Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), Heidelberg University, Heidelberg, Germany.
Clinic for Psychiatry and Psychotherapy, Brandenburg Medical School Theodor Fontane (MHB), Neuruppin, Germany.
J Physiol. 2020 Feb;598(4):633-650. doi: 10.1113/JP278362. Epub 2020 Feb 3.
We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ∼77 nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death.
Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35-42 postnatal days, we estimated ambient glutamate to be 72-83 nM resulting in a block of more than 82% of ES-NMDARs during a 5 min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling.
我们提出了一种新的方案来定量研究在成年大鼠急性脑切片中利用环境细胞外谷氨酸半选择性激活突触外受体的突触外 NMDA 受体功能。我们使用全细胞膜片钳技术来测量 NMDA 受体拮抗剂 MK-801 对突触和短暂局部激动剂应用引起的反应的影响。根据 NMDA 受体的持续活动，环境谷氨酸的水平估计为～77 nM，并用等效浓度的 NMDA 来估计我们的 MK-801 方案对突触外阻断的程度（＞82%）。从这些数据中可以数学上推导出总 NMDA 受体库的突触外成分，并估计在大鼠海马 CA1 区放射状层中的比例为 29-39%。这种技术可用于定量研究与突触外 NMDA 受体激活相关的神经元死亡的疾病的啮齿动物模型中的突触外 NMDA 受体功能。
在中枢神经系统的大多数兴奋性突触中，突触 NMDA 受体（NMDARs）在生存信号转导和突触可塑性中起着核心作用，而突触外 NMDARs（ES-NMDARs）则激活促凋亡途径，并与许多神经退行性疾病有关。在急性脑切片制备中，使用主要是不可逆的、活性依赖性的 NMDA 受体拮抗剂 MK-801 来阻断突触 NMDARs，从而对 ES-NMDARs 进行了表征。这种方法受到限制，因为同时 MK-801 阻断了由环境细胞外谷氨酸激活的 ES-NMDARs，由于附近谷氨酸转运体的高密度，这些谷氨酸大部分不存在于突触间隙中。在来自 35-42 日龄大鼠的急性海马切片中，我们估计环境谷氨酸为 72-83 nM，导致在 5 分钟的 MK-801 应用过程中，超过 82%的 ES-NMDARs 被阻断。本文描述了一种新的电生理学和数学方法，用于通过使用 MK-801 来优先阻断 ES-NMDARs，定量测定急性脑切片制备中局限区域内位于突触外位置的 NMDAR 比例。该方案使用全细胞膜片钳测量 NMDA 受体对突触刺激和短暂局部 NMDA 压力应用的反应，然后在应用 MK-801 前后进行测量。在对突触和突触外受体的相对阻断进行数学校正后，估计 ES-NMDARs 占海马 CA1 锥体神经元顶树突中总 NMDAR 库的 29-39%。这种新方法可能有助于准确量化与毒性 ES-NMDAR 信号增加相关的神经退行性疾病中的 NMDAR 分布。
