Longitudinal Analysis in Mecp2-het Female Mice Reveals Atypical Nociceptive Behaviours

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Herein, we present a study of longitudinal analyses, from 2- to 6-month of age, of Mecp 2 heterozygous ( Mecp2 -het) female mice to comprehensively explore pain perception in RTT. Interestingly, we found a significant variability in the timing and progression of symptom onset among Mecp2 -het females, with individuals classified as either early- or late-symptomatic based on the emergence of hallmark neurological features such as clasping and gait abnormalities. This variability pinpoints the heterogeneity of the disease model and highlights the need to stratify Mecp2 -het females by symptom onset in future studies to account for the diverse trajectories of disease progression. Additionally, our results reveal a shift from pre-symptomatic hypersensitivity in the von Frey test to apparent hyposensitivity, intricately linked with the onset of motor symptoms. Further, we found decreased neuronal activation in 6-month-old Mecp2 -het females after the hot plate test in the periaqueductal gray, as measured by FOS expression. Similarly, there is a lower expression of cannabinoid receptor 1 (CB1) in this area when compared to wild-type siblings. Taken together, our results suggest that both motor impairment and central deficits in the modulation of endogenous analgesia contribute to aberrant sensitivity in Mecp2 -het mice. Our study emphasises the presymptomatic phase as crucial for understanding sensory abnormalities in Mecp2 -het mice and highlights the challenges in identifying pain in RTT patients. Rett Syndrome Mecp2-het females pain endogenous analgesia endocannabinoid system nociception Figures Figure 1 Figure 2 Figure 3 Figure 4 KEY MESSAGES Mecp2 -het mice show early hypersensitivity to stimuli that shifts with age. Classification of Mecp2 -het mice by symptom onset reveals phenotypic variability. Mecp2 -het mice show lower PAG activity when facing a thermal stimulus. Mecp2 -het mice show decreased activity and CB1 receptor levels in the PAG. INTRODUCTION Rett Syndrome (RTT) is an X-linked rare genetic neurodevelopmental disorder that affects 1:10.000 alive female births [ 1 ]. It causes intellectual disability, language loss, orthopaedic and gastrointestinal issues, and epilepsy [ 2 ], some of which may cause varying levels of pain [ 3 ]. Motor and communicative impairments hinder pain assessment, leaving this aspect understudied [ 4 ]. The genetic basis of classical RTT is de novo mutations in the X-linked methyl-CpG binding protein 2 ( MECP2 ) gene, encoding the MeCP2 protein, an epigenetic reader essential for brain function [ 1 , 5 – 6 ]. Preclinical studies use a Mecp2 -deficient mouse models including Mecp2 -null males, exhibiting severe RTT features by 6–8 weeks of age, and Mecp2 -het females, showing delayed symptom onset starting at 3 months, with early abnormalities such as reflex delays, hypersensitivity, and atypical anxiety [ 7 – 13 ]. Nociceptive studies in RTT models report conflicting results. Mecp2 -null males show heightened mechanical [ 9 ] and thermal [ 14 ] sensitivity, while 6-month-old Mecp2 -het females display thermal hyposensitivity [ 8 ]. Contrastingly, RTT rats show hypersensitivity to mechanical pressure, viscerosensation, and cold, but hyposensitivity to heat [ 15 ]. Emerging evidence suggest that lack of MeCP2 results in alterations in the endocannabinoid system (ECS), which interacts with the endogenous opioid system (EOS) in pain processing [ 16 ]. Interestingly, studies of phytocannabinoid treatments in RTT mouse models reveal upregulated CB1 and CB2 receptors in Mecp2 -null males, with improved motor function, cognition, survival, and thermal pain perception after treatment [ 14 , 17 – 18 ]. This study examined Mecp2 -het females for symptom progression, focusing on weight gain, clasping, motor impairments, and mechanical sensitivity (Von Frey test) from 2 to 6 months. At 6 months, thermal sensitivity and neural activation (FOS expression) were assessed, along with CB1 and mu-opioid receptor (MOR) expression in the periaqueductal grey (PAG). A younger cohort (2 months old) was evaluated for presymptomatic thermal sensitivity and FOS expression. MATERIALS AND METHODS Animals For the longitudinal analysis, 24 female mice (WT, n = 11; Mecp2 -het, n = 13) from The Jackson Laboratory (B6.129(C)- Mecp2 tm1 .1Bird ) were tested at various ages depending on the experiment. The analysis included VFT, clasping test, footprint test, and body weight measurement. At 6 months, one week after completing the longitudinal tests, animals underwent the hot plate test (HPT). Half (WT females, n = 5; Mecp2 -het females, n = 7) were euthanised 90 minutes post-HPT for cFos immunohistochemical analysis, while the remaining (WT females, n = 6; Mecp2 -het females, n = 6) were euthanised for brain extraction and snap-freezing for CB1 receptor and MOR western blotting. To assess thermal sensitivity at 2 months, an additional 12 mice (WT: n = 6; Mecp2-het: n = 6) underwent HPT and were euthanised 90 minutes later for FOS analysis. Power calculations (beta = 0.8, alpha = 0.05), with effect sizes determined from pilot studies, were used to estimate sample size for each experiment. Whenever possible, animals/contrast/testing order were selected at random. Mice were housed in groups of 3–6 animals per litter in standard laboratory cages with controlled humidity and temperature (22°C), 12:12-h light/dark cycle, and water and food available ad libitum . Genotyping and histology procedures were carried out as described before [ 13 ]. All the procedures were carried out in strict accordance with the EU directive 2010/63/EU. Protocols were approved by the local veterinary and the Ethics in Animal Experimentation Committee of the University of Valencia. Behavioural phenotyping To monitor symptom onset, we measured body weight and clasping monthly from 2 to 6 months in the longitudinal study and once at 2 months in the younger cohort. Clasping was assessed by holding mice by their tail for up to 15 seconds. WT and pre-symptomatic Mecp2-het mice moved their paws to escape, while symptomatic Mecp2 -het mice clasped their hind and/or fore paws. Clasping is a common marker of neurological symptoms in this and other mouse models [ 7 , 19 ]. Paw print test To assess potential motor impairments, we analysed the gait of WT and Mecp2 -het females monthly from 4 to 6 months of age in a longitudinal study. Non-toxic washable tempera (black for hind paws, red for fore paws) was applied to the paws, and the animals ran through a delimited path on filter paper. Gait analysis included measuring the distances (mm) between hindlimbs, forelimbs, and same-side fore and hindlimbs over four consecutive steps per animal [ 20 – 21 ]. Von Frey test Mechanical sensitivity was measured by means of the VFT, which was performed monthly from the age of 2 to 6 months for the animals in the longitudinal study. The protocol started with a 30-min period of habituation of the behavioural boxes and the room where the test was performed. After this habituation period, we manually applied five filaments following a simplified up–down method, as previously described [ 22 ]. Results are expressed in grams (g). Hot plate test Animals underwent thermal sensitivity testing in a HPT at 6 months (longitudinal analysis) and 2 months (younger cohort) [ 8 ]. Mice were placed on a 52°C hot plate for 30 seconds, and their behaviour was recorded. The latency to paw licking was analysed. However, in the 6-month Mecp2 -het group, more than half displayed an abnormal backward walking movement (Supplementary Videos 1 and 2), not seen in WT controls (Supplementary Videos 3 and 4) or mice of the same genetic background. This movement, likely a failed avoidance attempts due to motor impairments (e.g., clasping, gait deficits), was assessed alongside paw-lick latency. Latencies to paw licking and backward walking were analysed separately and combined as the latency to the first avoidance response. Mice were euthanised 90 minutes post-test for histological and biochemical analyses. FOS immunohistochemistry Animals were deeply anaesthetised 90 min post-HPT with an overdose of pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde (0.1M PB, pH 7.4). Brains were removed, post-fixed in the same fixative for 4 h, and cryoprotected in 30% sucrose (PBS, pH 7.6). Frozen brains were sectioned (40 µm coronal) and stored in 30% sucrose with 0.02% sodium azide. Endogenous peroxidase was quenched with 1% H₂O₂ in TBS (pH 7.6). Sections were blocked with 3% NGS in 0.3% TBS-Tx, then incubated overnight with rabbit anti-c-FOS antibody (1:10,000, Santa Cruz). Secondary incubation used biotinylated horse anti-goat IgG (1:250, Vector). Sections were processed with ABC Elite (Vector) and visualised using DAB with H₂O₂ (25 min). FOS immunodetection was combined with Nissl staining and sections were mounted with Entellan. Photomicrographs were taken at selected bregma levels for Nucleus accumbens core (Acbc), paraventricular nucleus of the hypothalamus (PA) and thalamus (PV), central amygdala (Ce) and PAG. FOS-ir nuclei were quantified by splitting RGB channels to isolate the blue channel (avoiding Nissl interference) and manually counted using Image-J’s multipoint plugin. Results were reported as cFOS-ir cells per section. Western Blot We measured CB1 receptor and MOR expression in the PAG of WT and Mecp2 -het females using western blot as previously described [ 24 – 25 ]. Snap-frozen PAG tissue was homogenised in cold lysis buffer (1% IGEPAL CA-630, 20 mM Tris–HCl pH 8, 130 mM NaCl, 10 mM NaF, 1% protease inhibitor cocktail; Sigma) at 0.5 ml buffer per 250 mg tissue. After 30 minutes on ice, samples were centrifuged at 15,000 RCF for 15 minutes at 4°C. Supernatants were collected, and protein concentration was measured with a Bradford assay (Bio-Rad). Primary antibodies included anti-CB1 (1:1,000, Santa Cruz Biotechnology) [ 26 ] and anti-MOR (1:1,000, Abcam) [ 24 ], with GAPDH (1:2,000, Sigma) [ 27 ] as a loading control. Secondary antibody was goat anti-rabbit (1:1,000, Bio-Rad). Band intensities, normalised to GAPDH, were quantified in arbitrary units. All samples (20 µg each) were run in