. In a randomized, placebo-controlled pre-registered trial on 138 adult male subjects we investigated effects of single and repeated oxytocin treatment (24IU daily or alternatively days for 5 days). Primary neural outcomes assessed core therapeutic mechanisms of action, i.e. amygdala fear reactivity and amygdala-prefrontal intrinsic functional connectivity and modulation by oxytocin receptor polymorphisms (rs53576, rs2254298). The expected oxytocin-induced reduction in amygdala fear reactivity and associated anxious-arousal following single-dose administration was abolished after daily treatment but maintained when administered every other day. Oxytocin selectively reduced amygdala and arousal fear reactivity in AA homozygotes of rs53576 and A+ carriers of rs2254298. By contrast, oxytocin-enhanced intrinsic amygdala-prefrontal coupling was maintained independent of dose frequency and genotype. Together the findings provide the first evidence that infrequent rather than daily oxytocin administration protocols may be therapeutically most efficient and that its neural and behavioral anxiolytic actions are highly genotype-dependent. Introduction Intranasal oxytocin (OT) has been proposed as novel treatment to attenuate social dysfunctions and anxiety in autism spectrum disorder, social anxiety and schizophrenia ( Kendrick et al., 2017 ; Meyer-Lindenberg et al., 2011 ; Young and Barrett, 2015 ) as well as an augmentative strategy to facilitate fear extinction ( Eckstein et al., 2015 ) and working memory performance ( Zhao et al., 2018 ). Preclinical studies consistently suggest that effects on amygdala functioning (attenuated fear reactivity, increased amygdala-prefrontal intrinsic connectivity, ( Eckstein et al., 2015 ; Meyer-Lindenberg et al., 2011 ) represent the primary therapeutic-relevant neural mechanism of action of intranasal OT. The proposed neural mechanisms have been validated using single dose administration protocols (with 24 International Units (IU) being established as optimal ( Spengler et al., 2017 )), however, in contrast initial clinical trials with chronic (twice daily) administration protocols over longer intervals reported inconsistent, or at best modest therapeutic efficacy on social-emotional dysfunctions ( Guastella et al., 2015 ; Halverson et al., 2019 ; Jarskog et al., 2017 ; Kendrick et al., 2017 ; Watanabe et al., 2015 ; Yamasue et al., 2018 ). G-protein-coupled neuropeptide receptors typically exhibit internalization following even a relatively short period of constant exposure to their target peptides and cannot subsequently respond (desensitized) until they are recycled back the surface of the cell membrane ( Pierce et al., 2002 ; Lohse and Hofmann, 2015 ). Indeed, neuroendocrine neurons in the hypothalamus regulating pituitary peptide release tend to exhibit phasic discharge patterns resulting in a pulsatile pattern of release which may serve to reduce internalization of their target receptors ( Russell, 2018 ). Growing evidence from both in vitro and animal studies suggest there may be extensive internalization and subsequent desensitization of OT receptors (OXTR) following chronic OT administration ( Smith et al., 2006 ; Stoop, 2012 ). In terms of neural OXTR receptors those in the amygdala may be particularly susceptible to desensitization ( Terenzi and Ingram, 2005 ) and this region critically mediates many of the effects of intranasal OT on social cognition and anxiety ( Kendrick et al., 2017 ). Moreover, rodent models have reported that in contrast to single doses, chronic administration of OT can actually produce social impairment ( Bales et al., 2013 ; Du et al., 2017 ; Huang et al., 2014 ) in the context of reduced receptor expression in the amygdala and nucleus accumbens ( Du et al., 2017 ). Furthermore, an initial study reported that chronic versus single administration of OT results in divergent neurochemical changes in both the rodent and human frontal cortex ( Benner et al., 2018 ), emphasizing high translational relevance of the preclinical animal models for clinical trials employing chronic treatment protocols. Thus, empirically evaluated optimal treatment protocols for chronic intranasal OT administration in humans are urgently needed to determine the therapeutic potential of OT for social-emotional dysfunctions in psychiatric disorders. A further unresolved issue which may impede the therapeutic efficacy of intranasal OT is that different OT receptor (OXTR) genotypes have been associated with social behaviors and may also influence sensitivity of behavioral and neural responses to intranasal OT. In particular, OXTR polymorphisms rs53576 and rs2254298 have been associated with autism ( Cataldo et al., 2018 ) and deficits in social and emotional processing and anxiety ( Jurek and Neumann, 2018 ; Parker et al., 2014 ; Yang et al., 2017 ) as well as individual variations in behavioral and neural responses to intranasal OT ( Chen et al., 2015 ; Feng et al., 2015a ). Indeed, variants