Hippocampal dysregulation of neurofibromin-dependent pathways is associated with impaired spatial learning in engrailed 2 knock-out mice.

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2(-/-)) mice show anatomical and behavioral "ASD-like" features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2(-/-) mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2(-/-) mice before and after spatial learning testing. When compared with WT littermates, En2(-/-) mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2(-/-) mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2(-/-) mice after MWM. Treatment of En2(-/-) mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.

Face processing in animal models: implications for autism spectrum disorder.

Processing facial features is crucial to identify social partners (prey, predators, or conspecifics) and recognize and accurately interpret emotional expressions. Numerous studies in both human and non-human primates provided evidence promoting the notion of inherent mechanisms for detecting facial features. These mechanisms support a representation of faces independent of prior experiences and are vital for subsequent development in social and language domains. Moreover, deficits in processing faces are a reliable biomarker of autism spectrum disorder, appearing early and correlating with symptom severity. Face processing, however, is not only a prerogative of humans: other species also show remarkable face detection abilities. In this review, we present an overview of the current literature on face detection in vertebrate models that could be relevant to the study of autism.

Valproic acid exposure affects social visual lateralization and asymmetric gene expression in zebrafish larvae.

Cerebral asymmetry is critical for typical brain function and development; at the same time, altered brain lateralization seems to be associated with neuropsychiatric disorders. Zebrafish are increasingly emerging as model species to study brain lateralization, using asymmetric development of the habenula, a phylogenetically old brain structure associated with social and emotional processing, to investigate the relationship between brain asymmetry and social behavior. We exposed 5-h post-fertilization zebrafish embryos to valproic acid (VPA), a compound used to model the core signs of ASD in many vertebrate species, and assessed social interaction, visual lateralization and gene expression in the thalamus and the telencephalon. VPA-exposed zebrafish exhibit social deficits and a deconstruction of social visual laterality to the mirror. We also observe changes in the asymmetric expression of the epithalamic marker leftover and in the size of the dorsolateral part of the habenula in adult zebrafish. Our data indicate that VPA exposure neutralizes the animals' visual field bias, with a complete loss of the left-eye use bias in front of their own mirror image, and alters brain asymmetric gene expression and morphology, opening new perspectives to investigate brain lateralization and its link to atypical social cognitive development.

Embryonic Valproate Exposure Alters Mesencephalic Dopaminergic Neurons Distribution and Septal Dopaminergic Gene Expression in Domestic Chicks.

In recent years, the role of the dopaminergic system in the regulation of social behavior is being progressively outlined, and dysfunctions of the dopaminergic system are increasingly associated with neurodevelopmental disorders, including autism spectrum disorder (ASD). To study the role of the dopaminergic (DA) system in an animal model of ASD, we investigated the effects of embryonic exposure to valproic acid (VPA) on the postnatal development of the mesencephalic DA system in the domestic chick. We found that VPA affected the rostro-caudal distribution of DA neurons, without changing the expression levels of several dopaminergic markers in the mesencephalon. We also investigated a potential consequence of this altered DA neuronal distribution in the septum, a social brain area previously associated to social behavior in several vertebrate species, describing alterations in the expression of genes linked to DA neurotransmission. These findings support the emerging hypothesis of a role of DA dysfunction in ASD pathogenesis. Together with previous studies showing impairments of early social orienting behavior, these data also support the use of the domestic chick model to investigate the neurobiological mechanisms potentially involved in early ASD symptoms.

Light-induced asymmetries in embryonic retinal gene expression are mediated by the vascular system and extracellular matrix.

Left-right asymmetries in the nervous system (lateralisation) influence a broad range of behaviours, from social responses to navigation and language. The role and pathways of endogenous and environmental mechanisms in the ontogeny of lateralisation remains to be established. The domestic chick is a model of both endogenous and experience-induced lateralisation driven by light exposure. Following the endogenous rightward rotation of the embryo, the asymmetrical position in the egg results in a greater exposure of the right eye to environmental light. To identify the genetic pathways activated by asymmetric light stimulation, and their time course, we exposed embryos to different light regimes: darkness, 6 h of light and 24 h of light. We used RNA-seq to compare gene expression in the right and left retinas and telencephalon. We detected differential gene expression in right vs left retina after 6 h of light exposure. This difference was absent in the darkness condition and had already disappeared by 24 h of light exposure, suggesting that light-induced activation is a self-terminating phenomenon. This transient effect of light exposure was associated with a downregulation of the sensitive-period mediator gene DIO2 (iodothyronine deiodinase 2) in the right retina. No differences between genes expressed in the right vs. left telencephalon were detected. Gene networks associated with lateralisation were connected to vascularisation, cell motility, and the extracellular matrix. Interestingly, we know that the extracellular matrix-including the differentially expressed PDGFRB gene-is involved in morphogenesis, sensitive periods, and in the endogenous chiral mechanism of primary cilia, that drives lateralisation. Our data show a similarity between endogenous and experience-driven lateralisation, identifying functional gene networks that affect lateralisation in a specific time window.

