Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability

Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability | 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 Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability Oakley B. Morgan , View ORCID Profile Samuel Singleton , View ORCID Profile Sara Hestehave , Tim Sarter , Eva Wozniak , Charles A Mein , Felix Hausch , View ORCID Profile Christopher G. Bell , View ORCID Profile Sandrine M. Géranton doi: https://doi.org/10.1101/2025.01.07.631709 Oakley B. Morgan 1 Dept. of Cell & Developmental Biology, University College London , WC1E 6BT, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Samuel Singleton 2 School of Medicine, University of Dundee , Dundee, DD1 5EH, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samuel Singleton For correspondence: SSingleton001{at}dundee.ac.uk Sandrine.geranton{at}ucl.ac.uk Sara Hestehave 1 Dept. of Cell & Developmental Biology, University College London , WC1E 6BT, United Kingdom 3 Dept. Of Experimental Medicine, University of Copenhagen , Copenhagen N 2200, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sara Hestehave Tim Sarter 4 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universität München , Munich, 81377, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eva Wozniak 5 Genome Centre, Faculty of Medicine and Dentistry, Queen Mary University of London , London, E1 2AT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charles A Mein 5 Genome Centre, Faculty of Medicine and Dentistry, Queen Mary University of London , London, E1 2AT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Felix Hausch 6 Institute of Organic Chemistry and Biochemistry, Technical University Darmstadt , Darmstadt, 64287, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher G. Bell 7 William Harvey Research Institute, Barts & The London Faculty of Medicine, Charterhouse Square, Queen Mary University of London , London, EC1M 6BQ, United Kingdom 8 QMUL Centre for Epigenetics, Queen Mary University of London , London, E1 4NS, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher G. Bell Sandrine M. Géranton 1 Dept. of Cell & Developmental Biology, University College London , WC1E 6BT, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandrine M. Géranton For correspondence: SSingleton001{at}dundee.ac.uk Sandrine.geranton{at}ucl.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Stress is thought to contribute to the persistence of pain and comorbid anxiety, yet the underlying mechanisms remain unclear. In our pre-clinical model, sub-chronic stress exacerbated subsequently induced inflammatory pain and accelerated the development of comorbid anxiety. DNA methylation analysis of spinal cord tissue after stress exposure revealed hypomethylation in the Fkbp5 promoter site for the canonical FKBP51 transcript and other stress-related genes. However, most epigenetic changes in key regulatory regions did not correlate with changes in gene expression assessed by RNA sequencing, suggesting that stress exposure had remodeled the epigenome without altering gene activity and primed genes for hyper-responsiveness to future challenges. FKBP51 inhibition during stress exposure reduced the exacerbation of inflammatory pain by stress and reversed several stress-induced DNA methylation changes in promoter regions of genes associated with stress and nociception, including Rtn4, Cdk5 and Nrxn1 , but not Fkbp5 . These results indicate that sub-chronic stress leads to the hypomethylation of Fkbp5 and increased susceptibility to chronic pain driven by FKBP51, but reversing Fkbp5 hypomethylation is not necessary to prevent chronic pain vulnerability, which is likely driven by complex epigenetic regulation of multiple stress-regulated genes. 1. Introduction Chronic stress is a well-known modulator of physiological and behavioral responses, often leading to increased vulnerability to various pathological conditions 1 . Prolonged exposure to stress activates the hypothalamic-pituitary-adrenal (HPA) axis, leading to elevated levels of glucocorticoids, which can disrupt normal bodily functions 2 . This dysregulation affects immune responses, promotes inflammation and alters neural circuits, particularly in regions associated with mood. Consequently, chronic stress is linked to a heightened risk of developing disorders such as anxiety, depression, cardiovascular diseases and chronic pain 3 – 5 . Stressful experiences indeed have a profound impact on the manifestation of pain, both in humans and rodents. While short-lasting, intense stress experiences can trigger a physiological reaction that attenuates pain signalling and allows for expression of protective behaviours, sustained exposure to stress exacerbates hyperalgesic states 6 , 7 . Moreover, stress experience can prime for hyper-responsiveness to subsequent injury, as a result of long-lasting changes involving complex peripheral, central and systemic mechanisms 8 and stressful life events are considered a key environmental factor in increased risk for developing chronic pain 9 , 10 . FK 506 binding protein 51 (FKBP51) is a crucial element of the stress axis that regulates the glucocorticoid receptor (GR) sensitivity to glucocorticoids 11 , 12 . and FKBP5 genetic polymorphisms and DNA methylation landscape have both been associated with increased risk for developing mental health disorders in small-scale human studies 13 – 16 . This includes the observation of early life adversity in humans reducing FKBP5 DNA methylation (DNAm) at the promoter region, an epigenetic change that influences the gene’s expression, its responsiveness to stress and susceptibility to psychiatric disorders 14 , 17 . While these genetic findings have not, to date, been replicated in larger biobank-scale genome-wide association studies (GWAS), recent epigenome-wide association studies (EWAS) have identified blood-derived DNA methylation associations within the FKBP5 locus with neurodegenerative diseases 18 , as well as all-cause mortality 19 . Preclinical studies have recently shown that FKBP51 is a crucial driver of persistent pain states following physical injuries and traumatic stress exposure 20 – 22 . Moreover, FKBP51 is up-regulated at spinal cord level in the early days after injury and spinal FKBP51 specifically maintains persistent pain of neuropathic and inflammatory origin, as its genetic deletion and spinal pharmacological inhibition provide significant pain relief 20 , 23 . Crucially, we reported that early life trauma in mice leads to a reduction in spinal Fkbp5 DNAm and prolonged subsequent pain states when exposed to inflammation in adulthood 23 . We therefore hypothesised that stress exposure promotes the vulnerability to chronic pain in an FKBP51-dependent mechanism. Here, we tested this hypothesis using a model of sub-chronic stress exposure in adult mice. Our results suggest that following stress exposure, FKBP51 drives the susceptibility to persistent pain in male mice at least. This increased vulnerability is accompanied by changes in DNAm in nociceptive signaling and stress-regulated genes at spinal cord level, including a reduction in DNAm in Fkbp5. However, complete reversal of these DNAm changes is not necessary to reverse the primed state. 2. Results 2.1 Sub-chronic stress primes for hyper-responsiveness to inflammatory pain Male and female mice were exposed to the restraint stress paradigm (RS) 14 days before the injection of the inflammatory agent Complete Freund’s Adjuvant (CFA) ( Fig.1A ). Both male and female mice displayed mechanical hypersensitivity that peaked on the first day of testing (day 1 following last restraint = day 4) and were no different from control animals by day 10. ( Fig.1B ). There were no sex differences in the response to RS. On day 14, animals received an injection of CFA into the hindpaw. CFA induced a short-lasting hypersensitive state in control, non-stressed, mice (mechanical thresholds were no different from baseline thresholds by day 35 in male and female mice (one-way ANOVA: male controls, day 25 vs day 0: p=0.013, day 35 vs day 0: P>0.05; female controls, day 25 vs day 0: p=0.004, day 35 vs day 0: P>0.05)). However, stress exposure exacerbated the CFA-induced mechanical hypersensitivity in both male and female mice (RM-ANOVA: day 20 to day 80: RS mice vs Control mice: RS effect: F 1,12 =15.9, p=0.002; sex effect: F 1,12 =7.5, p=0.018; no RS effect x sex interactions). Mechanical thresholds were not only lower overall in both male and female mice exposed to RS (RS effects in males: day 20 to day 70: control vs RS: F 1,6 =8.6, p=0.026; in