NMDA dose-dependent increase in Jacob nuclear immunoreactivity of primary hippocampal neurons.

<p>Hippocampal primary neurons were maintained in 1 ml of neurobasal medium (NB). 12–16 hours prior stimulation the volume of NB medium was increased to 1.6 ml. Then 800 µl of conditioned NB medium was removed and NMDA applied. Five minutes later the NMDA-NB medium was replaced with the 800 µl conditioned NB medium and 25 minutes later the neurons were fixed. A) The diagram summarizes normalized nuclear Jacob immunofluorescence at DIV 18 after bath application of 20 µM or 50 µM NMDA. B) The bar diagram indicates the gain in the nuclear fluorescence signal after treatment with 20 µM or 50 µM NMDA in comparison to drug free conditions. Note the increased nuclear accumulation of Jacob at DIV 18. C) Representative images of Jacob nuclear immunoreactivity and DAPI staining in hippocampal primary neurons at DIV 23 under control, 20 µM and 50 µM NMDA conditions, respectively. ***p<0.001, **p<0.01. Scale bar is 10 µm.</p

Low frequency induction of LTD does not induce Jacob-GFP translocation into the nucleus.

<p>The time course of normalized fluorescence intensities is presented for the nucleus (red filled circle) and the proximal dendrite (dark blue diamond). The recordings were acquired in the presence of anisomycin (Anis.: 50 µM). The gray box indicates the time of LTD induction (900 bursts at 1 Hz, one burst consists of 3 stimuli at an interstimulus interval of 50 ms). Representative fEPSP-transients for indicated time points are shown. Vertical and horizontal scale bars indicate 0.5 mV and 2 ms, respectively. The other inlet depicts representative F/F_o*100% confocal images of a neuron expressing Jacob-eGFP before and after LTD induction. The vertical colored bar indicates green as 0, and dark blue as −20% and red as +20%. The image analysis was done as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017276#pone-0017276-g002" target="_blank">Figure 2</a>.</p

Time-lapse imaging of Jacob-GFP nuclear translocation in acute hippocampal slices in response to LTP induction.

<p>A) Schematic representation of data acquisition and image analysis. B) A representative 12 bit grayscale confocal image of a neuron expressing Jacob-eGFP (grayscale range: 0: black to 4095: white). Other images represent F/F₀ images that were acquired at the indicated time points. ROIs are indicated for nucleus, proximal dendrite and background area with white circles and squares (horizontal white scale bar: 10 µm). The colored vertical bar encodes green as 0, and dark blue as -20% and red as +20%. C) The time course of normalized fluorescence intensities is depicted for nuclei and proximal dendrites acquired under application with anisomycin (Anis., 50 µM) and Anis. + AP5 (40 µM). Brackets and asterisks (**, p≤0.05; Mann-Whitney U-Test) indicate the significance intervals between various groups (symbol pairs on bracket). The inlet shows fEPSP-transients before, and 5 minutes after the first tetanization for Anis. and Anis.+AP5 experiments. Scale bars indicate 0.5 mV for vertical and 2 ms for horizontal bar. Vertical arrows show the time point of 100 Hz/1-s tetanization.</p

Nuclear Jacob immunoreactivity at different culture ages following synaptic or extrasynaptic NMDA receptor activation.

<p>A1–A3 and C1–C3) Representative fluorescence images for anti-Jacob and DAPI stained hippocampal primary neurons at 9, 16 and 23 DIV. Cultures were stimulated with protocols activating synaptic (bicuculline, 4-AP) and extrasynaptic NMDA receptors (bicuculline, 4-AP, MK801; followed by 100 µM NMDA). Note the age dependent increase in Jacob nuclear immunoreactivity at basal conditions. Synaptic stimulation induced a Jacob translocation in cultures at DIV 9 (A1 and B1) but not after extrasynaptic stimulation (C1 and D1). B1–B3) The efficiency of synaptic stimulation on nuclear Jacob immunosignals decreased with culture age. D1–D3) At DIV 23 extrasynaptic NMDA receptor stimulation increased nuclear Jacob signal. E) TTX treatment for 2 hrs significantly reduced nuclear Jacob. **p<0.01 ***p<0.001. Scale bars are 10 µm.</p

Jacob translocates to the nucleus after induction of chemical LTP but not chemical LTD in adult primary hippocampal neurons.

