The autism spectrum disorder (ASD) is among the most devastating disorders of childhood in terms of prevalence, morbidity, outcome, impact on the family, and cost to society. According to recent epidemiological data, ϳ1 child in 166 is affected with ASD, a considerable increase compared with estimates compiled 15-20 years ago (Fombonne, 2003a,b). Although at one time considered an emotional disturbance resulting from early attachment experiences (Bettelheim, 1967), ASD is now recognized as a disorder of prenatal and postnatal brain development. Although ASD is primarily a genetic disorder involving multiple genes, insights into underlying mechanisms will require a multidisciplinary approach. Assessment of the earliest clinical signs and symptoms and the functional and structural networks by neuroimaging and neuropathology can be used to identify the underlying brain regions, neural networks, and cellular systems. In turn, the efforts of human and animal geneticists and neuroscientists are needed to define molecular and protein signaling pathways that mediate normal as well as abnormal development of language, social interaction, and cognitive and motor routines. In this review, we focus on several issues: the earliest manifestations of ASD, reported abnormalities of brain growth, functional neural networks, and neuropathology. We also consider the possible etiological factors and the challenges of creating animal models for this uniquely human behavioral disorder. Autism spectrum disorder: phenotypes and clinical diagnosisASD comprises several different disorders as defined by deficits in social behaviors and interactions. These deficits prevent the development of normal interpersonal relationships of affected patients with their parents, siblings, and other children. Deficits in nonverbal communication include reduced eye contact, facial expression, and body gestures (American Psychiatric Association, 1994). These disorders include prototypic autistic disorder, Asperger syndrome, and pervasive developmental disorder-not otherwise specified (PDD-NOS). Autistic disorder has three core symptom domains: deficits in communication, abnormal social interactions, and restrictive and/or repetitive interests and behaviors. Autistic disorder is typically noticed in the first or second year of life. The manifestations include delay or abnormality in language and play, repetitive behaviors, such as spinning things or lining up small objects, or unusual interests such as preoccupations with stop signs or ceiling fans. Asperger syndrome also involves social symptoms but language development and non-

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for more than 1–2% of cases. The clinical presentation, behavioural symptoms, imaging, and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using MRI based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were the parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus, and the striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2, and Fmr1; Nlgn3, BTBR, and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

Mounting evidence indicates that extracellular factors exert proliferative effects on neurogenetic precursors in vivo. Recently we found that systemic levels of basic fibroblast growth factor (bFGF) regulate neurogenesis in the brain of newborn rats, with factors apparently crossing the blood-brain barrier (BBB) to stimulate mitosis. To determine whether peripheral bFGF affects proliferation during adulthood, we focused on regions in which neurogenesis persists into maturity, the hippocampus and the forebrain subventricular zone (SVZ). In postnatal day 1 (P1) rats, 8 hr after subcutaneous injection (5 ng/gm body weight), bFGF increased [(3)H]thymidine incorporation 70% in hippocampal and SVZ homogenates and elicited twofold increases in mitotic nuclei in the dentate gyrus and the dorsolateral SVZ, detected by bromodeoxyuridine immunohistochemistry. Because approximately 25% of proliferating hippocampal cells stimulated in vivo expressed neuronal traits in culture, bFGF-induced mitosis may reflect increased neurogenesis. bFGF effects were not restricted to the perinatal period; hippocampal DNA synthesis was stimulated by peripheral factor in older animals (P7-P21), indicating the persistence of bFGF-responsive cells and activity of peripheral bFGF into late development. To begin defining underlying mechanisms, pharmacokinetic studies were performed in P28 rats; bFGF transferred from plasma to CSF rapidly, levels rising in both compartments in parallel, indicating that peripheral factor crosses the BBB during maturity. Consequently, we tested bFGF in adults; peripheral bFGF increased the number of mitotic nuclei threefold in the SVZ and olfactory tract, regions exhibiting persistent neurogenesis. Our observations suggest that bFGF regulates ongoing neurogenesis via a unique, endocrine-like pathway, potentially coordinating neuron number and body growth, and potentially providing new approaches for treating damaged brain during development and adulthood.

