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Maternal infections during pregnancy and the consequent maternal immune activation (MIA) are the major risk factors for autism spectrum disorder (ASD). Epidemiological evidence is corroborated by the preclinical models in which MIA leads to ASD-like behavioral abnormalities and altered neuroinflammatory profiles, with an increase in pro-inflammatory cytokines and microglial markers. In addition to neuroinflammatory response, an abnormal expression of endogenous retroviruses (ERVs) has been identified in neurodevelopmental disorders and have been found to correlate with disease severity. Our aim was to evaluate the transcriptional profile of several ERV families, ERV-related genes, and inflammatory mediators (by RT real-time PCR) in mouse offspring of both sexes, prenatally exposed to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3 that mimics viral maternal infection during pregnancy. We found that prenatal exposure to Poly I:C deregulated the expression of some ERVs and ERV-related genes both in the prefrontal cortex (PFC) and hippocampus, while no changes were detected in the blood. Interestingly, sex-related differences in the expression levels of some ERVs, ERV-related genes, and inflammatory mediators that were higher in females than in males emerged only in PFC. Our findings support the tissue specificity of ERV and ERV-related transcriptional profiles in MIA mice.

Autism Spectrum Disorder (ASD) is a sex-biased neurodevelopmental disorder with a male to female prevalence of 4:1, characterized by persistent deficits in social communication and interaction and restricted-repetitive patterns of behavior, interests or activities. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune dysregulation in ASD is further supported by evidence suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. The present study was aimed at characterizing the effects of MIA on behavior, gut microbiota and neuroinflammation in the mouse offspring also considering the impact of MIA in the two sexes. MIA offspring exhibited significant ASD-like behavioral alterations (i.e., deficits in sociability and sensorimotor gating, perseverative behaviors). The analysis of microbiota revealed changes in specific microbial taxa that recapitulated those seen in ASD children. In addition, molecular analyses indicated sex-related differences in the neuroinflammatory responses triggered by MIA, with a more prominent effect in the cerebellum. Our data suggest that both sexes should be included in the experimental designs of preclinical studies in order to identify those mechanisms that confer different vulnerability to ASD to males and females.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

Early life low-level lead (Pb) exposure is still an alarming child health issue. To date, animal studies investigating the effects of low doses of Pb since early stages of life to adulthood are scarce. We investigated in a mouse model the behavioral effects of developmental exposure to low-level Pb yielding blood levels similar to those observed in child clinical literature. CD1 outbred mouse dams received Pb (25- or 100-ppm) via drinking water from two weeks pre-mating until the end of lactation. Offspring of both sexes underwent a longitudinal assessment of motor, socio-emotional, and cognitive endpoints from neonatal to adult stage. Pb levels were determined in several matrices (blood, brain and bone) up to six months after the end of exposure. We found that new born pups exposed to Pb have slightly altered motor patterns and reduced preference for the nest odor. Offspring of both sexes exposed to the lowest Pb dose showed diminished interest for social novelty stimuli as adults. Moreover, sex-dependent effects of Pb exposure were observed in the spatial learning and memory task, where males were selectively impaired. Finally, blood, brain and bone Pb levels were elevated in a dose dependent fashion up to six months after termination of exposure. We observed marked accumulation of Pb in bones, with higher Pb levels in 100-ppm exposed females than in males at 7 months of age. In conclusion, developmental Pb exposure caused mild alterations in early- and late-life behavioral domains, particularly involving olfactory and cognitive responses. These findings confirm the importance of animal models to understand how early chronic low-level lead exposure impacts on health in a life-course perspective.

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content—i.e., optimal, sub-optimal, and deficient—and neurodevelopmental, neuroinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in sub-optimal offspring. In addition, sub-optimal, more than deficient supply, reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, deficient and sub-optimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabolism and homeostasis. The finding that the Se sub-optimal was more detrimental than Se deficient diet may suggest that maternal Se deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by sub-optimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development.

Mice produce ultrasonic vocalizations (USVs) in different social contexts across lifespan. There is ethological evidence that pup USVs elicit maternal retrieval and adult USVs facilitate social interaction with a conspecific. Analysis of mouse vocal and social repertoire across strains, sex and contexts remains not well explored. To address these issues, in inbred (C57BL/6, FVB) and outbred (CD-1) mouse strains, we recorded and evaluated USVs as neonates and during adult social encounters (male-female and female-female social interaction). We showed significant strain differences in the quantitative (call rate and duration of USVs) and qualitative vocal analysis (spectrographic characterization) from early stage to adulthood, in line with specific patterns of social behaviors. Inbred C57BL/6 mice produced a lower number of calls with less internal changes and shorter duration; inbred FVB mice displayed more social behaviors and produced more syllables with repeated internal changes; outbred CD-1 mice had an intermediate profile. Our results suggest specific vocal signatures in each mouse strain, thus helping to better define socio-communicative profiles of mouse strains and to guide the choice of an appropriate strain according to the experimental settings.

In rodent models of Autism Spectrum Disorders (ASD), the study of ultrasonic vocalizations has provided the unique opportunity to evaluate social communication and interaction in ethologically-appropriate contexts, behavioral domains relevant to the first core symptom of ASD. In the present review, we selected and evaluated ultrasonic vocalizations' data collected in rodent models of ASD in different experimental settings, either in the neonatal phase or in adulthood. Both quantitative (calling rates) and qualitative (range and shape of the vocal repertoire) abnormalities have been evidenced. The aim of our work was to highlight several promises and a few caveats in the use of ultrasonic vocalizations for behavioral phenotyping of ASD models and give some suggestions to maximize the translational value of these studies.

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.

Adolescence is a critical period for brain development. In most mammalian species, disturbances experienced during adolescence constitute a risk factor for several neuropsychiatric disorders. In this study, we compared the biochemical and behavioral profile induced by postweaning social isolation (PWSI) in inbred C57BL/6 N mice with that of BTBR mice, a rodent model of autism spectrum disorders. Male C57BL/6 N mice were either housed in groups of four or isolated from weaning (postnatal day 21) for four weeks before experimental analyses. After weaning, male BTBR mice were housed four per cage and analyzed at 48 days of age. PWSI reduced hippocampal levels of type 2 metabotropic glutamate (mGlu2) receptors, and glucocorticoid and mineralocorticoid receptors. A similar reduction was seen in group-housed BTBR mice. Plasma corticosterone levels in basal conditions were not influenced by PWSI, but were increased in BTBR mice. Social investigation (total and head sniffing) and the number of ultrasonic vocalizations were reduced in both PWSI mice and age-matched group-housed BTBR mice, indicating a lower social responsiveness in both groups of mice. These results suggest that absence of social stimuli during adolescence induces an endophenotype with social deficit features, which mimics the phenotype of a mouse model of autism spectrum disorders.

