Competitive blockade of group I and group II mGluRs with the LY341495 (100 μM) (Kingston et al., 1998) also failed to prevent bicuculline-induced downregulation of surface GluA1 and GluA2/3 (Figures S1C and S1D). The failure of CP and MCPG or LY341495 to block scaling to bicuculline suggests that mGluR activation is not due to glutamate

released at synapses or glutamate that might accumulate in the medium. To further examine this issue, we added glutamate-pyruvate transaminase (GPT) to the medium during Ibrutinib chronic bicuculline treatment at a concentration reported to prevent local accumulation of glutamate at synapses (Matthews et al., 2000 and Pula et al., 2004). The Kd of GPT for glutamate is ∼8 μM and this is close to the measured level of glutamate in the medium of our

cultures (∼7 μM). GPT did not alter the effect of chronic bicuculline to downregulate surface GluA1 and GluA2/3 (Figures S1E and S1F). Effects of group I mGluR GDC-0941 purchase antagonists on bicuculline-evoked scaling were examined in parallel using electrophysiological recordings. Chronic treatment with Bay and MPEP produced an increase in the miniature excitatory postsynaptic current (mEPSC) amplitudes (control 20.9 ± 1.1 pA; n = 24 cells versus Bay/MPEP 26.7 ± 1.9 pA; n = 21 cells; #p < 0.05), and blocked the effect of bicuculline (bic 14.1 ± 0.2 pA; n = 28 cells versus bic/Bay/MPEP 25.7 ± 2.0 pA; n = 18 cells) (Figures 1E and 1F). By contrast, chronic CP and MCPG did not block the effect of bicuculline (amp: 13.5 ± 0.4 pA, ∗∗p < 0.01; frequency: 22.8 ± 2.6 Hz, n = 22 cells), and did not produce an increase in the basal mEPSC amplitude (21.8 ± 0.9 pA, n = 11 cells, Figures 1E and 1F) or frequency (22.0 ± 2.8 Hz, n = 11 cells). The cumulative histogram below of mEPSCs indicates that Bay 36-7620 and MPEP produced a multiplicative increase of amplitudes, suggesting the antagonists produce a scaling up of relative synaptic weights (Turrigiano and Nelson, 2000). Chronic Bay and MPEP also increased the frequency of mEPSCs (control 23.4 ± 2.6 Hz;

n = 24 cells versus Bay/MPEP 36.4 ± 4.4 Hz; n = 21 cells; #p < 0.05). This is consistent with either a presynaptic action of group I mGluRs or the possibility that Bay and MPEP convert silent synapses to active synapses. To assess if Homer1a can activate group I mGluRs in cortical neurons to downregulate surface AMPARs, we expressed Homer1a transgene by Sindbis virus infection for 24 hr, and assayed surface AMPARs by biotinylation and IHC. We compared effects of Homer1a with that of Arc (Shepherd et al., 2006). Neurons were treated with tetrodotoxin (TTX, 1 μM) to reduce native expression of Homer1a and Arc, and thereby isolate the action of the transgenes. Surface GluA1 and GluA2/3 were reduced in neurons that expressed Homer1a or Arc, compared to GFP (Figures 2A and 2B).