Doublecortin-expressing cells are selectively altered in the piriform cortex but not in neurogenic areas of symptomatic Mecp2-heterozygous mice

Doublecortin-expressing cells are selectively altered in the piriform cortex but not in neurogenic areas of symptomatic Mecp2-heterozygous mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Doublecortin-expressing cells are selectively altered in the piriform cortex but not in neurogenic areas of symptomatic Mecp2-heterozygous mice Rafael Esteve-Pérez, Paloma Sevilla-Ferrer, Enrique Lanuza, Vicente Herranz-Pérez, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7269766/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2025 Read the published version in Neuroscience → Version 1 posted You are reading this latest preprint version Abstract Rett Syndrome (RTT) is a neurodevelopmental disorder which mainly affects girls, leading to profound impairments in motor function, loss of speech, intellectual disability, and epilepsy, among other symptoms. Most cases are caused by mutations in the X-linked MECP2 gene, which encodes the protein methyl-CpG-binding protein 2 (MeCP2), an epigenetic reader with a crucial function in the regulation of neural maturation. Previously, using the marker of immature neurons doublecortin (DCX), we showed that a population of embryonic-born neurons of the piriform cortex, which experience prolonged maturation throughout life, was increased in the piriform cortex of young adult (2 months old), symptomatic, Mecp2 -null male mice. By contrast, these cells were not affected in age matched Mecp2 -heterozygous female mice, who are pre-symptomatic at that age. To determine whether symptom onset would affect DCX-expressing neurons, in this study we analysed samples from 6 months old, symptomatic Mecp2 -heterozygous female mice. Our results show a specific increase in the density of DCX-positive neurons in the piriform cortex, consistent with observations in males. However, no differences were detected in the neurogenic niches of the dentate gyrus or the ventricular-subventricular zone when compared to their wild-type controls. Further, morphological analyses of the DCX-expressing cells of the piriform cortex reveal that they are smaller and show less complex dendritic branching in mutant mice. In conclusion, our findings support a role of MeCP2 in the maturation process of the embryonic-born DCX neurons in the piriform cortex and point to region-specific alterations in neuronal maturation in RTT. Neurodevelopmental disorders neurogenesis olfactory system Rett syndrome Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Rett syndrome (RTT) is a rare neurodevelopmental disorder that affects mainly females, characterised by a developmental regression occurring between 6 to 18 months of age, leading to loss of speech, intellectual disability, repetitive hand movements, breathing abnormalities, and motor impairment, among other symptoms (Amir et al. 1999 ). The main cause of classical RTT are loss-of-function mutations in the X-linked MECP2 gene, encoding the methyl-CpG-binding protein 2 (MeCP2) (Amir et al. 1999 ). In males, hemizygous loss-of-function MECP2 mutations are typically lethal, resulting in severe neonatal encephalopathy and often perinatal death (Santos et al. 2009 ), whereas mutations and variants not leading to complete loss of function in this gene have been linked to disorders causing intellectual disability, neuroendocrine dysfunction or psychiatric conditions (Moretti and Zoghbi 2006 ; Canton et al. 2023 ). Originally identified as a transcriptional repressor (Nan et al. 1997 ), MeCP2 is currently recognized as a multidomain epigenetic regulator that modulates gene expression in a context-dependent manner, both silencing and promoting transcription (Sharifi and Yasui 2021 ). Murine models with MeCP2 loss-of-function mutation serve as a valuable approach to understanding the implication of MeCP2 in the nervous system (Guy et al. 2001 ). In contrast to humans, male mice survive infancy but have a short lifespan and an early onset of symptoms, with animals of 2 months of age being fully symptomatic, whereas females display a variable onset of motor symptoms, being symptomatic by 2 to 6 months of age (Guy et al. 2001 ; Abellán-Álvaro et al. 2021 ; Cuitavi et al. 2025 ). The timing of phenotypic manifestations of RTT in humans and murine models suggests that the underlying cause of symptoms is not an altered developmental neurogenesis, but a disruption in the maintenance of a mature neuronal functionality (Shahbazian 2002 ; Kishi and Macklis 2004 ; Smrt et al. 2007 ). This is further supported by the observed pattern of MeCP2 protein expression, characterized by low levels during embryonic brain development and reaching the highest levels in more mature structures (Shahbazian 2002 ; Kishi and Macklis 2004 ). In agreement with a key role of MeCP2 for neuronal maturation, we previously showed that immature neurons, identified by using the marker doublecortin (DCX), were increased in specific brain regions of adolescent and young adult Mecp2 -null mice (Martínez-Rodríguez et al. 2019 ; Torres-Pérez et al. 2022 ). In the postnatal rodent brain, DCX-immunoreactive (DCX-ir) cells are present in neurogenic areas such as the dentate gyrus (DG) of the hippocampus and the ventricular-subventricular zone (V-SVZ), from which neuroblasts migrate mainly through the rostral migratory stream (RMS) to the olfactory bulb (OB) (Doetsch et al. 1999 ; Whitman and Greer 2009 ), but also to other locations such as the nucleus accumbens NAc (García-González et al. 2021 ). In addition, layer II of the piriform cortex (Pir) harbours a population of DCX-ir cells generated during embryonic development that persists into adulthood (Nacher et al. 2001 ; Rubio et al. 2016 ). These cells have been shown to mature gradually with age and integrate as glutamatergic neurons (Rotheneichner et al. 2018 ; Benedetti et al. 2020 ). Interestingly, while proliferative neurogenesis seems largely absent postnatally in mammals with larger brains, such as non-human and human primates (Paredes et al. 2016 ; Sorrells et al. 2018 ), cortical DCX-ir immature neurons of prenatal origin might be widespread in these species (La Rosa et al. 2020 ), including humans (Coviello et al. 2022 ), highlighting the importance of characterising this population in disease models. Our prior findings revealed that DCX-ir cells of the Pir, and adjacent olfactory tubercle (OT), but not the DG or OB, were significantly increased in young Mecp2 -null male mice as compared with age-matched wild-type (WT) controls (Martínez-Rodríguez et al. 2019 ). By contrast, we did not find any effect in any of those regions in young adult Mecp2 -heterozygous ( Mecp2 -het) females (Martínez-Rodríguez et al. 2019 ). Since young adult females are largely pre-symptomatic (Cuitavi et al. 2025 ), we hypothesised that alterations in DCX-ir cells might emerge later, when symptoms are already present in Mecp2 -het females. To test this hypothesis, here we analysed the density and morphological features of DCX-ir cells across several brain areas, in 6 months old Mecp2 -het female mice and their WT littermates. MATERIALS AND METHODS Animals For these experiments, we used spared samples from 6 months old female mice of the Mecp2 tm1.1Bird/J strain (WT, n = 5; Mecp2 -heterozygous, n = 8, Jackson Laboratory), obtained from symptomatic females used in a previous study (Cuitavi et al. 2025 ). Mice were housed in standard laboratory cages in groups of 2–5 animals, under controlled humidity and temperature (22ºC), with a 12:12-h light/dark cycle, and water and food available ad libitum . For genotyping, we obtained ear plugs at weaning, and after DNA extraction, we applied the protocol supplied for this strain by the Jackson Laboratory. The protocols were approved by the Animal Experimentation Ethics Committee of the University of Valencia, Protocol 2019/VSC/PEA/0027, and carried out in strict accordance with EU directive 2010/63/EU. Perfusion and histology Animals were deeply anaesthetized using pentobarbital (50 mg/Kg) and transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.4. Brains were carefully removed from the skull, postfixed overnight in the same fixative and transferred to 30% sucrose in 0.01M phosphate buffered saline (PBS, pH 7.6) until they sank. The brains were then frozen and cut in six series of 40-µm-thick coronal sections with a freezing microtome. Free-floating sections were collected in five parallel series and kept at -20 ºC in 30% sucrose in PB (0.1 M, pH 7.4) for future processing. Doublecortin immunohistochemistry We obtained permanent immunostained preparations for DCX in one out of the six parallel series, using the indirect avidin–biotin peroxidase complex/3,3’-diaminobenzidine-staining (ABC/DAB) procedure, as described in our previous work (Martínez-Rodríguez et al. 2019 a). In brief, brain slices were first incubated with 1% H 2 O 2 in 0.05M tris-buffered saline (TBS, 0.9% NaCl in TB) for 30 minutes at room temperature (RT) to block endogenous peroxidase activity. Subsequently, sections were incubated with 2% normal horse serum (NHS) and 0.3% Triton-X100 in 0.05M TBS pH 7.4 for one hour at RT, to block nonspecific binding. Brain slices were then incubated overnight at 4 o C with a goat primary antibody anti-DCX (C-18, sc:8066, Santa Cruz Biotechnology, Inc) diluted 1:500 in 0.05M TBS pH 7.4, with 2% NHS and 0.3% Triton-X100. The next day, sections were incubated for two hours at RT with biotinylated horse anti-Goat-IgG secondary antibody (BA-9500, Vector) diluted 1:200 in the same buffer. Afterwards, sections were incubated for 90 minutes at RT in ABC (Vector) in TBS with 0.3% Triton-X100. Sections were thoroughly washed in TBS (3 x 10 min.) between each step. To reveal peroxidase activity, sections were incubated for five minutes in DAB-SigmaFAST (Sigma) in TB. Sections were mounted using 0.2% gelatine in TB, dehydrated with alcohol, cleared with xylol and cover-slipped with Entellan. Quantification of DCX-immunoreactive cells We analysed the samples with a Leica Leitz DMRB microscope, equipped with a digital camera (Leica DFC495) and Motic Software at a priori selected Bregma levels (B), according to (Paxinos and Franklin 2012). For the piriform cortex (Pir), we analysed approximate bregma levels B0, B-1 and B-2 mm; for the dentate gyrus of the hippocampus (DG), B-1, B-2 and B-3 mm, and for the nucleus accumbens (NAc) we analysed from approximately B1.18 to B0.62 mm (Paxinos and Franklin 2019 ). In the Pir, an experimenter who was blind to the genotype of the mice inspected the sections and counted the number of cells in each area. DCX-ir cells have been previously classified into two populations based on size and morphology: (1) “simple”, small cells without neurites or with a rudimentary one and a smaller soma size; and (2) “complex”, larger cells with a larger and more developed dendritic tree (Rubio et al. 2016 ). Thus, in the Pir those DCX-ir cells with none or one neurite were classified as simple, whilst cells with two or more neurites were quantified as complex. To evaluate neurons originated in the V-SVZ, we counted manually DCX-ir neurons in the periglomerular region of the OB (since the intense labelling in the granular cell layer precluded the quantification of individual cells) and in the NAc, where cells were counted in the region between the ventricular-subventricular zone (V-SVZ) and the anterior commissure (aca). In addition, we measured the Euclidean distance from each DCX-ir cell located in the NAc to the nearest lateral ventricle wall as a measure of migratory capacity (García-González et