Inoue Shigeki, Murata Kaoru, Tanaka Aiko, Kakuta Eri, Tanemura Saori, Hatakeyama Shiori, Nakamura Atsunao, Yamamoto Chihiro, Hasebe Masaharu, Kosakai Kumiko, Yoshino Masami
Department of Biology, Tokyo Gakugei University, Tokyo, Japan.
Department of Biology, Tokyo Gakugei University, Tokyo, Japan.
J Insect Physiol. 2014 Sep;68:44-57. doi: 10.1016/j.jinsphys.2014.06.013. Epub 2014 Jul 1.
Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at -40 mV, peaked at -26 mV, and half-inactivated at -46.7 mV. The persistent sodium current (INaP) activated at -51 mV, peaked at -23 mV, and half-inactivated at -30.7 mV. Tetrodotoxin (TTX; 1 μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd(2+) (50 μM) potently blocked INaP with little effect on INaf. Riluzole (>20 μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at -30 mV, peaked at -11.3 mV, and half-inactivated at -34 mV. The Ca(2+) channel blocker verapamil (100 μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca(2+)-activated K(+) (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca(2+) or adding verapamil or nifedipine, and increased by adding the Ca(2+) agonist Bay K8644, indicating that Ca(2+) entry via the L-type Ca(2+) channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100 μM riluzole in the bath solution. These membrane oscillations disappeared with 1 μM TTX, 50 μM Cd(2+), replacement of external Na(+) with choline, and blockage of Na(+)-activated K(+) current (IKNa) with 50 μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca(2+)-free medium or in the presence of verapamil were eliminated by blocking INaP with 50 μM Cd(2+). Taken together, these results indicate that INaP and IKNa participate in the generation of membrane oscillations and that INaP additionally participates in the generation of plateau potentials and initiation of spontaneous action potentials. ICa, through L-type Ca(2+) channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels.
昆虫大脑蘑菇体内的内在神经元，即肯扬细胞，在嗅觉联想学习中发挥着重要作用。在本研究中，我们研究了介导双斑蟋蟀肯扬细胞内在兴奋性的离子机制。使用β-七叶皂苷的穿孔全细胞膜片钳研究表明存在几种内向和外向电流。鉴定出三种类型的内向电流（INaf、INaP和ICa）。瞬时钠电流（INaf）在-40 mV时激活，在-26 mV时达到峰值，在-46.7 mV时半失活。持续性钠电流（INaP）在-51 mV时激活，在-23 mV时达到峰值，在-30.7 mV时半失活。河豚毒素（TTX；1 μM）完全阻断INaf和INaP，但10 nM TTX对INaf的阻断作用比对INaP更强。Cd(2+)（50 μM）有效阻断INaP，对INaf影响很小。利鲁唑（>20 μM）非选择性阻断INaP和INaf。电压依赖性钙电流（ICa）在-30 mV时激活，在-11.3 mV时达到峰值，在-34 mV时半失活。Ca(2+)通道阻滞剂维拉帕米（100 μM）以使用依赖性方式阻断ICa。细胞贴附式膜片钳记录显示存在大电导Ca(2+)激活的K(+)（BK）通道，去除细胞外Ca(2+)或添加维拉帕米或硝苯地平可降低该通道的活性，添加Ca(2+)激动剂Bay K-8644可增加其活性，表明通过L型Ca(2+)通道进入的Ca(2+)调节BK通道活性。在电流钳条件下，在浴液中存在10 nM TTX或100 μM利鲁唑时，膜去极化产生膜振荡。这些膜振荡在加入1 μM TTX、50 μM Cd(2+)、用胆碱替代外部Na(+)以及用50 μM奎尼丁阻断Na(+)激活的K(+)电流（IKNa）时消失，表明膜振荡主要由INaP与IKNa协同介导。在无Ca(2+)培养基中或存在维拉帕米时观察到的平台电位，通过用50 μM Cd(2+)阻断INaP而消除。综上所述，这些结果表明INaP和IKNa参与膜振荡的产生，并且INaP还额外参与平台电位的产生和自发动作电位的起始。还发现通过L型Ca(2+)通道的ICa通过与BK通道的功能偶联在动作电位的快速膜复极化中发挥作用。