of rs2254298 may represent a trans-diagnostic biomarker for social dysfunctions ( Brüne, 2012 ). It is therefore imperative to establish whether OXTR polymorphisms critically determine sensitivity to the key neural mechanisms of intranasal OT in order to identify treatment-responsive individuals likely to exhibit therapeutic benefits. Although single doses of OT have been reported to produce numerous functional effects, those on amygdala functioning have been most consistently determined as promising key neural mechanisms: attenuated amygdala fear responses and increased resting state functional connectivity between the amygdala and medial prefrontal cortex ( Kendrick et al., 2017 ; Spengler et al., 2017 ). Both neural markers have been primarily associated with attenuated anxiety in terms of reduced responses to social threat and enhanced top-down control of emotion ( Zhao et al., 2019 ), although OT effects on the amygdala may also mediate its actions on a range of social cognition domains ( Kendrick et al., 2017 ; Meyer-Lindenberg et al., 2011 ; Spengler et al., 2017 ; Young and Barrett, 2015 ). In order to determine optimal treatment protocols for chronic intranasal OT administration the current pre-registered double-blind, randomized between subject placebo (PLC)-controlled pharmacological neuroimaging trial (see Fig. 1 ) in 138 adult male subjects therefore aimed at determining the effects of acute (single dose) and repeated doses (daily or every other day for 5 days) of 24IU OT versus PLC on amygdala-centered neural and behavioral (valence, arousal and intensity ratings of fear faces) mechanisms of OT. To identify individuals with the most promising treatment sensitivity we additionally assessed the influence of OXTR rs53576 and rs2254298 polymorphisms on acute and repeated dose OT effects. Download figure Open in new tab Fig.1 CONSORT flow diagram of the clinical trial. Results Acute effects of OT on neural responses to emotional faces In accordance with the primary outcome measure of the trial analyses focused on the neural responses to fearful faces, however no significant effects of OT were found for happy or angry faces. Comparison of single-dose OT-(combined OT 3 and OT 5 groups) and PLC-treated subjects (1 st day) revealed significantly decreased right amygdala reactivity towards fearful faces on the whole brain level (k = 78, p FWE = 0.043, x=27, y=-4, z=-13) ( Fig.2A ). Cytoarchitectonic probabilistic localization (Anatomy toolbox V1.8 ( Eickhoff et al., 2005 )) mapped the peak coordinate with >80% probability to the basolateral amygdala sub-region. In addition, OT also decreased fear-reactivity in the right superior frontal gyrus and bilateral primary visual cortex (see SI and Table S1 ). There were no significant differences between the OT 3 and OT 5 groups on the 1 st day. View this table: View inline View popup Download powerpoint Table S1 Whole brain acute effect (1st day) of intranasal OT on neural responses to fearful faces Download figure Open in new tab Fig.2 The effect of intranasal oxytocin (OT) treatment on neural responses to fearful faces on the 1st day. (A) The t-map of the treatment effect (FWE c = 78, t = 3.93, p = 0.043) showed an activated cluster peaking at the right amygdala (x = 27, y = −4, z = −13). (B) Parameter estimates extracted using a 6-mm radius sphere centered at the peak MNI coordinates at the right amygdala on the 1 st and 5 th day separately revealed that OT treatment on alternate days (OT 3 ) decreased amygdala responses on both the 1 st and 5 th days whereas for the daily OT treatment group (OT 5 ) group the decrease only occurred on the 1 st day. * p < 0.05, ** p < 0.01, two-tailed t-test. Bars indicate M ± SE. Primary outcome measures: effects of repeated doses on OT-evoked changes Mixed ANOVAs with treatment (PLC, OT 3, OT 5 ) and time point (1 st day, acute effects; 5 th day, chronic effects) as factors and right amygdala fear reactivity as dependent variable revealed a significant main effect of treatment (F 2, 135 = 7.85, p = 0.001, η 2 p = 0.104) and a treatment x time point interaction (F 2, 135 = 5.74, p = 0.004, η 2 p =0.078). Post-hoc Bonferroni corrected comparisons between groups on the 5 th day revealed that the OT 3 group exhibited a suppression of right amygdala fear reactivity relative to both the PLC (p = 0.001, Cohen’s d = 0.70, 95% CI, −0.833 to −0.224) and OT 5 groups (p = 0.018, d = 0.52, 95% CI, −0.662 to −0.062) but the OT 5 group did not (p = 0.266, relative to the PLC group). Within group comparisons showed that whereas amygdala reactivity did not change on day 1 relative to day 5 in the PLC group (p = 0.090, d = 0.29, 95% CI, −0.035 to 0.487), its suppression was significantly enhanced in the OT 3 group (p = 0.019, d = 0.40, 95% CI, 0.054 to 0.595) and attenuated in the OT 5 one (p = 0.044, d = 0.38, 95% CI, −0.514 to −0.007) ( Fig. 2B ). For the behavioral ratings mixed ANOVAs with treatment and time point as within-subject factors revealed a