miR-132/212 knockout cortical cultures exhibit normal morphology.

<p>Cultures of cortical neurons were established on coverslips from P0 pups from either wild-type or miR-132/212 knockouts. After 24, 48, 72 or 96 h in culture, neurons were fixed with PFA and stained with MAP2 primary mouse monoclonal then fluorescein-labelled secondary anti-mouse antibody. Morphology was examined using confocal fluorescence microscopy and the images were analysed using the FilamentTracer Module of the Imaris software (Bitplane, Switzerland). Dendrite length (A) and branching (B) were quantified and representative images are shown with MAP2 staining in green and DAPI in blue (C). A p value (students t-test) between wild-type and knockout of <0.05 is indicated by * or <0.01 by **. Alternatively cultures were stimulated with 20 µM NMDA for the indicated times and total RNA isolated. The levels of pri-miR-132/212, miR-132, miR-212 (D), p250-Gap and MeCP2 (E) were determined. Error bars represent the standard deviation of 4 cultures per genotype. Similar experiments were also performed but the level of MeCP2 was determined by immunoblotting (E).