al. 2021 ). Finally, in the DG, DCX-ir neurons were counted only in the granular layer, and their maximum somatic diameter was also measured manually. All quantifications were performed using Fiji software (NIH). Cell density was expressed as an average number of labelled cells per section per hemisphere. Sholl and skeleton analyses We examined the morphology of the DCX-ir cells from the Pir and the DG by means of Sholl and skeleton analysis using ImageJ SNT Plugin (default parameters, v4.2.1) and Analyze skeleton tool, respectively. First, cells from layer II of the Pir (complex only) and DG were drawn manually using a camera lucida attached to a Leica optic microscope. Neurons were selected only if they were relatively isolated from other neurons and without discernible breaks. Next, images were scanned and processed with ImageJ software to convert them to an 8-bit format and then skeletonized. We used Sholl method to quantify the number of intersections of the dendrites within concentric circles of increasing radius (radius steps set at 10 µm for Pir cells and 5 µm for DG cells, according to different neuronal arbour sizes) centred on the soma, and the total number of intersections. Complementary to Sholl, skeleton analysis was applied to examine several branching parameters, including total branch length, maximum branch length, number of junctions and number of branches. We analysed a total of 45 DCX-ir neurons of the Pir, and 70 DCX-ir neurons of the DG, with a minimum of 5 positive cells per animal. Fractal analysis To characterize structural complexity of DCX-ir neurons from the Pir (complex cells) and DG, we performed a fractal analysis. The binary images of outlined cells were then analysed using the ImageJ FracLac plug-in. Changes in neuronal structure complexity were assessed by calculating Fractal dimension (D B ) and Lacunarity (λ). Fractal dimension provides a measure of complexity, which in turn is how a pattern's detail changes with the scale at which it is considered. Thus, it reflects how completely a neuronal arbour fills a specific area. Lacunarity is a measure of heterogeneity (rotational invariance) as a complement to complexity in describing digital images. Box-counting method was used for D B and λ calculations. Default settings were left unchanged, except for Num G (the number of box counting grid orientations used during the scan), which was set at 5, following the recommended range indications from FracLac. Additional metrics, including Hull and circle metrics data (Density and Span ratio). Statistical Analyses Data were analysed using the GraphPad Prism 9 and R software. Data normality was tested with the Shapiro-Wilk test. To test statistical significance in multiple comparisons (number of intersections per radius in Sholl analysis), a two-way ANOVA followed by post hoc Bonferroni correction was employed. For two group comparisons, we used unpaired Student’s t-test if normality was confirmed; otherwise, the Mann-Whitney test was used. All values are reported as mean ± SEM. Significance level was set at p < 0.05. RESULTS Complex DCX-ir neurons are increased in the piriform cortex of Mecp2 -heterozygous symptomatic females, but not in the neurogenic niches of DG or V-SVZ As expected, we did not find qualitative differences in the distribution of DCX-ir cells between Mecp2 -het and WT female mice. Thus, DCX-ir cells were found in the Pir, OB, NAc and DG in both genotypes (Fig. 1 A). Considering the finding of a restricted population of DCX-ir cells in the olfactory tubercle, which were increased in Mecp2 -null male mice (Martínez-Rodríguez et al. 2019 ), we also examined the olfactory tubercle in these samples. However, none or very few DCX-ir cells were found in this region in either genotype, suggesting that this population may be transient. Quantitatively, we found no significant differences between genotypes in the total number of DCX-ir cells in the Pir (W = 10.5, p = 0.19; Fig. 2 B). However, when considering the classification on simple (more immature) and complex (more mature), we found a significant increase in the density of complex DCX-ir cells in Mecp2 -het females s (t = -2.6, p = 0.025), but not in simple DCX-ir cells (W = 11.5, p = 0.24). In contrast, the density of DCX-ir cells did not differ between genotypes in the OB (t = 0.12, p = 0.91; Fig. 1 C), in the NAc (t = -0.16, p = 0.87; Fig. 1 D) or in the DG (t = 0.72, p = 0.49; Fig. 1 E). In the NAc, we did not find significant differences in the mean distance from DCX-ir cells to the V-SVZ (W = 22.0, p = 0.83; Data not shown). Altogether, these results suggest a region-specific effect of the heterozygosity of Mecp2 in the Pir, without alterations in other neurogenic areas. Morphological analyses revealed decreased size and complexity in DCX-ir neurons of the piriform cortex We next investigated whether Mecp2 deficiency could affect the morphology and complexity of DCX-ir cells in the Pir. First, we measured the main somatic diameter of DCX-ir cells in the Pir, detecting a significant reduction in DCX-ir cells in Mecp2 -het females compared to WT (t = 2.24, p = 0.044; Fig. 2 C). This decrease might be largely due to a decrease in the main diameter of simple DCX-ir cells (t = 2.37, p = 0.03), while the main diameter of complex DCX-ir cells did not differ significantly between genotypes (t = 1.2, p = 0.25). Further, Sholl analysis revealed a significant reduction in the total number of intersections in DCX-ir complex cells of Mecp2 -het females when compared to their WT counterparts (t = 3.58; p = 0.004; Fig. 2 D). As expected, the reduction was consistent across the entire dendritic profile, with statistically significant differences at 5, 10, 15, 20 and 30 µm from the cell somata (all p < 0.05) (Fig. 2 E). Skeleton analysis further confirmed a reduced dendritic complexity in Mecp2 -het females, showing decreased total dendritic arbour length (t = 3.45, p = 0.006; Fig. 2 F), fewer junctions (t = 4.31, p = 0.001, Fig. 2 H), and fewer branches (t = 4.29, p = 0.001; Fig. 2 I) as compared to WT. Among the parameters measured by fractal and hull and circle analyses, only lacunarity, a measure of complexity of the dendritic tree, showed distinctive values between genotypes (t = 3.64, p = 0.004; Fig. 2 K) which was driven by a significantly lower value in the Mecp2 -het females. The rest of the measurement did not show differences between genotypes (all p > 0.05). Morphological analyses revealed no significant effect of Mecp2 deficiency in DCX-ir neurons of the dentate gyrus We performed the same morphological analyses in DCX cells in the DG of the hippocampus. In this case, none of the parameters evaluated by Sholl, skeleton or fractal analyses revealed significant differences between genotypes in DCX-ir cells of the DG (all p > 0.05, Fig. 3 ). DISCUSSION In this study, we analysed the effect of Mecp2 haploinsufficiency in distinct populations of immature neurons, namely the embryonic born DCX-ir cells of the Pir, and the continuously generated DCX-ir cells of the OB, the NAc and the the DG. We found a specific increase in the density of DCX-ir cells in the Pir, as well as a reduced soma diameter, and less complex, shorter dendritic trees of these cells in Mecp2 -het symptomatic females. In contrast, we did not find significant effects of Mecp2 deficiency in DCX-ir cells of the OB, NAc or the DG. Together with our previous study showing specific deficits in the Pir in symptomatic Mecp2 -null males but not pre-symptomatic Mecp2 -het females (Martínez-Rodríguez et al. 2019 ), our data suggest that Mecp2 deficiency contributes to arrested maturation of embryonic-born DCX-ir cells, which may be associated with the onset of overt symptoms. These results are in agreement with previous studies revealing region-specific effects of Mecp2 deficiency (Santos et al. 2010 ; Wang et al. 2013 ; Smith et al. 2019 ). Several factors may explain this regional specificity, including non-cell-autonomous effects of the function of MeCP2 (Kishi and Macklis 2004 ; Ballas et al. 2009 ), differential expression levels of MeCP2 in different regions (Cassel et al. 2004 ; Abellán-Álvaro et al. 2024 ) or regional differences in vulnerability to stress (Nacher et al. 2004 ; Abellán-Álvaro et al. 2024 ). Probably the most important difference to consider between the populations studied here is the different ontogeny of these immature neurons. Thus, whereas new neurons are continuously being incorporated in the OB, NAc and DG of young adult mice (Doetsch et al. 1999 ; García-González et al. 2021 ; Arellano et al. 2024 ), immature neurons in the Pir are generated during embryonic development and remain immature until intrinsic and/or environmental factors trigger their maturation (Gómez-Climent et al. 2011 ; Rotheneichner et al. 2018 ; Abellán-Álvaro et al. 2024 ; Esteve-Pérez et al. 2025 ). These findings support the idea that MeCP2 is more likely to influence neuronal differentiation and maturation than for proliferation (Shahbazian 2002 ; Kishi and Macklis 2004 ; Smrt et al. 2007 ). The olfactory system is a valuable system for studying neurodevelopment during healthy and pathological conditions, including RTT (Sweat and Cheetham 2024 ). Consequently, this system has been widely studied in both RTT patients and genetically modified mice. In RTT patients, nasal biopsies revealed fewer terminally differentiated olfactory receptor neurons and a notable increase in immature olfactory sensory neurons (Ronnett et al. 2003 ). In mouse models, previous studies in the olfactory system have found that delayed maturation caused by lack of Mecp2 results in a transient abnormal axonal projections in the olfactory bulb and subglomerular disorganisation (Matarazzo et al. 2004 ). These results, together with those that show a postnatal deficit in the olfactory refinement in Mecp2 -null males (Degano et al. 2014 ), suggest that an impairment in the RMS, which continuously brings newly generated neurons to the OB from the V-SVZ, might be occurring. However, we did not find significant effects of genotype in the density of DCX-ir cells in the periglomerular area Mecp2 -het symptomatic mice, suggesting that postnatal neurogenesis in the V-SVZ towards these area is not affected by heterozygosity of Mecp2 , despite the potential malfunctions in newly generated neurons in the OB (Matarazzo et al. 2004 ; Degano et al. 2014 ), or a subtle effect on transition to maturity such as the one we previously found in the Mecp2 -null males (Martínez-Rodríguez et al. 2019 ). On the other hand, immature neurons of the NAc, also arising from V-SVZ, have previously been related to chronic pain (García-González et al. 2021 ), and pain reception and behaviour is altered in both RTT patients and female rodent models (Downs et al. 2010 ; Cuitavi et al. 2025 ). The absence of differences in the density and migration distances of the V-SVZ in DCX-ir neurons in the NAc of Mecp2 -het females suggests that other mechanisms and areas of pain processing could be involved in these deficits. In addition to the increased density of complex DCX-ir cells in the Pir, we observed a reduction of dendritic arborization and complexity in the DCX-ir neurons of Mecp2 -het females, in agreement with previous reports showing a dendritic and synaptic impairment in cortical neurons from both mouse models (Kishi and Macklis 2004 ; Belichenko et al. 2009 ) and RTT patients (Bauman et al. 1995 ; Belichenko et al. 1996 ). Moreover, some studies propose that soma size as a phenotypic marker for of MeCP2 deficiency (Wang et al. 2013 ), and our data revealed that DCX-ir cells of the Pir of Mecp2 -het females also had a smaller soma