significant main effect of treatment for arousal, but not intensity ratings (arousal: F 1, 135 = 4.99, p = 0.008, η 2 p = 0.07, intensity: F 1, 135 = 2.60, p = 0.078, η 2 p = 0.04), and time point for both emotional arousal and intensity ratings (arousal: F 1, 135 = 12.09, p = 0.001, η 2 p = 0.082, intensity: F 1, 135 = 10.81, p = 0.001, η 2 p = 0.74) although no significant two-way interactions (arousal: F 1, 135 = 1.85, p = 0.162, η 2 p = 0.03, intensity: F 1, 135 = 2.97, p = 0.055, η 2 p = 0.04). An exploratory post hoc analysis showed that both emotional arousal and intensity ratings for fear faces in the OT 3 group were decreased on the 5 th day (arousal: p = 0.001 versus PLC, d = 0.63, 95% CI, −1.544 to −0.420, p = 0.033 versus OT 5 , d = 0.42, 95% CI, −1.158 to −0.051; intensity: p = 0.004 versus PLC, 95% CI, −1.299 to −0.247, d = 0.87, p = 0.088 versus OT 5 , d = 0.35, 95% CI, −0.968 to 0.068). Although there was a significant decrease in arousal ratings in the OT 3 group on the 1 st day compared to the PLC group there was no difference between the OT 3 and OT 5 groups (arousal: p = 0.015 versus PLC, d = 0.31, 95% CI, 0.129 to 1.175, p = 0.355 versus OT 5 , 95% CI, −0.268 to 0.744). If we combined the two OT groups on the 1 st day to increase statistical power there was a marginal effect of decreased arousal but not intensity ratings (arousal: p = 0.060 versus PLC, d =0.35; intensity p = 0.150) ( Fig.3 ). No significant main effects or interactions were found for valence ratings (all ps > 0.120). Download figure Open in new tab Fig.3 Oxytocin influenced intensity and arousal ratings. Oxytocin decreased intensity and arousal ratings of fearful faces only on the 5 th day of treatment in the group receiving OT on alternate days (OT 3 ) compared with placebo (PLC). *p< 0.05. **p<0.01. Bars depict M ± SE. Although not included in our primary outcome measures the (calcarine) visual cortex showed the same reduced responsivity to daily OT as the amygdala, and a similar pattern of association with behavioral intensity and arousal ratings, whereas the superior frontal gyrus did not (see SI ). Voxel based morphometry analysis revealed no evidence for acute or repeated dose effects of OT on gray matter volume (see SI ). Associations between amygdala responses and behavioral ratings to fear faces Significant associations between right amygdala activation and emotional arousal and intensity scores were observed in the PLC group on the 1 st day (arousal: r = 0.44, p = 0.003; intensity: r = 0.54, p < 0.001) demonstrating that greater amygdala activation by fear faces was associated with increased anxiety. This association was absent in both OT groups and significantly different from the PLC group on the 1 st day (arousal: OT 3 , r = −0.21 p = 0.181; Fisher’s z = 3.08, p = 0.002; OT 5 , r = 0.09, p = 0.555; z = 1.79, p = 0.073; intensity: OT 3 , r = −0.13 p = 0.399; Fisher’s z = 3.33, p < 0.001; OT 5 , r = 0.12, p = 0.413; z = 2.35, p = 0.024). The same effect was also seen on the 5 th day for arousal ratings (PLC: r = 0.33 p = 0.024; OT 3 , r = −0.21 p = 0.187; z = 2.52, p = 0.012; OT 5 group, r = −0.15 p = 0.317; z = 2.33, p = 0.020) although slightly weaker for intensity ratings (PLC: r = 0.25 p = 0.092; OT 3 , r = −0.29 p = 0.057; z = 2.53, p = 0.011; OT 5 group, r = −0.03 p = 0.847; z = 1.34, p = 0.180) ( Fig.4 ). Download figure Open in new tab Fig.4 Associations between intensity and arousal ratings of fear faces and amygdala activation in the three treatment groups (PLC, OT 3 and OT 5 ) on days 1 and 5. *p< 0.05. **p<0.01 Effects of repeated oxytocin doses on resting-state functional connectivity Right-amygdala seed-to-whole brain fMRI resting-state analysis by mixed-effect ANOVA revealed a main effect of treatment (OT 3 , OT 5 and PLC) on amygdala functional coupling with vmPFC (peak MNI x = −3, y =53, z = −19, F 2, 135 = 16.55, p FWE = 0.001, k = 88) before subjects underwent the face paradigm, whereas no brain regions showed a significant time point x treatment interaction. Post-hoc analyses demonstrated that right amygdala intrinsic connectivity with the vmPFC was stronger in both the OT 3 (k = 74, p FWE = 0.014, x = −3, y = 53, z = −19) and OT 5 groups (k = 127, p FWE = 0.001, x = −3, y = 53, z = −19) relative to PLC. Subsequent confirmatory analyses of resting state functional connectivity in this pathway acquired after the face task paradigm also revealed a significant main effect of treatment (F 2, 135 = 7.08, p = 0.001, η 2 p = 0.095). Post hoc comparisons showed both groups exhibited increased connectivity in this pathway after a single OT dose on the 1 st day (OT 3 , p = 0.001, d = 0.71; OT 5 , p = 0.063, d = 0.40; relative to PLC) and after repeated doses on the 5 th day (OT 3 p = 0.047, d = 0.46; OT 5 , p = 0.001, d = 0.69; relative to PLC). The OT 3 and OT 5 groups did not differ significantly on the 1 st (p = 0.140) or 5 th (p = 0.208) day ( Fig. 5B ). Download figure Open in new tab Fig.5 The effects of oxytocin (OT) on resting state functional connectivity (A) schematic showing functional connection between the right amygdala and ventromedial prefrontal cortex (vmPFC). Whole brain functional connectivity (FC) used the right amygdala as a region of interest (6mm sphere, x=27, y=-4, z=-13) and (B) revealed significant increased functional connectivity between vmPFC and right amygdala (p FWE <0.05, x=-3, y=53, z=-19) in both OT treatment groups (every day – OT and every other day - OT on both days 1 and 5). Data for the two resting state periods before and after the face emotion task were similar and are therefore showed here combined. Bar graph illustrates the extraction of parameter estimates from right amygdala connectivity with vmPFC (M ± SE). * p < 0.05, ** p < 0.01, two-tailed t-test. Bars indicate M ± SE. Associations with OXTR genotype The number of G-carriers and G-non-carriers of rs53576 as well as A-carriers and A-non-carriers of rs2254298 did not differ between the three groups and both SNPs satisfied the Hardy Weinberg Equilibrium (see Tables S2 and S3 ). With Bonferroni-correction for the multiple SNPs and alleles (i.e. 2 x 2 = 4), p < 0.0125 was considered significant. For amygdala responses there was a significant treatment x genotype interaction for rs53576 (F 2, 105 = 6.01, p = 0.003, η 2 p = 0.10). Post-hoc analysis revealed that OT-induced amygdala suppression on the 1 st day was only significant in G-non-carriers (p < 0.001, d = 0.99, 95% CI, 0.432 to 1.333) and also on the 5 th day in the OT 3 group (p < 0.001, d =1.41, 95% CI, 0.638 to 1.483). While the interaction between treatment and genotype did not achieve significance for rs2254298 (F 2, 105 = 1.48, p = 0.232, η 2 p = 0.03) an exploratory post hoc analysis revealed that OT-induced suppression on the 1 st day across treatment groups (OT 3 and OT 5 groups combined to increase power) was only significant in A-carriers (p = 0.001, d = 0.91, 95% CI, 0.337 to 1.244) and also on the 5 th day in the OT 3 group (p = 0.004, d =0.81, 95% CI, 0.218 to 1.122) (see Fig. 6 ). Similar patterns of genotype association were found for the visual cortex but not superior frontal gyrus responses to fear faces (see SI ). View this table: View inline View popup Download powerpoint Table S2 The number of A carriers (A+) and A non-carriers (A-) of rs2254298, G carriers (G+) and G non-carriers (G-) of rs53576 in each treatment group View this table: View inline View popup Download powerpoint Table S3 Distribution of Genotypes in the Sample of N=120 participants Download figure Open in new tab Fig.6 Influence of oxytocin receptor genotype on right amygdala responses to fearful faces (n = 111 subjects). (A)-(B): for rs 53576 only G-non carriers (i.e. AA) showed reduced amygdala response to fearful faces and for rs2254298 only A-carriers (i.e. AA and AG) in the groups with intranasal oxytocin treatment either daily (OT 5 ) or every other day (OT 3 ). (C)-(D): A similar pattern was found for rs2254298, but not rs53576 for arousal ratings of fearful faces although only on day 5 in the OT 3 group. (E)-(F): A similar pattern was found for rs2254298, but not rs53576 for intensity ratings of fearful faces although only on day 5 in the OT 3 group ** p < 0.005, * p < 0.0125, two-tailed t-test (Bonferroni corrected significance threshold of p = 0.0125). Bars indicate M ± SE. For both arousal and intensity ratings there was a significant treatment x genotype interaction for rs2254298 (arousal: F 2, 105 = 9.70, p < 0.001, η 2 p = 0.16, intensity: F 2, 105 = 7.42, p = 0.001, η 2 p = 0.12). To increase the statistical power, we combined the OT 3 and OT 5 groups on the 1 st day. Post hoc analysis showed a significant decrease in ratings on the 1 st day in the OT group only in A-carriers (arousal: p = 0.014 versus PLC, d = 0.64, 95% CI, 0.177 to 1.542; intensity: p = 0.024 versus PLC, d = 0.60, 95% CI, 0.109 to 1.478). There was a significant decrease in ratings on the 5 th day in the OT 3 group only in A-carriers (arousal: p < 0.001 versus PLC, d = 1.69, 95% CI, −2.780 to −1.231; p < 0.001 versus OT 5 , d = 1.10, 95% CI, −2.414 to −0.865; intensity: p < 0.001 versus PLC, d = 1.56, 95% CI, −2.531 to −1.075; p = 0.001 versus OT 5 , d = 0.97, 95% CI, −1.993 to −0.536). There was also a significant difference between the 1 st and 5 th days in the OT 3 group only in A-carriers (arousal: p=0.002, d =1.72, 95% CI, 0.201 to 0.862, intensity: p 0.06) and there was a significant difference between A-carriers and A-non carriers in the OT 3 group (arousal: p=0.001, d =1.07, 95% CI, 0.578 to 2.172, intensity: p=0.001, d =1.01, 95% CI, 0.483 to 1.982) and in PLC group (arousal: p=0.032, d =0.92, 95% CI, −1.783 to −0.084, intensity: p=0.021, d = 0.88, 95% CI, −1.742 to −0.144) on the 5 th day but not in OT 5 (ps>0.088) There was no significant treatment x genotype interaction for rs53576 (arousal: F 2, 105 = 1.28, p = 0.283, η 2 p = 0.02, intensity: F 2, 105 = 0.52, p = 0.599, η 2 p = 0.01). However, an exploratory