<p>10 minutes prior to stimulation the Neurobasal medium was replaced by magnesium free buffer solution containing TTX, strychnine and bicuculline. 200 µM glycine or co-application of 20 µM glycine and 20 µM NMDA were applied to modulate synaptic transmission; known to induce chemical LTP or chemical LTD, respectively. Five minutes after stimulation the conditioning medium was replaced and cells further incubated for additional 25 minutes and then fixed. A,C,E) Representative anti-Jacob and DAPI fluorescence images of 16, 18 and 23 DIV cultures for control, chemical LTP and chemical LTD, respectively, are shown. B, D, F) The diagrams summarize the relative deviation of nuclear Jacob fluorescence under different experimental conditions. Independent from the age of culture, chemical LTP induction induced a significant accumulation of Jacob, but induction of chemical LTD did not change Jacob immunosignal under all tested conditions. Statistical significance was determined by ANOVA with Bonferroni post-hoc t-test. *p<0.05. Scale bar is 5 µm.</p

The Jacob distribution remains unaltered after LTD induction.

<p>A) Averaged normalized fEPSP-slope values (black filled diamonds) are shown. fEPSP-slope values during LTD induction (horizontal black bar) are depicted as gray dots. LTD was induced by 900 bursts and one burst consists of 3 stimuli at an inter-stimulus interval of 50 ms. The color of the fEPSP-transients in the left inlet corresponds to time points indicted by the respective black, light gray and dark gray filled circles. The second inlet shows fEPSPs of an initial burst during LTD induction. Slices for the immunohistological analysis of Jacob translocation were taken 40 minutes after the last baseline recording. B) Representative digital fluorescence images of Jacob immunofluorescence for the conditions “no LTD” and “LTD” are presented. The white boxes indicate ROIs taken for the measurement of averaged intensities within the s.p. and s.r. field. The white horizontal bar represents 10 µm. C) The bar diagram summarizes the immunofluorescence analyses of hippocampal slices without (black) and with LTD (dark gray) induction. The ratio of s.p. to s.r. was calculated after subtracting background values (b; minimum value in dendritic area) from the averaged fluorescence intensity of s.r. (Fd: dendritic fluorescence) and s.p. (Fs: somatic fluorescence). After LTD induction the ratio was not different from the ratio of “no LTD” slices. The inlet indicates the time for the various activities.</p

Jacob translocation after induction of early (E)- and late (L)-LTP.

<p>Sets of three 400 µm thick hippocampal slices from 7-weeks-old rats were pre-incubated for 4 hours in an interface chamber. After recording of baseline PSs every hippocampal slice experienced one of the following tetanization paradigms: early-LTP: single tetanization; late-LTP: triple tetanization or no tetanization (control). A) The diagram summarizes the increase of PS-amplitude after early-LTP induction paradigm. B) This diagram indicates the PS-potentiation in response to the late-LTP induction protocol. Thirty minutes after the first tetanization the slices were fixed and processed for Jacob and MAP2 immunolabeling as described in methods. C) A merged image of Jacob (green) and MAP2 (red) is presented at low magnification. The white box indicates an analyzed CA1 area. To the right, higher magnification confocal images for MAP2, Hoechst (blue) and Jacob as well as a merged image are shown. D) Representative overlay images of Jacob, MAP2 and Hoechst for non-, single and triple tetanized slices (control, early-LTP and late-LTP, respectively) are depicted. The averaged fluorescence intensity of nuclear Jacob staining was corrected against proximal part of basal dendrites (small square) as shown in the corresponding anti-Jacob confocal images. E) More than 20 nuclei from each slice were analyzed and the intensities averaged resulting in 12 single values for each group. In the bar diagram the fluorescence intensities are presented as percentage deviation from the average of the control group values. The number of analyzed nuclei is indicated in the graph. Mann Whitney test: **p<0.01: between control and L-LTP.</p