Fetal blockade of nicotinic acetylcholine transmission causes autism-like impairment of biological motion preference in the neonatal chick.

Several environmental chemicals are suspected risk factors for autism spectrum disorder (ASD), including valproic acid (VPA) and pesticides acting on nicotinic acetylcholine receptors (nAChRs), if administered during pregnancy. However, their target processes in fetal neuro-development are unknown. We report that the injection of VPA into the fetus impaired imprinting to an artificial object in neonatal chicks, while a predisposed preference for biological motion (BM) remained intact. Blockade of nAChRs acted oppositely, sparing imprinting and impairing BM preference. Beside ketamine and tubocurarine, significant effects of imidacloprid (a neonicotinoid insecticide) appeared at a dose ≤1 ppm. In accord with the behavioral dissociations, VPA enhanced histone acetylation in the primary cell culture of fetal telencephalon, whereas ketamine did not. VPA reduced the brain weight and the ratio of NeuN-positive cells (matured neurons) in the telencephalon of hatchlings, whereas ketamine/tubocurarine did not. Despite the distinct underlying mechanisms, both VPA and nAChR blockade similarly impaired imprinting to biological image composed of point-light animations. Furthermore, both impairments were abolished by postnatal bumetanide treatment, suggesting a common pathology underlying the social attachment malformation. Neurotransmission via nAChR is thus critical for the early social bond formation, which is hindered by ambient neonicotinoids through impaired visual predispositions for animate objects.

Spontaneous Visual Preference for Face-Like Stimuli Is Impaired in Newly-Hatched Domestic Chicks Exposed to Valproic Acid During Embryogenesis.

Faces convey a great amount of socially relevant information related to emotional and mental states, identity and intention. Processing of face information is a key mechanism for social and cognitive development, such that newborn babies are already tuned to recognize and orient to faces and simple schematic face-like patterns since the first hours of life. Similar to neonates, also non-human primates and domestic chicks have been shown to express orienting responses to faces and schematic face-like patterns. More importantly, existing studies have hypothesized that early disturbances of these mechanisms represent one of the earliest biomarker of social deficits in autism spectrum disorders (ASD). We used VPA exposure to induce neurodevelopmental changes associated with ASD in domestic chicks and tested whether VPA could impact the expression of the animals' approach responses to schematic face-like stimuli. We found that VPA impairs the chicks' preference responses to these social stimuli. Based on the results shown here and on previous studies, we propose the domestic chick as animal model to investigate the biological mechanisms underlying face processing deficits in ASD.

Micromolar Valproic Acid Doses Preserve Survival and Induce Molecular Alterations in Neurodevelopmental Genes in Two Strains of Zebrafish Larvae.

Autism spectrum disorders (ASDs) comprise a genetically heterogeneous group of conditions characterized by a multifaceted range of impairments and multifactorial etiology. Epidemiological studies have identified valproic acid (VPA), an anticonvulsant used to treat epilepsy, as an environmental factor for ASDs. Based on these observations, studies using embryonic exposure to VPA have been conducted in many vertebrate species to model ASD. The zebrafish is emerging as a popular model in biomedical research to study the molecular pathways involved in nervous system disorders. VPA exposure in zebrafish larvae has been shown to produce a plethora of effects on social, motor and anxiety behavior, and several genetic pathways altered by VPA have been described. However, the doses and regimen of administration reported in the literature are very heterogenous, creating contradictory results and posing serious limits to the interpretation of VPA action on neurodevelopment. To shed light on the toxic effect of VPA, we tested micromolar concentrations of VPA, using exposure for 24 and 48 h in two different zebrafish strains. Our results show that micromolar doses of VPA mildly affect embryo survival but are sufficient to induce molecular alterations in neurodevelopmental genes previously shown to be influenced by VPA, with substantial differences between strains.

Altered Expression of GABAergic Markers in the Forebrain of Young and Adult Engrailed-2 Knockout Mice.