Lead (Pb) exposure in early life affects brain development resulting in cognitive and behavioral deficits. Epidemiologic and experimental evidence of sex as an effect modifier of developmental Pb exposure is emerging. In the present study, we investigated Pb effects on behavior and mechanisms of neuroplasticity in the hippocampus and potential sex differences. To this aim, dams were exposed, from one month pre-mating to offspring weaning, to Pb via drinking water at 5 mg/kg body weight per day. In the offspring of both sexes, the longitudinal assessment of motor, emotional, and cognitive end points was performed. We also evaluated the expression and synaptic distribution of N-methyl-D-Aspartate receptor (NMDA) and &alpha, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits at post-natal day (pnd) 23 and 70 in the hippocampus. Neonatal motor patterns and explorative behavior in offspring were affected in both sexes. Pb effects in emotional response and memory retention were observed in adult females only, preceded by increased levels of GluN2A and GluA1 subunits at the post-synapse at pnd 23. These data suggest that Pb exposure during development affects glutamatergic receptors distribution at the post-synaptic spine in females. These effects may contribute to alterations in selected behavioral domains.

Prenatal treatment with the antiepileptic drug valproic acid (VPA) is associated with a significant risk of somatic anomalies, neurodevelopmental delays, and 7-10× increase in the incidence of autism spectrum disorders (ASD) in children. Rodents exposed to VPA in pregnancy show birth defects, deficits in neurodevelopment, and cognitive/social anomalies resembling those of ASD children. Mechanisms of VPA neurobehavioral toxicity are still unclear but as VPA is a non-selective inhibitor of histone deacetylases, epigenetic modifications are likely involved. This study was aimed to evaluate the transgenerational impact of prenatal VPA exposure on mouse early behavioral development, studying F1, F2, and F3 generations after VPA challenge on gestational day (GD) 10.5. We also analyzed in brain and in peripheral blood mononuclear cells the expression levels of different endogenous retrovirus (ERV) families, potential biomarkers of derailed brain development, since human ERVs have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs) such as ASD. Somatic effects of VPA were evident only in F1 generation and more markedly in the female sex. Across F1 and F2 generations, VPA delayed righting reflex, increased motor activity, and reduced ultrasonic vocalizations. The behavioral changes in F3 are milder though in the same direction. VPA increased expression of most ERVs across the three generations in brain and blood. In utero VPA induced neurodevelopmental alterations more marked in the maternal lineage that persisted also in F3, suggesting ERVs as possible downstream effectors of the VPA epigenetic alterations.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by social communication deficits and repetitive/stereotyped behaviours. We evaluated the effects of a chronic treatment with the immunomodulator drug Fingolimod (FTY720 – a non-selective Sphingosine 1-Phosphate Receptor ligand) in an ASD model, the BTBR T+tf/J (BTBR) mouse strain. In adult BTBR males, chronic FTY720 treatment (4 weeks) increased social and vocal response during a male–female interaction and hippocampal expression of BDNF and Neuregulin 1, two trophic factors reduced in BTBR when compared to control C57 mice. FTY720 also re-established the expression of IL-1β and MnSOD in the hippocampus, whereas it did not modify IL-6 mRNA content. In addition to its central effect, FTY720 modulated the activation state of peripheral macrophages in the BTBR model, both in basal conditions and after stimulation with an immune challenge. Furthermore, IL-6 mRNA colonic content of BTBR mice, reduced when compared with C57 mice, was normalized by chronic treatment with FTY720. Our study, while indicating FTY720 as a tool to attenuate relevant alterations of the BTBR neurobehavioural phenotype, emphasizes the importance of gut mucosal immune evaluation as an additional target that deserve to be investigated in preclinical studies of anti-inflammatory therapeutic approaches in ASD.

Prenatal viral/bacterial infections are considered risk factors for autism spectrum disorders (ASD) and rodent models of maternal immune activation (MIA) have been developed and extensively used in preclinical studies. Poly inosinic-cytidylic acid (Poly I:C) was injected in C57BL6/J dams to mimic a viral infection on gestational day 12.5; the experimental design includes 10/12 litters in each treatment group and data were analysed always considering the litter-effect; neonatal (spontaneous motor behaviour and ultrasonic vocalizations) and adult [open field, marble burying, social approach, fear conditioning, prepulse inhibition (PPI)] offspring of both sexes were tested. In vivo magnetic resonance imaging/spectroscopy (MRI-MRS) and high-performance liquid chromatography (HPLC) to quantify both aminoacid and/or neurotransmitter concentration in cortical and striatal regions were also carried out. In both sexes high levels of repetitive motor responses and sensory gating deficits in PPI were the more striking effects of Poly I:C, whereas no alteration of social responses were evidenced. Poly I:C treatment did not affect mean values, but, intriguingly, increased variability in the levels of four aminoacids (aspartate glycine and GABA) selectively in males. As a whole prenatal Poly I:C induced relevant long-term alterations in explorative-stereotyped motor responses and in sensory gating, sparing cognitive and social competences. When systematically assessing differences between male and female siblings within each litter, no significant sex differences were evident except for increased variability of four aminoacid levels in male brains. As a whole, prenatal Poly I:C paradigms appear to be a useful tool to investigate the profound and translationally-relevant effects of developmental immune activation on brain and behavioural development, not necessarily recapitulating the full ASD symptomatology.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition with unknown etiology. Recent experimental evidences suggest the contribution of non-coding RNAs (ncRNAs) in the pathophysiology of ASD. In this work, we aimed to investigate the expression profile of the ncRNA class of circular RNAs (circRNAs) in the hippocampus of the BTBR T + tf/J (BTBR) mouse model and age-matched C57BL/6J (B6) mice. Alongside, we analyzed BTBR hippocampal gene expression profile to evaluate possible correlations between the differential abundance of circular and linear gene products. From RNA sequencing data, we identified circRNAs highly modulated in BTBR mice. Thirteen circRNAs and their corresponding linear isoforms were validated by RT-qPCR analysis. The BTBR-regulated circCdh9 was better characterized in terms of molecular structure and expression, highlighting altered levels not only in the hippocampus, but also in the cerebellum, prefrontal cortex, and amygdala. Finally, gene expression analysis of the BTBR hippocampus pinpointed altered biological and molecular pathways relevant for the ASD phenotype. By comparison of circRNA and gene expression profiles, we identified 6 genes significantly regulated at either circRNA or mRNA gene products, suggesting low overall correlation between circRNA and host gene expression. In conclusion, our results indicate a consistent deregulation of circRNA expression in the hippocampus of BTBR mice. ASD-related circRNAs should be considered in functional studies to identify their contribution to the etiology of the disorder. In addition, as abundant and highly stable molecules, circRNAs represent interesting potential biomarkers for autism.