duplicate. Statistical analysis Results are shown in figures as mean ± SEM (standard error of the mean). To perform the statistical analysis, we used R [ 28 ] and several libraries within it. For the analysis of the longitudinal data, we adopted a Bayesian perspective and fitted a Normal Linear Mixed Model LMM [ 29 ] to understand the differences in the evolution of different measurements with age controlling by relevant variables and considering repeated measurements for each female. The R library used with this purpose was rjags ( https://CRAN.R-project.org/package=rjags ) [ 30 ]. Results for the HPT, the immunohistochemistry and the western blot are shown as mean ± standard error of the mean (SEM). The HPT was further analysed by using a Kaplan-Meier survival approach. To perform the statistical analysis, the 26.0 version of the SPSS program was used. We first checked the data for normality (Shapiro–Wilk test) and homoscedasticity (Levene’s test). Next, we evaluated the differences between genotype using Student’s t test or Mann–Whitney U test when appropriate. Significance was set at p < 0.05. RESULTS Classification of Mecp2 -het females by the onset of symptomatology The timeline of the longitudinal evaluation can be found in Fig. 1 a. We measured monthly clasping and body weight of a cohort of 13 Mecp2 -het females and 11 WT controls from 2 to 6 months of age, taking clasping as a classic marker for the onset of an overtly symptomatic phase [ 7 ]. As shown in Fig. 1 b, the expression of clasping in Mecp2 -het females varies across individual subjects. Indeed, by the age of 2 months, less than 10% of the Mecp2 -het animals displayed clasping, whereas half of the animals displayed clasping by the age of 4 months. At 6 months of age, almost all Mecp2 -het females could be considered as symptomatic. Thus, we classified the animals as early- or late-symptomatic using 4 months old as a cut-off for upcoming analysis. As expected, none of the WT animals showed clasping behaviour. Mecp2 -het females show overweight as the pathology progresses We implemented a longitudinal analysis to assess body weight, incorporating the previously established classification of phenotypes and their interaction with time as explanatory variables. Figure 1 c illustrates a significant impact of this classification on both the initial point of measure at 2 month of age (intercept) and the trajectory (slope) of weight. The numerical summaries provided in Supplementary Table 1 indicate a high probability of the differences in slope being greater than 0, with a probability of 1. Furthermore, there is compelling evidence for a distinction in the body weight evolution between Mecp2 -het females with early and late onset of clasping, with a probability of 0.998. Mecp2- het symptomatic females show differences in gait pattern Gait pattern was measured in WT and Mecp2 -het females at 4, 5 and 6 months of age. While the results from the longitudinal model exhibited some degree of uncertainty, Fig. 1 d-g illustrates relevant differences in their motion patterns. Specifically, fore paws were affected in both early- and late-symptomatic Mecp2 -het females across testing, as opposed to hind paws, where late-symptomatic females were more similar to WT, in clear contrast to early-symptomatic females. This trend is further elucidated in the estimated values and probabilities of the designated parameters for fore paws as displayed in Supplementary Tables 2–5. Notably, highly probable negative values indicate a significant difference in slope from WT in both early- and late-symptomatic individuals. Conversely, for hind paws, the difference in slope from WT to late-symptomatic females are approximately 0, with probabilities hovering around 0.5, suggesting a lack of substantial deviation from the WT behaviour. The advance of symptoms masks mechanical hypersensitivity of Mecp2 -het females The longitudinal model applied to the VFT reveals notable distinctions among phenotypes, evident in both initial values and slopes. Figure 1 h show that while WT females demonstrate stable mechanical sensitivity throughout time, Mecp2 -het females exhibit an initial hypersensitivity that diminishes at later stages. Remarkably, at 6 months, late onset females display a sensitivity akin to WT, whereas those with early symptoms demonstrate an apparent hyposensitivity starting at 4 months. Examining the numerical results (Supplementary Table 6), we observe meaningful and negative differences in intercepts for both early- (P(β > 0│Data) = 0.021) and late- (P(β > 0│Data) = 0.015) symptomatic Mecp2 -het females. Regarding the slopes, significant distinctions emerge, with both mutant groups exhibiting larger slopes (P(β > 0│Data) = 0.986 for early- and P(β > 0│Data) = 0.946 for late-symptomatic) than WT. Interestingly, the model indicates similar slopes for late- and early-symptomatic females, with a mean difference of 0.059 (P(β > 0│Data) = 0.742). Thus, mechanical sensitivity shows discrepancies between early and late but has a similar progression. Mecp2 -het symptomatic females show aberrant thermal responses suggesting heat hypersensitivity We analysed the thermal sensitivity in 2-month-old and 6-month-old females by means of the hot HPT. First, we analysed the standard parameter that is taken as a measure of sensitivity in the HPT, namely the latency of animals to lick the paws [ 31 ]. When using this measure, 2 out of 13 6-month-old Mecp2 -het mice did not lick the paws before the cut-off time of 30 seconds, and therefore we analysed this measure by means of a Kaplan-Meier analysis. As shown in Fig. 2 a, this analysis did not reveal significant differences between genotypes (χ 2 (2) = 0.500, p = 0.779). However, we noticed that, probably due to the motor impairments caused by the progression of the disease (see above), more than half of Mecp2 -het females performed and abnormal walking backwards movement when placed in the HP as observed in Fig. 2 b (χ 2 (2) = 0.435, p = 0.114), a movement never seen in WT mice. Thus, we decided to measure the latency to first avoidance movement by combining either licking the paws or walking backwards for each animal. Results showed that 6-month-old females were different among them (χ 2 (2) = 11.610, p = 0.003, Fig. 2 c). Furthermore, the analysis showed significant differences when comparing either early (χ 2 (1) = 8.967, p = 0.003), or late (χ 2 (1) = 9.876, p = 0.002) onset Mecp2 -het individuals with WT animals. However, Kaplan-Meier failed to find any significant difference between early and late onset individuals (χ 2 (1) < 0.001, p = 0.985). Therefore, we performed a Kaplan-Meier test without dividing Mecp2 -het mice into early and late as shown in Fig. 2 d (χ 2 (1) = 11.590, p < 0.001). Interestingly, whereas all the presymptomatic 2-month-old did lick their paws in the HP (χ 2 (1) = 0.669, p = 0.4135, Fig. 2 g), some of them already presented with the abnormal walking backwards movement as shown in Fig. 2 f (χ 2 (1) = 3.669, p = 0.055). However, when using the latency to first avoidance parameter, the Kaplan-Meier analysis failed reveal any significant differences between genotypes (χ 2 (1) = 1.589, p = 0.208, Fig. 2 g). All these data were further analysed by Student t tests, which revealed a significant decrease in latency to avoidance in both late- (t = 3.389, p = 0.004, Fig. 2 h) and early-symptomatic females (t = 3.805, p = 0.002, Fig. 2 i) when compared to the WT females. This was maintained when early- and late- symptomatic females where collapsed (t = 4.101, p < 0.001, Fig. 2 j). However, this was not replicated in the younger cohort since no significant difference was found between Mecp2 -het and WT females (t = 1.438, p = 0.181, Fig. 2 k). FOS expression after the hot plate and CB1 expression are significantly reduced in PAG of Mecp2 -het females Half of the 6 months old mice and all the 2 months old mice were sacrificed 90 minutes after the HPT to check for possible alterations in the activation patterns in brain regions relevant for pain processing by means of FOS expression. We compared the number of cFos-ir cells in different brain regions and found no significant differences between genotypes in the group of 2 months old (Fig. 3 a-e). In 6-month-old mice, although no significant differences were observed in most of the areas tested (Fig. 3 g-j), a significant reduction in cFos-ir cells was detected in the dorsal PAG of Mecp2-het females compared to WT females, as shown in Fig. 2 f (Mann-Whitney U test, p = 0.004). This difference was maintained when analysing separately the group of het females in late- (Mann-Whitney U test, p = 0.04; Fig. 3 k) and early- (Mann-Whitney U test, p = 0.04; Fig. 3 l) symptomatic females. Having found specific decrease of activation in the PAG, we performed western blot against CB1 receptors and MOR in PAG punches of half of the mice 6 months old mice in this area. Student t-test analysis showed a significant reduction in Mecp2 -het females for the density of CB1 (t = 2.563, p = 0.028; Fig. 4 a) but not for MOR (Fig. 4 b). DISCUSSION Here, we carried out a longitudinal analysis in a cohort of Mecp2 -het females, assessing disease progression and somatosensory sensitivity. We found that Mecp2 -het females might experience hypersensitivity from pre-symptomatic age, which could be masked at later stages by the early onset of additional symptoms, highly variable among individuals. Further, we also identify “walking backwards” in the HPT as an atypical behavioural response to potentially painful stimulation [ 32 ]. Taken together, our data support that MeCP2 deficiency might cause heightened sensitivity to mechanical and thermal stimulation, albeit masked by the onset of motor symptoms. Further, our data and highlight the need to characterise atypical pain responses in mouse models of neurodevelopmental disorders and take into account the phenotypic heterogeneity displayed by Mecp2 -het females. Mecp2 -het females are hypersensitive to mechanical and thermal stimulation Hypersensitivity to somatosensory stimulation has been previously reported in Mecp2 -mutant mice. On the one hand, Orefice et al. (2016) demonstrated that Mecp2 -null males display increased sensitivity to mechanical stimulation, especially due to peripheral mechanisms [ 9 ]. On