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Knockdown of mental disorder susceptibility genes disrupts neuronal network physiology in vitro.

MacLaren EJ, Charlesworth P, Coba MP, Grant SG - Mol. Cell. Neurosci. (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: Genes to Cognition Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB101SA, UK.

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Related in: MedlinePlus

Experimental strategy. (a) A phase-contrast image of WT neurons grown on an MEA at 4 DIV. Scale bar is 200 μm. The table lists the seven network parameters quantified using MEA recordings. (b) Experimental design of study. Cultures were transfected at 4 DIV and recorded for 15 min daily until 12 DIV. (c) In control WT cultures, the total spikes and network size increase from 4 DIV to 12 DIV as the cultures mature. (d) Level of knockdown achieved as determined by real-time PCR or Western blot. In all cases, the level of target gene expression is significantly reduced compared to both untransfected and NTC cultures. The untransfected and NTC cultures did not demonstrate any differential expression of these genes compared to one another. *p < 0.05. (e) Model showing the synaptic localization of all the genes examined.
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f0005: Experimental strategy. (a) A phase-contrast image of WT neurons grown on an MEA at 4 DIV. Scale bar is 200 μm. The table lists the seven network parameters quantified using MEA recordings. (b) Experimental design of study. Cultures were transfected at 4 DIV and recorded for 15 min daily until 12 DIV. (c) In control WT cultures, the total spikes and network size increase from 4 DIV to 12 DIV as the cultures mature. (d) Level of knockdown achieved as determined by real-time PCR or Western blot. In all cases, the level of target gene expression is significantly reduced compared to both untransfected and NTC cultures. The untransfected and NTC cultures did not demonstrate any differential expression of these genes compared to one another. *p < 0.05. (e) Model showing the synaptic localization of all the genes examined.

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.


Knockdown of mental disorder susceptibility genes disrupts neuronal network physiology in vitro.

MacLaren EJ, Charlesworth P, Coba MP, Grant SG - Mol. Cell. Neurosci. (2011)

Experimental strategy. (a) A phase-contrast image of WT neurons grown on an MEA at 4 DIV. Scale bar is 200 μm. The table lists the seven network parameters quantified using MEA recordings. (b) Experimental design of study. Cultures were transfected at 4 DIV and recorded for 15 min daily until 12 DIV. (c) In control WT cultures, the total spikes and network size increase from 4 DIV to 12 DIV as the cultures mature. (d) Level of knockdown achieved as determined by real-time PCR or Western blot. In all cases, the level of target gene expression is significantly reduced compared to both untransfected and NTC cultures. The untransfected and NTC cultures did not demonstrate any differential expression of these genes compared to one another. *p < 0.05. (e) Model showing the synaptic localization of all the genes examined.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105225&req=5

f0005: Experimental strategy. (a) A phase-contrast image of WT neurons grown on an MEA at 4 DIV. Scale bar is 200 μm. The table lists the seven network parameters quantified using MEA recordings. (b) Experimental design of study. Cultures were transfected at 4 DIV and recorded for 15 min daily until 12 DIV. (c) In control WT cultures, the total spikes and network size increase from 4 DIV to 12 DIV as the cultures mature. (d) Level of knockdown achieved as determined by real-time PCR or Western blot. In all cases, the level of target gene expression is significantly reduced compared to both untransfected and NTC cultures. The untransfected and NTC cultures did not demonstrate any differential expression of these genes compared to one another. *p < 0.05. (e) Model showing the synaptic localization of all the genes examined.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Genes to Cognition Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB101SA, UK.

Show MeSH
Related in: MedlinePlus