females: day 20 to day 80: control vs RS: F 1,6 =15.5, p=0.009), but the duration of the CFA-induced mechanical hypersensitivity was also extended. Indeed, mechanical thresholds returned to baseline thresholds by day 80 in RS male mice (male RS, last significant difference on day 70: day 70 vs day 0: p=0.03; day 80 vs day 0: P>0.05) and never returned to baseline for RS female mice (female RS, day 80 vs day 0: p=0.007). Stress exposure also had a greater impact on female mice as the thresholds of stressed female mice were lower than stressed male mice (RM-ANOVA: Females RS vs Males RS, day 20 to day 80: F 1,6 =9.2, p=0.023). In another subset of male mice, we assessed whether RS could exacerbate the hypersensitive state induced by another inflammatory agent and, indeed, RS exacerbated the response to interleukin 6 (IL6) injected in the hindpaw (Fig.S1). Download figure Open in new tab Figure 1: Sub-chronic stress primes for hyper-responsiveness to inflammatory pain. ( A ) Experimental design. ( B ) Hind paw mechanical withdrawal thresholds assessed in male and female mice using von Frey filaments. All mice received intra-plantar CFA injection on day 14. Post-hoc analysis: *: p<0.05: control females vs RS females; #: p<0.05 control males vs RS males; $ p<0.05: RS females vs RS males. Green panel indicates the 3 days of RS paradigm. ( C ) C1 : number of c-Fos expressing cells in the ipsilateral superficial dorsal horn (lamina 1-2) 2 hours after CFA-injection; C2 : Representative images of ipsilateral lumbar spinal c-Fos staining. Scale bar: 200µm. ( D ) Anxiety-like behaviour was measured using the elevated plus maze. Measures were taken at day 14, then at two- and six-weeks post-CFA (day 28 and day 44). ( E ) Anxiety-like behavior data from panel D, presented as a % change from baseline. There were significant differences in percentage change over time from pre- to two-weeks post CFA but not at two- to six-weeks post CFA, suggesting that RS accelerated CFA-induced anxiety-like behaviour. ( F ) RTqPCR quantification of Fkbp5 mRNA levels. ( C-D ) *P<0.05. While RS did not exacerbate the peak hypersensitivity after CFA, i.e. when it reached its maximum at 6h after CFA (day 14 plus 6h), it did promote the expression of cFOS measured 2h after CFA in the superficial laminae of the dorsal horn ( Fig.1C ), suggesting that stress exposure could promote nociceptive signalling. We next assessed the development of comorbid anxiety-like behaviors, often observed following stress exposure. Stress exposure accelerated the development of anxiety like behavior following CFA injection ( Fig.1D , E). Animals who were not exposed to stress showed signs of anxiety only 6 weeks after CFA compared with stressed animals that showed signs at 2 weeks. Finally, we found that animals exposed to the RS paradigm had elevated levels of spinal Fkbp5 mRNA compared to control animals 48h after CFA injection (day 16) ( Fig.1F ). This finding was similar to our previous observations of elevated spinal Fkbp5 mRNA levels that occur in the first 48h of persistent injured state 21 , 24 ., suggesting that FKBP51 may be responsible for the longer lasting pain state observed in stressed mice. 2.2 FKPB51 drives stress-induced vulnerability to persistent pain in mice We next tested the hypothesis that FKBP51 is an important contributor to the mechanisms driving the increased vulnerability to persistent pain observed following stress exposure. For this, we first used Fkbp5 global knock out (KO) mice and exposed them to the RS plus CFA paradigm ( Fig.1A ). When mice were exposed to the RS paradigm, we observed that the Fkbp5 KO mice did not develop mechanical hypersensitivity as seen in WT mice. Both male and female KO mice had reduced RS-induced hypersensitivity (RM-ANOVA: day 0 to day 10: factor genotype: F 1,26 =15.9, p<0.001; no sex differences; post-hoc RM-ANOVA: day 0 to day 10: factor genotype: males only: F 1,13 =15.3, p=0.002; females only: F 1,13 =4.7, p=0.049). However, KO female mice were not different from WT female mice at any individual time point, suggesting that female KO were less resilient to RS than male KO. Following CFA injection, KO mice showed reduced mechanical hypersensitivity when compared to WT mice (RM-ANOVA, day 14 + 6h to day 60: factor genotype: F 1,26 =19.9, p<0.001; post-hoc males only: F 1,13 =22.0, p<0.001; post-hoc females only: F 1,13 =5.7, p=0.033). Unexpectedly, Fkbp5 KO females were no different from WT females from day 45. Moreover, female KOs did not return to their baseline threshold for the duration of the observations (day 0 vs day 60: KO females threshold: p=0.024) while KO males had returned to their baseline thresholds by day 40 (day 0 vs day 40: KO males threshold: p>0.05). Overall, these results indicated that FKBP51 may be driving the stress-induced increased vulnerability to persistent pain in male mice at least. Therefore, we further explored the mechanisms of FKBP51-driven persistent pain vulnerability in male mice only. 2.3 FKBP51 inhibition during stress exposure prevents stress-induced increased vulnerability to persistent pain To dissect the role of FKBP51 in promoting persistent pain during the stress phase and the injury phase of our paradigm, we used the specific FKBP51 inhibitor SAFit2. Our previous work had shown a delay in the initiation of FKBP51 antagonism when SAFit2 is encapsulated in a phospholipid gel for slow release (SAFit2-VPG). Under these conditions, SAFit2 is active for a maximum of 7 days 20 , 21 . SAFit2-VPG was injected 2 days before the start of the RS paradigm to ensure complete FKBP51 blockade during the RS paradigm but not at the time of the CFA injection. While FKBP51 inhibition did not prevent the RS-induced mechanical hypersensitivity, it prevented the exacerbation to subsequent CFA-induced mechanical hypersensitivity ( Fig.2B ). Vehicle treated mice had returned to their baseline threshold by day 80 (last significant difference on day 70; day 0 vs day 70: p=0.012) whereas SAFit2-treated mice had returned to baseline threshold by day 35 (last significant difference on day 30; day 0 vs day 30: p=0.0032), following a time course similar to the WT mice non-exposed to RS ( Fig.1B ). Moreover, there was a significant difference in CFA-induced mechanical hypersensitivity between SAFit2-VPG and vehicle treated animals ( Fig.2B ; RM-ANOVA: factor treatment; day 15 to day 80: F 1,10 =16.7, p=0.002). Download figure Open in new tab Figure 2: FKPB51 drives stress-induced vulnerability to persistent pain in mice. ( A ) Hind paw mechanical withdrawal thresholds measured using von Frey filaments in WT and KO Fkbp5 mice. All mice received intra-plantar CFA on day 14. Fkbp5 global deletion prevented the full development of RS hypersensitivity in male mice and to a lesser extent in female mice. Green panel indicates the 3 days of RS paradigm. Post-hoc analysis: *: WT females vs Fkbp5 KO females; #: WT males vs Fkbp5 males. ( B ) Hind paw mechanical withdrawal thresholds measured using von Frey filaments in male mice receiving either Vehicle- or SAFit2-VPG, administered two days prior to the onset of RS. Green panel indicates the 3 days of RS paradigm and red panel indicates the days of active SAFit2-VPG treatment. ( C ) Number of cFos expressing cells in the ipsilateral superficial dorsal horn (lamina 1-2) 2h after CFA injection. ( D, E ) Anxiety-like behaviour was measured using the elevated plus maze. (E) Anxiety-like behavior data from panel D, presented as a % change from baseline. **P<0.01, *P<0.05. As we were surprised by the absence of effect of SAFit2 in the RS phase of the paradigm, we repeated this experiment with another SAFit2 formulation, injected i.p., twice a day for 5 consecutive days, to inhibit FKBP51 during the RS phase, as before. This delivery method increases SAFit2 plasma concentration at peak (personal communication). Again, we observed no effects of FKBP51 inhibition on the RS-induced mechanical hypersensitivity, while SAFit2 did reduce the subsequent CFA-induced hypersensitivity (Fig.S2A). We next assessed the impact of FKBP51 inhibition during the RS phase on CFA-induced early nociceptive signaling. There was no difference between SAFit2-VPG and vehicle-VPG treated mice in cFos activation evoked by CFA ( Fig.2C ), implying that SAFit2 did not reduce the exacerbation of CFA-induced cFos by RS we had previously observed ( Fig.1C ). Finally, we looked at the impact of FKBP51 inhibition during RS on anxiety-like behavior and found that SAFit2-VPG delayed the development of anxiety-like behavior, previously observed in wild type mice injected with CFA after stress exposure ( Fig.1D , E and Fig.2D , E). Control experiments in naïve WT mice revealed that SAFit2-VPG alone did not modify anxiety-like behavior, measured using the EPM, or locomotion, as measured by the total distance travelled in the Open Field 5 day after SAFit2-VPG injection (Fig.S2B, 3C). 