Early life stress exacerbates behavioural and neuronal alterations in adolescent male mice lacking methyl-CpG binding protein 2 (Mecp2).

The methyl-CpG binding protein 2 gene (MECP2) encodes an epigenetic transcriptional regulator implicated in neuronal plasticity. Loss-of-function mutations in this gene are the primary cause of Rett syndrome and, to a lesser degree, of other neurodevelopmental disorders. Recently, we demonstrated that both Mecp2 haploinsuficiency and mild early life stress decrease anxiety-like behaviours and neuronal activation in brain areas controlling these responses in adolescent female mice. Here, we extend this work to males by using Mecp2-null and wild type adolescent mice subjected to maternal separation and their non-stressed controls. We assessed their behavioural responses in a battery of anxiety-provoking tests. Upon exposure to an elevated plus maze in aversive conditions, we evaluated changes in c-FOS expression in stress- and anxiety-related brain regions. In addition, we assessed the impact of maternal separation in neuronal maturation using doublecortin and reelin as surrogate markers. Mutant males showed reduced motor abilities, increased activation of the olfactory bulbs, probably due to breathing abnormalities, and decreased activation of the paraventricular thalamic nucleus, when compared to wild type mice. In addition, maternal separation increased the number of immature doublecortin-like neurons found in Mecp2-null animals. Moreover, this work shows for the first time that reelin is decreased in the mutant animals at the olfactory tubercle, piriform cortex and hippocampal dentate gyrus, an effect also associated to maternal separation. Taken together, our results suggest that maternal separation exacerbates some phenotypical alterations associated with lack of MeCP2 in adolescent males.

Male-specific features are reduced in Mecp2-null mice: analyses of vasopressinergic innervation, pheromone production and social behaviour.

Deficits in arginine vasopressin (AVP) and oxytocin (OT), two neuropeptides closely implicated in the modulation of social behaviours, have been reported in some early developmental disorders and autism spectrum disorders. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene are associated to Rett syndrome and other neuropsychiatric conditions. Thus, we first analysed AVP and OT expression in the brain of Mecp2-mutant mice by immunohistochemistry. Our results revealed no significant differences in these systems in young adult Mecp2-heterozygous females, as compared to WT littermates. By contrast, we found a significant reduction in the sexually dimorphic, testosterone-dependent, vasopressinergic innervation in several nuclei of the social brain network and oxytocinergic innervation in the lateral habenula of Mecp2-null males, as compared to WT littermates. Analysis of urinary production of pheromones shows that Mecp2-null males lack the testosterone-dependent pheromone darcin, strongly suggesting low levels of androgens in these males. In addition, resident-intruder tests revealed lack of aggressive behaviour in Mecp2-null males and decreased chemoinvestigation of the intruder. By contrast, Mecp2-null males exhibited enhanced social approach, as compared to WT animals, in a 3-chamber social interaction test. In summary, Mecp2-null males, which display internal testicles, display a significant reduction of some male-specific features, such as vasopressinergic innervation within the social brain network, male pheromone production and aggressive behaviour. Thus, atypical social behaviours in Mecp2-null males may be caused, at least in part, by the effect of lack of MeCP2 over sexual differentiation.

Lack of MeCP2 leads to region-specific increase of doublecortin in the olfactory system.

The protein doublecortin is mainly expressed in migrating neuroblasts and immature neurons. The X-linked gene MECP2, associated to several neurodevelopmental disorders such as Rett syndrome, encodes the protein methyl-CpG-binding protein 2 (MeCP2), a regulatory protein that has been implicated in neuronal maturation and refinement of olfactory circuits. Here, we explored doublecortin immunoreactivity in the brain of young adult female Mecp2-heterozygous and male Mecp2-null mice and their wild-type littermates. The distribution of doublecortin-immunoreactive somata in neurogenic brain regions was consistent with previous reports in rodents, and no qualitative differences were found between genotypes or sexes. Quantitatively, we found a significant increase in doublecortin cell density in the piriform cortex of Mecp2-null males as compared to WT littermates. A similar increase was seen in a newly identified population of doublecortin cells in the olfactory tubercle. In these olfactory structures, however, the percentage of doublecortin immature neurons that also expressed NeuN was not different between genotypes. By contrast, we found no significant differences between genotypes in doublecortin immunoreactivity in the olfactory bulbs. Nonetheless, in the periglomerular layer of Mecp2-null males, we observed a specific decrease of immature neurons co-expressing doublecortin and NeuN. Overall, no differences were evident between Mecp2-heterozygous and WT females. In addition, no differences could be detected between genotypes in the density of doublecortin-immunoreactive cells in the hippocampus or striatum of either males or females. Our results suggest that MeCP2 is involved in neuronal maturation in a region-dependent manner.