Wakita Masahito, Kotani Naoki, Akaike Norio
Research Division for Clinical Pharmacology, Medical Corporation, Jyuryokai, Kumamoto Kinoh Hospital, 6-8-1 Yamamuro, Kitaku, Kumamoto, 860-8518, Japan; Research Division for Life Science, Kumamoto Health Science University, 325 Izumi-machi, Kitaku, Kumamoto, 861-5598, Japan.
Research Division of Neurophysiology, Kitamoto Hospital, 3-7-6 Kawarasone, Koshigaya, 343-0821, Japan.
Toxicon. 2015 Sep;103:12-8. doi: 10.1016/j.toxicon.2015.05.003. Epub 2015 May 8.
The present study utilised a 'synaptic bouton' preparation of mechanically isolated rat hippocampal CA3 pyramidal neurons, which permits direct physiological and pharmacological quantitative analyses at the excitatory and inhibitory single synapse level. Evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs) were generated by focal paired-pulse electrical stimulation of single boutons. The sensitivity of eEPSC to tetrodotoxin (TTX) was higher than that of the voltage-dependent Na(+) channel whole-cell current (INa) in the postsynaptic CA3 soma membrane. The synaptic transmission was strongly inhibited by 3 nM TTX, at which concentration the INa was hardly suppressed. The IC50 values of eEPSC and INa for TTX were 2.8 and 37.9 nM, respectively, and complete inhibition was 3-10 nM for eEPSC and 1000 nM for INa. On the other hand, both eEPSC and eIPSC were equally and gradually inhibited by decreasing the external Na(+) concentration ([Na]o), which decreases the Na(+)gradient across the cell membrane. The results indicate that TTX at 3-10 nM could block most of voltage-dependent Na(+) channels on presynaptic nerve terminal, resulting in abruptly inhibition of action potential dependent excitatory neurotransmission.
本研究采用机械分离的大鼠海马CA3锥体神经元的“突触小体”标本，该标本可在兴奋性和抑制性单突触水平进行直接的生理和药理定量分析。通过对单个突触小体进行局灶性双脉冲电刺激来产生诱发兴奋性和抑制性突触后电流（eEPSCs和eIPSCs）。突触后CA3胞体膜中，eEPSC对河豚毒素（TTX）的敏感性高于电压依赖性Na(+)通道全细胞电流（INa）。3 nM TTX可强烈抑制突触传递，在此浓度下INa几乎未受抑制。eEPSC和INa对TTX的IC50值分别为2.8和37.9 nM，eEPSC完全抑制浓度为3 - 10 nM，INa为1000 nM。另一方面，降低细胞外Na(+)浓度（[Na]o）会使跨细胞膜的Na(+)梯度降低，从而使eEPSC和eIPSC均受到同等程度的逐渐抑制。结果表明，3 - 10 nM的TTX可阻断突触前神经末梢上的大多数电压依赖性Na(+)通道，导致依赖动作电位的兴奋性神经传递突然受到抑制。