2.4 Sub-chronic stress induces rapid changes to stress signaling at spinal cord level We next investigated whether stress exposure causes rapid changes to spinal cord signalling involved in the maintenance of long-term pain states. FKPB51 is upregulated by cortisol release and we found that in addition to mechanical hypersensitivity, the RS paradigm also led to an increase in blood serum glucocorticoids ( Fig.3A , B), together with an upregulation of spinal Fkbp5 mRNA measured 48h after the last restraint (day 5) ( Fig.3C ). There was also a downregulation of Nr3c1 , the gene encoding for the glucocorticoid receptor (GR), particularly of the beta isoform which is associated with glucocorticoid resistance 25 ( Fig.3C ). However, RS alone did not induce anxiety-like behaviour at the same time point (day 5 = 48h post RS; Fig.3D ) and at day 14 ( Fig.3E ). Download figure Open in new tab Figure 3: Sub-chronic stress induces rapid changes to stress signaling at spinal cord level. (A) Hind paw mechanical withdrawal thresholds assessed in male mice using Von Frey filaments. Green panel indicates the 3 days of RS paradigm. *p<0.05. Post-hoc one-way ANOVA. ( B ) Endogenous plasma CORT levels measured over the course of the RS paradigm. **P<0.01. ( C ) spinal mRNA levels assessed using RTqPCR at day 5. Graph shows percentage mRNA change normalized to control. ( D,E ) Anxiety-like behaviour assessed using EPM. 2.5 Sub-chronic stress induces long-lasting changes to the spinal methylome, including in the promoter of Fkbp5 We have previously shown that Fkbp 5 DNAm is reduced in the rodent spinal cord both after injury 20 and after early life adversity 23 , when reductions in DNAm may increase vulnerability to chronic pain later in life. We therefore assessed whether sub-chronic stress in adulthood modified the DNA methylome at spinal cord level and established to what extent the DNAm landscape of stress related genes such as Fkbp5 may be modified. Spinal cord dorsal halves ( i.e. ipsilateral + contralateral dorsal horns) were dissected on day 14 after the RS paradigm and prepared for both DNAm analysis and mRNA sequencing (RNAseq). At this time point, mice exposed to stress are no longer hypersensitive compared to control mice ( Fig.4A ), cortisol levels are no longer elevated ( Fig.4B ) but RS exposed mice are primed for hyper-responsiveness to CFA ( Fig.1B ). Differential DNA methylation analysis revealed that, of the 192,917 available post-QC CpGs, 11,644 probes were modulated by RS at a nominal significance of p < 0.05 (9,119 hypomethylated and 2,525 hypermethylated). However, no individual CpG surpassed an FDR threshold of p < 0.05 (Fig.S3A-B). Download figure Open in new tab Figure 4: Sub-chronic stress induces long-lasting changes in the spinal DNA methylome, including in Fkbp5 promoter region. ( A ) Hind paw mechanical withdrawal thresholds assessed in male mice using Von Frey filaments. RM ANOVA: T0 to T10: F 1,10 =45.4, p<0.001. Post-hoc analysis one-way ANOVA: **p<0.01 ( B ) Blood corticosterone levels from the same mice collected on day 14. ( C ) DNA methylation (DNAm) levels assessed at day 14, using the Infinium Mouse Methylation BeadChip arrays, at 2 independent CpG base. ( D ) Genomic region displaying the structure and regulatory elements associated with Fkbp5 . The horizontal axis represents the genomic coordinates, spanning from 28.4 to 28.5mb on chromosome 17. The gene structure is shown with blue boxes indicating exons and arrows showing transcription direction. Fkbp5 associated CpG sites present on the array beadchip are marked above the gene in red, highlighting potential methylation sites. A CpG (cg34791938_TC11), differentially methylated by RS, is indicated in green. Chromatin accessibility (ATAC sequencing), CG content, transcription factor binding sites and 15 state chromatin segmentation data derived from mouse neural tube tissue (E15.5) on the UCSC genome browser are displayed below the gene model. ( E ) DNA methylation levels in selected genes assessed using the Infinium Mouse Methylation BeadChip arrays. Y-axis: methylation levels (0-1). ( C,E ) * p<0.05 nominal significance. We annotated the location and chromatin state according to mouse neural tube tissue 26 of the 11,644 probes with nominal significance, to establish the importance that these probes may have on functional changes to gene expression. Hypomethylated CpGs in RS mice were enriched in active (Tss and TssFlnk) promoter sites (43.0% versus 24.1% array background, p <1.10 -4 ) whereas these regions were underrepresented in CpGs hypermethylated by RS (21.0%, p = 4.10 -4 ) (Fig.S3C). Consistent with these findings, Gene Set Enrichment via GREAT 27 with the 11,644 probes satisfying nominal significance further confirmed an enrichment (49.19% versus 38.51% array background; p 8 fold-enrichment, FDR p = 0.024), glutamate catabolism (>2.5 fold-enrichment, FDR p = 0.049) and retrograde axonal transport (>2 fold-enrichment, FDR p = 0.048). Additionally, heterotrimeric G-protein binding was identified as a molecular function (Table S1). We next focused on stress related genes, including Fkbp5 . A single probe (cg34791938_TC11) overlapping Fkbp5 was revealed to be hypomethylated in RS relative to control mice (median log2FC = -0.24, 2% raw change in DNAm; control β: 0.15 ± 0.01 vs RS β: 0.13 ± 0.01; p = 0.037; Fig.4C1). This CpG is located within a CpG shore at chr17:28,486,463 in an active promoter region 313 bases upstream from the nearest TSS and resides in close proximity to several transcription factor binding sites ( Fig.4D ). There were no other significant changes in any of the other 13 CpGs located in the Fkbp5 sequence (Table S2). However, a CpG (cg34792474_TC21) encoding a lincRNA (LOC102639076) associated with Fkbp5 by GREAT and located proximal to several transcription factor binding sites (PITX2, TFAP2C, ZIC1, ZIC4 and ZIC5), 37 Kb upstream to the canonical TSS of Fkbp5 (Chr17:28,486,150), was similarly hypomethylated in RS mice (median log2FC = -0.22; 2% raw change in DNAm; control β: 0.16 ± 0.01 vs RS β: 0.14 ± 0.01, p = 0.019; Fig.4C2). A number of HPA axis relevant genes also stood out as being differentially methylated by RS (at a nominal level), often in key regulatory regions, including Nr3c1 (Fig.4E1), Nr3c2 (Fig.4E2), Hsp90b1 (Fig.4E3) and Nfkb1 (Fig.4E4) ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1: Differentially Methylated Probes (DMPs) RS vs Control associated with genes linked to the HPA axis. CpGs are mapped to their closest gene, distance and chromatin state using mouse neural tube (E15.5) segmentation. FC: log2-fold change contrast. HMM: Chromatin state signatures established using hidden Markov model. Chr: Chromosome 2.6 Sub-chronic stress induced-changes in the spinal DNA methylome are not fully reversed by FKBP51 inhibition despite its behavioural impact As FKBP51 inhibition with the inhibitor SAFit2 prevented the exacerbation of CFA-induced mechanical hypersensitivity, we asked whether SAFit2 could prevent the RS-induced changes in spinal DNAm, including at Fkbp5 cg34791938_TC11. As previously observed, mice treated with SAFit2-VPG during the RS paradigm developed mechanical hypersensitivity to the same extent as vehicle treated mice ( Fig.5A ). There was no difference in Fkbp5 mRNA levels between vehicle and SAFit2-VPG treated mice at day 5 (48h post RS) ( Fig.5B ) suggesting that SAFit2-VPG did not prevent the upregulation of Fkbp5 mRNA induced by the RS paradigm we had previously observed ( Fig.3C ). Download figure Open in new tab Figure 5: FKBP51 inhibition during stress exposure reverses some, but not all, changes in sub-chronic stress induced changes in spinal DNA methylome. ( A ) Hind paw mechanical withdrawal thresholds assessed in male mice using von Frey filaments. Green panel indicates the 3 days of RS paradigm and red panel indicates the days of active SAFit2 treatment. ( B ) Fkbp5 mRNA levels in spinal cord superficial dorsal horn assessed by RTqPCR at day 5 (48h post RS). ( C ) Fkbp5 DNAm status assessed using the Infinium Mouse Methylation BeadChip array. ( D ) Venn Diagram showing the intersection of hypomethylation and hypermethylated CpGs across the 2 DNAm studies. ( E ) DNA methylation levels in selected genes assessed using the Infinium Mouse Methylation