The standard method was used. Hippocampal slices were perfused with ACSF (bubbled with 95% O2/5% CO2; 30°C) at the rate of 2 ml/min.
Cabozantinib Stimulating electrode was placed on the stratum radiatum in the CA2 area. Stimuli were delivered to the electrode at 20 s intervals. For field-recordings, recording pipettes (1–2 MΩ) were filled with the bath solution and placed in the CA1 region. Tetanic stimuli (100 pulses at 100 Hz, 2 trains at 20 s intervals) were delivered to induce LTP, while low-frequency stimulations (LFS; 900 pulses at 1 Hz) were delivered to induce LTD. For whole-cell recordings, 100 μM bicuculline was added to the ACSF to block GABAA receptors. The patch pipette (4–7 MΩ) solution is composed of (in mM) 130 cesium methanesulfonate, 8 NaCl, 4 Mg-ATP, 0.3 Na-GTP, 0.5 EGTA, 10 HEPES, and 5 QX-314 at pH 7.3. EPSCs of CA1 pyramidal cells were recorded at −70 mV. A 10 min-baseline recording was conducted www.selleckchem.com/products/pf-06463922.html prior to LTD induction. For whole-cell recordings in cultured hippocampal slices, 2 μM 2-chloroadenosine was added to the ACSF to prevent bursting. Simultaneous whole-cell recordings were obtained from pairs of nearby transfected and untransfected
CA1 neuron. LTD was induced by delivering low-frequency stimulations (LFS; 1 Hz) for 300 s at a holding potential of −45 mV. AMPA receptor-mediated EPSCs (EPSCAMPA) were measured at a holding potential of −70 mV. NMDA receptor-mediated EPSCs (EPSCNMDA) were measured 50–70 ms after the peak of EPSCAMPA at a holding potential of +40 mV (Terashima et al., 2004). The series resistance and input resistance were monitored on-line and analyzed with the Clampex program off-line. Only cells with a series resistance also of <25 MΩ and a <10% drift in both series resistance and input resistance during the recording period were included. Student's two-tailed t test was used for statistical analysis (p < 0.05 considered significant). Hippocampal and cortical neuron cultures were prepared from embryonic day (E) 18–19 rat or mouse embryos as previously described (Sala et al., 2001). Neurons
were seeded on poly-D-lysine (30 μg/ml) and laminin (2 μg/ml) coated coverslips or plates at a density of ∼750 cells/mm2. Cultures were grown in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamine and 12.5 mM glutamate. Hippocampal neurons were transfected with Lipofectamine 2000 (Invitrogen). The internalization assay was performed as described previously (Lee et al., 2002). Briefly, neurons were incubated with antibodies against the N terminus of GluR2 (Chemicon) for 15 min at 37°C, then stimulated with NMDA (30 μM) or left unstimulated for 5 min. Neurons were fixed with the fixation buffer (4% formaldehyde and 4% sucrose in PBS) immediately after the stimulation. Surface-remaining antibody-labeled GluR2 was saturated by incubation with Alexa Fluor 555-conjugated secondary antibody (Invitrogen).