Role of the locus coeruleus noradrenergic system in susceptibility and resilience following early life stress in male and female mice

Role of the locus coeruleus noradrenergic system in susceptibility and resilience following early life stress in male and female mice | 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 Role of the locus coeruleus noradrenergic system in susceptibility and resilience following early life stress in male and female mice View ORCID Profile Dea Slavova , Valentine Greffion , Lionel Granjon , Stéphanie De-Gois , Maud Blaise , View ORCID Profile Bruno Giros , Elsa Isngrini doi: https://doi.org/10.1101/2025.10.11.681820 Dea Slavova 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dea Slavova Valentine Greffion 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lionel Granjon 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stéphanie De-Gois 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maud Blaise 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bruno Giros 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France 2 Department of Psychiatry, McGill University , Montreal, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bruno Giros Elsa Isngrini 1 Université Paris Cité , INCC UMR 8002, CNRS, F-75006, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Isingrini.elsa{at}parisdescartes.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background Child adversity (CA), encompassing emotional, physical, and sexual maltreatment or abuse, affects a substantial number of children worldwide. Moreover, it is the leading predictor of psychiatric disorders such as major depressive disorder (MDD), anxiety, and suicidal behavior. Despite the robust link between CA and psychopathology, individual outcomes vary significantly, with some children demonstrating resilience. Resilience is an adaptive and dynamic process, which mitigates the long-term effects of CA, suggesting potential protective mechanisms that remain underexplored. This study investigates the role of the locus coeruleus-norepinephrine (LC-NE) system, a critical modulator of stress, cognition, and emotion, in mediating resilience and susceptibility following early life stress (ELS). Methods Using a maternal deprivation model combined with limited nesting and bedding, we examined behavioral, physiological, and neurobiological markers associated with ELS outcomes in mice of both sex. Results Behavioral clustering revealed distinct phenotypes: resilient, anxious, and depressive-like with sex-specific differences in distribution. Early markers, including body weight and ultrasonic vocalization (USV) patterns, predicted long-term susceptibility. Neuroanatomical analyses identified sex-specific LC-NE activation patterns associated with resilience and susceptibility, highlighting the caudal-dorsal LC as a critical region in males and females in different phenotypes, anxious in males and resilient in females. Conclusion These findings highlight the impact of ELS on the LC-NE system and its role in shaping adaptive and maladaptive trajectories, offering insights into potential interventions targeting resilience mechanisms in children exposed to CA. Introduction Child adversity (CA), encompassing emotional, physical, sexual abuse and neglect, affects around 25% of children worldwide, with neglect accounting for nearly 80% of cases ( 1 , 2 ). CA is the strongest predictors of psychiatric disorders, particularly major depressive disorder, anxiety, and suicidal behavior ( 3 – 8 ). In the U.S., maltreatment-related adverse childhood experiences contribute to 54% and 67% of the population attributable risk for depression and suicide attempts, respectively ( 9 ). CA is also associated with earlier onset, greater severity, and treatment resistance in depression ( 10 – 13 ). In anxiety, CA worsen outcomes, particularly by exacerbating social anxiety ( 14 , 15 ). However, outcomes vary: many children adapt successfully despite adversity, a process named resilience. Resilience is an active, dynamic adaptation rather than a simple absence of pathology ( 16 – 18 ). From a neurodevelopmental perspective, CA disrupts neurobiological networks involved in threat and reward processing, emotion and cognition ( 19 , 20 ). Imaging studies showed potential effects of CA on the developing brain at the structural, functional and connectivity level in the anterior cingulate cortex, prefrontal areas, and limbic regions such as the striatum, hippocampus, and amygdala ( 21 – 23 ). CA also produces lasting effects on stress-related systems, notably the hypothalamic-pituitary-adrenal (HPA) axis and norepinephrine (NE) pathways ( 24 – 26 ). These changes, persisting into adulthood, may sustain maladaptive coping and psychiatric risk. While much work has focused on vulnerability mechanisms, research on protective factors predicting positive outcomes is more recent ( 27 ). Resilience may be mediated by larger hippocampal volume, increased white matter integrity in the posterior cingulum, and enhanced prefrontal–limbic functional connectivity ( 28 – 32 ). Such findings highlight potential neural substrates of resilience, however, how this network is controlled by stress-integrative structures and upstream neuronal mechanisms remains largely unknown. The locus coeruleus (LC), the largest nucleus producing NE, is projecting throughout the entire brain ( 33 – 35 ). By modulating cortical and limbic regions, the LC-NE system regulates arousal, stress reactivity, cognition, mood, and emotion ( 36 – 38 ). It is among the first neurotransmitter systems to develop: in rodents, formation begins at gestational day 10–13 and continues up to three weeks postnatal ( 39 – 41 ), while in humans, catecholaminergic neurons emerge by five weeks of gestation ( 42 , 43 ). The LC-NE is thus crucial for brain development as it contributes to brain wiring during critical periods, whereby early life changes in NE signalling can permanently alter emotional and cognitive outcomes. For instance, altered gene expression regulating NE transmission during early development leads to long-term behavioral abnormalities in rodents ( 44 ). Moreover, early life stress (ELS) disrupt the LC-NE system both at the anatomical and functional level. Maternal separation or adolescent stress leads to hyper-activation of the LC, elevated NE release in the paraventricular nucleus of the hypothalamus and reduced α 2 -autoreceptors in the LC ( 45 – 47 ). Such changes may have lasting impact on cognitive and emotional functions throughout the lifespan, possibly sustaining psychopathological development. In recent years, the LC-NE system has emerged as a central player in resilience, both in humans ( 48 , 49 ) and animal models of chronic stress in adulthood ( 50 – 52 ). Most insights come from the chronic social defeat stress (CSDS) paradigm ( 53 ), widely used to study resilience ( 51 , 54 , 55 ). In this model, absence of NE neurotransmission promotes susceptibility, while optogenetic stimulation of LC-NE release in the VTA reversed susceptibility to induce resilience ( 50 ). Complementary imaging studies show LC-NE changes in