BeadChip arrays. Y-axis: methylation levels (0-1). *p<0.05 nominal significance. Using the same approach as in our first DNA methylation study, we found that 4,960 CpGs out of 191,134 total analytically robust CpGs were hypermethylated in SAFit2-VPG treated mice, whereas 5,086 CpGs were hypomethylated at a nominal p value threshold (p< 0.05; total 10,046 DMPs). There were similarly no individual CpGs surpassing a Benjamini-Hochberg FDR threshold of p < 0.05 (Fig.S3F-G). Probe location and chromatin segmentation in mouse neural tube tissue of the 10,046 CpGs did not significantly differ from the array background (Fig.S3H), nor were there any statistically significant terms identified by Gene Set enrichment via GREAT. Moreover, unlike the contrast between RS and control mice, which revealed enrichment of probes within 5 kb of the TSS (Fig.S3D-E), there was not an enrichment in probes within 5 kb of TSS for the 10,046 CpGs differing between RS mice treated with SAFit2 or vehicle (Fig.S4I-J). There was also no difference in DNA methylation at Fkbp5 cg34791938_TC11 (median log2FC = -0.031; <0.01% raw change in DNAm; vehicle β: 0.078 ± 0.001 vs SAFit2 β: 0.077 ± 0.006, p = 0.64; Fig.5C1) or cg34792474_TC21 (median log2FC = -0.077; <1% raw change in DNAm; vehicle β: 0.090 ± 0.001 vs SAFit2 β: 0.086 ± 0.006, p = 0.14; Fig.5C2) between vehicle and SAFit2-VPG treated mice exposed to RS. Taken together, these observations suggest that inhibition of FKBP51 during the RS phase may not revert changes to the Fkbp5 DNAm landscape induced by the RS paradigm. We also looked more widely at the intersection between the DMPs identified in the two DNAm studies to identify any DMPs regulated in opposite direction, that may contribute to the diverging pain phenotypes observed following RS with or without SAFit2 exposure. Intersections between hypermethylated and hypomethylated CpGs from each contrast revealed 366 CpGs regulated in opposite direction (250 CpGs hypomethylated RS vs Control and hypermethylated SAFit2 vs Vehicle and 116 hypermethylated RS vs Control and hypomethylated SAFit2 vs Vehicle, Fig.5D ). These associated with 284 distinct genes and many of these (108/284) were oppositely regulated in active promoter regions (Tss and TssFlnk). Of particular interest were genes related to nociceptive signalling ( Table 2 ), including Rtn4 (Fig.5E1), which encode the protein Nogo-A, a neurite outgrowth inhibitor, whose inhibition has been proposed a novel approach for pain management 28 ; Cdk5 (Fig.5E2), that encodes for the cyclin-dependent kinase 5, also considered a potential target for the management of chronic pain 29 ; Nrxn1 (Fig.5E3), which encodes the neurexin 1 protein, previously linked to hypersensitive states 30 and Gli2 (Fig.5E4), previously linked to neuropathic pain 31 . We also observed interesting DNAm reversal in regions linked to quiescent state, e.g. in Hk1 (Fig.5E5), that encodes the hemokinin 1, which binds to the NK1 receptor and is an important pain mediator 32 . View this table: View inline View popup Download powerpoint Table 2: Inversely Differentially Methylated Probes (DMPs) in RS vs Control and SAFit2 vs Vehicle studies, associated with genes relevant to priming mechanisms and nociceptive processing. CpGs are mapped to their closest gene, distance and chromatin state using mouse neural tube (E15.5) segmentation. FC: log2-fold change contrast. HMM: Chromatin state signatures established using hidden Markov model. Chr: Chromosome. *: p<0.05 nominal significance. 2.7 Following restraint stress (RS) the Fkbp5 loci CpGs cg34791938_TC11 and cg34792474_TC21 are hypomethylated and Fkbp5 mRNA is upregulated Differential gene expression (RS – Control) on log2-transformed RNA count intensities (n = 15533) revealed a difference in expression for 230 genes (153 downregulated genes and 77 upregulated genes) exceeding the criteria of FC > 1.2 (nominal p < 0.05). These genes were not significantly overrepresented in any biological process ontologies at an FDR corrected p value < 0.05. However, interestingly, Fkbp5 was among the genes upregulated (1.3-fold of control) by RS ( Fig.6A -C). These observations suggested that reduced DNAm after RS at both cg34791938_TC11 and cg34792474_TC21 could have some functional relevance to Fkbp5 expression. Download figure Open in new tab Figure 6: Fkbp5 mRNA is upregulated after RS when both cg34791938_TC11 and cg34792474_TC21 are hypomethylated. ( A ) Volcano plot visualising the differential expression contrasting RS mice versus controls. The x-axis denotes the log2 fold change (FC) in RNA expression between the two conditions, with positive values indicating higher expression in the RS group and negative values indicating higher expression in controls. The y-axis represents the -log10 transformed p-values. Data are adjusted for cell type heterogeneity. ( B ) Scatter plot visualising the intersection between CpGs occurring in promoter regions that are differentially methylated by RS and corresponding changes to RNA expression in the same samples. Y-axis: Log 2 RNA expression (RS/control). ( C ) Fkbp5 mRNA levels from sequencing data. ( D ) RTqPCR quantification of Fkbp5 mRNA in the ipsilateral dorsal horn of the spinal cord 48h after CFA injection. *p<0.05. ( E ) Correlation between mechanical threshold (average day 4 to 10) and DNAm at cg34791938_TC11 (E1) and cg34792474_TC21 (E2). ( F ) Correlation between DNAm at cg34791938_TC11 and Fkbp5 mRNA levels at day 14. To investigate whether this reduction in DNAm could also be mechanistically associated with the elevated levels of Fkbp5 mRNA seen in RS exposed animals 48h after CFA compared with non-stressed controls ( Fig.1F ), we compared spinal Fkbp5 mRNA levels between RS alone, CFA alone and RS + CFA exposed animals and found that RS potentiated the expression of Fkbp5 after CFA ( Fig.6D ), supporting the idea that RS exposure had primed the gene for responsiveness. We additionally found that the DNAm levels at cg34791938_TC11 correlated with the change in mechanical thresholds observed following the RS paradigm (cg34791938_TC11: r2 = 0.37, p < 0.05; cg34792474_TC21: r2=0.29, p=0.08, Fig.6E1, E2) but not with Fkbp5 mRNA levels at day 14 ( Fig.6F ). There was no correlation between the Fkbp5 mRNA levels at day 14 and the RS-induced change in mechanical thresholds, suggesting that long-term Fkbp5 mRNA levels after the stress exposure were not driven by the response to RS (Fig.S4). 2.8 Exposure to stress in adulthood leads to epigenetic changes at spinal cord levels often not associated with changes in gene expression The DNAm analysis for our study 1 (RS vs Control) indicated that 11,644 CpGs differed in RS mice compared with control and many (37.99%) occurred in actively transcribed promoter regions (Tss, TssFlnk) within their closest gene in mouse neural tube chromatin segmentation 26 . This included 509 of the 2,525 hypermethylated CpGs and 3,915 of the 9119 hypomethylated CpGs. When the CpGs occurring in promoters were paired against their corresponding RNA expression ( Fig.6B ), we found that 307 unique genes were associated with hypermethylated promoter CpGs and 2780 unique genes had hypomethylated promoter CpGs on day 14 after the RS procedure, when animal behaviour had returned to normal. However, only 1 of the hypermethylated genes ( Plagl1 ) was concurrently down-regulated at day 14, and 11 of the hypomethylated genes ( Acbd6 , Aifm2 , Ccs , Epha7 , Fhod3 , Fkbp5 , Mthfd2 , Pitpnb , Spock2 , Vwc2 and Zwint ) were up-regulated (out of a total of 77 up-regulated genes). Taken together, these results suggested that the RS-induced changes in the epigenomic landscape were more likely to modify gene responsiveness to future challenges, rather than maintaining genes in a persistent up-regulated or down-regulated state. 