Zhu Zhiyi, Zeng Xiao-Hui, Turecek Josef, Han Victor Z, Welsh John P
Center for Integrative Brain Research, Seattle Children's Research Institute, 1900 9th Avenue, Seattle, WA, 98101, USA.
J Mol Neurosci. 2015 Feb;55(2):416-29. doi: 10.1007/s12031-014-0353-0. Epub 2014 Jun 17.
RNA interference (RNAi) to knockdown N-methyl-D-aspartate receptor (NMDAR) function is being investigated to address disorders associated with pathological brain rhythms. A motivating finding has been that pharmacological block of NMDARs inhibited oscillations in neuronal membrane potential that entrain rhythmic bursts of action potentials. To determine whether transient effects of NMDAR antagonist drugs to inhibit neuronal rhythmicity can be stably induced with genetic specificity, we examined the effects of RNAi of GluN1 protein on the subthreshold oscillations (STOs) of neurons in the inferior olive (IO), a pacemaking nucleus necessary for motor and cognitive timing. Western blot of dissociated neurons demonstrated 90% knockdown of GluN1 after a strong in vivo transduction by a dual-microRNA lentiviral vector. GluN1 RNAi in whole-cell-patched IO neurons blocked both membrane depolarization and STOs typically induced by NMDAR activation for up to 54 days without affecting input resistance, membrane capacitance, action potential firing, high-threshold Ca(2+) spikes, the hyperpolarization-activated current Ih, or the activation of the low-threshold Ca(2+) current I(T). Although an off-target effect on Cav3 expression was ruled out also by BlastN query, we found that GluN1 RNAi chronically eliminated I(T)-dependent STOs at resting membrane potential, well below the activation threshold of the NMDAR channel. In the context of a recent report showing that NMDAR activation induces STOs as it strengthens electrical coupling, the long-term block of STOs by GluN1 RNAi may relate to the loss of an essential support mechanism. Lentivector-mediated RNAi of GluN1 provides a novel technique for future investigations of NMDAR involvement in electrical oscillations and behavior.
为解决与病理性脑节律相关的疾病，人们正在研究通过RNA干扰（RNAi）来降低N-甲基-D-天冬氨酸受体（NMDAR）的功能。一个引人关注的发现是，NMDAR的药理学阻断抑制了神经元膜电位的振荡，而这种振荡会引发动作电位的节律性爆发。为了确定能否通过基因特异性稳定诱导NMDAR拮抗剂药物抑制神经元节律性的短暂效应，我们研究了GluN1蛋白的RNAi对下橄榄核（IO）神经元阈下振荡（STOs）的影响，下橄榄核是运动和认知定时所必需的起搏核。对解离神经元进行的蛋白质免疫印迹分析表明，通过双微小RNA慢病毒载体进行强烈的体内转导后，GluN1的表达降低了90%。全细胞膜片钳记录的IO神经元中，GluN1的RNAi阻断了通常由NMDAR激活诱导的膜去极化和STOs，长达54天，且不影响输入电阻、膜电容、动作电位发放、高阈值Ca(2+)尖峰、超极化激活电流Ih或低阈值Ca(2+)电流I(T)的激活。尽管通过BlastN查询也排除了对Cav3表达的脱靶效应，但我们发现GluN1的RNAi在静息膜电位下长期消除了依赖I(T)的STOs，该电位远低于NMDAR通道的激活阈值。鉴于最近有报告显示NMDAR激活通过增强电耦合诱导STOs，GluN1的RNAi对STOs的长期阻断可能与一种重要支持机制的丧失有关。慢病毒载体介导的GluN1的RNAi为未来研究NMDAR参与电振荡和行为提供了一种新技术。