post hoc analysis found that the reductions in arousal and intensity ratings in the OT 3 group were only significant in G-non carriers on the 5 th day (arousal: p = 0.006 versus PLC, d = 0.87, 95% CI, −1.980 to −0.348; p = 0.012 versus OT 5 , d = 0.84, 95% CI, −2.102 to 0.265; intensity: p = 0.008 versus PLC, d = 0.72, 95% CI, −1.810 to −0.285; p = 0.193 versus OT 5 ) ( Fig. 6 ). Since comparable patterns of OT effects were observed on pre- and post-task resting state data right amygdala-vmPFC functional connectivity values were pooled to increase power ( Fig. 4B ). However, no significant main or interaction effects of genotype for either SNP (rs 2254298 all ps > 0.262; rs53576 all ps > 0.396) were found. Discussion Overall, our findings firstly validate the use of OT-reductions in amygdala responses to fear-faces and associated emotional intensity and arousal ratings together with increased resting state functional connectivity between the amygdala and vmPFC as robust markers for its putative therapeutic mechanisms of action. Intriguingly, these task-dependent and resting-state effects of OT show a markedly different sensitivity to repeated intranasal doses and OXTR genotype, although our results indicate that to achieve both maximal task and resting-state changes an optimal protocol using the standard 24IU dose may be to administer it every other day rather than daily. Indeed, both amygdala and behavioral anxiolytic responses to fearful faces were even more pronounced after 5 days when OT was administered every other day and with resting-state functional connectivity changes there was no advantage in terms of their magnitude when OT was administered daily as opposed to every other day. Our findings are therefore highly consistent with preclinical animal models demonstrating OXTR desensitization following repeated doses of OT in some brain regions ( Terenzi and Ingram, 2005 ; Smith et al., 2005; Stoop, 2012 ) and that chronic administration can reduce brain OXTR expression ( Du et al., 2017 ) and alter patterns of neurochemical release ( Benner et al., 2018 ). Importantly, chronic doses of OT in rodents fail to produce anxiolytic effects normally seen with single doses ( Du et al., 2017 ) which mirrors our present observations and suggests a translational mechanism of high clinical relevance. The apparent long-lasting desensitization effects of daily OT administration may be contributed to by the magnitude of the doses being administered and if so it is possible that lower daily doses might produce reduced effects. The amygdala is one of the main neural substrates mediating OT’s functional effects and its attenuation of fear of reactivity in this region is considered as a core therapeutic mechanism of action ( Jurek and Neumann, 2018 , 2018 ; Kendrick et al., 2017 ). Importantly, we have additionally demonstrated that the magnitude of amygdala responses to fear faces are positively associated with intensity and arousal ratings in the PLC group and that OT exerts an anxiolytic action by reducing both these ratings and abolishing their correlation with amygdala activation. Dysregulations in amygdala and behavioral responses to fear stimuli have been observed across major psychiatric disorders, particularly those characterized by marked social impairments and anxiety ( Hennessey et al., 2018 ; Neumann and Slattery, 2016 ). Furthermore, our finding that both neural and behavioral anxiolytic effects of OT are stronger after repeated compared to single doses in the group receiving OT every other day supports the assumption that optimal therapeutic effects should be obtained following chronic treatment. Our second major finding is that OT’s attenuations of neural and behavioral responses to fear faces are highly dependent on OXTR genotype. Only G-non-carriers of rs53576 (i.e. AA) and A-carriers of rs2254298 (i.e. AA and AG) and AA homozygotes for rs53576 and A+ carriers of rs2254298 were significantly responsive to OT. A-carriers of both SNPs have frequently, although not universally, been associated with social dysfunction in autism as well as social anxiety ( Cataldo et al., 2018 ; Jurek and Neumann, 2018 ). A recent haplotype-based analysis of OXTR SNPs including rs53576 and rs2254298 also indicated that individually they have some association with sensitivity to OT-effects on face recognition ( Chen et al., 2015 ). While the current study clearly indicates that OT modulation of amygdala and visual cortex responses to fear are strongly associated with the AA genotype (Cohen’s ds from 0.81-1.41), it remains to be seen whether other OT-dependent effects exhibit the same association. Limitations to the current findings are that subject numbers are still relatively low for establishing robust genetic associations and that the AA allele of rs53576 occurs more frequently in Asian compared with Caucasian populations ( Butovskaya et al., 2016 ). Also, rs53576 exhibits some sex-dependent differences in social cooperation effects of