post-traumatic stress disorders (PTSD), where heightened LC activity predicts greater symptom severity ( 56 , 57 ). Despite these evidences, the role of the LC-NE system in mediating resilience against ELS remains unexplored. Given its role in modulating key brain regions involved in both resilience networks and the impact of CA, we hypothesized that the LC-NE system could critically shape long-term outcomes of CA, promoting either psychiatric vulnerability or resilience. The objective of this study was to develop a maternal deprivation model combined with limited nesting and bedding as a paradigm of ELS, enabling segregation of susceptible and resilient mice based on their long-term anxio-depressive phenotypes. We further investigated whether early and late adaptive mechanisms contribute to resilience. Early emotional and physiological predictors were assessed through body weight and ultrasonic vocalization (USV), while long-term outcomes were examined via corticosterone levels and LC-NE neuronal activity in adulthood. Methods and materials Animals The Paris Cité University ethics committee (CEEA 40) approved all procedures, complying with the European Directive 2010/63/EU on animal protection. C57BL/6J mice (Janvier Labs) were housed under standard laboratory conditions (22 ± 1 °C, 60% humidity, 12h light/dark cycle) with food and water ad libitum . 8-week-old breeders were paired (two females/one male). Pregnancy was monitored twice weekly, and males were removed upon confirmation. Pregnant females were single-housed four days before parturition. Early life stress (ELS) paradigm ELS combined maternal deprivation (MD) with limited nesting (½ of standard material) and bedding (1/10 of standard bedding). Birth was designated as P0. From P2 to P14, pups were separated from dams for 3h/day. During separation, pups were placed individually on a heating pad in a different room to prevent communication. From P14 to P21, pups were reared under standard housing conditions. Control (CTL) litters remained undisturbed under standard conditions from P2 to P21. Body weight was measured at P2, P5, P7, P12, and P14, and weekly thereafter. At P21, pups were group-housed by sex and condition. Ultrasonic vocalizations (USV) recording Reflecting pups’ emotional state, USV were recorded at P2, P5, P7 and P12 ( 58 , 59 ). Pups were recorded individually for 5 min at 9:00 a.m. inside a styrofoam box, once daily for CTL and both before and after MD for ELS. For afternoon born litters, P2 recordings were adjusted to ensure age accuracy ( 59 ). Recordings were acquired using an Avisoft UltraSoundGate 116Hb system connected a CM16 microphone (Avisoft, 40011). Parameters were 250 kHz, 1024 FFT length, 16-bit resolution. Gain was adjusted to prevent signal overload. Analyses were performed using Hybrid Mouse software in MATLAB ( 60 ). Maternal Behavior Maternal behavior was assessed at P2, P7, and P14 before and after MD. Cages were habituated 30 min, then video-recorded for 15 min. Maternal behavior was quantified as total time in the nest. Splash Test At P21, dams were tested for stress-related behavior ( 61 ). A 10% sucrose solution was sprayed on the dorsal coat. Grooming was recorded for 5 min in the home cage as a measure of self-care. Anxiety and depressive like behavior in offspring At early adulthood (8–12 weeks), CTL and ELS mice underwent a battery of paradigms in order of increasing stressfulness, between 9:00 a.m. and 1:00 p.m. Males and females were tested on separate days. Three chamber test (3CH) Sociability was assessed using a Plexiglas box (68 × 22 × 24 cm) with two side (28 × 22 × 24 cm) and a central (12 × 22 × 24 cm) chambers ( 62 ). After 10 minutes habituation with empty wire-mesh in both chambers, an age- and sex-matched unfamiliar mouse was placed under one mesh, while the other remained empty. Social interaction was quantified as time spent within a 2-cm radius around each mesh. A social interaction index was calculated: [Time social / (Time social + Time empty)]. Novelty-suppressed feeding test (NSF) The NSF test was conducted in a 45 × 45 × 45 cm open field covered with bedding, after 24h food deprivation. Latency to eat a pellet on a white paper (12.5 cm diameter) placed in the center was recorded, with a maximum cutoff of 5 min. Food consumption was then measured for 3 min in the homecage. Sucrose preference test (SPT) Mice were individually housed overnight (ON) for two habituation days with two bottles of water. From days 3–6, one bottle contained 1% sucrose (SB) and the other water (WB). Bottle positions were alternated daily. Consumption was measured through bottle weight, and mean percentage of sucrose preference across four days calculated: [SB/ (SB+WB) ×100]. Forced swim test (FST) Mice were placed in a glass cylinder (height: 25 cm, diameter: 9 cm) filled with 21–23°C water. Immobility was recorded during 6 min as a measure of resignation. Immobility was defined as floating passively with minimal movements to keep the head above water. Estrous cycle Following behavioral tests (except SPT), vaginal smears were collected with 20µL sterile 0.9% NaCl. Samples, placed on a microscope slide, were stained 1 minute with crystal violet ( 63 ). Stages were identified microscopically by two experimenters. Corticosterone dosage Blood samples (100 µL) were collected from the submandibular vein at baseline (30 min pre-FST), immediately after, and 1h30 post-FST. Samples clotted at room temperature (RT) for 30 min were centrifuged (5,000 rpm, 10 min, 4°C). Collected supernatants was stored at −20°C. Corticosterone levels were measured using the DetectX EIA Kit (Arbor Assays). Results were expressed as mg/µL. Percentage changes were calculated: % Increase = [(Corticosterone post-FST – Corticosterone baseline ) / Corticosterone baseline ] x100 % Decrease = [(Corticosterone 1h30 post-FST – Corticosterone post-FST ) / Corticosterone post-FST ] x100 Tissue preparation and immunohistochemistry 1h30 post-FST, mice were perfused with 0.9% NaCl followed by Antigenfix (Diapath P0014). Brains were post-fixed ON in Antigenfix, stored in 0.1M PBS, embedded in 2% agarose and cut into 40-µm-thick coronal sections (Leica vibratome VT1000E). Free-floating sections were stored at −20°C in cryoprotective solution (30% glycerol, 30% ethylene glycol, 40% 0.1M PBS). Sections were washed in 0.1M PBS, incubated 30 min at RT in 1% NaBH4 and transferred in blocking solution (0.1M PBS, 0.3% Triton X-100, 5% donkey serum) for 30 min at RT. Section were incubated ON at 4°C under agitation with primary antibodies: Guinea pig anti-c-Fos (1:2500, Synaptic Systems 226004) and Mouse anti-TH (1:2000, Sigma-Aldrich MAB318) or Rabbit anti-TH (1:4000, Abcam 117112). After washes, sections were incubated for 2h at RT with the secondary antibodies: Cy3-conjugated donkey anti-guinea pig (1:500, Jackson Laboratory 706-165-148) and Alexa Fluor 488 donkey anti-mouse (1:500, Invitrogen A21202) or Alexa Fluor 488 donkey anti-rabbit (1:4000, Jackson Laboratory 711-546-152). Sections were washed, mounted on superfrost slides, and coverslipped with Fluoromount-G™ mounting medium. Image acquisition and analysis