the other hand, Mecp2 -het females with trimmed whiskers did not show the typical aberrant preference for open arms in the elevated plus maze found in this model, suggesting that sensory hypersensitivity could be an important contributor to their apparent reduced anxiety [ 11 ]. In our study, Mecp2 -het females showed a significant hypersensitivity, as compared to WT controls, in the VFT, only evident at pre-symptomatic stages. Thus, the mechanical nociception threshold, as measured by the withdrawal of the paw, of Mecp2 -het females significantly varies with age, from hypersensitivity in pre-symptomatic females at 2 month of age, to apparent hyposensitivity by 6. Interestingly, the timing of onset of the overt symptomatic phase in these animals is highly variable among individuals. When we segregated the group of Mecp2 -het females into early- and late-symptomatic, we found that the latter remained hypersensitive until later stages, whereas the former were apparently hyposensitive from 5 months of age onwards. In line with the other variables measured in our longitudinal analysis (clasping and gait analysis), it is plausible that early-symptomatic mice may not respond to the VFT filaments simply due to aspects likely to influence motility that are inherent to the disease's progression. The same might be true for thermal stimulation in 6-month-old Mecp2 -het females. Both overweight and gait abnormalities present in Mecp2 -het symptomatic females may interfere in their ability to lick their paws. Thus, at 6 months of age, when nearly all Mecp2 -het females were overtly symptomatic, we did not find significant differences in thermal nociception threshold when using the standard measure of paw licking. This result contrasts with a previous study reporting an increase in the latency to lick the paws in the HPT in 5-month-old Mecp2 -het female [ 8 ], which was regarded as hyposensitivity. However, we propose an alternative interpretation: due to concomitant factors intrinsic to the disease's progression (most likely impacting motility), Mecp2 -het females display an aberrant walking backwards movement when trying to lick their paws, which is already shown by the younger cohort. By taking this measure and combining it with the more standard paw licking, we found a significant reduction in the time to react in Mecp2 -het females, suggesting that Mecp2 -het females are also hypersensitive to thermal stimulation. In agreement, Vigli et al., (2021) reported increased sensitivity in the HPT in Mecp2 -null male mice. In agreement with this view on disease progression masking potential standard behavioural responses, our study on presymptomatic 2-month-old Mecp2 -het females displayed no significantly altered thermal sensitivity [ 14 ]. Together, these findings highlight the importance of monitoring symptom onset in Mecp2 -mutant models and the need for behavioural analyses that account for both standard and atypical responses. Neural and neurochemical substrate of hypersensitivity in Mecp2 -het females Thermal stimulation led to a significantly lower neuronal activation at the PAG, as measured by FOS immunoreactivity, of 6-months-old Mecp2 -het females when compared to WT controls. The PAG is an important structure in the pain descending pathway since its innervation to the spinal cord modulate pain sensitivity and endogenous analgesia [ 33 ]. Therefore, the activation of neurons within this brain area upon a painful stimulus is related to how the nervous system manages that stimulus [ 34 ]. The diminished neuronal activity that we report at the PAG of symptomatic Mecp2 -het females suggests a reduction of endogenous analgesia, which could contribute to a heightened sensitivity towards somatosensory stimulation. Géranton et al. (2008) demonstrated that, in rats, MeCP2 in descending serotonergic pathways can regulate gene expression in the dorsal horn and mechanical sensitivity associated with an inflammatory pain state [ 35 ]. Specifically, they found that a complete Freund’s adjuvant injection into WT hind paw (a model of inflammatory pain) significantly increased phosphorylated MeCP2 levels in Lamina I and II of the superficial dorsal horn, with a peak observed after 1 hour. The endocannabinoid system participates in in PAG-driven differential sensitivity [ 36 ]. In general, CB1 agonists seem to lead to antinociception [ 37 ], which is greatly mediated by receptors located within the PAG [ 38 ]. Therefore, it is not unexpected that our results show a decrease in CB1 expression levels, which correlate with decreased thermal nociception thresholds and decreased neuronal activity. Interestingly, pharmacological approaches targeting the endocannabinoid system have previously been used to ease RTT symptomatology in animal models [ 17 ], among which we find pain [ 14 ]. However, although overall upregulation of endocannabinoid system components might be reported in the above-mentioned studies, region-specific differences might arise due to Mecp2 deficiency [ 20 , 39 ]. Thus, future studies should investigate the possible region-specific alterations in the endocannabinoid and endogenous opioid systems in Mecp2 deficient models. CONCLUSIONS Taken together, our results reveal that pain might be largely hidden in mouse models of RTT and highlight the need of future studies to ameliorate pain conditions in RTT patients. Our results reflect the complexity of assessing aberrant behaviours in a RTT surrogate mouse model, given the sensory and motor impairments that characterise the disease. Our data supports the validity of Mecp2- het females from the pre-symptomatic phase to study RTT and the importance of assessing symptom progression to get a full understanding of the consequences derived from of Mecp2 deficiencies. STATEMENTS & DECLARATIONS ACKOWLEDGEMENTS Authors are indebted to Dr Enrique Lanuza for his continuous support and discussion of the data. Funding This work was supported by Ayudas FinRett para la Investigación del Síndrome de Rett 2019 and 2022 and the Spanish Ministry of Science and Innovation (PID2019-107322GB-C22 funded by MCIN/AEI/10.13039/501100011033) to CAP; the Spanish Ministry of Science and Innovation (PID2022-137803NB-I00 funded by MCIN/AEI/10.13039/501100011033) to LH; MAA obtained a postdoctoral fellowship from the Spanish Ministry of Universities of Spain (MS21-083) financed by Next Generation EU2; JC is supported by an Atracció de Talent Fellowship from the University of Valencia (UV-INV-PREDOC-1327981), JVTP by a Ramón y Cajal contract (RYC2021-034012-I) by the Spanish Ministry of Science and Innovation and the European Union “NextGenerationEU”/PRTR and the 2023 Pickford Award from the British Pharmacological Society, and MS by PT2020-Centro 2020 (CENTRO-01-0145-FEDER-000008). JVTP and MAA are also supported by the Conselleria de Educación, Universidades y Empleo from the Generalitat Valenciana with a Subvenciones a grupos de investigación emergentes (CIGE/2022/139). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Authors Contribution Javier Cuitavi performed research, analysed data, prepared figures and wrote the paper; Elena Martínez-Rodríguez performed research, analysed data, and wrote the paper; María Abellán-Álvaro performed research and analysed data; Moisés García-Arencibia participated in the design of the project; Mónica Santos participated in the design of the project; Anabel Forte analysed longitudinal data and provided general assessment on statistical analysis; Lucía Hipólito participated in the design of the study; Carmen Agustín-Pavón designed the study, supervised research and data analysis, contributed to writing and funding acquisition; Jose Vicente Torres-Pérez performed research, analysed data and contributed to writing and funding acquisition. All authors contributed to discuss results and read and approved the final manuscript. Data Availability Data are available upon request. 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Molecular pain , 4 , 1744-8069. https://doi.org/10.1186/1744-8069-4-35. Hohmann, A. G., Tsou, K., & Walker, J. M. (1999). Cannabinoid suppression of noxious heat-evoked activity in wide dynamic range neurons in the lumbar dorsal horn of the rat. Journal of Neurophysiology , 81 (2), 575-583. https://doi.org/10.1152/jn.1999.81.2.575. Corcoran, L., Roche, M., & Finn, D. P. (2015). The role of the brain's endocannabinoid system in pain and its modulation by stress. International review of neurobiology , 125 , 203-255. https://doi.org/10.1016/bs.irn.2015.10.003. Walker, J. M., Huang, S. M., Strangman, N. M., Tsou, K., & Sañudo-Peña, M. C. (1999). Pain modulation by release of the endogenous cannabinoid anandamide. Proceedings of the National Academy of Sciences , 96 (21), 12198-12203. https://doi.org/10.1073/pnas.96.21.12198. Martínez-Rodríguez, E., Martín-Sánchez, A., Coviello, S., Foiani, C., Kul, E., Stork, O., Martínez-García, F., Nacher, J., Lanuza, E., Santos, M., & Agustín-Pavón, C. (2019). Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system. Brain Structure and Function , 224 , 1647-1658. https://doi.org/10.1007/s00429-019-01860-6. Supplementary Files SupVideo15026Mecp2late.mp4 SupVideo25031Mecp2early.mp4 SupVideo35011WT.mp4 SupVideo45022WT.mp4 SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Journal of Molecular Medicine → Version 1 posted Editorial decision: Major Revisions Needed 27 Feb, 2025 Reviewers agreed at journal 30 Jan, 2025 Reviewers invited by journal 30 Jan, 2025 Editor assigned by journal 14 Jan, 2025 First submitted to journal 13 Jan, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-5804567","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408996278,"identity":"515eb0ff-623d-4a60-a53a-a5bf3ccb9542","order_by":0,"name":"Javier Cuitavi","email":"","orcid":"","institution":"Universitat de Valencia","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Cuitavi","suffix":""},{"id":408996279,"identity":"e5179416-a187-477b-a793-b952a572e735","order_by":1,"name":"Elena Martínez-Rodríguez","email":"","orcid":"","institution":"Universitat de Valencia","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Martínez-Rodríguez","suffix":""},{"id":408996280,"identity":"641484dd-8372-4ee1-908c-d12222046d72","order_by":2,"name":"María Abellán-Álvaro","email":"","orcid":"","institution":"Universitat de València: Universitat de Valencia","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Abellán-Álvaro","suffix":""},{"id":408996281,"identity":"8aabb108-5038-483e-9a9d-c672aee551f1","order_by":3,"name":"Moisés García-Arencibia","email":"","orcid":"","institution":"Consejería de Educación, Formación Profesional, Actividad Física y Deportes, Gobierno de Canarias","correspondingAuthor":false,"prefix":"","firstName":"Moisés","middleName":"","lastName":"García-Arencibia","suffix":""},{"id":408996282,"identity":"33eaad66-12db-40b2-83d0-6c0acec867bc","order_by":4,"name":"Mónica Santos","email":"","orcid":"","institution":"CNC: Universidade de Coimbra Instituto de Investigacao Clinica e Biomedica de Coimbra","correspondingAuthor":false,"prefix":"","firstName":"Mónica","middleName":"","lastName":"Santos","suffix":""},{"id":408996283,"identity":"ecf1b6d3-7aa8-480b-b3ca-57f2e78bf88c","order_by":5,"name":"Lucía Hipólito","email":"","orcid":"","institution":"Universitat de València: Universitat de Valencia","correspondingAuthor":false,"prefix":"","firstName":"Lucía","middleName":"","lastName":"Hipólito","suffix":""},{"id":408996284,"identity":"59bf482a-62e0-462e-8891-78e153c0f048","order_by":6,"name":"Anabel Forte","email":"","orcid":"","institution":"Universitat de València: Universitat de Valencia","correspondingAuthor":false,"prefix":"","firstName":"Anabel","middleName":"","lastName":"Forte","suffix":""},{"id":408996285,"identity":"76dbdb17-fb6c-4a9d-9a97-3a2e21253727","order_by":7,"name":"Carmen Agustín-Pavón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYAgACQZ+EnUkSDBINpCohYHB4AABNboNzA8/V+bY5DGwH78m+fOHRZ7xjRwDhg9/cGsxO8BmLHl2W1oxA09OmTRPgkSxGVAL48w2fFp4GCQbtx1ObGDISZMG+iVx2420BGZePB4CamH+2bjtf2ID/5s0yR9ALZtnALX8weswHjagLQcSGyTSj0kAHZa4QSL5ADMDGx4th9nMLBu3JSe2SbxhtuZJk0iccebxgYO9+PxyvPnxzcZtdon9/OkPb/6wqUvsb09sfPADj8MYmKE0GwOPAVzwAB4NyID9AZEKR8EoGAWjYKQBABJmT78r9wmFAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6725-6954","institution":"Universitat de València","correspondingAuthor":true,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Agustín-Pavón","suffix":""},{"id":408996286,"identity":"b59abc2d-48de-4955-b8cc-731d5454f490","order_by":8,"name":"Jose V. Torres-Pérez","email":"","orcid":"","institution":"Universitat de València: Universitat de Valencia","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"V.","lastName":"Torres-Pérez","suffix":""}],"badges":[],"createdAt":"2025-01-10 14:38:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5804567/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5804567/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00109-025-02628-8","type":"published","date":"2026-01-13T16:31:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75314373,"identity":"1e09f88e-7c4e-446c-a45a-04a3d85b8ef7","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLongitudinal study carried out in \u003c/em\u003eMecp2\u003cem\u003e-het females and WT controls. a) Timeline of the study. b) Time evolution of the percentage of \u003c/em\u003eMecp2\u003cem\u003e-het females displaying clasping. c) Time evolution of the weight of control and early and late onset \u003c/em\u003eMecp2\u003cem\u003e-het females. d) Time evolution of the ForeBase in the Paw print test in control and early and late onset \u003c/em\u003eMecp2\u003cem\u003e-het females. e) Time evolution of the ForeStride in the Paw print test in control and early and late onset \u003c/em\u003eMecp2\u003cem\u003e-het females. f) Time evolution of the HindBase in the Paw print test in control and early and late onset \u003c/em\u003eMecp2\u003cem\u003e-het females. g) Time evolution of the HindStride in the Paw print test in control and early and late onset \u003c/em\u003eMecp2\u003cem\u003e-het females. h) Time evolution of the mechanical nociception threshold measured by the Von Frey test in control and early and late onset \u003c/em\u003eMecp2\u003cem\u003e-het females. ForeBase: base distance between forepaws, ForeStride: stride distance between forepaws, HindBase: base distance between hind paws, HindStride: stride distance between hind paws, \u003c/em\u003eMecp2\u003cem\u003e: methyl CpG binding protein 2 gene, Het: Heterozygous, WT: wild type\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/29cb3bdaac13ccc5c594736f.png"},{"id":75314369,"identity":"91eab4ea-3678-43ae-91b3-2272f153e271","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThermal nociception and neuronal changes in the PAG in \u003c/em\u003eMecp2\u003cem\u003e-het females and WT controls. a) Survival curve of latency to lick the paws as a measure of thermal nociception threshold in the hot plate test in 6 months old \u003c/em\u003eMecp2\u003cem\u003e-het females (early and late onset) and controls. b) Survival curve of latency to walk backwards as a measure of thermal nociception in the hot plate test in 6 months old \u003c/em\u003eMecp2\u003cem\u003e-het females (early and late onset) and controls. c) Survival curve of latency to first avoidance movement (either lick the paws or walking backwards) as a measure of thermal nociception threshold in the hot plate test in 6 months old \u003c/em\u003eMecp2\u003cem\u003e-het females (early and late onset) and controls. d) Survival curve of latency first avoidance a measure of thermal nociception threshold in the hot plate test in 6 months old \u003c/em\u003eMecp2\u003cem\u003e-het females (all) and controls. e) Survival curve of latency to lick the paws as a measure of thermal nociception threshold in the hot plate test in 2 months old \u003c/em\u003eMecp2\u003cem\u003e-het females and controls. f) Survival curve of latency to walk backwards as a measure of thermal nociception in the hot plate test in 2 months old \u003c/em\u003eMecp2\u003cem\u003e-het females and controls. g) Survival curve of latency to first avoidance movement (either lick the paws or walking backwards) as a measure of thermal nociception threshold in the hot plate test in 2 months old \u003c/em\u003eMecp2\u003cem\u003e-het females. h) Bar chart of latency to first avoidance movement (either lick the paws or walking backwards) as a measure of thermal nociception threshold in the hot plate test in 6-month-old late-onset \u003c/em\u003eMecp2\u003cem\u003e-het females. i) Bar chart of latency to first avoidance movement (either lick the paws or walking backwards) as a measure of thermal nociception threshold in the hot plate test in 6-month-old early-onset \u003c/em\u003eMecp2\u003cem\u003e-het females. j) Bar chart of latency to first avoidance movement (either lick the paws or walking backwards) as a measure of thermal nociception threshold in the hot plate test in 6-month-old \u003c/em\u003eMecp2\u003cem\u003e-het females. k) Bar chart of latency to first avoidance movement (either lick the paws or walking backwards) as a measure of thermal nociception threshold in the hot plate test in 2-month-old\u003c/em\u003eMecp2\u003cem\u003e-het females. *p\u0026lt;0.05. \u003c/em\u003eMecp2\u003cem\u003e: methyl CpG binding protein 2 gene, Het: Heterozygous, WT: wild type\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/cb64e9555dd52ca1947a496b.png"},{"id":75314375,"identity":"94f78a34-8472-4796-81bd-9c841a4b70a5","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":542605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThermal stimulus-induced FOS-ir cells, graphs and representative pictures. a-e) 2-month-old females. f-l) 6-month-old females. *p\u0026lt;0.05. PAG: Periaqueductal gray, Ce: Central Amygdala, Acbc: Nucleus Accumbens core, PA: Paraventricular hypothalamic nucleus, PV: Paraventricular thalamic nucleus, ir: immunoreactive, \u003c/em\u003eMecp2\u003cem\u003e: methyl CpG binding protein 2 gene, Het: Heterozygous, WT: wild type\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHaving found specific decrease of activation in the PAG, we performed western blot against CB1 receptors and MOR in PAG punches of half of the mice 6 months old mice in this area. Student t-test analysis showed a significant reduction in \u003cem\u003eMecp2\u003c/em\u003e-het females for the density of CB1 (t=2.563, p=0.028; Fig4a) but not for MOR (Fig4b).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/1b11992d64ed7efe605fed71.png"},{"id":75315550,"identity":"6f251c03-45af-453b-9177-69a25d30420c","added_by":"auto","created_at":"2025-02-03 09:38:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChanges in the expression of the CB1 and MOR in the PAG of \u003c/em\u003eMecp2\u003cem\u003e-het females and controls. a) CB1. b) MOR. p\u0026lt;0.05. PAG: Periaqueductal gray, \u003c/em\u003eMecp\u003cem\u003e2: methyl CpG binding protein 2 gene, Het: Heterozygous, WT: wild type, CB1: Cannabinoid receptor 1, MOR: Mu-opioid receptor, ir: immunoreactive.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/3912f167d431695d46d7ff3d.png"},{"id":100614955,"identity":"37993348-8e15-45b8-9318-cb8c9d6e17ce","added_by":"auto","created_at":"2026-01-19 17:28:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1565852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/85acba2e-f87f-4345-b8ca-3117f889d696.pdf"},{"id":75314370,"identity":"2778ec87-8687-4082-894c-e25ceafc8825","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31460324,"visible":true,"origin":"","legend":"","description":"","filename":"SupVideo15026Mecp2late.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/b67c6ecf7c7c13040ddbb29e.mp4"},{"id":75314380,"identity":"d29294f4-330f-4e0e-abc0-930066f1088d","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31605110,"visible":true,"origin":"","legend":"","description":"","filename":"SupVideo25031Mecp2early.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/1aed3591c1e77ff9faa1f90a.mp4"},{"id":75314371,"identity":"064ca599-87f4-4bd6-a49a-e91d988dea18","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":30166747,"visible":true,"origin":"","legend":"","description":"","filename":"SupVideo35011WT.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/8345df540828650d97931407.mp4"},{"id":75314382,"identity":"55974adc-61fb-4397-8685-a3c79cd6a4ae","added_by":"auto","created_at":"2025-02-03 09:30:41","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":31384327,"visible":true,"origin":"","legend":"","description":"","filename":"SupVideo45022WT.