In hypertensive retinopathy, the retinal damage due to high blood pressure is accompanied by increased expression of Glial Fibrillary Acidic Protein (GFAP), which indicates a role of neuroinflammatory processes in such a retinopathy. Proteins belonging to the Rho GTPase family, particularly Rac1, are involved in the activation of Muller glia and in the progression of photoreceptor degeneration, and may thus represent a novel candidate for therapeutic intervention following central nervous system inflammation. In this paper, we have observed that topical administration as eye drops of Cytotoxic Necrotizing Factor 1 (CNF1), a Rho GTPase modulator, surprisingly improves electrophysiological and behavioral visual performances in aged spontaneously hypertensive rats. Furthermore, such functional improvement is accompanied by a reduction of Rac1 activity and retinal GFAP expression. Our results suggest that Rac1 inhibition through CNF1 topical administration may represent a new strategy to target retinal gliosis.

Retroelements, such as Human Endogenous Retroviruses (HERVs), have been implicated in many complex diseases, including neurological and neuropsychiatric disorders. Previously, we demonstrated a distinctive expression profile of specific HERV families in peripheral blood mononuclear cells from Autistic Spectrum Disorders (ASD) patients, suggesting their involvement in ASD. Here we used two distinct ASD mouse models: inbred BTBR T+tf/J mice and CD-1 outbred mice prenatally exposed to valproic acid. Whole embryos, blood and brain samples from the offspring were collected at different ages and the expression of several ERV families (ETnI, ETnII-α, ETnII-β, ETnII-γ, MusD and IAP), proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and Toll-like receptors (TLR3 and TLR4) was assessed. In the two distinct mouse models analysed, the transcriptional activity of the ERV families was significant higher in comparison with corresponding controls, in whole embryos, blood and brain samples. Also the expression levels of the proinflammatory cytokines and TLRs were significantly higher than controls. Current results are in agreement with our previous findings in ASD children, supporting the hypothesis that ERVs may serve as biomarkers of atypical brain development. Moreover, the changes in ERVs and proinflammatory cytokines expression could be related with the autistic-like traits acquisition in the two mouse models.

There is an increasing interest for analytical methods aimed to detect biological sulfur-containing amines, because of their involvement in human diseases and metabolic disorders. This work describes an improved HPLC method for the determination of sulfur containing amino acids and amines from different biological matrices. We optimized a pre-column derivatization procedure using dabsyl chloride, in which dabsylated products can be monitored spectrophotometrically at 460 nm. This method allows the simultaneous analysis of biogenic amines, amino acids and sulfo-amino compounds including carnosine, dopamine, epinephrine, glutathione, cysteine, taurine, lanthionine, and cystathionine in brain specimens, urines, plasma, and cell lysates. Moreover, the method is suitable for the study of physiological and non-physiological derivatives of taurine and glutathione such as hypotaurine, homotaurine, homocysteic acid and S-acetylglutathione. The present method displays good efficiency of derivatization, having the advantage to give rise to stable products compared to other derivatizing agents such as o-phthalaldehyde and dansyl chloride.With this method, we provide a tool to study sulfur cycle from a metabolic point of view in relation to the pattern of biological amino-compounds, allowing researchers to get a complete scenario of organic sulfur and amino metabolism in tissues and cells.

Rett syndrome (RTT) is a rare neurodevelopmental disorder usually caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Several Mecp2 mutant mouse lines have been developed recapitulating part of the clinical features. In particular, Mecp2-308 female heterozygous mice, bearing a truncating mutation, are a validated model of the disease. While recent data suggest a role for inflammation in RTT, little information on the inflammatory status in murine models of the disease is available. Here, we investigated the inflammatory status by proteomic 2-DE/MALDI-ToF/ToF analyses in symptomatic Mecp2-308 female mice. Ten differentially expressed proteins were evidenced in the Mecp2-308 mutated plasma proteome. In particular, 5 positive acute-phase response (APR) proteins increased (i.e., kininogen-1, alpha-fetoprotein, mannose-binding protein C, alpha-1-antitrypsin, and alpha-2-macroglobulin), and 3 negative APR reactants were decreased (i.e., serotransferrin, albumin, and apolipoprotein A1). CD5 antigen-like and vitamin D-binding protein, two proteins strictly related to inflammation, were also changed. These results indicate for the first time a persistent unresolved inflammation of unknown origin in the Mecp2-308 mouse model.

Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both −/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress., Highlights • Oxidative damage is demonstrated in the brain, and more specifically in the neurons, of Mecp2 mutant mouse models. • A direct evidence between enhanced oxidative stress and Mecp2 deficiency is provided. • Oxidative damage precedes the behavioral abnormalities in Mecp2 mutant mice. • Mecp2 is likely involved in the protection of the brain from oxidative stress.

Autism is a neurodevelopmental disorder characterized by social and communication impairments and repetitive behaviours. The inbred BTBR T+ tf/J (BTBR) strain, a putative mouse model of autism, exhibits lower social interactions, higher repetitive self-grooming levels and unusual pattern of vocalizations as compared to C57BL/6J strain. First aim of the present study was to evaluate at adolescence (postnatal days 30-35) male BTBR and C57BL/6J performances in two different tasks involving either investigation of social cues (same strain partners) or non social ones (inanimate objects). In the social interaction test, BTBR mice showed a reduction of investigation of the social partner, due to a selective reduction of head sniffing, associated with a decrease in ultrasonic vocalizations. By contrast, no strain differences were detected in object investigations. Second aim of the study was to evaluate adult male BTBR and C57BL/6J performances in a fear conditioning task. Strain differences were evident during contextual retest: these strain differences primarily suggested a lack of behavioural flexibility in BTBR mice (i.e., realizing the occurrence of changes in the experimental paradigm). Subsequent electrophysiological analysis in hippocampal slices from adult BTBR and C57BL/6J mice revealed a significant reduction of Brain Derived Neurotrophic Factor (BDNF)-induced potentiation of synaptic transmission in BTBR mice. BDNF and tyrosine kinase B (TrkB) protein levels measured in the hippocampal region were also lower in BTBR as compared to C57BL/6J mice. These data confirm the presence of low levels of direct interaction with social stimuli in BTBR mice at adolescence, in the absence of any strain difference as for investigation of physical objects. At adulthood in BTBR mice clear signs of behavioural inflexibility were evident whereas both biochemical and electrophysiological data point to decreased BDNF signalling (likely due to a reduction in TrkB levels) in the hippocampus of this mouse strain.

