Our findings now provide the characterization of the novel induction
mechanism underlying a physiological regulation of NR2 subunit composition. Our data suggest that this mechanism is widely used in cortical neurons, and it will be of great interest in future studies to determine if this mechanism Etoposide purchase is also involved in pathological changes in NMDAR subunit composition. Four- to nine-day-old Wistar rats were anesthetized with isoflurane and then decapitated in accordance with NIH animal care and use guidelines. Transverse hippocampal slices (400 μm thick) were cut in ice-cold artificial cerebrospinal fluid (ACSF) containing: 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 9 mM MgSO4, 1 mM NaH2PO4, 26.2 mM NaHCO3, 11 mM glucose equilibrated with 95% O2 and 5% CO2. Slices were then allowed to recover for at least 1 hr in ACSF at room temperature (composition as above except for 1.3 mM MgSO4). Whole-cell patch-clamp recordings were made from visually identified CA1 pyramidal neurons in the presence of 50 μM picrotoxin at room temperature. The whole-cell solution contained 115 mM OTX015 CsMeSO4, 20 mM CsCl2, 10 mM HEPES, 2.5 mM MgCl2, 4 mM NaATP, 0.4 mM NaGTP, 10 mM NaCreatine,
and 0.6 mM EGTA (pH 7.2). Preparation of hippocampal and cortical slices from mice was similar except that the ice-cold ACSF for cutting was of the following composition: 87 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl2, 25 mM NaHCO3, Ramoplanin 1.25 mM NaH2PO4, 25 mM glucose, 75 mM sucrose equilibrated with 95% O2 and 5% CO2. Slices were then placed at 35°C for 30 min
and allowed to recover for at least 1 hr in ACSF at room temperature. EPSCs were evoked by electrical stimulation of two independent populations of Schaffer collateral/commissural axons using two bipolar-stimulating electrodes placed in stratum radiatum of CA1 (0.1 Hz stimulation frequency). The stimulating electrodes were placed on opposite sides from recorded cell from each other. For layer 2/3 pyramidal cell recordings from the visual cortex, the stimulating electrode was placed in layer 4. NMDAR-mediated EPSCs were obtained in the presence of NBQX (5 μM) and picrotoxin (50 μM), while cells were voltage clamped at +40 mV. Recordings in which the access resistance changed by more than 10% were discarded and not included in our analysis. Recordings were performed using a MultiClamp 700B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA); signals were filtered at 4 kHz, digitized at 10 Hz, and displayed and analyzed online using pClamp 9.2 (Axon Instruments). To drive the activity-dependent switch in the subunit composition of synaptic NMDARs from rat slices, an LTP induction protocol was employed, in which cells were voltage clamped at 0 mV, while Schaffer collateral/commissural axons were stimulated at 1 Hz for 120 s, similar to that previously described (Bellone and Nicoll, 2007). Cells were then voltage clamped at −70 mV for 5 min following LTP induction.