OT ( Feng et al., 2015b ) and our current study focused only on males to avoid potential menstrual cycle effect issues. Interestingly, task-related and intrinsic network changes produced by OT showed a strikingly different sensitivity to repeated doses and OXTR genotype. However, importantly on the fifth day of treatment there was no advantage of giving OT daily as opposed to every other day. Intrinsic networks may be less influenced by repeated doses of OT or OXTR genotype than those activated by tasks, although arguably in the context of OT’s putative therapeutic effects its task-dependent impact of neural circuitry engaged during social interactions should be of greatest importance. Future studies should measure differential OT effects on both task-related and intrinsic networks. In conclusion, the current study provides the first evidence for an important influence of dose frequency and receptor genotype on the neural and behavioral actions of intranasal OT in response to fear faces in healthy human subjects. Dose frequency therefore requires further empirical evaluation in patient populations given that it can critically determine treatment efficacy in clinical trials employing chronic administration as an intervention in psychiatric disorders. Material and Methods Study design The main objectives of the study were to firstly investigate the effects of acute (single dose) and repeated doses (daily or every other day for 5 days) of 24IU OT versus PLC on two biomarkers: (1) amygdala and behavioral responses to fearful faces and (2) resting state functional connectivity between the amygdala and mPFC. Secondly we investigated modulatory influences of OXTR rs53576 and rs2254298 polymorphisms on sensitivity to intranasal OT. Participants A total of 147 healthy, right-handed healthy adult male subjects were enrolled according to common inclusion and exclusion criteria for human OT-administration studies. This subject number was determine a priori based on achieving 85% power for an expected medium effect size of 0.5. A total of 9 subjects were excluded due to failure to complete the study or excessive head movement (see Fig 1 ). Subjects were randomly assigned to repeated intranasal treatment (single daily dose on five consecutive days) of (1) placebo (PLC; n = 46, M ± SD, 22.46± 2.3 years), (2) oxytocin (OT 5; n = 49, M ± SD, 21.78 ± 2.3 years), or interleaved OT and PLC (OT on days 1, 3, 5, PLC on days 2, 4; OT 3 ; n = 43, M ± SD, 21.02 ± 2.0 years) (see SI ). To ensure compliance all subjects were required to come to the center every day and supervised during self-administration of nasal sprays. To control for between-group differences in potential confounders pre-treatment levels of anxiety, depression, and empathy and trait autism were assessed using validated scales. There were no significant differences between the OT and PLC groups ( Table S4 ). The study was approved by the local ethics committee (Institutional Review Board, University of Electronic Science and Technology of China) and subjects provided written informed consent. The study was in accordance with the latest revision of the Declaration of Helsinki, pre-registered at Clinical Trials.gov ( NCT03610919 - https://clinicaltrials.gov/ct2/show/NCT03610919 ) and in line with recommendations for trials in psychological experiments ( Guidi et al., 2018 ) (see Fig. 1 for Consort flow diagram). Subjects received monetary compensation for participation. View this table: View inline View popup Download powerpoint Table S4 Pre-treatment anxiety, depression, autism and empathy scores in both groups Experimental procedures The study employed a double-blind, randomized, placebo-controlled, between-subject design. The OT and PLC sprays used in the 3 groups were both supplied by Sichuan Meike Pharmaceutical Co. Ltd, Sichuan, China in identical dispenser bottles containing identical ingredients (glycerine and sodium chloride) other than OT. In line with recommended guidelines experiments started 45 minutes after intranasal administration ( Guastella et al., 2013 ). In post-treatment interviews collected on days 1 and 5 subjects were unable to identify better than chance whether they had received OT or PLC (χ 2 0.2, Table S5 ) confirming successful blinding over the entire study period. For OXTR genotyping subjects provided buccal swaps on the 1 st day for analysis of OXTR rs2254298, rs53576 SNPs (see SI and ( Montag et al., 2017 )). View this table: View inline View popup Download powerpoint Table S5 Chi Squared Test of post-experiment interviews where subjects were required to identify which treatment they received. In all cases subjects were unable to guess better than chance. For the implicit face-emotion processing task 208 grayscale facial stimuli displaying happy, neutral, angry or fearful facial expressions (n = 26 per category, 50% female) were used. Stimuli were initially rated with respect to arousal and valence by an independent group of subjects and two matched