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Membrane current was re-corded from single CA1 pyramidal neurons with the patch clamp technique in the whole-cell configuration, using an L/M-EPC7 amplifier The recording electrode was a glass micropipette filled with intracellular solution Capacitive currents were electronically cancelled and series resistance wascompensated by 60–80%. Data were acquired and a nalysed using EPC and Signal softwares 。 Immediately after the beginning of recording, the slice was per-fused with a Mg ++-free ACSF containing tetrodotoxin to prevent synaptic activity. N -methyl-D -aspartic acid (NMDA, 50lM, Sigma) was dissolved in Mg++ -free ACSF containingglycine (0.5 l M, Sigma) and TTX (1 lm) and was applied during 5 s using a computer-controlled electrovalve solution changer Compound 3d was dissolved in Mg++ -free ACSF (containing TTX, 1 l m) and applied by bath perfusion at a flow rate of 1 mL/min. The holding potential of CA1 pyramidal neurons was set at 60 mV and membrane current was continually recorded at an acquisition rate of 4 KHz. Several pulses of NMDA were applied for each neuron respectively in control conditions and during con-tinuous application of 3d at different doses (1, 10, and100 lM, 5 min) through the bath perfusion system. NMDA-induced current amplitude was measured as the difference between peak current amplitude and baseline. For each neuron studied, the amplitude of at least three consecutive NMDA-induced currents were mea-sured respectively in control conditions and in the presence of compound 3dand the two sets of raw values were compared using Student’s unpaired t -test (Graph Pad software). To illustrate the time course of NMDA-mediated current blockade by 3d (10 l M), current amplitude values were normalized (dividing by mean amplitude value, calculated from at least three NMDA responses in control conditions); normalized NMDA-mediated currents from all the responsive neurons were pooled (mean ± SEM) and repre-sented on a graphic as a function of time. [ 来自科研家族 药物化学家族 ] |
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3楼2012-03-29 15:49:11
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