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/601a6de18c44813a3a6c6c4e.mp4"},{"id":75315559,"identity":"48148bd4-2a14-4cbb-aa5b-10aa397be3f7","added_by":"auto","created_at":"2025-02-03 09:38:41","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":37749,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5804567/v1/ef71dc8e84798f2f7df7507e.docx"}],"financialInterests":"","formattedTitle":"Longitudinal Analysis in Mecp2-het Female Mice Reveals Atypical Nociceptive Behaviours","fulltext":[{"header":"KEY MESSAGES","content":"\u003cul\u003e\n \u003cli\u003e\u003cem\u003eMecp2\u003c/em\u003e-het mice show early hypersensitivity to stimuli that shifts with age.\u003c/li\u003e\n \u003cli\u003eClassification of \u003cem\u003eMecp2\u003c/em\u003e-het mice by symptom onset reveals phenotypic variability.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eMecp2\u003c/em\u003e-het mice show lower PAG activity when facing a thermal stimulus.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eMecp2\u003c/em\u003e-het mice show decreased activity and CB1 receptor levels in the PAG.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eRett Syndrome (RTT) is an X-linked rare genetic neurodevelopmental disorder that affects 1:10.000 alive female births [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It causes intellectual disability, language loss, orthopaedic and gastrointestinal issues, and epilepsy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], some of which may cause varying levels of pain [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Motor and communicative impairments hinder pain assessment, leaving this aspect understudied [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genetic basis of classical RTT is \u003cem\u003ede novo\u003c/em\u003e mutations in the X-linked methyl-CpG binding protein 2 (\u003cem\u003eMECP2\u003c/em\u003e) gene, encoding the MeCP2 protein, an epigenetic reader essential for brain function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Preclinical studies use a \u003cem\u003eMecp2\u003c/em\u003e-deficient mouse models including \u003cem\u003eMecp2\u003c/em\u003e-null males, exhibiting severe RTT features by 6\u0026ndash;8 weeks of age, and \u003cem\u003eMecp2\u003c/em\u003e-het females, showing delayed symptom onset starting at 3 months, with early abnormalities such as reflex delays, hypersensitivity, and atypical anxiety [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nociceptive studies in RTT models report conflicting results. \u003cem\u003eMecp2\u003c/em\u003e-null males show heightened mechanical [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and thermal [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] sensitivity, while 6-month-old \u003cem\u003eMecp2\u003c/em\u003e-het females display thermal hyposensitivity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Contrastingly, RTT rats show hypersensitivity to mechanical pressure, viscerosensation, and cold, but hyposensitivity to heat [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging evidence suggest that lack of MeCP2 results in alterations in the endocannabinoid system (ECS), which interacts with the endogenous opioid system (EOS) in pain processing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Interestingly, studies of phytocannabinoid treatments in RTT mouse models reveal upregulated CB1 and CB2 receptors in \u003cem\u003eMecp2\u003c/em\u003e-null males, with improved motor function, cognition, survival, and thermal pain perception after treatment [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study examined \u003cem\u003eMecp2\u003c/em\u003e-het females for symptom progression, focusing on weight gain, clasping, motor impairments, and mechanical sensitivity (Von Frey test) from 2 to 6 months. At 6 months, thermal sensitivity and neural activation (FOS expression) were assessed, along with CB1 and mu-opioid receptor (MOR) expression in the periaqueductal grey (PAG). A younger cohort (2 months old) was evaluated for presymptomatic thermal sensitivity and FOS expression.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eFor the longitudinal analysis, 24 female mice (WT, n\u0026thinsp;=\u0026thinsp;11; \u003cem\u003eMecp2\u003c/em\u003e-het, n\u0026thinsp;=\u0026thinsp;13) from The Jackson Laboratory (B6.129(C)-\u003cem\u003eMecp2\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1\u003c/em\u003e.1Bird\u003c/sup\u003e) were tested at various ages depending on the experiment. The analysis included VFT, clasping test, footprint test, and body weight measurement. At 6 months, one week after completing the longitudinal tests, animals underwent the hot plate test (HPT). Half (WT females, n\u0026thinsp;=\u0026thinsp;5; \u003cem\u003eMecp2\u003c/em\u003e-het females, n\u0026thinsp;=\u0026thinsp;7) were euthanised 90 minutes post-HPT for \u003cem\u003ecFos\u003c/em\u003e immunohistochemical analysis, while the remaining (WT females, n\u0026thinsp;=\u0026thinsp;6; \u003cem\u003eMecp2\u003c/em\u003e-het females, n\u0026thinsp;=\u0026thinsp;6) were euthanised for brain extraction and snap-freezing for CB1 receptor and MOR western blotting. To assess thermal sensitivity at 2 months, an additional 12 mice (WT: n\u0026thinsp;=\u0026thinsp;6; Mecp2-het: n\u0026thinsp;=\u0026thinsp;6) underwent HPT and were euthanised 90 minutes later for FOS analysis.\u003c/p\u003e \u003cp\u003ePower calculations (beta\u0026thinsp;=\u0026thinsp;0.8, alpha\u0026thinsp;=\u0026thinsp;0.05), with effect sizes determined from pilot studies, were used to estimate sample size for each experiment. Whenever possible, animals/contrast/testing order were selected at random. Mice were housed in groups of 3\u0026ndash;6 animals per litter in standard laboratory cages with controlled humidity and temperature (22\u0026deg;C), 12:12-h light/dark cycle, and water and food available \u003cem\u003ead libitum\u003c/em\u003e. Genotyping and histology procedures were carried out as described before [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. All the procedures were carried out in strict accordance with the EU directive 2010/63/EU. Protocols were approved by the local veterinary and the Ethics in Animal Experimentation Committee of the University of Valencia.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBehavioural phenotyping\u003c/h3\u003e\n\u003cp\u003eTo monitor symptom onset, we measured body weight and clasping monthly from 2 to 6 months in the longitudinal study and once at 2 months in the younger cohort. Clasping was assessed by holding mice by their tail for up to 15 seconds. WT and pre-symptomatic Mecp2-het mice moved their paws to escape, while symptomatic \u003cem\u003eMecp2\u003c/em\u003e-het mice clasped their hind and/or fore paws. Clasping is a common marker of neurological symptoms in this and other mouse models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePaw print test\u003c/h3\u003e\n\u003cp\u003eTo assess potential motor impairments, we analysed the gait of WT and \u003cem\u003eMecp2\u003c/em\u003e-het females monthly from 4 to 6 months of age in a longitudinal study. Non-toxic washable tempera (black for hind paws, red for fore paws) was applied to the paws, and the animals ran through a delimited path on filter paper. Gait analysis included measuring the distances (mm) between hindlimbs, forelimbs, and same-side fore and hindlimbs over four consecutive steps per animal [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eVon Frey test\u003c/h3\u003e\n\u003cp\u003eMechanical sensitivity was measured by means of the VFT, which was performed monthly from the age of 2 to 6 months for the animals in the longitudinal study. The protocol started with a 30-min period of habituation of the behavioural boxes and the room where the test was performed. After this habituation period, we manually applied five filaments following a simplified up\u0026ndash;down method, as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Results are expressed in grams (g).\u003c/p\u003e\n\u003ch3\u003eHot plate test\u003c/h3\u003e\n\u003cp\u003eAnimals underwent thermal sensitivity testing in a HPT at 6 months (longitudinal analysis) and 2 months (younger cohort) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mice were placed on a 52\u0026deg;C hot plate for 30 seconds, and their behaviour was recorded. The latency to paw licking was analysed. However, in the 6-month \u003cem\u003eMecp2\u003c/em\u003e-het group, more than half displayed an abnormal backward walking movement (Supplementary Videos 1 and 2), not seen in WT controls (Supplementary Videos 3 and 4) or mice of the same genetic background. This movement, likely a failed avoidance attempts due to motor impairments (e.g., clasping, gait deficits), was assessed alongside paw-lick latency. Latencies to paw licking and backward walking were analysed separately and combined as the latency to the first avoidance response. Mice were euthanised 90 minutes post-test for histological and biochemical analyses.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFOS immunohistochemistry\u003c/h2\u003e \u003cp\u003eAnimals were deeply anaesthetised 90 min post-HPT with an overdose of pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde (0.1M PB, pH 7.4). Brains were removed, post-fixed in the same fixative for 4 h, and cryoprotected in 30% sucrose (PBS, pH 7.6). Frozen brains were sectioned (40 \u0026micro;m coronal) and stored in 30% sucrose with 0.02% sodium azide. Endogenous peroxidase was quenched with 1% H₂O₂ in TBS (pH 7.6). Sections were blocked with 3% NGS in 0.3% TBS-Tx, then incubated overnight with rabbit anti-c-FOS antibody (1:10,000, Santa Cruz). Secondary incubation used biotinylated horse anti-goat IgG (1:250, Vector). Sections were processed with ABC Elite (Vector) and visualised using DAB with H₂O₂ (25 min). FOS immunodetection was combined with Nissl staining and sections were mounted with Entellan. Photomicrographs were taken at selected bregma levels for Nucleus accumbens core (Acbc), paraventricular nucleus of the hypothalamus (PA) and thalamus (PV), central amygdala (Ce) and PAG. FOS-ir nuclei were quantified by splitting RGB channels to isolate the blue channel (avoiding Nissl interference) and manually counted using Image-J\u0026rsquo;s multipoint plugin. Results were reported as cFOS-ir cells per section.