3 Discussion This study investigated the impact of stress exposure on subsequent inflammatory pain sensitivity using a sub-chronic stress paradigm (1h restraint on 3 consecutive days, RS) followed by injection of the inflammatory agent Complete Freund’s Adjuvant (CFA) into the hindpaw. The results demonstrate that exposure to stress primes both male and female mice for heightened and prolonged inflammatory pain, with a significant role played by the FKBP51 protein in mediating this stress-induced vulnerability to persistent pain, at least in male mice. Stress exposure induced numerous changes in DNAm at spinal cord level, including hypomethylation at a CpG overlapping the TSS of the gene Fkbp5 , but reversal of hypomethylation at this locus was not necessary to prevent the increased vulnerability to persistent pain. Nonetheless, pharmacological inhibition of FKBP51 prevented the priming and reversed a number of DNAm changes identified in regulatory sequences of stress and nociceptive signalling related genes, suggesting that FKBP51 and the activation of the HPA axis was key to the underlying mechanisms. Sex differences in stress-induced increased vulnerability to persistent pain Male and female mice exposed to sub-chronic stress showed mechanical hypersensitivity following stress and exacerbated hypersensitivity following CFA injection compared to non-stressed mice. However, the stress-induced increase in CFA-induced hypersensitivity was greater and longer-lasting in females, indicating a sex-dependent exacerbation of pain following stress. This aligns with existing literature suggesting that females often exhibit greater sensitivity to chronic pain and stress-related disorders 33 – 35 . Moreover, Fkpb5 KO females were less resilient than males to both stress-induced hypersensitivity and stress-induced increased persistent pain vulnerability. This was surprising as FKBP51 is known to drive stress persistent pain equally in both male and female mice 20 – 22 . Overall, our initial findings suggested that FKBP51 was likely to drive the increased susceptibility to persistent pain in male mice at least. As our statistical analysis did not indicate significant sex x treatment interactions, our study may have been under-powered to identify significant changes in female mice. FKBP51 as a mediator of stress-induced persistent pain vulnerability but not sub-chronic stress induced hypersensitivity To confirm the role of FKBP51 in the increased susceptibility to persistent pain, we used the specific inhibitor SAFit2, as before 20 , 21 . While both male and female Fkbp5 KO mice presented with reduced stress-induced hypersensitivity, pharmacological inhibition of FKBP51 with SAFit2 did not reduce the hypersensitivity induced by this sub-chronic stress exposure. These results were surprising, as SAFit2 can prevent the mechanical hypersensitivity following acute prolonged stress exposure in a rodent model of Post-Traumatic Stress Disorder 22 . In this paradigm, the stress exposure lasts 2h and does not re-occur on multiple days as in our paradigm. Our data therefore suggest that inhibition of FKBP51 alone cannot prevent the decrease in threshold induced by sub-chronic stress. Nonetheless, FKBP51 inhibition was sufficient to prevent the stress-induced exacerbation of the subsequent CFA-induced pain state. FKBP51 and Stress Signaling Stress exposure led to an increase in blood serum glucocorticoids. This was accompanied by an increase in spinal Fkbp5 mRNA and a decrease in spinal Nr3c1 mRNA, the gene encoding the glucocorticoid receptor (GR), 48h after the last exposure to stress. While changes in expression of stress-activated genes occur within minutes of stress exposure following the orchestrated release of numerous stress mediators 36–38 , we chose a later time point for our analysis of gene expression. This is because we were interested in the link with the persistent pain phenotype and had previously reported that Fkbp5 mRNA was elevated for at least up to 48h after the initiation of persistent pain states 20 , 24 . We had found that Nr3c1 mRNA was upregulated at this time point, while here we report a downregulation following stress exposure, suggesting differences in the regulation of the HPA axis between sub-chronic stress exposure and physical injury. The observations from the current study fit with previous reports of reduced GR expression in humans following NR3C1 increased DNAm in the promoter region upon substantial stress exposure 39 – 43 . We also report an increase in Nr3c1 DNAm at cg35635943_BC11 within 200 bp of TSS (-192 bases) following stress exposure, suggesting that this was likely to drive the early downregulation in Nr3c1 gene expression observed at day 5. Nr3c1 mRNA was no longer downregulated at the time point of the DNAm analysis, suggesting that the stress-induced change in epigenome was longer lasting than the change in gene expression. Crucially, the FKBP51 inhibition that prevented the primed state did not reverse the stress-induced change in Nr3c1 or in Fkbp5 DNAm. Stress exposure also led to an increase in CFA-induced cFos in Laminae I-II of the superficial dorsal horn, indicating enhanced nociceptive signalling. While FKBP51 inhibition prevented the RS-induced priming, it did not have any effect on cFos expression, suggesting that prevention of early cFos expression is not required to prevent the prolongation of persistent pain states. This aligns with our previous findings, showing that Fkbp5 KO mice have reduced mechanical hypersensitivity in persistent pain states but similar levels of spinal cFos expression to wild-type mice 2h after pain state induction 20 . These observations also confirmed the lack of correlation between cFos expression and pain behaviour reported by others 44 . It also suggests that Fkbp5 deletion and pharmacological inhibition does not reduce primary afferent input into the superficial dorsal horn following noxious stimulation, which confirms our hypothesis that FKBP51 drives persistent pain at the level of the central nervous system 20 . Fkbp5 DNAm and chronic pain vulnerability We and others have shown that early life trauma can modify Fkbp5 DNA methylation landscape. In particular, we reported that early life trauma could reduce Fkbp5 DNA methylation at spinal cord level, where FKBP51 has been shown to drive persistent pain states. Here, we report that exposure to sub-chronic stress in adulthood also leads to a reduction in DNAm in Fkbp5 (cg34791938_TC11; Chr17:28486463, -313 bases from TSS) at spinal cord level. This is an important observation, as it is currently unclear whether stress may have the same impact in young people and adults, with studies supporting the idea that adults may be more resilient to intense stress exposure 17 . It was also crucial for our hypothesis to demonstrate that stress exposure in adulthood can lead to DNAm changes at spinal cord level, a tissue rarely investigated in human studies. The nominal change in DNA methylation we report seems to prime Fkbp5 for hyper-responsiveness to subsequent challenges, as stressed mice had a higher level of Fkbp5 mRNA after CFA injection when compared with mice receiving only CFA and mice that were only stressed. Together, these observations would suggest that exposure to stress primes for chronic pain vulnerability through de-methylation of Fkbp5 . However, findings from this study also suggest that de-methylation of Fkbp5 alone is not sufficient to promote vulnerability, as inhibition of FKBP51 during the stress exposure period prevented the priming but not the change in DNAm in Fkbp5 . Nonetheless, FKBP51 inhibition with SAFit2 did reverse a number of stress-induced changes DNAm ( Table 2 ), notably in the pro-nociceptive genes Rtn4, Cdk5, Nrxn1 and Gli2 . Epigenetic changes and long-term pain vulnerability Our experiments suggest that stress exposure leads to more long-lasting changes to DNAm than mRNA expression levels. Indeed, we report 4,424 DMPs within promoter active chromatin overlapping 3,590 unique genes (3,087 of these available in corresponding RNA sequencing data), 14 days after stress exposure. At this time point, differences in gene expression between stress exposed and control mice were very few (n=230, with Fold-change > 1.2 and nominal p value < 0.05). These results suggested that the expression of a significant number of genes was not impacted by the changes to the DNAm landscape alone, even when potential cell heterogeneity effects were considered, and that they may have been primed by the sub-chronic stress exposure. This aligns with the observations that DNA methylation, chromatin dynamics and transcription factor occupancy work on differing time scales 45 and that DNA methylation may contribute to the stability of transcriptional regulation 46 . On the other hand, our findings support the idea that DNA methylation profiling may offer valuable insights into various biological processes and have the potential to reveal clinically relevant information, as methylation profiles may reflect not only the activity of transcription factors but also serve as potential biomarkers for disease states. Recent studies have demonstrated that even non-affected tissue can yield informative DNA methylation profiles, aiding in treatment decisions, clinical cohort stratification, and potentially guiding personalized medicine 47 . Large-scale cohort studies promise to deepen our understanding of how specific DNA methylation patterns relate to disease phenotypes, enhancing the potential for methylation to be used in disease prognosis 46 . Conclusion: implications for chronic pain and stress disorders Our findings indicate that sub-chronic stress primes both male and female mice for prolonged inflammatory pain, with FKBP51 proving crucial to this vulnerability, particularly in male mice. The molecular insights gained, including DNAm changes in pain-related regulatory gene sequences at spinal cord level, underscore FKBP51’s potential as a therapeutic target for chronic pain in humans. 