Köhr G, De Koninck Y, Mody I
Department of Neurology and Neurological Sciences, Stanford University School of Medicine, California 94305.
J Neurosci. 1993 Aug;13(8):3612-27. doi: 10.1523/JNEUROSCI.13-08-03612.1993.
The hyperexcitability accompanying chronic epileptiform activity may result from long-term alterations of ligand- and voltage-gated channels. Previous studies have indicated that NMDA responses and other electrophysiological characteristics of dentate gyrus granule cells are profoundly altered following chronic epilepsy (kindling). We have now investigated channels activated by NMDA using whole-cell patch-clamp and cell-attached single-channel recordings in granule cells acutely isolated from control and epileptic (kindled) rats. In control neurons, the amplitude of whole-cell NMDA currents was not sensitive to the presence of an intracellular ATP regeneration system, whereas NMDA currents in kindled cells showed a great variability, with larger amplitudes consistently recorded in the presence of intracellular high-energy phosphates. The ratio of peak to steady-state NMDA current (desensitization) was comparable (approximately 51%) in control and kindled neurons. Single-channel conductance determined from fluctuation analysis of whole-cell NMDA currents ranged between 21 and 35 pS in control and between 17 and 37 pS in kindled cells. Whole-cell NMDA channel noise power spectra yielded a single normal distribution of long channel lifetimes (mean, 4.3 msec) in control neurons, and the sum of two normal distributions (means, 4.6 and 7.1 msec) in kindled cells. The voltage-dependent Mg2+ block of NMDA channels was altered following kindling. From curves fitted to voltage-ramp-evoked currents in the presence of NMDA, the calculated affinity for Mg2+ of kindled channels at 0 mV was lower (12 mM) than that of controls (1.7 mM). Cell-attached recordings in the absence of Mg2+ have substantiated the lack of effect of kindling on single-channel conductance (approximately 50 pS), and have demonstrated large increases in mean open times (from 1.26 msec in control to 2.05 msec in kindled), burst lengths (from 1.91 msec to 4.18 msec), and cluster lengths (from 9.11 msec to 20.86 msec) of NMDA channels in kindled neurons. In summary, kindling, an NMDA receptor-dependent form of activity-dependent neuronal plasticity induced in vivo, results in lasting modifications in the function of single NMDA receptor channels that can be studied in acutely dissociated neurons. Kindling-induced epilepsy predominantly affects the mean open time, burst, and cluster duration of NMDA channels, their sensitivity to intracellular high-energy phosphates, and their block by Mg2+, but not the desensitization or single-channel conductance. Such alterations may reflect a change in the molecular structure of NMDA channels and may underlie the maintenance of the epileptic state.
伴随慢性癫痫样活动出现的兴奋性过高可能源于配体门控通道和电压门控通道的长期改变。先前的研究表明，慢性癫痫（点燃）后齿状回颗粒细胞的NMDA反应和其他电生理特性会发生深刻改变。我们现在使用全细胞膜片钳和细胞贴附式单通道记录技术，对从对照大鼠和癫痫（点燃）大鼠急性分离出的颗粒细胞中由NMDA激活的通道进行了研究。在对照神经元中，全细胞NMDA电流的幅度对细胞内ATP再生系统的存在不敏感，而点燃细胞中的NMDA电流表现出很大的变异性，在细胞内存在高能磷酸盐时始终记录到较大的幅度。对照神经元和点燃神经元中NMDA电流的峰值与稳态电流之比（脱敏）相当（约51%）。通过对全细胞NMDA电流的波动分析确定的单通道电导，在对照细胞中介于21至35 pS之间，在点燃细胞中介于17至37 pS之间。全细胞NMDA通道噪声功率谱在对照神经元中产生了单一的长通道寿命正态分布（平均值为4.3毫秒），在点燃细胞中产生了两个正态分布的总和（平均值分别为4.6和7.1毫秒）。点燃后NMDA通道的电压依赖性Mg2+阻断发生了改变。根据在存在NMDA的情况下对电压斜坡诱发电流拟合的曲线，计算得出点燃通道在0 mV时对Mg2+的亲和力（12 mM）低于对照通道（1.7 mM）。在不存在Mg2+的情况下进行的细胞贴附记录证实了点燃对单通道电导（约50 pS）没有影响，并证明了点燃神经元中NMDA通道的平均开放时间（从对照中的1.26毫秒增加到点燃中的2.05毫秒）、爆发长度（从1.91毫秒增加到4.18毫秒）和簇长度（从9.11毫秒增加到20.86毫秒）大幅增加。总之，点燃是一种在体内诱导的依赖NMDA受体的活动依赖性神经元可塑性形式，会导致单个NMDA受体通道功能的持久改变，这可以在急性解离的神经元中进行研究。点燃诱导的癫痫主要影响NMDA通道的平均开放时间、爆发和簇持续时间、它们对细胞内高能磷酸盐的敏感性以及它们被Mg2+的阻断，但不影响脱敏或单通道电导。这些改变可能反映了NMDA通道分子结构的变化，并可能是癫痫状态维持的基础。