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births; it represents the second most common cause of intellectual disability in females. Mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2) have been identified as clear etiological factors in more than 90% of classical RTT cases. Whereas the mechanisms leading to the severe, progressive and specific neurological dysfunctions when this gene is mutated still remain to be elucidated, a series of different mouse models have been generated, bearing different Mecp2 mutation. Neurobehavioural analysis in these mouse lines have been carried out and phenotyping analysis can be now utilised to preclinically evaluate the effects of potential RTT treatments. This review summarizes the different results achieved in this research field taking into account different key targets identified to ameliorate RTT phenotype in mouse models, including those not directly downstream of MeCP2 and those limited to the early phases of postnatal development. This article is part of the Special Issue entitled 'Neurodevelopmental Disorders'.

The complexity of the neuroendocrine level of investigation requires the assessment of behavioral patterns that extend beyond the reproductive functions, which are age- and sex-specific in rodents, described by defined clusters of behavioral items regulated by genetic, hormonal, and epigenetic factors. The study of social behavior in laboratory rodents reveals sex-dimorphic effects of environmental chemicals that may be undetected either by a traditional neurotoxicological approach or referring to the classical definition of endocrine disrupting chemicals. Here we review data on the neurobehavioral effects of developmental exposure to the non-persistent organophosphorus insecticide chlorpyrifos, whose neurotoxic activity at low doses is currently a matter of concern for children's health. In mice exposed to chlorpyrifos in utero and/or in early development social/emotional responses are differently affected in the two sexes in parallel with sex-dependent interference on hypothalamic neuroendocrine pathways regulating social behaviors (vasopressin, oxytocin, and steroid regulated systems). Through the analysis of complex sex-dimorphic behavioral patterns we show that neurotoxic and endocrine disrupting activities of CPF overlap. This widely diffused organophosphorus pesticide might thus be considered as a neuroendocrine disruptor possibly representing a risk factor for sex-biased neurodevelopmental disorders in children.

We have all experienced the pain of an injection at some point in our lives. This level of discomfort and stress is the threshold that the new European Directive on “the protection of animals used for scientific purposes” sets for its applicability: if a procedure is more painful than the insertion of a needle, then the legislation will apply. Published last October in the Official Journal of the European Union (EC, 2010), the new Directive is the culmination of about 10 years of work by the European Commission. Nevertheless, reaction to it will probably be mixed. There are those for whom it will represent yet another bureaucratic burden in the practice of experimentation on animals, whereas others will perceive it as merely an affirmation of the right of scientists to cause animals to suffer in the name of progress, with limited oversight or control. > …if a procedure is more painful than the insertion of a needle, then the legislation will apply In our opinion, the Directive (known as Directive 2010/63) represents a step forward in improving the quality of life of laboratory animals, while preserving the quality and reproducibility of experimental data. Furthermore, we believe that the Directive is a dynamic tool that provides the scientific community with the leeway to improve the living conditions of experimental animals. It therefore in no way represents the end of a scientific, ethical and legislative process, but instead functions as a starting point. Discussions about the details of how, when, where and why research using animals might be conducted are crucial and will continue to be so in the future. Our contribution should be seen in this light, and not simply as a critique of the work of the Commission. There are both altered and new elements of legislation throughout the Directive, all …

BTBR T+tf/J (BTBR) is an inbred mouse strain that displays social deficits and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. We previously reported an unusual pattern of ultrasonic vocalizations in BTBR pups that may represent a behavioral homolog to the second diagnostic symptom of autism, impaired communication. This study investigated the social and vocal repertoire in adult BTBR mice, to evaluate the role of ultrasonic vocalizations in multiple social situations at the adult stage of development. Three different social contexts were considered: male-female, male-male (resident-intruder) and female-female interactions. Behavioral responses and ultrasonic vocalizations were recorded for BTBR and for the highly social control strain C57BL/6J (B6). No episodes of overt fighting or mating were observed during the short durations of the three different experimental encounters. BTBR displayed lower levels of vocalizations and social investigation in all three social contexts as compared with B6. In addition, the correlation analyses between social investigation and ultrasonic vocalization emission rate showed that in B6 mice, the two variables were positively correlated in all the three different social settings, whereas in BTBR mice, the positive correlation was significant only in the male-female interactions. These findings strongly support the value of simultaneously recording two aspects of the mouse social repertoire: social motivation and bioacoustic communication. Moreover, our findings in adults are consistent with previous results in pups, showing an unusual vocal repertoire in BTBR as compared with B6.

The "cholinergic hypothesis" of dementia posits that the progressive loss of basal forebrain cholinergic neurons and the consequent decrease of acetylcholine levels in the deafferented projection sites are correlated with degree of cognitive decline in dementia. It has also been proposed that early dysfunction of the basal forebrain (BF) cholinergic system may be a risk factor for subsequent cognitive decline and possibly dementia. To characterize how age when BF cholinergic system is lesioned affects behavioral performances and morphology of neocortical neurons, seven-day-old rats were bilaterally i.c.v. injected with 192 IgG-saporin. In adulthood, these animals were subjected to spatial and associative tests. Subsequently, the morphology of parietal pyramidal neurons was assessed. The same behavioral and morphological evaluations were made in 80-day-old rats tested three weeks after bilateral i.c.v. injections of 192 IgG-saporin. The behavioral consequences of both cholinergic depletions were markedly similar. While both groups of lesioned animals exhibited very subtle deficits in the Morris water maze, they were significantly impaired in spatial discrimination in the open field and the radial maze. Paralleling behavioral data, the results of the morphological analysis revealed comparable increases in number and density of spines in apical and basal dendrites in layer-III parietal pyramidal neurons following both neonatal and adult cholinergic depletions. The present results demonstrate that the consequences of abnormal maturation of the cholinergic system are not substantially different from those evoked by cholinergic dysfunction in adulthood and provide a developmental psychobiological perspective of the neuronal foundations of the impaired cognitive functions.

