Whole-cell patch-clamp recordings were conducted as previously described in our laboratory (44). Male C57BL/6 mice (7 to 8 weeks old) were anesthetized and sacrificed for brain slice preparation. Transverse hippocampal slices (400 μm) were prepared (Leica VT1200S; Nussloch, Germany) in a cold sterile slice solution [2 mM CaCl2, 2 mM MgCl2, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM KH2PO4, 10 mM glucose, and 220 mM sucrose (pH 7.4) bubbled with 95% O2⁄5% CO2] and transferred to a storage chamber containing Mg2+-free artificial cerebrospinal fluid [ACSF; 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 26 mM NaHCO3, 1.25 mM KH2PO4, and 25 mM glucose (pH 7.4) bubbled with 95% O2/5% CO2] at 34°C for a recovery period of 1 hour before recording. For recordings, the slices were fully submerged in the same flowing Mg2+-free ACSF (4 ml/min) at room temperature (RT) (20 to 25°C).

For mIPSC recordings, glass pipette electrodes with a resistance of 3 to 5 megohms were filled with the following internal solution: 100 mM CsCl, 10 mM Hepes, 1 mM MgCl2, 1 mM EGTA, 5 mM MgATP, 0.5 mM Na3GTP, 12 mM phosphocreatine, and 30 mM N-methyl-d-glucamine (NMG) (pH 7.4), 280 to 290 mOsm. Voltage-clamp recordings were performed at a holding potential of −70 mV, and mIPSCs were recorded in the presence of 20 μM 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), 50 μM dl-2-amino-5-phosphonovaleric acid (D-APV), and 1 μM tetrodotoxin (TTX).

For mEPSC recordings, glass pipette electrodes with a resistance of 3 to 6 megohms were filled with the internal solution containing 130 mM CsMeSO4, 10 mM CsCl2, 10 mM Hepes, 4 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 5 mM MgATP, 0.5 mM Na3GTP, 12 mM phosphocreatine, and 5 mM NMG (pH 7.4), 280 to 290 mOsm. Membrane potential was held at −70 mV in voltage-clamp mode, and mEPSCs were recorded in the presence of 1 μM TTX and 100 μM picrotoxin (PTX) to block GABAA (γ-aminobutyric acid type A).

To evaluate NMDAR- and AMPAR-mediated EPSCs, evoked currents were generated using a 400-μs pulse at a rate of 0.1 Hz (intensity, 50 to 200 μA) delivered by a stimulation isolation unit using an S48 pulse generator (AstroMed). A bipolar stimulating electrode was positioned in the Schaffer collaterals. The evoked currents were measured in the presence of 100 μM PTX and collected at two holding potentials. At −70 mV, with application of the NMDAR-selective antagonist D-APV (50 μM), the peak amplitude of the evoked EPSCs was identified as the AMPAR-mediated current. At +40 mV and in the presence of the AMPAR-selective antagonist DNQX (20 μM), the amplitude of the evoked EPSC at 50 ms poststimulus was identified as the NMDAR-mediated current.

To record the PPRs of the NMDA-EPSCs, EPSCs were evoked by stimulating the Schaffer collaterals–CA1 pathway at a holding potential of +40 mV in the presence of 100 μM PTX and 20 μM DNQX. Each stimulation was 0.1 Hz for 40 μs, and paired pulses were delivered at a 50-ms interpulse interval. To record the PPRs of the AMPA-EPSCs, EPSCs were evoked at a holding potential of −70 mV in the presence of 100 μM PTX and 50 μM D-APV. The intervals of paired stimulations were set at 25, 50, and 100 ms. The values of the ratios were defined as [p2/p1], where p1 and p2 are the amplitudes of the EPSCs evoked by the first and second pulses, respectively (34, 35). To clarify the mechanism by which GPR40 affects NMDAR surface expression levels, the exocytosis blocker TeTx (0.1 μM; Sigma-Aldrich) and the endocytosis blocker dynasore (80 mM; Abcam) were added to the internal solution for NMDA-EPSC recordings (45, 46).

All recordings were initially conducted in Mg2+-free ACSF with 0.1% DMSO. After the baseline data were recorded, GW9508 (20 μM) or GW1100 (20 μM) in 0.1% DMSO was added to the perfusate. Signals were acquired using a MultiClamp 700B amplifier (Axon, USA), filtered at 2 kHz and digitized at 10 kHz, followed by recording using pClamp 9.2 software (Molecular Devices, Sunnyvale, CA, USA). The Mini Analysis Program (Synaptosoft, Leonia, NJ) was used to analyze synaptic activity. In whole-cell recording experiments, the series resistance was kept below 20 megohms and was not compensated. Cells were rejected if series resistance fluctuated by more than 25% of the initial value. Data were collected after the current was stable for 5 to 15 min.

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