Images were acquired with a Zeiss® LSM710 confocal microscope with a ×20 objective lens. Cell counting was performed manually in ImageJ ( 64 ). TH⁺, c-Fos⁺ and TH⁺/c-Fos⁺ cells were quantified in six sections per mouse across LC subregions. Cell densities were expressed as positive cells/µm². Proportion of activated NE cells was calculated: %(TH + c-Fos + )/TH + = (Number of TH + c-Fos + / Number of TH + ) x 100 Statistical analysis Normality was assessed with Shapiro-Wilk test and homogeneity of variance with Levene’s test. When assumptions were met, Student’s t-test, one- or two-way ANOVA were used, followed by Bonferroni post-hoc tests. If variances were unequal, Welch (W) correction was applied. If normality was violated, Mann-Whitney U (U) test was used. For repeated measures ANOVA, Mauchly’s test assessed sphericity; Greenhouse-Geisser correction was applied when violated. Mahalanobis distance was computed with MATLAB scripts, and k-means clustering with R scripts. Results are expressed as mean ± SEM (standard error of the mean) with significance set at p<0.05. Results ELS impact on maternal behavior and offspring development ( Figure 1A ) Maternal behavior, evaluated by nest time (P2, P7, P14), did not differ between CTL and ELS dams (Condition x Time x Day: F 2,72 =2.88, p=0.063; Figure 1B ). After weaning, corticosterone level (t 13 =1.37, p=0.193) and grooming duration (t 13 =0.23, p=0.818) were also unchanged ( Figure 1C-D ). Download figure Open in new tab Figure 1. Consequences of the maternal deprivation model combined with limiting nesting/bedding on maternal behavior and pups development. A. Timeline of the ELS paradigm applied from P2 to P14, combining maternal deprivation (3h/day) with reduced nesting (1/2) and bedding (1/10) materials. B. Maternal behavior was measured at P2, P7 and P14 before and after the deprivation. For the time spent on the nest, no significant interaction between post-natal day, time and condition was found (F 2,72 =2.88, p=0.063). C. Corticosterone level measured at P21 in dams, 30 minutes before the splash test, show no significant difference between groups (t 13 =1.37, p=0.193). D. Results of the splash test at P21 reveal no significant differences between CTL and ELS dams in total grooming duration (t 13 =0.23, p=0.818). E. The number of pups at birth did not differ significantly between CTL and ELS groups (t ­ =0.637, p=0.532). F. ELS survival rate dropped to 87.5% compared to 100% in CTL. G. The body weight (g) of male (Left) and female (Right) mice was analyzed from P2 to P70. In male, a significant interaction between postnatal day and condition (F ­­ =7.53, p<0.001) was found. Bonferroni post hoc tests revealed that ELS (n=25) had significantly lower weights compared to CTL (n=18) from P7 to P70 (P7: t=2.21, p = 0.034; P12: t=2.30, p=0.027; P14: t=2.44, p=0.02; P21: t=2.25, p=0.031; P28: t=3.20, p=0.003; P35: t=4.94, p<0.001; P42: t=2.91, p=0.006; P49: t=2.23, p=0.032; P56: t=3.11, p=0.004; P63 : t=2.83, p=0.008; P70: t=2.22, p=0.033). In female mice, no interaction was detected between postnatal day and condition (F 2.1,67.2 =0.67, p=0.52); CTL: n= 19, ELS n= 21). H. USVs were measured in both male (Left) and female (Right) pups at P2, P5, P7 and P12 before and after the deprivation in ELS. In males, a significant interaction was found between condition and postnatal day (F 3,195 =3.86, p=0.001). Bonferroni post hoc tests showed that USVs were significantly reduced in ELS compared to CTL at P7 pre- and post-deprivation (Pre: t=2.89, p=0.01; post: t=4.32, p<0.001) and at P12 post-deprivation (t=3.6, p=0.002). In females, a significant interaction between postnatal day and condition was also observed (F 5.03,143.34 =2.399, p=0.04), with a significant reduction in USVs pre-deprivation at P12 (t=2.56, p=0.039). Data are presented as mean ± SEM. Two-way ANOVA was followed by Bonferroni post hoc tests. The Greenhouse-geisser correction was applied if sphericity was violated. Statistical significance is indicated as: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CTL, control; ELS, early-life stress; P, post-natal day; USV, ultrasonic vocalisation. Regarding offspring development, litter size at P2 was similar across groups (t 18 =0.64, p=0.53), but survival dropped from 100% in CTL to 87.50% in ELS ( Figure 1E-F ) . In male, body weight reduction emerged at P7 and persisted until P70 (F 3.4,118.2 =7.53, p<0.001, P7: t=2.21, p=0.034; P12: t=2.30, p=0.027; P14: t=2.44, p=0.02; P21 : t=2.25, p=0.031; P28: t=3.20, p=0.003; P35: t=4.94, p<0.001; P42: t=2.91, p=0.006; P49: t=2.23, p=0.032; P56: t=3.11, p=0.004; P63: t=2.83, p=0.008; P70: t=2.22, p=0.033). Females weight was unaffected (F 2.1,67.2 =0.67, p=0.52; Figure 1G ). USV emissions were reduced in ELS male at P7 pre- and post-deprivation and P12 pre-deprivation (F 3,195 =3.86, p=0.001; P7pre: t=2.89, p=0.01; P7post: t=4.32, p<0.001; P12post: t=3.6, p=0.002). In female, ELS reduced USV at P12 pre-deprivation (F 5.03,143.34 =2.399, p=0.04, P12: t=2.56, p=0.039; Figure 1H ). Long-term ELS behavioral consequences: toward susceptibility and resilience Anxio-depressive paradigms were performed from P56 to P77 ( Figure 2A ). Anxiety-like behavior was assessed with the NSF test, sociability with the 3CH test, and anhedonia with the sucrose preference test. The FST, evaluated resignation, also served as an acute stressor to assess corticosterone response and LC-NE c-Fos activation. To account for cohort effect, data were normalized using z-score, calculated within CTL groups by cohort and sex. All subsequent analysis were conducted using z-score. Download figure Open in new tab Figure 2: Long-term consequences of ELS on behavior in males and females. ( A ) The timeline illustrates the early-life stress paradigm, which consisted of maternal deprivation (3 hours per day) combined with limited nesting (1/2) and bedding (1/10) from postnatal day P2 to P14. USVs were recorded at P2, P5, P7, and P12. Starting at P56, anxio-depressive behaviors were evaluated in male and female mice. At P77, mice were sacrificed 1h30 minutes following the FST. ( B ) In males, no significant differences between CTL and ELS groups were revealed for the social interaction index in the 3CH (t 41 =1.25, p=0.22), the latency to eat in the NSF (U ­ =189, p=0.38), the percentage of sucrose preference (SPT, U ­ =249, p=0.57) and the immobility time in the FST (t 41 =0.32, p=0.75). ( C ) In females, no significant differences between CTL and ELS groups were revealed for the social interaction index in the 3CH (U ­ =187, p=0.97), the latency to eat in the NSF (U ­ =175, p=0.7) and for the time immobile in the FST (t 37 =0.46, p=0.64). However, the percentage of sucrose preference was significantly reduced in ELS group compared to CTL (W 30.387 =3.04, p=0.005). All results are shown as mean +/− SEM. Statistical significance is indicated as follow: *p<0.05, **p<0.01, ***p<0.001. Abbreviations: CTL, control; ELS, early-life stress; FST, Forced swim test; NSF, novelty supressed-feeding test; 3CH, 3-chamber test; SPT, sucrose preference test. No significant differences emerged between CTL and