Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions

Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions Susanna Pietropaolo, Valeria Petroni, Enejda Subashi, Marika Premoli, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1372695/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Fragile X Syndrome (FXS) is the most common heritable form of mental retardation and the main monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 gene and is characterized by motor, cognitive and social alterations, mostly overlapping with ASD behavioral phenotypes. The severity of these symptoms and their timing may be exacerbated and/or advanced by environmental adversity interacting with the genetic mutation. We therefore tested the effects of the prenatal exposure to unpredictable chronic stress on the behavioral phenotype of juveniles of both sexes in the Fmr1 knock-out (KO) mouse model of FXS. Mice underwent behavioral tests at 7–8 weeks of age, that is, when most of the relevant behavioral alterations are absent or mild in Fmr1-KOs. Stress induced the early appearance of cognitive deficits in KO male mice, without exacerbating the behavioral phenotype of mutant females. In males stress also altered social interaction and communication, but mostly in WT mice, while in females it induced effects on locomotion and communication in mice of both genotypes. Our data therefore highlight the sex-dependent relevance of early environmental stressors to interact with genetic factors to influence the appearance of selected FXS- and ASD-like phenotypes. Unpredictable stress Fragile X syndrome sex differences mouse social behaviors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by multiple behavioral alterations, including mental retardation, hyperactivity, anxiety, cognitive and social deficits 1 . Autistic symptoms, including altered social interaction and communication, are also often detected in FXS patients 2 , 3 : FXS is indeed considered as the most common monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 human gene consisting in more than 250 CGG repetitions leading to the absence of FMRP protein 4 playing a major role in synaptic and neuronal functionality 5 . The lack of FMRP has been recapitulated by the Fmr1-KO mouse model of FXS together with several relevant behavioral alterations 6 . The FXS-like behavioral phenotypes of mutant mice are mostly evident at adulthood, i.e., at 3–6 months, that is, when most pre-clinical studies are carried out [as reviewed in 7 ]. Despite its clear and well-defined genetic origins, the FXS behavioral phenotype can be critically modulated by environmental factors, both in terms of its severity and of the timing of appearance. Environmental stimulation is for instance known to attenuate/delay the expression of behavioral alterations both in FXS patients and Fmr1-KO mice 8 , 9 . Conversely, exposure to stressful life events may exacerbate the behavioral deficits of FXS patients 10 , 11 especially when occurring during early life phases. Exposure to prenatal stress is a powerful tool to induce early adversity in a genetic mouse model and therefore to study the impact of gene-environment interactions in the expression of its behavioral phenotype. Indeed, prenatal stress exacerbates the behavioral alterations of genetic mouse models of Alzheimer disease, depression and schizophrenia 12 – 14 . Surprisingly, to our knowledge, the behavioral effects of prenatal stress have never been investigated in the Fmr1-KO mouse, or in other models of ASD. Furthermore, prenatal stress is known to induce marked long-term behavioral alterations in wild-type rodents, including cognitive, emotional, motor and social abnormalities [reviewed in 15 , 16 ]. These studies have pointed out in particular the relevance of the unpredictable chronic mild stress procedure, as the most suitable experimental approach to model early environmental adversity in laboratory rodents 17 – 19 . This procedure, combining multiple stressors of different nature, has also the advantage to minimize habituation and exclude pain or nutritional effects 20 , 21 . In most existing preclinical studies [reviewed in 15 , 16 ] stress exposure was implemented during the last week of gestation of the dams, as this phase is a preferential target to induce long-term brain and behavioral modifications in the offspring, because of its high environmental and stress sensitivity 22 , 23 . The inclusion of mice of both sexes in the behavioral analysis of the offspring is considered of critical relevance for preclinical studies on prenatal stress exposure. Several sex differences have been indeed described in the behavioral response to stress in rodents; these include differences in the severity of stress effects, but also in their specificity to selected behavioral domains 13 , 24 , 25 . The inclusion of subjects of both sexes is also important for studying FXS, both in human and preclinical research. Although FXS is more common in boys than girls, increasing attention has been devoted to heterozygous females, as they are the ones producing the affected offspring 26 , and they represent the majority of FXS female patients, as homozygous FMR1 mutations are extremely rare 27 . In humans, FXS female carriers present several behavioral symptoms, including hyperactivity 28 , mild cognitive impairments 29 , 30 and autistic behaviors 31 . In mice, similar behavioral abnormalities were described in Fmr1 mutant females, especially at adulthood [as reviewed in 7 ]. Here we therefore evaluated whether exposure to unpredictable chronic mild stress during the last prenatal week could advance and/or exacerbate the juvenile behavioral phenotype of Fmr1-KO offspring of both sexes (as schematized in Fig. 1 ). To this end, Fmr1-KO male (hemizygous, -/Y) and female (heterozygous, +/-) mice, together with their WT littermates, underwent behavioral tests for exploration, spatial memory, social interaction and communication at the juvenile age of 7–8 weeks, i.e., when most of the FXS-like behavioral alterations are absent or mild. At this age, Fmr1-KO males do not show any remarkable behavioral phenotype in the considered domains 7 , 32 , while mutant females displayed mild alterations in social interaction and communication 33 . This age partially overlaps with adolescence (occurring between 3 and 8 weeks of age in mice), a critical phase for brain and behavioral development in rodents and humans and largely involved in several neuropsychiatric disorders 34 . This phase has been also extensively studied for the expression of social behaviors in laboratory mice, with a special emphasis on the post-pubertal phase (i.e., approximately after the 5 weeks of age), since it is characterized by important changes in the patterns of intra-specific social interactions 35 . Late adolescence (7–9 weeks) is also of particular interest, since most behavioral abilities are already well developed in mice; it is therefore suitable to multiple behavioral testing, performing the same cognitive, emotional, and social tests done in adult mice and hence facilitating comparisons with data from adult subjects. Methods Ethics approval All experimental procedures were in accordance with ARRIVE guidelines ( https://arriveguidelines.org ), European Communities Council Di-rective 2010/63/EEC. Furthermore, there were approved by local ethical committee (“Comité d’Ethique pour l’experimentation animale de Bordeaux”, CE 50) and the French Ministry (“Ministere de l’enseignement superieur de la recherché et de l’innovation”). Breeding and stress procedure Twenty adult (12 ± 1 weeks-old) virgin Fmr1 heterozygous (+/-) females and 10 C57BL/6J adult wild type males [16 weeks-old; purchased from Janvier (Le Genest St Isle, France)] were used as breeders to generate the tested offspring. C57BL/6JFmr1 tm1Cgr/Nwu (B6) mice were originally obtained from Neuromice.org (Northwestern University) and maintained on the C57BL6/J background for more than 10 generations. They were bred as described previously 32 . Each half of the female breeders was assigned to one of the following groups in which they were kept during the last week of pregnancy: no-stress, i.e., kept undisturbed in their home-cage, or stress, i.e., exposed to the unpredictable stress procedure described below. The time line of the study is illustrated in Fig. 1 . The stress procedure included the following 2-day sequence of events that was repeated three consecutive times during the last week of gestation: Day 1: 30 minutes of restrain stress (3 times each day during the light phase, with a 4h-interval) in perforated conical tubes (3cm in diameter, 11.5cm long; Becton Dickinson Labware Europe, France), followed by overnight housing with wet bedding (50ml of water were added to floor sawdust of the home cage at the beginning of the dark phase). Day 2: multiple sawdust and cage changes (3 times each day during the light phase, with a 4h-interval), followed by overnight housing with novel objects (12 glass black beads, 1.5cm in diameter were added in the home cage at the beginning of the dark phase). Pregnant females were exposed to this sequence of events for 3 times during the last week before parturition: this procedure is based on previous studies [e.g., 23,36−38 ] and it is known to limit the habituation to stressful stimuli without using pain or nutritional manipulations. All breeders used for the study gave birth within 48hs after the last day of exposure to stress procedure. They were left undisturbed until weaning of the pups, i.e., on post-natal day (PND) 21. Animals and housing procedures At 3 weeks of age, all pups were weaned and housed in same-sex groups of 3–5 littermates in our animal facility 9 , 32 . On the same day, tail samples were collected for DNA extraction and subsequent PCR assessment of the genotypes as previously described 6 . Mice were then left undisturbed until the beginning of behavioral testing (i.e., at 7 weeks of age), except for the evaluation of body weight that was carried out once a week starting at 5 weeks of age (Fig. 1 ). Only litters including males and females of both mutant (KO) and wild-type (WT) genotypes were used for experiments, for a total of 14 litters. A total of 93 mice were subjected to behavioral testing: 45 males [25 WT and 20 KO (-/Y), n = 9–15 for stress condition) and 48 females [24 WT and 24 KO (+/-), n = 12 for stress condition]. Stimulus mice used for the direct social interaction test were adult (10 weeks of age) female NMRI mice, as this strain is commonly employed in social studies 39 , 40 , especially those using the Fmr1-KO mouse model 9 , 32 , 33 . This strain is often chosen since it is characterized by high levels of sociability, and it facilitates the behavioral analysis during social encounters with B6 mutants because of its albino phenotype. NMRI mice were purchased from Janvier (Le Genest-Saint-Isle, France), housed in groups of 3–4 per cage and left undisturbed for 2 weeks before being used in behavioral tests. Behavioral testing procedures Behavioral tests commenced at 7 weeks of age and were conducted as follows (see also Fig. 1 ). On day 1, an open field test for locomotion and exploration was administered, followed on day 3 by a spontaneous alternation test in a Y-maze, and on day 5 by a direct social interaction test and the females’ estrous cycle assessment. All behavioral tests were carried out during the light phase of the cycle (between 9 a.m. and 4 p.m.) by an experimenter who was blind to the group assignment of the subjects. All mice were habituated to the experimental room for at least 30 min before the beginning of each behavioral test. ----------------------FIGURE 1 ABOUT HERE------------------------- Open field The open field 32 consisted of a white plastic arena where the locomotion of each mouse was assessed during 10 minutes using automated tracking (Ethovision, Noldus, The Netherlands). Y maze The Y maze test (described in details before 32 ) was employed to assess spontaneous alternation through a 2-trial procedure, consisting of a 5-min habituation trial, followed by a 2-min test trial. Time spent in each arm during the habituation and testing phases was scored by automatic tracking and percent alternation rates during the test phase were derived as follows: 100 × (time in novel arm/time in all arms). Social interaction and ultrasonic communication Male experimental subjects were habituated to the testing apparatus 9 , 32 for 30 min prior to testing, while female subjects were isolated in the testing cage for 72hs, in order to induce a status of resident in adult females and therefore promote the emission of ultrasonic vocalizations (USVs) towards an adult female intruder 40 . An unfamiliar stimulus female mouse (an adult NMRI female) was then introduced into the testing cage of either male or female subjects and left there for 3 min. Previous studies have shown that in these experimental settings USVs are mainly emitted by the male mouse in the male-female interaction 41 – 43 , and by the female resident in the female-female interaction 40 , 44 . Testing sessions were recorded by a camera placed on the side of the cage and videos analyzed with Observer XT (Noldus, The Netherlands). One observer who was unaware of the genotype and sex of the animals scored the behavior of the test mice, quantifying the time spent performing affiliative behaviors 9 , 32 , 33 , and nonsocial activities as described in details before 9 , 32 . An ultrasonic microphone UltraSoundGate Condenser Microphone CM 16 (Avisoft Bioacoustics, Berlin, Germany) was mounted 2 cm above the cover of the testing cage. Recordings were then analyzed through Avisoft SASLab Pro (Version 5.20; Avisoft, Berlin, Germany) to compute the number of USVs as well as their mean duration, peak frequency and peak amplitude 9 , 32 . In addition density plots depicting the distribution of total calls for each genotype at peak frequency versus duration were obtained as described in details elsewhere 45 , 46 . Call subtypes were also determined for a more detailed qualitative analysis; for this purpose, USVs were automatically classified using the Sonotrack Call Classification Software (version 1.4.7, Metris B.V., The Netherlands), using categories previously described in details elsewhere 47 . The estrus phase of female mice was assessed by analysis of vaginal smears 48 performed on the testing day in both the experimental subjects and NMRI stimulus mice. The evaluation of Fmr1 WT and KO (+/-) females used as experimental subjects was conducted after their testing, in order to minimize the potential stress effects of the manipulation necessary for determining the estrous phase. Stimulus NMRI females were approximately half in diestrus and half in estrus phases, and their assignment to social encounters was equally distributed between experimental groups. The estrus phase of experimental female subjects included pro-estrus, estrus and diestrus, following a distribution that was balanced across genotypes and stress conditions. Statistical analysis All data were separately analyzed in males and females. This was due to sex differences in (i) the X-linked Fmr1-mutation (i.e., hemizygous in males, heterozygous in females), (ii) in some behavioral testing procedures (such as different duration of pre-testing isolation necessary for USV assessment), (iii) in most of the behavioral phenotypes measured here. The latter sex differences were further confirmed in our data set, through a preliminary ANOVA showing overall sex effects in basically all measured variables (data not shown). Data from each sex were analyzed with a 2x2 ANOVA with genotype and stress as the between subject factors. Within-subject factors were included when appropriate (e.g., testing time for body weight). Alternation rates from the Y-maze test were instead analyzed for differences from the chance level (with a t-test), in line with previous studies 49 . Post-hoc comparisons were performed using Fisher’s LSD test when a significant interaction was detected. Separate ANOVAs were also conducted when appropriate. Data from the density plots of ultrasonic calls did not undergo statistical analysis, but were used to obtain a qualitative three-dimensional evaluation of USV data 45 , 46 . Analyses were conducted using the software Statview and SPSS and α was set at 0.05. Results are expressed as mean ± SEM throughout the text. The exact number of mice is indicated in the legend of each figure; differences may be due to technical reasons (e.g., loss of behavioral video recordings) or to the exclusion of outliers (using Grubbs' ESD test adapted for small sample size) or of non-vocalizing mice for USV assessment. Results Body weight Body weight was assessed once a week between 5 and 7 weeks of age (Fig.2). In males, there was an expected body weight gain with time [testing time effect: F(2,82)=982.57, p<0.0001; Fig.2-A] and this was more marked in WT mice than KOs [interaction genotype x time: F(2,82)=9.22, P<0.001]. Nonetheless, this was mainly due to the overall higher body weight of WT-stressed males, as demonstrated by separate ANOVAs showing a significant effect of stress in WT mice only [F(1,23)=4.2, p=0.05; in KO: n.s.; Fig.2-B]. A similar pattern was found in females, where body weight also increased over weeks as expected [time effect: F(2,88)=768.32, p<0.0001; Fig.2-C], and this gain did not differ between genotype or stress conditions [all interactions with time, ns]. In females also, stress increased the overall body weight, but equally in both WT and KO mice [main stress effect: F(1,44)=9.17, p<0.01; Fig.2-D]. ----------------------FIGURE 2 ABOUT HERE------------------------- Open field In males, there was no difference among experimental groups in locomotor activity in the open field [genotype, stress effects and their interaction: all n.s.; Fig. 3-A]. In females, a tendency to a decrease in locomotor activity following stress was observed in mice of both genotypes [stress effect: F(1,44)=3.87, p=0.060; Fig. 3-B]. Y-maze All male and female mice equally explored the maze arms during the habituation phase, and no differences among experimental groups were detected (data not shown). During the test phase, all males displayed spontaneous alternation, except stressed KO mice that showed a performance not significantly different from the chance level: (t=2.16, ns; in other groups, all ts>4, p<0.01; Fig.3-C). In females, none of the four experimental groups showed significant levels of spontaneous alternation (t-tests: all ns; Fig.3-D), suggesting that this cognitive ability is not sufficiently expressed in Fmr1 WT and KO female mice at this juvenile age. Social interaction In males, WT stressed mice showed higher levels of affiliative behaviors towards the WT female stimulus [interaction genotype x stress: F(1,38)=4.47, p<0.05; post-hoc: WT-no stress versus WT-stressed, p<0.05; Fig.3-E]. In females, KO mice showed enhanced levels of affiliation towards the WT female intruder compared to their WT littermates, but this genotype difference disappeared following stress, since stress tended to increase affiliative levels in WT mice [interaction genotype x stress: F(1,44)=4.19, p<0.05; post-hoc: WT-no stress versus KO-no stress, p<0.05; WT-no stress versus WT-stressed, p=0.06; Fig.3-F]. No significant effects were found for any non-social behaviors in both sexes (data not shown). ----------------------FIGURE 3 ABOUT HERE------------------------- Ultrasonic vocalizations (USVs) In males, the number of USVs and their mean duration did not differ among experimental groups [genotype, stress effects and their interaction: all ns; Fig.4-A and C]. Stress decreased the mean peak frequency in mice of both genotypes [F(1,32)=4.50, p<0.05; Fig.4-E] and contribute to the emergence of a significant genotype difference in the mean peak amplitude, due to the highest values of WT-stressed mice [interaction genotype x stress: F(1,30)=5.22, p<0.05; post-hoc: WT-no stress versus WT-stressed, p<0.05; Fig.4-G]. In females, KO mice emitted more and longer USVs compared to WT animals, and this effect was not altered by stress exposure [genotype effect on number (sqrt-transformed) and mean duration, respectively: F(1,43)= 6.65, 23.42, p<0.05 and 0.0001 (Fig. 4-B and D); all other effect and interactions: ns]. USVs produced by KO females were also characterized by a significant lower mean peak frequency [genotype effect: F(1,43)= 5.38, p<0.05; Fig.4-F] and by lower peak amplitude, but only under no stress conditions [genotype x stress interaction: F(1,41)= 4.81, p<0.05; post-hoc WT-no stress versus KO-no stress, p<0.05; Fig.4-H]. ----------------------FIGURE 4 ABOUT HERE------------------------- The inspection of the density plots (Fig.5) extended the results previously obtained from the quantitative analyses of the ultrasonic spectrograms. In both males and females, stress tended to increase the occurrence of unusual long USVs (mean duration>60msec, Fig. 5) an effect that appeared especially marked in KO mice. In KO-stressed mice there was an increased variability in the duration of the calls, an effect that was particularly dramatic in females (Fig.5 lower panel). ----------------------FIGURE 5 ABOUT HERE------------------------- The analysis of call subtypes 47 revealed no major difference in the composition of the calls emitted by males [genotype, stress effects and their interaction, all n.s.; Fig.6). In contrast, a clear genotype difference emerged in female mice, irrespectively of their stress conditions (Fig. 6). Female Fmr1-KOs emitted less simple calls, i.e., based on one or two components [genotype effects, respectively: F(1,42)=18.06 and 14.59, p<0.001], and more complex calls, i.e., containing 