Gibbs J W, Sombati S, DeLorenzo R J, Coulter D A
Department of Neurology, Medical College of Virginia, Richmond 23298-0599, USA.
J Neurophysiol. 1997 Apr;77(4):2139-52. doi: 10.1152/jn.1997.77.4.2139.
Cultured rat hippocampal neurons previously exposed to a media containing no added Mg2+ for 3 h begin to spontaneously trigger recurrent epileptiform discharges following return to normal medium, and this altered population epileptiform activity persisted for the life of the neurons in culture (> 2 wk). Neurons in "epileptic" cultures appeared similar in somatic and dendritic morphology and cellular density to control, untreated cultures. In patch-clamp recordings from hippocampal pyramidal cells from "epileptic," low Mg2+ pretreated hippocampal cultures, a rapid (within 2 h of treatment), permanent (lasting > or = 8 days) and statistically significant 50-65% reduction in the current density of functional gamma-aminobutyric acid-A (GABA(A)) receptors was evident when the GABA responses of these cells were compared with control neurons. Functional GABA receptor current density was calculated by determining the maximal response of a cell to GABA 1 mM application and normalizing this response to cellular capacitance. Despite the marked GABA efficacy differences noted above, the potency of GABA in activating chloride currents was not significantly different when the responses to control and "epileptic" pyramidal cells to multiple concentrations of GABA were compared. The EC50 for GABA was 4.5 +/- 0.2 (mean +/- SE) for control neurons and 3.5 +/- 0.4 microM, 5.2 +/- 0.5 microM, 3.7 +/- 0.3 microM, and 4.6 +/- 0.3 microM for epileptic neurons 2 h, 2 days, 3 days, and 8 days after low Mg2+ pretreatment, respectively. Modulation of GABA responses by the benzodiazepine, clonazepam, was significantly reduced in epileptic neurons compared with controls. The kinetically determined clonazepam 100 nM GABA augmentation efficacy decreased from 44.1% in control neurons to 9.3% augmentation in neurons recorded from cultures 10 days posttreatment. The kinetics of GABA current block by the noncompetitive antagonist picrotoxin were determined in hippocampal cultured neurons, and an IC50 of 14 microM determined. Bath application of picrotoxin at half of the IC50 concentration (7 microM) induced epileptiform activity in control cultures and this activity appeared very similar to the epileptiform activity induced by prior low Mg2+ treatment. This concentration of picrotoxin was determined experimentally to block 30% of the GABA(A)-mediated receptor responses in these cultures, and this level of block was sufficient to trigger spontaneous epileptiform activity. The 50% reduction of GABA responses induced as a permanent consequence of low Mg2+ treatment therefore was determined to be sufficient in and of itself to induce the spontaneous epileptiform activity, which was also a consequence of this treatment.
先前在不含添加镁离子的培养基中培养3小时的大鼠海马神经元，在恢复到正常培养基后开始自发触发反复的癫痫样放电，并且这种改变的群体癫痫样活动在培养的神经元存活期内持续存在（>2周）。“癫痫”培养物中的神经元在体细胞和树突形态以及细胞密度方面与对照未处理培养物相似。在来自“癫痫”、低镁预处理海马培养物的海马锥体细胞的膜片钳记录中，当将这些细胞的GABA反应与对照神经元进行比较时，功能性γ-氨基丁酸-A（GABA(A)）受体的电流密度迅速（在处理后2小时内）、永久性（持续≥8天）且统计学上显著降低50-65%。功能性GABA受体电流密度通过确定细胞对1 mM GABA应用的最大反应并将该反应归一化到细胞电容来计算。尽管有上述明显的GABA功效差异，但当比较对照和“癫痫”锥体细胞对多种浓度GABA的反应时，GABA激活氯离子电流的效能没有显著差异。对照神经元的GABA EC50为4.5±0.2（平均值±标准误），低镁预处理后2小时、2天、3天和8天的癫痫神经元的GABA EC50分别为3.5±0.4 microM、5.2±0.5 microM、3.7±0.3 microM和4.6±0.3 microM。与对照相比，癫痫神经元中苯二氮䓬类药物氯硝西泮对GABA反应的调节作用显著降低。动力学测定的100 nM氯硝西泮增强GABA的功效从对照神经元中的44.1%降至处理后10天培养物中记录的神经元中的9.3%增强。在海马培养神经元中测定了非竞争性拮抗剂苦味毒阻断GABA电流的动力学，并确定IC50为14 microM。在对照培养物中，以IC50浓度的一半（7 microM）浴用苦味毒可诱导癫痫样活动，并且这种活动与先前低镁处理诱导的癫痫样活动非常相似。通过实验确定该浓度的苦味毒可阻断这些培养物中30%的GABA(A)介导的受体反应，并且这种阻断水平足以触发自发癫痫样活动。因此，低镁处理作为永久性后果导致的GABA反应降低50%被确定本身就足以诱导自发癫痫样活动，这也是该处理的一个后果。
