Whole cell patch-clamp recordings were made from CA1 stratum oriens inhibitory neurons of rat hippocampal slices in vitro to determine their contribution to the epileptiform activity elicited by elevating the extracellular potassium ion concentration ([K+]o) from 3.5 to 8.5 mM. 2. Under current-clamp conditions, spontaneous action potential activity in inhibitory neurons normally occurs in a sustained repetitive firing mode paced by nonsynaptic, intrinsic mechanisms. On elevation of [K+]o to 8.5 mM the pattern of activity is altered such that clusters of action potentials occur interrupted by periods of silence without an appreciable after hyperpolarization (AHP). In addition, elevation of [K+]o caused a large reduction in the action potential AHP amplitude and duration concomitant with a 20-mV shift in the reversal potential of the AHP. 3. In voltage clamp a small persistent inward current was observed after the introduction of elevated potassium concomitant with an increase in the frequency of spontaneous excitatory post-synaptic currents (EPSCs) in all interneurons studied. After a short period of time (approximately 1 min) temporal summation of synchronously occurring EPSCs contributed a periodic inward current (PIC; 10-40 pA, 0.8 Hz) that persisted for the duration of the [K+]o elevation. Analysis of the charge transfer associated with the PIC suggests that they comprise the temporal summation of approximately 35 EPSCs. This PIC was synchronous with the extracellular field potential recorded from the CA1 pyramidal neuron layer. 4. The PIC was responsible for the clustering of action potential activity because blockade of EPSC activity by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) abolished PICs and reverted action potential activity to single sustained firing, despite the continued application of 8.5 mM [K+]o. Antagonists of N-methyl-D-aspartate receptors were without effect on either the PICs or the action potential activity. 5. Addition of the metabotropic glutamate receptor (mGluR) antagonist (+)-2-methyl-4-carboxyphenylglycine (MCPG) reversibly abolished the PIC without affecting the increase in EPSC frequency. 6. Recordings from CA3 pyramidal neurons in 8.5 mM [K+]o demonstrated that interictal activity occurred at a frequency identical to the PICs observed in interneurons. Interictal activity in CA3 pyramidal neurons was attenuated but never abolished by MCPG, suggesting a role for mGluR receptors in the maintenance of interictal activity in area CA3.(ABSTRACT TRUNCATED AT 400 WORDS)
摘要
采用全细胞膜片钳记录技术,对体外培养的大鼠海马脑片CA1海马下托抑制性神经元进行记录,以确定其在将细胞外钾离子浓度([K+]o)从3.5 mM升高至8.5 mM所引发的癫痫样活动中的作用。2. 在电流钳条件下,抑制性神经元中的自发动作电位活动通常以由非突触性内在机制驱动的持续重复放电模式发生。当[K+]o升高至8.5 mM时,活动模式发生改变,动作电位簇出现,其间穿插着沉默期,且无明显的超极化后电位(AHP)。此外,[K+]o升高导致动作电位AHP幅度和时程大幅降低,同时AHP的反转电位发生20 mV的偏移。3. 在电压钳中,引入高钾后观察到一个小的持续性内向电流,同时在所研究的所有中间神经元中,自发兴奋性突触后电流(EPSCs)频率增加。短时间(约1分钟)后,同步出现的EPSCs的时间总和产生一个周期性内向电流(PIC;10 - 40 pA,0.8 Hz),该电流在[K+]o升高期间持续存在。对与PIC相关的电荷转移分析表明,它们由约35个EPSCs的时间总和组成。该PIC与从CA1锥体神经元层记录的细胞外场电位同步。4. PIC是动作电位活动簇集的原因,因为α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体拮抗剂6,7-二硝基喹喔啉-2,3-二酮(DNQX)阻断EPSC活动可消除PIC,并使动作电位活动恢复为单个持续放电,尽管持续施加8.5 mM [K+]o。N-甲基-D-天冬氨酸受体拮抗剂对PIC或动作电位活动均无影响。5. 添加代谢型谷氨酸受体(mGluR)拮抗剂(+)-2-甲基-4-羧基苯基甘氨酸(MCPG)可可逆地消除PIC,而不影响EPSC频率的增加。6. 在8.5 mM [K+]o条件下对CA3锥体神经元的记录表明,发作间期活动的频率与在中间神经元中观察到的PIC频率相同。MCPG可减弱但从未消除CA3锥体神经元的发作间期活动,提示mGluR受体在维持CA3区发作间期活动中起作用。(摘要截取自400字)