4 Material and Methods Extended methods can be found in the supplementary file. 4.1 Animals ARRIVE guidelines were followed. Wild-type (WT) experiments were performed using 8–10-week-old C57BL/6 mice of both sexes (N=146; Charles River, UK). Male and female Fkbp5 +/- mice (N=16) and their WT littermates (N=14) were bred in house and genotyped according to Maiaru et al. 20 . Mice were housed in individually ventilated home cages in a temperature-controlled environment with a 12-hour light-dark cycle, with food and water available ad libitum. All procedures were carried out in accordance with the United Kingdom Animal Scientific Procedures Act 1986. 4.2 Sub Chronic Stress (restraint stress) Mice were restrained for 1 hour/day for three consecutive days in 50ml falcon tubes, adapted to allow ventilation. Restraint onset was standardised to 10am to control natural fluctuations in circulating CORT 48 that occur through the day 49 . Where possible, restrained mice were separated from controls to prevent social transmission of stress through odour 50 . 4.3 Models of inflammation Acute hind paw inflammation was induced by injection of the pro-inflammatory cytokine interleukin 6 (IL-6; Sigma), while a longer lasting inflammatory pain state was induced by intra-plantar administration of Complete Freund’s Adjuvant (CFA; Sigma). Briefly, either 25µl of 0.1ng IL6 or undiluted CFA, was injected subcutaneously into the plantar surface of the left hind paw, under light anaesthetic (2% isoflurane in oxygen), in line with previous work from the lab 20 . 4.4 Drugs The FKBP51 inhibitor, SAFit2, was synthesised as previously described 51 . The drug was used at a concentration of 200mg/kg, dissolved in two vehicles (see supplementary for vehicle and delivery methods) 21 . SAFit2-VPG was administered two days prior to the first restraint. According to previous experiments in the lab, the gel formulation can provide slow drug release lasting up to 7 days, thus SAFit2-VPG administration supplied FKBP51 inhibition for the duration of the restraint 21 , 52 . Control mice received injection of the vehicle formulation only. Drug group was blinded by a member of the lab. 4.5 Mechanical withdrawal thresholds Hind paw mechanical withdrawal thresholds were assessed using Von Frey fibres applied to the plantar region of the left hind paw via the up-down method 53 , 54 ., at a starting force of 0.6g (Ugo Basile SRL). Mice were habituated for 45 minutes before testing and measures were completed at the same time each day. Thresholds have been log-transformed in accordance with Mills et al. 55 , to account for the logarithmic distribution of fibres. 4.6 Elevated plus Maze (EPM) The elevated plus maze 56–58 was used to measure anxiety-like behaviour. Mice were placed on the centre of the EPM (Ugo Basile SRL), and exploratory behaviour was recorded and tracked for 5 minutes using EthoVision XT14 software (Noldus Information Technology). Open arm time was chosen as a proxy measure for anxiety-like behaviours and was normalised to controls for all experiments, including analysis across time. 4.7 Immunohistochemistry Mice were euthanised with an overdose of pentobarbital (Dolethal; Vetoquinol) and perfused transcardially with heparinised saline (5000IU/ml) followed by 4% paraformaldehyde (PFA). Brain and spinal cord tissue was dissected and post-fixed in 4% PFA for 2 hours, transferred to 30% sucrose and kept at 4°C until ready for sectioning. For c-fos immunostaining, free floating spinal cord sections of 40µm were blocked in 3% normal goat serum and incubated in anti-c-fos (1:5000; Synaptic Systems) for three nights. After staining slides were imaged using the Zeiss Axioscan slide scanner and cFos expressing cells were counted using Image J. 4.8 RT-qPCR gene expression analysis Fresh tissue was collected following euthanasia with CO 2. For the RS tissue experiments, mouse spinal cords were dissected into quadrants and the 2 dorsal quadrants were pooled for analysis. Cords of intra-plantar CFA injected mice were separated into ipsilateral and contralateral quadrants to site of injury and ipsilateral dorsal quadrants were processed. Total RNA was extracted using an acid phenol extraction method and processed using the RNeasy mini kit (Qiagen). 500ng of total RNA was reverse transcribed to cDNA via a two-step method, described in detail in the supplementary methods. cDNA was stored at -20°C until further processing. RT-qPCR reactions were run using the DNA Engine and the SYBR Green JumpStart Taq Ready Mix (Sigma), in the standard 3-step SYBR green heat cycle. Primer sequences for gene targets are listed in Table S3. The ratio of the relative expression of target genes to HGPRT expression was calculated using the 2ΔCt formula (Further details in supplementary methods). 4.9 ELISA Blood CORT levels End of life blood was collected and processed, and circulating levels of corticosterone were measured using an ELISA kit (Abcam) as before 20 . 4.10 Statistical Analysis of Behavioural and Molecular Data Statistical analyses were performed using Graph Pad Prism (Version 9.5) and SPSS IBM SPSS Statistics (Version 29). Analysis was performed with either T-tests, Analysis of Variance (ANOVA) (repeated measures when appropriate) or via Simple Linear Regression. Post hoc analysis was dependent on the test employed and performed by either Tukey’s multiple comparison test (2-way ANOVA), Holm-Sidak’s multiple comparison test (1-way ANOVA), or Sidak’s multiple comparison test (2-way ANOVA) as appropriate. Significance level was set at p<0.05. Data is presented as mean ±SEM, with or without single data points, as appropriate. 4.11 RNA and DNA extraction/library preps DNA and RNA was extracted from the same lumbar spinal cord samples (L4 to L6) quadrants using the Qiagen All Prep DNA/RNA/miRNA kit as per manufacturer’s instructions. DNA and RNA were quantified using a Nanodrop 8000 spectrophotometer. RNA integrity was measured using the Agilent 2100 Bioanalyser. All samples passed quality control with RIN scores >7. mRNA libraries were prepared from total RNA using NEBNext Ultra II Directional Library Preparation Kit. mRNA fragmentation and first strand cDNA synthesis were performed according to manufacturer’s recommendations for an insert size of 300bp (94°C for 10 minutes) and amplified for 13 cycles of PCR. Resulting libraries were quantified using the Qubit 2.0 spectrophotometer and average fragment size assessed using the Agilent 2200 Tapestation. A final sequencing pool was created using equimolar quantities of each sample library. 75bp paired-end reads were generated for each library using a NextSeq®500 in conjunction with the NextSeq®500 v2 High-output 150-cycle kit (Illumina). DNA samples were assessed for integrity using the Agilent Genomic DNA ScreenTape and reagents. Bisulphite conversion for DNAm analysis was performed using the Zymo EZ-DNA Methylation Kit using 500ng of gDNA as input. 4.12 RNA sequencing FASTQ sequencing reads were assessed for quality scores (QS) and trimmed using a sliding window operation (average QS > 20). Transcripts per million of mouse mm10 reference alignment features (n = 27179) were normalised using the trimmed mean of M-values (median library size of 15.6 million counts) and filtered by expression among each group ≥10 counts. Differential gene expression was assessed by linear modelling in RStudio (>version 4.3.1) using cell type composition estimates of each RNA sample as covariates, established by querying analytically robust RNA count matrices against the mouse single-cell atlas 59 ). Bulk RNA most closely resembled 4 single cell types: myelinating oligodendrocytes, oligodendrocyte precursor cells, neurones and astrocytes (all with r > 0.6). There was no apparent difference between RS and control mice in cell types (Fig.S3). Genes exceeding a 1.2-fold change in expression and nominal p values < 0.05 were considered differentially expressed among groups. 