The organophosphate chlorpyrifos (CPF) is a pesticide largely used worldwide. Studies from animal models indicate that CPF exposure during development at low doses can target different neurotransmitter systems in the absence of overt cholinergic effects.Late gestational exposure (gestational days 14-17) to CPF at the dose of 6 mg/kg was evaluated in CD-1 mice at adulthood. Neurobehavioural effects likely involving serotonin (5-hydroxytryptamine, 5HT) transmission were assessed both in males and females, through the light-dark exploration test to assess CPF effects on anxiety profiles and the forced swimming test to evaluate the response to the 5HT transporter (5HTT) inhibitor fluvoxamine (30 mg/kg). In females only, we evaluated the effects of gestational exposure to CPF on maternal aggression, under basal condition or after injection of fluvoxamine.Gestational CPF exposure increased anxiety levels only in female mice, as shown by the augmented thigmotaxis behaviour and the lower latency to enter in the dark compartment. In the forced swimming test, no differences between CPF and control mice were found when assessed under basal condition (saline administration), but both male and female CPF mice missed to show the typical behavioural effects of the 5HTT inhibitor fluvoxamine. During maternal aggression, CPF females showed lower propensity to and intensity of aggressive behaviour, together with mild decreased responsiveness to fluvoxamine administration.Overall, the present results confirm a specific and sex-dependent vulnerability of affective/emotional domains to developmental CPF exposure. Furthermore, data provide clear indication on the disrupting effects of prenatal CPF on serotoninergic transmission.

Organophosphorus insecticides, as Chlorpyrifos (CPF), are widely used in agriculture and against household pests; these compounds receive an increasing consideration as potential endocrine disrupters. The aim of the present study was to examine the potential short- and long-term effects of CPF on thyroid and adrenal glands in CD1 mice following exposure at dose levels not inducing brain acetyl cholinesterase (AchE) inhibition, during gestational and/or postnatal vulnerable phases. Pregnant dams were treated with 0, 3, 6 mg/kg bw/day of CPF on gestational days 15-18. After delivery, pups were treated subcutaneously on postnatal days (PND) 11-14 with: 0, 1, 3 mg/kg bw/day of CPF. Serum thyroxin (T4), thyroid and adrenals histology and histomorphometry were evaluated in dams and in F1 mice. In dams at 6 mg/kg, decreased T4 levels and increased cell height in thyroid were observed, and adrenal histology showed a slightly increased vacuolization in the X-zone. In the F1, short-term morphological modifications (reduced follicular size at PND 2) and long-term morphological (increased necrotic follicular cells) and biochemical alterations (reduced serum T4 levels) were found at PND 150 with an apparent higher vulnerability of males. For the first time these results indicate that CPF exposure at dose levels not inducing brain AchE inhibition causes thyroid alterations in dams and in F1 CD1 mice. Thyroid may be a sensitive target to CPF developmental exposure possibly leading to long-term effects on thyroid function. Because thyroid plays a pivotal role in mammalian development, these findings can be relevant to humans.

CD-1 mice were exposed to the organophosphate pesticide chlorpyrifos (CPF) throughout postnatal days (PND) 11-14 at the subtoxic dose of 3 mg/kg. At adolescent age, females and males underwent a sociability test in which level of sociability and social preference were measured. At adulthood only females' behavior was analyzed. Maternal behavior of CPF-exposed females was assessed on postpartum day 1 after removal of the pups for 1 h, while anxiety levels were measured in a 5 min dark-light test on postpartum day 2. Nest defense response to an unfamiliar male intruder was assessed on postpartum day 7. In addition, from birth to postpartum day 7 a detailed analysis of nest building activity was carried out. Neonatal CPF exposure does not interfere with social behavior and social preferences at adolescence, whereas at adulthood it induces significant behavioral alterations in lactating females. Motivation to build and defend the nest was decreased in CPF females that were also less anxious than controls in the dark-light paradigm. These results confirm that developmental exposure to CPF induces long-lasting alterations in selected sexual-dimorphic responses of the adult social repertoire, and suggest that early exposure to CPF might interfere with hypothalamic neuroendocrine mechanisms regulating social responses. © 2008 Elsevier Inc. All rights reserved.

Rett syndrome (RTT) is a neurodevelopmental disorder, primarily affecting girls. RTT causes severe cognitive, social, motor and physiological impairments and no cure currently exists. The discovery of a monogenic origin for RTT and the subsequent generation of RTT mouse models provided a major breakthrough for RTT research. Although the characterization of these mutant mice is far from complete, they recapitulate several RTT symptoms. This review provides an overview of the behavioural domains so far investigated in these models, including the very few mouse data concerning the developmental course of RTT. Both clinical and animal studies support the presence of early defects and highlight the importance of probing the presymptomatic phase for both the precocious identification of biomarkers and the early assessment of potential therapies. Preclinical evaluations of pharmacological and nonpharmacological interventions so far carried out are also illustrated. In addition, genetic manipulations are reported that demonstrate rescue from the damage caused by the absence of the methyl-CpG-binding protein 2 (MeCP2) gene even at a mature stage. Given the rare occurrence of RTT cases, transnational collaborative networks are expected to provide a deeper understanding of aetiopathology and the development of new therapeutic approaches.

BACKGROUND: Increasing evidence from animal and human studies indicates that chlorpyrifos (CPF), similar to other organophosphorus insecticides still widely used, is a developmental neurotoxicant. Developmental exposure to CPF in rodents induces sex-dimorphic behavioral changes at adulthood, including social and agonistic responses, which suggests that CPF may interfere with maturation of neuroendocrine mechanisms. OBJECTIVES: We assessed the hypothesis that CPF affects the levels of neurohypophyseal hormones acting as modulators of social behavior in mammals, such as oxytocin (OT), arginine vasopressin (AVP), and prolactin (PRL). METHODS: Pregnant female mice were orally administered with either vehicle (peanut oil) or 3 or 6 mg/kg CPF on gestational day (GD) 15 to GD18, and offspring were treated subcutaneously with either vehicle or 1 or 3 mg/kg CPF on postnatal days (PNDs) 11 to PND14. Dose levels were chosen to avoid systemic toxicity and inhibition of brain acetylcholinesterase. Offspring were sacrificed at 5 months of age, and expression of OT, AVP, and PRL was analyzed in the hypothalamus by Western blot or enzyme-linked immunosorbent assay (ELISA) analysis. RESULTS: Both male and female mice showed dose-related enhancement of OT expression, with males presenting the more intense effect. AVP expression was significantly reduced in male mice at the higher prenatal and postnatal dose. We observed no significant effect on PRL expression in either sex. Overall, outcomes were mainly attributable to fetal exposure, whereas postnatal doses appeared to potentiate the prenatal effects. CONCLUSIONS: Our data indicate that developmental exposure to CPF may permanently interfere with specific key signaling proteins of the hypothalamic peptidergic system, with time-, dose-, and sex-related effects still evident at adulthood.