3, 4, 5 or more components, than their WT littermates [genotype effects, respectively: F(1,42)=57.21, 58.48, 35.16 and 26.26, p<0.0001]. This is in line with the results of the density plots, since complex calls typically correspond to longer USVs. ----------------------FIGURE 6 ABOUT HERE------------------------- Discussion Our findings highlighted the impact of several gene-environment interactions on the behavioral phenotype of juvenile Fmr1 mutant mice that varies according to the sex of the animals, as summarized by Table 1 . Overall, prenatal exposure to stress was able to induce several effects that were mostly dependent on sex differences and the considered behavioral domain. Our hypothesis, i.e., that stress exposure may advance/exacerbate the emergence of the behavioral alterations of Fmr1-KO mice was only partially confirmed, i.e., in the cognitive domain and in male mice (Table 1 ). Table 1 Summary of the results Variables measured ♂ ♀ KO genotype effect Stress effect KO genotype effect Stress effect Body Weight (Fig. 2 ) ----- ↑ only in WT ----- ↑ in WT and KO Locomotion (Fig. 3 -A, B) ----- ----- ----- ↓ in WT and KO Spontaneous alternation (Fig. 3 -C, D) ----- ↓ only in KO ----- ----- Social interaction (Fig. 3 -E, F) ----- ↑ only in WT ↑ in no stress ↑ only in WT Ultrasonic communication (Figs. 4 , 5 and 6 ) ----- ↓peak frequency in WT and KO ↑ peak amplitude only in WT ↑ call number and duration, ↓peak frequency ↑complex calls ↓ simple calls ----- ----- ↑ peak amplitude only in KO All gene-environment interactions are marked in grey; dark grey highlight refers to interactions inducing the emergence of a novel KO phenotype (i.e., different from WT) under stressed conditions. As expected from previous reports 7 , our results confirmed that the behavioral phenotype of our juvenile Fmr1-KO mutants was almost undistinguishable from their WT littermates. This is the reason why we chose this testing age as it provided the optimal baseline conditions to evaluate a potential exacerbating/anticipating impact of prenatal stress avoiding floor or ceiling effects. In male KO mice, no alteration emerged in any of the considered behavioral domains under no stress conditions, supporting the view that FXS- and ASD-like behavioral abnormalities, such as hyperactivity, cognitive deficits and social alterations, appear only at adulthood 7 . In females, an hyper-social phenotype was the only one detected in our juvenile mutants, including enhanced affiliation levels, increased number of ultrasonic calls and their duration (with qualitative alterations). Once again, these results were in agreement with our previous reports: interestingly, these communicative and social abnormalities were observed only at the juvenile age as they disappeared in adult mutant females 33 . These hyper-social phenotypes may seem surprising in view of the ASD-like alterations shown by FXS patients, consisting mostly of social avoidance and reduced social interest. Nonetheless, the more abundant and longer USVs emitted by mutant juvenile females could be also interpreted as autistic-like phenotypes, since several studies have described excessive talking and repetitive speech as major autistic communicative alterations in FXS patients [see for example 28 ]. Furthermore, our analysis of the ultrasonic call types revealed a different composition of the USV repertoire of Fmr1-KO females (Fig. 6 ), with a prevalence of complex multi-component calls compared to WT littermates. Although little is still known about the social meaning of different call types 47 , it is possible that Fmr1-KO females may emit more and longer USVs, but with less appropriate or adaptive communicative properties. The increased levels of affiliations could also be interpreted as an inappropriate social attitude since they are directed toward an intruder, i.e., a potential threat for the resident female. This testing context was indeed necessary to allow the detection of USVs in female mice 40 . It is therefore still possible that a different social phenotype may appear in a different testing context, e.g., in a neutral environment; indeed, when Fmr1 mutant juvenile females were assessed for their social interest in the three compartment test no sign of increased sociability was observed 33 . On this basis of genotype differences, prenatal exposure to stress was able to induce the appearance of a cognitive deficit in the spontaneous alternation Y maze test, although only in males. KO stressed male mice were indeed the only experimental group displaying a performance similar to the chance level (Fig. 3 -C; Table 1 ). In females, stress instead seemed to eliminate the hyper-social phenotype of mutant mice (Fig. 3 -E) without affecting their ultrasonic communication profile (Fig. 4 ). Nonetheless, these effects in females were actually due to a selective effect of stress in WT mice, rendering the WT phenotype similar to that of mutants. Hence, our data suggest that exposure to prenatal stress does not dramatically advance the appearance of pathological behavioral phenotypes in male and female mutants, juvenile stressed KO mice being mostly comparable to their WT littermates, as in no-stress conditions. Indeed, with the exception of the Y maze effect in males, no selective effect of stress on mutant behavioral phenotypes was detected (Table 1 ). Our findings suggest therefore a higher sensitivity of the cognitive domain to the effects of stress in the male sex, in line with clinical data describing a positive correlation between stress levels and cognitive deficits in FXS boys 50 . Interestingly, stress did interact with genotype on several behavioral measures, but mostly by inducing its effects in WT mice only. This may suggest a reduced sensitivity of Fmr1-KO mice to stress that could be interpreted as a deficit in the adaptive response to stressors, as already proposed by others 51 . Previous studies have indeed described a reduced behavioral and endocrine sensitivity of adult Fmr1-KO mice (though only males were investigated) to the adult exposure to chronic stressors 51 , 52 . Here the genotype-specific effects of stress were characterized by clear sex differences: in males, stress enhanced body weight (Fig. 2 -A), affiliative behaviors (Fig. 3 -E) and peak amplitude (Fig. 4 -G) in WT only, while it reduced peak frequency in both genotypes (Fig. 4 -E). In females, stress enhanced affiliative behaviors in WT only (Fig. 3 -F), while it enhanced body weight (Fig. 2 -B) and reduced locomotion in both WT and mutant mice (Fig. 3 -B). Furthermore, in female KOs stress increased USV peak frequency (Fig. 4 -F). Overall, not the magnitude, but the behavioral specificity of the effects of stress differed between sexes, in line with most of the previous reports 53 – 57 . Despite the overall agreement of the behavioral effects described in WT mice by our findings, an important difference between our and others’ studies on prenatal stress should be underlined, that is the genotype of our breeders exposed to prenatal stress. The dams exposed in our study to prenatal stress are indeed heterozygous Fmr1 mutant females and not WT as in previous similar studies: it is therefore possible that the sensitivity to stress of our female breeders may be different [as previously demonstrated for Fmr1-KO males with adult post-natal stress 51 , 52 ] and result in specific sex-dependent effects on the offspring behaviors. Studies comparing the behavioral and endocrine response to stress of Fmr1 mutant and WT dams should be performed in the future in order to clarify this issue; also, it would be interesting to evaluate the maternal behavior of stressed and no-stress dams to investigate whether the effects of stress on the Fmr1 offspring behavior could be mediated by alterations in the maternal care received. Similarly to other manipulations of the early environment [e.g., early enrichment 58 ], prenatal stress may induce its effects on the offspring both at the prenatal level, i.e., directly affecting pups’ embryonic development, and during the early post-natal phase, i.e., interfering with normal mother-pup interactions and altering maternal behaviors 59 . In conclusion, our findings demonstrate for the first time the impact of prenatal stress on the juvenile FXS- and ASD-like behavioral phenotype of Fmr1 mice, underlying the relevance of including sex differences and assessing multiple behavioral domains in mouse studies on FXS and ASD. These data therefore highlight the importance of complex gene-environment interactions in the etiopathology of neurodevelopmental disorders, also for a syndrome of clear genetic origins, such as FXS. Our results focused on the juvenile age, which is critical for the early detection of behavioral abnormalities and their early therapeutic rescuing; this research focus could be extended in future studies by investigating the effects of prenatal stress on a longer term, for instance on the behavioral phenotype of Fmr1 mice at the adult age, i.e., when the behavioral alterations of mutants are more marked and well-established. Declarations ACKNOWLEDGEMENTS This work was supported by CNRS and the University of Bordeaux. S. Pietropaolo and V. Petroni received funding from the Fondation pour l’Audition (FPA-RD-2020-8) and the Association Autour de Williams. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflict of interest to report in relation to the work described. DATA AVAILABILITY STATEMENT The datasets used and analysed during the current study are available from the corresponding author on reasonable request. References Hagerman, R. J. & Hagerman, P. J. Fragile X syndrome: Diagnosis, treatment, and research . (Taylor & Francis US, 2002). Bailey, D. B., Jr. et al. Autistic behavior in young boys with fragile X syndrome. J Autism Dev Disord 28 , 499-508 (1998). Hagerman, R. J. Lessons from fragile X regarding neurobiology, autism, and neurodegeneration. J Dev Behav Pediatr 27 , 63-74 (2006). 