Journal: Journal of neurochemistry

Article Title: Adenosine A₂A receptors permit mGluR5-evoked tyrosine phosphorylation of NR2B (Tyr1472) in rat hippocampus: a possible key mechanism in NMDA receptor modulation.

doi: 10.1111/jnc.13291

Figure Lengend Snippet: Fig. 1 Stimulation of mGluR5 induces phosphorylation of NR2B subunit at Tyr1472. Western blot analysis of the phosphorylation level of (a) NR2B (Tyr1472) subunit and (b) NR2B (Ser1303) subunit of NMDAR, normalized with the respective total protein levels in dorsal hippocampal slices in different animal groups, according to the drug treatment: [control], [CHPG 30], [CHPG 50], [CHPG 100], [CHPG 300], [CHPG 500]. Tubulin was used as loading control. Incubation of hippocampal slices with the selective agonist of mGluR5 receptor, CHPG, at different doses, induced a dose-dependent increase in the phosphorylation level of the NR2B (Tyr1472), but not of the NR2B (Ser1303) subunit of NMDAR. Results are shown in means SEM values (number of animals, N = 8) per group of representative western blots. *p < 0.05 significantly different compared with the control group. #p < 0.05 significantly different compared with the [CHPG 30] group. ^p < 0.05 significantly different compared with all other drug treated groups. Statistical analysis used ANOVA followed by Tukey post hoc test.

Article Snippet: After 1 h of blocking in 10% non-fat dried milk at 21°C, the nitrocellulose was incubated overnight at 4°C with the following antibodies: rabbit antiPhosphoNR2B (ser1303) polyclonal antibody (1 : 750, Millipore Corporation, Bedford, MA, USA) and rabbit anti-PhosphoNR2B (tyr1472) polyclonal antibody (1 : 500, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Incubation

Journal: Journal of neurochemistry

Article Title: Adenosine A₂A receptors permit mGluR5-evoked tyrosine phosphorylation of NR2B (Tyr1472) in rat hippocampus: a possible key mechanism in NMDA receptor modulation.

doi: 10.1111/jnc.13291

Figure Lengend Snippet: Fig. 3 Temporal coincidence between molecular and electrophysiolog- ical actions of NMDAR/mGluR5 synergy. Time course of the effects of co-activation of NMDAR and mGluR5 on: (a) the post-synaptic excitability [PS/excitatory post-synaptic potential (EPSP)], (b) the phosphorylation levels of NR2B (Tyr1472) and (c) the synaptic trans- mission (EPSP/Fv). Experiments were performed in CA1 minislices. Measures of electrophysiological parameters were obtained before and during co-application of 50 lΜ NMDA and 50 lΜ CHPG. Measures of the three parameters were made on the same CA1 minislices. Repre- sentative examples of field recordings from the st. radiatum and st. pyramidale are shown on the top of the collective plots for PS/EPSP and EPSP/Fv, respectively. Representative examples of phosphorylation of NR2B (Tyr1472) are shown in the corresponding collective plot. The drug-induced increase in post-synaptic excitability was expressed by the remarkable appearance of multiple PSs despite the decline in the synaptic transmission (EPSP/Fv). Note the temporal coincidence of the effects of co-activation of NMDARs and mGluR5 on the neuronal excitability and the phosphorylation of NR2B (Tyr1472). Asterisks denote statistically significant differences compared with control values at *p < 0.05 (Wilcoxon test and ANOVA with Tukey post hoc test).

Article Snippet: After 1 h of blocking in 10% non-fat dried milk at 21°C, the nitrocellulose was incubated overnight at 4°C with the following antibodies: rabbit antiPhosphoNR2B (ser1303) polyclonal antibody (1 : 750, Millipore Corporation, Bedford, MA, USA) and rabbit anti-PhosphoNR2B (tyr1472) polyclonal antibody (1 : 500, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Transmission Assay, Control

Journal: Journal of neurochemistry

Article Title: Adenosine A₂A receptors permit mGluR5-evoked tyrosine phosphorylation of NR2B (Tyr1472) in rat hippocampus: a possible key mechanism in NMDA receptor modulation.

doi: 10.1111/jnc.13291

Figure Lengend Snippet: Fig. 5 The effects of co-activation of NMDAR and mGluR5 on the post-synaptic excitability [PS/excitatory post-synaptic potential (EPSP)], on the phosphorylation of the NR2B (Tyr1472) and on the excitatory synaptic transmission (EPSP/Fv) outscored the effects of only NMDAR’s activation and required the activity of adenosine A2ARs, and the Src family kinases. Histograms of the effects of various pharmacological conditions on the: (a) PS/EPSP (b) phosphorylation of NR2B (Tyr1472) and (c) EPSP/Fv are shown. Examples of field potential recordings obtained before and during drug application (thin and thick line traces, respectively) are shown on the top of the columns of the PS/EPSP and EPSP/Fv histograms. Scale bars for recordings shown in (a and c): 1 mV, 5 ms. Measures of electrophysiological parameters were obtained during the time period when the NMDA- CHPG drug cocktail had maximal effects. Note that the increase in the post-synaptic excitability (PS/EPSP) was expressed as an increase in the amplitude of PS and/or the appearance of additional PSs. Asterisks denote statistically significant differences at *p < 0.05 or **p < 0.01 (Wilcoxon test). Statistically significant differences in the drug effects are indicated by diesis at #p < 0.05 or ##p < 0.01 (Mann–Whitney U test and ANOVA with Tukey post hoc test). Blockade of mGluR5 by 200 lM MTEP, blockade of A2ARs by 1 lM ZM 241385 and inhibition of Src family kinases by 30 lM PP1 occluded the potentiating effect of mGluR5 activation on the NMDA-induced responses.

Article Snippet: After 1 h of blocking in 10% non-fat dried milk at 21°C, the nitrocellulose was incubated overnight at 4°C with the following antibodies: rabbit antiPhosphoNR2B (ser1303) polyclonal antibody (1 : 750, Millipore Corporation, Bedford, MA, USA) and rabbit anti-PhosphoNR2B (tyr1472) polyclonal antibody (1 : 500, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Transmission Assay, Activity Assay, MANN-WHITNEY, Inhibition