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eWe measured CB1 receptor and MOR expression in the PAG of WT and \u003cem\u003eMecp2\u003c/em\u003e-het females using western blot as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Snap-frozen PAG tissue was homogenised in cold lysis buffer (1% IGEPAL CA-630, 20 mM Tris\u0026ndash;HCl pH 8, 130 mM NaCl, 10 mM NaF, 1% protease inhibitor cocktail; Sigma) at 0.5 ml buffer per 250 mg tissue. After 30 minutes on ice, samples were centrifuged at 15,000 RCF for 15 minutes at 4\u0026deg;C. Supernatants were collected, and protein concentration was measured with a Bradford assay (Bio-Rad). Primary antibodies included anti-CB1 (1:1,000, Santa Cruz Biotechnology) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and anti-MOR (1:1,000, Abcam) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], with GAPDH (1:2,000, Sigma) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] as a loading control. Secondary antibody was goat anti-rabbit (1:1,000, Bio-Rad). Band intensities, normalised to GAPDH, were quantified in arbitrary units. All samples (20 \u0026micro;g each) were run in duplicate.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are shown in figures as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). To perform the statistical analysis, we used R [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and several libraries within it. For the analysis of the longitudinal data, we adopted a Bayesian perspective and fitted a Normal Linear Mixed Model LMM [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] to understand the differences in the evolution of different measurements with age controlling by relevant variables and considering repeated measurements for each female. The R library used with this purpose was \u003cem\u003erjags\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://CRAN.R-project.org/package=rjags\u003c/span\u003e\u003cspan address=\"https://CRAN.R-project.org/package=rjags\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Results for the HPT, the immunohistochemistry and the western blot are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The HPT was further analysed by using a Kaplan-Meier survival approach.\u003c/p\u003e \u003cp\u003eTo perform the statistical analysis, the 26.0 version of the SPSS program was used. We first checked the data for normality (Shapiro\u0026ndash;Wilk test) and homoscedasticity (Levene\u0026rsquo;s test). Next, we evaluated the differences between genotype using Student\u0026rsquo;s t test or Mann\u0026ndash;Whitney U test when appropriate. Significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eClassification of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het females by the onset of symptomatology\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe timeline of the longitudinal evaluation can be found in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. We measured monthly clasping and body weight of a cohort of 13 \u003cem\u003eMecp2\u003c/em\u003e-het females and 11 WT controls from 2 to 6 months of age, taking clasping as a classic marker for the onset of an overtly symptomatic phase [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, the expression of clasping in \u003cem\u003eMecp2\u003c/em\u003e-het females varies across individual subjects. Indeed, by the age of 2 months, less than 10% of the \u003cem\u003eMecp2\u003c/em\u003e-het animals displayed clasping, whereas half of the animals displayed clasping by the age of 4 months. At 6 months of age, almost all \u003cem\u003eMecp2\u003c/em\u003e-het females could be considered as symptomatic. Thus, we classified the animals as early- or late-symptomatic using 4 months old as a cut-off for upcoming analysis. As expected, none of the WT animals showed clasping behaviour.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het females show overweight as the pathology progresses\u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe implemented a longitudinal analysis to assess body weight, incorporating the previously established classification of phenotypes and their interaction with time as explanatory variables. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec illustrates a significant impact of this classification on both the initial point of measure at 2 month of age (intercept) and the trajectory (slope) of weight.\u003c/p\u003e \u003cp\u003eThe numerical summaries provided in Supplementary Table\u0026nbsp;1 indicate a high probability of the differences in slope being greater than 0, with a probability of 1. Furthermore, there is compelling evidence for a distinction in the body weight evolution between \u003cem\u003eMecp2\u003c/em\u003e-het females with early and late onset of clasping, with a probability of 0.998.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2-\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehet symptomatic females show differences in gait pattern\u003c/span\u003e \u003c/p\u003e \u003cp\u003eGait pattern was measured in WT and \u003cem\u003eMecp2\u003c/em\u003e-het females at 4, 5 and 6 months of age. While the results from the longitudinal model exhibited some degree of uncertainty, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-g illustrates relevant differences in their motion patterns. Specifically, fore paws were affected in both early- and late-symptomatic \u003cem\u003eMecp2\u003c/em\u003e-het females across testing, as opposed to hind paws, where late-symptomatic females were more similar to WT, in clear contrast to early-symptomatic females.\u003c/p\u003e \u003cp\u003eThis trend is further elucidated in the estimated values and probabilities of the designated parameters for fore paws as displayed in Supplementary Tables\u0026nbsp;2\u0026ndash;5. Notably, highly probable negative values indicate a significant difference in slope from WT in both early- and late-symptomatic individuals. Conversely, for hind paws, the difference in slope from WT to late-symptomatic females are approximately 0, with probabilities hovering around 0.5, suggesting a lack of substantial deviation from the WT behaviour.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe advance of symptoms masks mechanical hypersensitivity of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het females\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe longitudinal model applied to the VFT reveals notable distinctions among phenotypes, evident in both initial values and slopes. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh show that while WT females demonstrate stable mechanical sensitivity throughout time, \u003cem\u003eMecp2\u003c/em\u003e-het females exhibit an initial hypersensitivity that diminishes at later stages. Remarkably, at 6 months, late onset females display a sensitivity akin to WT, whereas those with early symptoms demonstrate an apparent hyposensitivity starting at 4 months.\u003c/p\u003e \u003cp\u003eExamining the numerical results (Supplementary Table\u0026nbsp;6), we observe meaningful and negative differences in intercepts for both early- (P(β\u0026thinsp;\u0026gt;\u0026thinsp;0│Data)\u0026thinsp;=\u0026thinsp;0.021) and late- (P(β\u0026thinsp;\u0026gt;\u0026thinsp;0│Data)\u0026thinsp;=\u0026thinsp;0.015) symptomatic \u003cem\u003eMecp2\u003c/em\u003e-het females. Regarding the slopes, significant distinctions emerge, with both mutant groups exhibiting larger slopes (P(β\u0026thinsp;\u0026gt;\u0026thinsp;0│Data)\u0026thinsp;=\u0026thinsp;0.986 for early- and P(β\u0026thinsp;\u0026gt;\u0026thinsp;0│Data)\u0026thinsp;=\u0026thinsp;0.946 for late-symptomatic) than WT. Interestingly, the model indicates similar slopes for late- and early-symptomatic females, with a mean difference of 0.059 (P(β\u0026thinsp;\u0026gt;\u0026thinsp;0│Data)\u0026thinsp;=\u0026thinsp;0.742). Thus, mechanical sensitivity shows discrepancies between early and late but has a similar progression.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het symptomatic females show aberrant thermal responses suggesting heat hypersensitivity\u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe analysed the thermal sensitivity in 2-month-old and 6-month-old females by means of the hot HPT. First, we analysed the standard parameter that is taken as a measure of sensitivity in the HPT, namely the latency of animals to lick the paws [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. When using this measure, 2 out of 13 6-month-old \u003cem\u003eMecp2\u003c/em\u003e-het mice did not lick the paws before the cut-off time of 30 seconds, and therefore we analysed this measure by means of a Kaplan-Meier analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, this analysis did not reveal significant differences between genotypes (χ\u003csup\u003e2\u003c/sup\u003e(2)\u0026thinsp;=\u0026thinsp;0.500, p\u0026thinsp;=\u0026thinsp;0.779). However, we noticed that, probably due to the motor impairments caused by the progression of the disease (see above), more than half of \u003cem\u003eMecp2\u003c/em\u003e-het females performed and abnormal walking backwards movement when placed in the HP as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb (χ\u003csup\u003e2\u003c/sup\u003e(2)\u0026thinsp;=\u0026thinsp;0.435, p\u0026thinsp;=\u0026thinsp;0.114), a movement never seen in WT mice. Thus, we decided to measure the latency to first avoidance movement by combining either licking the paws or walking backwards for each animal. Results showed that 6-month-old females were different among them (χ\u003csup\u003e2\u003c/sup\u003e(2)\u0026thinsp;=\u0026thinsp;11.610, p\u0026thinsp;=\u0026thinsp;0.003, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Furthermore, the analysis showed significant differences when comparing either early (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;=\u0026thinsp;8.967, p\u0026thinsp;=\u0026thinsp;0.003), or late (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;=\u0026thinsp;9.876, p\u0026thinsp;=\u0026thinsp;0.002) onset \u003cem\u003eMecp2\u003c/em\u003e-het individuals with WT animals. However, Kaplan-Meier failed to find any significant difference between early and late onset individuals (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;=\u0026thinsp;0.985). Therefore, we