compared to WT females. Both the morphological impairment in these immature neurons of Pir and the increase of the density of complex DCX-ir cells in this region might compromise the reserve of cortical plasticity and glutamatergic input provided by these immature neurons represent for olfactory and associative functions (Rotheneichner et al. 2018 ). Finally, MeCP2 deficiency has been associated with structural and functional deficits in the hippocampus. For example, in RTT patients, impairment in the expression of MECP2 has been associated with lower dendritic spine density in hippocampal mature cells (Chapleau et al. 2009 ; Nerli et al. 2020 ), whereas analogous studies in rodents have found how lack of MECP2 induces morphological and synaptic alterations in mature neurons that leads to memory and learning deficits (Moretti and Zoghbi 2006 ), although without changes in the levels of proliferation in the DG (Smrt et al. 2007 ). As to newly generated neurons, a study showed that, in young adult male mice, Mecp2 -KO new neurons of the DG that are already integrating in circuits within a niche of MECP2 -expressing neurons exhibited less dendritic complexity and development (Sun et al. 2019 ). On the contrary, in our study, we failed to show morphological alterations in DCX-ir neurons in the DG of Mecp2 -het females, likely because of the higher immaturity of these cells. Limitations Although our study reports quantitative and morphological differences in the DCX-ir population of the Pir between Mecp2 -het and WT mice, we do not provide behavioural, electrophysiological or circuit-level data to back up functional interpretations. As such, ours is a descriptive study; however, together with our previous study in young Mecp2 -mutant mice, our study supports the notion that MeCP2 is involved in the maturation process of neurons, and that deficits in this process are linked to the onset of an overt symptomatic phase in the mouse model of Rett. In conclusion, our findings support the notion that the protracted maturation process of immature populations of embryonic origin is especially vulnerable to MeCP2 deficiency, whereas proliferative neurogenic sites might be less susceptible, at least in terms of density of DCX-ir cells. Future studies are needed to clarify the molecular causes of these local impairments and the potential consequences of these deficits in the neural networks. This way, we may contribute to identify new therapeutic targets aimed at promoting neuronal maturation in Rett syndrome. Declarations Funding Funded by Ayudas a la investigación en Síndrome de Rett, FinRett 2019 and 2022 to C.A-P. and Conselleria de Educación, Cultura, Universidades y Empleo from the Generalitat Valenciana (CIAICO/2023/027). JVTP is funded by the Spanish Ministry of Science, Innovation and Universities (MCIN/AEI/https://doi.org/10.13039/501100011033) and the European Union “NextGenerationEU”/PRTR with a Ramón y Cajal contract (grant RYC2021-034012-I); JVTP is also supported by the Conselleria de Educación, Cultura, Universidades y Empleo from the Generalitat Valenciana with a Subvencion a grupos de investigación emergentes (grant CIGE/2024/73). Proyecto desarrollado en el marco del programa propio del Vicerrectorado de Investigación de la UV, convocatoria de Acciones Especiales, expediente UV-INV-AE-3651656. This work was also supported by the Conselleria de Educación, Cultura, Universidades y Empleo from the Generalitat Valenciana (CIPROM/2023/053) to VH-P. R.E-P is supported by a predoctoral fellowship from Conselleria de Educación, Cultura, Universidades y Empleo from the Generalitat Valenciana (ACIF2022/387). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions R.E-P and P.S-F. performed experimental procedures, acquired images, analysed and interpreted data, prepared figures and wrote the manuscript; E.L. contributed to data interpretation; V.H-P. contributed to data analysis and interpretation; J.V.T-P. contributed to data interpretation and writing. C.A-P. obtained funding, designed the study, supervised experimental procedures, performed data analysis and wrote the manuscript. Final version of this manuscript was discussed and approved by all authors. Data Availability The datasets generated during the current study are available from the corresponding author on reasonable request. Ethics approval The study was approved by the Animal Experimentation Ethics Committee of the University of Valencia, Protocol 2019/VSC/PEA/0027, and carried out in strict accordance with EU directive 2010/63/EU. Acknowledgements Authors are indebted to Dr Elena Martínez-Rodríguez and Josep Pardo-García for technical assistance, and to Dr María Martínez de Lagrán and Dr Emilio Varea for suggestions about Sholl analysis. References Abellán-Álvaro M, Stork O, Agustín-Pavón C, Santos M (2021) MeCP2 haplodeficiency and early-life stress interaction on anxiety-like behavior in adolescent female mice. J Neurodevelop Disord 13:59. https://doi.org/10.1186/s11689-021-09409-7 Abellán-Álvaro M, Teruel-Sanchis A, Madeira MF, et al (2024) Doublecortin-immunoreactive neurons in the piriform cortex are sensitive to the long lasting effects of early life stress. Front Neurosci 18:1446912. https://doi.org/10.3389/fnins.2024.1446912 Amir RE, Van Den Veyver IB, Wan M, et al (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188. https://doi.org/10.1038/13810 Arellano JI, Duque A, Rakic P (2024) A coming-of-age story: adult neurogenesis or adolescent neurogenesis in rodents? Front Neurosci 18:1383728. https://doi.org/10.3389/fnins.2024.1383728 Ballas N, Lioy DT, Grunseich C, Mandel G (2009) Non–cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Nat Neurosci 12:311–317. https://doi.org/10.1038/nn.2275 Bauman M, Kemper Th, Arin D (1995) Microscopic Observations of the Brain in Rett Syndrome. Neuropediatrics 26:105–108. https://doi.org/10.1055/s-2007-979737 Belichenko PV, Fedorov AA, Dahlström AB (1996) Quantitative analysis of immunofluorescence and lipofuscin distribution in human cortical areas by dual-channel confocal laser scanning microscopy. Journal of Neuroscience Methods 69:155–161. https://doi.org/10.1016/S0165-0270(96)00035-0 Belichenko PV, Wright EE, Belichenko NP, et al (2009) Widespread changes in dendritic and axonal morphology in Mecp2 ‐mutant mouse models of rett syndrome: Evidence for disruption of neuronal networks. J of Comparative Neurology 514:240–258. https://doi.org/10.1002/cne.22009 Benedetti B, Dannehl D, König R, et al (2020) Functional Integration of Neuronal Precursors in the Adult Murine Piriform Cortex. Cerebral Cortex 30:1499–1515. https://doi.org/10.1093/cercor/bhz181 Canton APM, Tinano FR, Guasti L, et al (2023) Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study. The Lancet Diabetes & Endocrinology 11:545–554. https://doi.org/10.1016/S2213-8587(23)00131-6 Cassel S, Revel M-O, Kelche C, Zwiller J (2004) Expression of the methyl-CpG-binding protein MeCP2 in rat brain. An ontogenetic study. Neurobiology of Disease 15:206–211. https://doi.org/10.1016/j.nbd.2003.10.011 Chapleau CA, Calfa GD, Lane MC, et al (2009) Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations. Neurobiology of Disease 35:219–233. https://doi.org/10.1016/j.nbd.2009.05.001 Coviello S, Gramuntell Y, Klimczak P, et al (2022) Phenotype and Distribution of Immature Neurons in the Human Cerebral Cortex Layer II. Front Neuroanat 16:851432. https://doi.org/10.3389/fnana.2022.851432 Cuitavi J, Martínez-Rodríguez E, Abellán-Álvaro M, et al (2025) Longitudinal Analysis in Mecp2-het Female Mice Reveals Atypical Nociceptive Behaviours Degano AL, Park MJ, Penati J, et al (2014) MeCP2 is required for activity-dependent refinement of olfactory circuits. Molecular and Cellular Neuroscience 59:63–75. https://doi.org/10.1016/j.mcn.2014.01.005 Doetsch F, Caillé I, Lim DA, et al (1999) Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult Mammalian Brain. Cell 97:703–716. https://doi.org/10.1016/S0092-8674(00)80783-7 Downs J, Géranton SM, Bebbington A, et al (2010) Linking MECP2 and pain sensitivity: The example of Rett syndrome. American J of Med Genetics Pt A 152A:1197–1205. https://doi.org/10.1002/ajmg.a.33314 Esteve-Pérez R, Abellán-Álvaro M, Navarro-Moreno C, et al (2025) The density of doublecortin cells in the piriform cortex is affected by transition to adulthood but not first pregnancy in mice. Brain Struct Funct 230:94. https://doi.org/10.1007/s00429-025-02957-x García-González D, Dumitru I, Zuccotti A, et al (2021) Neurogenesis of medium spiny neurons in the nucleus accumbens continues into adulthood and is enhanced by pathological pain. Mol Psychiatry 26:4616–4632. https://doi.org/10.1038/s41380-020-0823-4 Gómez-Climent MÁ, Hernández-González S, Shionoya K, et al (2011) Olfactory bulbectomy, but not odor conditioned aversion, induces the differentiation of immature neurons in the adult rat piriform cortex. Neuroscience 181:18–27. https://doi.org/10.1016/j.neuroscience.2011.03.004 Guy J, Hendrich B, Holmes M, et al (2001) A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet 27:322–326. https://doi.org/10.1038/85899 Kishi N, Macklis JD (2004) MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Molecular and Cellular Neuroscience 27:306–321. https://doi.org/10.1016/j.mcn.2004.07.006 La Rosa C, Cavallo F, Pecora A, et al (2020) Phylogenetic variation in cortical layer II immature neuron reservoir of mammals. eLife 9:e55456. https://doi.org/10.7554/eLife.55456 Martínez-Rodríguez E, Martín-Sánchez A, Coviello S, et al (2019) Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system. Brain Struct Funct 224:1647–1658. https://doi.org/10.1007/s00429-019-01860-6 Matarazzo V, Cohen D, Palmer AM, et al (2004) The transcriptional repressor Mecp2 regulates terminal neuronal differentiation. Molecular and Cellular Neuroscience 27:44–58. https://doi.org/10.1016/j.mcn.2004.05.005 Moretti P, Zoghbi HY (2006) MeCP2 dysfunction in Rett syndrome and related disorders. Current Opinion in Genetics & Development 16:276–281. https://doi.org/10.1016/j.gde.2006.04.009 Nacher J, Crespo C, McEwen BS (2001) Doublecortin expression in the adult rat telencephalon. Eur J of Neuroscience 14:629–644. https://doi.org/10.1046/j.0953-816x.2001.01683.x Nacher J, Pham K, Gil-Fernandez V, McEwen BS (2004) Chronic restraint stress and chronic corticosterone treatment modulate differentially the expression of molecules related to structural plasticity in the adult rat piriform cortex. Neuroscience 126:503–509. https://doi.org/10.1016/j.neuroscience.2004.03.038 Nan X, Campoy FJ, Bird A (1997) MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin. Cell 88:471–481. https://doi.org/10.1016/S0092-8674(00)81887-5 Nerli E, Roggero OM, Baj G, Tongiorgi E (2020) In vitro modeling of dendritic atrophy in Rett syndrome: determinants for phenotypic drug screening in neurodevelopmental disorders. Sci Rep 10:2491. https://doi.org/10.1038/s41598-020-59268-w Paredes MF, Sorrells SF, Garcia‐Verdugo JM, Alvarez‐Buylla A (2016) Brain size and limits to adult neurogenesis. J of Comparative Neurology 524:646–664. https://doi.org/10.1002/cne.23896 Paxinos G, Franklin KBJ (2019) Paxinos and Franklin’s The mouse brain in stereotaxic coordinates, Fifth edition. Academic Press, an imprint of Elsevier, London Ronnett GV, Leopold D, Cai X, et al (2003) Olfactory biopsies demonstrate a defect in neuronal development in Rett’s syndrome. Annals of Neurology 54:206–218. https://doi.org/10.1002/ana.10633 Rotheneichner P, Belles M, Benedetti B, et al (2018) Cellular Plasticity in the Adult Murine Piriform Cortex: Continuous Maturation of Dormant Precursors Into Excitatory Neurons. Cerebral Cortex 