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Effect of the fragile X status categories and the fragile X mental retardation protein levels on executive functioning in males and females with fragile X. Neuropsychology 17 , 646-657 (2003). Mazzocco, M. M., Kates, W. R., Baumgardner, T. L., Freund, L. S. & Reiss, A. L. Autistic behaviors among girls with fragile X syndrome. J Autism Dev Disord 27 , 415-435 (1997). Gaudissard, J. et al. Behavioral abnormalities in the Fmr1-KO2 mouse model of fragile X syndrome: The relevance of early life phases. Autism Res 10 , 1584-1596, doi:10.1002/aur.1814 (2017). Gauducheau, M. et al. Age-specific autistic-like behaviors in heterozygous Fmr1-KO female mice. Autism Res 10 , 1067-1078, doi:10.1002/aur.1743 (2017). Spear, L. P. The adolescent brain and age-related behavioral manifestations. Neurosci Biobehav Rev 24 , 417-463 (2000). Terranova, M. L., Laviola, G. & Alleva, E. Ontogeny of amicable social behavior in the mouse: gender differences and ongoing isolation outcomes. Dev Psychobiol 26 , 467-481 (1993). Negroni, J. et al. Chronic ultra-mild stress improves locomotor performance of B6D2F1 mice in a motor risk situation. Behav Brain Res 155 , 265-273, doi:10.1016/j.bbr.2004.04.023 (2004). Pardon, M., Perez-Diaz, F., Joubert, C. & Cohen-Salmon, C. Age-dependent effects of a chronic ultramild stress procedure on open-field behaviour in B6D2F1 female mice. Physiol Behav 70 , 7-13, doi:10.1016/s0031-9384(00)00216-x (2000). Pardon, M. C., Perez-Diaz, F., Joubert, C. & Cohen-Salmon, C. Influence of a chronic ultramild stress procedure on decision-making in mice. J Psychiatry Neurosci 25 , 167-177 (2000). Moles, A. & D'Amato F, R. Ultrasonic vocalization by female mice in the presence of a conspecific carrying food cues. Anim Behav 60 , 689-694 (2000). Moles, A., Costantini, F., Garbugino, L., Zanettini, C. & D'Amato, F. R. Ultrasonic vocalizations emitted during dyadic interactions in female mice: a possible index of sociability? Behav Brain Res 182 , 223-230 (2007). Wang, H., Liang, S., Burgdorf, J., Wess, J. & Yeomans, J. Ultrasonic vocalizations induced by sex and amphetamine in M2, M4, M5 muscarinic and D2 dopamine receptor knockout mice. PLoS One 3 , e1893 (2008). Warburton, V. L., Sales, G. D. & Milligan, S. R. The emission and elicitation of mouse ultrasonic vocalizations: the effects of age, sex and gonadal status. Physiol Behav 45 , 41-47 (1989). Whitney, G., Coble, J. R., Stockton, M. D. & Tilson, E. F. Ultrasonic emissions: do they facilitate courtship of mice. J Comp Physiol Psychol 84 , 445-452 (1973). Maggio, J. C. & Whitney, G. Ultrasonic vocalizing by adult female mice (Mus musculus). J Comp Psychol 99 , 420-436 (1985). Wohr, M. Ultrasonic vocalizations in Shank mouse models for autism spectrum disorders: detailed spectrographic analyses and developmental profiles. Neurosci Biobehav Rev 43 , 199-212, doi:10.1016/j.neubiorev.2014.03.021 (2014). Mosienko, V., Beis, D., Alenina, N. & Wohr, M. Reduced isolation-induced pup ultrasonic communication in mouse pups lacking brain serotonin. Mol Autism 6 , 13, doi:10.1186/s13229-015-0003-6 (2015). Caruso, A., Ricceri, L. & Scattoni, M. L. Ultrasonic vocalizations as a fundamental tool for early and adult behavioral phenotyping of Autism Spectrum Disorder rodent models. Neurosci Biobehav Rev 116 , 31-43, doi:10.1016/j.neubiorev.2020.06.011 (2020). Caligioni, C. S. Assessing reproductive status/stages in mice. Curr Protoc Neurosci Appendix 4 , Appendix 4I, doi:10.1002/0471142301.nsa04is48 (2009). Vandesquille, M. et al. Working memory deficits and related disinhibition of the cAMP/PKA/CREB are alleviated by prefrontal alpha4beta2*-nAChRs stimulation in aged mice. Neurobiol Aging 34 , 1599-1609 (2013). Scherr, J. F., Hahn, L. J., Hooper, S. R., Hatton, D. & Roberts, J. E. HPA axis function predicts development of working memory in boys with FXS. Brain and cognition 102 , 80-90, doi:10.1016/j.bandc.2015.12.002 (2016). Qin, M., Xia, Z., Huang, T. & Smith, C. B. Effects of chronic immobilization stress on anxiety-like behavior and basolateral amygdala morphology in Fmr1 knockout mice. Neuroscience 194 , 282-290 (2011). Lemaire-Mayo, V., Subashi, E., Henkous, N., Beracochea, D. & Pietropaolo, S. Behavioral effects of chronic stress in the Fmr1 mouse model for fragile X syndrome. Behav Brain Res 320 , 128-135, doi:10.1016/j.bbr.2016.11.051 (2017). Advani, T., Koek, W. & Hensler, J. G. Gender differences in the enhanced vulnerability of BDNF+/- mice to mild stress. Int J Neuropsychopharmacol 12 , 583-588, doi:10.1017/S1461145709000248 (2009). Hodes, G. E. et al. Sex Differences in Nucleus Accumbens Transcriptome Profiles Associated with Susceptibility versus Resilience to Subchronic Variable Stress. J Neurosci 35 , 16362-16376, doi:10.1523/JNEUROSCI.1392-15.2015 (2015). Meng, F. et al. Brain-derived neurotrophic factor in 5-HT neurons regulates susceptibility to depression-related behaviors induced by subchronic unpredictable stress. J Psychiatr Res 126 , 55-66, doi:10.1016/j.jpsychires.2020.05.003 (2020). Mueller, B. R. & Bale, T. L. Early prenatal stress impact on coping strategies and learning performance is sex dependent. Physiol Behav 91 , 55-65, doi:10.1016/j.physbeh.2007.01.017 (2007). Schwendener, S., Meyer, U. & Feldon, J. Deficient maternal care resulting from immunological stress during pregnancy is associated with a sex-dependent enhancement of conditioned fear in the offspring. J Neurodev Disord 1 , 15-32, doi:10.1007/s11689-008-9000-9 (2009). Branchi, I. The mouse communal nest: investigating the epigenetic influences of the early social environment on brain and behavior development. Neurosci Biobehav Rev 33 , 551-559 (2009). Moles, A., Rizzi, R. & D'Amato, F. R. Postnatal stress in mice: does "stressing" the mother have the same effect as "stressing" the pups? Dev Psychobiol 44 , 230-237 (2004). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 17 Mar, 2022 Reviews received at journal 13 Mar, 2022 Reviewers agreed at journal 07 Mar, 2022 Reviewers invited by journal 02 Mar, 2022 Editor assigned by journal 02 Mar, 2022 Editor invited by journal 23 Feb, 2022 Submission checks completed at journal 23 Feb, 2022 First submitted to journal 18 Feb, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1372695","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":85999438,"identity":"b9e47461-5c01-4ad9-a07e-f95c7cbb8ec2","order_by":0,"name":"Susanna Pietropaolo","email":"data:image/png;base64,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","orcid":"","institution":"INCIA, UMR5287, Bordeaux University and CNRS","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Susanna","middleName":"","lastName":"Pietropaolo","suffix":""},{"id":85999432,"identity":"be17fea3-9c15-49df-be63-76546fad18ce","order_by":1,"name":"Valeria Petroni","email":"","orcid":"","institution":"INCIA, UMR5287, Bordeaux University and CNRS","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Valeria","middleName":"","lastName":"Petroni","suffix":""},{"id":85999433,"identity":"13c9421e-a520-4b4c-9a32-f980c5630627","order_by":2,"name":"Enejda Subashi","email":"","orcid":"","institution":"INCIA, UMR5287, Bordeaux University and CNRS","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Enejda","middleName":"","lastName":"Subashi","suffix":""},{"id":85999434,"identity":"5e84ce28-ae3e-4542-811d-ad2e710dd8e5","order_by":3,"name":"Marika Premoli","email":"","orcid":"","institution":"University of Brescia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marika","middleName":"","lastName":"Premoli","suffix":""},{"id":85999435,"identity":"b346c6a8-004e-45f5-a1a1-a34e883fa55e","order_by":4,"name":"Markus Wöhr","email":"","orcid":"","institution":"Philipps-University of Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Markus","middleName":"","lastName":"Wöhr","suffix":""},{"id":85999436,"identity":"3a9858fb-846e-4148-ba7f-a5f39348d62c","order_by":5,"name":"Wim E Crusio","email":"","orcid":"","institution":"INCIA, UMR5287, Bordeaux University and CNRS","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wim","middleName":"E","lastName":"Crusio","suffix":""},{"id":85999437,"identity":"f38b45fb-e16f-47ab-8bb0-523357e83524","order_by":6,"name":"Valerie Lemaire","email":"","orcid":"","institution":"INCIA, UMR5287, Bordeaux University and CNRS","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Valerie","middleName":"","lastName":"Lemaire","suffix":""}],"badges":[],"createdAt":"2022-02-18 10:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1372695/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1372695/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18624856,"identity":"777a1867-c08d-44ec-a04f-0c7c59c6cccc","added_by":"auto","created_at":"2022-02-25 17:47:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":163774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the experimental design of the study and its timeline. \u003c/strong\u003eUnpredictable mild stress consisted of\u003cstrong\u003e \u003c/strong\u003ethe following 2 day-sequence that was repeated three consecutive times during the last week of gestation: on day 1, 3 sessions of 30-min restrain stress during the light phase, at 4h intervals were followed by overnight housing with wet bedding, while on day 2, 3 sessions of sawdust and cage changes during the light phase, at 4h-intervals, were followed by overnight housing with novel glass black beads. Control mice were left undisturbed during all pregnancy. Behavioral tests were conducted between 7 and 8 weeks of age, with 48hs interval between consecutive tests. GD=gestational day; PND=postnatal day; BW=body weight; OF=open field; YM=Y maze; SI=social interaction; USVs=ultrasonic vocalizations.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/9f5329769a39d8a3691dbc1a.jpg"},{"id":18624654,"identity":"40589d3d-ef37-43b4-b980-facf318c55ab","added_by":"auto","created_at":"2022-02-25 17:44:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":176005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of prenatal stress in juvenile mice on body weight. \u003c/strong\u003eBody weight was assessed during the last two weeks before behavioral testing, i.e., at 7 weeks of age. Time course illustrates the expected weight gain in males and females (A-C), while overall group differences are shown by the mean weight values averaged across time-points in each sex (B-D). * p\u0026lt;0.05. N for males: 15 WT-no stress, 10 WT-stress, 9 KO-no stress, 11 KO-stress; N for females: 12 in all groups. KO refers to -/Y in males, +/- in females. Data are expressed as mean±SEM.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/e6cc22be5df94beffeb4409e.jpg"},{"id":18624659,"identity":"fee32665-50b3-47e1-8905-a7a198b52312","added_by":"auto","created_at":"2022-02-25 17:44:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":347112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBehavioral effects of prenatal stress in juvenile mice. \u003c/strong\u003eLocomotion was assessed in the open field test (A-B), while spontaneous alternation was evaluated in the Y maze (C-D). Social interaction was measured during a 3-min encounter with an adult NMRI WT female (E-F). * p\u0026lt;0.05; * p=0.06; # p\u0026lt;0.05 versus chance level (indicated by dotted line). N for males: 14 (A and E) or 13 (C) WT-no stress, 9 (A, C and E) WT-stress, 8 (A and E) or 9 (C) KO-no stress, 11 (A and E) or 10 (C) KO-stress; N for females: 12 in all groups (B, D, F). KO refers to -/Y in males, +/- in females. Data are expressed as mean±SEM.