Summary Peripheral nerve injury causes neuropathic pain accompanied by remarkable microgliosis in the spinal cord dorsal horn. However, it is still debated whether infiltrated monocytes contribute to injury-induced expansion of the microglial population. Here we found that spinal microgliosis predominantly results from local proliferation of resident microglia but not from infiltrating monocytes after spinal nerve transection (SNT), using two genetic mouse models (CCR2RFP/+:CX3CR1GFP/+ and CX3CR1creER/+:R26tdTomato/+ mice) as well as specific staining of microglia and macrophages. Pharmacological inhibition of SNT-induced microglial proliferation correlated with attenuated neuropathic pain hypersensitivities. Microglial proliferation is partially controlled by purinergic and fractalkine signaling, as CX3CR1−/− and P2Y12−/− mice show reduced spinal microglial proliferation and neuropathic pain. These results suggest that local microglial proliferation is the sole source of spinal microgliosis, which represents a potential therapeutic target for neuropathic pain management.

Our previous research involving 167 nuclear families from the Autism Genetic Resource Exchange (AGRE) demonstrated that two intronic SNPs, rs1861972 and rs1861973, in the homeodomain transcription factor gene ENGRAILED 2 (EN2) are significantly associated with autism spectrum disorder (ASD). In this study, significant replication of association for rs1861972 and rs1861973 is reported for two additional data sets: an independent set of 222 AGRE families (rs1861972-rs1861973 haplotype, P=.0016) and a separate sample of 129 National Institutes of Mental Health families (rs1861972-rs1861973 haplotype, P=.0431). Association analysis of the haplotype in the combined sample of both AGRE data sets (389 families) produced a P value of .0000033, whereas combining all three data sets (518 families) produced a P value of .00000035. Population-attributable risk calculations for the associated haplotype, performed using the entire sample of 518 families, determined that the risk allele contributes to as many as 40% of ASD cases in the general population. Linkage disequilibrium (LD) mapping with the use of polymorphisms distributed throughout the gene has shown that only intronic SNPs are in strong LD with rs1861972 and rs1861973. Resequencing and association analysis of all intronic SNPs have identified alleles associated with ASD, which makes them candidates for future functional analysis. Finally, to begin defining the function of EN2 during development, mouse En2 was ectopically expressed in cortical precursors. Fewer En2-transfected cells than controls displayed a differentiated phenotype. Together, these data provide further genetic evidence that EN2 might act as an ASD susceptibility locus, and they suggest that a risk allele that perturbs the spatial/temporal expression of EN2 could significantly alter normal brain development.

ABSTRACT

ENGRAILED 2 (En2) , a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2 , we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by in situ hybridization that genes for the neuropeptide pituitary adenylate cyclaseactivating peptide (PACAP) and one of its high-affinity receptors (PAC 1 ) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and f loor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the sonic hedgehog-and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.Recent studies suggest that phenotypic determination in the developing nervous system results from interactions of patterning genes conserved through evolution (1). For example, sonic hedgehog (shh) is one of three mammalian homologs to the segment polarity gene hedgehog (hh). shh has been implicated as a notochord-and floor plate-secreted factor that controls dorsal͞ventral patterning in the vertebrate neural tube (1-4). Abundant genetic and molecular evidence in flies (5), fish (6), and mice (7) indicates that hh and its homologs act by antagonizing cAMP-dependent protein kinase A (PKA) signaling. Although shh may act in the ventral tube by blocking constitutive PKA activity, it is possible that physiological activators of the cAMP͞PKA pathway are important in patterning.We considered that pituitary adenylate cyclase-activating peptide (PACAP) might be involved in patterning for several reasons. First, although PACAP originally was discovered as a hypothalamic factor that potently increased cAMP in the pituitary through G protein-coupled receptors (8), peptide expression later was localized to many central and peripheral neuronal populations as well as the developing embryo (9). Second, the 27-aa form of the peptide, PACAP-27, is conserved 100% in species ranging from fish to humans. Finally, our tissue culture studies indicated that PACAP and a closely related peptide vasoactive intestinal peptide stimulate cAMP production, regulating proliferation, differentiation, and͞or survival of multiple neuronal precursors (10-14). The current studies indicate that a functional PACAP ligand͞receptor cAMP signaling system is expressed in the neural tube at the onset of neurogenesis, raising the possibility that PACAP might be involved in neural tube patterning. MATERIALS AND METHODS In Situ Hybridi...