Analysis of the functional role of the hyperpolarization-activated cation cunert within the centaral nervous system.

本研究利用膜片钳技术研究了大鼠脑切片后脑区神经元的膜性质、形态和化学敏感性。结果总结如下。1)超极化激活的阳离子电流（I_h）在约62%的区域后睡眠神经元中被激活。胞外Cs +离子（2 mM）和选择性I_h通道拮抗剂ZD7288 （100 mM）阻断I_h并诱导膜电位超极化，表明I_h在静息电位附近持续激活，并且I_h在去极化水平上对维持膜电位有贡献。ZD7288通过延长两个尖峰之间的缓慢去极化来降低自发放电率，而对后超极化或动作电位波形的幅度影响最小。I_h稳定了反弹动作电位的潜伏期。外源性8-溴腺苷3,5、环单磷酸（1 mM）或…More forskolin （50-100 mM）对I_h的激活作用较弱，表明细胞内cAMP可调节区域后置细胞的I_h通道亚型。我们的研究结果表明，I_h参与大鼠脑后区神经元的阈下膜和放电特性，并可能调节其静息膜电位和放电模式。2)mEPSCs和诱发EPSCs在含有非nmda离子型受体拮抗剂CNQX （10 μM）的培养基中被完全阻断，表明EPSC是谷氨酸事件。3)约78%（35/45）的细胞对尼古丁（50 μM）有兴奋性反应。反应细胞包括显示超极化激活阳离子电流（Ih）的细胞和不显示Ih的细胞。尼古丁的抑制作用从未被发现。我们得出结论，大鼠脑后区域的尼古丁受体可以通过(1)直接的突触后和/或突触外机制激活细胞；(2)谷氨酸释放的间接增强。4)血清素（5-HT, 50 μM）或苯基双胍（PBA, 50 μM，一种有效的5-HT_3受体激动剂）在83个神经元中增加了35个（42%）自发兴奋性突触后电流（sEPSCs）或微型EPSCs （mEPSCs）的频率。这些增加发生在所有电生理细胞类别中。没有细胞EPSC频率下降。这些结果表明，突触前5-HT_3受体激活可增加脑后区域谷氨酸释放。此外，我们提供的证据表明，5-HT_3受体激活可能能够直接从末端释放谷氨酸，绕过电压依赖性钙进入末端的要求。这一机制可能与脑后区域神经元的化学敏感功能有关。5)尽管异丙酚对脑后区神经元Ih的抑制作用与海马CA1神经元相似，呈剂量依赖性，但脑后区神经元（38 mM） Ih的IC_<50>比海马CA1神经元（235 mM）的IC_<50>要小6倍以上。我们的结论是，大鼠脑后皮层神经元对异丙酚非常敏感。考虑到Ih的降低与神经元兴奋性降低有关，我们认为异丙酚麻醉的止吐作用至少部分是直接作用于区域后睡眠神经元以降低其兴奋性的结果。少

This study elucidated the membrane properties, morphology and chemosensitivities of area postrema neurons using a patch-clamp technique in rat brain slices. Results are summarized as below.1)The activation of the hyperpolarization-activated cation current (I_h) was identified in approximately 62% of area postrema neurons tested. Extracellular Cs^+ ions (2 mM) and ZD7288 (100 mM), a potent selective I_h channel antagonist, blocked I_h and induced a membrane potential hyperpolarization, suggesting the sustained activation of I_h near the resting potential and a contribution from I_h to membrane potential maintenance at more depolarized levels. ZD7288 decreased the spontaneous discharge rate by prolonging the slow depolarization between two spikes, with minimal effect on the amplitude of the afterhyperpolarization or action potential waveforms. I_h stabilized the latency of rebound action potentials. I_h was weakly activated by external 8-bromoadenosine 3,5, cyclic monophosphate (1 mM) or … More forskolin (50-100 mM), indicating that the I_h channel subtypes in area postrema cells could be modulated by intracellular cAMP. Our findings indicate that I_h contributes to the subthreshold membrane and firing properties of rat area postrema neurons and may regulate their resting membrane potential and firing patterns.2)mEPSCs and evoked EPSCs were completely blocked in media containing the non-NMDA ionotropic receptor antagonist, CNQX (10 μM), indicating that EPSC were glutamate events.3)Excitatory responses to the bath application of nicotine (50 μM) were found in approximately 78% (35/45) of all cells tested. Responsive cells included both the cells that display the hyperpolarization-activated cation current (Ih) and cells that do not display Ih. An inhibitory effect of nicotine was never seen. We conclude that nicotinic receptors in the rat area postrema can excite cells via (1)a direct post- and/or extrasynaptic mechanism ; and (2)an indirect enhancement of glutamate release.4)The bath application of serotonin (5-HT, 50 μM) or phenylbiguanide (PBA, 50 μM, a potent 5-HT_3 receptor agonist) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) or miniature EPSCs (mEPSCs) in 35 of 83 neurons (42%). These increases occurred in all electrophysiological cell classes. No cells exhibited a decrease in EPSC frequency. These results suggest that glutamate release is increased in the area postrema by presynaptic 5-HT_3 receptor activation. Further, we present evidence that 5-HT_3 receptor activation may be able to directly release glutamate from terminals, by-passing a requirement for voltage-dependent calcium entry into terminals. Such a mechanism may contribute to the chemosensitive function of area postrema neurons.5)Although propofol suppressed Ih of area postrema neurons in a dose-dependent manner that was similar to what we observed for the hippocampal CA1 neurons, the IC_<50>, for I_h in area postrema neurons (38 mM) was more than six times less than that found for hippocampal CA1 neurons (235 mM). We conclude that rat area postrema neurons are exquisitely sensitive to propofol. Given that reductions of Ih are associated with decreased excitability in neurons, we believe that the known antiemetic effects of propofol anesthesia are at least partly a result of a direct action on area postrema neurons to lower their excitability. Less