JNK dependent AMPA-R regulation GM Thomas et al of NMDA, which speciﬁcally activates NMDA-type glutamate receptors, caused rapid and dramatic dephosphorylation of both GluR2L-Thr912 and GluR4-Thr855 (Figure 5A). Both sites were also dephosphorylated following brief treatment with bicuculline (which blocks inhibitory transmission, thus increasing synaptic glutamate release; Figure 5B), withdrawal of the NMDA-R antagonist APV or treatment with AMPA (Supplementary Figure S6). The treatments that decreased GluR2L-Thr912 and GluR4- Thr855 phosphorylation act in distinct ways, but share the ability to increase glutamatergic signaling. We therefore wondered whether reagents that act in an opposite manner (i.e. to inhibit glutamatergic signaling) might differentially affect GluR2L/GluR4 phosphorylation. Indeed, inhibiting neuronal activity with TTX clearly increased phosphorylation of JNK, GluR2L-Thr912 and GluR4-Thr855 (Figure 5B). This suggests that phosphorylation of synaptic JNK targets is regulated by a novel mechanism and that the JNK pathway, and the phosphatases that oppose it, function as a rapid detector of changes in neuronal activity. dephosphorylation The rapid dephosphorylation of GluR2L-Thr912 and GluR4-Thr855 triggered by NMDA or bicuculline (Figure 5A and B) occurred without changes in phosphoJNK (Figure 5B and C) suggesting that dephosphorylation is due not to decreased JNK activity but to increased protein phosphatase activity. Indeed, addition of okadaic acid immediately prior to NMDA treatment prevented NMDA-induced GluR2L-Thr912 and GluR4-Thr855 dephosphorylation (Figure 5A). NMDA- induced was not prevented by low okadaic acid concentrations that selectively inhibit PP2A- type phosphatases (Supplementary Figure S6), suggesting that dephosphorylation of GluR2L-Thr912 and GluR4- Thr855 is regulated by a PP1 family phosphatase. NMDA treatment also triggers dephosphorylation of GluR1, another long-tailed AMPA-R subunit, at Ser845, and this dephosphorylation accompanies GluR1 internalization Figure 5 Neuronal activity changes rapidly and bi-directionally modulate GluR2L-Thr912 and GluR4-Thr855 phosphorylation in neurons. (A) Cortical neurons were incubated in aCSF for 1 h, treated for 10 min with 1 mM okadaic acid or DMSO vehicle, and then stimulated for 10 min with 30 mM NMDA, or were left unstimulated (Con). GluR2L immunoprecipitates were blotted for phosphoThr912 (top) or GluR2L (lower panel). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) NMDA: 36.879.0%, N ¼ 6; NMDA þ OA: 92.1712%, N ¼ 6. GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel) or GluR4 (bottom). GluR4-Thr855 phosphoryla- tion relative to total GluR4 (normalized to untreated control, 100%) NMDA: 28.576.3%, N ¼ 6; NMDA þ OA: 89.3717.4%, N ¼ 6. (B) Cortical neurons were stimulated with 20 mM Bicuculline (Bic) or 1 mM Tetrodotoxin (TTX) for 20 min. Immunoprecipitates were prepared and blotted as in (A). Lysates were blotted to detect phosphoJNK. GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) Bic: 61.9712.8%, N ¼ 4; TTX: 142.2716.9%, N ¼ 4. GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) Bic: 42.5721.5%, N ¼ 4; TTX: 176.1740.4%, N ¼ 4. (C) Cortical neurons were washed into aCSF in the presence ( þ ) or absence ( ) of 10 mM SP600125 for 1 h prior to stimulation for 5 min with 20 mM NMDA. Cells were lysed or washed into aCSF in the absence of NMDA for a further 40 min prior to lysis. Control cells were left unstimulated. GluR2L immunoprecipitates were blotted for phosphoThr912 (top), or GluR2L (second panel). GluR2L-Thr912 phosphorylation relative to total GluR2L (normalized to control, 100%) Con/SP: 75.273.3%, N ¼ 4; NMDA: 58.477.0%, N ¼ 4; NMDA/SP: 30.171.8%, N ¼ 4; Rec: 85.0712.2%, N ¼ 4; Rec/SP: 27.57 6.6%, N ¼ 4. GluR4 immunoprecipitates were blotted for phosphoThr855 (third panel), or GluR4 (bottom). GluR4-Thr855 phosphorylation relative to total GluR4 (normalized to control, 100%) Con/SP: 65.6722.0%, N ¼ 4; NMDA: 49.3710.8%, N ¼ 4; NMDA/SP: 30.279.7%, N ¼ 4; Rec: 87.4710.7%, N ¼ 4; Rec/SP: 38.379.9%, N ¼ 4. Lysates were blotted for phosphoJNK (bottom). Note that the effect of SP600125 on GluR2L-Thr912 and GluR4-Thr855 phosphorylation is more marked after NMDA washout than under basal conditions (compare lanes 1,2 with 3,4 and 9,10 with 11,12). 366 The EMBO Journal VOL 27 | NO 2 | 2008 & 2008 European Molecular Biology Organization