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/517fc5ce42dfa29d3acaf34f.jpg"},{"id":18624857,"identity":"03673c84-1945-4d53-88a6-b8e2d2e38982","added_by":"auto","created_at":"2022-02-25 17:47:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":370134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of prenatal stress on ultrasonic communication in juvenile mice. \u003c/strong\u003eUltrasonic vocalizations (USVs) were assessed during the direct social interaction test with an adult NMRI WT female in Fmr1 mice of both sexes. The following parameters were measured through spectrographic analysis of the calls: total number (A-B), mean duration (C-D), mean peak frequency (E-F) and amplitude (G-H). The number of the calls was subjected to square-root (sqrt) transformation in order to meet the normality assumptions of parametric ANOVA. * p\u0026lt;0.05. N for males: 10 (A,C,E,G) WT-no stress, 8 (A, C and E) and 7 (G) WT-stress, 8 (A, C and E) and 11 (G) KO-no stress, 10 (A,C,E,G) KO-stress; N for females: 11 (B,D,F,H) WT-no stress, 12 (B,D,F,H) WT-stress, 12 (B, D and F) and 11 (H) KO-no stress, 12 (B, D and F) and 11 (H)\u0026nbsp;KO-stress. KO refers to -/Y in males, +/- in females. Data are expressed as mean±SEM.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/278ca1d24a86c99bb41f099c.jpg"},{"id":18624657,"identity":"0e3122ee-6d1c-4926-8241-b5fb031ec8be","added_by":"auto","created_at":"2022-02-25 17:44:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":427420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDensity plots of individual ultrasonic calls.\u0026nbsp;\u003c/strong\u003eDensity plots depict the distribution of individual USV emitted during 3-min social interaction with a NMRI adult stimulus female, plotted by frequency in kHz and duration in ms. Color coding reflects frequency in percentages.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/0bc7400e439f6d9297be9b06.jpg"},{"id":18624656,"identity":"dcf7340b-a2ac-4cb8-84e1-3f88facc0b10","added_by":"auto","created_at":"2022-02-25 17:44:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":651263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComposition of ultrasonic call types. \u003c/strong\u003ePie charts of different call types automatically classified by Sonotrack software. Call categories are expressed as percentages over the total number of USVs for each experimental group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/7b0be825dc023392eb47b30e.jpg"},{"id":18923253,"identity":"e57ccd80-00b5-4602-a68b-1e27a630e1a6","added_by":"auto","created_at":"2022-03-07 14:38:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1051232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1372695/v1/c4c00021-441a-4b11-babe-8d93f6eba1dc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFragile X syndrome (FXS) is a neurodevelopmental disorder characterized by multiple behavioral alterations, including mental retardation, hyperactivity, anxiety, cognitive and social deficits \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Autistic symptoms, including altered social interaction and communication, are also often detected in FXS patients \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e: FXS is indeed considered as the most common monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 human gene consisting in more than 250 CGG repetitions leading to the absence of FMRP protein \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e playing a major role in synaptic and neuronal functionality \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The lack of FMRP has been recapitulated by the Fmr1-KO mouse model of FXS together with several relevant behavioral alterations \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The FXS-like behavioral phenotypes of mutant mice are mostly evident at adulthood, i.e., at 3\u0026ndash;6 months, that is, when most pre-clinical studies are carried out [as reviewed in \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e].\u003c/p\u003e \u003cp\u003eDespite its clear and well-defined genetic origins, the FXS behavioral phenotype can be critically modulated by environmental factors, both in terms of its severity and of the timing of appearance. Environmental stimulation is for instance known to attenuate/delay the expression of behavioral alterations both in FXS patients and Fmr1-KO mice \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Conversely, exposure to stressful life events may exacerbate the behavioral deficits of FXS patients \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e especially when occurring during early life phases. Exposure to prenatal stress is a powerful tool to induce early adversity in a genetic mouse model and therefore to study the impact of gene-environment interactions in the expression of its behavioral phenotype. Indeed, prenatal stress exacerbates the behavioral alterations of genetic mouse models of Alzheimer disease, depression and schizophrenia \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Surprisingly, to our knowledge, the behavioral effects of prenatal stress have never been investigated in the Fmr1-KO mouse, or in other models of ASD.\u003c/p\u003e \u003cp\u003eFurthermore, prenatal stress is known to induce marked long-term behavioral alterations in wild-type rodents, including cognitive, emotional, motor and social abnormalities [reviewed in \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e]. These studies have pointed out in particular the relevance of the unpredictable chronic mild stress procedure, as the most suitable experimental approach to model early environmental adversity in laboratory rodents \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This procedure, combining multiple stressors of different nature, has also the advantage to minimize habituation and exclude pain or nutritional effects \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In most existing preclinical studies [reviewed in \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e] stress exposure was implemented during the last week of gestation of the dams, as this phase is a preferential target to induce long-term brain and behavioral modifications in the offspring, because of its high environmental and stress sensitivity \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe inclusion of mice of both sexes in the behavioral analysis of the offspring is considered of critical relevance for preclinical studies on prenatal stress exposure. Several sex differences have been indeed described in the behavioral response to stress in rodents; these include differences in the severity of stress effects, but also in their specificity to selected behavioral domains \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The inclusion of subjects of both sexes is also important for studying FXS, both in human and preclinical research. Although FXS is more common in boys than girls, increasing attention has been devoted to heterozygous females, as they are the ones producing the affected offspring \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and they represent the majority of FXS female patients, as homozygous \u003cem\u003eFMR1\u003c/em\u003e mutations are extremely rare \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In humans, FXS female carriers present several behavioral symptoms, including hyperactivity \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, mild cognitive impairments \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and autistic behaviors \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In mice, similar behavioral abnormalities were described in Fmr1 mutant females, especially at adulthood [as reviewed in \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e].\u003c/p\u003e \u003cp\u003eHere we therefore evaluated whether exposure to unpredictable chronic mild stress during the last prenatal week could advance and/or exacerbate the juvenile behavioral phenotype of Fmr1-KO offspring of both sexes (as schematized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To this end, Fmr1-KO male (hemizygous, -/Y) and female (heterozygous, +/-) mice, together with their WT littermates, underwent behavioral tests for exploration, spatial memory, social interaction and communication at the juvenile age of 7\u0026ndash;8 weeks, i.e., when most of the FXS-like behavioral alterations are absent or mild. At this age, Fmr1-KO males do not show any remarkable behavioral phenotype in the considered domains \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, while mutant females displayed mild alterations in social interaction and communication \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. This age partially overlaps with adolescence (occurring between 3 and 8 weeks of age in mice), a critical phase for brain and behavioral development in rodents and humans and largely involved in several neuropsychiatric disorders \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This phase has been also extensively studied for the expression of social behaviors in laboratory mice, with a special emphasis on the post-pubertal phase (i.e., approximately after the 5 weeks of age), since it is characterized by important changes in the patterns of intra-specific social interactions \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Late adolescence (7\u0026ndash;9 weeks) is also of particular interest, since most behavioral abilities are already well developed in mice; it is therefore suitable to multiple behavioral testing, performing the same cognitive, emotional, and social tests done in adult mice and hence facilitating comparisons with data from adult subjects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eAll experimental procedures were in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003c/span\u003e), European Communities Council Di-rective 2010/63/EEC. Furthermore, there were approved by local ethical committee (\u0026ldquo;Comit\u0026eacute; d\u0026rsquo;Ethique pour l\u0026rsquo;experimentation animale de Bordeaux\u0026rdquo;, CE 50) and the French Ministry (\u0026ldquo;Ministere de l\u0026rsquo;enseignement superieur de la recherch\u0026eacute; et de l\u0026rsquo;innovation\u0026rdquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBreeding and stress procedure\u003c/h2\u003e \u003cp\u003eTwenty adult (12\u0026thinsp;\u0026plusmn;\u0026thinsp;1 weeks-old) virgin Fmr1 heterozygous (+/-) females and 10 C57BL/6J adult wild type males [16 weeks-old; purchased from Janvier (Le Genest St Isle, France)] were used as breeders to generate the tested offspring. C57BL/6JFmr1\u003csup\u003etm1Cgr/Nwu\u003c/sup\u003e (B6) mice were originally obtained from Neuromice.org (Northwestern University) and maintained on the C57BL6/J background for more than 10 generations.\u003c/p\u003e \u003cp\u003eThey were bred as described previously\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Each half of the female breeders was assigned to one of the following groups in which they were kept during the last week of pregnancy: no-stress, i.e., kept undisturbed in their home-cage, or stress, i.e., exposed to the unpredictable stress procedure described below.