Impaired function of GABAergic interneurons, and the subsequent alteration of excitation/inhibition balance, is thought to contribute to autism spectrum disorders (ASD). Altered numbers of GABAergic interneurons and reduced expression of GABA receptors has been detected in the brain of ASD subjects and mouse models of ASD. We previously showed a reduced expression of GABAergic interneuron markers parvalbumin (PV) and somatostatin (SST) in the forebrain of adult mice lacking the Engrailed2 gene (En2-/- mice). Here, we extended this analysis to postnatal day (P) 30 by using in situ hybridization, immunohistochemistry, and quantitative RT-PCR to study the expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of En2-/- and wild type (WT) mice. In addition, GABA receptor subunit mRNA expression was investigated by quantitative RT-PCR in the same brain regions of P30 and adult En2-/- and WT mice. As observed in adult animals, PV and SST expression was decreased in En2-/- forebrain of P30 mice. The expression of GABA receptor subunits (including the ASD-relevant Gabrb3) was also altered in young and adult En2-/- forebrain. Our results suggest that GABAergic neurotransmission deficits are already evident at P30, confirming that neurodevelopmental defects of GABAergic interneurons occur in the En2 mouse model of ASD.

Embryonic Exposure to Valproic Acid Affects Social Predispositions for Dynamic Cues of Animate Motion in Newly-Hatched Chicks.

Early predispositions to preferentially orient toward cues associated with social partners have been documented in several vertebrate species including human neonates and domestic chicks. Human newborns at high familiar risk of Autism Spectrum Disorder (ASD) show differences in their attention toward these predisposed stimuli, suggesting potential impairments in the social-orienting mechanisms in ASD. Using embryonic exposure to valproic acid (VPA) we modeled ASD behavioral deficits in domestic chicks. To investigate social predispositions toward animate motion in domestic chicks, we focused on self-propulsion, using two video-animations representing a simple red circle moving at constant speed (speed-constant) or one that was changing its speed (accelerating and decelerating; speed-change). Using a spontaneous choice test for the two stimuli, we compared spontaneous preferences for stimuli that autonomously change speed between VPA- and vehicle-injected chicks. We found that the preference for speed changes was abolished in VPA-injected chicks compared to vehicle-injected controls. These results add to previous findings indicating similar impairments for static social stimuli and suggest a specific effect of VPA on the development of mechanisms that enhance orienting toward animate stimuli. These findings strengthen the hypothesis of an early impairment of predispositions in the early development of ASD. Hence, early predispositions are a potentially useful tool to detect early ASD symptoms in human neonates and to investigate the molecular and neurobiological mechanisms underlying the onset of this neurodevelopmental disorder.

Characterization of the Engrailed mutant mice as experimental models for Parkinson's disease.

Engrailed genes are homeodomain-containing transcription factors necessary for the development and maintenance of mesencephalic dopaminergic neurons. Deletion in the Engrailed genes has been shown to affect the survival of mesencephalic dopaminergic neurons both during development and in the adult. Here we describe for the first time a significant reduction in striatal dopamine levels in En1+/-;En2+/- mice compared with their En2+/- littermates, accompanied by a modest reduction in the number of nigral DA neurons. Our results strengthen previous evidence indicating Engrailed genes as survival factors for mature dopaminergic neurons. Furthermore, our data suggest a role for these transcription factors in the maintenance of synaptic dopaminergic neurotransmission in adult neurons.

Embryonic Exposure to Valproic Acid Impairs Social Predispositions of Newly-Hatched Chicks.

Biological predispositions to attend to visual cues, such as those associated with face-like stimuli or with biological motion, guide social behavior from the first moments of life and have been documented in human neonates, infant monkeys and domestic chicks. Impairments of social predispositions have been recently reported in neonates at high familial risk of Autism Spectrum Disorder (ASD). Using embryonic exposure to valproic acid (VPA), an anticonvulsant associated to increased risk of developing ASD, we modeled ASD behavioral deficits in domestic chicks. We then assessed their spontaneous social predispositions by comparing approach responses to a stimulus containing a face configuration, a stuffed hen, vs. a scrambled version of it. We found that this social predisposition was abolished in VPA-treated chicks, whereas experience-dependent mechanisms associated with filial imprinting were not affected. Our results suggest a specific effect of VPA on the development of biologically-predisposed social orienting mechanisms, opening new perspectives to investigate the neurobiological mechanisms involved in early ASD symptoms.

Physiological and behavioral responses in Drosophila melanogaster to odorants present at different plant maturation stages.