independent sets of stimuli were produced for use on the 1 st and 5 th days (all ps > 0.3, see SI for details and Table S6 ). The presentation order of the two sets was counter balanced. To ensure attentive processing subjects were required to identify the gender of each face picture (see Figure S2 ). View this table: View inline View popup Download powerpoint Table S6 Ratings for each of the two sets of different face emotion stimuli used on the 1 st day and 5 th day in a counterbalanced design. There were no significant differences between the two sets for valence and arousal ratings. View this table: View inline View popup Download powerpoint Table S7 Group Comparison of Mean frame-wise displacement Primary outcomes and analysis plan Face emotion-related amygdala responses were assessed using the event-related implicit face processing fMRI paradigm on treatment days 1 and 5. Intrinsic amygdala connectivity was assessed by means of two resting state fMRI assessments (before and after the task-paradigm). Valence, arousal and intensity ratings (scale: 1-9) for the facial stimuli were collected immediately after MRI acquisition as additional behavioral outcomes (further details see SI ). In line with the main aim of the study changes in amygdala fear reactivity and amygdala intrinsic connectivity between the 1 st and 5 th day served as primary outcome measures. Changes in valence, arousal and intensity ratings for the fear faces represented an associated behavioral outcome measure. Imaging acquisition and analysis MRI data was acquired using standard sequences on a 3T GE MR750 system. In addition to the functional time series high resolution T1-weighted structural MRI data was acquired at both time points to improve normalization and control for acute and chronic effects of treatment on brain structure. MRI data was preprocessed using validated procedures in SPM12 ( Friston et al., 1994 ) (Statistical Parametric Mapping; http://www.fil.ion.ucl.ac.uk/spm ) and Data Processing Assistant or Resting-State fMRI( Yan, 2010 ) (DPARSFA; http://rfmri.org/DPARSF ) (for details see SI ). First level General Linear Models (GLM) for the task-related fMRI data included separate regressors for the four emotional conditions, gender identity rating period and 6 movement parameters and appropriate contrasts were subjected to a second level random effects analysis. Statistical analyses To identify OT-sensitive regions and confirm previous findings on suppression of amygdala reactivity following single-dose OT-administration ( Young and Barrett, 2015 ) a voxel-wise whole-brain two-sample t-test was conducted in SPM comparing neural activity in OT- and PLC-treated subjects on the1 st day. For all subsequent analyses a 6-mm sphere centered at the maximum t-values of the acute OT effects in the amygdala served as target region to determine different trajectories of amygdala functioning following acute versus repeated treatment (for similar approach see ( Spengler et al., 2017 )). Primary outcomes: amygdala and behavioral fear-face reactivity and intrinsic connectivity Differences in stimulus-induced amygdala and behavioral responses to fear faces were examined by means of mixed ANOVAs with treatment (PLC, OT 3, OT 5 ) as between-subject factor, time point (1 st day, acute effects; 5 th day, chronic effects) as within-subject factor. Dependent variables were extracted target region amygdala-reactivity towards (parameter estimates extracted using Marsbar, http://marsbar.sourceforge.net ) and subsequent valence, intensity and arousal ratings of fearful faces. Bonferroni-corrected post-hoc comparisons were used to explore significant interactions. Associations between amygdala activation in response to fear faces and subsequent behavioral ratings of their valence, intensity and arousal were assessed using Pearson correlation and group differences calculated using Fishers-z tests. Differences in resting state amygdala functional connectivity were determined using a voxel-wise seed-to-whole-brain ANOVA as implemented in the SPM flexible factorial design with treatment (PLC, OT 3, OT 5 ) as between-subject factor, time point (1 st day, acute effects; 5 th day, chronic effects) as within-subject factor and amygdala connectivity maps as dependent variable. To account for potential effects of task-engagement on intrinsic amygdala connectivity the primary analysis focused on pre-task resting state maps. A subsequent ROI analysis on the post-task data (focusing on extracted estimates from a 6mm sphere centered at vmPFC determined by the pre-task data) served as a replication and further validation. Secondary outcome measures: influence of OXTR genotype For rs2254298 subjects were divided into A+ carriers (AA and AG) and A-non-carriers (GG) and rs53576 into G+ carriers (GG and GA) and G-non-carriers (AA). To explore effects of genotype as a secondary outcome measure, OXTR group was included as additional between-subject factor in the corresponding ANOVAs and