\u003c/p\u003e \u003cp\u003eThe time line of the study is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The stress procedure included the following 2-day sequence of events that was repeated three consecutive times during the last week of gestation:\u003c/p\u003e \u003cp\u003eDay 1: 30 minutes of restrain stress (3 times each day during the light phase, with a 4h-interval) in perforated conical tubes (3cm in diameter, 11.5cm long; Becton Dickinson Labware Europe, France), followed by overnight housing with wet bedding (50ml of water were added to floor sawdust of the home cage at the beginning of the dark phase).\u003c/p\u003e \u003cp\u003eDay 2: multiple sawdust and cage changes (3 times each day during the light phase, with a 4h-interval), followed by overnight housing with novel objects (12 glass black beads, 1.5cm in diameter were added in the home cage at the beginning of the dark phase).\u003c/p\u003e \u003cp\u003ePregnant females were exposed to this sequence of events for 3 times during the last week before parturition: this procedure is based on previous studies [e.g., \u003csup\u003e23,36\u0026minus;38\u003c/sup\u003e] and it is known to limit the habituation to stressful stimuli without using pain or nutritional manipulations. All breeders used for the study gave birth within 48hs after the last day of exposure to stress procedure. They were left undisturbed until weaning of the pups, i.e., on post-natal day (PND) 21.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and housing procedures\u003c/h2\u003e \u003cp\u003eAt 3 weeks of age, all pups were weaned and housed in same-sex groups of 3\u0026ndash;5 littermates in our animal facility \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. On the same day, tail samples were collected for DNA extraction and subsequent PCR assessment of the genotypes as previously described \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Mice were then left undisturbed until the beginning of behavioral testing (i.e., at 7 weeks of age), except for the evaluation of body weight that was carried out once a week starting at 5 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only litters including males and females of both mutant (KO) and wild-type (WT) genotypes were used for experiments, for a total of 14 litters. A total of 93 mice were subjected to behavioral testing: 45 males [25 WT and 20 KO (-/Y), n\u0026thinsp;=\u0026thinsp;9\u0026ndash;15 for stress condition) and 48 females [24 WT and 24 KO (+/-), n\u0026thinsp;=\u0026thinsp;12 for stress condition].\u003c/p\u003e \u003cp\u003eStimulus mice used for the direct social interaction test were adult (10 weeks of age) female NMRI mice, as this strain is commonly employed in social studies \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, especially those using the Fmr1-KO mouse model \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. This strain is often chosen since it is characterized by high levels of sociability, and it facilitates the behavioral analysis during social encounters with B6 mutants because of its albino phenotype. NMRI mice were purchased from Janvier (Le Genest-Saint-Isle, France), housed in groups of 3\u0026ndash;4 per cage and left undisturbed for 2 weeks before being used in behavioral tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral testing procedures\u003c/h2\u003e \u003cp\u003eBehavioral tests commenced at 7 weeks of age and were conducted as follows (see also Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On day 1, an open field test for locomotion and exploration was administered, followed on day 3 by a spontaneous alternation test in a Y-maze, and on day 5 by a direct social interaction test and the females\u0026rsquo; estrous cycle assessment. All behavioral tests were carried out during the light phase of the cycle (between 9 a.m. and 4 p.m.) by an experimenter who was blind to the group assignment of the subjects. All mice were habituated to the experimental room for at least 30 min before the beginning of each behavioral test.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e----------------------FIGURE 1 ABOUT HERE-------------------------\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section4\"\u003e \u003ch2\u003eOpen field\u003c/h2\u003e \u003cp\u003eThe open field \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e consisted of a white plastic arena where the locomotion of each mouse was assessed during 10 minutes using automated tracking (Ethovision, Noldus, The Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003eY maze\u003c/h2\u003e \u003cp\u003eThe Y maze test (described in details before \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e) was employed to assess spontaneous alternation through a 2-trial procedure, consisting of a 5-min habituation trial, followed by a 2-min test trial. Time spent in each arm during the habituation and testing phases was scored by automatic tracking and percent alternation rates during the test phase were derived as follows: 100 \u0026times; (time in novel arm/time in all arms).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003eSocial interaction and ultrasonic communication\u003c/h2\u003e \u003cp\u003eMale experimental subjects were habituated to the testing apparatus \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e for 30 min prior to testing, while female subjects were isolated in the testing cage for 72hs, in order to induce a status of resident in adult females and therefore promote the emission of ultrasonic vocalizations (USVs) towards an adult female intruder \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. An unfamiliar stimulus female mouse (an adult NMRI female) was then introduced into the testing cage of either male or female subjects and left there for 3 min. Previous studies have shown that in these experimental settings USVs are mainly emitted by the male mouse in the male-female interaction \u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and by the female resident in the female-female interaction \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTesting sessions were recorded by a camera placed on the side of the cage and videos analyzed with Observer XT (Noldus, The Netherlands). One observer who was unaware of the genotype and sex of the animals scored the behavior of the test mice, quantifying the time spent performing affiliative behaviors \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and nonsocial activities as described in details before \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn ultrasonic microphone UltraSoundGate Condenser Microphone CM 16 (Avisoft Bioacoustics, Berlin, Germany) was mounted 2 cm above the cover of the testing cage. Recordings were then analyzed through Avisoft SASLab Pro (Version 5.20; Avisoft, Berlin, Germany) to compute the number of USVs as well as their mean duration, peak frequency and peak amplitude \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition density plots depicting the distribution of total calls for each genotype at peak frequency versus duration were obtained as described in details elsewhere \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Call subtypes were also determined for a more detailed qualitative analysis; for this purpose, USVs were automatically classified using the Sonotrack Call Classification Software (version 1.4.7, Metris B.V., The Netherlands), using categories previously described in details elsewhere \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe estrus phase of female mice was assessed by analysis of vaginal smears\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e performed on the testing day in both the experimental subjects and NMRI stimulus mice. The evaluation of Fmr1 WT and KO (+/-) females used as experimental subjects was conducted after their testing, in order to minimize the potential stress effects of the manipulation necessary for determining the estrous phase. Stimulus NMRI females were approximately half in diestrus and half in estrus phases, and their assignment to social encounters was equally distributed between experimental groups. The estrus phase of experimental female subjects included pro-estrus, estrus and diestrus, following a distribution that was balanced across genotypes and stress conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were separately analyzed in males and females. This was due to sex differences in (i) the X-linked Fmr1-mutation (i.e., hemizygous in males, heterozygous in females), (ii) in some behavioral testing procedures (such as different duration of pre-testing isolation necessary for USV assessment), (iii) in most of the behavioral phenotypes measured here. The latter sex differences were further confirmed in our data set, through a preliminary ANOVA showing overall sex effects in basically all measured variables (data not shown).\u003c/p\u003e \u003cp\u003eData from each sex were analyzed with a 2x2 ANOVA with genotype and stress as the between subject factors. Within-subject factors were included when appropriate (e.g., testing time for body weight). Alternation rates from the Y-maze test were instead analyzed for differences from the chance level (with a t-test), in line with previous studies \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Post-hoc comparisons were performed using Fisher\u0026rsquo;s LSD test when a significant interaction was detected. Separate ANOVAs were also conducted when appropriate. Data from the density plots of ultrasonic calls did not undergo statistical analysis, but were used to obtain a qualitative three-dimensional evaluation of USV data \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnalyses were conducted using the software Statview and SPSS and α was set at 0.05. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM throughout the text. The exact number of mice is indicated in the legend of each figure; differences may be due to technical reasons (e.g., loss of behavioral video recordings) or to the exclusion of outliers (using Grubbs' ESD test adapted for small sample size) or of non-vocalizing mice for USV assessment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cem\u003eBody weight\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eBody weight was assessed once a week between 5 and 7 weeks of age (Fig.2). In males, there was an expected body weight gain with time [testing time effect: F(2,82)=982.57, p\u0026lt;0.0001; Fig.2-A] and this was more marked in WT mice than KOs [interaction genotype x time: F(2,82)=9.22, P\u0026lt;0.001]. Nonetheless, this was mainly due to the overall higher body weight of WT-stressed males, as demonstrated by separate ANOVAs showing a significant effect of stress in WT mice only [F(1,23)=4.2, p=0.05; in KO: n.s.; Fig.2-B]. A similar pattern was found in females, where body weight also increased over weeks as expected [time effect: F(2,88)=768.32, p\u0026lt;0.0001; Fig.2-C], and this gain did not differ between genotype or stress conditions [all interactions with time, ns]. In females also, stress increased the overall body weight, but equally in both WT and KO mice [main stress effect: F(1,44)=9.17, p\u0026lt;0.01; Fig.2-D].