Autism spectrum disorder (ASD) is a pervasive neurodevelopmental syndrome with a high human and economic burden. The pathophysiology of ASD is largely unclear, thus hampering development of pharmacological treatments for the core symptoms of the disorder. Abnormalities in glutamate and GABA signaling have been hypothesized to underlie ASD symptoms, and may form a therapeutic target, but it is not known whether these abnormalities are recapitulated in humans with ASD, as well as in rodent models of the disorder. We used translational proton magnetic resonance spectroscopy ([1H]MRS) to compare glutamate and GABA levels in adult humans with ASD and in a panel of six diverse rodent ASD models, encompassing genetic and environmental etiologies. [1H]MRS was performed in the striatum and the medial prefrontal cortex, of the humans, mice, and rats in order to allow for direct cross-species comparisons in specific cortical and subcortical brain regions implicated in ASD. In humans with ASD, glutamate concentration was reduced in the striatum and this was correlated with the severity of social symptoms. GABA levels were not altered in either brain region. The reduction in striatal glutamate was recapitulated in mice prenatally exposed to valproate, and in mice and rats carrying Nlgn3 mutations, but not in rodent ASD models with other etiologies. Our findings suggest that glutamate/GABA abnormalities in the corticostriatal circuitry may be a key pathological mechanism in ASD; and may be linked to alterations in the neuroligin–neurexin signaling complex.

SUMMARYWiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching. Nogo-A KO mice were generated by homologous recombination of exons 2 and 3 in the Nogo-A gene, as described previously (Dimou et al., 2006;Simonen et al., 2003). Antibodies11C7 antibody was raised against an 18-amino acid Nogo-A peptide corresponding to the rat sequence of amino acids 623-640 (Oertle et al., 2003). 7B12 antibody has an epitope in the C-terminal part of the Nogo-A-specific region (aa 763-820) (Oertle et al., 2003). Both antibodies are function-blocking antibodies (Liebscher et al., 2005) and are monospecific for Nogo-A (Dodd et al., 2005;Oertle et al., 2003). Polyclonal rabbit antibody Rb173A (Laura) recognizes the Nogo-A-specific region (aa 174-979) and the antibody Rb1 (Bianca) is specific for the N terminus of Nogo-A and Nogo-B (aa 1-172) (Dodd et al., 2005;Oertle et al., 2003). Nogo-A receptor complex components were blocked by anti-NgR (R&D Systems) or anti-Lingo1 (Abcam) antibodies. Compound Y27632 was used to inhibit ROCK (Sigma). Rabbit anti-neurofilament 160 antibody (Chemicon) was used for whole-mount staining and mouse anti--tubulin III (Abcam) was used for staining of dissociated DRG cultures. DRG cultures DRG explant culturesDRGs of newborn rats, wild-type and Nogo-A KO mice were plated on 20 g/ml poly-L-lysine (PLL)-coated four-well tissue culture plates. Cultures were incubated for 5-7 days at 37°C and 5% CO 2 atmosphere in F12 medium (Invitrogen) supplemented with 10% foetal bovine serum (Sigma), 100 g/ml nerve growth factor (NGF) and 10 g/ml gentamycin (Sigma). Cytosine arabinoside was added to inhibit mitosis of non-neuronal cells. Control, monoclonal mouse IgG antibody directed against wheat auxin, anti-Nogo-A antibodies 11C7 or 7B12, antibodies against NgR-1 or Lingo1 or the ROCK blocker Y27632 were added to the culture medium at a concentration of 10 g/ml at the beginnin...

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