Journal: PloS one

Article Title: Nuclear translocation of jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.

doi: 10.1371/journal.pone.0017276

Figure Lengend Snippet: Figure 6. Jacob translocates to the nucleus after induction of chemical LTP but not chemical LTD in adult primary hippocampal neurons. 10 minutes prior to stimulation the Neurobasal medium was replaced by magnesium free buffer solution containing TTX, strychnine and bicuculline. 200 mM glycine or co-application of 20 mM glycine and 20 mM 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 mm. doi:10.1371/journal.pone.0017276.g006

Article Snippet: To activate synaptic NMDA receptors the hippocampal cultures were treated with 50 mM bicuculline (Tocris) with supplementation of the weak potassium-channel blocker 4-aminopyridine (4-AP, 2.5 mM, Sigma-Aldrich).

Techniques: Transmission Assay, Incubation, Fluorescence, Control

Journal: PloS one

Article Title: Nuclear translocation of jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.

doi: 10.1371/journal.pone.0017276

Figure Lengend Snippet: Figure 8. Nuclear Jacob immunoreactivity at different culture ages following synaptic or extrasynaptic NMDA receptor activation. 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 mM 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 mm. doi:10.1371/journal.pone.0017276.g008

Article Snippet: To activate synaptic NMDA receptors the hippocampal cultures were treated with 50 mM bicuculline (Tocris) with supplementation of the weak potassium-channel blocker 4-aminopyridine (4-AP, 2.5 mM, Sigma-Aldrich).

Techniques: Activation Assay, Fluorescence, Staining, Translocation Assay

Journal: PloS one

Article Title: Nuclear translocation of jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.

doi: 10.1371/journal.pone.0017276

Figure Lengend Snippet: Figure 9. NMDA dose-dependent increase in Jacob nuclear immunoreactivity of primary hippocampal neurons. 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 ml of conditioned NB medium was removed and NMDA applied. Five minutes later the NMDA-NB medium was replaced with the 800 ml 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 mM or 50 mM NMDA. B) The bar diagram indicates the gain in the nuclear fluorescence signal after treatment with 20 mM or 50 mM 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 mM and 50 mM NMDA conditions, respectively. ***p,0.001, **p,0.01. Scale bar is 10 mm. doi:10.1371/journal.pone.0017276.g009

Article Snippet: To activate synaptic NMDA receptors the hippocampal cultures were treated with 50 mM bicuculline (Tocris) with supplementation of the weak potassium-channel blocker 4-aminopyridine (4-AP, 2.5 mM, Sigma-Aldrich).

Techniques: Immunofluorescence, Fluorescence, Comparison, Staining, Control

Journal: The Journal of Neuroscience

Article Title: Activation of Early Silent Synapses by Spontaneous Synchronous Network Activity Limits the Range of Neocortical Connections

doi: 10.1523/JNEUROSCI.3803-04.2005

Figure Lengend Snippet: Activation of silent synapses by synchronous network activity. A, Cultures were raised in BMI from 5 to 9 DIV and transferred to 0 mm Mg2+ Ringer's solution before fixation. A significant change in the percentage of colocalized NR1/GluR2/3 clusters was evident after 15 min, and control levels were reached after 30 min of 0 mm Mg2+ stimulation. Total number of clusters examined: control (contr.), n = 287; BMI, n = 400; 0 Mg2+ at increasing delays, n = 582, 398, 436, 441, 453; the second graph in A shows the percentage of active neurons imaged in sister cultures in the presence of BMI (n = 340) or in a 0 mm Mg2+ Ringer's solution (n = 478). Although the 9-d-old cultures presented no synchronicity between the spontaneously active neurons under BMI, strong synchronized activity occurred under 0 mm Mg2+ stimulation. B, The fraction of NR1/GluR2/3 clusters was determined in 9 DIV cultures cultivated in the presence of the GABAA receptor antagonist BMI (gray columns; n = 667) and compared with control cultures (black column; n = 556). Three hours before fixation, three sets of cultures were transferred to 0 mm Mg2+ Ringer's solution (third column; n = 519) or to 0 mm Mg2+ Ringer's solution containing either the NMDA receptor antagonist APV (fourth column; n = 561) or the AMPA receptor antagonist CNQX (fifth column; n = 669). Unstimulated cultures raised in the presence of BMI (no synchronous network activity) or stimulated cultures in the presence of APV (blocked NMDA receptors) showed a significantly lower fraction of NR1/GluR2/3 clusters compared with control cultures. Conversely, the fraction of NR1/GluR2/3 clusters in cultures raised in BMI and stimulated with 0 mm Mg2+ in either the absence or presence of CNQX before fixation did not differ significantly from control cultures. The stimulation in A and B was performed in BMI-treated cultures after removal of BMI (***p ≤ 0.001; χ2 test; 18 analyzed fields from 2 cultures per time point in all cases; for imaging, 5 fields from 1 culture were analyzed per time point). n.s., Not significant.

Article Snippet: For the complete block of glutamate- and GABA-mediated synaptic transmission, a mixture of glutamate receptor antagonists CNQX (10 μ m ; Tocris Cookson) and APV (50 μ m ; Tocris Cookson) and GABA A receptor (GABA A R) antagonists BMI (20 μ m ; Sigma/RBI) and picrotoxin (PTX) (10 μ m ; Tocris Cookson) were added to the cultures at 5 DIV and refreshed at 9 and 11 DIV.

Techniques: Activation Assay, Activity Assay, Control, Imaging