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e----------------------FIGURE 2 ABOUT HERE-------------------------\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eOpen field\u0026nbsp;\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn males, there was no difference among experimental groups in locomotor activity in the open field [genotype, stress effects and their interaction: all n.s.; Fig. 3-A]. In females, a tendency to a decrease in locomotor activity following stress was observed in mice of both genotypes [stress effect: F(1,44)=3.87, p=0.060; Fig. 3-B].\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eY-maze\u0026nbsp;\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eAll male and female mice equally explored the maze arms during the habituation phase, and no differences among experimental groups were detected (data not shown). During the test phase, all males displayed spontaneous alternation, except stressed KO mice that showed a performance not significantly different from the chance level: (t=2.16, ns; in other groups, all ts\u0026gt;4, p\u0026lt;0.01; Fig.3-C). In females, none of the four experimental groups showed significant levels of spontaneous alternation (t-tests: all ns; Fig.3-D), suggesting that this cognitive ability is not sufficiently expressed in \u003cem\u003eFmr1\u003c/em\u003e WT and KO female mice at this juvenile age.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eSocial interaction\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn males, WT stressed mice showed higher levels of affiliative behaviors towards the WT female stimulus [interaction genotype x stress: F(1,38)=4.47, p\u0026lt;0.05; post-hoc: WT-no stress versus WT-stressed, p\u0026lt;0.05; Fig.3-E]. In females, KO mice showed enhanced levels of affiliation towards the WT female intruder compared to their WT littermates, but this genotype difference disappeared following stress, since stress tended to increase affiliative levels in WT mice [interaction genotype x stress: F(1,44)=4.19, p\u0026lt;0.05; post-hoc: WT-no stress versus KO-no stress, p\u0026lt;0.05; WT-no stress versus WT-stressed, p=0.06; Fig.3-F]. No significant effects were found for any non-social behaviors in both sexes (data not shown).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e----------------------FIGURE 3 ABOUT HERE-------------------------\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eUltrasonic vocalizations (USVs)\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn males, the number of USVs and their mean duration did not differ among experimental groups [genotype, stress effects and their interaction: all ns; Fig.4-A and C]. Stress decreased the mean peak frequency in mice of both genotypes [F(1,32)=4.50, p\u0026lt;0.05; Fig.4-E] and contribute to the emergence of a significant genotype difference in the mean peak amplitude, due to the highest values of WT-stressed mice [interaction genotype x stress: F(1,30)=5.22, p\u0026lt;0.05; post-hoc: WT-no stress versus WT-stressed, p\u0026lt;0.05; Fig.4-G]. In females, KO mice emitted more and longer USVs compared to WT animals, and this effect was not altered by stress exposure [genotype effect on number (sqrt-transformed) and mean duration, respectively: F(1,43)= 6.65, 23.42, p\u0026lt;0.05 and 0.0001 (Fig. 4-B and D); all other effect and interactions: ns]. USVs produced by KO females were also characterized by a significant lower mean peak frequency [genotype effect: F(1,43)= 5.38, p\u0026lt;0.05; Fig.4-F] and by lower peak amplitude, but only under no stress conditions [genotype x stress interaction: F(1,41)= 4.81, p\u0026lt;0.05; post-hoc WT-no stress versus KO-no stress, p\u0026lt;0.05; Fig.4-H].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e----------------------FIGURE 4 ABOUT HERE-------------------------\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe inspection of the density plots (Fig.5) extended the results previously obtained from the quantitative analyses of the ultrasonic spectrograms. In both males and females, stress tended to increase the occurrence of unusual long USVs (mean duration\u0026gt;60msec, Fig. 5) an effect that appeared especially marked in KO mice. In KO-stressed mice there was an increased variability in the duration of the calls, an effect that was particularly dramatic in females (Fig.5 lower panel).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e----------------------FIGURE 5 ABOUT HERE-------------------------\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of call subtypes \u003csup\u003e47\u003c/sup\u003e revealed no major difference in the composition of the calls emitted by males [genotype, stress effects and their interaction, all n.s.; Fig.6). In contrast, a clear genotype difference emerged in female mice, irrespectively of their stress conditions (Fig. 6). Female Fmr1-KOs emitted less simple calls, i.e., based on one or two components [genotype effects, respectively: F(1,42)=18.06 and 14.59, p\u0026lt;0.001], and more complex calls, i.e., containing 3, 4, 5 or more components, than their WT littermates [genotype effects, respectively: F(1,42)=57.21, 58.48, 35.16 and 26.26, p\u0026lt;0.0001]. This is in line with the results of the density plots, since complex calls typically correspond to longer USVs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e----------------------FIGURE 6 ABOUT HERE-------------------------\u003c/em\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings highlighted the impact of several gene-environment interactions on the behavioral phenotype of juvenile Fmr1 mutant mice that varies according to the sex of the animals, as summarized by Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Overall, prenatal exposure to stress was able to induce several effects that were mostly dependent on sex differences and the considered behavioral domain. Our hypothesis, i.e., that stress exposure may advance/exacerbate the emergence of the behavioral alterations of Fmr1-KO mice was only partially confirmed, i.e., in the cognitive domain and in male mice (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of the results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eVariables measured\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e♂\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003e♀\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eKO genotype effect\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eStress effect\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eKO genotype effect\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eStress effect\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eBody Weight\u003c/p\u003e\n \u003cp\u003e(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003eonly in WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003e in WT and KO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLocomotion\u003c/p\u003e\n \u003cp\u003e(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-A, B)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026darr;\u003c/strong\u003ein WT and KO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eSpontaneous alternation\u003c/p\u003e\n \u003cp\u003e(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-C, D)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026darr;\u003c/strong\u003eonly in KO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eSocial interaction (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-E, F)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003eonly in WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003e in no stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003e only in WT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eUltrasonic communication\u003c/p\u003e\n \u003cp\u003e(Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026darr;peak frequency in WT and KO\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003e peak amplitude only in WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026uarr;\u003c/strong\u003ecall number and duration,\u003c/p\u003e\n \u003cp\u003e\u0026darr;peak frequency\u003c/p\u003e\n \u003cp\u003e\u0026uarr;complex calls\u003c/p\u003e\n \u003cp\u003e\u0026darr; simple calls\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003cp\u003e\u0026uarr; peak amplitude only in KO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"17\"\u003eAll gene-environment interactions are marked in grey; dark grey highlight refers to interactions inducing the emergence of a novel KO phenotype (i.e., different from WT) under stressed conditions.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eAs expected from previous reports \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, our results confirmed that the behavioral phenotype of our juvenile Fmr1-KO mutants was almost undistinguishable from their WT littermates. This is the reason why we chose this testing age as it provided the optimal baseline conditions to evaluate a potential exacerbating/anticipating impact of prenatal stress avoiding floor or ceiling effects. In male KO mice, no alteration emerged in any of the considered behavioral domains under no stress conditions, supporting the view that FXS- and ASD-like behavioral abnormalities, such as hyperactivity, cognitive deficits and social alterations, appear only at adulthood \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In females, an hyper-social phenotype was the only one detected in our juvenile mutants, including enhanced affiliation levels, increased number of ultrasonic calls and their duration (with qualitative alterations). Once again, these results were in agreement with our previous reports: interestingly, these communicative and social abnormalities were observed only at the juvenile age as they disappeared in adult mutant females \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These hyper-social phenotypes may seem surprising in view of the ASD-like alterations shown by FXS patients, consisting mostly of social avoidance and reduced social interest. Nonetheless, the more abundant and longer USVs emitted by mutant juvenile females could be also interpreted as autistic-like phenotypes, since several studies have described excessive talking and repetitive speech as major autistic communicative alterations in FXS patients [see for example \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e]. Furthermore, our analysis of the