Changes in tyrosine phosphorylation of GluA2 have previously been implicated in insulin-dependent LTD (Ahmadian et al., 2004) and mGluR-LTD (Gladding et al., 2009). The involvement of regulated tyrosine phosphorylation of GluA2 in homeostatic scaling demonstrates a convergent molecular mechanism that may be differentially evoked either locally or cell-wide to produce Hebbian or non-Hebbian plasticity. We examined the hypothesis that group I mGluR activity contributes to homeostatic scaling using antagonists of mGluR1 and mGluR5. The pharmacology of group I mGluR antagonists is notable in that it includes agents that

competitively inhibit the glutamate binding pocket in the N terminus (O’Hara et al., 1993), as well as agents that noncompetitively bind within the transmembrane domain (Pagano et al., 2000). Noncompetitive Selleck Perifosine agents can act as inverse agonists if they block agonist-independent activity, or as neutral antagonists if they do not block agonist-independent activity (Milligan, 2003). Days in vitro (DIV) 14 cortical neurons in culture were chronically treated with bicuculline (bic, 40 μM) in the presence or absence of group I mGluR antagonists, and scaling of AMPARs was monitored by surface biotinylation and immunohistochemistry (IHC). As expected, chronic bicuculline

treatment reduced the levels of GluA1 (GluR1) and GluA2/3 (GluR2/3) on the cell surface. Inhibition of mGluR1 and mGluR5 with inverse agonists Bay 36-7620 (Bay, 10 μM) and 2-methyl-6-(phenylethynyl)-pyridine find protocol (MPEP, 5 μM) prevented the effect of bicuculline to reduce surface GluA1 and GluA2/3 (Figures 1A and 1B). Figure 1A illustrates a representative western blot at a single exposure, while quantitative data (Figure 1B) were obtained from additional exposures to assure signals were in the linear range. (The same approach is used for western data in MycoClean Mycoplasma Removal Kit all figures.) A similar effect was observed with the structurally different mGluR1 inverse agonist, LY367385 (LY, 100 μM) (Pula et al., 2004), together

with MPEP (Figures 1A and 1B). Single treatment with Bay, LY, or MPEP did not prevent bicuculline-induced downregulation of surface AMPAR (data not shown), indicating that inhibition of both mGluR1 and mGluR5 is required. Competitive or neutral antagonists of group I mGluR did not block bicuculline-induced downregulation of GluA1 and GluA2/3 as shown by inhibition of mGluR1 with the neutral antagonist CPCCOEt (CP, 100 μM), and mGluR5 with the competitive antagonist (S)-MCPG (500 μM) (Figures 1A and 1B). This combination was verified to block the effect of group I mGluR signaling on ERK phosphorylation to a similar extent as Bay and MPEP (Figures S1A and S1B available online). CP (100 μM) combined with higher dose (S)-MCPG (2.5 mM) also failed to block bicuculline-induced downregulation of GluA1 and GluA2/3 (Figures S1C and S1D).