28:2610–2621. https://doi.org/10.1093/cercor/bhy087 Rubio A, Belles M, Belenguer G, et al (2016) Characterization and isolation of immature neurons of the adult mouse piriform cortex. Developmental Neurobiology 76:748–763. https://doi.org/10.1002/dneu.22357 Santos M, Summavielle T, Teixeira-Castro A, et al (2010) Monoamine deficits in the brain of methyl-CpG binding protein 2 null mice suggest the involvement of the cerebral cortex in early stages of Rett syndrome. Neuroscience 170:453–467. https://doi.org/10.1016/j.neuroscience.2010.07.010 Santos M, Temudo T, Kay T, et al (2009) Mutations in the MECP2 Gene Are Not a Major Cause of Rett Syndrome-Like or Related Neurodevelopmental Phenotype in Male Patients. J Child Neurol 24:49–55. https://doi.org/10.1177/0883073808321043 Shahbazian MD (2002) Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Human Molecular Genetics 11:115–124. https://doi.org/10.1093/hmg/11.2.115 Sharifi O, Yasui DH (2021) The Molecular Functions of MeCP2 in Rett Syndrome Pathology. Front Genet 12:624290. https://doi.org/10.3389/fgene.2021.624290 Smith ES, Smith DR, Eyring C, et al (2019) Altered trajectories of neurodevelopment and behavior in mouse models of Rett syndrome. Neurobiology of Learning and Memory 165:106962. https://doi.org/10.1016/j.nlm.2018.11.007 Smrt RD, Eaves-Egenes J, Barkho BZ, et al (2007) Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons. Neurobiology of Disease 27:77–89. https://doi.org/10.1016/j.nbd.2007.04.005 Sorrells SF, Paredes MF, Cebrian-Silla A, et al (2018) Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature 555:377–381. https://doi.org/10.1038/nature25975 Sun Y, Gao Y, Tidei JJ, et al (2019) Loss of MeCP2 in immature neurons leads to impaired network integration. Human Molecular Genetics 28:245–257. https://doi.org/10.1093/hmg/ddy338 Sweat SC, Cheetham CEJ (2024) Deficits in olfactory system neurogenesis in neurodevelopmental disorders. Genesis 62:e23590. https://doi.org/10.1002/dvg.23590 Torres-Pérez JV, Martínez-Rodríguez E, Forte A, et al (2022) Early life stress exacerbates behavioural and neuronal alterations in adolescent male mice lacking methyl-CpG binding protein 2 (Mecp2). Front Behav Neurosci 16:974692. https://doi.org/10.3389/fnbeh.2022.974692 Wang I-TJ, Reyes A-RS, Zhou Z (2013) Neuronal morphology in MeCP2 mouse models is intrinsically variable and depends on age, cell type, and Mecp2 mutation. Neurobiology of Disease 58:3–12. https://doi.org/10.1016/j.nbd.2013.04.020 Whitman MC, Greer CA (2009) Adult neurogenesis and the olfactory system. Progress in Neurobiology 89:162–175. https://doi.org/10.1016/j.pneurobio.2009.07.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 06 Dec, 2025 Read the published version in Neuroscience → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7269766","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":500710318,"identity":"01d11aa9-e6dc-4890-a9b8-77dcb7b4ac8e","order_by":0,"name":"Rafael Esteve-Pérez","email":"","orcid":"","institution":"Universitat de València","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Esteve-Pérez","suffix":""},{"id":500710319,"identity":"c7d10a14-9c8b-47b2-b2a9-d77099201097","order_by":1,"name":"Paloma Sevilla-Ferrer","email":"","orcid":"","institution":"Universitat de València","correspondingAuthor":false,"prefix":"","firstName":"Paloma","middleName":"","lastName":"Sevilla-Ferrer","suffix":""},{"id":500710321,"identity":"e45e45be-4aff-4bc0-8e5b-a9d67c6846b0","order_by":2,"name":"Enrique Lanuza","email":"","orcid":"","institution":"Universitat de València","correspondingAuthor":false,"prefix":"","firstName":"Enrique","middleName":"","lastName":"Lanuza","suffix":""},{"id":500710322,"identity":"7028031d-ac64-481b-9c52-cd4c3a7e04b2","order_by":3,"name":"Vicente Herranz-Pérez","email":"","orcid":"","institution":"Universitat de València","correspondingAuthor":false,"prefix":"","firstName":"Vicente","middleName":"","lastName":"Herranz-Pérez","suffix":""},{"id":500710324,"identity":"7f2112f4-af14-4d33-881e-39b14c97101d","order_by":4,"name":"Jose V. Torres-Pérez","email":"","orcid":"","institution":"Universitat de València","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"V.","lastName":"Torres-Pérez","suffix":""},{"id":500710326,"identity":"d7bf109a-5b3c-45c1-88f8-e71dd51f9a87","order_by":5,"name":"Carmen Agustín-Pavón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAk0lEQVRIiWNgGAWjYNCCCtK1nCFZB2MbKarN2c8YPi6cdzixgb39AXFaLHtyjI1nbgNq4TljQJwWgwNpadK82w4bM0jkEOkwg/PPgFrmALXIPyfSYQY3ko9J8zYclmOQYCDSYZYzHh825jmWLsfGk0OkFnP+xMbHPDXWPPzsx4l1GIzBRpx6ZC2jYBSMglEwCnACAAK7JUoA3OsvAAAAAElFTkSuQmCC","orcid":"","institution":"Universitat de València","correspondingAuthor":true,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Agustín-Pavón","suffix":""}],"badges":[],"createdAt":"2025-08-01 09:38:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7269766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7269766/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.neuroscience.2025.12.010","type":"published","date":"2025-12-07T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89282165,"identity":"9c2094da-82ae-445b-a56a-313856b6e4b2","added_by":"auto","created_at":"2025-08-18 10:41:21","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3207158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution and density of DCX-ir cells in WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMecp2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-het female mice. (A)\u003c/strong\u003e Representative images of DCX-ir neurons in the piriform cortex (Pir), olfactory bulbs (OB), nucleus accumbens (NAc) and dentate gyrus (DG). Scale bar: 50 µm (inset: 20 µm). (\u003cstrong\u003eB, C, D, E)\u003c/strong\u003e Bar graphs comparing density of DCX-ir cells between WT (gray) and \u003cem\u003eMecp2\u003c/em\u003e-heterozygous (red) females in the Pir \u003cstrong\u003e(B)\u003c/strong\u003e, OB \u003cstrong\u003e(C)\u003c/strong\u003e, NAc \u003cstrong\u003e(D)\u003c/strong\u003e and DG. The comparison of DCX-ir cells in the Pir \u003cstrong\u003e(B)\u003c/strong\u003e has been performed considering all labelled cells (Total), and then separately considering the classification into simple (more immature), and complex (more mature), which are significantly increased in \u003cem\u003eMecp2\u003c/em\u003e-het females with respect to WT. ML: molecular layer; GL: granular layer; H: hilus; SVZ: subventricular zone; ac: anterior commissure. All graphs represent mean ± Standard Error Mean (SEM), and individual values (dots). *: p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7269766/v1/7aa13ee4dea09df0e68a0073.jpeg"},{"id":89282164,"identity":"b82ac753-50b4-465c-b3e6-bac535eae26c","added_by":"auto","created_at":"2025-08-18 10:41:21","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1568534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of DCX-ir cells in the Pir. A, B) \u003c/strong\u003eImages depicting \u003cem\u003ecamera lucida\u003c/em\u003e drawings of DCX-ir cells of the Pir, along with their respective hull (blue) and circle (magenta) diagrams in WT \u003cstrong\u003e(A)\u003c/strong\u003e and \u003cem\u003eMecp2\u003c/em\u003e-het \u003cstrong\u003e(B)\u003c/strong\u003e mice. \u003cstrong\u003eC\u003c/strong\u003e) Main diameter of total, simple and complex DCX-ir neurons of the Pir revealed a significant decrease of this measure in \u003cem\u003eMecp2\u003c/em\u003e-het mice. \u003cstrong\u003eSholl analysis \u003c/strong\u003ewas performed measuring (\u003cstrong\u003eD)\u003c/strong\u003e the total number of intersections for DCX-ir neurons and \u003cstrong\u003e(E)\u003c/strong\u003e the number of intersections versus distance from the soma. \u003cstrong\u003eSkeleton analysis\u003c/strong\u003e was performed measuring \u003cstrong\u003e(F)\u003c/strong\u003e total dendritic length, \u003cstrong\u003e(G)\u003c/strong\u003e maximum branch length, \u003cstrong\u003e(H)\u003c/strong\u003e number of junctions and \u003cstrong\u003e(I)\u003c/strong\u003e number of branches. Finally, \u003cstrong\u003efractal analysis\u003c/strong\u003ewas assessed in terms of fractal dimension \u003cstrong\u003e(J)\u003c/strong\u003e, lacunarity \u003cstrong\u003e(K)\u003c/strong\u003e, density \u003cstrong\u003e(L)\u003c/strong\u003e and span ratio \u003cstrong\u003e(M)\u003c/strong\u003e calculations. These analyses reveal a shorter and less complex dendritic branching of DCX-ir cells of \u003cem\u003eMecp2\u003c/em\u003e-het females. All graphs represent mean ± Standard Error Mean (SEM). *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.005.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7269766/v1/1fa586d5e009934d36fe4140.jpeg"},{"id":89282746,"identity":"d66e1158-5054-4c73-ad02-6a3e0e095460","added_by":"auto","created_at":"2025-08-18 10:49:21","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1563649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of DCX-ir cells in the hippocampal DG region. A, B) \u003c/strong\u003eImages depicting camera lucida drawings of DCX-ir DG cells, along with their respective hull (blue) and circle (magenta) diagrams in \u003cstrong\u003e(A)\u003c/strong\u003eWT and \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eMecp2\u003c/em\u003e-het mice. \u003cstrong\u003eC)\u003c/strong\u003e Main diameter of DCX-ir neurons of the DG. \u003cstrong\u003eSholl analysis \u003c/strong\u003ewas performed measuring (\u003cstrong\u003eD)\u003c/strong\u003e the total number of intersections for DCX-ir neurons and \u003cstrong\u003e(E)\u003c/strong\u003e the number of intersections versus distance from the soma. \u003cstrong\u003eSkeleton analysis\u003c/strong\u003e was performed measuring \u003cstrong\u003e(F)\u003c/strong\u003e total dendritic length, \u003cstrong\u003e(G)\u003c/strong\u003e maximum branch length, \u003cstrong\u003e(H)\u003c/strong\u003e number of junctions and \u003cstrong\u003e(I)\u003c/strong\u003e number of branches. Finally, \u003cstrong\u003efractal analysis\u003c/strong\u003e was assessed in terms of fractal dimension \u003cstrong\u003e(J)\u003c/strong\u003e, lacunarity \u003cstrong\u003e(K)\u003c/strong\u003e, density \u003cstrong\u003e(L)\u003c/strong\u003e and span ratio \u003cstrong\u003e(M)\u003c/strong\u003ecalculations. All graphs represent mean ± Standard Error Mean (SEM).