ultrasonic call types revealed a different composition of the USV repertoire of Fmr1-KO females (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), with a prevalence of complex multi-component calls compared to WT littermates. Although little is still known about the social meaning of different call types \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, it is possible that Fmr1-KO females may emit more and longer USVs, but with less appropriate or adaptive communicative properties. The increased levels of affiliations could also be interpreted as an inappropriate social attitude since they are directed toward an intruder, i.e., a potential threat for the resident female. This testing context was indeed necessary to allow the detection of USVs in female mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. It is therefore still possible that a different social phenotype may appear in a different testing context, e.g., in a neutral environment; indeed, when Fmr1 mutant juvenile females were assessed for their social interest in the three compartment test no sign of increased sociability was observed \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOn this basis of genotype differences, prenatal exposure to stress was able to induce the appearance of a cognitive deficit in the spontaneous alternation Y maze test, although only in males. KO stressed male mice were indeed the only experimental group displaying a performance similar to the chance level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-C; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In females, stress instead seemed to eliminate the hyper-social phenotype of mutant mice (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-E) without affecting their ultrasonic communication profile (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Nonetheless, these effects in females were actually due to a selective effect of stress in WT mice, rendering the WT phenotype similar to that of mutants. Hence, our data suggest that exposure to prenatal stress does not dramatically advance the appearance of pathological behavioral phenotypes in male and female mutants, juvenile stressed KO mice being mostly comparable to their WT littermates, as in no-stress conditions. Indeed, with the exception of the Y maze effect in males, no selective effect of stress on mutant behavioral phenotypes was detected (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Our findings suggest therefore a higher sensitivity of the cognitive domain to the effects of stress in the male sex, in line with clinical data describing a positive correlation between stress levels and cognitive deficits in FXS boys \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eInterestingly, stress did interact with genotype on several behavioral measures, but mostly by inducing its effects in WT mice only. This may suggest a reduced sensitivity of Fmr1-KO mice to stress that could be interpreted as a deficit in the adaptive response to stressors, as already proposed by others \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Previous studies have indeed described a reduced behavioral and endocrine sensitivity of adult Fmr1-KO mice (though only males were investigated) to the adult exposure to chronic stressors \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Here the genotype-specific effects of stress were characterized by clear sex differences: in males, stress enhanced body weight (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-A), affiliative behaviors (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-E) and peak amplitude (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e-G) in WT only, while it reduced peak frequency in both genotypes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e-E). In females, stress enhanced affiliative behaviors in WT only (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-F), while it enhanced body weight (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-B) and reduced locomotion in both WT and mutant mice (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e-B). Furthermore, in female KOs stress increased USV peak frequency (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e-F). Overall, not the magnitude, but the behavioral specificity of the effects of stress differed between sexes, in line with most of the previous reports \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDespite the overall agreement of the behavioral effects described in WT mice by our findings, an important difference between our and others\u0026rsquo; studies on prenatal stress should be underlined, that is the genotype of our breeders exposed to prenatal stress. The dams exposed in our study to prenatal stress are indeed heterozygous Fmr1 mutant females and not WT as in previous similar studies: it is therefore possible that the sensitivity to stress of our female breeders may be different [as previously demonstrated for Fmr1-KO males with adult post-natal stress \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e] and result in specific sex-dependent effects on the offspring behaviors. Studies comparing the behavioral and endocrine response to stress of Fmr1 mutant and WT dams should be performed in the future in order to clarify this issue; also, it would be interesting to evaluate the maternal behavior of stressed and no-stress dams to investigate whether the effects of stress on the Fmr1 offspring behavior could be mediated by alterations in the maternal care received. Similarly to other manipulations of the early environment [e.g., early enrichment \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e], prenatal stress may induce its effects on the offspring both at the prenatal level, i.e., directly affecting pups\u0026rsquo; embryonic development, and during the early post-natal phase, i.e., interfering with normal mother-pup interactions and altering maternal behaviors \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings demonstrate for the first time the impact of prenatal stress on the juvenile FXS- and ASD-like behavioral phenotype of Fmr1 mice, underlying the relevance of including sex differences and assessing multiple behavioral domains in mouse studies on FXS and ASD. These data therefore highlight the importance of complex gene-environment interactions in the etiopathology of neurodevelopmental disorders, also for a syndrome of clear genetic origins, such as FXS. Our results focused on the juvenile age, which is critical for the early detection of behavioral abnormalities and their early therapeutic rescuing; this research focus could be extended in future studies by investigating the effects of prenatal stress on a longer term, for instance on the behavioral phenotype of Fmr1 mice at the adult age, i.e., when the behavioral alterations of mutants are more marked and well-established.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\n\u003cp\u003eThis work was supported by CNRS and the University of Bordeaux. S. Pietropaolo and V. Petroni received funding from the Fondation pour l\u0026rsquo;Audition (FPA-RD-2020-8) and the Association Autour de Williams. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflict of interest to report in relation to the work described.\u003c/p\u003e\n\u003ch2\u003eDATA AVAILABILITY STATEMENT\u003c/h2\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e Hagerman, R. J. \u0026amp; Hagerman, P. J. \u003cem\u003eFragile X syndrome: Diagnosis, treatment, and research\u003c/em\u003e. (Taylor \u0026amp; Francis US, 2002).\u003c/li\u003e\n \u003cli\u003e Bailey, D. B., Jr.\u003cem\u003eet al.\u003c/em\u003e Autistic behavior in young boys with fragile X syndrome. \u003cem\u003eJ Autism Dev Disord\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 499-508 (1998).\u003c/li\u003e\n \u003cli\u003e Hagerman, R. J. 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Postnatal stress in mice: does \u0026quot;stressing\u0026quot; the mother have the same effect as \u0026quot;stressing\u0026quot; the pups?\u003cem\u003eDev Psychobiol\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 230-237 (2004).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Unpredictable stress, Fragile X syndrome, sex differences, mouse, social behaviors","lastPublishedDoi":"10.21203/rs.3.rs-1372695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1372695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFragile X Syndrome (FXS) is the most common heritable form of mental retardation and the main monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 gene and is characterized by motor, cognitive and social alterations, mostly overlapping with ASD behavioral phenotypes. The severity of these symptoms and their timing may be exacerbated and/or advanced by environmental adversity interacting with the genetic mutation. We therefore tested the effects of the prenatal exposure to unpredictable chronic stress on the behavioral phenotype of juveniles of both sexes in the Fmr1 knock-out (KO) mouse model of FXS. Mice underwent behavioral tests at 7\u0026ndash;8 weeks of age, that is, when most of the relevant behavioral alterations are absent or mild in Fmr1-KOs. Stress induced the early appearance of cognitive deficits in KO male mice, without exacerbating the behavioral phenotype of mutant females. In males stress also altered social interaction and communication, but mostly in WT mice, while in females it induced effects on locomotion and communication in mice of both genotypes. Our data therefore highlight the sex-dependent relevance of early environmental stressors to interact with genetic factors to influence the appearance of selected FXS- and ASD-like phenotypes.\u003c/p\u003e","manuscriptTitle":"Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-25 17:44:56","doi":"10.21203/rs.3.rs-1372695/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-03-18T03:25:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-03-13T17:43:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0ae1c177-0ce5-420b-941a-dfaef913202f","date":"2022-03-07T19:01:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-02T12:13:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-02T12:02:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-02-23T12:16:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-02-23T12:14:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-02-18T10:49:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0a9c5a7-b653-42f4-ab00-11a564754c01","owner":[],"postedDate":"February 25th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-04-11T06:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-25 17:44:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1372695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1372695","identity":"rs-1372695","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}