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. 2008 Feb 26;6(2):e34. doi: 10.1371/journal.pbio.0060034

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© 2008 Dieterich et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Primary hippocampal neurons were transfected at DIV11, fixed the next day, and stained with MAP2-specific antibodies (Cy5), pCREB (Ser133; Alexa568), and mounted with DAPI-containing (blue) medium. Gradient lookup tables applied to determine the dynamic range of pCREB are included to visualize pixel intensity differences as indicated with the scale from 0 to 255. Scale bar indicates 40 μm.

(A and B) Overexpression of ΔMyr-Jacob-GFP decreases the basal level of CREB phosphorylation in nonstimulated primary hippocampal cultures (DIV12). No effect as compared to nontransfected control neurons was seen after the transfection of a Jacob-GFP construct encoding the C-terminal half of Jacob. Quantification is shown in (B). A single asterisk (*) indicates p < 0.01.

(C and D) Overexpression of ΔMyr-Jacob-EGFP using a Semliki Forest virus vector significantly reduced the level of pCREB at resting conditions in comparison to EGFP-infected and noninfected cortical primary neurons. The diagram (D) represents the data from four to five independent experiments normalized to β-Tubulin III. Total CREB levels were not affected by infection of the cultures.

(E and F) Knockdown of nuclear Jacob utilizing the construct RNAi-NLS-GFP increases basal pCREB levels and prevents CREB shut-off after stimulation of extrasynaptic NMDA receptors. Prior to bath application of NMDA, cultures were stimulated with bicuculline in the presence of MK801. Arrows indicate RNAi-NLS-GFP–transfected neurons. Cultures were fixed 30 min after the stimulation with either bicuculline or the subsequent bath application with NMDA. Triple asterisks (***) indicate p < 0.001.