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7269766/v1/fcf2f82f4d6eac6c8c150aa3.jpeg"},{"id":97727604,"identity":"98fa811d-adbb-4c4d-811f-3c3afe1dfcca","added_by":"auto","created_at":"2025-12-08 16:50:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21960532,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7269766/v1/6910b1b5-031a-4f43-a45b-905478de43b4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Doublecortin-expressing cells are selectively altered in the piriform cortex but not in neurogenic areas of symptomatic Mecp2-heterozygous mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRett syndrome (RTT) is a rare neurodevelopmental disorder that affects mainly females, characterised by a developmental regression occurring between 6 to 18 months of age, leading to loss of speech, intellectual disability, repetitive hand movements, breathing abnormalities, and motor impairment, among other symptoms (Amir et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The main cause of classical RTT are loss-of-function mutations in the X-linked \u003cem\u003eMECP2\u003c/em\u003e gene, encoding the methyl-CpG-binding protein 2 (MeCP2) (Amir et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In males, hemizygous loss-of-function \u003cem\u003eMECP2\u003c/em\u003e mutations are typically lethal, resulting in severe neonatal encephalopathy and often perinatal death (Santos et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), whereas mutations and variants not leading to complete loss of function in this gene have been linked to disorders causing intellectual disability, neuroendocrine dysfunction or psychiatric conditions (Moretti and Zoghbi \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Canton et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOriginally identified as a transcriptional repressor (Nan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), MeCP2 is currently recognized as a multidomain epigenetic regulator that modulates gene expression in a context-dependent manner, both silencing and promoting transcription (Sharifi and Yasui \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Murine models with MeCP2 loss-of-function mutation serve as a valuable approach to understanding the implication of MeCP2 in the nervous system (Guy et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In contrast to humans, male mice survive infancy but have a short lifespan and an early onset of symptoms, with animals of 2 months of age being fully symptomatic, whereas females display a variable onset of motor symptoms, being symptomatic by 2 to 6 months of age (Guy et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Abell\u0026aacute;n-\u0026Aacute;lvaro et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cuitavi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The timing of phenotypic manifestations of RTT in humans and murine models suggests that the underlying cause of symptoms is not an altered developmental neurogenesis, but a disruption in the maintenance of a mature neuronal functionality (Shahbazian \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kishi and Macklis \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Smrt et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This is further supported by the observed pattern of MeCP2 protein expression, characterized by low levels during embryonic brain development and reaching the highest levels in more mature structures (Shahbazian \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kishi and Macklis \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn agreement with a key role of MeCP2 for neuronal maturation, we previously showed that immature neurons, identified by using the marker doublecortin (DCX), were increased in specific brain regions of adolescent and young adult \u003cem\u003eMecp2\u003c/em\u003e-null mice (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Torres-P\u0026eacute;rez et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the postnatal rodent brain, DCX-immunoreactive (DCX-ir) cells are present in neurogenic areas such as the dentate gyrus (DG) of the hippocampus and the ventricular-subventricular zone (V-SVZ), from which neuroblasts migrate mainly through the rostral migratory stream (RMS) to the olfactory bulb (OB) (Doetsch et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Whitman and Greer \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), but also to other locations such as the nucleus accumbens NAc (Garc\u0026iacute;a-Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, layer II of the piriform cortex (Pir) harbours a population of DCX-ir cells generated during embryonic development that persists into adulthood (Nacher et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rubio et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These cells have been shown to mature gradually with age and integrate as glutamatergic neurons (Rotheneichner et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Benedetti et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Interestingly, while proliferative neurogenesis seems largely absent postnatally in mammals with larger brains, such as non-human and human primates (Paredes et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sorrells et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), cortical DCX-ir immature neurons of prenatal origin might be widespread in these species (La Rosa et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), including humans (Coviello et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), highlighting the importance of characterising this population in disease models.\u003c/p\u003e\u003cp\u003eOur prior findings revealed that DCX-ir cells of the Pir, and adjacent olfactory tubercle (OT), but not the DG or OB, were significantly increased in young \u003cem\u003eMecp2\u003c/em\u003e-null male mice as compared with age-matched wild-type (WT) controls (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By contrast, we did not find any effect in any of those regions in young adult \u003cem\u003eMecp2\u003c/em\u003e-heterozygous (\u003cem\u003eMecp2\u003c/em\u003e-het) females (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since young adult females are largely pre-symptomatic (Cuitavi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we hypothesised that alterations in DCX-ir cells might emerge later, when symptoms are already present in \u003cem\u003eMecp2\u003c/em\u003e-het females. To test this hypothesis, here we analysed the density and morphological features of DCX-ir cells across several brain areas, in 6 months old \u003cem\u003eMecp2\u003c/em\u003e-het female mice and their WT littermates.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor these experiments, we used spared samples from 6 months old female mice of the \u003cem\u003eMecp2\u003c/em\u003e\u003csup\u003etm1.1Bird/J\u003c/sup\u003e strain (WT, n\u0026thinsp;=\u0026thinsp;5; \u003cem\u003eMecp2\u003c/em\u003e-heterozygous, n\u0026thinsp;=\u0026thinsp;8, Jackson Laboratory), obtained from symptomatic females used in a previous study (Cuitavi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Mice were housed in standard laboratory cages in groups of 2\u0026ndash;5 animals, under controlled humidity and temperature (22\u0026ordm;C), with a 12:12-h light/dark cycle, and water and food available \u003cem\u003ead libitum\u003c/em\u003e. For genotyping, we obtained ear plugs at weaning, and after DNA extraction, we applied the protocol supplied for this strain by the Jackson Laboratory. The protocols were approved by the Animal Experimentation Ethics Committee of the University of Valencia, Protocol 2019/VSC/PEA/0027, and carried out in strict accordance with EU directive 2010/63/EU.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePerfusion and histology\u003c/b\u003e\u003c/p\u003e\u003cp\u003e Animals were deeply anaesthetized using pentobarbital (50 mg/Kg) and transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.4. Brains were carefully removed from the skull, postfixed overnight in the same fixative and transferred to 30% sucrose in 0.01M phosphate buffered saline (PBS, pH 7.6) until they sank. The brains were then frozen and cut in six series of 40-\u0026micro;m-thick coronal sections with a freezing microtome. Free-floating sections were collected in five parallel series and kept at -20 \u0026ordm;C in 30% sucrose in PB (0.1 M, pH 7.4) for future processing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDoublecortin immunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe obtained permanent immunostained preparations for DCX in one out of the six parallel series, using the indirect avidin\u0026ndash;biotin peroxidase complex/3,3\u0026rsquo;-diaminobenzidine-staining (ABC/DAB) procedure, as described in our previous work (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003ea). In brief, brain slices were first incubated with 1% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 0.05M tris-buffered saline (TBS, 0.9% NaCl in TB) for 30 minutes at room temperature (RT) to block endogenous peroxidase activity. Subsequently, sections were incubated with 2% normal horse serum (NHS) and 0.3% Triton-X100 in 0.05M TBS pH 7.4 for one hour at RT, to block nonspecific binding. Brain slices were then incubated overnight at 4\u003csup\u003eo\u003c/sup\u003eC with a goat primary antibody anti-DCX (C-18, sc:8066, Santa Cruz Biotechnology, Inc) diluted 1:500 in 0.05M TBS pH 7.4, with 2% NHS and 0.3% Triton-X100. The next day, sections were incubated for two hours at RT with biotinylated horse anti-Goat-IgG secondary antibody (BA-9500, Vector) diluted 1:200 in the same buffer. Afterwards, sections were incubated for 90 minutes at RT in ABC (Vector) in TBS with 0.3% Triton-X100. Sections were thoroughly washed in TBS (3 x 10 min.) between each step. To reveal peroxidase activity, sections were incubated for five minutes in DAB-SigmaFAST (Sigma) in TB. Sections were mounted using 0.2% gelatine in TB, dehydrated with alcohol, cleared with xylol and cover-slipped with Entellan.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification of DCX-immunoreactive cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe analysed the samples with a Leica Leitz DMRB microscope, equipped with a digital camera (Leica DFC495) and Motic Software at \u003cem\u003ea priori\u003c/em\u003e selected Bregma levels (B), according to (Paxinos and Franklin 2012). For the piriform cortex (Pir), we analysed approximate bregma levels B0, B-1 and B-2 mm; for the dentate gyrus of the hippocampus (DG), B-1, B-2 and B-3 mm, and for the nucleus accumbens (NAc) we analysed from approximately B1.18 to B0.62 mm (Paxinos and Franklin \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the Pir, an experimenter who was blind to the genotype of the mice inspected the sections and counted the number of cells in each area. DCX-ir cells have been previously classified into two populations based on size and morphology: (1) \u0026ldquo;simple\u0026rdquo;, small cells without neurites or with a rudimentary one and a smaller soma size; and (2) \u0026ldquo;complex\u0026rdquo;, larger cells with a larger and more developed dendritic tree (Rubio et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, in the Pir those DCX-ir cells with none or one neurite were classified as simple, whilst cells with two or more neurites were quantified as complex. To evaluate neurons originated in the V-SVZ, we counted manually DCX-ir neurons in the periglomerular region of the OB (since the intense labelling in the granular cell layer precluded the quantification of individual cells) and in the NAc, where cells were counted in the region between the ventricular-subventricular zone (V-SVZ) and the anterior commissure (aca). In addition, we measured the Euclidean distance from each DCX-ir cell located in the NAc to the nearest lateral ventricle wall as a measure of migratory capacity (Garc\u0026iacute;a-Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Finally, in the DG, DCX-ir neurons were counted only in the granular layer, and their maximum somatic diameter was also measured manually. All quantifications were performed using Fiji software (NIH). Cell density was expressed as an average number of labelled cells per section per hemisphere.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSholl and skeleton analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe examined the morphology of the DCX-ir cells from the Pir and the DG by means of Sholl and skeleton analysis using ImageJ SNT Plugin (default parameters, v4.2.1) and Analyze skeleton tool, respectively. First, cells from layer II of the Pir (complex only) and DG were drawn manually using a \u003cem\u003ecamera lucida\u003c/em\u003e attached to a Leica optic microscope. Neurons were selected only if they were relatively isolated from other neurons and without discernible breaks. Next, images were scanned and processed with ImageJ software to convert them to an 8-bit format and then skeletonized. We used Sholl method to quantify the number of intersections of the dendrites within concentric circles of increasing radius (radius steps set at 10 \u0026micro;m for Pir cells and 5 \u0026micro;m for DG cells, according to different neuronal arbour sizes) centred on the soma, and the total number of intersections. Complementary to Sholl, skeleton analysis was applied to examine several branching parameters, including total branch length, maximum branch length, number of junctions and number of branches. We analysed a total of 45 DCX-ir neurons of the Pir, and 70 DCX-ir neurons of the DG, with a minimum of 5 positive cells per animal.