4.13 DNA methylation analysis DNA methylation was assessed using Infinium Mouse Methylation (285k) BeadChip (Illumina, USA) processed via standard protocols at the QMUL Genome Centre, Blizard Institute, Queen Mary University, London, UK. Bisulfite converted DNA was hybridised to the array and read via the Illumina iScan using the manufacturer’s standard protocol to generate red/green channel idat files. Bisulfite conversion success was confirmed prior to analysis using the mean signal intensities of CpCs over TpCs in the green channel i.e., GCT score (Green CpC to TpC) > 1 46 . Data pre-processing Probes on the sex chromosomes X and Y, and those previously identified as cross reactive,with polymorphic CpGs, or common SNVs under probe binding sites 60 , were removed. Poor samples were detected and removed if >=10% of probes possessed 0.01). Probes with poor detection P values (>0.01 in >=10% samples), and bead counts (=10% samples) were eliminated. Dye and probe type biases were normalised using the subset-quantile within array method and DNA methylation status (β) calculated for all remaining probes (n=192917 and n=191134 probes in RS vs control and SAFit2 vs vehicle, respectively). Differential DNA methylation was performed using linear modelling on log2 transformed β value ratios (M-value) 61 using estimates of cell type composition established from bulk RNA sequencing data from the same samples and surrogate variables established using SVA [PMID:17907809] as covariates. Differentially methylated probes (DMPs) with nominal significance of p < 0.05 were advanced for additional exploratory analysis using GREAT v4.0.4 27 . Default GREAT (Mouse: GRCm38) association parameters (basal proximal 5kb upstream and 1kb downstream, plus distal extension <=1Mb) were used with the location of the post-QC mouse array CpGs uploaded as the background regions set for comparisons. CpGs were annotated using the mouse 285k (mm10) manifest file and according to mouse neural tube (E15.5) chromatin segmentation data using the Know Your CG knowledgebase available through SeSAMe 26 , 60 . 4.14 Intersection between DNAm and RNAseq All cited mouse genomic co-ordinates are build GRCm38 (mm10). CpGs occurring in active promoter-like chromatin states (Tss, TssFlnk) in neural tube tissue were grouped by gene derived from the mouse 285k (mm10) manifest file and the average fold-change calculated from the differential methylation analysis. These were mapped to the average fold-change in corresponding RNA count matrices derived from the same samples 26 , 60 . Study approval All experiments were carried out under the Home Office License P8F6ECC28, ARRIVE guidelines were followed, and all efforts were made to minimize animal suffering and to reduce the number of animals used (UK Animal Act, 1986) Data availability Data generated in this study will be made available upon completion of further analyses. Once fully processed and validated, the datasets will be accessible upon reasonable request from the corresponding authors. Author contributions Study conception and experimental designs: OM and SMG. Data collection: All behavioural studies were done by OM; SMG contributed to tissue collection; TS prepared the SAFit2-VPG. EW and CAM ran the sequencing and DNA methylation arrays. Data analysis and interpretation: OM and SMG analysed and interpreted all data. SS ran all RNA sequencing and DNA methylome analysis guided by CGB. SH provided critical insights throughout the study. Manuscript preparation : OM, SS and SG wrote the initial draft of the manuscript. CGB and SH provided critical comments and revisions on the draft and all authors read and approved the final manuscript. Funding his project was funded by a Brain Research UK PhD studentship to OM. SS is a member of the Advanced Pain Discovery Platform and supported by a UKRI and Versus Arthritis grant (MR/W002566/1). Competing interests The authors have no competing interests related to the results or compounds in this work. Acknowledgments The authors thank Greg Dussor, UT Dallas, for discussion on the RS model. References 1. ↵ Chrousos , G. P . Stress and disorders of the stress system . Nat Rev Endocrinol 5 , 374 – 381 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ McEwen , B. S . Physiology and neurobiology of stress and adaptation: central role of the brain . Physiol Rev 87 , 873 – 904 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 3. ↵ Woda , A. , Picard , P. & Dutheil , F . Dysfunctional stress responses in chronic pain . Psychoneuroendocrinology 71 , 127 – 135 ( 2016 ). OpenUrl CrossRef PubMed 4. Myers , B. & Ulrich-Lai , Y. M . The impact of psychological stress on cardiovascular function and health . Physiology & Behavior 172 , 1 – 2 ( 2017 ). OpenUrl CrossRef PubMed 5. ↵ Ross , R. A. , Foster , S. L. & Ionescu , D. F . The Role of Chronic Stress in Anxious Depression . Chronic Stress 1 , 2470547016689472 ( 2017 ). 6. ↵ Butler , R. K. & Finn , D. P. Stress-induced analgesia . Prog Neurobiol 88 , 184 – 202 ( 2009 ). OpenUrl CrossRef PubMed 7. ↵ Olango , W. M. & Finn , D. P. Neurobiology of Stress-Induced Hyperalgesia. in Behavioral Neurobiology of Chronic Pain (eds. Taylor , B. K. & Finn , D. P. ) vol. 20 251 – 280 ( Springer Berlin Heidelberg, Berlin, Heidelberg , 2014 ). 8. ↵ Reichling , D. B. & Levine , J. D . Critical role of nociceptor plasticity in chronic pain . Trends Neurosci . 32 , 611 – 618 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 9. ↵ Vachon-Presseau , E. et al. The Emotional Brain as a Predictor and Amplifier of Chronic Pain . Journal of Dental Research 95 , 605 – 612 ( 2016 ). OpenUrl CrossRef PubMed 10. ↵ Singaravelu , S. K. et al. Persistent muscle hyperalgesia after adolescent stress is exacerbated by a mild-nociceptive input in adulthood and is associated with microglia activation . Sci Rep 12 , 18324 ( 2022 ). 11. ↵ Touma , C. et al. FK506 binding protein 5 shapes stress responsiveness: modulation of neuroendocrine reactivity and coping behavior . Biol. Psychiatry 70 , 928 – 936 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 12. ↵ Fries , G. , Gassen , N. & Rein , T . The FKBP51 Glucocorticoid Receptor Co-Chaperone: Regulation , Function, and Implications in Health and Disease. IJMS 18 , 2614 ( 2017 ). OpenUrl PubMed 13. ↵ Binder , E. B . The role of FKBP5, a co-chaperone of the glucocorticoid receptor in the pathogenesis and therapy of affective and anxiety disorders . Psychoneuroendocrinology 34 Suppl 1 , S186 – 195 ( 2009 ). OpenUrl CrossRef PubMed 14. ↵ Klengel , T. & Binder , E. B . FKBP5 Allele-Specific Epigenetic Modification in Gene by Environment Interaction . Neuropsychopharmacology 40 , 244 – 246 ( 2015 ). OpenUrl CrossRef PubMed 15. Zannas , A. S. , Wiechmann , T. , Gassen , N. C. & Binder , E. B . Gene–Stress–Epigenetic Regulation of FKBP5: Clinical and Translational Implications . Neuropsychopharmacology 41 , 261 – 274 ( 2016 ). OpenUrl CrossRef PubMed 16. ↵ Matosin , N. , Halldorsdottir , T. & Binder , E. B . Understanding the Molecular Mechanisms Underpinning Gene by Environment Interactions in Psychiatric Disorders: The FKBP5 Model . Biol Psychiatry 83 , 821 – 830 ( 2018 ). OpenUrl CrossRef PubMed 17. ↵ Klengel , T. et al. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions . Nat Neurosci 16 , 33 – 41 ( 2013 ). OpenUrl CrossRef PubMed 18. ↵ Nabais , M. F. et al. Meta-analysis of genome-wide DNA methylation identifies shared associations across neurodegenerative disorders . Genome Biol 22 , 90 ( 2021 ). 19. ↵ Bernabeu , E. et al. Refining epigenetic prediction of chronological and biological age . Genome Med 15 , 12 ( 2023 ). 20. ↵ Maiarù , M. et al. The stress regulator FKBP51 drives chronic pain by modulating spinal glucocorticoid signaling . Sci. Transl. Med . 8 , ( 2016 ). 21. ↵ Maiarù , M. et al. The stress regulator FKBP51: a novel and promising druggable target for the treatment of persistent pain states across sexes . Pain 159 , 1224 – 1234 ( 2018 ). OpenUrl CrossRef PubMed 22. ↵ Wanstrath , B. J. et al. Duration of Reduction in Enduring Stress-Induced Hyperalgesia Via FKBP51 Inhibition Depends on Timing of Administration Relative to Traumatic Stress Exposure . J Pain 23 , 1256 – 1267 ( 2022 ). OpenUrl CrossRef PubMed 23. ↵ Maiarù , M. , et al. Using the DNA Methylation Profile of the Stress Driver Gene FKBP5 for Chronic Pain Diagnosis . http://biorxiv.org/lookup/doi/10.1101/2022.12.22.521573 ( 2022 ) doi: 10.1101/2022.12.22.521573 . OpenUrl Abstract / FREE Full Text 24. ↵ Geranton , S. M. , Morenilla-Palao , C. & Hunt , S. P . A Role for Transcriptional Repressor Methyl-CpG-Binding Protein 2 and Plasticity-Related Gene Serum- and Glucocorticoid-Inducible Kinase 1 in the Induction of Inflammatory Pain States . Journal of Neuroscience 27 , 6163 – 6173 ( 2007 ). OpenUrl Abstract / FREE Full Text 25. ↵ Lewis-Tuffin , L. J. & Cidlowski , J. A . The physiology of human glucocorticoid receptor beta (hGRbeta) and glucocorticoid resistance . Ann. N. Y. Acad. Sci . 