Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.

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Autism spectrum disorder (ASD) is a complex group of clinically heterogeneous neurodevelopmental disorders with unclear etiology and pathogenesis. Genetic studies have identified numerous candidate genetic variants, including de novo mutated ASD-associated genes; however, the function of these de novo mutated genes remains unclear despite extensive bioinformatics resources. Accordingly, it is not easy to assign priorities to numerous candidate ASD-associated genes for further biological analysis. Here we developed a convenient system for identifying an experimental evidence-based annotation of candidate ASD-associated genes. We performed trio-based whole-exome sequencing in 30 sporadic cases of ASD and identified 37 genes with de novo single-nucleotide variations (SNVs). Among them, 5 of those 37 genes, POGZ, PLEKHA4, PCNX, PRKD2 and HERC1, have been previously reported as genes with de novo SNVs in ASD; and consultation with in silico databases showed that only HERC1 might be involved in neural function. To examine whether the identified gene products are involved in neural functions, we performed small hairpin RNA-based assays using neuroblastoma cell lines to assess neurite development. Knockdown of 8 out of the 14 examined genes significantly decreased neurite development (P<0.05, one-way analysis of variance), which was significantly higher than the number expected from gene ontology databases (P=0.010, Fisher's exact test). Our screening system may be valuable for identifying the neural functions of candidate ASD-associated genes for further analysis and a substantial portion of these genes with de novo SNVs might have roles in neuronal systems, although further detailed analysis might eliminate false positive genes from identified candidate ASD genes.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.

Schizophrenia is a chronic psychiatric disorder with complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair of monozygotic twin cases with treatment-resistant schizophrenia, in which one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activities including modulation of synaptic plasticity and memory, hippocampal neurogenesis, and neuroprotection, most of which are shared with brain-derived neurotrophic factor (BDNF). Therefore, the aim of this study was to compare morphological effects of PACAP and BDNF on primary cultured hippocampal neurons. At days in vitro (DIV) 3, PACAP increased neurite length and number to similar levels by BDNF, but vasoactive intestinal polypeptide showed much lower effects. In addition, PACAP increased axon, but not dendrite, length, and soma size at DIV 3 similarly to BDNF. The PACAP antagonist PACAP6–38 completely blocked the PACAP-induced increase in axon, but not dendrite, length. Interestingly, the BDNF-induced increase in axon length was also inhibited by PACAP6–38, suggesting a mechanism involving PACAP signaling. K252a, a TrkB receptor inhibitor, inhibited axon outgrowth induced by PACAP and BDNF without affecting dendrite length. These results indicate that in primary cultured hippocampal neurons, PACAP shows morphological actions via its cognate receptor PAC1, stimulating neurite length and number, and soma size to a comparable extent as BDNF, and that the increase in total neurite length is ascribed to axon outgrowth.

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancerdriven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders. Keywords: embryonic stem cells, in vitro differentiation, induced pluripotent stem cells, noggin, psychiatric disorders, serotonergic neurons. Address correspondence and reprint requests to Hitoshi Hashimoto, PhD, Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: hasimoto@phs.osaka-u.ac.jp 1 Present address: School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe, Hyogo 650-8530, Japan.Abbreviations used: 5-HT, 5-hydroxytryptamine; BMP, bone morphogenetic protein; EGFP, enhanced green fluorescent protein; ePet, the enhancer of the mouse Pet-1 (Fev) gene; ES, embryonic stem; EYFP, enhanced yellow fluorescent protein; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FGF, fibroblast growth factor; Gad1, glutamate decarboxylase 1; iPS, induced pluripotent stem; MAP2, microtubule-associated protein 2; MEM, minimum essential medium; SDIA, stromal cell-derived inducing activity; Sert, serotonin transporter; Shh, sonic hedgehog; TH, tyrosine hydroxylase; Tph2, tryptophan hydroxylase 2; TuJ1, mouse monoclonal anti-class III-tublin antibody. [approximately 20 000 tryptophan hydroxylase 2 (Tph2; EC 1.14.16.4)-positive cells in the rat]. These cells are exclusively located in the raphe nuclei with a vast axonal network innervating most other areas in the brain and spinal cord (Alenina et al. 2006). In the brain, the serotonergic projections innervate multiple cortical brain regions to regulate a wide repertoire of behaviors including cognition and mood (Martinowich and Lu 2008). In addition to its canonical role, 5-HT plays an important role in brain development with respect to neurogenesis, neurite outgrowth, synaptogenesis, and cell survival (Gaspar et al. 2003). It has been shown that disturbances in 5-HT signaling and homeost...