Etiological and molecular studies on the sporadic form of Alzheimer's disease have yet to determine the underlying mechanisms of neurodegeneration. Hyperhomocysteinemia is associated with Alzheimer's disease, and has been hypothesized to promote neurodegeneration, by inhibiting brain methylation activity. The aim of this work was to determine whether a combined folate, B12 and B6 dietary deficiency, would induce amyloid-beta overproduction, and to study the mechanisms linking vitamin deficiency, hyperhomocysteinemia and amyloidogenesis in TgCRND8 and 129Sv mice. We confirmed that B-vitamin deprivation induces hyperhomocysteinemia and imbalance of S-adenosylmethionine and S-adenosylhomocysteine. This effect was associated with PS1 and BACE up-regulation and amyloid-beta deposition. Finally, we detected intraneuronal amyloid-beta and a slight cognitive impairment in a water maze task at a pre-plaque age, supporting the hypothesis of early pathological function of intracellular amyloid. Collectively, these findings are consistent with the hypothesis that abnormal methylation in association with hyperhomocysteinemia may contribute to Alzheimer's disease.

The present study investigated the effects of intracebroventricular injections of 192 IgG-saporin in 7-day-old rats on (i) ultrasound vocalizations (USVs) on postnatal day (pnd) 13 following isolation and reunion with the mother and (ii) fear conditioning on pnd 18-19 recording both freezing and other behavioural responses as well as USVs. On pnd 13 lesioned and control pups showed comparable USV baseline values; a brief reunion with the mother induced a significant increase in USVs in all rats (maternal potentiation). On pnd 18, during the fear conditioning training, 192 IgG-saporin rats emitted a lower number of USVs. On pnd 19 all rats showed a stronger conditioned response (with full inhibition of locomotion) to auditory than to contextual cues. Surprisingly, lesioned rats showed a stronger fear-conditioned response to contextual cues than controls. These results suggest that early selective removal of the cholinergic basal forebrain paradoxically enhances hippocampally dependent fear-conditioned responses on pnd 19.

BACKGROUND: Chlorpyrifos (CPF) is one of the most widely used organophosphate pesticides worldwide. Epidemiological studies on pregnant women and their children suggest a link between in utero CPF exposure and delay in psychomotor and cognitive maturation. A large number of studies in animal models have shown adverse effects of CPF on developing brain and more recently on endocrine targets. Our aim was to determine if developmental exposure to CPF affects social responsiveness and associated molecular neuroendocrine markers at adulthood. METHOD: Pregnant CD1 outbred mice were fed from gestational day 15 to lactation day 14 with either a CPF-added (equivalent to 6 mg/kg/bw/day during pregnancy) or a standard diet. We then assessed in the offspring the long-term effects of CPF exposure on locomotion, social recognition performances and gene expression levels of selected neurondocrine markers in amygdala and hypothalamus. RESULTS: No sign of CPF systemic toxicity was detected. CPF induced behavioral alterations in adult offspring of both sexes: CPF-exposed males displayed enhanced investigative response to unfamiliar social stimuli, whereas CPF-exposed females showed a delayed onset of social investigation and lack of reaction to social novelty. In parallel, molecular effects of CPF were sex dimorphic: in males CPF increased expression of estrogen receptor beta in hypothalamus and decreased oxytocin expression in amygdala; CPF increased vasopressin 1a receptor expression in amygdala in both sexes. CONCLUSIONS: These data indicate that developmental CPF affects mouse social behavior and interferes with development of sex-dimorphic neuroendocrine pathways with potential disruptive effects on neuroendocrine axes homeostasis. The route of exposure selected in our study corresponds to relevant human exposure scenarios, our data thus supports the view that neuroendocrine effects, especially in susceptible time windows, should deserve more attention in risk assessment of OP insecticides.

Autism spectrum disorders are characterized by impaired social and communicative skills and repetitive behaviors. Emerging evidence supported the hypothesis that these neurodevelopmental disorders may result from a combination of genetic susceptibility and exposure to environmental toxins in early developmental phases. This study assessed the effects of prenatal exposure to chlorpyrifos (CPF), a widely diffused organophosphate insecticide endowed with developmental neurotoxicity at sub-toxic doses, in the BTBR T+tf/J mouse strain, a validated model of idiopathic autism that displays several behavioral traits relevant to the autism spectrum. To this aim, pregnant BTBR mice were administered from gestational day 14 to 17 with either vehicle or CPF at a dose of 6 mg/kg/bw by oral gavages. Offspring of both sexes underwent assessment of early developmental milestones, including somatic growth, motor behavior and ultrasound vocalization. To evaluate the potential long-term effects of CPF, two different social behavior patterns typically altered in the BTBR strain (free social interaction with a same-sex companion in females, or interaction with a sexually receptive female in males) were also examined in the two sexes at adulthood. Our findings indicate significant effects of CPF on somatic growth and neonatal motor patterns. CPF treated pups showed reduced weight gain, delayed motor maturation (i.e., persistency of immature patterns such as pivoting at the expenses of coordinated locomotion) and a trend to enhanced ultrasound vocalization. At adulthood, CPF associated alterations were found in males only: the altered pattern of investigation of a sexual partner, previously described in BTBR mice, was enhanced in CPF males, and associated to increased ultrasonic vocalization rate. These findings strengthen the need of future studies to evaluate the role of environmental chemicals in the etiology of neurodevelopment disorders.

We examined somatic growth, somatosensory reflexes, and ultrasonic calls from postnatal day 3 to day 18 in Mecp2 mutant mice, a mouse model of Rett syndrome. Both Mecp2 null male and Mecp2 heterozygous female mice exhibited normal somatic growth, but transient delays in the development of some reflexes relative to sex-matched wild-type mice. Both Mecp2 null male and heterozygous female mice exhibited dramatic increases in ultrasonic vocalizations in response to social isolation; these differences were evident as early as postnatal day 5. These results suggest very early abnormalities in sensory reflex development and behavioral responsiveness in the Mecp2 mutants that may provide a target for early therapeutic intervention.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and highlight CNF1 efficacy in counteracting RTT-related mitochondrial defects.