performed a Kaplan-Meier test without dividing \u003cem\u003eMecp2\u003c/em\u003e-het mice into early and late as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;=\u0026thinsp;11.590, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Interestingly, whereas all the presymptomatic 2-month-old did lick their paws in the HP (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;=\u0026thinsp;0.669, p\u0026thinsp;=\u0026thinsp;0.4135, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), some of them already presented with the abnormal walking backwards movement as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;=\u0026thinsp;3.669, p\u0026thinsp;=\u0026thinsp;0.055). However, when using the latency to first avoidance parameter, the Kaplan-Meier analysis failed reveal any significant differences between genotypes (χ\u003csup\u003e2\u003c/sup\u003e(1)\u0026thinsp;=\u0026thinsp;1.589, p\u0026thinsp;=\u0026thinsp;0.208, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eAll these data were further analysed by Student t tests, which revealed a significant decrease in latency to avoidance in both late- (t\u0026thinsp;=\u0026thinsp;3.389, p\u0026thinsp;=\u0026thinsp;0.004, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) and early-symptomatic females (t\u0026thinsp;=\u0026thinsp;3.805, p\u0026thinsp;=\u0026thinsp;0.002, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei) when compared to the WT females. This was maintained when early- and late- symptomatic females where collapsed (t\u0026thinsp;=\u0026thinsp;4.101, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). However, this was not replicated in the younger cohort since no significant difference was found between \u003cem\u003eMecp2\u003c/em\u003e-het and WT females (t\u0026thinsp;=\u0026thinsp;1.438, p\u0026thinsp;=\u0026thinsp;0.181, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFOS expression after the hot plate and CB1 expression are significantly reduced in PAG of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het females\u003c/span\u003e\u003c/p\u003e \u003cp\u003eHalf of the 6 months old mice and all the 2 months old mice were sacrificed 90 minutes after the HPT to check for possible alterations in the activation patterns in brain regions relevant for pain processing by means of FOS expression. We compared the number of cFos-ir cells in different brain regions and found no significant differences between genotypes in the group of 2 months old (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-e). In 6-month-old mice, although no significant differences were observed in most of the areas tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-j), a significant reduction in cFos-ir cells was detected in the dorsal PAG of Mecp2-het females compared to WT females, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef (Mann-Whitney U test, p\u0026thinsp;=\u0026thinsp;0.004). This difference was maintained when analysing separately the group of het females in late- (Mann-Whitney U test, p\u0026thinsp;=\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek) and early- (Mann-Whitney U test, p\u0026thinsp;=\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el) symptomatic females.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHaving found specific decrease of activation in the PAG, we performed western blot against CB1 receptors and MOR in PAG punches of half of the mice 6 months old mice in this area. Student t-test analysis showed a significant reduction in \u003cem\u003eMecp2\u003c/em\u003e-het females for the density of CB1 (t\u0026thinsp;=\u0026thinsp;2.563, p\u0026thinsp;=\u0026thinsp;0.028; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) but not for MOR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we carried out a longitudinal analysis in a cohort of \u003cem\u003eMecp2\u003c/em\u003e-het females, assessing disease progression and somatosensory sensitivity. We found that \u003cem\u003eMecp2\u003c/em\u003e-het females might experience hypersensitivity from pre-symptomatic age, which could be masked at later stages by the early onset of additional symptoms, highly variable among individuals. Further, we also identify \u0026ldquo;walking backwards\u0026rdquo; in the HPT as an atypical behavioural response to potentially painful stimulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Taken together, our data support that MeCP2 deficiency might cause heightened sensitivity to mechanical and thermal stimulation, albeit masked by the onset of motor symptoms. Further, our data and highlight the need to characterise atypical pain responses in mouse models of neurodevelopmental disorders and take into account the phenotypic heterogeneity displayed by \u003cem\u003eMecp2\u003c/em\u003e-het females.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het females are hypersensitive to mechanical and thermal stimulation\u003c/span\u003e \u003c/p\u003e \u003cp\u003eHypersensitivity to somatosensory stimulation has been previously reported in \u003cem\u003eMecp2\u003c/em\u003e-mutant mice. On the one hand, Orefice et al. (2016) demonstrated that \u003cem\u003eMecp2\u003c/em\u003e-null males display increased sensitivity to mechanical stimulation, especially due to peripheral mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. On the other hand, \u003cem\u003eMecp2\u003c/em\u003e-het females with trimmed whiskers did not show the typical aberrant preference for open arms in the elevated plus maze found in this model, suggesting that sensory hypersensitivity could be an important contributor to their apparent reduced anxiety [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, \u003cem\u003eMecp2\u003c/em\u003e-het females showed a significant hypersensitivity, as compared to WT controls, in the VFT, only evident at pre-symptomatic stages. Thus, the mechanical nociception threshold, as measured by the withdrawal of the paw, of \u003cem\u003eMecp2\u003c/em\u003e-het females significantly varies with age, from hypersensitivity in pre-symptomatic females at 2 month of age, to apparent hyposensitivity by 6. Interestingly, the timing of onset of the overt symptomatic phase in these animals is highly variable among individuals. When we segregated the group of \u003cem\u003eMecp2\u003c/em\u003e-het females into early- and late-symptomatic, we found that the latter remained hypersensitive until later stages, whereas the former were apparently hyposensitive from 5 months of age onwards. In line with the other variables measured in our longitudinal analysis (clasping and gait analysis), it is plausible that early-symptomatic mice may not respond to the VFT filaments simply due to aspects likely to influence motility that are inherent to the disease's progression.\u003c/p\u003e \u003cp\u003eThe same might be true for thermal stimulation in 6-month-old \u003cem\u003eMecp2\u003c/em\u003e-het females. Both overweight and gait abnormalities present in \u003cem\u003eMecp2\u003c/em\u003e-het symptomatic females may interfere in their ability to lick their paws. Thus, at 6 months of age, when nearly all \u003cem\u003eMecp2\u003c/em\u003e-het females were overtly symptomatic, we did not find significant differences in thermal nociception threshold when using the standard measure of paw licking. This result contrasts with a previous study reporting an increase in the latency to lick the paws in the HPT in 5-month-old \u003cem\u003eMecp2\u003c/em\u003e-het female [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which was regarded as hyposensitivity. However, we propose an alternative interpretation: due to concomitant factors intrinsic to the disease's progression (most likely impacting motility), \u003cem\u003eMecp2\u003c/em\u003e-het females display an aberrant walking backwards movement when trying to lick their paws, which is already shown by the younger cohort. By taking this measure and combining it with the more standard paw licking, we found a significant reduction in the time to react in \u003cem\u003eMecp2\u003c/em\u003e-het females, suggesting that \u003cem\u003eMecp2\u003c/em\u003e-het females are also hypersensitive to thermal stimulation. In agreement, Vigli et al., (2021) reported increased sensitivity in the HPT in \u003cem\u003eMecp2\u003c/em\u003e-null male mice. In agreement with this view on disease progression masking potential standard behavioural responses, our study on presymptomatic 2-month-old \u003cem\u003eMecp2\u003c/em\u003e-het females displayed no significantly altered thermal sensitivity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Together, these findings highlight the importance of monitoring symptom onset in \u003cem\u003eMecp2\u003c/em\u003e-mutant models and the need for behavioural analyses that account for both standard and atypical responses.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNeural and neurochemical substrate of hypersensitivity in\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMecp2\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-het females\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThermal stimulation led to a significantly lower neuronal activation at the PAG, as measured by FOS immunoreactivity, of 6-months-old \u003cem\u003eMecp2\u003c/em\u003e-het females when compared to WT controls. The PAG is an important structure in the pain descending pathway since its innervation to the spinal cord modulate pain sensitivity and endogenous analgesia [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, the activation of neurons within this brain area upon a painful stimulus is related to how the nervous system manages that stimulus [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The diminished neuronal activity that we report at the PAG of symptomatic \u003cem\u003eMecp2\u003c/em\u003e-het females suggests a reduction of endogenous analgesia, which could contribute to a heightened sensitivity towards somatosensory stimulation. G\u0026eacute;ranton et al. (2008) demonstrated that, in rats, MeCP2 in descending serotonergic pathways can regulate gene expression in the dorsal horn and mechanical sensitivity associated with an inflammatory pain state [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Specifically, they found that a complete Freund\u0026rsquo;s adjuvant injection into WT hind paw (a model of inflammatory pain) significantly increased phosphorylated MeCP2 levels in Lamina I and II of the superficial dorsal horn, with a peak observed after 1 hour.