1069 , 1 – 9 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 26. ↵ van der Velde , A. et al. Annotation of chromatin states in 66 complete mouse epigenomes during development . Commun Biol 4 , 239 ( 2021 ). 27. ↵ McLean , C. Y. et al. GREAT improves functional interpretation of cis-regulatory regions . Nat Biotechnol 28 , 495 – 501 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 28. ↵ Hu , F. et al. Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron . The FASEB Journal 33 , 668 – 682 ( 2019 ). OpenUrl CrossRef PubMed 29. ↵ Gomez , K. et al. The role of cyclin-dependent kinase 5 in neuropathic pain . Pain 161 , 2674 – 2689 ( 2020 ). OpenUrl CrossRef PubMed 30. ↵ Taylor , C. P. & Harris , E. W . Analgesia with Gabapentin and Pregabalin May Involve N -Methyl-d-Aspartate Receptors, Neurexins, and Thrombospondins . J Pharmacol Exp Ther 374 , 161 – 174 ( 2020 ). OpenUrl Abstract / FREE Full Text 31. ↵ Meng , L. , Zhang , Y. , He , X. & Hu , C . LncRNA H19 modulates neuropathic pain through miR-141/GLI2 axis in chronic constriction injury (CCI) rats . Transplant Immunology 71 , 101526 ( 2022 ). 32. ↵ Hunyady , Á. et al. Hemokinin-1 is an important mediator of pain in mouse models of neuropathic and inflammatory mechanisms . Brain Res Bull 147 , 165 – 173 ( 2019 ). OpenUrl CrossRef PubMed 33. ↵ Fillingim , R. B . Sex, gender, and pain: women and men really are different . Curr Rev Pain 4 , 24 – 30 ( 2000 ). OpenUrl CrossRef PubMed 34. Fillingim , R. B . Individual differences in pain: understanding the mosaic that makes pain personal . Pain 158 Suppl 1 , S11 – S18 ( 2017 ). OpenUrl CrossRef PubMed 35. ↵ Casale , R. et al. Pain in Women: A Perspective Review on a Relevant Clinical Issue that Deserves Prioritization . Pain Ther 10 , 287 – 314 ( 2021 ). OpenUrl CrossRef PubMed 36. Droste , S. K. et al. Corticosterone Levels in the Brain Show a Distinct Ultradian Rhythm but a Delayed Response to Forced Swim Stress . Endocrinology 149 , 3244 – 3253 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 37. Roszkowski , M. et al. Rapid stress-induced transcriptomic changes in the brain depend on beta-adrenergic signaling . Neuropharmacology 107 , 329 – 338 ( 2016 ). OpenUrl CrossRef PubMed 38. McKibben , L. A. et al. Transcriptional changes across tissue and time provide molecular insights into a therapeutic window of opportunity following traumatic stress exposure. Preprint at doi: 10.1101/2024.11.01.621484 ( 2024 ). OpenUrl Abstract / FREE Full Text 39. ↵ McGowan , P. O. et al. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse . Nat. Neurosci . 12 , 342 – 348 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 40. Van Der Knaap , L. J. et al. Glucocorticoid receptor gene (NR3C1) methylation following stressful events between birth and adolescence. The TRAILS study . Transl Psychiatry 4 , e381 – e381 ( 2014 ). OpenUrl CrossRef 41. Palma-Gudiel , H. , Córdova-Palomera , A. , Leza , J. C. & Fañanás , L . Glucocorticoid receptor gene (NR3C1) methylation processes as mediators of early adversity in stress-related disorders causality: A critical review . Neurosci Biobehav Rev 55 , 520 – 535 ( 2015 ). OpenUrl CrossRef PubMed 42. van der Knaap , L. J. , Oldehinkel , A. J. , Verhulst , F. C. , van Oort , F. V. A. & Riese , H . Glucocorticoid receptor gene methylation and HPA-axis regulation in adolescents. The TRAILS study . Psychoneuroendocrinology 58 , 46 – 50 ( 2015 ). OpenUrl CrossRef PubMed 43. ↵ Mourtzi , N. , Sertedaki , A. & Charmandari , E . Glucocorticoid Signaling and Epigenetic Alterations in Stress-Related Disorders . Int J Mol Sci 22 , 5964 ( 2021 ). OpenUrl CrossRef PubMed 44. ↵ Gao , Y.-J. & Ji , R . -R. c-Fos or pERK, Which is a Better Marker for Neuronal Activation and Central Sensitization After Noxious Stimulation and Tissue Injury? TOPAINJ 2 , 11 – 17 ( 2009 ). OpenUrl CrossRef 45. ↵ Guerin , L. N. et al. Temporally discordant chromatin accessibility and DNA demethylation define short and long-term enhancer regulation during cell fate specification. Preprint at doi: 10.1101/2024.08.27.609789 ( 2024 ). OpenUrl Abstract / FREE Full Text 46. ↵ Schübeler , D . Function and information content of DNA methylation . Nature 517 , 321 – 326 ( 2015 ). OpenUrl CrossRef PubMed Web of Science 47. ↵ Yousefi , P. D. et al. DNA methylation-based predictors of health: applications and statistical considerations . Nat Rev Genet 23 , 369 – 383 ( 2022 ). OpenUrl CrossRef PubMed 48. ↵ Bartlang , M. S. et al. Time matters: pathological effects of repeated psychosocial stress during the active, but not inactive, phase of male mice . J Endocrinol 215 , 425 – 437 ( 2012 ). OpenUrl Abstract / FREE Full Text 49. ↵ D’Agostino , J. , Vaeth , G. F. & Henning , S. J . Diurnal rhythm of total and free concentrations of serum corticosterone in the rat . Acta Endocrinol (Copenh ) 100 , 85 – 90 ( 1982 ). OpenUrl Abstract / FREE Full Text 50. ↵ Sterley , T.-L. et al. Social transmission and buffering of synaptic changes after stress . Nat Neurosci 21 , 393 – 403 ( 2018 ). OpenUrl CrossRef PubMed 51. ↵ Gaali , S. et al. Selective inhibitors of the FK506-binding protein 51 by induced fit . Nat. Chem. Biol . 11 , 33 – 37 ( 2015 ). OpenUrl CrossRef PubMed 52. ↵ Buffa , V. et al. Analysis of the Selective Antagonist SAFit2 as a Chemical Probe for the FK506-Binding Protein 51 . ACS Pharmacol Transl Sci 6 , 361 – 371 ( 2023 ). OpenUrl CrossRef PubMed 53. ↵ Dixon , W. J . Efficient Analysis of Experimental Observations . Annu. Rev. Pharmacol. Toxicol . 20 , 441 – 462 ( 1980 ). OpenUrl CrossRef PubMed Web of Science 54. ↵ Chaplan , S. R. , Bach , F. W. , Pogrel , J. W. , Chung , J. M. & Yaksh , T. L . Quantitative assessment of tactile allodynia in the rat paw . J. Neurosci. Methods 53 , 55 – 63 ( 1994 ). OpenUrl CrossRef PubMed Web of Science 55. ↵ Mills , C. et al. Estimating Efficacy and Drug ED50’s Using von Frey Thresholds: Impact of Weber’s Law and Log Transformation . The Journal of Pain 13 , 519 – 523 ( 2012 ). OpenUrl CrossRef PubMed 56. Handley , S. L. & Mithani , S . Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of ’fear’-motivated behaviour . Naunyn Schmiedebergs Arch Pharmacol 327 , 1 – 5 ( 1984 ). OpenUrl CrossRef PubMed Web of Science 57. Pellow , S. , Chopin , P. , File , S. E. & Briley , M . Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat . J Neurosci Methods 14 , 149 – 167 ( 1985 ). OpenUrl CrossRef PubMed Web of Science 58. Hestehave , S. , et al. Predicting Hypersensitivity and Comorbid Depressive-like Behavior in Late Stages of Joint Disease Using Early Weight Bearing Deficit . http://biorxiv.org/lookup/doi/10.1101/2023.11.29.569246( 2023 ) doi: 10.1101/2023.11.29.569246 . OpenUrl Abstract / FREE Full Text 59. ↵ Han , X. et al. Mapping the Mouse Cell Atlas by Microwell-Seq . Cell 172 , 1091 – 1107 .e17 ( 2018 ). OpenUrl CrossRef PubMed 60. ↵ Zhou , W. et al. DNA methylation dynamics and dysregulation delineated by high-throughput profiling in the mouse . Cell Genom 2 , 100144 ( 2022 ). 61. ↵ Du , P. et al. Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis . BMC Bioinformatics 11 , 587 ( 2010 ). View the discussion thread. Back to top Previous Next Posted January 07, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability Oakley B. Morgan , Samuel Singleton , Sara Hestehave , Tim Sarter , Eva Wozniak , Charles A Mein , Felix Hausch , Christopher G. Bell , Sandrine M. Géranton bioRxiv 2025.01.07.631709; doi: https://doi.org/10.1101/2025.01.07.631709 Share This Article: Copy Citation Tools Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability Oakley B. Morgan , Samuel Singleton , Sara Hestehave , Tim Sarter , Eva Wozniak , Charles A Mein , Felix Hausch , Christopher G. Bell , Sandrine M. Géranton bioRxiv 2025.01.07.631709; doi: https://doi.org/10.1101/2025.01.07.631709 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 (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41970) Biophysics (21463) Cancer Biology (18605) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19911) Epidemiology (2067) Evolutionary Biology (24329) Genetics (15615) Genomics (22514) Immunology (17743) Microbiology (40424) Molecular Biology (17194) Neuroscience (88650) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4827) Physiology (7648) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)