Analysis of the functional and biological roles of the hyperpolarization-activated cation current in the central nervous system,

我们采用全细胞膜片钳记录区域后睡眠神经元，以研究超极化激活阳离子电流（Ih）的功能和生物学作用。1)形态学特征根据电生理标准，将AP神经元分为三组：1)同时具有超极化激活阳离子电流（I_h）和快速瞬态外向电流（I_ < ~ >）；2)单元格只显示快速的I_< ~ >；3)细胞只显示缓慢的Ito。所有AP神经元都有一个明显比细胞树突细的轴突。在树突或轴突的方向上，各组间没有发现系统性差异。第3组神经元的细胞大小和电容平均值明显大于其他各组。有趣的是，1组和3组的许多细胞将树突延伸到孤束核（NTS），而2组则没有，这表明AP神经元可以在NTS内的树突末端接收迷走神经传入输入。虽然AP含有形状均匀的神经元，但这项研究表明，AP神经元存在显著不同的亚群，这些亚群不仅具有电生理特征，而且具有形态学特征。2)对ATP的敏感性：大多数区域的后脑神经元对ATP有反应，且大多数是兴奋性的。电压钳记录显示三种不同类型的反应：1)突触后或突触外兴奋反应（向内电流，n=26/51个细胞），2)突触前兴奋反应（只有小的直接突触后电流的微型兴奋性突触后电流频率增加，n=24/51个细胞），或3)突触后抑制反应（向外电流，n=1/51）。在显示I_h和不显示I_h的两种主要电生理细胞类别中均发现兴奋性反应，而仅在不显示I_h的细胞中发现抑制反应。电流钳记录显示atp诱导的膜电位去极化（n=13/15）或超极化（n=2/15）调节了动作电位的频率。在CNQX存在的情况下，mEPSCs被消除，而使用ATP的细胞不产生mEPSCs，这表明谷氨酸的释放是通过激活突触前定位的ATP受体来促进的。我们对ATP、a - βme-ATP、βyme-ATP和PPADS的药理研究结果表明，P2X_2、P2X_5和P2X_7是最有可能组成P2X受体的亚基，位于突触后区和/或突触外区。我们得出结论，半数的突触前P2X受体很可能由P2X_2、P2X_5和P2X_7亚基组成，而其余的也含有P2X_1亚基。众所周知，P2X_7亚基仅形成同质P2X受体。最后，本研究表明嘌呤受体的激活可能有助于控制区域后睡眠神经元的几种自主神经功能。少

We performed whole-cell patch-clamp recordings from area postrema neurons to investigate the functional and biological roles of the hyperpolarization-activated cation current (Ih).1) Morphological propertiesUsing electrophysiological criteria, AP neurons were subdivided into three groups : 1) cells displaying both the hyperpolarization-activated cation current (I_h) and the fast transient outward current (fast I_<to>) ; 2) cells displaying only the fast I_<to> ; and 3) cells displaying only the slow Ito. All AP neurons had a single axon that was distinctly thinner than the cells' dendrites. No systematic differences, across groups, in the orientation of dendrites or axons were identified. Mean values of cell size and capacitance of neurons from group 3 were significantly larger than those of the other groups. Interestingly, a number of cells from group 1 and 3 but not group 2 were found to extend their dendrites into the nucleus tractus solitarius (NTS), suggesting that AP neurons coul … More d receive vagal afferent inputs at their dendritic termini within the NTS. Although the AP have been implicated to contain uniformly shaped neurons, this study indicates the presence of significantly different subpopulations of AP neurons, which were characterized not only electrophysiologically but also morphologically.2) Sensitivity to ATPMost area postrema neurons responded to ATP application, and most responses were excitatory. Voltage-clamp recordings showed three different types of response : 1) a postsynaptic or extrasynaptic excitatory response (inward currents ; n=26/51 cells), 2) a presynaptic excitatory response (increased frequency of miniature excitatory postsynaptic currents with only a small direct postsynaptic current; n=24/51 cells, or 3) a postsynaptic inhibitory response (outward current ; n=1/51). The excitatory responses were found in both of the two major electrophysiological cell classes, i.e. cells displaying I_h and cells not displaying I_h, while the inhibitory responses were found in only cells not displaying I_h. Current-clamp recordings showed ATP-induced depolarization (n=13/15) or hyperpolarization (n=2/15) of membrane potential that modulated the frequency of action potentials. In the presence of CNQX, mEPSCs were abolished and bath applied ATP did not generate mEPSCs, indicating that glutamate release was facilitated by the activation of presynaptically located ATP receptors. Our pharmacological results from studies with ATP, aβme-ATP, βyme-ATP and PPADS indicate that P2X_2, P2X_5 and P2X_7 are the most likely subunits which compose the P2X receptor in the post-and/or extrasynaptic regions. We conclude that half of the presynaptic P2X receptors are most likely composed of P2X_2, P2X_5 and P2X_7 subunits while the others also contain P2X_1 subunits. It is well known that P2X_7 subunit forms only homomultimeric P2X receptors. Finally, the present study suggests that purinoceptor activation may contribute to the control of several autonomic functions by area postrema neurons. Less