Rho GTPases are molecules critically involved in neuronal plasticity and cognition. We have previously reported that modulation of brain Rho GTPases by the bacterial toxin CNF1 rescues the neurobehavioral phenotype in MeCP2-308 male mice, a model of Rett syndrome (RTT). RTT is a rare X-linked neurodevelopmental disorder and a genetic cause of intellectual disability, for which no effective therapy is available. Mitochondrial dysfunction has been proposed to be involved in the mechanism of the disease pathogenesis. Here we demonstrate that modulation of Rho GTPases by CNF1 rescues the reduced mitochondrial ATP production via oxidative phosphorylation in the brain of MeCP2-308 heterozygous female mice, the condition which more closely recapitulates that of RTT patients. In RTT mouse brain, CNF1 also restores the alterations in the activity of the mitochondrial respiratory chain (MRC) complexes and of ATP synthase, the molecular machinery responsible for the majority of cell energy production. Such effects were achieved through the upregulation of the protein content of those MRC complexes subunits, which were defective in RTT mouse brain. Restored mitochondrial functionality was accompanied by the rescue of deficits in cognitive function (spatial reference memory in the Barnes maze), synaptic plasticity (long-term potentiation) and Tyr1472 phosphorylation of GluN2B, which was abnormally enhanced in the hippocampus of RTT mice. Present findings bring into light previously unknown functional mitochondrial alterations in the brain of female mice modeling RTT and provide the first evidence that RTT brain mitochondrial dysfunction can be rescued by modulation of Rho GTPases.

The immunotoxin 192 IgG saporin (192 IgG-sap) offers a valuable tool to investigate the role of the developing basal forebrain cholinergic system in modulating behavioral functions in developing, as well as adult rats. After neonatal 192 IgG-sap lesions, rats display reduced ultrasonic vocalizations as neonates, deficits in passive avoidance learning as juveniles, and altered reactions to spatial novelty as adults. These data suggest that neonatal cholinergic depletion affects cognitive performance in juvenile and adult rats. Additionally, neonatal cholinergic depletion alters ultrasonic vocalizations, which could then alter establishing normal mother-infant relationships, and thus compound the pup's cognitive deficits. These findings underscore the importance of assessing behavior during ontogeny, as well as in adulthood.

We investigated the effects of Nerve Growth Factor (NGF) administration on the maturation of reactivity to spatial and non-spatial novelty in developing mice. CD-1 mice of both sexes received intracerebral administration of NGF on postnatal day (pnd) 15, and their response to object displacement (spatial novelty) and object substitution (object novelty) were assessed in a spatial open-field with four objects on pnd 18 or 28. On pnd 18, NGF induced only in males precocious appearance of spatial novelty discrimination, while increasing choline acetyltransferase activity in neocortex and hippocampus of both sexes. The behavioral and neurochemical effects disappeared by pnd 28. NGF triggers adult-like responding to spatial novelty in developing mice and such effect is gender-specific.

Several pieces of evidence from animal and human studies indicate that the organophosphate insecticide chlorpyrifos (CPF) acts as a developmental neurotoxicant at environmentally relevant doses, and it is possibly endowed with endocrine-disrupting activity. Data collected in rodent models show that developmental exposure to CPF at sub-toxic doses induces long-lasting and sex-dimorphic changes in social and investigative responses in exposed offspring. The aim of this study was to evaluate the effects of gestational CPF treatment on social and olfactory discrimination in adult mice of both sexes. Pregnant CD1 out-bred mice were exposed to CPF per os on gestational days (GD) 14–17 at the sub-toxic dose of 6 mg/kg/bw. At adulthood, male and female offspring underwent the same experimental paradigms, namely i) a social discrimination test where mice were presented with a simultaneous binary choice between a novel conspecific and a familiar one, and ii) an olfactory habituation/dishabituation test to evaluate their capability to discriminate between odors with different eco-ethological salience (non-social vs. social odors). Results showed that in the social discrimination test prenatal CPF primarily affected the female sex by raising the investigation time in females to the same levels as found in vehicle- and CPF-exposed males. The ability to discriminate between a familiar and a novel social mate was not affected by CPF in either sex. In the olfactory habituation/dishabituation test, mice of both sexes successfully discriminated non-social from social odors regardless of the prenatal treatment received. These results confirm previous evidence indicating that developmental exposure to CPF causes long-lasting and sex-dimorphic changes in responsiveness to social cues, in the absence of significant impairment of social and olfactory discrimination capacity. These findings are discussed within the framework of recent data pointing to the limbic/hypothalamic circuitry and steroid hormonal regulations as possible targets for CPF neurotoxicity.

Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioral and physiological symptoms. Mutations in the methyl CpG-binding protein 2 gene (MECP2) cause >95% of classic cases, and currently there is no cure for this devastating disorder. The serotonin receptor 7 (5-HT7R) is linked to neuro-physiological regulation of circadian rhythm, mood, cognition, and synaptic plasticity. We presently report that 5-HT7R density is consistently reduced in cortical and hippocampal brain areas of symptomatic MeCP2–308 male mice, a RTT model. Systemic repeated treatment with LP-211 (0.25 mg/kg once/day for 7 days), a brain-penetrant selective 5-HT7R agonist, was able to rescue RTT-related defective performance: anxiety-related profiles in a Light/Dark test, motor abilities in a Dowel test, the exploratory behavior in the Marble Burying test, as well as memory in the Novelty Preference task. In the brain of RTT mice, LP-211 also reversed the abnormal activation of PAK and cofilin (key regulators of actin cytoskeleton dynamics) and of the ribosomal protein (rp) S6, whose reduced activation in MECP2 mutant neurons by mTOR is responsible for the altered protein translational control. Present findings indicate that pharmacological targeting of 5-HT7R improves specific behavioral and molecular manifestations of RTT, thus representing a first step toward the validation of an innovative systemic treatment. Beyond RTT, the latter might be extended to other disorders associated with intellectual disability.

Free-living female laboratory mice, adapted to outdoor life in large pens providing a naturalistic environment, were tested for their ability to modify their foraging habits to controlled food supply. An automatic feeder box delivered a small portion of the daily quantity of seeds to each individual mouse. Eight such boxes were placed into an outdoor pen. Each day, mice had to visit all boxes to gather the daily amount of food and were rewarded only at the first visit to each box. Mice were individually recognised by an implanted microchip. Throughout a 16-day period, feeding activity concentrated in an interval time around the beginning of the daily session. During the same period, the number of different feeders visited every day by mice increased irrespective of variation in exploratory activity. The experimental set-up allowed detecting temporal and spatial adaptations to the food restriction, as well as behavioural differences due to territorial and social factors. These data permit the design of novel tests assessing behavioural changes, memory and learning in normal and genetically modified mice, both in the laboratory and in naturalistic settings.

