Figure 4. Reduced membrane excitabilities in shLuc-transfected CA1 pyramidal neurons. A, Effect of shRNA overexpression on neuronal excitability. An empty vector (pSup), shLuc, or shScr was transfected together with pCAG-EGFP. Top, Sample traces from untransfected and transfected CA1 pyramidal neurons in organotypic hippocampal slice cultures. The superimposed traces were elicited by current injections of 0, 100, and 500 pA for 200 ms. Bottom, Summary graph of the frequency of APs in untransfected and transfected neurons. The input–output relationship [number of spikes elicited versus amount of current injection (200-ms duration)] was plotted for untransfected and transfected neurons. Neurons were held at resting membrane potentials (−56.8 mV ± 0.57, n = 100 cells, 9 mice). Number of cells tested: untrans, 45 cells from eight mice (45/8); pSup, 13/7; shScr, 20/5; and shLuc, 22/9. B, Effect of shRNA overexpression on AP kinetics. Top, Sample traces from CA1 pyramidal neurons untransfected and transfected with the three different plasmids. Single APs were induced by current injection (100 pA for 4 ms) and threshold (horizontal arrows) and half width of AP determined by vertical double arrow heads in trans- and untransfected neurons were measured. Bottom, Summary graph of the threshold (left) and half width (right) of single AP in untransfected and transfected neurons. Number of cells tested: untrans, 45 cells/8 mice (45/8); pSup, 13/7; shScr, 20/5; shLuc, 22/9. C, top, Sample traces of sodium currents recorded from untransfected and transfected CA1 pyramidal neurons. Note that these currents were completely blocked by TTX (right). Bottom, Summary graph of the sodium currents in untransfected and transfected neurons. Neurons were voltage-clamped at –80 mV and depolarized from –80 to 30 mV (10-ms duration). Number of cells: untrans, 16 cells/5 mice; pSup, 10/5; shScr, 10/5; shLuc, 14/6. D, top, Sample traces of potassium currents recorded from untransfected and transfected CA1 pyramidal neurons. Note that these upward currents were completely blocked by TEA and 4-AP (right). Bottom, Summary graph of the potassium currents in untransfected and transfected neurons. Neurons were voltage-clamped at –80 mV and depolarized from –80 to 30 mV (10-ms duration). Number of cells tested: untrans, 15 cells/4 mice; pSup, 13/3; shScr, 7/3; shLuc, 9/3.