The fruit fly Drosophila melanogaster feeds and oviposits on fermented fruit, hence its physiological and behavioral responses are expected to be tuned to odorants abundant during later stages of fruit maturation. We used a population of about two-hundred isogenic lines of D. melanogaster to assay physiological responses (electroantennograms (EAG)) and behavioral correlates (preferences and choice ratio) to odorants found at different stages of fruit maturation. We quantified electrophysiological and behavioral responses of D. melanogaster for the leaf compound ß-cyclocitral, as well as responses to odorants mainly associated with later fruit maturation stages. Electrophysiological and behavioral responses were modulated by the odorant dose. For the leaf compound we observed a steep dose-response curve in both EAG and behavioral data and shallower curves for odorants associated with later stages of maturation. Our data show the connection between sensory and behavioral responses and are consistent with the specialization of D. melanogaster on fermented fruit and avoidance of high doses of compounds associated with earlier stages of maturation. Odor preferences were modulated in a non-additive way when flies were presented with two alternative odorants, and combinations of odorants elicited higher responses than single compounds.

Hippocampal dysregulation of FMRP/mGluR5 signaling in engrailed-2 knockout mice: a model of autism spectrum disorders.

Many evidences indicate that mice lacking the homeobox transcription factor engrailed-2 (En2(-/-) mice) represent a reliable model to investigate neurodevelopmental basis and gene expression changes relevant to autism spectrum disorders. Dysfunctions in fragile X mental retardation protein (FMRP), metabotropic glutamate receptor 5 (mGluR5), and GABAergic signaling pathways have been proposed as a possible pathogenic mechanism of autism spectrum disorders. Here, we exploited En2(-/-) mice to investigate hippocampal expression of FMRP, mGluR5, and GABA(A) receptor ß3 subunit (GABRB3). Quantitative reverse-transcription PCR showed that all these mRNAs were significantly downregulated in En2(-/-) mice compared with wild-type littermates. Western blot and immunohistochemistry confirmed the downregulation of FMRP and GABRB3 proteins, while showing a significant increase of mGluR5 protein in the En2(-/-) hippocampus. Our results suggest that the dysregulation of FMRP-mGluR5 signaling pathway, accompanied with a downregulation of GABRB3 expression, may contribute to the 'autistic-like' features observed in En2 mice, providing possible molecular targets for future pharmacological studies.

Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors.

Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system and are associated with one of the most prominent human neurological disorders, Parkinson's disease. During development, they are induced in the ventral midbrain by an interaction between two diffusible factors, SHH and FGF8. The local identity of this part of the midbrain is probably determined by the combinatorial expression of three transcription factors, Otx2, Pax2, and Pax5. After the last cell division, the neurons start to express transcription factors that control further differentiation and the manifestation of cellular properties characteristic for adult dopaminergic neurons of the substantia nigra compacta and the ventral tegmentum. The first to appear is the LIM-homeodomain transcription factor, Lmx1b. It is essential for the survival of these neurons, and it regulates the expression of another transcription factor, Pitx3, an activator of tyrosine hydroxylase. Lmx1b is followed by the orphan steroid receptor Nurr1. It is essential for the expression of the dopaminergic phenotype. Several genes involved in dopamine synthesis, transport, release, and reuptake are regulated by Nurr1. This requirement is specific to the midbrain dopaminergic neurons, since other populations of the same neurotransmitter phenotype develop normally in absence of the gene. A day after Nurr1, two homeodomain transcription factors, engrailed-1 and -2, are expressed. In animals deficient in the two genes, the midbrain dopaminergic neurons are generated, but then fail to differentiate and disappear very rapidly. Interestingly, alpha-synuclein, a gene recently linked to familial forms of Parkinson's disease, is regulated by engrailed-1 and -2.

GH Dysfunction in Engrailed-2 Knockout Mice, a Model for Autism Spectrum Disorders.

Insulin-like growth factor 1 (IGF-1) signaling promotes brain development and plasticity. Altered IGF-1 expression has been associated to autism spectrum disorders (ASD). IGF-1 levels were found increased in the blood and decreased in the cerebrospinal fluid of ASD children. Accordingly, IGF-1 treatment can rescue behavioral deficits in mouse models of ASD, and IGF-1 trials have been proposed for ASD children. IGF-1 is mainly synthesized in the liver, and its synthesis is dependent on growth hormone (GH) produced in the pituitary gland. GH also modulates cognitive functions, and altered levels of GH have been detected in ASD patients. Here, we analyzed the expression of GH, IGF-1, their receptors, and regulatory hormones in the neuroendocrine system of adult male mice lacking the homeobox transcription factor Engrailed-2 (En2 (-/-) mice). En2 (-/-) mice display ASD-like behaviors (social interactions, defective spatial learning, increased seizure susceptibility) accompanied by relevant neuropathological changes (loss of cerebellar and forebrain inhibitory neurons). Recent studies showed that En2 modulates IGF-1 activity during postnatal cerebellar development. We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in En2 (-/-) mice, as compared to wild-type controls. In mutant mice, GH mRNA levels were significantly increased in the pituitary gland, blood, and liver, whereas decreased levels were detected in the hippocampus. These changes were paralleled by decreased levels of GH protein in the hippocampus but not other tissues of En2 (-/-) mice. IGF-1 mRNA was significantly up-regulated in the liver and down-regulated in the En2 (-/-) hippocampus, but no differences were detected in the levels of IGF-1 protein between the two genotypes. Our data strengthen the notion that altered GH levels in the hippocampus may be involved in learning disabilities associated to ASD.