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFractal analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo characterize structural complexity of DCX-ir neurons from the Pir (complex cells) and DG, we performed a fractal analysis. The binary images of outlined cells were then analysed using the ImageJ FracLac plug-in. Changes in neuronal structure complexity were assessed by calculating Fractal dimension (D\u003csub\u003eB\u003c/sub\u003e) and Lacunarity (λ). Fractal dimension provides a measure of complexity, which in turn is how a pattern's detail changes with the scale at which it is considered. Thus, it reflects how completely a neuronal arbour fills a specific area. Lacunarity is a measure of heterogeneity (rotational invariance) as a complement to complexity in describing digital images. Box-counting method was used for D\u003csub\u003eB\u003c/sub\u003e and λ calculations. Default settings were left unchanged, except for Num G (the number of box counting grid orientations used during the scan), which was set at 5, following the recommended range indications from FracLac. Additional metrics, including Hull and circle metrics data (Density and Span ratio).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData were analysed using the GraphPad Prism 9 and R software. Data normality was tested with the Shapiro-Wilk test. To test statistical significance in multiple comparisons (number of intersections per radius in Sholl analysis), a two-way ANOVA followed by \u003cem\u003epost hoc\u003c/em\u003e Bonferroni correction was employed. For two group comparisons, we used unpaired Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e if normality was confirmed; otherwise, the Mann-Whitney test was used. All values are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eComplex DCX-ir neurons are increased in the piriform cortex of\u003c/b\u003e \u003cb\u003eMecp2\u003c/b\u003e\u003cb\u003e-heterozygous symptomatic females, but not in the neurogenic niches of DG or V-SVZ\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs expected, we did not find qualitative differences in the distribution of DCX-ir cells between \u003cem\u003eMecp2\u003c/em\u003e-het and WT female mice. Thus, DCX-ir cells were found in the Pir, OB, NAc and DG in both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Considering the finding of a restricted population of DCX-ir cells in the olfactory tubercle, which were increased in \u003cem\u003eMecp2\u003c/em\u003e-null male mice (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), we also examined the olfactory tubercle in these samples. However, none or very few DCX-ir cells were found in this region in either genotype, suggesting that this population may be transient.\u003c/p\u003e\u003cp\u003eQuantitatively, we found no significant differences between genotypes in the total number of DCX-ir cells in the Pir (W\u0026thinsp;=\u0026thinsp;10.5, p\u0026thinsp;=\u0026thinsp;0.19; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, when considering the classification on simple (more immature) and complex (more mature), we found a significant increase in the density of complex DCX-ir cells in \u003cem\u003eMecp2\u003c/em\u003e-het females s (t = -2.6, p\u0026thinsp;=\u0026thinsp;0.025), but not in simple DCX-ir cells (W\u0026thinsp;=\u0026thinsp;11.5, p\u0026thinsp;=\u0026thinsp;0.24). In contrast, the density of DCX-ir cells did not differ between genotypes in the OB (t\u0026thinsp;=\u0026thinsp;0.12, p\u0026thinsp;=\u0026thinsp;0.91; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), in the NAc (t = -0.16, p\u0026thinsp;=\u0026thinsp;0.87; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) or in the DG (t\u0026thinsp;=\u0026thinsp;0.72, p\u0026thinsp;=\u0026thinsp;0.49; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In the NAc, we did not find significant differences in the mean distance from DCX-ir cells to the V-SVZ (W\u0026thinsp;=\u0026thinsp;22.0, p\u0026thinsp;=\u0026thinsp;0.83; Data not shown). Altogether, these results suggest a region-specific effect of the heterozygosity of \u003cem\u003eMecp2\u003c/em\u003e in the Pir, without alterations in other neurogenic areas.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological analyses revealed decreased size and complexity in DCX-ir neurons of the piriform cortex\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next investigated whether \u003cem\u003eMecp2\u003c/em\u003e deficiency could affect the morphology and complexity of DCX-ir cells in the Pir. First, we measured the main somatic diameter of DCX-ir cells in the Pir, detecting a significant reduction in DCX-ir cells in \u003cem\u003eMecp2\u003c/em\u003e-het females compared to WT (t\u0026thinsp;=\u0026thinsp;2.24, p\u0026thinsp;=\u0026thinsp;0.044; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This decrease might be largely due to a decrease in the main diameter of simple DCX-ir cells (t\u0026thinsp;=\u0026thinsp;2.37, p\u0026thinsp;=\u0026thinsp;0.03), while the main diameter of complex DCX-ir cells did not differ significantly between genotypes (t\u0026thinsp;=\u0026thinsp;1.2, p\u0026thinsp;=\u0026thinsp;0.25).\u003c/p\u003e\u003cp\u003eFurther, Sholl analysis revealed a significant reduction in the total number of intersections in DCX-ir complex cells of \u003cem\u003eMecp2\u003c/em\u003e-het females when compared to their WT counterparts (t\u0026thinsp;=\u0026thinsp;3.58; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As expected, the reduction was consistent across the entire dendritic profile, with statistically significant differences at 5, 10, 15, 20 and 30 \u0026micro;m from the cell somata (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSkeleton analysis further confirmed a reduced dendritic complexity in \u003cem\u003eMecp2\u003c/em\u003e-het females, showing decreased total dendritic arbour length (t\u0026thinsp;=\u0026thinsp;3.45, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), fewer junctions (t\u0026thinsp;=\u0026thinsp;4.31, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), and fewer branches (t\u0026thinsp;=\u0026thinsp;4.29, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) as compared to WT.\u003c/p\u003e\u003cp\u003eAmong the parameters measured by fractal and hull and circle analyses, only lacunarity, a measure of complexity of the dendritic tree, showed distinctive values between genotypes (t\u0026thinsp;=\u0026thinsp;3.64, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK) which was driven by a significantly lower value in the \u003cem\u003eMecp2\u003c/em\u003e-het females. The rest of the measurement did not show differences between genotypes (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological analyses revealed no significant effect of\u003c/b\u003e \u003cb\u003eMecp2\u003c/b\u003e \u003cb\u003edeficiency in DCX-ir neurons of the dentate gyrus\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe performed the same morphological analyses in DCX cells in the DG of the hippocampus. In this case, none of the parameters evaluated by Sholl, skeleton or fractal analyses revealed significant differences between genotypes in DCX-ir cells of the DG (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we analysed the effect of \u003cem\u003eMecp2\u003c/em\u003e haploinsufficiency in distinct populations of immature neurons, namely the embryonic born DCX-ir cells of the Pir, and the continuously generated DCX-ir cells of the OB, the NAc and the the DG. We found a specific increase in the density of DCX-ir cells in the Pir, as well as a reduced soma diameter, and less complex, shorter dendritic trees of these cells in \u003cem\u003eMecp2\u003c/em\u003e-het symptomatic females. In contrast, we did not find significant effects of \u003cem\u003eMecp2\u003c/em\u003e deficiency in DCX-ir cells of the OB, NAc or the DG. Together with our previous study showing specific deficits in the Pir in symptomatic \u003cem\u003eMecp2\u003c/em\u003e-null males but not pre-symptomatic \u003cem\u003eMecp2\u003c/em\u003e-het females (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), our data suggest that \u003cem\u003eMecp2\u003c/em\u003e deficiency contributes to arrested maturation of embryonic-born DCX-ir cells, which may be associated with the onset of overt symptoms.\u003c/p\u003e\u003cp\u003eThese results are in agreement with previous studies revealing region-specific effects of \u003cem\u003eMecp2\u003c/em\u003e deficiency (Santos et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several factors may explain this regional specificity, including non-cell-autonomous effects of the function of MeCP2 (Kishi and Macklis \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Ballas et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), differential expression levels of MeCP2 in different regions (Cassel et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Abell\u0026aacute;n-\u0026Aacute;lvaro et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) or regional differences in vulnerability to stress (Nacher et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Abell\u0026aacute;n-\u0026Aacute;lvaro et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eProbably the most important difference to consider between the populations studied here is the different ontogeny of these immature neurons. Thus, whereas new neurons are continuously being incorporated in the OB, NAc and DG of young adult mice (Doetsch et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Garc\u0026iacute;a-Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Arellano et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), immature neurons in the Pir are generated during embryonic development and remain immature until intrinsic and/or environmental factors trigger their maturation (G\u0026oacute;mez-Climent et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rotheneichner et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Abell\u0026aacute;n-\u0026Aacute;lvaro et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Esteve-P\u0026eacute;rez et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These findings support the idea that MeCP2 is more likely to influence neuronal differentiation and maturation than for proliferation (Shahbazian \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kishi and Macklis \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Smrt et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe olfactory system is a valuable system for studying neurodevelopment during healthy and pathological conditions, including RTT (Sweat and Cheetham \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, this system has been widely studied in both RTT patients and genetically modified mice. In RTT patients, nasal biopsies revealed fewer terminally differentiated olfactory receptor neurons and a notable increase in immature olfactory sensory neurons (Ronnett et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In mouse