\u003c/p\u003e \u003cp\u003eThe endocannabinoid system participates in in PAG-driven differential sensitivity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In general, CB1 agonists seem to lead to antinociception [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which is greatly mediated by receptors located within the PAG [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, it is not unexpected that our results show a decrease in CB1 expression levels, which correlate with decreased thermal nociception thresholds and decreased neuronal activity. Interestingly, pharmacological approaches targeting the endocannabinoid system have previously been used to ease RTT symptomatology in animal models [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], among which we find pain [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, although overall upregulation of endocannabinoid system components might be reported in the above-mentioned studies, region-specific differences might arise due to \u003cem\u003eMecp2\u003c/em\u003e deficiency [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Thus, future studies should investigate the possible region-specific alterations in the endocannabinoid and endogenous opioid systems in \u003cem\u003eMecp2\u003c/em\u003e deficient models.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eTaken together, our results reveal that pain might be largely hidden in mouse models of RTT and highlight the need of future studies to ameliorate pain conditions in RTT patients. Our results reflect the complexity of assessing aberrant behaviours in a RTT surrogate mouse model, given the sensory and motor impairments that characterise the disease. Our data supports the validity of \u003cem\u003eMecp2-\u003c/em\u003ehet females from the pre-symptomatic phase to study RTT and the importance of assessing symptom progression to get a full understanding of the consequences derived from of \u003cem\u003eMecp2\u003c/em\u003e deficiencies.\u003c/p\u003e"},{"header":"STATEMENTS \u0026 DECLARATIONS","content":"\u003cp\u003e\u003cstrong\u003eACKOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are indebted to Dr Enrique Lanuza for his continuous support and discussion of the data.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Ayudas FinRett para la Investigaci\u0026oacute;n del S\u0026iacute;ndrome de Rett 2019 and 2022 and the Spanish Ministry of Science and Innovation (PID2019-107322GB-C22 funded by MCIN/AEI/10.13039/501100011033) to CAP; the Spanish Ministry of Science and Innovation (PID2022-137803NB-I00 funded by MCIN/AEI/10.13039/501100011033) to LH; MAA obtained a postdoctoral fellowship from the Spanish Ministry of Universities of Spain (MS21-083) financed by Next Generation EU2; JC is supported by\u0026nbsp;an\u0026nbsp;Atracció de Talent Fellowship from the University of Valencia (UV-INV-PREDOC-1327981), JVTP by a Ram\u0026oacute;n y Cajal contract (RYC2021-034012-I) by the Spanish Ministry of Science and Innovation and the European Union \u0026ldquo;NextGenerationEU\u0026rdquo;/PRTR and the 2023 Pickford Award from the British Pharmacological Society, and MS by PT2020-Centro 2020 (CENTRO-01-0145-FEDER-000008). JVTP and MAA are also supported by the \u003cem\u003eConselleria de Educaci\u0026oacute;n, Universidades y Empleo\u003c/em\u003e from the \u003cem\u003eGeneralitat Valenciana\u003c/em\u003e with a \u003cem\u003eSubvenciones a grupos de investigaci\u0026oacute;n emergentes\u003c/em\u003e (CIGE/2022/139).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJavier Cuitavi\u003c/strong\u003e performed research, analysed data, prepared figures and wrote the paper; \u0026nbsp;\u003cstrong\u003eElena Mart\u0026iacute;nez-Rodr\u0026iacute;guez\u003c/strong\u003e performed research, analysed data, and wrote the paper; \u003cstrong\u003eMar\u0026iacute;a Abell\u0026aacute;n-\u0026Aacute;lvaro\u003c/strong\u003e performed research and analysed data; \u003cstrong\u003eMois\u0026eacute;s Garc\u0026iacute;a-Arencibia\u0026nbsp;\u003c/strong\u003eparticipated in the design of the project;\u003cstrong\u003e\u0026nbsp;M\u0026oacute;nica Santos\u0026nbsp;\u003c/strong\u003eparticipated in the design of the project; \u003cstrong\u003eAnabel Forte\u003c/strong\u003e analysed longitudinal data and provided general assessment on statistical analysis; \u003cstrong\u003eLuc\u0026iacute;a Hip\u0026oacute;lito\u003c/strong\u003e participated in the design of the study; \u003cstrong\u003eCarmen Agust\u0026iacute;n-Pav\u0026oacute;n\u003c/strong\u003e designed the study, supervised research and data analysis, contributed to writing and funding acquisition;\u003cstrong\u003e\u0026nbsp;Jose Vicente Torres-P\u0026eacute;rez\u003c/strong\u003e performed research, analysed data and contributed to writing and funding acquisition. All authors contributed to discuss results and read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAnimal protocols followed in this work were approved by the Animal Care Committee of the University of Valencia and were strictly adhered to in compliance with the EEC Council Directive 63/2010, Spanish laws (RD 53/2013) and animal protection policies.\u003c/p\u003e"},{"header":"REFERENCES","content":"\u003col\u003e\n\u003cli\u003eAmir, R. E., \u0026amp; Zoghbi, H. Y. (2000). Rett syndrome: Methyl‐CpG‐binding protein 2 mutations and phenotype\u0026ndash;genotype correlations. \u003cem\u003eAmerican journal of medical genetics\u003c/em\u003e, \u003cem\u003e97\u003c/em\u003e(2), 147-152. https://doi.org/10.1002/1096-8628(200022)97:2\u0026lt;147::aid-ajmg6\u0026gt;3.0.co;2-o.\u003c/li\u003e\n\u003cli\u003eGold, W. A., Krishnarajy, R., Ellaway, C., \u0026amp; Christodoulou, J. (2018). Rett syndrome: a genetic update and clinical review focusing on comorbidities. \u003cem\u003eACS chemical neuroscience\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(2), 167-176. https://doi.org/10.1021/acschemneuro.7b00346.\u003c/li\u003e\n\u003cli\u003eSymons, F. J., Byiers, B., Tervo, R., \u0026amp; Beisang, A. (2013). Parent reported pain in rett syndrome. \u003cem\u003eThe Clinical journal of pain\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(8), 744. https://doi.org/10.1097/AJP.0b013e318274b6bd.\u003c/li\u003e\n\u003cli\u003eHagberg, B. (2002). 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Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system. \u003cem\u003eBrain Structure and Function\u003c/em\u003e, \u003cem\u003e224\u003c/em\u003e, 1647-1658. https://doi.org/10.1007/s00429-019-01860-6.\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rett Syndrome, Mecp2-het females, pain, endogenous analgesia, endocannabinoid system, nociception","lastPublishedDoi":"10.21203/rs.3.rs-5804567/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5804567/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRett Syndrome (RTT), a neurodevelopmental disorder predominantly affecting females, is characterised by evolving symptoms impacting motor and sensory domains. Herein, we present a study of longitudinal analyses, from 2- to 6-month of age, of \u003cem\u003eMecp\u003c/em\u003e2 heterozygous (\u003cem\u003eMecp2\u003c/em\u003e-het) female mice to comprehensively explore pain perception in RTT. Interestingly, we found a significant variability in the timing and progression of symptom onset among \u003cem\u003eMecp2\u003c/em\u003e-het females, with individuals classified as either early- or late-symptomatic based on the emergence of hallmark neurological features such as clasping and gait abnormalities. This variability pinpoints the heterogeneity of the disease model and highlights the need to stratify \u003cem\u003eMecp2\u003c/em\u003e-het females by symptom onset in future studies to account for the diverse trajectories of disease progression. Additionally, our results reveal a shift from pre-symptomatic hypersensitivity in the von Frey test to apparent hyposensitivity, intricately linked with the onset of motor symptoms. Further, we found decreased neuronal activation in 6-month-old \u003cem\u003eMecp2\u003c/em\u003e-het females after the hot plate test in the periaqueductal gray, as measured by FOS expression. Similarly, there is a lower expression of cannabinoid receptor 1 (CB1) in this area when compared to wild-type siblings. Taken together, our results suggest that both motor impairment and central deficits in the modulation of endogenous analgesia contribute to aberrant sensitivity in \u003cem\u003eMecp2\u003c/em\u003e-het mice. Our study emphasises the presymptomatic phase as crucial for understanding sensory abnormalities in \u003cem\u003eMecp2\u003c/em\u003e-het mice and highlights the challenges in identifying pain in RTT patients.\u003c/p\u003e","manuscriptTitle":"Longitudinal Analysis in Mecp2-het Female Mice Reveals Atypical Nociceptive Behaviours","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-03 09:30:35","doi":"10.21203/rs.3.rs-5804567/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-02-27T14:54:08+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-01-30T09:05:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-30T08:55:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-14T13:40:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2025-01-13T10:13:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"33e6895e-09b6-442f-8275-c6b19ee56935","owner":[],"postedDate":"February 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:51:25+00:00","versionOfRecord":{"articleIdentity":"rs-5804567","link":"https://doi.org/10.1007/s00109-025-02628-8","journal":{"identity":"journal-of-molecular-medicine","isVorOnly":false,"title":"Journal of Molecular Medicine"},"publishedOn":"2026-01-13 16:31:01","publishedOnDateReadable":"January 13th, 2026"},"versionCreatedAt":"2025-02-03 09:30:35","video":"","vorDoi":"10.1007/s00109-025-02628-8","vorDoiUrl":"https://doi.org/10.1007/s00109-025-02628-8","workflowStages":[]},"version":"v1","identity":"rs-5804567","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5804567","identity":"rs-5804567","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}