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首页> 美国卫生研究院文献>Journal of Visualized Experiments : JoVE >Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons
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Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

机译：干细胞源性中枢神经系统神经元网络化培养中生物神经毒素的功能评估

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Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.
机译：突触前靶向的梭菌神经毒素（CNTs）的治疗和机理研究受到对可扩展的，基于细胞的模型的需求的限制，该模型可产生功能性突触并接受中毒的生理反应。在这里，我们描述了一种简单而强大的方法，可以有效地将鼠类胚胎干细胞（ESC）区分为突触活性，网络神经元的定义谱系。经过8天的分化方案后，小鼠胚胎干细胞衍生的神经元（ESN）迅速表达和分隔神经型蛋白，形成神经元形态并产生内在的电响应。分化后18天（DIV 18），ESN表现出活跃的谷氨酸能和γ-氨基丁酸（GABA）能突触，并出现了以兴奋性：抑制性平衡为特征的新兴网络行为。为了确定CNT的中毒功能是否拮抗突触神经传递，从而复制负责肉毒中毒或破伤风临床表现的体内病理生理学，全细胞膜片钳电生理学用于量化ESNs中的自发性微型兴奋性突触后电流（mEPSCs）。暴露于破伤风神经毒素（TeNT）或肉毒杆菌神经毒素（BoNT）血清型/ A- / G。在所有情况下，ESN在20小时内都表现出几乎完全丧失的突触活性。静息的膜电位和固有的电反应表明，醉人的神经元仍然具有活力。为了进一步表征这种方法的敏感性，在加入BoNT / A后20小时，测定了中毒对突触活性的剂量依赖性作用。 0.005 pM BoNT / A的中毒导致mEPSC明显减少，中位抑制浓度（IC50）为0.013 pM。中值剂量的比较表明，与SNARE裂解测定或小鼠致死率测定相比，突触抑制的功能测定更快，更特异性且更灵敏。这些数据验证了突触偶联干细胞来源的神经元在CNTs的高特异性和灵敏检测中的应用。

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