models, previous studies in the olfactory system have found that delayed maturation caused by lack of \u003cem\u003eMecp2\u003c/em\u003e results in a transient abnormal axonal projections in the olfactory bulb and subglomerular disorganisation (Matarazzo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These results, together with those that show a postnatal deficit in the olfactory refinement in \u003cem\u003eMecp2\u003c/em\u003e-null males (Degano et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggest that an impairment in the RMS, which continuously brings newly generated neurons to the OB from the V-SVZ, might be occurring. However, we did not find significant effects of genotype in the density of DCX-ir cells in the periglomerular area \u003cem\u003eMecp2\u003c/em\u003e-het symptomatic mice, suggesting that postnatal neurogenesis in the V-SVZ towards these area is not affected by heterozygosity of \u003cem\u003eMecp2\u003c/em\u003e, despite the potential malfunctions in newly generated neurons in the OB (Matarazzo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Degano et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), or a subtle effect on transition to maturity such as the one we previously found in the \u003cem\u003eMecp2\u003c/em\u003e-null males (Mart\u0026iacute;nez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the other hand, immature neurons of the NAc, also arising from V-SVZ, have previously been related to chronic pain (Garc\u0026iacute;a-Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and pain reception and behaviour is altered in both RTT patients and female rodent models (Downs et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cuitavi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The absence of differences in the density and migration distances of the V-SVZ in DCX-ir neurons in the NAc of \u003cem\u003eMecp2\u003c/em\u003e-het females suggests that other mechanisms and areas of pain processing could be involved in these deficits.\u003c/p\u003e\u003cp\u003eIn addition to the increased density of complex DCX-ir cells in the Pir, we observed a reduction of dendritic arborization and complexity in the DCX-ir neurons of \u003cem\u003eMecp2\u003c/em\u003e-het females, in agreement with previous reports showing a dendritic and synaptic impairment in cortical neurons from both mouse models (Kishi and Macklis \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Belichenko et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and RTT patients (Bauman et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Belichenko et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Moreover, some studies propose that soma size as a phenotypic marker for of MeCP2 deficiency (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and our data revealed that DCX-ir cells of the Pir of \u003cem\u003eMecp2\u003c/em\u003e-het females also had a smaller soma compared to WT females. Both the morphological impairment in these immature neurons of Pir and the increase of the density of complex DCX-ir cells in this region might compromise the reserve of cortical plasticity and glutamatergic input provided by these immature neurons represent for olfactory and associative functions (Rotheneichner et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFinally, MeCP2 deficiency has been associated with structural and functional deficits in the hippocampus. For example, in RTT patients, impairment in the expression of \u003cem\u003eMECP2\u003c/em\u003e has been associated with lower dendritic spine density in hippocampal mature cells (Chapleau et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Nerli et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), whereas analogous studies in rodents have found how lack of \u003cem\u003eMECP2\u003c/em\u003e induces morphological and synaptic alterations in mature neurons that leads to memory and learning deficits (Moretti and Zoghbi \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), although without changes in the levels of proliferation in the DG (Smrt et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As to newly generated neurons, a study showed that, in young adult male mice, \u003cem\u003eMecp2\u003c/em\u003e-KO new neurons of the DG that are already integrating in circuits within a niche of \u003cem\u003eMECP2\u003c/em\u003e-expressing neurons exhibited less dendritic complexity and development (Sun et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the contrary, in our study, we failed to show morphological alterations in DCX-ir neurons in the DG of \u003cem\u003eMecp2\u003c/em\u003e-het females, likely because of the higher immaturity of these cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough our study reports quantitative and morphological differences in the DCX-ir population of the Pir between \u003cem\u003eMecp2\u003c/em\u003e-het and WT mice, we do not provide behavioural, electrophysiological or circuit-level data to back up functional interpretations. As such, ours is a descriptive study; however, together with our previous study in young \u003cem\u003eMecp2\u003c/em\u003e-mutant mice, our study supports the notion that MeCP2 is involved in the maturation process of neurons, and that deficits in this process are linked to the onset of an overt symptomatic phase in the mouse model of Rett.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings support the notion that the protracted maturation process of immature populations of embryonic origin is especially vulnerable to MeCP2 deficiency, whereas proliferative neurogenic sites might be less susceptible, at least in terms of density of DCX-ir cells. Future studies are needed to clarify the molecular causes of these local impairments and the potential consequences of these deficits in the neural networks. This way, we may contribute to identify new therapeutic targets aimed at promoting neuronal maturation in Rett syndrome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunded by Ayudas a la investigaci\u0026oacute;n en S\u0026iacute;ndrome de Rett, FinRett 2019 and 2022 to C.A-P. and Conselleria de Educaci\u0026oacute;n, Cultura, Universidades y Empleo from the Generalitat Valenciana (CIAICO/2023/027). JVTP is funded by the Spanish Ministry of Science, Innovation and Universities (MCIN/AEI/https://doi.org/10.13039/501100011033) and the European Union \u0026ldquo;NextGenerationEU\u0026rdquo;/PRTR with a Ram\u0026oacute;n y Cajal contract (grant RYC2021-034012-I); JVTP is also supported by the Conselleria de Educaci\u0026oacute;n, Cultura, Universidades y Empleo from the Generalitat Valenciana with a Subvencion a grupos de investigaci\u0026oacute;n emergentes (grant CIGE/2024/73). Proyecto desarrollado en el marco del programa propio del Vicerrectorado de Investigaci\u0026oacute;n de la UV, convocatoria de Acciones Especiales, expediente UV-INV-AE-3651656. This work was also supported by the Conselleria de Educaci\u0026oacute;n, Cultura, Universidades y Empleo from the Generalitat Valenciana \u0026nbsp;(CIPROM/2023/053) to VH-P. R.E-P is supported by a predoctoral fellowship from Conselleria de Educaci\u0026oacute;n, Cultura, Universidades y Empleo from the Generalitat Valenciana (ACIF2022/387).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.E-P and P.S-F. performed experimental procedures, acquired images, analysed and interpreted data, prepared figures and wrote the manuscript; E.L. contributed to data interpretation; V.H-P. contributed to data analysis and interpretation; J.V.T-P. contributed to data interpretation and writing. \u0026nbsp;C.A-P. obtained funding, designed the study, supervised experimental procedures, performed data analysis and wrote the manuscript. Final version of this manuscript was discussed and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Animal Experimentation Ethics Committee of the University of Valencia, Protocol 2019/VSC/PEA/0027, and carried out in strict accordance with EU directive 2010/63/EU.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are indebted to Dr Elena Mart\u0026iacute;nez-Rodr\u0026iacute;guez and Josep Pardo-Garc\u0026iacute;a for technical assistance, and to Dr Mar\u0026iacute;a Mart\u0026iacute;nez de Lagr\u0026aacute;n and Dr Emilio Varea for suggestions about Sholl analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbell\u0026aacute;n-\u0026Aacute;lvaro M, Stork O, Agust\u0026iacute;n-Pav\u0026oacute;n C, Santos M (2021) MeCP2 haplodeficiency and early-life stress interaction on anxiety-like behavior in adolescent female mice. J Neurodevelop Disord 13:59. https://doi.org/10.1186/s11689-021-09409-7\u003c/li\u003e\n\u003cli\u003eAbell\u0026aacute;n-\u0026Aacute;lvaro M, Teruel-Sanchis A, Madeira MF, et al (2024) Doublecortin-immunoreactive neurons in the piriform cortex are sensitive to the long lasting effects of early life stress. Front Neurosci 18:1446912. https://doi.org/10.3389/fnins.2024.1446912\u003c/li\u003e\n\u003cli\u003eAmir RE, Van Den Veyver IB, Wan M, et al (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185\u0026ndash;188. https://doi.org/10.1038/13810\u003c/li\u003e\n\u003cli\u003eArellano JI, Duque A, Rakic P (2024) A coming-of-age story: adult neurogenesis or adolescent neurogenesis in rodents? Front Neurosci 18:1383728. https://doi.org/10.3389/fnins.2024.1383728\u003c/li\u003e\n\u003cli\u003eBallas N, Lioy DT, Grunseich C, Mandel G (2009) Non\u0026ndash;cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Nat Neurosci 12:311\u0026ndash;317. https://doi.org/10.1038/nn.2275\u003c/li\u003e\n\u003cli\u003eBauman M, Kemper Th, Arin D (1995) Microscopic Observations of the Brain in Rett Syndrome. Neuropediatrics 26:105\u0026ndash;108. https://doi.org/10.1055/s-2007-979737\u003c/li\u003e\n\u003cli\u003eBelichenko PV, Fedorov AA, Dahlstr\u0026ouml;m AB (1996) Quantitative analysis of immunofluorescence and lipofuscin distribution in human cortical areas by dual-channel confocal laser scanning microscopy. Journal of Neuroscience Methods 69:155\u0026ndash;161. https://doi.org/10.1016/S0165-0270(96)00035-0\u003c/li\u003e\n\u003cli\u003eBelichenko PV, Wright EE, Belichenko NP, et al (2009) Widespread changes in dendritic and axonal morphology in \u003cem\u003eMecp2\u003c/em\u003e ‐mutant mouse models of rett syndrome: Evidence for disruption of neuronal networks. J of Comparative Neurology 514:240\u0026ndash;258. https://doi.org/10.1002/cne.22009\u003c/li\u003e\n\u003cli\u003eBenedetti B, Dannehl D, K\u0026ouml;nig R, et al (2020) Functional Integration of Neuronal Precursors in the Adult Murine Piriform Cortex. Cerebral Cortex 30:1499\u0026ndash;1515. https://doi.org/10.1093/cercor/bhz181\u003c/li\u003e\n\u003cli\u003eCanton APM, Tinano FR, Guasti L, et al (2023) Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study. The Lancet Diabetes \u0026amp; Endocrinology 11:545\u0026ndash;554. https://doi.org/10.1016/S2213-8587(23)00131-6\u003c/li\u003e\n\u003cli\u003eCassel S, Revel M-O, Kelche C, Zwiller J (2004) Expression of the methyl-CpG-binding protein MeCP2 in rat brain. An ontogenetic study. Neurobiology of Disease 15:206\u0026ndash;211. https://doi.org/10.1016/j.nbd.2003.10.011\u003c/li\u003e\n\u003cli\u003eChapleau CA, Calfa GD, Lane MC, et al (2009) Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations. Neurobiology of Disease 35:219\u0026ndash;233. https://doi.org/10.1016/j.nbd.2009.05.001\u003c/li\u003e\n\u003cli\u003eCoviello S, Gramuntell Y, Klimczak P, et al (2022) Phenotype and Distribution of Immature Neurons in the Human Cerebral Cortex Layer II. Front Neuroanat 16:851432. https://doi.org/10.3389/fnana.2022.851432\u003c/li\u003e\n\u003cli\u003eCuitavi