Whole-cell patch-clamp recordings of NMDA receptor-mediated EPSCs.

A hippocampal slice was transferred to a recording chamber (RC-26GLP, Warner Instruments, Hamden, CT) mounted on a SliceScope Pro 2000 (Scientifica, Clarksburg, NJ) and continuously perfused at a rate of 2–3 ml/min with oxygenated ACSF solution containing (in mM) 130 NaCl, 3 KCl, 24 NaHCO₃, 1.25 NaH₂PO₄, 10 glucose, 2 CaCl₂, and 1 MgSO₄, maintained at 32°C using dual in-line and heated platform temperature control (TC-344A, Warner Instruments). Recording electrodes with a tip resistance of 2–4 MΩ were fabricated using a micropipette puller (P-1000, Sutter Instruments, Novato, CA) and filled by internal solution containing (in mM): 120 Cs-methanesulfonate, 15 CsCl, 10 tetraethylammonium chloride, 10 HEPES, 8 NaCl, 3 Mg-ATP, 1.5 MgCl₂, 10 QX-314, 0.3 Na-GTP, and 0.2 EGTA, pH 7.3, 295–305 mOsm. Whole-cell recordings were made using a Multiclamp 700B amplifier (Molecular Devices) with filtering at 4 kHz (Bessel) and digitized at 20 kHz using Digidata 1440A with pClamp 10 software (Molecular Devices).

Excitatory postsynaptic currents (EPSCs) from hippocampal CA1 pyramidal cells or interneurons in the CA1 stratum pyramidale (s.p.) and stratum radiatum/lucidum (s.r./s.l.m.) layers, respectively, were recorded at +40 mV by stimulation of Schaffer collaterals with a bipolar electrode (MX211EW(PC3), FHC, Bowdoin, ME). The stimulation (0.1 ms; 20–500 μA) was delivered every 30 s (0.033 Hz) using a Master-8 (A.M.P.I., Jerusalem, Israel) with a stimulus isolator (A365, World Precision Instruments, Sarasota, FL). DNQX (20 μM) and gabazine (10 μM) were included in ACSF solution during recordings to block AMPA/kainate receptors and GABA_A receptors, resulting in a DMSO concentration of 0.03%. After 5–10 min stable baseline EPSC recordings, test compounds (CP-101,606, NAB-14, MPX-004, or spermine) were delivered for 10–15 min, followed by 400 μM DL-APV for full inhibition of NMDA receptor-mediated EPSCs. Stock solutions of test compounds were prepared to achieve an added DMSO concentration of 0.01% (i.e. vehicle is 0.01% DMSO) when added to the ACSF recording solution at the concentrations used. Series resistance (typically <20 MΩ) was monitored throughout the experiment and the recording was excluded from analysis if changes of >20% were observed.