ELS groups in social (3CH M: t 41 =1.25, p=0.22; F: U 37 =187, p=0.97), anxiety-like (NSF, M: U 41 =189, p=0.38; F: U 37 =175, p=0.7), and resignation (FST, M: t 41 =0.32, p=0.75; F: t 37 =0.46, p=0.64) behaviors. However, ELS female displayed increased anhedonia, measured by reduced sucrose preference compared to CTL (W 30.387 =3.04, p=0.005). This effect was absent in male (U 41 =249, p=0.57; Figure 2B-C ) . ELS effects on corticosterone were sex-dependent, with elevated baseline level in male (t 41 =3.3, p=0.002), and no effects in females, while LC-NE functional anatomy was unchanged in both sexes ( Supplementary Figure 1 - 2 ). Download figure Open in new tab Supplementary figure 1: Long-term consequences of ELS on corticosterone level in males and females. ( A ) In males, ELS induced a significant increase in baseline corticosterone level compared to CTL (t 41 =3.3, p=0.002), but had no effect immediately after the FST (post-FST: t ­ =1.06, p=0.30; % increase: U ­ =164, p=0.14) or 1h30 later (1h30 post-FST: U ­ =231, p=0.89; % decrease: U ­ =218, p=0.87). ( B ) In females, corticosterone levels did not differ between ELS and CTL animals at baseline (U 37 =150, p=0.28), post-FST (t 37 =0.8, p=0.43), or 1h30 post-FST (t ­ =–0.66, p=0.52). Similarly, the relative increase (U ­ =238, p=0.17) and subsequent decrease (W 32.13 =–1.16, p=0.25) were unaffected by ELS. Data are presented as mean ± SEM. Student’s t-test was used, with Welch’s correction applied when homogeneity of variance was violated. Mann–Whitney U test was used when normality assumptions were not met. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CTL, control; ELS, early-life stress; FST, forced swim test. Download figure Open in new tab Supplementary Figure 2. Long-term consequences of ELS on LC-NE functional anatomy in males and females. Along the rostro-caudal and dorso-ventral axis of the LC, neither the density of activated LC-NE neurons (TH + c-Fos + /µm²; M: F 2.5,103.1 =0.92, p=0.42; F: F 2.4,90.7 =2.49, p=0.076) nor the proportion of activated LC-NE neurons (TH + c-Fos + /TH + ; M: F 3,123 =1.86, p=0.14; F: F 3,111 =1.83, p=0.15) were significantly altered by ELS compared to CTL, in either males ( A ) or females ( B ). Data are presented as mean ± SEM. Two-way ANOVA followed by Bonferroni’s multiple comparisons test was performed, with Greenhouse–Geisser correction applied when sphericity was violated. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: LC, locus coeruleus; TH, tyrosine hydroxylase; NE, noradrenergic; CTL, control; ELS, early-life stress. Oestrus cycle did not influence social interaction or immobility (3CH: F 1,35 =0.51, p=0.48; FST: F 1,35 =2.05, p=0.16). However, in the NSF, ELS females in P/E showed a tendency for longer latency to eat than CTL (F 1,35 =5.72, p=0.022; P/E: t 35 =−2.51, p=0.1), an effect absent in D/M (t 35 =0.69, p=1; Supplementary Figure 3A ). Although cycle had no effect on corticosterone, D/M females displayed higher LC-NE activation density and proportion than P/E females, regardless of CTL or ELS status ( Supplementary figure 4 ). Download figure Open in new tab Supplementary Figure 3. Impact of the oestrus cycle on behavior in females. ( A ) The oestrus cycle stage (proestrus/estrus, P/E vs diestrus/metestrus, D/M), determined after the 3CH and FST, had no effect on the social interaction index or immobility time in either CTL or ELS groups (3CH: F 1,35 =0.51, p=0.48; FST: F 1,35 =2.05, p=0.16). However, when the cycle stage was determined after the NSF, females in the P/E stage displayed a tendency toward longer latency to eat in the ELS group compared to CTL (F 1,35 =5.72, p=0.022; P/E: t 35 =−2.51, p=0.1), whereas no difference was observed in the D/M stage (t ­ =0.69, p=1). ( B ) The Mahalanobis distance was significantly higher in ELS compared to CTL animals, independently of cycle stage (P/E or D/M) (CTL vs ELS; 3CH: F ­ =19.53, p<0.001; NSF: F ­ =20.1, p<0.001; FST: F ­ =22.06, p<0.001. ( C ) K-means clustering analysis revealed that the distribution of P/E and D/M stages was similar across all clusters when determined after the 3CH. In contrast, when cycle stage was determined after the NSF and FST, there was a trend toward a higher proportion of D/M-stage females in the DEP cluster (proportion tested against 0.5, P/E vs D/M: NSF p = 0.1; FST p = 0.1), while proportions remained similar across CTL, RES, and ANX clusters. Moreover, when comparing CTL and DEP clusters based on cycle stage determined after the FST, a significant difference was observed: 60% of CTL females were in P/E, whereas only 20% of DEP females were in P/E (X 2 =4.37, p=0.037). No such differences were detected when comparing CTL with RES or ANX clusters. Data are presented as mean ± SEM. Contingency tables with chi-square tests were used for comparisons with the CTL group, and binomial tests were applied for P/E vs D/M distributions. Statistical significance: # p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CTL, control; ELS, early-life stress; P/E, proestrus/estrus; D/M, diestrus/metestrus, ANX, anxiety-like cluster; RES, resilient cluster; DEP, depression-like cluster; 3CH, 3-chambers test; NSF, novelty-supressed feeding test; FST, forced swim test. Download figure Open in new tab Supplementary figure 4: Impact of the oestrus cycle in female on corticosterone levels and LC-NE functional anatomy. ( A ) The oestrus cycle stage (proestrus/estrus, P/E vs diestrus/metestrus, D/M), determined after the FST, had no effect on corticosterone levels at baseline (F ­ =0.94, p=0.34), immediately post-FST (F 1,35 =1.09, p=0.30), or 1h30 post-FST (F 1,35 =0.40, p=0.53) in either CTL or ELS groups. However, baseline corticosterone levels were significantly higher in ELS compared to CTL animals, independently of cycle stage (F ­ =6.97, p=0.012). The relative corticosterone increase post-FST (F ­ =0.30, p=0.14) and decrease at 1h30 post-FST (F 1,35 =0.65, p=0.42) were unaffected by cycle stage in both groups. ( B ) The density of activated LC-NE neurons (TH+c-Fos+/µm², left) did not differ across cycle stages or conditions in the rostral LC, but was significantly affected in the caudal LC by both condition (structure × condition: F 3,105 =4.22, p=0.007) and cycle stage (structure × cycle: F ­ =3.12, p=0.029). Post-hoc tests revealed an increased density of activated neurons in D/M compared to P/E stages in the caudal-dorsal LC (t 35 =–2.313, p=0.027), independent of condition. In addition, ELS animals showed a reduced density compared to CTL, regardless of cycle stage (t ­ =2.63, p=0.013). The proportion of activated LC-NE neurons (% TH+c-Fos+/TH+, right) was unchanged across cycle stages or conditions in the rostral LC, but was significantly reduced in the caudal LC of ELS animals compared to CTL (structure × condition: F ­ =3.12, p=0.029; post-hoc: t ­ =2.99, p=0.005), independently of cycle stage. Data are presented as mean ± SEM. Three-way ANOVA followed by Bonferroni’s multiple comparisons test was used. Statistical significance: *p<0.05, **p<0.01, ***p<0.001. Abbreviations: LC, locus coeruleus; TH, tyrosine hydroxylase; NE, noradrenergic; CTL, control; ELS, early-life stress; P/E, proestrus/estrus; D/M, diestrus/metestrus. To further distinguish susceptible and resilient individuals, we applied two analyses, Mahalanobis distance and k-means clustering, using key behavioral measures: latency to eat (NSF), social interaction index (3CH), % sucrose preference (SPT), and immobility time (FST), separately in males and females. Mahalanobis distance quantified each mouse’s deviation from the CTL distribution, with greater distance reflecting higher susceptibility. In parallel, k-means clustering assigned individuals to clusters by minimizing within-clusters variances. 