J, Mart\u0026iacute;nez-Rodr\u0026iacute;guez E, Abell\u0026aacute;n-\u0026Aacute;lvaro M, et al (2025) Longitudinal Analysis in Mecp2-het Female Mice Reveals Atypical Nociceptive Behaviours\u003c/li\u003e\n\u003cli\u003eDegano AL, Park MJ, Penati J, et al (2014) MeCP2 is required for activity-dependent refinement of olfactory circuits. Molecular and Cellular Neuroscience 59:63\u0026ndash;75. https://doi.org/10.1016/j.mcn.2014.01.005\u003c/li\u003e\n\u003cli\u003eDoetsch F, Caill\u0026eacute; I, Lim DA, et al (1999) Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult Mammalian Brain. Cell 97:703\u0026ndash;716. https://doi.org/10.1016/S0092-8674(00)80783-7\u003c/li\u003e\n\u003cli\u003eDowns J, G\u0026eacute;ranton SM, Bebbington A, et al (2010) Linking \u003cem\u003eMECP2\u003c/em\u003e and pain sensitivity: The example of Rett syndrome. American J of Med Genetics Pt A 152A:1197\u0026ndash;1205. https://doi.org/10.1002/ajmg.a.33314\u003c/li\u003e\n\u003cli\u003eEsteve-P\u0026eacute;rez R, Abell\u0026aacute;n-\u0026Aacute;lvaro M, Navarro-Moreno C, et al (2025) The density of doublecortin cells in the piriform cortex is affected by transition to adulthood but not first pregnancy in mice. Brain Struct Funct 230:94. https://doi.org/10.1007/s00429-025-02957-x\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Gonz\u0026aacute;lez D, Dumitru I, Zuccotti A, et al (2021) Neurogenesis of medium spiny neurons in the nucleus accumbens continues into adulthood and is enhanced by pathological pain. Mol Psychiatry 26:4616\u0026ndash;4632. https://doi.org/10.1038/s41380-020-0823-4\u003c/li\u003e\n\u003cli\u003eG\u0026oacute;mez-Climent M\u0026Aacute;, Hern\u0026aacute;ndez-Gonz\u0026aacute;lez S, Shionoya K, et al (2011) Olfactory bulbectomy, but not odor conditioned aversion, induces the differentiation of immature neurons in the adult rat piriform cortex. Neuroscience 181:18\u0026ndash;27. https://doi.org/10.1016/j.neuroscience.2011.03.004\u003c/li\u003e\n\u003cli\u003eGuy J, Hendrich B, Holmes M, et al (2001) A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet 27:322\u0026ndash;326. https://doi.org/10.1038/85899\u003c/li\u003e\n\u003cli\u003eKishi N, Macklis JD (2004) MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Molecular and Cellular Neuroscience 27:306\u0026ndash;321. https://doi.org/10.1016/j.mcn.2004.07.006\u003c/li\u003e\n\u003cli\u003eLa Rosa C, Cavallo F, Pecora A, et al (2020) Phylogenetic variation in cortical layer II immature neuron reservoir of mammals. eLife 9:e55456. https://doi.org/10.7554/eLife.55456\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-Rodr\u0026iacute;guez E, Mart\u0026iacute;n-S\u0026aacute;nchez A, Coviello S, et al (2019) Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system. Brain Struct Funct 224:1647\u0026ndash;1658. https://doi.org/10.1007/s00429-019-01860-6\u003c/li\u003e\n\u003cli\u003eMatarazzo V, Cohen D, Palmer AM, et al (2004) The transcriptional repressor Mecp2 regulates terminal neuronal differentiation. Molecular and Cellular Neuroscience 27:44\u0026ndash;58. https://doi.org/10.1016/j.mcn.2004.05.005\u003c/li\u003e\n\u003cli\u003eMoretti P, Zoghbi HY (2006) MeCP2 dysfunction in Rett syndrome and related disorders. Current Opinion in Genetics \u0026amp; Development 16:276\u0026ndash;281. https://doi.org/10.1016/j.gde.2006.04.009\u003c/li\u003e\n\u003cli\u003eNacher J, Crespo C, McEwen BS (2001) Doublecortin expression in the adult rat telencephalon. Eur J of Neuroscience 14:629\u0026ndash;644. https://doi.org/10.1046/j.0953-816x.2001.01683.x\u003c/li\u003e\n\u003cli\u003eNacher J, Pham K, Gil-Fernandez V, McEwen BS (2004) Chronic restraint stress and chronic corticosterone treatment modulate differentially the expression of molecules related to structural plasticity in the adult rat piriform cortex. Neuroscience 126:503\u0026ndash;509. https://doi.org/10.1016/j.neuroscience.2004.03.038\u003c/li\u003e\n\u003cli\u003eNan X, Campoy FJ, Bird A (1997) MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin. Cell 88:471\u0026ndash;481. https://doi.org/10.1016/S0092-8674(00)81887-5\u003c/li\u003e\n\u003cli\u003eNerli E, Roggero OM, Baj G, Tongiorgi E (2020) In vitro modeling of dendritic atrophy in Rett syndrome: determinants for phenotypic drug screening in neurodevelopmental disorders. Sci Rep 10:2491. https://doi.org/10.1038/s41598-020-59268-w\u003c/li\u003e\n\u003cli\u003eParedes MF, Sorrells SF, Garcia‐Verdugo JM, Alvarez‐Buylla A (2016) Brain size and limits to adult neurogenesis. J of Comparative Neurology 524:646\u0026ndash;664. https://doi.org/10.1002/cne.23896\u003c/li\u003e\n\u003cli\u003ePaxinos G, Franklin KBJ (2019) Paxinos and Franklin\u0026rsquo;s The mouse brain in stereotaxic coordinates, Fifth edition. Academic Press, an imprint of Elsevier, London\u003c/li\u003e\n\u003cli\u003eRonnett GV, Leopold D, Cai X, et al (2003) Olfactory biopsies demonstrate a defect in neuronal development in Rett\u0026rsquo;s syndrome. Annals of Neurology 54:206\u0026ndash;218. https://doi.org/10.1002/ana.10633\u003c/li\u003e\n\u003cli\u003eRotheneichner P, Belles M, Benedetti B, et al (2018) Cellular Plasticity in the Adult Murine Piriform Cortex: Continuous Maturation of Dormant Precursors Into Excitatory Neurons. Cerebral Cortex 28:2610\u0026ndash;2621. https://doi.org/10.1093/cercor/bhy087\u003c/li\u003e\n\u003cli\u003eRubio A, Belles M, Belenguer G, et al (2016) Characterization and isolation of immature neurons of the adult mouse piriform cortex. Developmental Neurobiology 76:748\u0026ndash;763. https://doi.org/10.1002/dneu.22357\u003c/li\u003e\n\u003cli\u003eSantos M, Summavielle T, Teixeira-Castro A, et al (2010) Monoamine deficits in the brain of methyl-CpG binding protein 2 null mice suggest the involvement of the cerebral cortex in early stages of Rett syndrome. Neuroscience 170:453\u0026ndash;467. https://doi.org/10.1016/j.neuroscience.2010.07.010\u003c/li\u003e\n\u003cli\u003eSantos M, Temudo T, Kay T, et al (2009) Mutations in the \u003cem\u003eMECP2\u003c/em\u003e Gene Are Not a Major Cause of Rett Syndrome-Like or Related Neurodevelopmental Phenotype in Male Patients. J Child Neurol 24:49\u0026ndash;55. https://doi.org/10.1177/0883073808321043\u003c/li\u003e\n\u003cli\u003eShahbazian MD (2002) Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Human Molecular Genetics 11:115\u0026ndash;124. https://doi.org/10.1093/hmg/11.2.115\u003c/li\u003e\n\u003cli\u003eSharifi O, Yasui DH (2021) The Molecular Functions of MeCP2 in Rett Syndrome Pathology. Front Genet 12:624290. https://doi.org/10.3389/fgene.2021.624290\u003c/li\u003e\n\u003cli\u003eSmith ES, Smith DR, Eyring C, et al (2019) Altered trajectories of neurodevelopment and behavior in mouse models of Rett syndrome. Neurobiology of Learning and Memory 165:106962. https://doi.org/10.1016/j.nlm.2018.11.007\u003c/li\u003e\n\u003cli\u003eSmrt RD, Eaves-Egenes J, Barkho BZ, et al (2007) Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons. Neurobiology of Disease 27:77\u0026ndash;89. https://doi.org/10.1016/j.nbd.2007.04.005\u003c/li\u003e\n\u003cli\u003eSorrells SF, Paredes MF, Cebrian-Silla A, et al (2018) Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature 555:377\u0026ndash;381. https://doi.org/10.1038/nature25975\u003c/li\u003e\n\u003cli\u003eSun Y, Gao Y, Tidei JJ, et al (2019) Loss of MeCP2 in immature neurons leads to impaired network integration. Human Molecular Genetics 28:245\u0026ndash;257. https://doi.org/10.1093/hmg/ddy338\u003c/li\u003e\n\u003cli\u003eSweat SC, Cheetham CEJ (2024) Deficits in olfactory system neurogenesis in neurodevelopmental disorders. Genesis 62:e23590. https://doi.org/10.1002/dvg.23590\u003c/li\u003e\n\u003cli\u003eTorres-P\u0026eacute;rez JV, Mart\u0026iacute;nez-Rodr\u0026iacute;guez E, Forte A, et al (2022) Early life stress exacerbates behavioural and neuronal alterations in adolescent male mice lacking methyl-CpG binding protein 2 (Mecp2). Front Behav Neurosci 16:974692. https://doi.org/10.3389/fnbeh.2022.974692\u003c/li\u003e\n\u003cli\u003eWang I-TJ, Reyes A-RS, Zhou Z (2013) Neuronal morphology in MeCP2 mouse models is intrinsically variable and depends on age, cell type, and Mecp2 mutation. Neurobiology of Disease 58:3\u0026ndash;12. https://doi.org/10.1016/j.nbd.2013.04.020\u003c/li\u003e\n\u003cli\u003eWhitman MC, Greer CA (2009) Adult neurogenesis and the olfactory system. Progress in Neurobiology 89:162\u0026ndash;175. https://doi.org/10.1016/j.pneurobio.2009.07.003\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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neurodevelopmental disorders, neurogenesis, olfactory system, Rett syndrome","lastPublishedDoi":"10.21203/rs.3.rs-7269766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7269766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRett Syndrome (RTT) is a neurodevelopmental disorder which mainly affects girls, leading to profound impairments in motor function, loss of speech, intellectual disability, and epilepsy, among other symptoms. Most cases are caused by mutations in the X-linked \u003cem\u003eMECP2\u003c/em\u003e gene, which encodes the protein methyl-CpG-binding protein 2 (MeCP2), an epigenetic reader with a crucial function in the regulation of neural maturation. Previously, using the marker of immature neurons doublecortin (DCX), we showed that a population of embryonic-born neurons of the piriform cortex, which experience prolonged maturation throughout life, was increased in the piriform cortex of young adult (2 months old), symptomatic, \u003cem\u003eMecp2\u003c/em\u003e-null male mice. By contrast, these cells were not affected in age matched \u003cem\u003eMecp2\u003c/em\u003e-heterozygous female mice, who are pre-symptomatic at that age. To determine whether symptom onset would affect DCX-expressing neurons, in this study we analysed samples from 6 months old, symptomatic \u003cem\u003eMecp2\u003c/em\u003e-heterozygous female mice. Our results show a specific increase in the density of DCX-positive neurons in the piriform cortex, consistent with observations in males. However, no differences were detected in the neurogenic niches of the dentate gyrus or the ventricular-subventricular zone when compared to their wild-type controls. Further, morphological analyses of the DCX-expressing cells of the piriform cortex reveal that they are smaller and show less complex dendritic branching in mutant mice. In conclusion, our findings support a role of MeCP2 in the maturation process of the embryonic-born DCX neurons in the piriform cortex and point to region-specific alterations in neuronal maturation in RTT.\u003c/p\u003e","manuscriptTitle":"Doublecortin-expressing cells are selectively altered in the piriform cortex but not in neurogenic areas of symptomatic Mecp2-heterozygous mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-18 10:41:17","doi":"10.21203/rs.3.rs-7269766/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33e6895e-09b6-442f-8275-c6b19ee56935","owner":[],"postedDate":"August 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:50:34+00:00","versionOfRecord":{"articleIdentity":"rs-7269766","link":"https://doi.org/10.1016/j.neuroscience.2025.12.010","journal":{"identity":"neuroscience","isVorOnly":true,"title":"Neuroscience"},"publishedOn":"2025-12-07 00:00:00","publishedOnDateReadable":"December 7th, 2025"},"versionCreatedAt":"2025-08-18 10:41:17","video":"","vorDoi":"10.1016/j.neuroscience.2025.12.010","vorDoiUrl":"https://doi.org/10.1016/j.neuroscience.2025.12.010","workflowStages":[]},"version":"v1","identity":"rs-7269766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7269766","identity":"rs-7269766","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}