Knockdown of mental disorder susceptibility genes disrupts neuronal network physiology in vitro.
Bottom Line: Measurement of multiple neural network parameters demonstrated phenotypes for these genes compared with controls.Moreover, the different genes disrupted network properties and showed distinct and overlapping effects.These data show multiple susceptibility genes for complex psychiatric disorders, regulate neural network physiology and demonstrate a new assay system with wide application.
Affiliation: Genes to Cognition Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB101SA, UK.Show MeSH
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Mentions: Hippocampal neurons from E17.5 mouse embryos were cultured on MEAs (Fig. 1a) and transfected after 4 days in vitro (DIV 4). The four target genes were tested using three different conditions: transfection of siRNAs targeting the gene of interest and two negative controls, untransfected controls and transfection of non-targeting siRNA (NTC). The network activity of the neurons was recorded for 15 min daily beginning on DIV 5 and continuing until DIV 12 (Fig. 1b). Knockdown for all genes was assayed at DIV 7 (72 h post-transfection) and ranged from 60 to 84% compared with control cultures (Fig. 1d), which is in the range relevant to the levels of expression occurring in the human hemizygous mutations (Kristiansen et al., 2006; Millar et al., 2005). Because the mRNA and protein were harvested from the entire culture, and are derived both from neurons transfected with the siRNAs and untransfected cells, the level of expression knockdown can be taken as a lower limit of the transfection efficiency. That is, if the siRNA pool targeting Dctn5 reduces mRNA expression by 85% overall in the culture, 85% of the cells in that culture have been transfected at a minimum. Thus, the siRNAs used to target the genes in this study are transfected into the majority of the cells in the cultures.
Affiliation: Genes to Cognition Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB101SA, UK.