Altered GABAergic markers, increased binocularity and reduced plasticity in the visual cortex of Engrailed-2 knockout mice.

The maturation of the GABAergic system is a crucial determinant of cortical development during early postnatal life, when sensory circuits undergo a process of activity-dependent refinement. An altered excitatory/inhibitory balance has been proposed as a possible pathogenic mechanism of autism spectrum disorders (ASD). The homeobox-containing transcription factor Engrailed-2 (En2) has been associated to ASD, and En2 knockout (En2 (-/-)) mice show ASD-like features accompanied by a partial loss of cortical GABAergic interneurons. Here we studied GABAergic markers and cortical function in En2 (-/-) mice, by exploiting the well-known anatomical and functional features of the mouse visual system. En2 is expressed in the visual cortex at postnatal day 30 and during adulthood. When compared to age-matched En2 (+/+) controls, En2 (-/-) mice showed an increased number of parvalbumin (PV(+)), somatostatin (SOM(+)), and neuropeptide Y (NPY(+)) positive interneurons in the visual cortex at P30, and a decreased number of SOM(+) and NPY(+) interneurons in the adult. At both ages, the differences in distinct interneuron populations observed between En2 (+/+) and En2 (-/-) mice were layer-specific. Adult En2 (-/-) mice displayed a normal eye-specific segregation in the retino-geniculate pathway, and in vivo electrophysiological recordings showed a normal development of basic functional properties (acuity, response latency, receptive field size) of the En2 (-/-) primary visual cortex. However, a significant increase of binocularity was found in P30 and adult En2 (-/-) mice, as compared to age-matched controls. Differently from what observed in En2 (+/+) mice, the En2 (-/-) primary visual cortex did not respond to a brief monocular deprivation performed between P26 and P29, during the so-called "critical period." These data suggest that altered GABAergic circuits impact baseline binocularity and plasticity in En2 (-/-) mice, while leaving other visual functional properties unaffected.

Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders.

BACKGROUND: Transcriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. METHODS: Cerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes. RESULTS: Given the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays. CONCLUSIONS: Despite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.

Loss of GABAergic neurons in the hippocampus and cerebral cortex of Engrailed-2 null mutant mice: implications for autism spectrum disorders.

The homeobox-containing transcription factor Engrailed-2 (En2) is involved in patterning and neuronal differentiation of the midbrain/hindbrain region, where it is prominently expressed. En2 mRNA is also expressed in the adult mouse hippocampus and cerebral cortex, indicating that it might also function in these brain areas. Genome-wide association studies revealed that En2 is a candidate gene for autism spectrum disorders (ASD), and mice devoid of its expression (En2(-/-) mice) display anatomical, behavioral and clinical "autistic-like" features. Since reduced GABAergic inhibition has been proposed as a possible pathogenic mechanism of ASD, we hypothesized that the phenotype of En2(-/-) mice might include defective GABAergic innervation in the forebrain. Here we show that the Engrailed proteins are present in postnatal GABAergic neurons of the mouse hippocampus and cerebral cortex, and adult En2(-/-) mice show reduced expression of GABAergic marker mRNAs in these areas. In addition, reduction in parvalbumin (PV), somatostatin (SOM) and neuropeptide Y (NPY) expressing interneurons is detected in the hippocampus and cerebral cortex of adult En2(-/-) mice. Our results raise the possibility of a link between altered function of En2, anatomical deficits of GABAergic forebrain neurons and the pathogenesis of ASD.

Mutant mouse models of autism spectrum disorders.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental diseases characterized by a triad of specific behavioral traits: abnormal social interactions, communication deficits and stereotyped or repetitive behaviors. Several recent studies showed that ASDs have a strong genetic basis, contributing to the discovery of a number of ASD-associated genes. Due to the genetic complexity of these disorders, mouse strains with targeted deletion of ASD genes have become an essential tool to investigate the molecular and neurodevelopmental mechanisms underlying ASD. Here we will review the most relevant genetic mouse models developed by targeted inactivation of ASD-associated genes, and discuss their importance for the development of novel pharmacological therapies of these disorders.