Electrophysiology

Organotypic hippocampal slices were prepared from P7-8 mice as described previously (Stoppini et al., 1991) and used at 7–12 days in vitro. Where indicated, slices were infected with Sindbis virus expressing GFP- or SEP-tagged rat GluA3 (flip) 20–28 hr prior to experiments. During recordings, slices were perfused with artificial cerebrospinal fluid (ACSF; in mM): 118 NaCl, 2.5 KCl, 26 NaHCO₃, 1 NaH₂PO₄, supplemented with 4 MgCl₂, 4 CaCl₂, 20 glucose. Patch recording pipettes were filled with internal solution containing (in mM): 115 CsMeSO₃, 20 CsCl, 10 HEPES, 2.5 MgCl₂, 4 Na_2-ATP, 0.4 Na-GTP, 10 Na-Phosphocreatine, 0.6 EGTA. Outside-out recordings were made with 3–5 MΩ pipettes and the bath contained the desensitization blockers PEPA (80 µM; Tocris) and cyclothiazide (100 µM; Tocris) to exclude variations due to differences in desensitization properties. Every 20 s a 100 ms puff of 100 μM S-AMPA was delivered with a Picospritzer III (Parker, Hollis, USA). Single channel recordings were measured under cell-attached configuration with 6–8 MΩ pipettes filled with internal solution to which S-AMPA (100 μM; Tocris) was added. Whole-cell recordings in organotypic slice cultures were made with 3–5 MΩ pipettes (R_access < 20 MΩ, and R_input > 10× R_access). During mEPSC recordings, TTX (1 μM; Tocris) and picrotoxin (100 μM; Sigma) were added to the bath. Where indicated, the following drugs were added to the perfusion solution: forskolin (50 μM; Sigma), IBMX (50 μM; Tocris), KT5720 (4 μM; Tocris), PKI (2 μM; Calbiochem), ESI05 (10 μM; Biolog); Salirasib (10 μM; Tocris); or inside the recording pipette: cAMP (100 μM; Sigma), N002 (100 μM; Biolog), 8-CPT (20 μM; Tocris). During evoked recordings, a cut was made between CA1 and CA3, and picrotoxin (50 μM) and 2-chloroadenosine (4 μM; Tocris) were added to the bath. Two stimulating electrodes, (two-contact Pt/Ir cluster electrode, Frederick Haer), were placed 100 μm apart between 100 and 300 μm down the apical dendrite and 200 μm apart laterally. AMPAR-mediated EPSCs were measured as the peak inward current at −60 mV directly after stimulation. Paired pulse ratios were determined with an inter pulse interval of 50 ms. NMDAR-mediated EPSC were measured as the mean outward current between 40 and 90 ms after the stimulation at +40 mV, and corrected by the current at 0 mV. Rectification was calculated as the ratio of the peak AMPAR current at −60 and +40 mV, corrected by the current at 0 mV, in the presence of D-APV (100 μM; Tocris) in the bath and Spermine (0.1 mM; Sigma) in the intracellular solution. EPSC amplitudes were obtained from an average of at least 30 sweeps at each holding potential. Acute hippocampal slices were prepared from 3 to 5 week-old mice. Dissection was done in ice-cold sucrose cutting solution containing (in mM): 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 10 D-glucose, 230 Sucrose, 0.5 CaCl₂, 10 MgSO₄, bubbled with 95%O₂/5%CO₂. Brain slices (400 μm) were cut using a vibratome (Thermo Scientific) and placed in a holding chamber containing ACSF supplemented with (in mM) 1 MgCl₂, 2 CaCl₂, 20 glucose and bubbled with 95%O₂/5%CO₂. They were allowed to recover at 34°C for 40 min then at room temperature for at least 40 min. Whole-cell recordings (3–5 MΩ pipettes, R_access < 26 MΩ, and R_input > 10 x R_access) were made in ACSF containing TTX (1 μM) and picrotoxin (50 μM) at 28°C. To block Ras, 3 μg/ml the OP01 Anti-v-H-Ras (Ab-1) Rat mAb (Y13-259, Millipore; RRID:AB_565094) or Rat IgG1 isotype control (MA1-90035, Invitrogen; RRID:AB_10984952) was included in the intracellular solution. After obtaining whole-cell configuration the antibody was allowed to diffuse in the cell for 5 min before recording. After 10 min FSK was added to the perfusion and allowed to wash in for 5 min. Data was acquired using a Multiclamp 700B amplifier (Molecular Devices). mEPSC data are based on at least 100 events or 5 min of recording, with exception of Figure 6—figure supplement 1 (1 min). Data were analyzed with MiniAnalysis (Synaptosoft). Individual events above a 5 pA threshold were manually selected. Evoked recordings were analyzed using pClamp 10 software (Molecular Devices).

Non-stationary noise analysis of outside-out patches traces was carried out following previously described methods (Alvarez et al., 2002; Hartveit and Veruki, 2007). Peak aligned AMPA-evoked currents recorded over 10–15 sweeps per outside out patch, were binned in 10 equally sized bins of 150 ms each and for each bin, the mean amplitude and variance was calculated. The data distribution resulting after plotting amplitude versus variance was fitted with the following equation: σ2=iI−I2N+σb2, where the variance (σ²) of the amplitude of the current (I) obtained at each time point is explained as a function of the single unitary current (i) and the number of functionally conducting channels (N) with an offset given by the variance of the baseline noise (σ²_b). From the derivative at I=0, the relative number of functional channels was extracted as well as the single channel conductance which was calculated by dividing the unitary current by the applied voltage with respect to the reversal potential (V_holding-E_reversal, −60 mV and 0 mV respectively). The peak open probability (P_o), corresponding to the fraction of available functional channels open at the time of the peak current (I_peak), can be calculated from the following equation: P0=Ipeak/Nmax, where N_max represents the theoretical maximum of available channels opened at the point where the theoretical maximum amplitude reaches the minimum variability (σ²_b) in the given parabola fit. Single channel activity was analyzed using ClampFit (Molecular Devices). Three detection thresholds were used to detect O1 (1.5 pA), O2 (3 pA) and O3 (4.5 pA) openings in single channel AMPARs in steady baseline recordings (no holding current fluctuations). Events with latency shorter than 0.3 ms were ignored to prevent noise from being recognized as openings. Non-stationary noise analyses for the mEPSC events were based on peak scaled mEPSCs.