Glutamate and kainate (1 mM), CNQX (20 μM), and LY404187 (3 μM) were applied where indicated and cyclothiazide (CTZ; 100 or 200 μM) was added to the external for potentiation experiments. The recording from primary cultured neurons was performed on the coverslips where the neurons had grown with the 16-barrel pipette array positioned 200–500 μm away from the recorded neurons. Unless otherwise indicated (Figure 2), resensitization percentage was calculated as: IGlu-Resens/IGlu-SS×100,where IGlu-Resens is the

current this website that accrues from the trough of desensitization (Figure 1A). Kainate/glutamate ratios were calculated as: IKA-ss/IGlu-ss,IKA-ss/IGlu-ss,where IKA-ss and IGlu-ss are the steady state responses evoked by kainate and glutamate application, respectively. CTZ potentiation of kainate-evoked responses was calculated as: ((IKA+CTZ/IKA)×100)−100,where IKA + CTZ is the steady state current amplitude recorded during kainate + CTZ application and IKA is the

steady state current amplitude recorded during kainate application. Spontaneous AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) from transfected and untransfected cultured primary hippocampal neurons (>14 DIV) were recorded in the presence of 10 μM bicuculline, 50 μM picotoxin, 10 μM CPP, 300 nM 7-CK, and 3 μM TTX using an internal solution containing (in mM): 95 CsF, 25 CsCl, 10 Cs-HEPES pH 7.4, 10 EGTA, 2 NaCl, 1 MgCl2, 10 QX-314, and 5 TEA-Cl adjusted ISRIB molecular weight to ∼290 mOsm with Mg-ATP. mEPSCs used for analysis were collected from a 2 min period immediately after a 3 min recording solution equilibrium period, were inspected visually

and were selected with a lower limit amplitude cutoff of greater Fossariinae than 15 pA to eliminate any possible contamination from noise and holding current oscillation. Analyses and curve fitting were performed using MiniAnal software (Synaptosoft, Decatur, GA). Patch-clamp recordings from cerebellar granule cells (DIV7–10) were made in external solution containing (in mM): 10 HEPES, 140 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 2.7 MgCl2, and 10 glucose. Patch pipettes were filled with recording solution (pH 7.2, 320 mOsm) that contained (in mM): 130 cesium methanesulfonate, 5 HEPES, 5 Mg-ATP, 0.2 Na-GTP, 20 TEA, and 5 EGTA. All recordings were performed at room temperature. To isolate and record AMPA receptor-mediated mEPSCs, tetrodotoxin (0.5 μM), AP-5 (50 μM), and picrotoxin (100 μM) were added to the external solution. mEPSCs were recorded from cerebellar granule cells in whole-cell configuration at a holding potential of −70 mV. The current was analog low-pass filtered at 3 kHz and digitally sampled at 25 kHz. Sampling traces were further filtered with eight-pole low-pass Bessell filter (1 KHz, −3 dB) for demonstration purposes. Amplitude and frequency of events were analyzed using Minianalysis (Synaptosoft).