1. The Mahalanobis distance analysis Mahalanobis distance analysis, combined with correlation, identified bio-behavioral markers of susceptibility ( Figure 3 ) . While the distance was significantly higher in ELS than CTL groups, oestrus cycle stage in females had no effect ( Supplementary Figure 3B ). Download figure Open in new tab Figure 3. Mahalanobis distance analysis reveals long-term behavioral susceptibility to ELS. ( A ) Susceptibility to ELS, quantified by the Mahalanobis distance, is shown for males (top) and females (bottom), based on four behavioral measures: social interaction index in the three-chamber test (3CH), latency to eat in the novelty-suppressed feeding test (NSF), sucrose preference (%), and immobility time in the forced swim test (FST). ( B ) Pearson correlations between Mahalanobis distance and behavioral measures. In males (top), Mahalanobis distance was negatively correlated with latency to eat (R = −0.78, p < 0.001) and sucrose preference (R = −0.33, p = 0.032). In females (bottom), a significant negative correlation was found with sucrose preference (R = −0.679, p < 0.001), while no association was observed with latency to eat (R = 0.146, p = 0.376). ( C ) Volcano plots illustrating −log(p) values as a function of Pearson’s r for males (top) and females (bottom). Parameters include body weight (P2–P70 and mean weight), USV (P2–P12), mean pre- and post-deprivation, corticosterone (baseline, post-FST, 1h30 post-FST, % increase, % decrease), and LC activity markers (density of TH + c-Fos + cells and %TH + c-Fos + /TH + across the rostro-caudal and dorso-ventral axes). Orange points indicate significance at −log(p) > 1.3 (p 1 (p < 0.1). Abbreviations: CTL, control; ELS, early-life stress; P, post-natal day; USV, ultrasonic vocalisation; FST, Forced swim test; NSF, novelty supressed-feeding test; 3CH, 3-chamber test; SPT, sucrose preference test. Behavioral correlates In both sexes, higher Mahalanobis distance correlated with lower sucrose preference (M: R=−0.33, p=0.032; F: R=−0.679, p<0.001). In males, it was also associated with longer latency to eat in the NSF (R=−0.78, p<0.001), a link absent in females (R=0.146, p=0.376; Figure 3B ). No correlation emerged with the social interaction index (M: R=−0.235, p=0.13; F: R=−0.205, p=0.21) or the immobility time (FST, M: R=0.053, p=0.74; F: R=−0.118, p=0.47). Early-life predictors Lower mean body weight from P2 to P14 predicted higher susceptibility in both males (R=−0.38, p=0.012) and females (R=−0.37, p=0.020). Post-deprivation USV emissions at P2 were positively correlated with susceptibility in both sexes (M: R=0.35, p=0.020; F: R=0.32, p=0.049), suggesting heightened vocalization after the first deprivation as an early predictor. In male, a trend toward decrease post-deprivation USVs at P7 predicted susceptibility (R=−0.28, p=0.068), while females displayed a trend for lower mean pre-deprivation USVs (R=−0.28, p=0.088; Figure 3C ). Late-onset markers Corticosterone responses to stress showed no association with Mahalanobis distance in either sex ( Figure 3C ) at baseline (M: R=0.061, p=0.70; F: R=0.27, p=0.10), post-FST (M: R=–0.25, p=0.11; F: R=–0.19, p=0.24), or 1h30 post-FST (M: R=–0.094, p=0.55; F: R=0.074, p=0.65). Similarly, neither the relative increase (M: R=–0.087, p=0.58; F: R=0.23, p=0.16) nor the subsequent decrease (M: R=–0.047, p=0.77; F: R=0.20, p=0.22) correlated with susceptibility. However, anatomical analyses revealed sex-specific LC-NE activation patterns ( Figure 3C ). In males, reduced activation of NE neurons in the caudal-dorsal LC predicted susceptibility (%TH + c-Fos + /TH + ; R=–0.52, p<0.001), while females exhibited a significant increased activation in the rostral-ventral LC as a predictor of greater susceptibility (TH + c-Fos + /µm²; R=0.41, p=0.008). 2. K-means clustering analysis to reveal resilience K-means clustering of ELS mice allowed identifying three clusters in both sex: anxious-like (ANX) , characterized by increased NSF latency (M: F 3,14.4 =4.33, p=0.025; CTL vs ANX: t=– 4.22, p<0.001; F: F 3,35 =6.99, p<0.001; CTL vs ANX: t=–4.02, p=0.002) and reduced social interaction in males only (M: F 3,39 =7.88, p<0.001; CTL vs ANX: t=3.79, p=0.003; F: F 3,35 =4.43, p=0.01; CTL vs ANX: t=2.71, p=0.06); depressive-like (DEP) , defined by reduced sucrose preference (M: F 3,39 =19.71, p<0.001; CTL vs. DEP: t=6.98, p<0.001; F: F 3,35 =31.52, p<0.001; CTL vs. DEP: t=8.75, p<0.001); and resilient (RES) , showing no behavioral alterations relative to CTL ( Figure 4A-B ). Download figure Open in new tab Figure 4. K-means clustering analysis reveals long-term behavioral susceptibility and resilience to ELS. (A) Cluster plots from k-means analysis (k = 3) in males (left) and females (right), based on four behavioral measures: social interaction index in the three-chamber test (3CH), latency to eat in the novelty-suppressed feeding test (NSF), sucrose preference (%) in the sucrose preference test (SPT), and immobility time in the forced swim test (FST). Three clusters were identified in both sexes: ANX (anxiety-like, orange), DEP (depression-like, red), and RES (resilient, green). ( B ) Boxplots for each behavioral test comparing ANX, DEP, and RES clusters to controls (CTL), in males [(left): 3CH (F ­ =7.88, p<0.001; Post hoc CTL vs ANX: t=3.79, p=0.003), NSF (F ­14.4 =4.33, p=0.025; CTL vs ANX: t=–4.22, p<0.001), SPT (F 3, 39 =19.71, p<0.001; CTL vs DEP: t=6.98, p<0.001), FST (F ­ =0.84, p=0.48)] and in females [(right): 3CH (F ­ =4.43, p=0.01; CTL vs ANX: t=2.71, p=0.06), NSF (F 3, 35 =6.99, p<0.001; CTL vs ANX: t=–4.02, p=0.002), SPT (F 3, 35 =31.52, p<0.001; CTL vs DEP: t=8.75, p<0.001), FST (F ­ =0.49, p=0.69)]. ( C ) Distribution of animals across RES, ANX, and DEP clusters in males (M) and females (F). Data are presented as mean ± SEM. One-way ANOVA (with Welch’s correction when Levene’s test indicated unequal variances) was followed by Bonferroni post hoc tests. Statistical significance is indicated as: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CTL, control; ELS, early-life stress; ANX, anxiety-like cluster; RES, resilient cluster; DEP, depression-like cluster; FST, Forced swim test; NSF, novelty supressed-feeding test; 3CH, 3-chamber test; SPT, sucrose preference test. Although behavioral phenotypes were consistent across sexes, cluster distribution differed ( Figure 4C ). The proportion of resilient mice was nearly identical (males: 24%; females: 23.8%). However, males were more frequently assigned to the ANX cluster (48%) than to the DEP (24%), whereas female showed the reverse, with more in DEP (47.6%) than in ANX (28.6%). These findings mirror the Mahalanobis distance, reinforcing sex-specific patterns of susceptibility to ELS. In females, P/E and D/M stage distribution was similar across clusters after the 3CH. After the NSF and FST, there was a trend toward more D/M-stage females in the DEP cluster only (proportion compared to 0.5: NSF p=0.1; FST p=0.1). Notably, after the FST, CTL and DEP differed significantly, with 60% of CTL vs 20% of DEP females in P/E (χ²=4.37, p=0.037; Supplementary figure 3C ). Early-life predictors In males, the ANX cluster displayed reduced body weight from P5 to P14 compared to CTL ( Figure 5A ; Condition × Day: F 5.7, 101.6 =5.42, p<0.001; Post hoc : P5: t=5.88, p<0.001; P7: t=4.90, p<0.001; P12: t=4.63, p<0.001; P14: t=4.28, p<0.001). Consistently, mean early weight was significantly lower in ANX (F 3,15.2 =10.69, p<0.001; t=4.61, p<0.001). In females, both RES and DEP clusters showed persistently reduced body weight from P5 to P14 ( Figure 5A ; F 6.3,73.8 =10.7, p<0.001; RES vs CTL: P5: t=2.78, p=0.035; P7: t=3.78, p=0.003; P12: t=4.21, p=0.001; P14: t=4.71, p<0.001; DEP vs CTL: P5: t=2.52, p=0.049; P7: t=3.12, p=0.011; P12: t=2.80, p=0.034; P14: t=2.73, p=0.04). However, only the RES cluster differed significantly from CTL in mean early weight (F 3.11,165 =10.67, p=0.001; post hoc: t=3.51, p=0.007). Download figure Open in new tab Figure 5. Early predictors of susceptibility and resilience to the long-term effects of early-life stress identified by k-means clustering analysis. (A) Body weight trajectories during ELS (P2–P14). Weight development was significantly affected in both males (F 5.7, 101.6 = 5.42, p < 0.001) and females (F 6.3, 73.8 = 10.7, p < 0.001). Left : In males, the ANX cluster displayed reduced body weight from P5 to P14 compared to CTL (P5: t = 5.88, p < 0.001; P7: t = 4.90, p < 0.001; P12: t = 4.63, p < 0.001; P14: t = 4.28, p < 0.001). Consistently, mean early weight was significantly lower in the ANX cluster compared to both CTL (F ­ = 10.69, p < 0.001. t = 4.61, p < 0.001) and DEP (t = −4.26, p < 0.001). Right: In females, both RES and DEP clusters showed reduced body weight from P5 to P14 compared to CTL (RES vs. CTL: P5: t = 2.78, p = 0.035; P7: t = 3.78, p = 0.003; P12: t = 4.21, p = 0.001; P14: t = 4.71, p < 0.001; DEP vs. CTL: P5: t = 2.52, p = 0.049; P7: t = 3.12, p = 0.011; P12: t = 2.80, p = 0.034; P14: t = 2.73, p < 0.04). For mean early weight, only the RES cluster differed significantly from CTL (F 3.11, 165 = 10.67, p = 0.001; t = 3.51, p = 0.007). ( B ) Pre-deprivation USV emissions . USVs prior to maternal deprivation were significantly affected by ELS in males (F 6.8, 88.4 = 5.22, p < 0.001) and females (F 7.8, 91.6 = 3.8, p < 0.001). Left: In males, both DEP and RES clusters showed significantly fewer USVs at P12 compared to CTL (DEP: t = 2.79, p = 0.024; RES: t = 8.48, p < 0.001), with a stronger reduction in the RES cluster (RES vs. DEP: t = −4.42, p < 0.001). Mean pre-deprivation USVs were significantly reduced in the RES cluster compared to CTL (F 3, 15.6 = 4.44, p = 0.019; t = 3.31, p = 0.012). Right : In females, the RES cluster showed higher USV emissions at P7 compared to CTL (t = −3.76, p = 0.003). ( C ) Post-deprivation USV emissions . Left : In males, USV emissions in the RES cluster were significantly reduced compared to CTL at P12 (F 7.8, 101.6 = 5.23, p < 0.001; t=5.40, p<0.001). Moreover, ANX displayed elevated post-deprivation USVs at P2 compared to CTL (t=–3.18, p=0.017). Mean post-deprivation USVs were reduced in both RES and DEP compared to CTL (F 3,17.3 =12.15, p<0.001; RES: t=3.25, p=0.015; DEP: t=2.90, p=0.036). Right : In females, no significant cluster differences were detected (F 6.5, 76.2 = 2.45, p = 0.028). Data are presented as mean ± SEM. Two-way ANOVA followed by Bonferroni’s multiple comparisons test was used, with Greenhouse–Geisser correction applied when sphericity was violated. Statistical significance is indicated as: *p < 0.05, **p < 0.01, ***p < 0.001. Colored asterisks indicate significant differences from the CTL group. Abbreviations: CTL, control; ELS, early-life stress; ANX, anxiety-like cluster; RES, resilient cluster; DEP, depression-like cluster. Cluster-specific USV patterns also emerged ( Figure 5B-C ). In males, RES and DEP showed reduced pre-deprivation USVs at P12 (F 6.8,88.4 =5.22, p<0.001; DEP: t=2.79, p=0.024; RES: t=8.48, p<0.001) with a stronger effect in RES (RES vs DEP: t=–4.42, p<0.001). Post-deprivation, this P12 reduction persisted only in RES (t=5.40, p<0.001). Consistently, mean pre-deprivation USV levels were reduced only in RES (F 3,15.6 =4.44, p=0.019; t=3.31, p=0.012), while mean post-deprivation USVs were reduced in both RES and DEP compared to CTL (F 3,17.3 =12.15, p<0.001; RES: t=3.25, p=0.015; DEP: t=2.90, p=0.036) and ANX (RES: t=3.75, p=0.003; DEP: t=3.41, p=0.009). Conversely, ANX displayed elevated post-deprivation USVs at P2 compared to CTL (Condition × Day: F 7.8,101.6 =5.23, p<0.001; Post hoc : t=–3.18, p=0.017). In females, the only effect was a transient pre-deprivation increase in USVs at P7 in RES (Pre: F 7.8,91.6 =3.8, p<0.001; P7, CTL vs RES: t=–3.76, p=0.003; Post: F 6.5,76.2 =2.45, p=0.028, post hoc ns ). Late-onset markers In males, despite lower baseline corticosterone in RES (F 3,17.7 =7.78, p=0.002; CTL vs RES: t=4.98, p<0.001; ANX vs RES: t=3.36, p=0.01; Figure 6A ), a greater stress-induced increase was observed (F 3,15 =4.32, p=0.022; CTL vs RES: t=–4.62, p<0.001; CTL vs ANX: t=–3.57, p=0.006; Figure 6B ). No cluster differences compared to CTL emerged post-FST (F 3,15.6 =1.82, p=0.19), 1h30 post-FST (F 3,39 =3.71, p=0.019; Supplementary Figure 5A-B ) or for percentage reduction (F 3.39 =0.78, p=0.51, Figure 6C ). In females, corticosterone levels or percentage changes did not differ across clusters at any time point (Baseline: F 3, 9.1 =3.44, p=0.13; Post-FST: F 3,35 =0.55, p=0.65; 1h30 post-FST: F 3,35 =2.06, p=0.12; %Increase; F 3,9.2 =1.48, p=0.28; %Decrease: F 3,10.6 =2.46, p=0.12). Download figure Open in new tab Supplementary figure 5: Long-term consequences of ELS on corticosterone level and LC-NE functional anatomy in males and females. ( A ) Corticosterone level post-FST was similar between clusters in male ( Top , F ­ =1,82, p=0.19) and female ( Bottom , F ­ =0.55, p=0.65). ( B ) Corticosterone level 1h30 post-FST, was similar between clusters in female ( Bottom , F 3,35 =2.06, p=0.12), however, in male, an increase was observed in the DEP compared to the ANX cluster ( Top , F ­ =3.71, p=0.019; t ­ =−2.97, p=0.031). ( C )The density of activated LC-NE neurons (TH+c-Fos+/µm 2 ) was similar between cluster across the rostro-caudal and dorso-ventral axis of the LC both in male ( Top , F 7.2,97.3 =1.37, p=0.22) and female ( Bottom , F 7.5,87.6 =1.96, p=0.065). Data are presented as mean ± SEM. Two-way ANOVA followed by Bonferroni’s multiple comparisons test was used, with Greenhouse– Geisser correction applied when sphericity was violated. Statistical significance: *p<0.05, **p<0.01, ***p<0.001. Abbreviations: LC, locus coeruleus; TH, tyrosine hydroxylase; NE, noradrenergic; CTL, control; ELS, early-life stress; P/E, proestrus/estrus; D/M, diestrus/metestrus. Download figure Open in new tab Figure 6. Physiological predictors of susceptibility and resilience to the long-term effects of early-life stress identified by k-means clustering analysis. ( A ) Baseline corticosterone levels. In males ( top ), baseline corticosterone levels were significantly lower in the RES cluster compared to CTL and ANX (F ­ = 7.78, p = 0.002; CTL vs RES: t = 4.98, p < 0.001; ANX vs RES: t = 3.36, p = 0.01). In females ( bottom ), no significant cluster effects were observed (F 3, 9.1 = 3.44, p = 0.13). ( B ) Percentage increase in corticosterone following FST . In males ( top ), the percentage increase in corticosterone after FST was greater in the RES cluster compared to CTL and ANX (F 3, 15 = 4.32, p = 0.022; CTL vs RES: t = −4.62, p < 0.001; CTL vs ANX: t = −3.57, p = 0.006). In females ( bottom ), no significant cluster effect was detected (F ­ = 1.48, p = 0.28). ( C ) Percentage decrease in corticosterone levels 1h30 after FST. No significant cluster effects were observed in either males (F ­ = 0.78, p = 0.51) or females (F ­ = 2.46, p = 0.12). Data are presented as mean ± SEM. One-way ANOVA (with Welch’s correction when Levene’s test indicated unequal variances) was followed by Bonferroni’s post hoc test. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CTL, control; ELS, early-life stress; ANX, anxiety-like cluster; RES, resilient cluster; DEP, depression-like cluster; FST, forced swim test. LC-NE analyses revealed sex-specific patterns ( Figure 7A-B ). In males, ANX showed reduced proportion of activated NE cells (%(TH + c-Fos + )/TH + ) selectively in the caudal-dorsal LC (F 9,117 =2.15, p=0.031; CTL vs ANX: t=2.81, p=0.046; DEP vs ANX: t=−2.69, p=0.05, while in females, this reduction was observed in RES (F 9,105 =2.52, p=0.012; CTL vs RES: t=3.34, p<0.001; DEP vs RES: t=−3.58, p=0.005). No significant differences emerged across phenotypes for the density of activated NE cells along LC sub-regions ( Supplementary figure 5C ). Download figure Open in new tab Figure 7. Functional anatomy of LC-NE activity in susceptibility and resilience to the long-term effects of early-life stress identified by k-means clustering. ( A ) Schematic representation of the locus coeruleus (LC, left ) and representative immunofluorescence image ( right ) showing TH-positive neurons (green), c-Fos expression (red), and colocalization of both markers (yellow), indicating activated noradrenergic (NE) cells. ( B ) Proportion of activated NE neurons (%TH + C-Fos + /TH + ) along the rostro-caudal and dorso-ventral axes of the LC . In males ( left ), the proportion of activated NE neurons was significantly reduced in the ANX cluster compared to CTL and DEP, specifically in the caudal-dorsal LC (F ­ = 2.15, p = 0.031; CTL vs ANX: t = 2.81, p = 0.046; DEP vs ANX: t = −2.69, p = 0.05). In females ( right ), a similar reduction was observed in the RES cluster relative to CTL and DEP (F 9, 105 = 2.52, p = 0.012; CTL vs RES: t = 3.34, p < 0.001; DEP vs RES: t = −3.58, p = 0.005). Data are presented as mean ± SEM. Two-way ANOVA followed by Bonferroni’s multiple comparisons test was used, with Greenhouse–Geisser correction applied when sphericity was violated. Statistical significance is indicated as: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: LC, locus coeruleus; TH, tyrosine hydroxylase; NE, noradrenergic; CTL, control; ELS, early-life stress; ANX, anxiety-like cluster; RES, resilient cluster; DEP, depression-like cluster. Discussion This study investigated how ELS shapes anxio-depressive-like behaviors in mice, with emphasis on resilience in the context of CA. Since CA and ELS are major psychiatric risk factors, uncovering resilience mechanisms is critical. The LC-NE system, a modulator of resilience ( 48 , 50 , 52 , 65 – 67 ), has not been studied in relation to ELS. Using a paradigm combining maternal deprivation and limited bedding/nesting, we defined resilient versus susceptible phenotypes (ANX, DEP) and identified early and long-term markers. Findings revealed sex-specific mechanisms ( Figure 8 ). In males, reduced body weight with increased first post-deprivation USV predicted susceptibility, especially anxiety-like phenotype, whereas prolonged USV reductions until P12 characterized resilience. In females, reduced body weight emerged in both resilient and depressive-like phenotypes but was greater in resilience. Increased USV emissions predicted resilience, while decreased pre-deprivation USVs indicated susceptibility. Later trajectories also diverged by sex. Male resilience was associated with lower baseline corticosterone and enhanced corticosterone response to acute stress, while anxiety-like correlated with reduced LC-NE activity in the caudal-dorsal LC. Conversely, in females, this reduction marked resilience rather than susceptibility. Download figure Open in new tab Figure 8. Summary of main findings in each phenotype in both males and females In the maternal deprivation with limited bedding/nesting model, dams’ behavior and stress response were evaluated as factors shaping pup development. This variant was designed to replicate CA by introducing maternal stress, counteracting the increased care often observed in deprivation alone. In our study, maternal behavior and long-lasting stress response remained unaffected ( 61 , 68 ). Additional real-time analyses ( 69 ) could have refined these findings. However, ELS reduced pup survival, especially in large litters, an effect rarely reported in deprivation paradigms ( 62 , 70 – 73 ). Litter equalization was avoided to reduce disturbance ( 74 ). Body weight was unaffected in females but reduced in ELS males from P7– P14, persisting into adulthood ( Figure 1G-H ). ELS also reduced USVs, particularly in males. We did not replicate the pre/post-separation increase observed in females at P7 in the study of Yin et al. (2016) ( 75 ), possibly due to methodological differences ( 60 ). Together, these findings highlight sex-specific early effects of ELS, with males showing greater vulnerability. Opposing sex-specific outcomes emerged in the long-term ( Figure 2 ). In males, ELS had no impact on anxio-depressive behaviors, while in females it increased anhedonia. These findings align with the resilience of C57BL/6 mice in ELS paradigms and variability across models ( 76 , 77 ). For instance, maternal deprivation associated with limited nesting showed no effect on anhedonia in females ( 78 ) but produces social deficits in males ( 79 ). To standardize comparisons, we assessed both sexes across NSF, 3CH, SPT, and FST, covering anxiety-and depression-like dimensions ( 80 , 81 ). To identify susceptible from resilient individuals, we employed Mahalanobis distance and k-means clustering analysis. Mahalanobis distance, typically used to detect outliers ( 82 ), identified behaviors deviating most from controls, defined as susceptibility, while clustering ( 83 ), classified mice into anxious-like (ANX), depressive-like (DEP), and resilient-like (RES) phenotypes ( 84 ). This framework captured stress-induced phenotypes beyond task-specific effects. In males, susceptibility involved anxiety and anhedonia, while in females it was marked only by anhedonia. Clustering confirmed this, showing higher prevalence of anhedonic phenotypes in females and anxiety-like phenotypes in males. These results parallel clinical evidence, where women are twice as likely to develop depression ( 85 , 86 ). Resilience prevalence was similar in both sexes (∼25%), consistent with adult chronic stress models ( 50 , 87 – 89 ). In humans, resilience rates following CA varies widely (10–50%) depending on definitions, age, sex, and type of abuse ( 90 – 92 ). Here, resilience, defined as absence of social, anxiety, or anhedonia deficits, was equally likely in both sexes, underscoring the need for multidimensional approaches. In females, oestrus stage had little impact on bio-behavioral markers, with only a trend indicating that those in proestrus/oestrus during the NSF and FST were more often classified as depressive-like. Resilience is increasingly recognized as a dynamic and adaptive process fluctuating across life rather than a fixed trait ( 18 , 93 ). Human studies rarely clarify whether biobehavioral factors act as predictors or consequences of resilience. Animal models help address this gap by revealing phenotypic changes across multiple levels. In this study, we examined body weight and USVs during the stress period as potential early predictors ( Figures 3 - 5 ). In males, both lower body weight and reduced USV emission linked to susceptibility in the Mahalanobis distance and confirmed by clustering, which tied them to an anxiety-like phenotype. Early low body weight predicted persistent anxiety-like behavior, with reduced growth extending into adulthood. While USV reductions appeared across clusters, they persisted only in resilient males until P12. Notably, anxiety-linked decrease followed an initial rise after the first deprivation, a pattern absent in resilient mice, suggesting early adaptive dampening of emotional responses. In females, weight loss occurred in both resilient and depressive-like clusters, but its association with increased USVs predicted resilience, whereas decreased USVs predicted susceptibility. These findings highlight sex-specific physiological and emotional markers as early predictors of long-term ELS outcomes. ELS models often fail to capture human-like long-term effects, partly because resilience is overlooked. Our analysis highlighted that excluding the ∼25% resilient individuals revealed anxio-depressive outcomes. Beyond this, we identified physiological and anatomical signatures linked to both susceptible and resilient phenotypes ( Figure 6 - 7 ). Corticosterone levels were not predictive in the Mahalanobis analysis, but clustering revealed phenotype-specific regulation, suggesting efficient adaptation in resilient males. This contrasts with some human data ( 94 ), underscoring the complexity of resilience markers: both patterns have been observed in human, making challenging to establish clear conclusions ( 90 ). ELS impacts on LC-NE functional anatomy differed by sex. In males, Mahalanobis analysis linked susceptibility to reduced NE neuron activation in the caudal-dorsal LC, associated with anxiety-like traits in clustering. No adaptations were observed in resilient males. In females, susceptibility involved greater NE activation in rostral-ventral LC as shown in the Mahalanobis distance, whereas reduced caudal-dorsal NE activation characterized resilience. Since peri-LC and dorso-medial regions contain GABAergic neurons known to inhibit NE activity ( 33 , 95 , 96 ), sex-specific modulation may underlie resilience. In males, GABA neurons may buffer NE activity in resilient individuals, supporting adaptive stress responses. In females, resilience directly involved decreased caudal-dorsal NE activation, suggesting sex-specific regulation. Whether these functional changes are late-onset, progressive, or predictive remains unclear. Future longitudinal recordings will be required to clarify these adaptations. This study highlights sex-specific mechanisms underlying susceptibility and resilience to ELS, identifying early emotional and physiological predictors and distinct LC-NE system adaptations. In males, reduced caudal-dorsal LC-NE activation was linked to anxiety-like behaviors, whereas in females, it’s linked to resilience. Examining resilience is essential to understand individual differences and guide therapeutic strategies aimed at enhancing resilience and mitigating ELS consequences. Author contributions EI and DS designed the experiments. DS and VG performed the behavioral and physiological experiments and analysis. DS and VG performed the anatomical experiments and VG the counting analysis. DS, VG and EI performed the statistical analysis. LG performed the matlab script for the mahalonobis distance analysis. DS and EI wrote the publication, which was edited by BG. EI supervised this research. Acknowledgements The authors express their gratitude to the financial support of the ANR – FRANCE (French National Research Agency, ANR-21-CE16-004), the FRM (Fondation pour la recherche médicale, EQU202203014667), the Sissley Fundation, the IDEX Emergence grant and the doctoral school MCTI (ED 563, Médicament, Toxicologie, Chimie, Imageries) from the University Paris Cité. The project was carried out with the support of the ERIE Foundation, a fund hosted by the King Baudouin Foundation. Image acquisitions were performed at the SCM Imaging facility and Corticosterone dosage at the Cyto2BM facility (BioMedTech Facilities, INSERM US36 | CNRS UAR2009 | Université Paris Cité, https://biomedicale.u-paris.fr/biomedtech-facilities/ ). Funder Information Declared French National Research Agency , ANR-21-CE16-004 Fondation pour la recherche médicale , EQU202203014667 References 1. ↵ Brown CL , Yilanli M , Rabbitt AL ( 2024 ): Child Physical Abuse and Neglect . StatPearls . 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Share Role of the locus coeruleus noradrenergic system in susceptibility and resilience following early life stress in male and female mice Dea Slavova , Valentine Greffion , Lionel Granjon , Stéphanie De-Gois , Maud Blaise , Bruno Giros , Elsa Isngrini bioRxiv 2025.10.11.681820; doi: https://doi.org/10.1101/2025.10.11.681820 Share This Article: Copy Citation Tools Role of the locus coeruleus noradrenergic system in susceptibility and resilience following early life stress in male and female mice Dea Slavova , Valentine Greffion , Lionel Granjon , Stéphanie De-Gois , Maud Blaise , Bruno Giros , Elsa Isngrini bioRxiv 2025.10.11.681820; doi: https://doi.org/10.1101/2025.10.11.681820 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 (7618) Biochemistry (17633) Bioengineering (13856) Bioinformatics (41841) Biophysics (21399) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24282) Genetics (15582) Genomics (22462) Immunology (17700) Microbiology (40295) Molecular Biology (17140) Neuroscience (88419) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)