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

我们用全细胞膜片钳记录的方法研究了超极化激活阳离子电流(Ih)的功能和生物学作用。1)形态特征根据电生理指标，AP神经元分为三组：1)同时显示超极化激活阳离子电流(I_H)和快速瞬时外向电流(FAST)的细胞；(2)只显示快速Ito的细胞；所有AP神经元都有一个轴突，明显比细胞的树突细。在不同组之间，树突或轴突的取向没有系统性差异。第3组神经元的细胞大小和电容的平均值显著大于其他组。有趣的是，在第1组和第3组中，发现许多细胞的树突伸入孤束核(Nts)，提示AP神经元可以…。更多的d在NTS内的树突终末接受迷走神经传入。尽管AP含有形状一致的神经元，但本研究表明AP神经元的亚群存在显著不同，这些神经元不仅在电生理上而且在形态上也具有特征。2)对ATPM的敏感性最后区神经元对ATP的应用有反应，大多数反应是兴奋性的。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性反应(内向电流；n=26/51个细胞)；2)突触前兴奋性反应(微小的兴奋性突触后电流频率增加，只有少量的突触后电流；n=24/51个细胞；或3)突触后抑制反应(外向电流；n=1/51)。在两类主要的电生理细胞，即显示i_h的细胞和不显示i_h的细胞中都有兴奋反应，而只有不显示i_h的细胞出现抑制反应，电流钳记录显示ATP引起的膜电位去极化(n=13/15)或超极化(n=2/15)调节动作电位的频率。在CNQX存在的情况下，mEPSCs被取消，而沐浴的ATP不产生mEPSCs，这表明突触前定位的ATP受体的激活促进了谷氨酸的释放。我们的药理研究结果表明，在突触后和/或突触外区域，最有可能构成P2X受体的亚基是P2X_2、P2X_5和P2X_7。我们对β、aβMe-ATP、PPADs的药理研究结果表明，在突触后和/或突触外区域，最有可能构成P2X受体的亚基是P2X_2、P2X_5和P2X_7。我们认为，突触前P2X受体中有一半很可能由P2X_2、P2X_5和P2X_7亚基组成，其余的也可能含有P2X_1亚基。众所周知，P2X_7亚基只形成同源多聚体的P2X受体。最后，本研究提示，嘌呤能受体的激活可能参与最后区神经元对多种自主神经功能的调控。较少

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