The processing of beta-amyloid precursor protein (betaAPP) and its metabolites plays an important role in the pathogenesis of Alzheimer's disease (AD) and Down's syndrome. The authors have reported elsewhere that a targeted mutation resulting in low expression of a shortened betaAPP protein (betaAPP(delta/delta)) entails reduced learning abilities. Here the authors investigate whether these effects were caused by postnatal developmental actions of the altered protein. The authors examined 35 mice carrying the betaAPP(delta/delta) mutation for somatic growth and sensorimotor development during the first 4 postnatal weeks (pw) and compared them with 31 wildtype litter-mates. Thereafter, the same mice were tested at about 10 weeks of age for openfield behavior and for swimming navigation learning. Mutant mice showed both transient and long-lasting deficits in development. Body weight deficit started to emerge at postnatal day (pd) 12, peaked with a 15.1% deficit at pd 27 and lasted until pw 33-37. Significant transient deficits in mutant mice during sensorimotor development were observed in three time windows (pd 3-10, pd 11-19 and pd 20-27), long-lasting effects, manifest at pw 8-12 and pw 33-37, emerged at any of the three periods. In the adult mice, exploratory activity of betaAPP mutants in the openfield arena was severely reduced. In the Morris water maze task, mutant mice showed moderate escape performance deficits during the acquisition period but no impairment in spatial memory. The authors conclude that a defective betaAPP gene impairs postnatal somatic development, associated with transient as well as long-lasting neurobehavioral retardation and muscular weakness. Comparison with earlier data suggests that early postnatal handling may attenuate some of the non-cognitive performance deficits in the water maze. Further, the manifestation and time course of behavioral yet not neuropathological symptoms in betaAPP mutant mice resemble in some aspects those of the human Down's syndrome.

The aim of this study was to investigate the effects of postnatal choline supplementation on neurochemical and behavioral parameters in preweanling BALB/cByJ mice. Mouse pups were injected daily subcutaneously with choline chloride (0.85 mM/g body weight) from Postnatal Day (PND) 1 to PND 16. Pups performed a passive avoidance (PA) learning task on PND 17-18 and a 30-min locomotor activity test on PND 19. The choline treatment affected retention of the PA task on PND 18. The treatment also increased locomotor activity in females, but not in males, on PND 19. Choline acetyltransferase (ChAT) enzymatic activity was measured on PND 20 and revealed that choline administration in the first 2 weeks of postnatal life selectively affects male pups. Choline's effect, as seen in previous rat experiments, was to decrease ChAT activity in the hippocampal region.

Passive avoidance learning and retention, as well as locomotor and exploratory behaviors, were assessed in rats after intraventricular 192 immunoglobulin G-saporin injections on either Postnatal Day 1 (PND1) or PND7. PND1-lesioned rats were not significantly impaired on acquisition or retention of passive avoidance. PND7-lesioned rats acquired the task slower than controls, but retention was not affected. PND7-lesioned rats were less exploratory than controls and showed reduced wall rearing. Histological analysis of PND1- and PND7-lesioned rats revealed no neuronal degeneration in hippocampus or cortex. There was a marked reduction of choline acetyltransferase (ChAT) activity in the hippocampus, cortex, and septum in the PND7-lesioned rats and a slight but significant ChAT depletion in the cortex of PND1-lesioned rats. These data suggest that the cholinergic system is critical for the learning of passive avoidance and exploratory behaviors in the developing rat.

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and compared with respect to their ability to generate membrane potential and to produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency has been successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics, will significantly expand the study of mitochondrial defects in neurological pathologies allowing large comparative studies and favoring inter-laboratory and interdisciplinary analyses.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder, primarily affecting girls. RTT causes a wide variety of debilitating symptoms and no cure currently exists. Mouse models bearing mutations in the Mecp2 gene recapitulate most physiological and behavioural RTT-related abnormalities. Stimulating neonatal environments (e.g. brief maternal separations or maternal low-dose corticosterone supplementation) reduce stress and fear responses at adulthood. The present study investigated whether impacting early in development the hypothalamic-pituitary-adrenal axis, by exposing Mecp2-308 mutant pups to a low dose of corticosterone (50 µg/ml, during the 1st week of life) may contrast RTT-related abnormalities in neuroendocrine regulation and behavioural adaptation at adulthood. In line with previous reports, when fully symptomatic, MeCP2-308 mice showed a reduction in the regular nocturnal hyperactivity in the home-cage and increased anxiety-like behaviours and plasma corticosterone (CORT) levels in response to restraint stress. An abnormal elevation in mRNA levels of mineralocorticoid receptors ( MR ) and BDNF gene was also evident in the hippocampus of fully symptomatic mutant mice. Neonatal CORT modulated MR gene expression and behavioural reactivity towards a novel object, also restoring wt-like levels of locomotor/exploratory behaviour in mutant mice. Enhanced sensitivity to the neonatal treatment (in terms of increase in GR and MR mRNA levels), was also evident in the hippocampus of MeCP2-308 mice compared to wt littermates. Present results corroborate the hypothesis that targeting the glucocorticoid system may prove valid in contrasting at least some of the RTT-related symptoms and provide evidence that pharmacological interventions during critical early time windows can persistently improve the behavioural phenotype of RTT mice. Current data also support the emerging role played by Mecp2 in mediating the epigenetic programming induced by early life events and indicate that, in the absence of functional MeCP2, programming of the central nervous system in response to early environmental stimuli is abnormally regulated. This article is part of the Special Issue entitled ‘Neurodevelopmental Disorders’.

The aim of this study was to assess in developing mice whether the neutralization of endogenous NGF following ICV administration of anti-NGF antibodies (50 micrograms/2 microliters) on postnatal days 3, 6, 9, and 12 affected locomotor activity, exploratory behavior, and response to the cholinergic blocker scopolamine. In Experiments 1 and 2 activity and age-typical scopolamine effects were evaluated on PND 13 or 17 in an automated apparatus. No significant main effect of anti-NGF treatment was found at either age. On day 13 scopolamine (0.2, 1, or 2 mg/kg) decreased locomotion in both anti-NGF and control animals. In Experiment 3, locomotion and exploratory behavior were analyzed in an open field arena or in a hole board apparatus on PND 16. No significant effects of anti-NGF treatment on general motor activity and investigation of a novel object in the open field was found, though anti-NGF animals tended to be less active than controls. In the hole board anti-NGF pups showed a different pattern of head dipping behavior from controls, exploring mainly the holes located in the periphery of the apparatus.