amygdala fear reactivity, amygdala-vmPFC intrinsic functional connectivity and behavior rating response served as dependent variables. Control for treatment effects on brain structure To control for potential confounding effects of single- and repeated OT-administration on brain structure, a voxel-based morphometry (VBM) analysis was conducted on the T1-weighted images acquired on both testing days using SPM12 standard procedures ( Ashburner, 2007 ; Ashburner and Friston, 2005 ). Effects of single- and repeated dose administration were explored concordant with the fMRI analyses (see SI ). Thresholding and statistical implementation Voxel-wise whole brain analyses in SPM were thresholded at a cluster-based Family-wise error (FWE) corrected level (p < .05) with an initial cluster forming threshold of p < .001 (in line with recent recommendations ( Eklund et al., 2016 ; Mueller et al., 2017 )). Behavioral and neural (extracted parameter estimates) indices were analyzed using SPSS 22.0 and appropriate ANOVA models. Partial eta squared ( η 2 p ) and Cohen’s d were computed as measures of effect size. Group differences in parameter estimates extracted from significant main effect in SPM were further evaluated using two-sample t tests. All reported p values were Bonferroni-corrected, two-tailed, and p ≤ 0.05 considered significant. Funding This project was supported by National Natural Science Foundation of Science (NSFC) grant numbers 31530032 (KMK) and 91632117 (BB). Author contributions JK and KMK designed the experiment. JK, YZ and CS carried out the experiment. JK, KMK, CM, FZ and BB analyzed the experiment and JK, KMK, CM and BB wrote the paper. All authors contributed to the conception of the study and approved the paper. Competing interests The authors declare that they have no competing interests. Download figure Open in new tab Fig.S1. Treatment protocol. Participants were randomly assigned to have nasal spray of oxytocin (OT) for 5 days 24 IU per day (OT 5 group) or have OT or placebo nasal spray on alternate days during the 5 days (OT on the 1 st , 3 rd and 5 th day), 24 IU per day (OT 3 group) or have daily nasal spray of PLC for 5 days (PLC group). Download figure Open in new tab Fig.S2 Face emotion task procedure. Face stimuli appeared on the screen for 3 seconds, and then on the subsequent response screen participants were required to press the left or right response key to identify the gender of the face they had seen. Download figure Open in new tab Fig.S3 The influence of combined rs53576 and rs2254298 genotypes on oxytocin acute treatment effect on brain activation and behavioral response (A) right amygdala and (B) bilateral calcarine gyrus responses to fear faces in the placebo (PLC) and combined oxytocin (OT) treatment groups on the 1 st treatment day and (C) the same for arousal ratings (D) and intensity ratings of fear faces. ** p <0.001 and # p <0.05. After Bonferroni correction p <0.0125 considered significant. Subject numbers for combined rs 2254298 and rs 53576: A+ & G-13 (PLC), 24 (OT); A- & G+ 8 (PLC), 18 (OT); A- & G-8 (PLC), 14 (OT); A+ & G+ 7 (PLC), 19 (OT). Download figure Open in new tab Fig.S4 Associations between calcarine cortex (visual cortex) responses to fear faces and arousal and intensity ratings in the three treatment groups (PLC, OT 3 and OT 5 ). * p <0.05. # p<0.1. 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Share Genotype and dose-frequency may critically determine the therapeutic efficacy of chronic oxytocin treatment in humans Juan Kou , Yingying Zhang , Feng Zhou , Cornelia Sindermann , Christian Montag , Benjamin Becker , Keith M Kendrick bioRxiv 493387; doi: https://doi.org/10.1101/493387 Share This Article: Copy Citation Tools Genotype and dose-frequency may critically determine the therapeutic efficacy of chronic oxytocin treatment in humans Juan Kou , Yingying Zhang , Feng Zhou , Cornelia Sindermann , Christian Montag , Benjamin Becker , Keith M Kendrick bioRxiv 493387; doi: https://doi.org/10.1101/493387 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (8013) Biochemistry (18737) Bioengineering (14888) Bioinformatics (44415) Biophysics (22597) Cancer Biology (19722) Cell Biology (26899) Clinical Trials (138) Developmental Biology (13964) Ecology (21005) Epidemiology (2067) Evolutionary Biology (25454) Genetics (16165) Genomics (23505) Immunology (18703) Microbiology (42483) Molecular Biology (18058) Neuroscience (93440) Paleontology (700) Pathology (2977) Pharmacology and Toxicology (5094) Physiology (8114) Plant Biology (15999) Scientific Communication and Education (2095) Synthetic Biology (4560) Systems Biology (10235) Zoology (2391) window.__CF$cv$params={r:'a3dd663fa81adb75',t:'MTc4OTg3MTMzNA==',u:'01a0bca52e7c7456bfdd3197192343e8',ut:'HZN4jQURPzMAaksIxNuKw8OBEseEKPgE3MiQO0KDqvM-1789871337-1.2.1.1-sUWdEj_UC3_20FdTt0A_uFKV7oaxEyrsJstFo0eYxQRiKmbNdo3N4fY0yZeFdwi2UXTD2unkfX3b8osds6IBv4gVCh8fKoc9XNwWBf0_VN4',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();